Functional	O
interactions	O
of	O
transforming	B-Gene_or_gene_product
growth	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
beta	I-Gene_or_gene_product
-	I-Gene_or_gene_product
activated	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
1	I-Gene_or_gene_product
with	O
IkappaB	B-Gene_or_gene_product
kinases	I-Gene_or_gene_product
to	O
stimulate	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
activation	O
.	O

Several	O
mitogen	B-Gene_or_gene_product
-	I-Gene_or_gene_product
activated	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
kinases	I-Gene_or_gene_product
play	O
critical	O
roles	O
in	O
nuclear	B-Complex
factor	I-Complex
-	I-Complex
kappaB	I-Complex
(	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
)	O
activation	O
.	O

We	O
recently	O
reported	O
that	O
the	O
overexpression	O
of	O
transforming	B-Gene_or_gene_product
growth	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
-	I-Gene_or_gene_product
beta	I-Gene_or_gene_product
-	I-Gene_or_gene_product
activated	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
1	I-Gene_or_gene_product
(	O
TAK1	B-Gene_or_gene_product
)	O
,	O
a	O
member	O
of	O
the	O
mitogen	B-Gene_or_gene_product
-	I-Gene_or_gene_product
activated	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
family	I-Gene_or_gene_product
,	O
together	O
with	O
its	O
activator	O
TAK1	B-Gene_or_gene_product
-	I-Gene_or_gene_product
binding	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
1	I-Gene_or_gene_product
(	O
TAB1	B-Gene_or_gene_product
)	O
stimulates	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
activation	O
.	O

Here	O
we	O
investigated	O
the	O
molecular	O
mechanism	O
of	O
TAK1	B-Gene_or_gene_product
-	O
induced	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
activation	O
.	O

Dominant	O
negative	O
mutants	O
of	O
IkappaB	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
(	I-Gene_or_gene_product
IKK	I-Gene_or_gene_product
)	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
and	O
IKKbeta	B-Gene_or_gene_product
inhibited	O
TAK1	B-Gene_or_gene_product
-	O
induced	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
activation	O
.	O

TAK1	B-Gene_or_gene_product
activated	O
IKKalpha	B-Gene_or_gene_product
and	O
IKKbeta	B-Gene_or_gene_product
in	O
the	O
presence	O
of	O
TAB1	B-Gene_or_gene_product
.	O

IKKalpha	B-Gene_or_gene_product
and	O
IKKbeta	B-Gene_or_gene_product
were	O
coimmunoprecipitated	O
with	O
TAK1	B-Gene_or_gene_product
in	O
the	O
absence	O
of	O
TAB1	B-Gene_or_gene_product
.	O

TAB1	B-Gene_or_gene_product
-	O
induced	O
TAK1	B-Gene_or_gene_product
activation	O
promoted	O
the	O
dissociation	O
of	O
active	O
forms	O
of	O
IKKalpha	B-Gene_or_gene_product
and	O
IKKbeta	B-Gene_or_gene_product
from	O
active	O
TAK1	B-Gene_or_gene_product
,	O
whereas	O
the	O
IKK	B-Gene_or_gene_product
mutants	O
remained	O
to	O
interact	O
with	O
active	O
TAK1	B-Gene_or_gene_product
.	O

Furthermore	O
,	O
tumor	B-Gene_or_gene_product
necrosis	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
activated	O
endogenous	O
TAK1	B-Gene_or_gene_product
,	O
and	O
the	O
kinase	O
-	O
negative	O
TAK1	B-Gene_or_gene_product
acted	O
as	O
a	O
dominant	O
negative	O
inhibitor	O
against	O
tumor	B-Gene_or_gene_product
necrosis	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
-	O
induced	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
activation	O
.	O

These	O
results	O
demonstrated	O
a	O
novel	O
signaling	O
pathway	O
to	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
activation	O
through	O
TAK1	B-Gene_or_gene_product
in	O
which	O
TAK1	B-Gene_or_gene_product
may	O
act	O
as	O
a	O
regulatory	O
kinase	O
of	O
IKKs	B-Gene_or_gene_product
.	O

Phosphorylation	O
of	O
axin	B-Gene_or_gene_product
,	O
a	O
Wnt	B-Gene_or_gene_product
signal	O
negative	O
regulator	O
,	O
by	O
glycogen	B-Gene_or_gene_product
synthase	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
-	I-Gene_or_gene_product
3beta	I-Gene_or_gene_product
regulates	O
its	O
stability	O
.	O

Axin	B-Gene_or_gene_product
forms	O
a	O
complex	O
with	O
glycogen	B-Gene_or_gene_product
synthase	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
-	I-Gene_or_gene_product
3beta	I-Gene_or_gene_product
(	O
GSK	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3beta	I-Gene_or_gene_product
)	O
and	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
and	O
promotes	O
GSK	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3beta	I-Gene_or_gene_product
-	O
dependent	O
phosphorylation	O
of	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
,	O
thereby	O
stimulating	O
the	O
degradation	O
of	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
.	O

Because	O
GSK	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3beta	I-Gene_or_gene_product
also	O
phosphorylates	O
Axin	B-Gene_or_gene_product
in	O
the	O
complex	O
,	O
the	O
physiological	O
significance	O
of	O
the	O
phosphorylation	O
of	O
Axin	B-Gene_or_gene_product
was	O
examined	O
.	O

Treatment	O
of	O
COS	O
cells	O
with	O
LiCl	O
,	O
a	O
GSK	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3beta	I-Gene_or_gene_product
inhibitor	O
,	O
and	O
okadaic	B-Simple_chemical
acid	I-Simple_chemical
,	O
a	O
protein	O
phosphatase	O
inhibitor	O
,	O
decreased	O
and	O
increased	O
,	O
respectively	O
,	O
the	O
cellular	O
protein	O
level	O
of	O
Axin	B-Gene_or_gene_product
.	O

Pulse	O
-	O
chase	O
analyses	O
showed	O
that	O
the	O
phosphorylated	O
form	O
of	O
Axin	B-Gene_or_gene_product
was	O
more	O
stable	O
than	O
the	O
unphosphorylated	O
form	O
and	O
that	O
an	O
Axin	B-Gene_or_gene_product
mutant	O
,	O
in	O
which	O
the	O
possible	O
phosphorylation	O
sites	O
for	O
GSK	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3beta	I-Gene_or_gene_product
were	O
mutated	O
,	O
exhibited	O
a	O
shorter	O
half	O
-	O
life	O
than	O
wild	O
type	O
Axin	B-Gene_or_gene_product
.	O

Dvl	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
,	O
which	O
was	O
genetically	O
shown	O
to	O
function	O
upstream	O
of	O
GSK	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3beta	I-Gene_or_gene_product
,	O
inhibited	O
the	O
phosphorylation	O
of	O
Axin	B-Gene_or_gene_product
by	O
GSK	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3beta	I-Gene_or_gene_product
in	O
vitro	O
.	O

Furthermore	O
,	O
Wnt	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3a	I-Gene_or_gene_product
-	O
containing	O
conditioned	O
medium	O
down	O
-	O
regulated	O
Axin	B-Gene_or_gene_product
and	O
accumulated	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
in	O
L	O
cells	O
and	O
expression	O
of	O
Dvl	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
(	I-Gene_or_gene_product
DeltaPDZ	I-Gene_or_gene_product
)	I-Gene_or_gene_product
,	O
in	O
which	O
the	O
PDZ	O
domain	O
was	O
deleted	O
,	O
suppressed	O
this	O
action	O
of	O
Wnt	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3a	I-Gene_or_gene_product
.	O

These	O
results	O
suggest	O
that	O
the	O
phosphorylation	O
of	O
Axin	B-Gene_or_gene_product
is	O
important	O
for	O
the	O
regulation	O
of	O
its	O
stability	O
and	O
that	O
Wnt	B-Gene_or_gene_product
down	O
-	O
regulates	O
Axin	B-Gene_or_gene_product
through	O
Dvl	B-Gene_or_gene_product
.	O

Activation	O
of	O
p53	B-Gene_or_gene_product
and	O
its	O
target	O
genes	O
p21	B-Gene_or_gene_product
(	O
WAF1	B-Gene_or_gene_product
/	O
Cip1	B-Gene_or_gene_product
)	O
and	O
PAG608	B-Gene_or_gene_product
/	O
Wig	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
in	O
ischemic	O
preconditioning	O
.	O

A	O
brief	O
,	O
3	O
min	O
period	O
of	O
global	O
forebrain	O
ischemia	O
in	O
the	O
rat	O
,	O
induced	O
by	O
bilateral	O
common	O
carotid	O
occlusion	O
combined	O
with	O
hypotension	O
,	O
confers	O
resistance	O
to	O
hippocampal	O
pyramidal	O
neurons	O
against	O
a	O
subsequent	O
10	O
min	O
ischemia	O
,	O
which	O
is	O
normally	O
lethal	O
to	O
these	O
cells	O
.	O

The	O
molecular	O
mechanisms	O
underlying	O
this	O
ischemic	O
preconditioning	O
,	O
or	O
tolerance	O
,	O
are	O
poorly	O
understood	O
.	O

The	O
tumor	O
suppressor	O
p53	B-Gene_or_gene_product
is	O
a	O
transcription	O
factor	O
implicated	O
in	O
neuronal	O
death	O
following	O
various	O
insults	O
,	O
including	O
cerebral	O
ischemia	O
.	O

p53	B-Gene_or_gene_product
is	O
activated	O
in	O
response	O
to	O
cellular	O
stress	O
,	O
e	O
.	O
g	O
.	O
hypoxia	O
and	O
DNA	O
damage	O
.	O

Using	O
in	O
situ	O
hybridization	O
,	O
we	O
investigated	O
the	O
hippocampal	O
mRNA	O
expression	O
of	O
p53	B-Gene_or_gene_product
,	O
and	O
two	O
of	O
its	O
target	O
genes	O
,	O
p21	B-Gene_or_gene_product
(	O
WAF1	B-Gene_or_gene_product
/	O
Cip1	B-Gene_or_gene_product
)	O
and	O
the	O
recently	O
cloned	O
PAG608	B-Gene_or_gene_product
/	O
Wig	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
,	O
in	O
a	O
two	O
-	O
vessel	O
occlusion	O
model	O
of	O
ischemic	O
preconditioning	O
.	O

We	O
also	O
evaluated	O
changes	O
in	O
the	O
protein	O
levels	O
of	O
p53	B-Gene_or_gene_product
and	O
PAG608	B-Gene_or_gene_product
/	O
Wig	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
using	O
immunohistochemistry	O
.	O

The	O
mRNA	O
levels	O
of	O
all	O
three	O
genes	O
increased	O
in	O
the	O
ischemia	O
sensitive	O
CA1	O
region	O
both	O
following	O
3	O
min	O
(	O
non	O
-	O
lethal	O
)	O
preconditioning	O
and	O
10	O
min	O
of	O
(	O
lethal	O
)	O
nonconditioned	O
ischemia	O
.	O

In	O
contrast	O
,	O
after	O
10	O
min	O
of	O
ischemia	O
preconditioned	O
by	O
a	O
3	O
min	O
ischemic	O
insult	O
48	O
h	O
earlier	O
,	O
no	O
upregulation	O
of	O
these	O
genes	O
was	O
detected	O
in	O
the	O
CA1	O
.	O

Following	O
10	O
min	O
of	O
nonconditioned	O
ischemia	O
,	O
increased	O
neuronal	O
immunostaining	O
of	O
p53	B-Gene_or_gene_product
and	O
PAG608	B-Gene_or_gene_product
/	O
Wig	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
was	O
observed	O
in	O
the	O
hippocampus	O
,	O
which	O
was	O
less	O
pronounced	O
following	O
3	O
min	O
of	O
preconditioning	O
ischemia	O
and	O
10	O
min	O
of	O
preconditioned	O
ischemia	O
.	O

Our	O
results	O
demonstrate	O
that	O
activation	O
of	O
p53	B-Gene_or_gene_product
and	O
its	O
response	O
genes	O
p21	B-Gene_or_gene_product
(	O
WAF1	B-Gene_or_gene_product
/	O
Cip1	B-Gene_or_gene_product
)	O
and	O
PAG608	B-Gene_or_gene_product
/	O
Wig	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
occurs	O
in	O
the	O
brain	O
following	O
lethal	O
as	O
well	O
as	O
non	O
-	O
lethal	O
ischemic	O
insults	O
,	O
and	O
that	O
ischemic	O
preconditioning	O
markedly	O
diminishes	O
this	O
activation	O
.	O

A	O
GSK3beta	B-Gene_or_gene_product
phosphorylation	O
site	O
in	O
axin	B-Gene_or_gene_product
modulates	O
interaction	O
with	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
and	O
Tcf	B-Gene_or_gene_product
-	O
mediated	O
gene	O
expression	O
.	O

Upon	O
binding	O
of	O
a	O
Wnt	B-Gene_or_gene_product
to	O
its	O
receptor	O
,	O
GSK3beta	B-Gene_or_gene_product
is	O
inhibited	O
through	O
an	O
unknown	O
mechanism	O
involving	O
Dishevelled	B-Gene_or_gene_product
(	O
Dsh	B-Gene_or_gene_product
)	O
,	O
resulting	O
in	O
the	O
dephosphorylation	O
and	O
stabilization	O
of	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
,	O
which	O
translocates	O
to	O
the	O
nucleus	B-Cellular_component
and	O
interacts	O
with	O
Lef	B-Gene_or_gene_product
/	O
Tcf	B-Gene_or_gene_product
transcription	O
factors	O
to	O
activate	O
target	O
gene	O
expression	O
.	O

Axin	B-Gene_or_gene_product
is	O
a	O
scaffold	O
protein	O
which	O
binds	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
and	O
GSK3beta	B-Gene_or_gene_product
(	O
as	O
well	O
as	O
several	O
other	O
proteins	O
)	O
and	O
thus	O
promotes	O
the	O
phosphorylation	O
of	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
.	O

Here	O
we	O
report	O
that	O
Axin	B-Gene_or_gene_product
is	O
phosphorylated	O
on	O
Ser	B-Simple_chemical
and	O
Thr	B-Simple_chemical
residues	O
in	O
several	O
regions	O
in	O
vivo	O
,	O
while	O
only	O
one	O
region	O
(	O
amino	B-Simple_chemical
acids	I-Simple_chemical
600	I-Simple_chemical
-	I-Simple_chemical
672	I-Simple_chemical
)	O
is	O
efficiently	O
phosphorylated	O
by	O
GSK3beta	B-Gene_or_gene_product
in	O
vitro	O
.	O

Site	O
-	O
directed	O
mutagenesis	O
,	O
together	O
with	O
in	O
vitro	O
and	O
in	O
vivo	O
phosphorylation	O
assays	O
,	O
demonstrates	O
that	O
Axin	B-Gene_or_gene_product
residues	O
T609	B-Simple_chemical
and	O
S614	B-Simple_chemical
are	O
physiological	O
GSK3beta	B-Gene_or_gene_product
targets	O
.	O

Substitutions	O
for	O
one	O
or	O
more	O
of	O
these	O
residues	O
,	O
which	O
lie	O
within	O
a	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
binding	O
site	O
,	O
reduce	O
the	O
ability	O
of	O
Axin	B-Gene_or_gene_product
to	O
modulate	O
Wnt	B-Gene_or_gene_product
-	O
induced	O
signaling	O
in	O
a	O
Lef	B-Gene_or_gene_product
/	O
Tcf	B-Gene_or_gene_product
reporter	O
assay	O
.	O

These	O
amino	O
acid	O
substitutions	O
also	O
reduce	O
the	O
binding	O
between	O
Axin	B-Gene_or_gene_product
and	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
.	O

We	O
propose	O
a	O
model	O
in	O
which	O
inhibition	O
of	O
GSK3beta	B-Gene_or_gene_product
activity	O
upon	O
Wnt	B-Gene_or_gene_product
signaling	O
leads	O
to	O
the	O
dephosphorylation	O
of	O
GSK3beta	B-Gene_or_gene_product
sites	O
in	O
Axin	B-Gene_or_gene_product
,	O
resulting	O
in	O
the	O
release	O
of	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
from	O
the	O
phosphorylation	O
complex	O
.	O

Novel	O
inositol	B-Gene_or_gene_product
polyphosphate	I-Gene_or_gene_product
5	I-Gene_or_gene_product
-	I-Gene_or_gene_product
phosphatase	I-Gene_or_gene_product
localizes	O
at	O
membrane	B-Cellular_component
ruffles	I-Cellular_component
.	O

We	O
have	O
cloned	O
a	O
novel	O
inositol	B-Gene_or_gene_product
polyphosphate	I-Gene_or_gene_product
5	I-Gene_or_gene_product
-	I-Gene_or_gene_product
phosphatase	I-Gene_or_gene_product
from	O
the	O
rat	O
brain	O
cDNA	O
library	O
.	O

It	O
contains	O
two	O
highly	O
conserved	O
5	B-Gene_or_gene_product
-	I-Gene_or_gene_product
phosphatase	I-Gene_or_gene_product
motifs	O
,	O
both	O
of	O
which	O
are	O
essential	O
for	O
its	O
enzymatic	O
activity	O
.	O

Interestingly	O
,	O
the	O
proline	B-Simple_chemical
content	O
of	O
this	O
protein	O
is	O
high	O
and	O
concentrated	O
in	O
its	O
N	O
-	O
and	O
C	O
-	O
terminal	O
regions	O
.	O

One	O
putative	O
SH3	O
-	O
binding	O
motif	O
and	O
six	O
14	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3	I-Gene_or_gene_product
-	I-Gene_or_gene_product
3	I-Gene_or_gene_product
zeta	I-Gene_or_gene_product
-	O
binding	O
motifs	O
were	O
found	O
in	O
the	O
amino	O
acid	O
sequence	O
.	O

This	O
enzyme	O
hydrolyzed	O
phosphate	B-Simple_chemical
at	O
the	O
D	O
-	O
5	O
position	O
of	O
inositol	B-Simple_chemical
1	I-Simple_chemical
,	I-Simple_chemical
4	I-Simple_chemical
,	I-Simple_chemical
5	I-Simple_chemical
-	I-Simple_chemical
trisphosphate	I-Simple_chemical
,	O
inositol	B-Simple_chemical
1	I-Simple_chemical
,	I-Simple_chemical
3	I-Simple_chemical
,	I-Simple_chemical
4	I-Simple_chemical
,	I-Simple_chemical
5	I-Simple_chemical
-	I-Simple_chemical
tetrakisphosphate	I-Simple_chemical
,	O
and	O
phosphatidylinositol	B-Simple_chemical
4	I-Simple_chemical
,	I-Simple_chemical
5	I-Simple_chemical
-	I-Simple_chemical
bisphosphate	I-Simple_chemical
,	O
consistent	O
with	O
the	O
substrate	O
specificity	O
of	O
type	B-Gene_or_gene_product
II	I-Gene_or_gene_product
5	I-Gene_or_gene_product
-	I-Gene_or_gene_product
phosphatase	I-Gene_or_gene_product
,	O
OCRL	B-Gene_or_gene_product
,	O
synaptojanin	B-Gene_or_gene_product
and	O
synaptojanin	B-Gene_or_gene_product
2	I-Gene_or_gene_product
,	O
already	O
characterized	O
5	B-Gene_or_gene_product
-	I-Gene_or_gene_product
phosphatases	I-Gene_or_gene_product
.	O

When	O
the	O
Myc	B-Gene_or_gene_product
-	O
epitope	O
-	O
tagged	O
enzyme	O
was	O
expressed	O
in	O
COS	O
-	O
7	O
cells	O
and	O
stained	O
with	O
anti	O
-	O
Myc	B-Gene_or_gene_product
polyclonal	O
antibody	O
,	O
a	O
signal	O
was	O
observed	O
at	O
ruffling	B-Cellular_component
membranes	I-Cellular_component
and	O
in	O
the	O
cytoplasm	B-Cellular_component
.	O

We	O
prepared	O
several	O
deletion	O
mutants	O
and	O
demonstrated	O
that	O
the	O
123	O
N	O
-	O
terminal	O
amino	O
acids	O
(	O
311	O
-	O
433	O
)	O
and	O
a	O
C	O
-	O
terminal	O
proline	B-Simple_chemical
-	O
rich	O
region	O
containing	O
277	O
amino	O
acids	O
(	O
725	O
-	O
1001	O
)	O
were	O
essential	O
for	O
its	O
localization	O
to	O
ruffling	B-Cellular_component
membranes	I-Cellular_component
.	O

This	O
enzyme	O
might	O
regulate	O
the	O
level	O
of	O
inositol	B-Simple_chemical
and	O
phosphatidylinositol	B-Simple_chemical
polyphosphates	I-Simple_chemical
at	O
membrane	B-Cellular_component
ruffles	I-Cellular_component
.	O

The	O
I	B-Complex
kappa	I-Complex
B	I-Complex
kinase	I-Complex
(	O
IKK	B-Complex
)	O
and	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
:	O
key	O
elements	O
of	O
proinflammatory	O
signalling	O
.	O

NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
is	O
a	O
heterodimeric	O
transcription	O
factor	O
that	O
plays	O
a	O
key	O
role	O
in	O
inflammatory	O
and	O
immune	O
responses	O
.	O

In	O
nonstimulated	O
cells	O
,	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
dimers	O
are	O
maintained	O
in	O
the	O
cytoplasm	B-Cellular_component
through	O
interaction	O
with	O
inhibitory	O
proteins	O
,	O
the	O
I	B-Gene_or_gene_product
kappa	I-Gene_or_gene_product
Bs	I-Gene_or_gene_product
.	O

In	O
response	O
to	O
cell	O
stimulation	O
,	O
mainly	O
by	O
proinflammatory	O
cytokines	O
,	O
a	O
multisubunit	O
protein	O
kinase	O
,	O
the	O
I	B-Complex
kappa	I-Complex
B	I-Complex
kinase	I-Complex
(	O
IKK	B-Complex
)	O
,	O
is	O
rapidly	O
activated	O
and	O
phosphorylates	O
two	O
critical	O
serines	O
in	O
the	O
N	O
-	O
terminal	O
regulatory	O
domain	O
of	O
the	O
I	B-Gene_or_gene_product
kappa	I-Gene_or_gene_product
Bs	I-Gene_or_gene_product
.	O

Phosphorylated	O
I	B-Gene_or_gene_product
kappa	I-Gene_or_gene_product
Bs	I-Gene_or_gene_product
are	O
recognized	O
by	O
a	O
specific	O
E3	O
ubiquitin	B-Gene_or_gene_product
ligase	O
complex	O
and	O
undergo	O
polyubiquitination	O
which	O
targets	O
them	O
for	O
rapid	O
degradation	O
by	O
the	O
26S	B-Cellular_component
proteasome	I-Cellular_component
.	O

NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
dimers	O
,	O
which	O
are	O
spared	O
from	O
degradation	O
,	O
translocate	O
to	O
the	O
nucleus	B-Cellular_component
to	O
activate	O
gene	O
transcription	O
.	O

There	O
is	O
strong	O
biochemical	O
and	O
genetic	O
evidence	O
that	O
the	O
IKK	B-Complex
complex	O
,	O
which	O
consists	O
of	O
two	O
catalytic	O
subunits	O
,	O
IKK	B-Gene_or_gene_product
alpha	I-Gene_or_gene_product
and	O
IKK	B-Gene_or_gene_product
beta	I-Gene_or_gene_product
,	O
and	O
a	O
regulatory	O
subunit	O
,	O
IKK	B-Gene_or_gene_product
gamma	I-Gene_or_gene_product
,	O
is	O
the	O
master	O
regulator	O
of	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
-	O
mediated	O
innate	O
immune	O
and	O
inflammatory	O
responses	O
.	O

In	O
the	O
absence	O
of	O
IKK	B-Gene_or_gene_product
gamma	I-Gene_or_gene_product
,	O
which	O
normally	O
connects	O
IKK	B-Complex
to	O
upstream	O
activators	O
,	O
no	O
IKK	B-Complex
or	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
activation	O
can	O
occur	O
.	O

Surprisingly	O
,	O
however	O
,	O
of	O
the	O
two	O
catalytic	O
subunits	O
,	O
only	O
IKK	B-Gene_or_gene_product
beta	I-Gene_or_gene_product
is	O
essential	O
for	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
activation	O
in	O
response	O
to	O
proinflammatory	O
stimuli	O
.	O

The	O
second	O
catalytic	O
subunit	O
,	O
IKK	B-Gene_or_gene_product
alpha	I-Gene_or_gene_product
,	O
plays	O
a	O
critical	O
role	O
in	O
developmental	O
processes	O
,	O
in	O
particular	O
formation	O
and	O
differentiation	O
of	O
the	O
epidermis	O
.	O

Forkhead	B-Gene_or_gene_product
genes	O
in	O
transcriptional	O
silencing	O
,	O
cell	O
morphology	O
and	O
the	O
cell	O
cycle	O
.	O

Overlapping	O
and	O
distinct	O
functions	O
for	O
FKH1	B-Gene_or_gene_product
and	O
FKH2	B-Gene_or_gene_product
in	O
Saccharomyces	O
cerevisiae	O
.	O

The	O
SIR1	B-Gene_or_gene_product
gene	O
is	O
one	O
of	O
four	O
specialized	O
genes	O
in	O
Saccharomyces	O
cerevisiae	O
required	O
for	O
repressing	O
transcription	O
at	O
the	O
silent	O
mating	O
-	O
type	O
cassettes	O
,	O
HMLalpha	B-Gene_or_gene_product
and	O
HMRa	B-Gene_or_gene_product
,	O
by	O
a	O
mechanism	O
known	O
as	O
silencing	O
.	O

Silencing	O
requires	O
the	O
assembly	O
of	O
a	O
specialized	O
chromatin	B-Cellular_component
structure	O
analogous	O
to	O
heterochromatin	B-Cellular_component
.	O

FKH1	B-Gene_or_gene_product
was	O
isolated	O
as	O
a	O
gene	O
that	O
,	O
when	O
expressed	O
in	O
multiple	O
copies	O
,	O
could	O
substitute	O
for	O
the	O
function	O
of	O
SIR1	B-Gene_or_gene_product
in	O
silencing	O
HMRa	B-Gene_or_gene_product
.	O

FKH1	B-Gene_or_gene_product
(	O
Forkhead	B-Gene_or_gene_product
Homologue	I-Gene_or_gene_product
One	I-Gene_or_gene_product
)	O
was	O
named	O
for	O
its	O
homology	O
to	O
the	O
forkhead	O
family	O
of	O
eukaryotic	O
transcription	O
factors	O
classified	O
on	O
the	O
basis	O
of	O
a	O
conserved	O
DNA	O
binding	O
domain	O
.	O

Deletion	O
of	O
FKH1	B-Gene_or_gene_product
caused	O
a	O
defect	O
in	O
silencing	O
HMRa	B-Gene_or_gene_product
,	O
indicating	O
that	O
FKH1	B-Gene_or_gene_product
has	O
a	O
positive	O
role	O
in	O
silencing	O
.	O

Significantly	O
,	O
deletion	O
of	O
both	O
FKH1	B-Gene_or_gene_product
and	O
its	O
closest	O
homologue	O
in	O
yeast	O
,	O
FKH2	B-Gene_or_gene_product
,	O
caused	O
a	O
form	O
of	O
yeast	O
pseudohyphal	O
growth	O
,	O
indicating	O
that	O
the	O
two	O
genes	O
have	O
redundant	O
functions	O
in	O
controlling	O
yeast	O
cell	O
morphology	O
.	O

By	O
several	O
criteria	O
,	O
fkh1Delta	B-Gene_or_gene_product
fkh2Delta	B-Gene_or_gene_product
-	O
induced	O
pseudohyphal	O
growth	O
was	O
distinct	O
from	O
the	O
nutritionally	O
induced	O
form	O
of	O
pseudohyphal	O
growth	O
observed	O
in	O
some	O
strains	O
of	O
S	O
.	O
cerevisiae	O
.	O

Although	O
FKH2	B-Gene_or_gene_product
is	O
redundant	O
with	O
FKH1	B-Gene_or_gene_product
in	O
controlling	O
pseudohyphal	O
growth	O
,	O
the	O
two	O
genes	O
have	O
different	O
functions	O
in	O
silencing	O
HMRa	B-Gene_or_gene_product
.	O

High	O
-	O
copy	O
expression	O
of	O
CLB2	B-Gene_or_gene_product
,	O
a	O
G2	O
/	O
M	O
-	O
phase	O
cyclin	B-Gene_or_gene_product
,	O
prevented	O
fkh1Delta	B-Gene_or_gene_product
fkh2Delta	B-Gene_or_gene_product
-	O
induced	O
pseudohyphal	O
growth	O
and	O
modulated	O
some	O
of	O
the	O
fkhDelta	B-Gene_or_gene_product
-	O
induced	O
silencing	O
phenotypes	O
.	O

Interestingly	O
,	O
deletions	O
in	O
either	O
FKH1	B-Gene_or_gene_product
or	O
FKH2	B-Gene_or_gene_product
alone	O
caused	O
subtle	O
but	O
opposite	O
effects	O
on	O
cell	O
-	O
cycle	O
progression	O
and	O
CLB2	B-Gene_or_gene_product
mRNA	O
expression	O
,	O
consistent	O
with	O
a	O
role	O
for	O
each	O
of	O
these	O
genes	O
in	O
modulating	O
the	O
cell	O
cycle	O
and	O
having	O
opposing	O
effects	O
on	O
silencing	O
.	O

The	O
differences	O
between	O
Fkh1p	B-Gene_or_gene_product
and	O
Fkh2p	B-Gene_or_gene_product
in	O
vivo	O
were	O
not	O
attributable	O
to	O
differences	O
in	O
their	O
DNA	O
binding	O
domains	O
.	O

Analysis	O
of	O
promoter	O
binding	O
by	O
the	O
E2F	B-Gene_or_gene_product
and	O
pRB	B-Gene_or_gene_product
families	O
in	O
vivo	O
:	O
distinct	O
E2F	B-Gene_or_gene_product
proteins	O
mediate	O
activation	O
and	O
repression	O
.	O

The	O
E2F	B-Gene_or_gene_product
transcription	O
factor	O
plays	O
a	O
pivotal	O
role	O
in	O
the	O
timely	O
activation	O
of	O
gene	O
expression	O
during	O
mammalian	O
cell	O
cycle	O
progression	O
,	O
whereas	O
pRB	B-Gene_or_gene_product
and	O
related	O
proteins	O
control	O
cell	O
growth	O
in	O
part	O
through	O
the	O
ability	O
to	O
block	O
the	O
action	O
of	O
E2F	B-Gene_or_gene_product
.	O

To	O
identify	O
physiologically	O
important	O
E2F	B-Gene_or_gene_product
-	O
responsive	O
promoters	O
and	O
to	O
study	O
their	O
occupancy	O
and	O
histone	B-Gene_or_gene_product
acetylation	O
state	O
in	O
vivo	O
,	O
we	O
have	O
taken	O
advantage	O
of	O
a	O
cross	O
-	O
linking	O
approach	O
in	O
synchronized	O
,	O
living	O
cells	O
.	O

We	O
find	O
that	O
the	O
pattern	O
of	O
E2F	B-Gene_or_gene_product
and	O
pRB	B-Gene_or_gene_product
-	O
related	O
polypeptides	O
recruited	O
to	O
these	O
promoters	O
changes	O
in	O
a	O
strikingly	O
dynamic	O
fashion	O
as	O
cells	O
progress	O
from	O
quiescence	O
into	O
G	O
(	O
1	O
)	O
and	O
S	O
phase	O
:	O
Repression	O
of	O
each	O
promoter	O
in	O
quiescent	O
cells	O
is	O
associated	O
with	O
recruitment	O
of	O
E2F	B-Gene_or_gene_product
-	I-Gene_or_gene_product
4	I-Gene_or_gene_product
and	O
p130	B-Gene_or_gene_product
and	O
low	O
levels	O
of	O
histone	B-Gene_or_gene_product
acetylation	O
,	O
but	O
by	O
late	O
G	O
(	O
1	O
)	O
,	O
these	O
proteins	O
are	O
replaced	O
largely	O
by	O
E2F	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
and	O
E2F	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3	I-Gene_or_gene_product
,	O
in	O
concert	O
with	O
acetylation	O
of	O
histones	B-Gene_or_gene_product
H3	I-Gene_or_gene_product
and	O
H4	B-Gene_or_gene_product
and	O
gene	O
activation	O
.	O

These	O
findings	O
suggest	O
that	O
repression	O
and	O
activation	O
of	O
E2F	B-Gene_or_gene_product
-	O
responsive	O
genes	O
may	O
occur	O
through	O
distinct	O
E2F	B-Complex
heterodimers	I-Complex
that	O
direct	O
the	O
sequential	O
recruitment	O
of	O
enzymes	O
able	O
to	O
deacetylate	O
and	O
then	O
acetylate	O
core	O
histones	B-Gene_or_gene_product
.	O

Analysis	O
of	O
p73	B-Gene_or_gene_product
in	O
human	O
borderline	O
and	O
invasive	O
ovarian	O
tumor	O
.	O

p73	B-Gene_or_gene_product
is	O
a	O
novel	O
gene	O
that	O
has	O
high	O
sequence	O
homology	O
and	O
similar	O
gene	O
structure	O
to	O
the	O
tumor	O
suppressor	O
gene	O
p53	B-Gene_or_gene_product
.	O

We	O
analysed	O
p73	B-Gene_or_gene_product
in	O
seven	O
ovarian	O
carcinoma	O
cell	O
lines	O
and	O
a	O
total	O
of	O
63	O
human	O
borderline	O
and	O
invasive	O
ovarian	O
tumor	O
samples	O
.	O

Loss	O
of	O
heterozygosity	O
at	O
this	O
locus	O
was	O
observed	O
in	O
50	O
%	O
of	O
invasive	O
tumors	O
but	O
in	O
none	O
of	O
the	O
borderline	O
tumors	O
.	O

Biallelic	O
expression	O
of	O
the	O
gene	O
was	O
observed	O
in	O
the	O
heterozygous	O
tumor	O
tissues	O
.	O

Direct	O
sequencing	O
and	O
single	O
-	O
strand	O
conformation	O
polymorphism	O
analyses	O
of	O
the	O
p73	B-Gene_or_gene_product
cDNA	O
sequence	O
homologous	O
to	O
the	O
highly	O
mutatable	O
region	O
of	O
p53	B-Gene_or_gene_product
did	O
not	O
reveal	O
any	O
mutations	O
.	O

When	O
compared	O
to	O
the	O
primary	O
cultures	O
of	O
normal	O
human	O
ovarian	O
surface	O
epithelial	O
cells	O
and	O
immortalized	O
cell	O
lines	O
,	O
four	O
of	O
the	O
seven	O
ovarian	O
carcinoma	O
cell	O
lines	O
,	O
71	O
%	O
of	O
the	O
invasive	O
tumors	O
,	O
and	O
92	O
%	O
of	O
the	O
borderline	O
tumor	O
tissues	O
express	O
elevated	O
levels	O
of	O
p73	B-Gene_or_gene_product
transcript	O
.	O

Except	O
for	O
the	O
OVCA3	O
cell	O
line	O
,	O
Western	O
blot	O
analysis	O
of	O
the	O
nuclear	B-Cellular_component
extracts	O
prepared	O
from	O
the	O
cell	O
lines	O
showed	O
concordant	O
levels	O
of	O
p73	B-Gene_or_gene_product
protein	O
.	O

Our	O
analysis	O
also	O
demonstrated	O
the	O
expression	O
of	O
a	O
spliced	O
variant	O
of	O
p73	B-Gene_or_gene_product
transcript	O
with	O
the	O
omission	O
of	O
exon	O
2	O
solely	O
in	O
the	O
cancer	O
cell	O
lines	O
and	O
invasive	O
tumor	O
tissues	O
.	O

This	O
exon	O
2	O
-	O
spliced	O
transcript	O
would	O
give	O
rise	O
to	O
a	O
truncated	O
p73	B-Gene_or_gene_product
protein	O
without	O
the	O
N	O
-	O
terminal	O
transactivation	O
domain	O
.	O

In	O
reminiscence	O
of	O
the	O
dominant	O
negative	O
phenotype	O
of	O
the	O
N	O
-	O
terminal	O
truncated	O
variants	O
of	O
another	O
p53	B-Gene_or_gene_product
-	O
related	O
gene	O
,	O
p63	B-Gene_or_gene_product
,	O
the	O
expression	O
of	O
the	O
truncated	O
p73	B-Gene_or_gene_product
variant	O
form	O
in	O
ovarian	O
tumors	O
may	O
play	O
an	O
important	O
role	O
in	O
the	O
pathogenesis	O
of	O
ovarian	O
cancer	O
.	O

Phosphorylation	O
of	O
the	O
MEKK	B-Gene_or_gene_product
Ste11p	B-Gene_or_gene_product
by	O
the	O
PAK	B-Gene_or_gene_product
-	O
like	O
kinase	O
Ste20p	B-Gene_or_gene_product
is	O
required	O
for	O
MAP	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
signaling	O
in	O
vivo	O
.	O

BACKGROUND	O
:	O
Many	O
signals	O
are	O
transduced	O
from	O
the	O
cell	B-Cellular_component
surface	I-Cellular_component
to	O
the	O
nucleus	B-Cellular_component
through	O
mitogen	B-Gene_or_gene_product
-	I-Gene_or_gene_product
activated	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
(	I-Gene_or_gene_product
MAP	I-Gene_or_gene_product
)	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
cascades	O
.	O

Activation	O
of	O
MAP	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
requires	O
phosphorylation	O
by	O
MEK	B-Gene_or_gene_product
,	O
which	O
in	O
turn	O
is	O
controlled	O
by	O
Raf	B-Gene_or_gene_product
,	O
Mos	B-Gene_or_gene_product
or	O
a	O
group	O
of	O
structurally	O
related	O
kinases	O
termed	O
MEKKs	B-Gene_or_gene_product
.	O

It	O
is	O
not	O
understood	O
how	O
MEKKs	B-Gene_or_gene_product
are	O
regulated	O
by	O
extracellular	O
signals	O
.	O

In	O
yeast	O
,	O
the	O
MEKK	B-Gene_or_gene_product
Ste11p	B-Gene_or_gene_product
functions	O
in	O
multiple	O
MAP	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
cascades	O
activated	O
in	O
response	O
to	O
pheromones	O
,	O
high	O
osmolarity	O
and	O
nutrient	O
starvation	O
.	O

Genetic	O
evidence	O
suggests	O
that	O
the	O
p21	B-Gene_or_gene_product
-	I-Gene_or_gene_product
activated	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
(	O
PAK	B-Gene_or_gene_product
)	O
Ste20p	B-Gene_or_gene_product
functions	O
upstream	O
of	O
Ste11p	B-Gene_or_gene_product
,	O
and	O
Ste20p	B-Gene_or_gene_product
has	O
been	O
shown	O
to	O
phosphorylate	O
Ste11p	B-Gene_or_gene_product
in	O
vitro	O
.	O

RESULTS	O
:	O
Ste20p	B-Gene_or_gene_product
phosphorylated	O
Ste11p	B-Gene_or_gene_product
on	O
Ser302	B-Simple_chemical
and	O
/	O
or	O
Ser306	B-Simple_chemical
and	O
Thr307	B-Simple_chemical
in	O
yeast	O
,	O
residues	O
that	O
are	O
conserved	O
in	O
MEKKs	B-Gene_or_gene_product
of	O
other	O
organisms	O
.	O

Mutating	O
these	O
sites	O
to	O
non	O
-	O
phosphorylatable	O
residues	O
abolished	O
Ste11p	B-Gene_or_gene_product
function	O
,	O
whereas	O
changing	O
them	O
to	O
aspartic	B-Simple_chemical
acid	I-Simple_chemical
to	O
mimic	O
the	O
phosphorylated	O
form	O
constitutively	O
activated	O
Ste11p	B-Gene_or_gene_product
in	O
vivo	O
in	O
a	O
Ste20p	B-Gene_or_gene_product
-	O
independent	O
manner	O
.	O

The	O
amino	O
-	O
terminal	O
regulatory	O
domain	O
of	O
Ste11p	B-Gene_or_gene_product
interacted	O
with	O
its	O
catalytic	O
domain	O
,	O
and	O
overexpression	O
of	O
a	O
small	O
amino	O
-	O
terminal	O
fragment	O
of	O
Ste11p	B-Gene_or_gene_product
was	O
able	O
to	O
inhibit	O
signaling	O
in	O
response	O
to	O
pheromones	O
.	O

Mutational	O
analysis	O
suggested	O
that	O
this	O
interaction	O
was	O
regulated	O
by	O
phosphorylation	O
and	O
dependent	O
on	O
Thr596	B-Simple_chemical
,	O
which	O
is	O
located	O
in	O
the	O
substrate	O
cleft	O
of	O
the	O
catalytic	O
domain	O
.	O

CONCLUSIONS	O
:	O
Our	O
results	O
suggest	O
that	O
,	O
in	O
response	O
to	O
multiple	O
extracellular	O
signals	O
,	O
phosphorylation	O
of	O
Ste11p	B-Gene_or_gene_product
by	O
Ste20p	B-Gene_or_gene_product
removes	O
an	O
amino	O
-	O
terminal	O
inhibitory	O
domain	O
,	O
leading	O
to	O
activation	O
of	O
the	O
Ste11	B-Gene_or_gene_product
protein	O
kinase	O
.	O

This	O
mechanism	O
may	O
serve	O
as	O
a	O
paradigm	O
for	O
the	O
activation	O
of	O
mammalian	O
MEKKs	B-Gene_or_gene_product
.	O

GLUT8	B-Gene_or_gene_product
is	O
a	O
glucose	B-Simple_chemical
transporter	O
responsible	O
for	O
insulin	B-Gene_or_gene_product
-	O
stimulated	O
glucose	B-Simple_chemical
uptake	O
in	O
the	O
blastocyst	O
.	O

Mammalian	O
preimplantation	O
blastocysts	O
exhibit	O
insulin	B-Gene_or_gene_product
-	O
stimulated	O
glucose	B-Simple_chemical
uptake	O
despite	O
the	O
absence	O
of	O
the	O
only	O
known	O
insulin	B-Gene_or_gene_product
-	O
regulated	O
transporter	O
,	O
GLUT4	B-Gene_or_gene_product
.	O

We	O
describe	O
a	O
previously	O
unidentified	O
member	O
of	O
the	O
mammalian	O
facilitative	O
GLUT	B-Gene_or_gene_product
superfamily	O
that	O
exhibits	O
approximately	O
20	O
-	O
25	O
%	O
identity	O
with	O
other	O
murine	O
facilitative	O
GLUTs	B-Gene_or_gene_product
.	O

Insulin	B-Gene_or_gene_product
induces	O
a	O
change	O
in	O
the	O
intracellular	B-Cellular_component
localization	O
of	O
this	O
protein	O
,	O
which	O
translates	O
into	O
increased	O
glucose	B-Simple_chemical
uptake	O
into	O
the	O
blastocyst	O
,	O
a	O
process	O
that	O
is	O
inhibited	O
by	O
antisense	O
oligoprobes	O
.	O

Presence	O
of	O
this	O
transporter	O
may	O
be	O
necessary	O
for	O
successful	O
blastocyst	O
development	O
,	O
fuel	O
metabolism	O
,	O
and	O
subsequent	O
implantation	O
.	O

Moreover	O
,	O
the	O
existence	O
of	O
an	O
alternative	O
transporter	O
may	O
explain	O
examples	O
in	O
other	O
tissues	O
of	O
insulin	B-Gene_or_gene_product
-	O
regulated	O
glucose	B-Simple_chemical
transport	O
in	O
the	O
absence	O
of	O
GLUT4	B-Gene_or_gene_product
.	O

Regulation	O
of	O
the	O
yeast	O
transcriptional	O
factor	O
PHO2	B-Gene_or_gene_product
activity	O
by	O
phosphorylation	O
.	O

The	O
induction	O
of	O
yeast	O
Saccharomyces	O
cerevisiae	O
gene	O
PHO5	B-Gene_or_gene_product
expression	O
is	O
mediated	O
by	O
transcriptional	O
factors	O
PHO2	B-Gene_or_gene_product
and	O
PHO4	B-Gene_or_gene_product
.	O

PHO4	B-Gene_or_gene_product
protein	O
has	O
been	O
reported	O
to	O
be	O
phosphorylated	O
and	O
inactivated	O
by	O
a	O
cyclin	B-Complex
-	I-Complex
CDK	I-Complex
(	O
cyclin	B-Gene_or_gene_product
-	I-Gene_or_gene_product
dependent	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
)	O
complex	O
,	O
PHO80	B-Complex
-	I-Complex
PHO85	I-Complex
.	O

We	O
report	O
here	O
that	O
PHO2	B-Gene_or_gene_product
can	O
also	O
be	O
phosphorylated	O
.	O

A	O
Ser	B-Simple_chemical
-	I-Simple_chemical
230	I-Simple_chemical
to	O
Ala	B-Simple_chemical
mutation	O
in	O
the	O
consensus	O
sequence	O
(	O
SPIK	O
)	O
recognized	O
by	O
cdc2	B-Gene_or_gene_product
/	I-Gene_or_gene_product
CDC28	I-Gene_or_gene_product
-	I-Gene_or_gene_product
related	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
in	O
PHO2	B-Gene_or_gene_product
protein	O
led	O
to	O
complete	O
loss	O
of	O
its	O
ability	O
to	O
activate	O
the	O
transcription	O
of	O
PHO5	B-Gene_or_gene_product
gene	O
.	O

Further	O
investigation	O
showed	O
that	O
the	O
Pro	B-Simple_chemical
-	I-Simple_chemical
231	I-Simple_chemical
to	O
Ser	B-Simple_chemical
mutation	O
inactivated	O
PHO2	B-Gene_or_gene_product
protein	O
as	O
well	O
,	O
whereas	O
the	O
Ser	B-Simple_chemical
-	I-Simple_chemical
230	I-Simple_chemical
to	O
Asp	B-Simple_chemical
mutation	O
did	O
not	O
affect	O
PHO2	B-Gene_or_gene_product
activity	O
.	O

Since	O
the	O
PHO2	B-Gene_or_gene_product
Asp	B-Simple_chemical
-	I-Simple_chemical
230	I-Simple_chemical
mutant	O
mimics	O
Ser	B-Simple_chemical
-	I-Simple_chemical
230	I-Simple_chemical
-	O
phosphorylated	O
PHO2	B-Gene_or_gene_product
,	O
we	O
postulate	O
that	O
only	O
phosphorylated	O
PHO2	B-Gene_or_gene_product
protein	O
could	O
activate	O
the	O
transcription	O
of	O
PHO5	B-Gene_or_gene_product
gene	O
.	O

Two	O
hybrid	O
assays	O
showed	O
that	O
yeast	O
CDC28	B-Gene_or_gene_product
could	O
interact	O
with	O
PHO2	B-Gene_or_gene_product
.	O

CDC28	B-Gene_or_gene_product
immunoprecipitate	O
derived	O
from	O
the	O
YPH499	O
strain	O
grown	O
under	O
low	O
phosphate	B-Simple_chemical
conditions	O
phosphorylated	O
GST	B-Gene_or_gene_product
-	I-Gene_or_gene_product
PHO2	I-Gene_or_gene_product
in	O
vitro	O
.	O

A	O
phosphate	B-Simple_chemical
switch	O
regulates	O
the	O
transcriptional	O
activation	O
activity	O
of	O
PHO2	B-Gene_or_gene_product
,	O
and	O
mutations	O
of	O
the	O
(	O
SPIK	O
)	O
site	O
affect	O
the	O
transcriptional	O
activation	O
activity	O
of	O
PHO2	B-Gene_or_gene_product
and	O
the	O
interaction	O
between	O
PHO2	B-Gene_or_gene_product
and	O
PHO4	B-Gene_or_gene_product
.	O

BIAcore	O
(	O
R	O
)	O
analysis	O
indicated	O
that	O
the	O
negative	O
charge	O
in	O
residue	B-Simple_chemical
230	I-Simple_chemical
of	O
PHO2	B-Gene_or_gene_product
was	O
sufficient	O
to	O
help	O
PHO2	B-Gene_or_gene_product
interact	O
with	O
PHO4	B-Gene_or_gene_product
in	O
vitro	O
.	O

Requirement	O
for	O
glycogen	B-Gene_or_gene_product
synthase	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
-	I-Gene_or_gene_product
3beta	I-Gene_or_gene_product
in	O
cell	O
survival	O
and	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
activation	O
.	O

Glycogen	B-Gene_or_gene_product
synthase	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
-	I-Gene_or_gene_product
3	I-Gene_or_gene_product
(	I-Gene_or_gene_product
GSK	I-Gene_or_gene_product
-	I-Gene_or_gene_product
3	I-Gene_or_gene_product
)	I-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
and	O
-	B-Gene_or_gene_product
beta	I-Gene_or_gene_product
are	O
closely	O
related	O
protein	O
-	O
serine	B-Simple_chemical
kinases	O
,	O
which	O
act	O
as	O
inhibitory	O
components	O
of	O
Wnt	B-Gene_or_gene_product
signalling	O
during	O
embryonic	O
development	O
and	O
cell	O
proliferation	O
in	O
adult	O
tissues	O
.	O

Insight	O
into	O
the	O
physiological	O
function	O
of	O
GSK	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3	I-Gene_or_gene_product
has	O
emerged	O
from	O
genetic	O
analysis	O
in	O
Drosophila	O
,	O
Dictyostelium	O
and	O
yeast	O
.	O

Here	O
we	O
show	O
that	O
disruption	O
of	O
the	O
murine	O
GSK	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3beta	I-Gene_or_gene_product
gene	O
results	O
in	O
embryonic	O
lethality	O
caused	O
by	O
severe	O
liver	O
degeneration	O
during	O
mid	O
-	O
gestation	O
,	O
a	O
phenotype	O
consistent	O
with	O
excessive	O
tumour	B-Gene_or_gene_product
necrosis	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
(	O
TNF	B-Gene_or_gene_product
)	O
toxicity	O
,	O
as	O
observed	O
in	O
mice	O
lacking	O
genes	O
involved	O
in	O
the	O
activation	O
of	O
the	O
transcription	O
factor	O
activation	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
.	O

GSK	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3beta	I-Gene_or_gene_product
-	O
deficient	O
embryos	O
were	O
rescued	O
by	O
inhibition	O
of	O
TNF	B-Gene_or_gene_product
using	O
an	O
anti	O
-	O
TNF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
antibody	O
.	O

Fibroblasts	O
from	O
GSK	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3beta	I-Gene_or_gene_product
-	O
deficient	O
embryos	O
were	O
hypersensitive	O
to	O
TNF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
and	O
showed	O
reduced	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
function	O
.	O

Lithium	B-Simple_chemical
treatment	O
(	O
which	O
inhibits	O
GSK	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3	I-Gene_or_gene_product
;	O
refs	O
8	O
,	O
9	O
)	O
sensitized	O
wild	O
-	O
type	O
fibroblasts	O
to	O
TNF	B-Gene_or_gene_product
and	O
inhibited	O
transactivation	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
.	O

The	O
early	O
steps	O
leading	O
to	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
activation	O
(	O
degradation	O
of	O
I	B-Gene_or_gene_product
-	I-Gene_or_gene_product
kappaB	I-Gene_or_gene_product
and	O
translocation	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
to	O
the	O
nucleus	B-Cellular_component
)	O
were	O
unaffected	O
by	O
the	O
loss	O
of	O
GSK	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3beta	I-Gene_or_gene_product
,	O
indicating	O
that	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
is	O
regulated	O
by	O
GSK	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3beta	I-Gene_or_gene_product
at	O
the	O
level	O
of	O
the	O
transcriptional	O
complex	O
.	O

Thus	O
,	O
GSK	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3beta	I-Gene_or_gene_product
facilitates	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
function	O
.	O

Marathon	O
running	O
transiently	O
increases	O
c	B-Gene_or_gene_product
-	I-Gene_or_gene_product
Jun	I-Gene_or_gene_product
NH2	I-Gene_or_gene_product
-	I-Gene_or_gene_product
terminal	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
and	O
p38	B-Gene_or_gene_product
activities	O
in	O
human	O
skeletal	O
muscle	O
.	O

We	O
examined	O
the	O
pattern	O
of	O
activation	O
and	O
deactivation	O
of	O
the	O
stress	B-Gene_or_gene_product
-	I-Gene_or_gene_product
activated	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
signalling	O
molecules	O
c	B-Gene_or_gene_product
-	I-Gene_or_gene_product
Jun	I-Gene_or_gene_product
NH2	I-Gene_or_gene_product
-	I-Gene_or_gene_product
terminal	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
(	O
JNK	B-Gene_or_gene_product
)	O
and	O
p38	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
in	O
skeletal	O
muscle	O
in	O
response	O
to	O
prolonged	O
strenuous	O
running	O
exercise	O
in	O
human	O
subjects	O
.	O

Male	O
subjects	O
(	O
n	O
=	O
14	O
;	O
age	O
32	O
+	O
/	O
-	O
2	O
years	O
;	O
VO2	O
,	O
max	O
60	O
+	O
/	O
-	O
2	O
ml	O
kg	O
-	O
1	O
min	O
-	O
1	O
)	O
completed	O
a	O
42	O
.	O
2	O
km	O
marathon	O
(	O
mean	O
race	O
time	O
3	O
h	O
35	O
min	O
)	O
.	O

Muscle	O
biopsies	O
were	O
obtained	O
10	O
days	O
prior	O
to	O
the	O
marathon	O
,	O
immediately	O
following	O
the	O
race	O
,	O
and	O
1	O
,	O
3	O
and	O
5	O
days	O
after	O
the	O
race	O
.	O

The	O
activation	O
of	O
JNK	B-Gene_or_gene_product
and	O
p38	B-Gene_or_gene_product
,	O
including	O
both	O
p38alpha	B-Gene_or_gene_product
and	O
p38gamma	B-Gene_or_gene_product
,	O
was	O
measured	O
with	O
immune	O
complex	O
assays	O
.	O

The	O
phosphorylation	O
state	O
of	O
p38	B-Gene_or_gene_product
(	O
alpha	B-Gene_or_gene_product
and	O
gamma	B-Gene_or_gene_product
)	O
and	O
the	O
upstream	O
regulators	O
of	O
JNK	B-Gene_or_gene_product
and	O
p38	B-Gene_or_gene_product
,	O
mitogen	B-Gene_or_gene_product
-	I-Gene_or_gene_product
activated	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
4	I-Gene_or_gene_product
(	O
MKK4	B-Gene_or_gene_product
)	O
and	O
mitogen	B-Gene_or_gene_product
-	I-Gene_or_gene_product
activated	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
6	I-Gene_or_gene_product
(	O
MKK6	B-Gene_or_gene_product
)	O
,	O
were	O
assessed	O
using	O
phosphospecific	O
antibodies	O
.	O

JNK	B-Gene_or_gene_product
activity	O
increased	O
7	O
-	O
fold	O
over	O
basal	O
level	O
immediately	O
post	O
-	O
exercise	O
,	O
but	O
decreased	O
back	O
to	O
basal	O
levels	O
1	O
,	O
3	O
and	O
5	O
days	O
after	O
the	O
exercise	O
.	O

p38gamma	B-Gene_or_gene_product
phosphorylation	O
(	O
4	O
-	O
fold	O
)	O
and	O
activity	O
(	O
1	O
.	O
5	O
-	O
fold	O
)	O
increased	O
immediately	O
post	O
-	O
exercise	O
and	O
returned	O
to	O
basal	O
levels	O
at	O
1	O
,	O
3	O
and	O
5	O
days	O
following	O
exercise	O
.	O

In	O
contrast	O
,	O
p38alpha	B-Gene_or_gene_product
phosphorylation	O
and	O
activity	O
did	O
not	O
change	O
over	O
the	O
time	O
course	O
studied	O
.	O

MKK4	B-Gene_or_gene_product
and	O
MKK6	B-Gene_or_gene_product
phosphorylation	O
increased	O
and	O
decreased	O
in	O
a	O
trend	O
similar	O
to	O
that	O
observed	O
with	O
JNK	B-Gene_or_gene_product
activity	O
and	O
p38gamma	B-Gene_or_gene_product
phosphorylation	O
.	O

Prolonged	O
running	O
exercise	O
did	O
not	O
affect	O
JNK	B-Gene_or_gene_product
,	O
p38alpha	B-Gene_or_gene_product
,	O
or	O
p38gamma	B-Gene_or_gene_product
protein	O
expression	O
in	O
the	O
days	O
following	O
the	O
race	O
.	O

This	O
study	O
demonstrates	O
that	O
both	O
JNK	B-Gene_or_gene_product
and	O
p38	B-Gene_or_gene_product
intracellular	B-Cellular_component
signalling	O
cascades	O
are	O
robustly	O
,	O
yet	O
transiently	O
increased	O
following	O
prolonged	O
running	O
exercise	O
.	O

The	O
differential	O
activation	O
of	O
the	O
p38	B-Gene_or_gene_product
isoforms	O
with	O
exercise	O
in	O
human	O
skeletal	O
muscle	O
indicates	O
that	O
these	O
proteins	O
may	O
have	O
distinct	O
functions	O
in	O
vivo	O
.	O

Ataxia	B-Gene_or_gene_product
telangiectasia	I-Gene_or_gene_product
-	I-Gene_or_gene_product
mutated	I-Gene_or_gene_product
phosphorylates	O
Chk2	B-Gene_or_gene_product
in	O
vivo	O
and	O
in	O
vitro	O
.	O

The	O
protein	O
kinase	O
Chk2	B-Gene_or_gene_product
,	O
the	O
mammalian	O
homolog	O
of	O
the	O
budding	O
yeast	O
Rad53	B-Gene_or_gene_product
and	O
fission	O
yeast	O
Cds1	B-Gene_or_gene_product
checkpoint	O
kinases	O
,	O
is	O
phosphorylated	O
and	O
activated	O
in	O
response	O
to	O
DNA	O
damage	O
by	O
ionizing	O
radiation	O
(	O
IR	O
)	O
,	O
UV	O
irradiation	O
,	O
and	O
replication	O
blocks	O
by	O
hydroxyurea	B-Simple_chemical
(	O
HU	B-Simple_chemical
)	O
.	O

Phosphorylation	O
and	O
activation	O
of	O
Chk2	B-Gene_or_gene_product
are	O
ataxia	B-Gene_or_gene_product
telangiectasia	I-Gene_or_gene_product
-	I-Gene_or_gene_product
mutated	I-Gene_or_gene_product
(	O
ATM	B-Gene_or_gene_product
)	O
dependent	O
in	O
response	O
to	O
IR	O
,	O
whereas	O
Chk2	B-Gene_or_gene_product
phosphorylation	O
is	O
ATM	B-Gene_or_gene_product
-	O
independent	O
when	O
cells	O
are	O
exposed	O
to	O
UV	O
or	O
HU	B-Simple_chemical
.	O

Here	O
we	O
show	O
that	O
in	O
vitro	O
,	O
ATM	B-Gene_or_gene_product
phosphorylates	O
the	O
Ser	B-Simple_chemical
-	O
Gln	B-Simple_chemical
/	O
Thr	B-Simple_chemical
-	O
Gln	B-Simple_chemical
(	O
SQ	B-Simple_chemical
/	O
TQ	B-Simple_chemical
)	O
cluster	O
domain	O
(	O
SCD	O
)	O
on	O
Chk2	B-Gene_or_gene_product
,	O
which	O
contains	O
seven	O
SQ	B-Simple_chemical
/	O
TQ	B-Simple_chemical
motifs	O
,	O
and	O
Thr68	B-Simple_chemical
is	O
the	O
major	O
in	O
vitro	O
phosphorylation	O
site	O
by	O
ATM	B-Gene_or_gene_product
.	O

ATM	B-Gene_or_gene_product
-	I-Gene_or_gene_product
and	I-Gene_or_gene_product
Rad3	I-Gene_or_gene_product
-	I-Gene_or_gene_product
related	I-Gene_or_gene_product
also	O
phosphorylates	O
Thr68	B-Simple_chemical
in	O
addition	O
to	O
Thr26	B-Simple_chemical
and	O
Ser50	B-Simple_chemical
,	O
which	O
are	O
not	O
phosphorylated	O
to	O
a	O
significant	O
extent	O
by	O
ATM	B-Gene_or_gene_product
in	O
vitro	O
.	O

In	O
vivo	O
,	O
Thr68	B-Simple_chemical
is	O
phosphorylated	O
in	O
an	O
ATM	B-Gene_or_gene_product
-	O
dependent	O
manner	O
in	O
response	O
to	O
IR	O
,	O
but	O
not	O
in	O
response	O
to	O
UV	O
or	O
HU	B-Simple_chemical
.	O

Substitution	O
of	O
Thr68	B-Simple_chemical
with	O
Ala	B-Simple_chemical
reduced	O
the	O
extent	O
of	O
phosphorylation	O
and	O
activation	O
of	O
Chk2	B-Gene_or_gene_product
in	O
response	O
to	O
IR	O
,	O
and	O
mutation	O
of	O
all	O
seven	O
SQ	B-Simple_chemical
/	O
TQ	B-Simple_chemical
motifs	O
blocked	O
all	O
phosphorylation	O
and	O
activation	O
of	O
Chk2	B-Gene_or_gene_product
after	O
IR	O
.	O

These	O
results	O
suggest	O
that	O
in	O
vivo	O
,	O
Chk2	B-Gene_or_gene_product
is	O
directly	O
phosphorylated	O
by	O
ATM	B-Gene_or_gene_product
in	O
response	O
to	O
IR	O
and	O
that	O
Chk2	B-Gene_or_gene_product
is	O
regulated	O
by	O
phosphorylation	O
of	O
the	O
SCD	O
.	O

Structure	O
of	O
PAK1	B-Gene_or_gene_product
in	O
an	O
autoinhibited	O
conformation	O
reveals	O
a	O
multistage	O
activation	O
switch	O
.	O

The	O
p21	B-Gene_or_gene_product
-	I-Gene_or_gene_product
activated	I-Gene_or_gene_product
kinases	I-Gene_or_gene_product
(	O
PAKs	B-Gene_or_gene_product
)	O
,	O
stimulated	O
by	O
binding	O
with	O
GTP	B-Simple_chemical
-	O
liganded	O
forms	O
of	O
Cdc42	B-Gene_or_gene_product
or	O
Rac	B-Gene_or_gene_product
,	O
modulate	O
cytoskeletal	B-Cellular_component
actin	B-Gene_or_gene_product
assembly	O
and	O
activate	O
MAP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
pathways	O
.	O

The	O
2	O
.	O
3	O
A	O
resolution	O
crystal	O
structure	O
of	O
a	O
complex	O
between	O
the	O
N	O
-	O
terminal	O
autoregulatory	O
fragment	O
and	O
the	O
C	O
-	O
terminal	O
kinase	O
domain	O
of	O
PAK1	B-Gene_or_gene_product
shows	O
that	O
GTPase	O
binding	O
will	O
trigger	O
a	O
series	O
of	O
conformational	O
changes	O
,	O
beginning	O
with	O
disruption	O
of	O
a	O
PAK1	B-Complex
dimer	I-Complex
and	O
ending	O
with	O
rearrangement	O
of	O
the	O
kinase	O
active	O
site	O
into	O
a	O
catalytically	O
competent	O
state	O
.	O

An	O
inhibitory	O
switch	O
(	O
IS	O
)	O
domain	O
,	O
which	O
overlaps	O
the	O
GTPase	O
binding	O
region	O
of	O
PAK1	B-Gene_or_gene_product
,	O
positions	O
a	O
polypeptide	O
segment	O
across	O
the	O
kinase	O
cleft	O
.	O

GTPase	O
binding	O
will	O
refold	O
part	O
of	O
the	O
IS	O
domain	O
and	O
unfold	O
the	O
rest	O
.	O

A	O
related	O
switch	O
has	O
been	O
seen	O
in	O
the	O
Wiskott	B-Gene_or_gene_product
-	I-Gene_or_gene_product
Aldrich	I-Gene_or_gene_product
syndrome	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
(	O
WASP	B-Gene_or_gene_product
)	O
.	O

Activation	O
of	O
the	O
IkappaB	B-Complex
kinase	I-Complex
complex	O
by	O
TRAF6	B-Gene_or_gene_product
requires	O
a	O
dimeric	O
ubiquitin	B-Gene_or_gene_product
-	O
conjugating	O
enzyme	O
complex	O
and	O
a	O
unique	O
polyubiquitin	B-Gene_or_gene_product
chain	O
.	O

TRAF6	B-Gene_or_gene_product
is	O
a	O
signal	O
transducer	O
in	O
the	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
pathway	O
that	O
activates	O
IkappaB	B-Complex
kinase	I-Complex
(	O
IKK	B-Complex
)	O
in	O
response	O
to	O
proinflammatory	O
cytokines	O
.	O

We	O
have	O
purified	O
a	O
heterodimeric	O
protein	O
complex	O
that	O
links	O
TRAF6	B-Gene_or_gene_product
to	O
IKK	B-Complex
activation	O
.	O

Peptide	O
mass	O
fingerprinting	O
analysis	O
reveals	O
that	O
this	O
complex	O
is	O
composed	O
of	O
the	O
ubiquitin	B-Gene_or_gene_product
conjugating	O
enzyme	O
Ubc13	B-Gene_or_gene_product
and	O
the	O
Ubc	B-Gene_or_gene_product
-	O
like	O
protein	O
Uev1A	B-Gene_or_gene_product
.	O

We	O
find	O
that	O
TRAF6	B-Gene_or_gene_product
,	O
a	O
RING	O
domain	O
protein	O
,	O
functions	O
together	O
with	O
Ubc13	B-Gene_or_gene_product
/	O
Uev1A	B-Gene_or_gene_product
to	O
catalyze	O
the	O
synthesis	O
of	O
unique	O
polyubiquitin	B-Gene_or_gene_product
chains	O
linked	O
through	O
lysine	B-Simple_chemical
-	I-Simple_chemical
63	I-Simple_chemical
(	O
K63	B-Simple_chemical
)	O
of	O
ubiquitin	B-Gene_or_gene_product
.	O

Blockade	O
of	O
this	O
polyubiquitin	B-Gene_or_gene_product
chain	O
synthesis	O
,	O
but	O
not	O
inhibition	O
of	O
the	O
proteasome	B-Cellular_component
,	O
prevents	O
the	O
activation	O
of	O
IKK	B-Complex
by	O
TRAF6	B-Gene_or_gene_product
.	O

These	O
results	O
unveil	O
a	O
new	O
regulatory	O
function	O
for	O
ubiquitin	B-Gene_or_gene_product
,	O
in	O
which	O
IKK	B-Complex
is	O
activated	O
through	O
the	O
assembly	O
of	O
K63	B-Simple_chemical
-	O
linked	O
polyubiquitin	B-Gene_or_gene_product
chains	O
.	O

TAK1	B-Gene_or_gene_product
regulates	O
multiple	O
protein	O
kinase	O
cascades	O
activated	O
by	O
bacterial	O
lipopolysaccharide	B-Simple_chemical
.	O

During	O
inflammation	O
the	O
balance	O
between	O
cell	O
activation	O
and	O
cell	O
death	O
is	O
determined	O
by	O
the	O
tight	O
regulation	O
of	O
multiple	O
intracellular	B-Cellular_component
enzyme	O
cascades	O
.	O

Key	O
regulatory	O
steps	O
often	O
involve	O
protein	O
kinases	O
.	O

We	O
show	O
that	O
the	O
prototypical	O
pro	O
-	O
inflammatory	O
molecule	O
,	O
bacterial	O
lipopolysaccharide	B-Simple_chemical
,	O
activates	O
multiple	O
protein	O
kinases	O
such	O
as	O
p38	B-Gene_or_gene_product
,	O
JNK	B-Gene_or_gene_product
,	O
IKK	B-Gene_or_gene_product
-	I-Gene_or_gene_product
beta	I-Gene_or_gene_product
,	O
and	O
PKB	B-Gene_or_gene_product
/	O
Akt	B-Gene_or_gene_product
via	O
transforming	B-Gene_or_gene_product
growth	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
beta	I-Gene_or_gene_product
-	I-Gene_or_gene_product
activated	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
(	O
TAK1	B-Gene_or_gene_product
)	O
.	O

We	O
also	O
show	O
that	O
TAK1	B-Gene_or_gene_product
plays	O
an	O
important	O
role	O
in	O
similar	O
activation	O
pathways	O
triggered	O
by	O
interleukin	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
.	O

Thus	O
TAK1	B-Gene_or_gene_product
must	O
be	O
considered	O
as	O
an	O
important	O
component	O
of	O
intracellular	B-Cellular_component
pathways	O
in	O
cells	O
involved	O
in	O
host	O
responses	O
to	O
physiological	O
and	O
/	O
or	O
environmental	O
stress	O
signals	O
during	O
inflammation	O
.	O

Cloning	O
,	O
Golgi	B-Cellular_component
localization	O
,	O
and	O
enzyme	O
activity	O
of	O
the	O
full	O
-	O
length	O
heparin	B-Simple_chemical
/	O
heparan	B-Simple_chemical
sulfate	I-Simple_chemical
-	O
glucuronic	B-Simple_chemical
acid	I-Simple_chemical
C5	B-Gene_or_gene_product
-	I-Gene_or_gene_product
epimerase	I-Gene_or_gene_product
.	O

While	O
studying	O
the	O
cellular	O
localization	O
and	O
activity	O
of	O
enzymes	O
involved	O
in	O
heparan	B-Simple_chemical
sulfate	I-Simple_chemical
biosynthesis	O
,	O
we	O
discovered	O
that	O
the	O
published	O
sequence	O
for	O
the	O
glucuronic	B-Simple_chemical
acid	I-Simple_chemical
C5	B-Gene_or_gene_product
-	I-Gene_or_gene_product
epimerase	I-Gene_or_gene_product
responsible	O
for	O
the	O
interconversion	O
of	O
d	B-Simple_chemical
-	I-Simple_chemical
glucuronic	I-Simple_chemical
acid	I-Simple_chemical
and	O
l	B-Simple_chemical
-	I-Simple_chemical
iduronic	I-Simple_chemical
acid	I-Simple_chemical
residues	O
encodes	O
a	O
truncated	O
protein	O
.	O

Genome	O
analysis	O
and	O
5	O
'	O
-	O
rapid	O
amplification	O
of	O
cDNA	O
ends	O
was	O
used	O
to	O
clone	O
the	O
full	O
-	O
length	O
cDNA	O
from	O
a	O
mouse	O
mastocytoma	O
cell	O
line	O
.	O

The	O
extended	O
cDNA	O
encodes	O
for	O
an	O
additional	O
174	O
amino	O
acids	O
at	O
the	O
amino	O
terminus	O
of	O
the	O
protein	O
.	O

The	O
murine	O
sequence	O
is	O
95	O
%	O
identical	O
to	O
the	O
human	O
epimerase	B-Gene_or_gene_product
identified	O
from	O
genomic	O
sequences	O
and	O
fits	O
with	O
the	O
general	O
size	O
and	O
structure	O
of	O
the	O
gene	O
from	O
Drosophila	O
melanogaster	O
and	O
Caenorhabditis	O
elegans	O
.	O

Full	O
-	O
length	O
epimerase	B-Gene_or_gene_product
is	O
predicted	O
to	O
have	O
a	O
type	O
II	O
transmembrane	B-Cellular_component
topology	O
with	O
a	O
17	O
-	O
amino	O
acid	O
transmembrane	B-Cellular_component
domain	O
and	O
an	O
11	O
-	O
amino	O
acid	O
cytoplasmic	B-Cellular_component
tail	O
.	O

An	O
assay	O
with	O
increased	O
sensitivity	O
was	O
devised	O
that	O
detects	O
enzyme	O
activity	O
in	O
extracts	O
prepared	O
from	O
cultured	O
cells	O
and	O
in	O
recombinant	O
proteins	O
.	O

Unlike	O
other	O
enzymes	O
involved	O
in	O
glycosaminoglycan	B-Simple_chemical
biosynthesis	O
,	O
the	O
addition	O
of	O
a	O
c	B-Gene_or_gene_product
-	I-Gene_or_gene_product
myc	I-Gene_or_gene_product
tag	O
or	O
green	B-Gene_or_gene_product
fluorescent	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
to	O
the	O
highly	O
conserved	O
COOH	O
-	O
terminal	O
portion	O
of	O
the	O
protein	O
inhibits	O
its	O
activity	O
.	O

The	O
amino	O
-	O
terminally	O
truncated	O
epimerase	B-Gene_or_gene_product
does	O
not	O
localize	O
to	O
any	O
cellular	B-Cellular_component
compartment	I-Cellular_component
,	O
whereas	O
the	O
full	O
-	O
length	O
enzyme	O
is	O
in	O
the	O
Golgi	B-Cellular_component
,	O
where	O
heparan	B-Simple_chemical
sulfate	I-Simple_chemical
synthesis	O
is	O
thought	O
to	O
occur	O
.	O

Human	O
immunodeficiency	O
virus	O
type	O
1	O
Tat	B-Gene_or_gene_product
protein	O
decreases	O
cyclic	B-Simple_chemical
AMP	I-Simple_chemical
synthesis	O
in	O
rat	O
microglia	O
cultures	O
.	O

We	O
have	O
studied	O
the	O
modulation	O
of	O
cyclic	B-Simple_chemical
AMP	I-Simple_chemical
(	O
cAMP	B-Simple_chemical
)	O
accumulation	O
by	O
the	O
human	O
immunodeficiency	O
virus	O
type	O
1	O
(	O
HIV	O
1	O
)	O
protein	O
Tat	B-Gene_or_gene_product
in	O
microglia	O
and	O
astrocyte	O
cultures	O
obtained	O
from	O
neonatal	O
rat	O
brain	O
.	O

Pretreatment	O
of	O
microglia	O
with	O
recombinant	O
Tat	B-Gene_or_gene_product
resulted	O
in	O
a	O
dose	O
-	O
and	O
time	O
-	O
dependent	O
decrease	O
of	O
cAMP	B-Simple_chemical
accumulation	O
induced	O
by	O
subsequent	O
exposure	O
to	O
isoproterenol	B-Simple_chemical
(	O
1	O
microM	O
)	O
.	O

The	O
inhibitory	O
action	O
of	O
100	O
ng	O
/	O
mL	O
Tat	B-Gene_or_gene_product
approached	O
50	O
%	O
after	O
4	O
h	O
of	O
preincubation	O
and	O
reached	O
a	O
maximum	O
of	O
70	O
%	O
after	O
24	O
h	O
.	O

The	O
Tat	B-Gene_or_gene_product
-	O
induced	O
time	O
-	O
and	O
dose	O
-	O
dependent	O
decrease	O
of	O
cAMP	B-Simple_chemical
accumulation	O
was	O
observed	O
also	O
when	O
microglial	O
cultures	O
were	O
stimulated	O
with	O
the	O
adenylyl	B-Gene_or_gene_product
cyclase	I-Gene_or_gene_product
activator	O
forskolin	B-Simple_chemical
(	O
100	O
microM	O
)	O
.	O

In	O
both	O
cases	O
,	O
Tat	B-Gene_or_gene_product
inhibitory	O
action	O
was	O
70	O
%	O
reverted	O
by	O
a	O
specific	O
monoclonal	O
anti	O
-	O
Tat	B-Gene_or_gene_product
antibody	O
,	O
but	O
was	O
not	O
prevented	O
either	O
by	O
the	O
phosphodiesterase	B-Gene_or_gene_product
inhibitor	O
3	B-Simple_chemical
-	I-Simple_chemical
isobutyl	I-Simple_chemical
-	I-Simple_chemical
1	I-Simple_chemical
-	I-Simple_chemical
methyl	I-Simple_chemical
-	I-Simple_chemical
xantine	I-Simple_chemical
(	O
100	O
microM	O
)	O
or	O
by	O
a	O
16	O
-	O
h	O
pretreatment	O
of	O
microglial	O
cultures	O
with	O
the	O
Gi	B-Gene_or_gene_product
protein	O
inhibitor	O
pertussis	B-Simple_chemical
toxin	I-Simple_chemical
(	O
10	O
ng	O
/	O
mL	O
)	O
.	O

All	O
these	O
results	O
suggested	O
that	O
the	O
viral	O
protein	O
acts	O
at	O
a	O
step	O
of	O
the	O
cAMP	B-Simple_chemical
transduction	O
pathway	O
other	O
than	O
receptors	O
,	O
G	B-Complex
proteins	I-Complex
and	O
phosphodiesterases	B-Simple_chemical
.	O

The	O
target	O
of	O
Tat	B-Gene_or_gene_product
appeared	O
to	O
be	O
adenylyl	B-Gene_or_gene_product
cyclase	I-Gene_or_gene_product
,	O
whose	O
activity	O
was	O
markedly	O
reduced	O
(	O
up	O
to	O
60	O
%	O
)	O
in	O
membranes	B-Cellular_component
prepared	O
from	O
Tat	B-Gene_or_gene_product
-	O
treated	O
microglial	O
cells	O
,	O
both	O
in	O
basal	O
conditions	O
and	O
after	O
stimulation	O
with	O
isoproterenol	B-Simple_chemical
and	O
forskolin	B-Simple_chemical
.	O

The	O
inability	O
of	O
the	O
competitive	O
inhibitor	O
of	O
nitric	B-Gene_or_gene_product
oxide	I-Gene_or_gene_product
synthase	I-Gene_or_gene_product
N	B-Simple_chemical
(	I-Simple_chemical
G	I-Simple_chemical
)	I-Simple_chemical
-	I-Simple_chemical
monometyl	I-Simple_chemical
-	I-Simple_chemical
L	I-Simple_chemical
-	I-Simple_chemical
arginine	I-Simple_chemical
(	O
20	O
and	O
200	O
microM	O
)	O
to	O
revert	O
Tat	B-Gene_or_gene_product
action	O
on	O
forskolin	B-Simple_chemical
-	O
induced	O
cAMP	B-Simple_chemical
accumulation	O
,	O
and	O
of	O
two	O
potent	O
nitric	B-Simple_chemical
oxide	I-Simple_chemical
donors	O
,	O
PAPA	B-Simple_chemical
and	O
DETA	B-Simple_chemical
(	O
0	O
.	O
1	O
-	O
2	O
m	O
M	O
)	O
,	O
to	O
alter	O
forskolin	B-Simple_chemical
-	O
induced	O
cAMP	B-Simple_chemical
accumulation	O
,	O
excluded	O
an	O
involvement	O
of	O
nitric	B-Simple_chemical
oxide	I-Simple_chemical
in	O
Tat	B-Gene_or_gene_product
-	O
induced	O
adenylyl	B-Gene_or_gene_product
cyclase	I-Gene_or_gene_product
inhibition	O
.	O

On	O
the	O
contrary	O
,	O
two	O
inhibitors	O
of	O
nuclear	B-Complex
factor	I-Complex
kappaB	I-Complex
activation	O
,	O
N	B-Simple_chemical
-	I-Simple_chemical
tosyl	I-Simple_chemical
-	I-Simple_chemical
(	I-Simple_chemical
L	I-Simple_chemical
)	I-Simple_chemical
-	I-Simple_chemical
phenylalanine	I-Simple_chemical
chloromethyl	I-Simple_chemical
ketone	I-Simple_chemical
(	O
10	O
microM	O
)	O
and	O
SN50	B-Simple_chemical
(	O
25	O
microM	O
)	O
,	O
markedly	O
prevented	O
the	O
reduction	O
of	O
forskolin	B-Simple_chemical
-	O
evoked	O
cAMP	B-Simple_chemical
accumulation	O
by	O
Tat	B-Gene_or_gene_product
,	O
suggesting	O
a	O
possible	O
role	O
for	O
this	O
nuclear	B-Cellular_component
transcriptional	O
factor	O
in	O
the	O
regulation	O
of	O
adenylyl	B-Gene_or_gene_product
cyclase	I-Gene_or_gene_product
by	O
Tat	B-Gene_or_gene_product
in	O
microglia	O
.	O

This	O
assumption	O
was	O
strengthened	O
by	O
the	O
ability	O
of	O
lipopolysaccharide	B-Simple_chemical
(	O
100	O
ng	O
/	O
mL	O
,	O
4	O
h	O
)	O
to	O
mimic	O
the	O
inhibitory	O
effect	O
of	O
the	O
viral	O
protein	O
.	O

Conversely	O
,	O
astrocyte	O
cAMP	B-Simple_chemical
accumulation	O
was	O
unaffected	O
by	O
the	O
viral	O
protein	O
,	O
as	O
tested	O
at	O
various	O
concentrations	O
and	O
time	O
points	O
.	O

Finally	O
,	O
Tat	B-Gene_or_gene_product
inhibition	O
of	O
microglial	O
adenylyl	B-Gene_or_gene_product
cyclase	I-Gene_or_gene_product
was	O
not	O
due	O
to	O
non	O
-	O
specific	O
cytotoxicity	O
.	O

As	O
cAMP	B-Simple_chemical
has	O
been	O
reported	O
to	O
exert	O
a	O
neuroprotective	O
role	O
in	O
several	O
in	O
vivo	O
and	O
in	O
vitro	O
models	O
of	O
brain	O
pathologies	O
,	O
and	O
microglia	O
is	O
believed	O
to	O
mediate	O
Tat	B-Gene_or_gene_product
-	O
induced	O
neurotoxicity	O
,	O
these	O
results	O
suggest	O
that	O
the	O
ability	O
of	O
Tat	B-Gene_or_gene_product
to	O
inhibit	O
cAMP	B-Simple_chemical
synthesis	O
in	O
microglia	O
may	O
contribute	O
to	O
neuronal	O
degeneration	O
and	O
cell	O
death	O
associated	O
with	O
HIV	O
infection	O
.	O

Biosynthesis	O
of	O
N	B-Simple_chemical
-	I-Simple_chemical
acetylneuraminic	I-Simple_chemical
acid	I-Simple_chemical
in	O
cells	O
lacking	O
UDP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
N	I-Gene_or_gene_product
-	I-Gene_or_gene_product
acetylglucosamine	I-Gene_or_gene_product
2	I-Gene_or_gene_product
-	I-Gene_or_gene_product
epimerase	I-Gene_or_gene_product
/	I-Gene_or_gene_product
N	I-Gene_or_gene_product
-	I-Gene_or_gene_product
acetylmannosamine	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
.	O

The	O
first	O
two	O
steps	O
in	O
mammalian	O
biosynthesis	O
of	O
N	B-Simple_chemical
-	I-Simple_chemical
acetylneuraminic	I-Simple_chemical
acid	I-Simple_chemical
,	O
an	O
important	O
carbohydrate	B-Simple_chemical
moiety	O
in	O
biological	O
recognition	O
systems	O
,	O
are	O
performed	O
by	O
the	O
bifunctional	O
enzyme	O
UDP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
N	I-Gene_or_gene_product
-	I-Gene_or_gene_product
acetylglucosamine	I-Gene_or_gene_product
2	I-Gene_or_gene_product
-	I-Gene_or_gene_product
epimerase	I-Gene_or_gene_product
/	I-Gene_or_gene_product
N	I-Gene_or_gene_product
-	I-Gene_or_gene_product
acetylmannosamine	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
.	O

A	O
subclone	O
of	O
the	O
human	O
B	O
lymphoma	O
cell	O
line	O
BJA	O
-	O
B	O
K20	O
,	O
lacking	O
UDP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
N	I-Gene_or_gene_product
-	I-Gene_or_gene_product
acetylglucosamine	I-Gene_or_gene_product
2	I-Gene_or_gene_product
-	I-Gene_or_gene_product
epimerase	I-Gene_or_gene_product
/	I-Gene_or_gene_product
N	I-Gene_or_gene_product
-	I-Gene_or_gene_product
acetylmannosamine	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
mRNA	O
as	O
well	O
as	O
epimerase	O
activity	O
,	O
displayed	O
hyposialylated	O
,	O
functionally	O
impaired	O
cell	B-Cellular_component
surface	I-Cellular_component
glycoconjugates	O
.	O

Here	O
we	O
show	O
that	O
this	O
cell	O
line	O
surprisingly	O
still	O
retains	O
N	B-Gene_or_gene_product
-	I-Gene_or_gene_product
acetylmannosamine	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
activity	O
.	O

A	O
gel	O
filtration	O
analysis	O
of	O
BJA	O
-	O
B	O
K88	O
control	O
cells	O
,	O
which	O
express	O
UDP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
N	I-Gene_or_gene_product
-	I-Gene_or_gene_product
acetylglucosamine	I-Gene_or_gene_product
2	I-Gene_or_gene_product
-	I-Gene_or_gene_product
epimerase	I-Gene_or_gene_product
/	I-Gene_or_gene_product
N	I-Gene_or_gene_product
-	I-Gene_or_gene_product
acetylmannosamine	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
,	O
revealed	O
two	O
N	B-Gene_or_gene_product
-	I-Gene_or_gene_product
acetylmannosamine	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
activity	O
peaks	O
,	O
one	O
co	O
-	O
eluting	O
with	O
UDP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
N	I-Gene_or_gene_product
-	I-Gene_or_gene_product
acetylglucosamine	I-Gene_or_gene_product
2	I-Gene_or_gene_product
-	I-Gene_or_gene_product
epimerase	I-Gene_or_gene_product
activity	O
and	O
one	O
co	O
-	O
eluting	O
with	O
N	B-Gene_or_gene_product
-	I-Gene_or_gene_product
acetylglucosamine	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
.	O

For	O
this	O
enzyme	O
previous	O
studies	O
already	O
showed	O
a	O
ManNAc	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
activity	O
in	O
vitro	O
.	O

In	O
contrast	O
,	O
the	O
hyposialylated	O
BJA	O
-	O
B	O
K20	O
subclone	O
displayed	O
only	O
the	O
N	B-Gene_or_gene_product
-	I-Gene_or_gene_product
acetylmannosamine	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
peak	O
,	O
co	O
-	O
migrating	O
with	O
N	B-Gene_or_gene_product
-	I-Gene_or_gene_product
acetylglucosamine	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
.	O

The	O
CMP	B-Simple_chemical
-	I-Simple_chemical
N	I-Simple_chemical
-	I-Simple_chemical
acetylneuraminic	I-Simple_chemical
acid	I-Simple_chemical
content	O
of	O
both	O
K88	O
and	O
K20	O
cells	O
and	O
the	O
sialylation	O
of	O
cell	B-Cellular_component
surface	I-Cellular_component
glycoconjugates	O
of	O
K20	O
cells	O
could	O
be	O
significantly	O
increased	O
by	O
supplementing	O
the	O
medium	O
with	O
N	B-Simple_chemical
-	I-Simple_chemical
acetylmannosamine	I-Simple_chemical
.	O

This	O
N	B-Simple_chemical
-	I-Simple_chemical
acetylmannosamine	I-Simple_chemical
-	O
induced	O
increase	O
was	O
drastically	O
reduced	O
by	O
co	O
-	O
supplementation	O
with	O
N	B-Simple_chemical
-	I-Simple_chemical
acetylglucosamine	I-Simple_chemical
only	O
in	O
K20	O
cells	O
.	O

We	O
therefore	O
propose	O
the	O
phosphorylation	O
of	O
N	B-Simple_chemical
-	I-Simple_chemical
acetylmannosamine	I-Simple_chemical
as	O
a	O
hitherto	O
unrecognized	O
role	O
of	O
N	B-Gene_or_gene_product
-	I-Gene_or_gene_product
acetylglucosamine	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
in	O
living	O
cells	O
.	O

Overlapping	O
and	O
distinct	O
roles	O
of	O
the	O
duplicated	O
yeast	O
transcription	O
factors	O
Ace2p	B-Gene_or_gene_product
and	O
Swi5p	B-Gene_or_gene_product
.	O

The	O
yeast	O
transcription	O
factors	O
Ace2p	B-Gene_or_gene_product
and	O
Swi5p	B-Gene_or_gene_product
regulate	O
the	O
expression	O
of	O
several	O
target	O
genes	O
involved	O
in	O
mating	O
type	O
switching	O
,	O
exit	O
from	O
mitosis	O
and	O
cell	B-Cellular_component
wall	I-Cellular_component
function	O
.	O

We	O
describe	O
the	O
analysis	O
of	O
12	O
novel	O
targets	O
,	O
some	O
regulated	O
by	O
Ace2p	B-Gene_or_gene_product
or	O
Swi5p	B-Gene_or_gene_product
alone	O
and	O
some	O
by	O
both	O
.	O

We	O
show	O
that	O
Ace2p	B-Gene_or_gene_product
is	O
the	O
major	O
regulator	O
of	O
four	O
genes	O
(	O
CTS1	B-Gene_or_gene_product
,	O
YHR143W	B-Gene_or_gene_product
,	O
SCW11	B-Gene_or_gene_product
and	O
YER124C	B-Gene_or_gene_product
)	O
.	O

Expression	O
of	O
all	O
four	O
is	O
inhibited	O
by	O
Swi5p	B-Gene_or_gene_product
.	O

Like	O
Cts1p	B-Gene_or_gene_product
and	O
Scw11p	B-Gene_or_gene_product
,	O
the	O
two	O
new	O
Ace2p	B-Gene_or_gene_product
targets	O
are	O
associated	O
with	O
cell	B-Cellular_component
wall	I-Cellular_component
metabolism	O
.	O

Yhr143p	B-Gene_or_gene_product
is	O
localized	O
to	O
the	O
cell	B-Cellular_component
wall	I-Cellular_component
,	O
and	O
deletion	O
affects	O
cell	O
separation	O
and	O
enhances	O
pseudohyphal	O
growth	O
.	O

Deleting	O
YER124C	B-Gene_or_gene_product
also	O
affects	O
cell	O
separation	O
and	O
sensitivity	O
to	O
drugs	O
targeted	O
against	O
the	O
cell	B-Cellular_component
wall	I-Cellular_component
.	O

Expression	O
of	O
PIR1	B-Gene_or_gene_product
,	O
YPL158C	B-Gene_or_gene_product
and	O
YNL046W	B-Gene_or_gene_product
is	O
dependent	O
on	O
Swi5p	B-Gene_or_gene_product
alone	O
.	O

In	O
contrast	O
,	O
expression	O
of	O
YBR158W	B-Gene_or_gene_product
,	O
YNL078W	B-Gene_or_gene_product
and	O
YOR264W	B-Gene_or_gene_product
is	O
minimized	O
when	O
both	O
ace2	B-Gene_or_gene_product
and	O
swi5	B-Gene_or_gene_product
are	O
disrupted	O
.	O

We	O
propose	O
that	O
,	O
although	O
Ace2p	B-Gene_or_gene_product
and	O
Swi5p	B-Gene_or_gene_product
co	O
-	O
operate	O
to	O
induce	O
the	O
expression	O
of	O
a	O
subset	O
of	O
genes	O
,	O
some	O
functional	O
divergence	O
has	O
occurred	O
.	O

This	O
results	O
in	O
a	O
delay	O
in	O
the	O
expression	O
of	O
those	O
genes	O
predominantly	O
regulated	O
by	O
Ace2p	B-Gene_or_gene_product
,	O
compared	O
with	O
those	O
predominantly	O
regulated	O
by	O
Swi5p	B-Gene_or_gene_product
.	O

Binding	O
of	O
double	O
-	O
stranded	O
RNA	O
to	O
protein	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
PKR	I-Gene_or_gene_product
is	O
required	O
for	O
dimerization	O
and	O
promotes	O
critical	O
autophosphorylation	O
events	O
in	O
the	O
activation	O
loop	O
.	O

Protein	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
PKR	I-Gene_or_gene_product
is	O
activated	O
by	O
double	O
-	O
stranded	O
RNA	O
(	O
dsRNA	O
)	O
and	O
phosphorylates	O
translation	B-Gene_or_gene_product
initiation	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
2alpha	I-Gene_or_gene_product
to	O
inhibit	O
protein	O
synthesis	O
in	O
virus	O
-	O
infected	O
mammalian	O
cells	O
.	O

PKR	B-Gene_or_gene_product
contains	O
two	O
dsRNA	O
binding	O
motifs	O
(	O
DRBMs	O
I	O
and	O
II	O
)	O
required	O
for	O
activation	O
by	O
dsRNA	O
.	O

There	O
is	O
strong	O
evidence	O
that	O
PKR	B-Gene_or_gene_product
activation	O
requires	O
dimerization	O
,	O
but	O
the	O
role	O
of	O
dsRNA	O
in	O
dimer	O
formation	O
is	O
controversial	O
.	O

By	O
making	O
alanine	B-Simple_chemical
substitutions	O
predicted	O
to	O
remove	O
increasing	O
numbers	O
of	O
side	O
chain	O
contacts	O
between	O
the	O
DRBMs	O
and	O
dsRNA	O
,	O
we	O
found	O
that	O
dimerization	O
of	O
full	O
-	O
length	O
PKR	B-Gene_or_gene_product
in	O
yeast	O
was	O
impaired	O
by	O
the	O
minimal	O
combinations	O
of	O
mutations	O
required	O
to	O
impair	O
dsRNA	O
binding	O
in	O
vitro	O
.	O

Mutation	O
of	O
Ala	B-Simple_chemical
-	I-Simple_chemical
67	I-Simple_chemical
to	O
Glu	B-Simple_chemical
in	O
DRBM	O
-	O
I	O
,	O
reported	O
to	O
abolish	O
dimerization	O
without	O
affecting	O
dsRNA	O
binding	O
,	O
destroyed	O
both	O
activities	O
in	O
our	O
assays	O
.	O

By	O
contrast	O
,	O
deletion	O
of	O
a	O
second	O
dimerization	O
region	O
that	O
overlaps	O
the	O
kinase	O
domain	O
had	O
no	O
effect	O
on	O
PKR	B-Gene_or_gene_product
dimerization	O
in	O
yeast	O
.	O

Human	O
PKR	B-Gene_or_gene_product
contains	O
at	O
least	O
15	O
autophosphorylation	O
sites	O
,	O
but	O
only	O
Thr	B-Simple_chemical
-	I-Simple_chemical
446	I-Simple_chemical
and	O
Thr	B-Simple_chemical
-	I-Simple_chemical
451	I-Simple_chemical
in	O
the	O
activation	O
loop	O
were	O
found	O
here	O
to	O
be	O
critical	O
for	O
kinase	O
activity	O
in	O
yeast	O
.	O

Using	O
an	O
antibody	O
specific	O
for	O
phosphorylated	O
Thr	B-Simple_chemical
-	I-Simple_chemical
451	I-Simple_chemical
,	O
we	O
showed	O
that	O
Thr	B-Simple_chemical
-	I-Simple_chemical
451	I-Simple_chemical
phosphorylation	O
is	O
stimulated	O
by	O
dsRNA	O
binding	O
.	O

Our	O
results	O
provide	O
strong	O
evidence	O
that	O
dsRNA	O
binding	O
is	O
required	O
for	O
dimerization	O
of	O
full	O
-	O
length	O
PKR	B-Gene_or_gene_product
molecules	O
in	O
vivo	O
,	O
leading	O
to	O
autophosphorylation	O
in	O
the	O
activation	O
loop	O
and	O
stimulation	O
of	O
the	O
eIF2alpha	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
function	O
of	O
PKR	B-Gene_or_gene_product
.	O

IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
induced	O
chemokine	O
production	O
through	O
the	O
association	O
of	O
Syk	B-Gene_or_gene_product
with	O
TNF	B-Gene_or_gene_product
receptor	I-Gene_or_gene_product
-	I-Gene_or_gene_product
associated	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
-	I-Gene_or_gene_product
6	I-Gene_or_gene_product
in	O
nasal	O
fibroblast	O
lines	O
.	O

The	O
fibroblasts	O
stimulated	O
by	O
cytokines	O
released	O
the	O
chemokine	O
and	O
recruited	O
the	O
infiltrating	O
cells	O
,	O
including	O
eosinophils	O
,	O
that	O
play	O
a	O
key	O
role	O
in	O
the	O
pathogenesis	O
of	O
airway	O
disease	O
.	O

We	O
established	O
the	O
human	O
fibroblast	O
lines	O
showing	O
high	O
Syk	B-Gene_or_gene_product
expression	O
and	O
the	O
lines	O
showing	O
low	O
Syk	B-Gene_or_gene_product
expression	O
from	O
pieces	O
of	O
nasal	O
polyp	O
.	O

IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
induces	O
the	O
interaction	O
of	O
TNFR	B-Gene_or_gene_product
-	I-Gene_or_gene_product
associated	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
(	I-Gene_or_gene_product
TRAF	I-Gene_or_gene_product
)	I-Gene_or_gene_product
6	I-Gene_or_gene_product
with	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1R	I-Gene_or_gene_product
-	I-Gene_or_gene_product
associated	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
,	O
which	O
is	O
rapidly	O
recruited	O
to	O
the	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1R	I-Gene_or_gene_product
after	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
induction	O
,	O
whereas	O
TRAF2	B-Gene_or_gene_product
participates	O
in	O
TNF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
-	O
signaling	O
.	O

In	O
the	O
present	O
study	O
,	O
we	O
found	O
that	O
Syk	B-Gene_or_gene_product
played	O
a	O
different	O
role	O
in	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
-	O
and	O
TNF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
-	O
induced	O
chemokine	O
production	O
through	O
a	O
signaling	O
complex	O
involving	O
Syk	B-Gene_or_gene_product
and	O
TRAF6	B-Gene_or_gene_product
.	O

Overexpression	O
of	O
wild	O
-	O
type	O
Syk	B-Gene_or_gene_product
by	O
gene	O
transfer	O
enhanced	O
RANTES	B-Gene_or_gene_product
production	O
from	O
nasal	O
fibroblasts	O
stimulated	O
with	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
.	O

The	O
decrease	O
of	O
Syk	B-Gene_or_gene_product
expression	O
by	O
the	O
administration	O
of	O
Syk	B-Gene_or_gene_product
antisense	O
inhibited	O
RANTES	B-Gene_or_gene_product
production	O
in	O
response	O
to	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
.	O

However	O
,	O
the	O
change	O
of	O
Syk	B-Gene_or_gene_product
expression	O
did	O
not	O
affect	O
RANTES	B-Gene_or_gene_product
production	O
by	O
TNF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
stimulation	O
.	O

We	O
concluded	O
that	O
Syk	B-Gene_or_gene_product
is	O
required	O
for	O
the	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
-	O
induced	O
chemokine	O
production	O
through	O
the	O
association	O
with	O
TRAF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
6	I-Gene_or_gene_product
in	O
fibroblasts	O
of	O
nasal	O
polyps	O
.	O

Identification	O
and	O
functional	O
analysis	O
of	O
splice	O
variants	O
of	O
the	O
germ	O
cell	O
soluble	O
adenylyl	B-Gene_or_gene_product
cyclase	I-Gene_or_gene_product
.	O

In	O
mammalian	O
germ	O
cells	O
,	O
cAMP	B-Simple_chemical
signaling	O
is	O
dependent	O
on	O
two	O
forms	O
of	O
adenylyl	B-Gene_or_gene_product
cyclase	I-Gene_or_gene_product
,	O
the	O
conventional	O
membrane	B-Cellular_component
-	O
bound	O
ACIII	B-Gene_or_gene_product
and	O
a	O
soluble	O
form	O
of	O
adenylyl	B-Gene_or_gene_product
cyclase	I-Gene_or_gene_product
(	O
sAC	B-Gene_or_gene_product
)	O
.	O

Recent	O
elucidation	O
of	O
the	O
sAC	B-Gene_or_gene_product
sequence	O
indicates	O
that	O
this	O
enzyme	O
is	O
phylogenetically	O
distinct	O
from	O
the	O
membrane	B-Cellular_component
-	O
bound	O
AC	B-Gene_or_gene_product
,	O
does	O
not	O
interact	O
with	O
G	B-Gene_or_gene_product
proteins	I-Gene_or_gene_product
,	O
and	O
its	O
activity	O
is	O
regulated	O
by	O
bicarbonate	B-Simple_chemical
ions	I-Simple_chemical
.	O

Here	O
we	O
have	O
investigated	O
the	O
properties	O
and	O
regulation	O
of	O
this	O
enzyme	O
during	O
spermatogenesis	O
.	O

Two	O
different	O
transcripts	O
encoding	O
a	O
full	O
-	O
length	O
and	O
truncated	O
sAC	B-Gene_or_gene_product
were	O
identified	O
by	O
reverse	B-Gene_or_gene_product
transcriptase	I-Gene_or_gene_product
-	O
polymerase	O
chain	O
reaction	O
and	O
RNase	B-Gene_or_gene_product
protection	O
analysis	O
.	O

The	O
truncated	O
sAC	B-Gene_or_gene_product
transcript	O
lacks	O
exon	O
11	O
with	O
a	O
premature	O
termination	O
of	O
the	O
open	O
reading	O
frame	O
after	O
the	O
catalytic	O
domain	O
.	O

Reverse	B-Gene_or_gene_product
transcriptase	I-Gene_or_gene_product
-	O
polymerase	O
chain	O
reaction	O
with	O
testis	O
RNA	O
from	O
adult	O
mouse	O
and	O
rat	O
of	O
different	O
ages	O
,	O
as	O
well	O
as	O
RNase	B-Gene_or_gene_product
protection	O
,	O
showed	O
that	O
both	O
transcripts	O
are	O
absent	O
at	O
11	O
days	O
of	O
age	O
,	O
appear	O
between	O
20	O
and	O
30	O
days	O
of	O
age	O
,	O
and	O
are	O
retained	O
in	O
the	O
adult	O
testis	O
.	O

The	O
presence	O
of	O
corresponding	O
proteins	O
in	O
testis	O
,	O
germ	O
cells	O
,	O
and	O
spermatozoa	O
was	O
demonstrated	O
by	O
fast	O
protein	O
liquid	O
chromatography	O
and	O
differential	O
immunoprecipitation	O
with	O
full	O
-	O
length	O
sAC	B-Gene_or_gene_product
-	O
specific	O
antibodies	O
.	O

Bicarbonate	B-Simple_chemical
ions	I-Simple_chemical
activated	O
both	O
sAC	B-Gene_or_gene_product
forms	O
and	O
increased	O
cAMP	B-Simple_chemical
levels	O
in	O
germ	O
cells	O
isolated	O
from	O
25	O
-	O
and	O
50	O
-	O
day	O
-	O
old	O
rats	O
and	O
adult	O
rats	O
in	O
a	O
concentration	O
-	O
dependent	O
manner	O
.	O

These	O
findings	O
provide	O
evidence	O
that	O
full	O
-	O
length	O
and	O
truncated	O
sAC	B-Gene_or_gene_product
are	O
generated	O
by	O
alternate	O
splicing	O
.	O

Both	O
forms	O
are	O
active	O
in	O
spermatids	O
,	O
and	O
the	O
bicarbonate	B-Simple_chemical
present	O
in	O
the	O
seminiferous	O
tubule	O
may	O
be	O
a	O
signal	O
that	O
regulates	O
cAMP	B-Simple_chemical
levels	O
in	O
these	O
cells	O
.	O

Functional	O
analysis	O
of	O
the	O
hydrophobic	O
patch	O
on	O
nuclear	B-Gene_or_gene_product
transport	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
2	I-Gene_or_gene_product
involved	O
in	O
interactions	O
with	O
the	O
nuclear	B-Cellular_component
pore	I-Cellular_component
in	O
vivo	O
.	O

Nuclear	B-Gene_or_gene_product
transport	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
2	I-Gene_or_gene_product
(	O
NTF2	B-Gene_or_gene_product
)	O
is	O
a	O
small	O
homodimeric	O
protein	O
that	O
interacts	O
simultaneously	O
with	O
both	O
RanGDP	B-Gene_or_gene_product
and	O
FxFG	B-Gene_or_gene_product
nucleoporins	I-Gene_or_gene_product
.	O

The	O
interaction	O
between	O
NTF2	B-Gene_or_gene_product
and	O
Ran	B-Gene_or_gene_product
is	O
essential	O
for	O
the	O
import	O
of	O
Ran	B-Gene_or_gene_product
into	O
the	O
nucleus	B-Cellular_component
.	O

Here	O
we	O
use	O
mutational	O
analysis	O
to	O
dissect	O
the	O
in	O
vivo	O
role	O
of	O
the	O
interaction	O
between	O
NTF2	B-Gene_or_gene_product
and	O
nucleoporins	B-Gene_or_gene_product
.	O

We	O
identify	O
a	O
series	O
of	O
surface	O
residues	O
that	O
form	O
a	O
hydrophobic	O
patch	O
on	O
NTF2	B-Gene_or_gene_product
,	O
which	O
when	O
mutated	O
disrupt	O
the	O
NTF2	B-Gene_or_gene_product
-	O
nucleoporin	B-Gene_or_gene_product
interaction	O
.	O

Analysis	O
of	O
these	O
mutants	O
in	O
vivo	O
demonstrates	O
that	O
the	O
strength	O
of	O
this	O
interaction	O
can	O
be	O
significantly	O
reduced	O
without	O
affecting	O
cell	O
viability	O
.	O

However	O
,	O
cells	O
cease	O
to	O
be	O
viable	O
if	O
the	O
interaction	O
between	O
NTF2	B-Gene_or_gene_product
and	O
nucleoporins	B-Gene_or_gene_product
is	O
abolished	O
completely	O
,	O
indicating	O
that	O
this	O
interaction	O
is	O
essential	O
for	O
the	O
function	O
of	O
NTF2	B-Gene_or_gene_product
in	O
vivo	O
.	O

In	O
addition	O
,	O
we	O
have	O
isolated	O
a	O
dominant	O
negative	O
mutant	O
of	O
NTF2	B-Gene_or_gene_product
,	O
N77Y	B-Gene_or_gene_product
,	O
which	O
has	O
increased	O
affinity	O
for	O
nucleoporins	B-Gene_or_gene_product
.	O

Overexpression	O
of	O
the	O
N77Y	B-Gene_or_gene_product
protein	O
blocks	O
nuclear	B-Cellular_component
protein	O
import	O
and	O
concentrates	O
Ran	B-Gene_or_gene_product
at	O
the	O
nuclear	B-Cellular_component
rim	I-Cellular_component
.	O

These	O
data	O
support	O
a	O
mechanism	O
in	O
which	O
NTF2	B-Gene_or_gene_product
interacts	O
transiently	O
with	O
FxFG	B-Gene_or_gene_product
nucleoporins	I-Gene_or_gene_product
to	O
translocate	O
through	O
the	O
pore	B-Cellular_component
and	O
import	O
RanGDP	B-Gene_or_gene_product
into	O
the	O
nucleus	B-Cellular_component
.	O

Expression	O
and	O
properties	O
of	O
human	O
liver	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
ureidopropionase	I-Gene_or_gene_product
.	O

A	O
cDNA	O
encoding	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
ureidopropionase	I-Gene_or_gene_product
(	O
BUP	B-Gene_or_gene_product
)	O
was	O
isolated	O
from	O
a	O
human	O
liver	O
cDNA	O
library	O
,	O
expressed	O
in	O
E	O
.	O
coli	O
,	O
and	O
purified	O
from	O
the	O
culture	O
extract	O
.	O

The	O
2	O
,	O
006	O
bp	O
cDNA	O
contained	O
a	O
1	O
,	O
152	O
bp	O
open	O
reading	O
frame	O
encoding	O
a	O
protein	O
of	O
384	O
amino	O
acids	O
with	O
a	O
molecular	O
weight	O
of	O
43	O
,	O
165	O
Da	O
.	O

The	O
subunit	O
molecular	O
weight	O
of	O
the	O
enzyme	O
expressed	O
was	O
about	O
43	O
,	O
000	O
Da	O
.	O

The	O
enzyme	O
was	O
inhibited	O
by	O
1	O
mM	O
propionate	B-Simple_chemical
,	O
but	O
not	O
by	O
10	O
mM	O
beta	B-Simple_chemical
-	I-Simple_chemical
alanine	I-Simple_chemical
.	O

Chemical	O
analysis	O
of	O
the	O
purified	O
human	O
BUP	B-Gene_or_gene_product
showed	O
0	O
.	O
54	O
zinc	B-Simple_chemical
atoms	O
per	O
subunit	O
,	O
and	O
the	O
sequence	O
of	O
BUP	B-Gene_or_gene_product
cDNA	O
contained	O
one	O
putative	O
zinc	B-Simple_chemical
-	O
binding	O
site	O
motif	O
.	O

The	O
purified	O
enzyme	O
had	O
a	O
pI	O
of	O
5	O
.	O
65	O
,	O
and	O
exhibited	O
positive	O
cooperativity	O
with	O
N	B-Simple_chemical
-	I-Simple_chemical
carbamoyl	I-Simple_chemical
-	I-Simple_chemical
beta	I-Simple_chemical
-	I-Simple_chemical
alanine	I-Simple_chemical
as	O
the	O
substrate	O
with	O
a	O
Hill	O
coefficient	O
2	O
.	O
0	O
.	O

These	O
properties	O
of	O
human	O
BUP	B-Gene_or_gene_product
,	O
except	O
the	O
inhibition	O
by	O
beta	B-Simple_chemical
-	I-Simple_chemical
alanine	I-Simple_chemical
,	O
were	O
similar	O
to	O
the	O
rat	O
liver	O
purified	O
enzyme	O
.	O

Beta	B-Simple_chemical
-	I-Simple_chemical
alanine	I-Simple_chemical
inhibits	O
rats	O
BUP	B-Gene_or_gene_product
activity	O
.	O

The	O
complex	O
regulatory	O
function	O
and	O
the	O
negative	O
cooperative	O
mechanism	O
of	O
BUP	B-Gene_or_gene_product
by	O
beta	B-Simple_chemical
-	I-Simple_chemical
alanine	I-Simple_chemical
have	O
been	O
observed	O
in	O
rats	O
.	O

This	O
kind	O
of	O
mechanism	O
may	O
not	O
exist	O
in	O
humans	O
,	O
because	O
beta	B-Simple_chemical
-	I-Simple_chemical
alanine	I-Simple_chemical
did	O
not	O
inhibit	O
human	O
BUP	B-Gene_or_gene_product
.	O

Cell	O
cycle	O
-	O
dependent	O
recruitment	O
of	O
HDAC	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
correlates	O
with	O
deacetylation	O
of	O
histone	B-Gene_or_gene_product
H4	I-Gene_or_gene_product
on	O
an	O
Rb	B-Gene_or_gene_product
-	O
E2F	B-Gene_or_gene_product
target	O
promoter	O
.	O

The	O
transcription	O
factor	O
E2F	B-Gene_or_gene_product
,	O
which	O
is	O
a	O
key	O
element	O
in	O
the	O
control	O
of	O
cell	O
proliferation	O
,	O
is	O
repressed	O
by	O
Rb	B-Gene_or_gene_product
and	O
other	O
pocket	O
proteins	O
in	O
growth	O
-	O
arrested	O
differentiating	O
cells	O
,	O
as	O
well	O
as	O
in	O
proliferating	O
cells	O
when	O
they	O
progress	O
through	O
early	O
G1	O
.	O

It	O
is	O
not	O
known	O
whether	O
similar	O
mechanisms	O
are	O
operative	O
in	O
the	O
two	O
situations	O
.	O

A	O
body	O
of	O
data	O
suggests	O
that	O
E2F	B-Gene_or_gene_product
repression	O
by	O
pocket	O
proteins	O
involves	O
class	B-Gene_or_gene_product
I	I-Gene_or_gene_product
histone	I-Gene_or_gene_product
deacetylases	I-Gene_or_gene_product
(	O
HDACs	B-Gene_or_gene_product
)	O
.	O

It	O
has	O
been	O
hypothesized	O
that	O
these	O
enzymes	O
are	O
recruited	O
to	O
E2F	B-Gene_or_gene_product
target	O
promoters	O
where	O
they	O
deacetylate	O
histones	O
.	O

Here	O
we	O
have	O
tested	O
this	O
hypothesis	O
directly	O
by	O
using	O
formaldehyde	B-Simple_chemical
cross	O
-	O
linked	O
chromatin	B-Cellular_component
immunoprecipitation	O
(	O
XChIP	O
)	O
assays	O
to	O
evaluate	O
HDAC	B-Gene_or_gene_product
association	O
in	O
living	O
cells	O
.	O

Our	O
data	O
show	O
that	O
a	O
histone	B-Gene_or_gene_product
deacetylase	I-Gene_or_gene_product
,	O
HDAC	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
,	O
is	O
stably	O
bound	O
to	O
an	O
E2F	B-Gene_or_gene_product
target	O
promoter	O
during	O
early	O
G1	O
in	O
proliferating	O
cells	O
and	O
released	O
at	O
the	O
G1	O
-	O
S	O
transition	O
.	O

In	O
addition	O
,	O
our	O
results	O
reveal	O
an	O
inverse	O
correlation	O
between	O
HDAC	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
recruitment	O
and	O
histone	B-Gene_or_gene_product
H4	I-Gene_or_gene_product
acetylation	O
on	O
specific	O
lysines	B-Simple_chemical
.	O

Transcriptional	O
coregulation	O
by	O
the	O
cell	O
integrity	O
mitogen	B-Gene_or_gene_product
-	I-Gene_or_gene_product
activated	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
Slt2	B-Gene_or_gene_product
and	O
the	O
cell	O
cycle	O
regulator	O
Swi4	B-Gene_or_gene_product
.	O

In	O
Saccharomyces	O
cerevisiae	O
,	O
the	O
heterodimeric	O
transcription	O
factor	O
SBF	B-Complex
(	O
for	O
SCB	B-Complex
binding	I-Complex
factor	I-Complex
)	O
is	O
composed	O
of	O
Swi4	B-Gene_or_gene_product
and	O
Swi6	B-Gene_or_gene_product
and	O
activates	O
gene	O
expression	O
at	O
the	O
G	O
(	O
1	O
)	O
/	O
S	O
-	O
phase	O
transition	O
of	O
the	O
mitotic	O
cell	O
cycle	O
.	O

Cell	O
cycle	O
commitment	O
is	O
associated	O
not	O
only	O
with	O
major	O
alterations	O
in	O
gene	O
expression	O
but	O
also	O
with	O
highly	O
polarized	O
cell	O
growth	O
;	O
the	O
mitogen	B-Gene_or_gene_product
-	I-Gene_or_gene_product
activated	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
(	O
MAPK	B-Gene_or_gene_product
)	O
Slt2	B-Gene_or_gene_product
is	O
required	O
to	O
maintain	O
cell	B-Cellular_component
wall	I-Cellular_component
integrity	O
during	O
periods	O
of	O
polarized	O
growth	O
and	O
cell	B-Cellular_component
wall	I-Cellular_component
stress	O
.	O

We	O
describe	O
experiments	O
aimed	O
at	O
defining	O
the	O
regulatory	O
pathway	O
involving	O
the	O
cell	O
cycle	O
transcription	O
factor	O
SBF	B-Complex
and	O
Slt2	B-Gene_or_gene_product
-	I-Gene_or_gene_product
MAPK	I-Gene_or_gene_product
.	O

Gene	O
expression	O
assays	O
and	O
chromatin	B-Cellular_component
immunoprecipitation	O
experiments	O
revealed	O
Slt2	B-Gene_or_gene_product
-	O
dependent	O
recruitment	O
of	O
SBF	B-Complex
to	O
the	O
promoters	O
of	O
the	O
G	O
(	O
1	O
)	O
cyclins	B-Gene_or_gene_product
PCL1	B-Gene_or_gene_product
and	O
PCL2	B-Gene_or_gene_product
after	O
activation	O
of	O
the	O
Slt2	B-Gene_or_gene_product
-	I-Gene_or_gene_product
MAPK	I-Gene_or_gene_product
pathway	O
.	O

We	O
performed	O
DNA	O
microarray	O
analysis	O
and	O
identified	O
other	O
genes	O
whose	O
expression	O
was	O
reduced	O
in	O
both	O
SLT2	B-Gene_or_gene_product
and	O
SWI4	B-Gene_or_gene_product
deletion	O
strains	O
.	O

Genes	O
that	O
are	O
sensitive	O
to	O
both	O
Slt2	B-Gene_or_gene_product
and	O
Swi4	B-Gene_or_gene_product
appear	O
to	O
be	O
uniquely	O
regulated	O
and	O
reveal	O
a	O
role	O
for	O
Swi4	B-Gene_or_gene_product
,	O
the	O
DNA	O
-	O
binding	O
component	O
of	O
SBF	B-Complex
,	O
which	O
is	O
independent	O
of	O
the	O
regulatory	O
subunit	O
Swi6	B-Gene_or_gene_product
.	O

Some	O
of	O
the	O
Swi4	B-Gene_or_gene_product
-	O
and	O
Slt2	B-Gene_or_gene_product
-	O
dependent	O
genes	O
do	O
not	O
require	O
Swi6	B-Gene_or_gene_product
for	O
either	O
their	O
expression	O
or	O
for	O
Swi4	B-Gene_or_gene_product
localization	O
to	O
their	O
promoters	O
.	O

Consistent	O
with	O
these	O
results	O
,	O
we	O
found	O
a	O
direct	O
interaction	O
between	O
Swi4	B-Gene_or_gene_product
and	O
Slt2	B-Gene_or_gene_product
.	O

Our	O
results	O
establish	O
a	O
new	O
Slt2	B-Gene_or_gene_product
-	O
dependent	O
mode	O
of	O
Swi4	B-Gene_or_gene_product
regulation	O
and	O
suggest	O
roles	O
for	O
Swi4	B-Gene_or_gene_product
beyond	O
its	O
prominent	O
role	O
in	O
controlling	O
cell	O
cycle	O
transcription	O
.	O

Regulation	O
of	O
the	O
anaphase	B-Complex
-	I-Complex
promoting	I-Complex
complex	I-Complex
by	O
the	O
dual	O
specificity	O
phosphatase	O
human	O
Cdc14a	B-Gene_or_gene_product
.	O

Two	O
forms	O
of	O
the	O
anaphase	B-Complex
-	I-Complex
promoting	I-Complex
complex	I-Complex
(	O
APC	B-Complex
)	O
mediate	O
the	O
degradation	O
of	O
critical	O
cell	O
cycle	O
regulators	O
.	O

APC	B-Complex
(	I-Complex
Cdc20	I-Complex
)	I-Complex
promotes	O
sister	B-Cellular_component
-	I-Cellular_component
chromatid	I-Cellular_component
separation	O
by	O
ubiquitinating	O
securin	B-Gene_or_gene_product
,	O
whereas	O
APC	B-Complex
(	I-Complex
Cdh1	I-Complex
)	I-Complex
ubiquitinates	O
mitotic	O
cyclins	B-Gene_or_gene_product
,	O
allowing	O
the	O
exit	O
from	O
mitosis	O
.	O

Here	O
we	O
show	O
that	O
phosphorylation	O
of	O
human	O
Cdh1	B-Gene_or_gene_product
(	O
hCdh1	B-Gene_or_gene_product
)	O
by	O
cyclin	B-Complex
B	I-Complex
-	I-Complex
Cdc2	I-Complex
alters	O
the	O
conformation	O
of	O
hCdh1	B-Gene_or_gene_product
and	O
prevents	O
it	O
from	O
activating	O
APC	B-Complex
.	O

A	O
human	O
homologue	O
of	O
yeast	O
Cdc14	B-Gene_or_gene_product
,	O
human	O
Cdc14a	B-Gene_or_gene_product
(	O
hCdc14a	B-Gene_or_gene_product
)	O
,	O
dephosphorylates	O
hCdh1	B-Gene_or_gene_product
and	O
activates	O
APC	B-Complex
(	I-Complex
Cdh1	I-Complex
)	I-Complex
.	O

In	O
contrast	O
,	O
hCdc14a	B-Gene_or_gene_product
does	O
not	O
affect	O
the	O
activity	O
of	O
APC	B-Complex
(	I-Complex
Cdc20	I-Complex
)	I-Complex
.	O

hCdc14a	B-Gene_or_gene_product
is	O
a	O
major	O
phosphatase	O
for	O
hCdh1	B-Gene_or_gene_product
and	O
localizes	O
to	O
centrosomes	B-Cellular_component
in	O
HeLa	O
cells	O
.	O

Therefore	O
,	O
hCdc14a	B-Gene_or_gene_product
may	O
promote	O
the	O
activation	O
of	O
APC	B-Complex
(	I-Complex
Cdh1	I-Complex
)	I-Complex
and	O
exit	O
from	O
mitosis	O
in	O
mammalian	O
cells	O
.	O

Physiological	O
substrates	O
for	O
human	O
lysosomal	B-Cellular_component
beta	B-Complex
-	I-Complex
hexosaminidase	I-Complex
S	I-Complex
.	O

Human	O
lysosomal	B-Cellular_component
beta	B-Complex
-	I-Complex
hexosaminidases	I-Complex
remove	O
terminal	O
beta	O
-	O
glycosidically	O
bound	O
N	B-Simple_chemical
-	I-Simple_chemical
acetylhexosamine	I-Simple_chemical
residues	O
from	O
a	O
number	O
of	O
glycoconjugates	O
.	O

Three	O
different	O
isozymes	O
composed	O
of	O
two	O
noncovalently	O
linked	O
subunits	O
alpha	O
and	O
beta	O
exist	O
:	O
Hex	B-Complex
A	I-Complex
(	O
alphabeta	O
)	O
,	O
Hex	B-Complex
B	I-Complex
(	O
betabeta	O
)	O
,	O
and	O
Hex	B-Complex
S	I-Complex
(	O
alphaalpha	O
)	O
.	O

While	O
the	O
role	O
of	O
Hex	B-Complex
A	I-Complex
and	O
B	B-Complex
for	O
the	O
degradation	O
of	O
several	O
anionic	O
and	O
neutral	O
glycoconjugates	O
has	O
been	O
well	O
established	O
,	O
the	O
physiological	O
significance	O
of	O
labile	O
Hex	B-Complex
S	I-Complex
has	O
remained	O
unclear	O
.	O

However	O
,	O
the	O
striking	O
accumulation	O
of	O
anionic	O
oligosaccharides	B-Simple_chemical
in	O
double	O
knockout	O
mice	O
totally	O
deficient	O
in	O
hexosaminidase	B-Complex
activity	O
but	O
not	O
in	O
mice	O
expressing	O
Hex	B-Complex
S	I-Complex
(	O
Sango	O
,	O
K	O
.	O
,	O
McDonald	O
,	O
M	O
.	O
P	O
.	O
,	O
Crawley	O
,	O
J	O
.	O
N	O
.	O
,	O
Mack	O
,	O
M	O
.	O
L	O
.	O
,	O
Tifft	O
,	O
C	O
.	O
J	O
.	O
,	O
Skop	O
,	O
E	O
.	O
,	O
Starr	O
,	O
C	O
.	O
M	O
.	O
,	O
Hoffmann	O
,	O
A	O
.	O
,	O
Sandhoff	O
,	O
K	O
.	O
,	O
Suzuki	O
,	O
K	O
.	O
,	O
and	O
Proia	O
,	O
R	O
.	O
L	O
.	O
,	O
(	O
1996	O
)	O
Nat	O
.	O
Genet	O
.	O
14	O
,	O
348	O
-	O
352	O
)	O
prompted	O
us	O
to	O
reinvestigate	O
the	O
substrate	O
specificity	O
of	O
Hex	B-Complex
S	I-Complex
.	O

To	O
identify	O
physiological	O
substrates	O
of	O
Hex	B-Complex
S	I-Complex
,	O
anionic	O
and	O
neutral	O
oligosaccharides	B-Simple_chemical
excreted	O
in	O
the	O
urine	O
of	O
the	O
double	O
knockout	O
mice	O
were	O
isolated	O
and	O
analyzed	O
.	O

Using	O
ESI	O
-	O
MS	O
/	O
MS	O
and	O
glycosidase	B-Gene_or_gene_product
digestion	O
the	O
anionic	O
glycans	B-Simple_chemical
were	O
identified	O
as	O
products	O
of	O
incomplete	O
dermatan	B-Simple_chemical
sulfate	I-Simple_chemical
degradation	O
whereas	O
the	O
neutral	O
storage	O
oligosaccharides	B-Simple_chemical
were	O
found	O
to	O
be	O
fragments	O
of	O
N	B-Simple_chemical
-	I-Simple_chemical
glycan	I-Simple_chemical
degradation	O
.	O

In	O
vitro	O
,	O
recombinant	O
Hex	B-Complex
S	I-Complex
was	O
highly	O
active	O
on	O
water	B-Simple_chemical
-	O
soluble	O
and	O
amphiphilic	O
glycoconjugates	O
including	O
artificial	O
substrates	O
,	O
sulfated	O
GAG	O
fragments	O
,	O
and	O
the	O
sulfated	O
glycosphingolipid	B-Simple_chemical
SM2	I-Simple_chemical
.	O

Hydrolysis	O
of	O
membrane	B-Cellular_component
-	O
bound	O
SM2	B-Simple_chemical
by	O
the	O
recombinant	O
Hex	B-Complex
S	I-Complex
was	O
synergistically	O
stimulated	O
by	O
the	O
GM2	B-Gene_or_gene_product
activator	O
protein	O
and	O
the	O
lysosomal	B-Cellular_component
anionic	B-Simple_chemical
phospholipid	I-Simple_chemical
bis	B-Simple_chemical
(	I-Simple_chemical
monoacylglycero	I-Simple_chemical
)	I-Simple_chemical
phosphate	I-Simple_chemical
.	O

C	B-Gene_or_gene_product
-	I-Gene_or_gene_product
TAK1	I-Gene_or_gene_product
regulates	O
Ras	B-Gene_or_gene_product
signaling	O
by	O
phosphorylating	O
the	O
MAPK	B-Gene_or_gene_product
scaffold	O
,	O
KSR1	B-Gene_or_gene_product
.	O

Kinase	B-Gene_or_gene_product
suppressor	I-Gene_or_gene_product
of	I-Gene_or_gene_product
Ras	I-Gene_or_gene_product
(	O
KSR	B-Gene_or_gene_product
)	O
is	O
a	O
conserved	O
component	O
of	O
the	O
Ras	B-Gene_or_gene_product
pathway	O
that	O
interacts	O
directly	O
with	O
MEK	B-Gene_or_gene_product
and	O
MAPK	B-Gene_or_gene_product
.	O

Here	O
we	O
show	O
that	O
KSR1	B-Gene_or_gene_product
translocates	O
from	O
the	O
cytoplasm	B-Cellular_component
to	O
the	O
cell	B-Cellular_component
surface	I-Cellular_component
in	O
response	O
to	O
growth	O
factor	O
treatment	O
and	O
that	O
this	O
process	O
is	O
regulated	O
by	O
Cdc25C	B-Gene_or_gene_product
-	I-Gene_or_gene_product
associated	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
1	I-Gene_or_gene_product
(	O
C	B-Gene_or_gene_product
-	I-Gene_or_gene_product
TAK1	I-Gene_or_gene_product
)	O
.	O

C	B-Gene_or_gene_product
-	I-Gene_or_gene_product
TAK1	I-Gene_or_gene_product
constitutively	O
associates	O
with	O
mammalian	O
KSR1	B-Gene_or_gene_product
and	O
phosphorylates	O
serine	B-Simple_chemical
392	I-Simple_chemical
to	O
confer	O
14	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3	I-Gene_or_gene_product
-	I-Gene_or_gene_product
3	I-Gene_or_gene_product
binding	O
and	O
cytoplasmic	B-Cellular_component
sequestration	O
of	O
KSR1	B-Gene_or_gene_product
in	O
unstimulated	O
cells	O
.	O

In	O
response	O
to	O
signal	O
activation	O
,	O
the	O
phosphorylation	O
state	O
of	O
S392	B-Simple_chemical
is	O
reduced	O
,	O
allowing	O
the	O
KSR1	B-Gene_or_gene_product
complex	O
to	O
colocalize	O
with	O
activated	O
Ras	B-Gene_or_gene_product
and	O
Raf	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
at	O
the	O
plasma	B-Cellular_component
membrane	I-Cellular_component
,	O
thereby	O
facilitating	O
the	O
phosphorylation	O
reactions	O
required	O
for	O
the	O
activation	O
of	O
MEK	B-Gene_or_gene_product
and	O
MAPK	B-Gene_or_gene_product
.	O

TIP41	B-Gene_or_gene_product
interacts	O
with	O
TAP42	B-Gene_or_gene_product
and	O
negatively	O
regulates	O
the	O
TOR	B-Gene_or_gene_product
signaling	O
pathway	O
.	O

In	O
Saccharomyces	O
cerevisiae	O
,	O
the	O
rapamycin	B-Simple_chemical
-	O
sensitive	O
TOR	B-Gene_or_gene_product
kinases	O
negatively	O
regulate	O
the	O
type	O
2A	O
-	O
related	O
phosphatase	O
SIT4	B-Gene_or_gene_product
by	O
promoting	O
the	O
association	O
of	O
this	O
phosphatase	O
with	O
the	O
inhibitor	O
TAP42	B-Gene_or_gene_product
.	O

Here	O
,	O
we	O
describe	O
TIP41	B-Gene_or_gene_product
,	O
a	O
conserved	O
TAP42	B-Gene_or_gene_product
-	O
interacting	O
protein	O
involved	O
in	O
the	O
regulation	O
of	O
SIT4	B-Gene_or_gene_product
.	O

Deletion	O
of	O
the	O
TIP41	B-Gene_or_gene_product
gene	O
confers	O
rapamycin	B-Simple_chemical
resistance	O
,	O
suppresses	O
a	O
tap42	B-Gene_or_gene_product
mutation	O
,	O
and	O
prevents	O
dissociation	O
of	O
SIT4	B-Gene_or_gene_product
from	O
TAP42	B-Gene_or_gene_product
.	O

Furthermore	O
,	O
a	O
TIP41	B-Gene_or_gene_product
deletion	O
prevents	O
SIT4	B-Gene_or_gene_product
-	O
dependent	O
events	O
such	O
as	O
dephosphorylation	O
of	O
the	O
kinase	O
NPR1	B-Gene_or_gene_product
and	O
nuclear	B-Cellular_component
translocation	O
of	O
the	O
transcription	O
factor	O
GLN3	B-Gene_or_gene_product
.	O

Thus	O
,	O
TIP41	B-Gene_or_gene_product
negatively	O
regulates	O
the	O
TOR	B-Gene_or_gene_product
pathway	O
by	O
binding	O
and	O
inhibiting	O
TAP42	B-Gene_or_gene_product
.	O

The	O
binding	O
of	O
TIP41	B-Gene_or_gene_product
to	O
TAP42	B-Gene_or_gene_product
is	O
stimulated	O
upon	O
rapamycin	B-Simple_chemical
treatment	O
via	O
SIT4	B-Gene_or_gene_product
-	O
dependent	O
dephosphorylation	O
of	O
TIP41	B-Gene_or_gene_product
,	O
suggesting	O
that	O
TIP41	B-Gene_or_gene_product
is	O
part	O
of	O
a	O
feedback	O
loop	O
that	O
rapidly	O
amplifies	O
SIT4	B-Gene_or_gene_product
phosphatase	O
activity	O
under	O
TOR	B-Gene_or_gene_product
-	O
inactivating	O
conditions	O
.	O

Pseudosubstrate	O
regulation	O
of	O
the	O
SCF	B-Complex
(	I-Complex
beta	I-Complex
-	I-Complex
TrCP	I-Complex
)	I-Complex
ubiquitin	B-Gene_or_gene_product
ligase	O
by	O
hnRNP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
U	I-Gene_or_gene_product
.	O

beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
TrCP	I-Gene_or_gene_product
/	O
E3RS	B-Gene_or_gene_product
(	O
E3RS	B-Gene_or_gene_product
)	O
is	O
the	O
F	O
-	O
box	O
protein	O
that	O
functions	O
as	O
the	O
receptor	O
subunit	O
of	O
the	O
SCF	B-Complex
(	I-Complex
beta	I-Complex
-	I-Complex
TrCP	I-Complex
)	I-Complex
ubiquitin	B-Gene_or_gene_product
ligase	O
(	O
E3	O
)	O
.	O

Surprisingly	O
,	O
although	O
its	O
two	O
recognized	O
substrates	O
,	O
IkappaB	B-Gene_or_gene_product
(	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
)	I-Gene_or_gene_product
and	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
,	O
are	O
present	O
in	O
the	O
cytoplasm	B-Cellular_component
,	O
we	O
have	O
found	O
that	O
E3RS	B-Gene_or_gene_product
is	O
located	O
predominantly	O
in	O
the	O
nucleus	B-Cellular_component
.	O

Here	O
we	O
report	O
the	O
isolation	O
of	O
the	O
major	O
E3RS	B-Gene_or_gene_product
-	O
associated	O
protein	O
,	O
hnRNP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
U	I-Gene_or_gene_product
,	O
an	O
abundant	O
nuclear	B-Cellular_component
phosphoprotein	O
.	O

This	O
protein	O
occupies	O
E3RS	B-Gene_or_gene_product
in	O
a	O
specific	O
and	O
stoichiometric	O
manner	O
,	O
stabilizes	O
the	O
E3	O
component	O
,	O
and	O
is	O
likely	O
responsible	O
for	O
its	O
nuclear	B-Cellular_component
localization	O
.	O

hnRNP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
U	I-Gene_or_gene_product
binding	O
was	O
abolished	O
by	O
competition	O
with	O
a	O
pIkappaB	B-Gene_or_gene_product
(	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
)	I-Gene_or_gene_product
peptide	O
,	O
or	O
by	O
a	O
specific	O
point	O
mutation	O
in	O
the	O
E3RS	B-Gene_or_gene_product
WD	O
region	O
,	O
indicating	O
an	O
E3	O
-	O
substrate	O
-	O
type	O
interaction	O
.	O

However	O
,	O
unlike	O
pI	B-Gene_or_gene_product
(	I-Gene_or_gene_product
kappa	I-Gene_or_gene_product
)	I-Gene_or_gene_product
Balpha	I-Gene_or_gene_product
,	O
which	O
is	O
targeted	O
by	O
SCF	B-Complex
(	I-Complex
beta	I-Complex
-	I-Complex
TrCP	I-Complex
)	I-Complex
for	O
degradation	O
,	O
the	O
E3	O
-	O
bound	O
hnRNP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
U	I-Gene_or_gene_product
is	O
stable	O
and	O
is	O
,	O
therefore	O
,	O
a	O
pseudosubstrate	O
.	O

Consequently	O
,	O
hnRNP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
U	I-Gene_or_gene_product
engages	O
a	O
highly	O
neddylated	O
active	O
SCF	B-Complex
(	I-Complex
beta	I-Complex
-	I-Complex
TrCP	I-Complex
)	I-Complex
,	O
which	O
dissociates	O
in	O
the	O
presence	O
of	O
a	O
high	O
-	O
affinity	O
substrate	O
,	O
resulting	O
in	O
ubiquitination	O
of	O
the	O
latter	O
.	O

Our	O
study	O
points	O
to	O
a	O
novel	O
regulatory	O
mechanism	O
,	O
which	O
secures	O
the	O
localization	O
,	O
stability	O
,	O
substrate	O
binding	O
threshold	O
,	O
and	O
efficacy	O
of	O
a	O
specific	O
protein	O
-	O
ubiquitin	B-Gene_or_gene_product
ligase	O
.	O

Chromatin	B-Complex
-	I-Complex
IgG	I-Complex
complexes	O
activate	O
B	O
cells	O
by	O
dual	O
engagement	O
of	O
IgM	B-Gene_or_gene_product
and	O
Toll	B-Gene_or_gene_product
-	I-Gene_or_gene_product
like	I-Gene_or_gene_product
receptors	I-Gene_or_gene_product
.	O

Autoreactive	O
B	O
cells	O
are	O
present	O
in	O
the	O
lymphoid	O
tissues	O
of	O
healthy	O
individuals	O
,	O
but	O
typically	O
remain	O
quiescent	O
.	O

When	O
this	O
homeostasis	O
is	O
perturbed	O
,	O
the	O
formation	O
of	O
self	O
-	O
reactive	O
antibodies	O
can	O
have	O
serious	O
pathological	O
consequences	O
.	O

B	O
cells	O
expressing	O
an	O
antigen	O
receptor	O
specific	O
for	O
self	O
-	O
immunoglobulin	B-Gene_or_gene_product
-	I-Gene_or_gene_product
gamma	I-Gene_or_gene_product
(	O
IgG	B-Gene_or_gene_product
)	O
make	O
a	O
class	O
of	O
autoantibodies	O
known	O
as	O
rheumatoid	B-Gene_or_gene_product
factor	I-Gene_or_gene_product
(	O
RF	B-Gene_or_gene_product
)	O
.	O

Here	O
we	O
show	O
that	O
effective	O
activation	O
of	O
RF	B-Gene_or_gene_product
+	O
B	O
cells	O
is	O
mediated	O
by	O
IgG2a	B-Gene_or_gene_product
-	O
chromatin	O
immune	O
complexes	O
and	O
requires	O
the	O
synergistic	O
engagement	O
of	O
the	O
antigen	O
receptor	O
and	O
a	O
member	O
of	O
the	O
MyD88	B-Gene_or_gene_product
-	O
dependent	O
Toll	B-Gene_or_gene_product
-	I-Gene_or_gene_product
like	I-Gene_or_gene_product
receptor	I-Gene_or_gene_product
(	O
TLR	B-Gene_or_gene_product
)	O
family	O
.	O

Inhibitor	O
studies	O
implicate	O
TLR9	B-Gene_or_gene_product
.	O

These	O
data	O
establish	O
a	O
critical	O
link	O
between	O
the	O
innate	O
and	O
adaptive	O
immune	O
systems	O
in	O
the	O
development	O
of	O
systemic	O
autoimmune	O
disease	O
and	O
explain	O
the	O
preponderance	O
of	O
autoantibodies	O
reactive	O
with	O
nucleic	O
acid	O
-	O
protein	O
particles	O
.	O

The	O
unique	O
features	O
of	O
this	O
dual	O
-	O
engagement	O
pathway	O
should	O
facilitate	O
the	O
development	O
of	O
therapies	O
that	O
specifically	O
target	O
autoreactive	O
B	O
cells	O
.	O

Kremen	B-Gene_or_gene_product
proteins	O
are	O
Dickkopf	B-Gene_or_gene_product
receptors	O
that	O
regulate	O
Wnt	B-Gene_or_gene_product
/	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
signalling	O
.	O

The	O
Wnt	B-Gene_or_gene_product
family	O
of	O
secreted	O
glycoproteins	O
mediate	O
cell	O
cell	O
interactions	O
during	O
cell	O
growth	O
and	O
differentiation	O
in	O
both	O
embryos	O
and	O
adults	O
.	O

Canonical	O
Wnt	B-Gene_or_gene_product
signalling	O
by	O
way	O
of	O
the	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
pathway	O
is	O
transduced	O
by	O
two	O
receptor	O
families	O
.	O

Frizzled	B-Gene_or_gene_product
proteins	O
and	O
lipoprotein	B-Gene_or_gene_product
-	I-Gene_or_gene_product
receptor	I-Gene_or_gene_product
-	I-Gene_or_gene_product
related	I-Gene_or_gene_product
proteins	I-Gene_or_gene_product
5	I-Gene_or_gene_product
and	O
6	B-Gene_or_gene_product
(	O
LRP5	B-Gene_or_gene_product
/	O
6	B-Gene_or_gene_product
)	O
bind	O
Wnts	B-Gene_or_gene_product
and	O
transmit	O
their	O
signal	O
by	O
stabilizing	O
intracellular	B-Cellular_component
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
.	O

Wnt	B-Gene_or_gene_product
/	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
signalling	O
is	O
inhibited	O
by	O
the	O
secreted	O
protein	O
Dickkopf1	B-Gene_or_gene_product
(	O
Dkk1	B-Gene_or_gene_product
)	O
,	O
a	O
member	O
of	O
a	O
multigene	O
family	O
,	O
which	O
induces	O
head	O
formation	O
in	O
amphibian	O
embryos	O
.	O

Dkk1	B-Gene_or_gene_product
has	O
been	O
shown	O
to	O
inhibit	O
Wnt	B-Gene_or_gene_product
signalling	O
by	O
binding	O
to	O
and	O
antagonizing	O
LRP5	B-Gene_or_gene_product
/	O
6	B-Gene_or_gene_product
.	O

Here	O
we	O
show	O
that	O
the	O
transmembrane	O
proteins	O
Kremen1	B-Gene_or_gene_product
and	O
Kremen2	B-Gene_or_gene_product
are	O
high	O
-	O
affinity	O
Dkk1	B-Gene_or_gene_product
receptors	O
that	O
functionally	O
cooperate	O
with	O
Dkk1	B-Gene_or_gene_product
to	O
block	O
Wnt	B-Gene_or_gene_product
/	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
signalling	O
.	O

Kremen2	B-Gene_or_gene_product
forms	O
a	O
ternary	O
complex	O
with	O
Dkk1	B-Gene_or_gene_product
and	O
LRP6	B-Gene_or_gene_product
,	O
and	O
induces	O
rapid	O
endocytosis	O
and	O
removal	O
of	O
the	O
Wnt	B-Gene_or_gene_product
receptor	O
LRP6	B-Gene_or_gene_product
from	O
the	O
plasma	B-Cellular_component
membrane	I-Cellular_component
.	O

The	O
results	O
indicate	O
that	O
Kremen1	B-Gene_or_gene_product
and	O
Kremen2	B-Gene_or_gene_product
are	O
components	O
of	O
a	O
membrane	B-Cellular_component
complex	O
modulating	O
canonical	O
Wnt	B-Gene_or_gene_product
signalling	O
through	O
LRP6	B-Gene_or_gene_product
in	O
vertebrates	O
.	O

Expression	O
of	O
p73	B-Gene_or_gene_product
and	O
Reelin	B-Gene_or_gene_product
in	O
the	O
developing	O
human	O
cortex	O
.	O

Cajal	O
-	O
Retzius	O
(	O
CR	O
)	O
cells	O
of	O
the	O
developing	O
neocortex	O
secrete	O
Reelin	B-Gene_or_gene_product
(	O
Reln	B-Gene_or_gene_product
)	O
,	O
a	O
glycoprotein	O
involved	O
in	O
neuronal	O
migration	O
.	O

CR	O
cells	O
selectively	O
express	O
p73	B-Gene_or_gene_product
,	O
a	O
p53	B-Gene_or_gene_product
family	O
member	O
implicated	O
in	O
cell	O
survival	O
and	O
apoptosis	O
.	O

Immunocytochemistry	O
in	O
prenatal	O
human	O
telencephalon	O
reveals	O
a	O
complex	O
sequence	O
of	O
migration	O
waves	O
of	O
p73	B-Gene_or_gene_product
-	O
and	O
Reln	B-Gene_or_gene_product
-	O
immunoreactive	O
(	O
IR	O
)	O
neurons	O
into	O
the	O
cortical	O
marginal	O
zone	O
(	O
MZ	O
)	O
.	O

At	O
early	O
preplate	O
stages	O
,	O
p73	B-Gene_or_gene_product
/	O
Reln	B-Gene_or_gene_product
-	O
IR	O
cells	O
arise	O
in	O
distinct	O
sectors	O
of	O
the	O
telencephalon	O
,	O
including	O
cortical	O
primordium	O
and	O
ganglionic	O
eminences	O
.	O

After	O
the	O
appearance	O
of	O
the	O
cortical	O
plate	O
,	O
further	O
p73	B-Gene_or_gene_product
/	O
Reln	B-Gene_or_gene_product
-	O
IR	O
cells	O
originate	O
in	O
the	O
medial	O
periolfactory	O
forebrain	O
.	O

In	O
addition	O
,	O
p73	B-Gene_or_gene_product
marks	O
a	O
novel	O
cell	O
population	O
that	O
appears	O
at	O
the	O
choroid	O
-	O
cortical	O
junction	O
or	O
cortical	O
hem	O
before	O
the	O
emergence	O
of	O
the	O
dorsal	O
hippocampus	O
.	O

A	O
pronounced	O
mediolateral	O
gradient	O
in	O
the	O
density	O
of	O
p73	B-Gene_or_gene_product
/	O
Reln	B-Gene_or_gene_product
-	O
IR	O
neurons	O
in	O
the	O
neocortical	O
MZ	O
at	O
8	O
gestational	O
weeks	O
suggests	O
that	O
a	O
subset	O
of	O
CR	O
cells	O
migrate	O
tangentially	O
from	O
cortical	O
hem	O
and	O
taenia	O
tecta	O
into	O
neocortical	O
territory	O
.	O

This	O
hypothesis	O
is	O
supported	O
by	O
the	O
absence	O
of	O
p73	B-Gene_or_gene_product
-	O
transcripts	O
in	O
prospective	O
neocortex	O
of	O
p73	B-Gene_or_gene_product
-	O
/	O
-	O
mice	O
at	O
embryonic	O
day	O
12	O
(	O
E12	O
)	O
,	O
whereas	O
they	O
are	O
present	O
in	O
cortical	O
hem	O
and	O
taenia	O
tecta	O
.	O

In	O
the	O
p73	B-Gene_or_gene_product
-	O
/	O
-	O
preplate	O
,	O
Reln	B-Gene_or_gene_product
is	O
faintly	O
expressed	O
in	O
a	O
calretinin	B-Gene_or_gene_product
-	O
positive	O
cell	O
population	O
,	O
not	O
present	O
in	O
this	O
form	O
in	O
the	O
E12	O
wild	O
-	O
type	O
cortex	O
.	O

At	O
P2	O
,	O
Reln	B-Gene_or_gene_product
-	O
IR	O
CR	O
cells	O
are	O
undetectable	O
in	O
the	O
p73	B-Gene_or_gene_product
-	O
/	O
-	O
cortex	O
,	O
whereas	O
Reln	B-Gene_or_gene_product
-	O
expression	O
in	O
interneurons	O
is	O
unchanged	O
.	O

Our	O
results	O
point	O
to	O
a	O
close	O
association	O
between	O
p73	B-Gene_or_gene_product
and	O
Reln	B-Gene_or_gene_product
in	O
CR	O
cells	O
of	O
the	O
developing	O
neocortex	O
,	O
with	O
a	O
partial	O
dissociation	O
in	O
early	O
preplate	O
and	O
basal	O
telencephalon	O
,	O
and	O
to	O
a	O
p73	B-Gene_or_gene_product
-	O
mediated	O
role	O
of	O
the	O
cortical	O
hem	O
in	O
neocortical	O
development	O
.	O

Apaf	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
is	O
a	O
mediator	O
of	O
E2F	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
-	O
induced	O
apoptosis	O
.	O

E2F	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
is	O
capable	O
of	O
promoting	O
both	O
cell	O
cycle	O
progression	O
and	O
apoptosis	O
.	O

The	O
latter	O
is	O
important	O
for	O
suppressing	O
untoward	O
expansion	O
of	O
proliferating	O
cells	O
.	O

In	O
this	O
study	O
,	O
we	O
investigated	O
its	O
underlying	O
mechanisms	O
.	O

E2F	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
-	O
induced	O
apoptosis	O
was	O
accompanied	O
by	O
caspase	B-Gene_or_gene_product
-	I-Gene_or_gene_product
9	I-Gene_or_gene_product
activation	O
and	O
inhibited	O
by	O
a	O
specific	O
inhibitor	O
of	O
caspase	B-Gene_or_gene_product
-	I-Gene_or_gene_product
9	I-Gene_or_gene_product
in	O
K562	O
sublines	O
overexpressing	O
E2F	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
.	O

E2F	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
enhanced	O
the	O
expression	O
of	O
Apaf	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
without	O
the	O
cytosolic	B-Cellular_component
accumulation	O
of	O
cytochrome	B-Gene_or_gene_product
c	I-Gene_or_gene_product
.	O

Apaf	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
-	O
deficient	O
melanoma	O
cell	O
lines	O
were	O
resistant	O
to	O
E2F	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
,	O
indicating	O
that	O
Apaf	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
is	O
an	O
essential	O
element	O
of	O
E2F	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
-	O
mediated	O
apoptosis	O
.	O

Finally	O
,	O
we	O
isolated	O
the	O
promoter	O
region	O
of	O
the	O
Apaf	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
gene	O
and	O
found	O
a	O
putative	O
binding	O
site	O
for	O
E2F	B-Gene_or_gene_product
.	O

A	O
chromatin	B-Cellular_component
immunoprecipitation	O
assay	O
revealed	O
that	O
E2F	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
bound	O
to	O
Apaf	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
promoter	O
upon	O
E2F	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
overexpression	O
,	O
suggesting	O
that	O
Apaf	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
is	O
under	O
transcriptional	O
regulation	O
of	O
E2F	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
.	O

These	O
data	O
demonstrate	O
a	O
novel	O
mechanism	O
of	O
apoptosis	O
in	O
which	O
an	O
increase	O
in	O
Apaf	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
levels	O
results	O
in	O
direct	O
activation	O
of	O
caspase	B-Gene_or_gene_product
-	I-Gene_or_gene_product
9	I-Gene_or_gene_product
without	O
mitochondrial	B-Cellular_component
damage	O
,	O
leading	O
to	O
the	O
initiation	O
of	O
a	O
caspase	B-Gene_or_gene_product
cascade	O
.	O

Dual	O
regulation	O
of	O
the	O
met4	B-Gene_or_gene_product
transcription	O
factor	O
by	O
ubiquitin	B-Gene_or_gene_product
-	O
dependent	O
degradation	O
and	O
inhibition	O
of	O
promoter	O
recruitment	O
.	O

The	O
ubiquitin	B-Gene_or_gene_product
system	O
has	O
been	O
recently	O
implicated	O
in	O
various	O
aspects	O
of	O
transcriptional	O
regulation	O
,	O
including	O
proteasome	B-Cellular_component
-	O
dependent	O
degradation	O
of	O
transcriptional	O
activators	O
.	O

In	O
yeast	O
,	O
the	O
activator	O
Met4	B-Gene_or_gene_product
is	O
inhibited	O
by	O
the	O
SCF	B-Complex
(	I-Complex
Met30	I-Complex
)	I-Complex
ubiquitin	B-Gene_or_gene_product
ligase	O
,	O
which	O
recognizes	O
and	O
oligo	O
-	O
ubiquitylates	O
Met4	B-Gene_or_gene_product
.	O

Here	O
,	O
we	O
demonstrate	O
that	O
in	O
minimal	O
media	O
,	O
Met4	B-Gene_or_gene_product
is	O
ubiquitylated	O
and	O
rapidly	O
degraded	O
in	O
response	O
to	O
methionine	B-Simple_chemical
excess	O
,	O
whereas	O
in	O
rich	O
media	O
,	O
Met4	B-Gene_or_gene_product
is	O
oligo	O
-	O
ubiquitylated	O
but	O
remains	O
stable	O
.	O

In	O
the	O
latter	O
growth	O
condition	O
,	O
oligo	O
-	O
ubiquitylated	O
Met4	B-Gene_or_gene_product
is	O
not	O
recruited	O
to	O
MET	O
gene	O
promoters	O
,	O
but	O
is	O
recruited	O
to	O
the	O
SAM	O
genes	O
,	O
which	O
are	O
required	O
for	O
production	O
of	O
S	B-Simple_chemical
-	I-Simple_chemical
adenosylmethionine	I-Simple_chemical
,	O
an	O
unstable	O
metabolite	O
that	O
is	O
not	O
present	O
in	O
rich	O
medium	O
.	O

Thus	O
,	O
ubiquitylation	O
not	O
only	O
regulates	O
Met4	B-Gene_or_gene_product
by	O
distinct	O
degradation	O
-	O
dependent	O
and	O
-	O
independent	O
mechanisms	O
,	O
but	O
also	O
controls	O
differential	O
recruitment	O
of	O
a	O
single	O
transcription	O
factor	O
to	O
distinct	O
promoters	O
,	O
thereby	O
diversifying	O
transcriptional	O
activator	O
specificity	O
.	O

Genetic	O
and	O
epigenetic	O
alterations	O
of	O
the	O
INK4a	B-Gene_or_gene_product
-	O
ARF	B-Gene_or_gene_product
pathway	O
in	O
cholangiocarcinoma	O
.	O

The	O
INK4a	B-Gene_or_gene_product
-	O
ARF	B-Gene_or_gene_product
locus	O
,	O
located	O
on	O
chromosome	B-Cellular_component
9p21	O
,	O
encodes	O
two	O
cell	O
-	O
cycle	O
regulatory	O
proteins	O
,	O
p16	B-Gene_or_gene_product
(	O
INK4a	B-Gene_or_gene_product
)	O
and	O
p14	B-Gene_or_gene_product
(	O
ARF	B-Gene_or_gene_product
)	O
,	O
acting	O
through	O
the	O
Rb	B-Gene_or_gene_product
-	O
CDK4	B-Gene_or_gene_product
and	O
p53	B-Gene_or_gene_product
pathways	O
.	O

To	O
study	O
the	O
contribution	O
of	O
each	O
pathway	O
in	O
the	O
tumourigenesis	O
of	O
cholangiocarcinoma	O
,	O
the	O
alterations	O
of	O
p14	B-Gene_or_gene_product
(	O
ARF	B-Gene_or_gene_product
)	O
,	O
p16	B-Gene_or_gene_product
(	O
INK4a	B-Gene_or_gene_product
)	O
,	O
p53	B-Gene_or_gene_product
,	O
and	O
pRb	B-Gene_or_gene_product
were	O
analysed	O
.	O

After	O
microdissection	O
,	O
DNAs	O
from	O
51	O
cholangiocarcinomas	O
were	O
analysed	O
by	O
methylation	O
-	O
specific	O
PCR	O
(	O
MSP	O
)	O
,	O
restriction	O
-	O
enzyme	O
related	O
polymerase	O
chain	O
reaction	O
(	O
RE	O
-	O
PCR	O
)	O
,	O
microsatellite	O
analysis	O
,	O
mRNA	O
expression	O
,	O
and	O
DNA	O
sequencing	O
.	O

Immunohistochemistry	O
of	O
p14	B-Gene_or_gene_product
(	O
ARF	B-Gene_or_gene_product
)	O
,	O
p16	B-Gene_or_gene_product
(	O
INK4a	B-Gene_or_gene_product
)	O
,	O
p53	B-Gene_or_gene_product
,	O
and	O
pRb	B-Gene_or_gene_product
was	O
also	O
performed	O
.	O

Promoter	O
methylation	O
of	O
p14	B-Gene_or_gene_product
(	O
ARF	B-Gene_or_gene_product
)	O
was	O
found	O
in	O
13	O
/	O
51	O
cases	O
(	O
25	O
%	O
)	O
and	O
p16	B-Gene_or_gene_product
(	O
INK4a	B-Gene_or_gene_product
)	O
showed	O
aberrant	O
promoter	O
methylation	O
in	O
39	O
/	O
51	O
cases	O
(	O
76	O
%	O
)	O
which	O
correlated	O
with	O
loss	O
of	O
mRNA	O
transcription	O
.	O

Two	O
tumours	O
(	O
4	O
%	O
)	O
had	O
homozygous	O
deletion	O
of	O
the	O
INK4a	B-Gene_or_gene_product
-	O
ARF	B-Gene_or_gene_product
locus	O
.	O

Specific	O
mutations	O
of	O
both	O
exons	O
were	O
not	O
detected	O
.	O

p14	B-Gene_or_gene_product
(	O
ARF	B-Gene_or_gene_product
)	O
inactivation	O
appeared	O
in	O
the	O
context	O
of	O
an	O
unmethylated	O
p16	B-Gene_or_gene_product
(	O
INK4a	B-Gene_or_gene_product
)	O
promoter	O
in	O
eight	O
of	O
13	O
cases	O
(	O
61	O
%	O
)	O
of	O
the	O
carcinomas	O
methylated	O
at	O
p14	B-Gene_or_gene_product
(	O
ARF	B-Gene_or_gene_product
)	O
.	O

Mutations	O
of	O
p53	B-Gene_or_gene_product
were	O
found	O
in	O
19	O
of	O
51	O
tumours	O
(	O
37	O
%	O
)	O
,	O
and	O
four	O
of	O
them	O
(	O
21	O
%	O
)	O
harboured	O
p14	B-Gene_or_gene_product
(	O
ARF	B-Gene_or_gene_product
)	O
inactivation	O
.	O

The	O
pRb	B-Gene_or_gene_product
protein	O
was	O
detected	O
in	O
30	O
/	O
51	O
(	O
59	O
%	O
)	O
tumours	O
examined	O
.	O

The	O
absence	O
of	O
pRB	B-Gene_or_gene_product
protein	O
did	O
not	O
correlate	O
with	O
any	O
of	O
the	O
examined	O
parameters	O
.	O

Alterations	O
of	O
the	O
INK4a	B-Gene_or_gene_product
-	O
ARF	B-Gene_or_gene_product
locus	O
,	O
pRB	B-Gene_or_gene_product
or	O
p53	B-Gene_or_gene_product
status	O
could	O
not	O
be	O
established	O
as	O
independent	O
prognostic	O
factors	O
in	O
these	O
tumours	O
.	O

These	O
findings	O
indicate	O
that	O
the	O
INK4a	B-Gene_or_gene_product
-	O
ARF	B-Gene_or_gene_product
locus	O
is	O
frequently	O
inactivated	O
in	O
cholangiocarcinoma	O
of	O
the	O
liver	O
and	O
occurs	O
independently	O
of	O
the	O
status	O
of	O
p53	B-Gene_or_gene_product
or	O
pRb	B-Gene_or_gene_product
.	O

The	O
human	O
L	B-Gene_or_gene_product
-	I-Gene_or_gene_product
threonine	I-Gene_or_gene_product
3	I-Gene_or_gene_product
-	I-Gene_or_gene_product
dehydrogenase	I-Gene_or_gene_product
gene	O
is	O
an	O
expressed	O
pseudogene	O
.	O

BACKGROUND	O
:	O
L	B-Simple_chemical
-	I-Simple_chemical
threonine	I-Simple_chemical
is	O
an	O
indispensable	O
amino	O
acid	O
.	O

One	O
of	O
the	O
major	O
L	B-Simple_chemical
-	I-Simple_chemical
threonine	I-Simple_chemical
degradation	O
pathways	O
is	O
the	O
conversion	O
of	O
L	B-Simple_chemical
-	I-Simple_chemical
threonine	I-Simple_chemical
via	O
2	B-Simple_chemical
-	I-Simple_chemical
amino	I-Simple_chemical
-	I-Simple_chemical
3	I-Simple_chemical
-	I-Simple_chemical
ketobutyrate	I-Simple_chemical
to	O
glycine	B-Simple_chemical
.	O

L	B-Gene_or_gene_product
-	I-Gene_or_gene_product
threonine	I-Gene_or_gene_product
dehydrogenase	I-Gene_or_gene_product
(	O
EC	B-Gene_or_gene_product
1	I-Gene_or_gene_product
.	I-Gene_or_gene_product
1	I-Gene_or_gene_product
.	I-Gene_or_gene_product
1	I-Gene_or_gene_product
.	I-Gene_or_gene_product
103	I-Gene_or_gene_product
)	O
is	O
the	O
first	O
enzyme	O
in	O
the	O
pathway	O
and	O
catalyses	O
the	O
reaction	O
:	O
L	B-Simple_chemical
-	I-Simple_chemical
threonine	I-Simple_chemical
+	O
NAD	B-Simple_chemical
+	I-Simple_chemical
=	O
2	B-Simple_chemical
-	I-Simple_chemical
amino	I-Simple_chemical
-	I-Simple_chemical
3	I-Simple_chemical
-	I-Simple_chemical
ketobutyrate	I-Simple_chemical
+	O
NADH	B-Simple_chemical
.	O

The	O
murine	O
and	O
porcine	O
L	B-Gene_or_gene_product
-	I-Gene_or_gene_product
threonine	I-Gene_or_gene_product
dehydrogenase	I-Gene_or_gene_product
genes	O
(	O
TDH	B-Gene_or_gene_product
)	O
have	O
been	O
identified	O
previously	O
,	O
but	O
the	O
human	O
gene	O
has	O
not	O
been	O
identified	O
.	O

RESULTS	O
:	O
The	O
human	O
TDH	B-Gene_or_gene_product
gene	O
is	O
located	O
at	O
8p23	O
-	O
22	O
and	O
has	O
8	O
exons	O
spanning	O
10	O
kb	O
that	O
would	O
have	O
been	O
expected	O
to	O
encode	O
a	O
369	O
residue	O
ORF	O
.	O

However	O
,	O
2	O
cDNA	O
TDH	B-Gene_or_gene_product
transcripts	O
encode	O
truncated	O
proteins	O
of	O
157	O
and	O
230	O
residues	O
.	O

These	O
truncated	O
proteins	O
are	O
the	O
result	O
of	O
3	O
mutations	O
within	O
the	O
gene	O
.	O

There	O
is	O
a	O
SNP	O
,	O
A	O
to	O
G	O
,	O
present	O
in	O
the	O
genomic	O
DNA	O
sequence	O
of	O
some	O
individuals	O
which	O
results	O
in	O
the	O
loss	O
of	O
the	O
acceptor	O
splice	O
site	O
preceding	O
exon	O
4	O
.	O

The	O
acceptor	O
splice	O
site	O
preceding	O
exon	O
6	O
was	O
lost	O
in	O
all	O
23	O
individuals	O
genotyped	O
and	O
there	O
is	O
an	O
in	O
-	O
frame	O
stop	O
codon	O
in	O
exon	O
6	O
(	O
CGA	O
to	O
TGA	O
)	O
resulting	O
in	O
arginine	B-Simple_chemical
-	I-Simple_chemical
214	I-Simple_chemical
being	O
replaced	O
by	O
a	O
stop	O
codon	O
.	O

These	O
truncated	O
proteins	O
would	O
be	O
non	O
-	O
functional	O
since	O
they	O
have	O
lost	O
part	O
of	O
the	O
NAD	B-Simple_chemical
+	I-Simple_chemical
binding	O
motif	O
and	O
the	O
COOH	O
terminal	O
domain	O
that	O
is	O
thought	O
to	O
be	O
involved	O
in	O
binding	O
L	B-Simple_chemical
-	I-Simple_chemical
threonine	I-Simple_chemical
.	O

TDH	B-Gene_or_gene_product
mRNA	O
was	O
present	O
in	O
all	O
tissues	O
examined	O
.	O

CONCLUSIONS	O
:	O
The	O
human	O
L	B-Gene_or_gene_product
-	I-Gene_or_gene_product
threonine	I-Gene_or_gene_product
3	I-Gene_or_gene_product
-	I-Gene_or_gene_product
dehydrogenase	I-Gene_or_gene_product
gene	O
is	O
an	O
expressed	O
pseudogene	O
having	O
lost	O
the	O
splice	O
acceptor	O
site	O
preceding	O
exon	O
6	O
and	O
codon	O
arginine	B-Simple_chemical
-	I-Simple_chemical
214	I-Simple_chemical
(	O
CGA	O
)	O
is	O
mutated	O
to	O
a	O
stop	O
codon	O
(	O
TGA	O
)	O
.	O

Human	O
T	O
-	O
lymphotropic	O
virus	O
type	O
I	O
tax	B-Gene_or_gene_product
activates	O
I	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
kinase	I-Complex
by	O
inhibiting	O
I	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
kinase	I-Complex
-	O
associated	O
serine	B-Gene_or_gene_product
/	I-Gene_or_gene_product
threonine	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
phosphatase	I-Gene_or_gene_product
2A	I-Gene_or_gene_product
.	O

I	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
kinase	I-Complex
(	O
IKK	B-Complex
)	O
is	O
a	O
serine	B-Simple_chemical
/	O
threonine	B-Simple_chemical
kinase	O
that	O
phosphorylates	O
I	B-Gene_or_gene_product
-	I-Gene_or_gene_product
kappa	I-Gene_or_gene_product
B	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
and	O
I	B-Gene_or_gene_product
-	I-Gene_or_gene_product
kappa	I-Gene_or_gene_product
B	I-Gene_or_gene_product
beta	I-Gene_or_gene_product
and	O
targets	O
them	O
for	O
polyubiquitination	O
and	O
proteasome	B-Cellular_component
-	O
mediated	O
degradation	O
.	O

IKK	B-Complex
consists	O
of	O
two	O
highly	O
related	O
catalytic	O
subunits	O
,	O
alpha	B-Gene_or_gene_product
and	O
beta	B-Gene_or_gene_product
,	O
and	O
a	O
regulatory	O
gamma	B-Gene_or_gene_product
subunit	O
,	O
which	O
becomes	O
activated	O
after	O
serine	B-Simple_chemical
phosphorylation	O
of	O
the	O
activation	O
loops	O
of	O
the	O
catalytic	O
domains	O
.	O

The	O
human	O
T	O
-	O
lymphotropic	O
retrovirus	O
type	O
-	O
I	O
trans	O
-	O
activator	O
,	O
Tax	B-Gene_or_gene_product
,	O
has	O
been	O
shown	O
to	O
interact	O
directly	O
with	O
IKK	B-Gene_or_gene_product
gamma	I-Gene_or_gene_product
and	O
activates	O
IKK	B-Complex
via	O
a	O
mechanism	O
not	O
fully	O
understood	O
.	O

Here	O
we	O
demonstrate	O
that	O
IKK	B-Complex
binds	O
serine	B-Gene_or_gene_product
/	I-Gene_or_gene_product
threonine	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
phosphatase	I-Gene_or_gene_product
2A	I-Gene_or_gene_product
(	O
PP2A	B-Gene_or_gene_product
)	O
,	O
and	O
via	O
a	O
tripartite	O
protein	O
-	O
protein	O
interaction	O
,	O
Tax	B-Gene_or_gene_product
,	O
IKK	B-Gene_or_gene_product
gamma	I-Gene_or_gene_product
,	O
and	O
PP2A	B-Gene_or_gene_product
form	O
a	O
stable	O
ternary	O
complex	O
.	O

In	O
vitro	O
,	O
PP2A	B-Gene_or_gene_product
down	O
-	O
regulates	O
active	O
IKK	B-Complex
prepared	O
from	O
Tax	B-Gene_or_gene_product
-	O
producing	O
MT4	O
cells	O
.	O

In	O
the	O
presence	O
of	O
Tax	B-Gene_or_gene_product
,	O
however	O
,	O
the	O
ability	O
of	O
PP2A	B-Gene_or_gene_product
to	O
inactivate	O
IKK	B-Complex
is	O
diminished	O
.	O

Despite	O
their	O
interaction	O
with	O
IKK	B-Gene_or_gene_product
gamma	I-Gene_or_gene_product
,	O
PP2A	B-Gene_or_gene_product
-	O
interaction	O
-	O
defective	O
Tax	B-Gene_or_gene_product
mutants	O
failed	O
to	O
activate	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
.	O

Our	O
data	O
support	O
the	O
notion	O
that	O
IKK	B-Gene_or_gene_product
gamma	I-Gene_or_gene_product
-	O
associated	O
PP2A	B-Gene_or_gene_product
is	O
responsible	O
for	O
the	O
rapid	O
deactivation	O
of	O
IKK	B-Complex
,	O
and	O
inhibition	O
of	O
PP2A	B-Gene_or_gene_product
by	O
Tax	B-Gene_or_gene_product
in	O
the	O
context	O
of	O
IKK	B-Complex
x	I-Complex
PP2A	I-Complex
x	I-Complex
Tax	I-Complex
ternary	O
complex	O
leads	O
to	O
constitutive	O
IKK	B-Complex
and	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
activation	O
.	O

E2F	B-Gene_or_gene_product
mediates	O
sustained	O
G2	O
arrest	O
and	O
down	O
-	O
regulation	O
of	O
Stathmin	B-Gene_or_gene_product
and	O
AIM	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
expression	O
in	O
response	O
to	O
genotoxic	O
stress	O
.	O

Exposure	O
of	O
cells	O
to	O
genotoxic	O
agents	O
results	O
in	O
activation	O
of	O
checkpoint	O
pathways	O
leading	O
to	O
cell	O
cycle	O
arrest	O
.	O

These	O
arrest	O
pathways	O
allow	O
repair	O
of	O
damaged	O
DNA	O
before	O
its	O
replication	O
and	O
segregation	O
,	O
thus	O
preventing	O
accumulation	O
of	O
mutations	O
.	O

The	O
tumor	O
suppressor	O
retinoblastoma	B-Gene_or_gene_product
(	O
RB	B-Gene_or_gene_product
)	O
is	O
required	O
for	O
the	O
G	O
(	O
1	O
)	O
/	O
S	O
checkpoint	O
function	O
.	O

In	O
addition	O
,	O
regulation	O
of	O
the	O
G	O
(	O
2	O
)	O
checkpoint	O
by	O
the	O
tumor	O
suppressor	O
p53	B-Gene_or_gene_product
is	O
RB	B-Gene_or_gene_product
-	O
dependent	O
.	O

However	O
,	O
the	O
molecular	O
mechanism	O
underlying	O
the	O
involvement	O
of	O
RB	B-Gene_or_gene_product
and	O
its	O
related	O
proteins	O
p107	B-Gene_or_gene_product
and	O
p130	B-Gene_or_gene_product
in	O
the	O
G	O
(	O
2	O
)	O
checkpoint	O
is	O
not	O
fully	O
understood	O
.	O

We	O
show	O
here	O
that	O
sustained	O
G	O
(	O
2	O
)	O
/	O
M	O
arrest	O
induced	O
by	O
the	O
genotoxic	O
agent	O
doxorubicin	B-Simple_chemical
is	O
E2F	B-Gene_or_gene_product
-	O
dependent	O
and	O
involves	O
a	O
decrease	O
in	O
expression	O
of	O
two	O
mitotic	O
regulators	O
,	O
Stathmin	B-Gene_or_gene_product
and	O
AIM	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
.	O

Abrogation	O
of	O
E2F	B-Gene_or_gene_product
function	O
by	O
dominant	O
negative	O
E2F	B-Gene_or_gene_product
abolishes	O
the	O
doxorubicin	B-Simple_chemical
-	O
induced	O
down	O
-	O
regulation	O
of	O
Stathmin	B-Gene_or_gene_product
and	O
AIM	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
and	O
leads	O
to	O
premature	O
exit	O
from	O
G	O
(	O
2	O
)	O
.	O

Expression	O
of	O
the	O
E7	B-Gene_or_gene_product
papilloma	O
virus	O
protein	O
,	O
which	O
dissociates	O
complexes	O
containing	O
E2F	B-Gene_or_gene_product
and	O
RB	B-Gene_or_gene_product
family	O
members	O
,	O
also	O
prevents	O
the	O
down	O
-	O
regulation	O
of	O
these	O
mitotic	O
genes	O
and	O
leads	O
to	O
premature	O
exit	O
from	O
G	O
(	O
2	O
)	O
after	O
genotoxic	O
stress	O
.	O

Furthermore	O
,	O
genotoxic	O
stress	O
increases	O
the	O
levels	O
of	O
nuclear	B-Cellular_component
E2F	B-Gene_or_gene_product
-	I-Gene_or_gene_product
4	I-Gene_or_gene_product
and	O
p130	B-Gene_or_gene_product
as	O
well	O
as	O
their	O
in	O
vivo	O
binding	O
to	O
the	O
Stathmin	B-Gene_or_gene_product
promoter	O
.	O

Thus	O
,	O
functional	O
complexes	O
containing	O
E2F	B-Gene_or_gene_product
and	O
RB	B-Gene_or_gene_product
family	O
members	O
appear	O
to	O
be	O
essential	O
for	O
repressing	O
expression	O
of	O
critical	O
mitotic	O
regulators	O
and	O
maintaining	O
the	O
G	O
(	O
2	O
)	O
/	O
M	O
checkpoint	O
.	O

Cell	O
cycle	O
-	O
dependent	O
nuclear	B-Cellular_component
export	O
of	O
Cdh1p	B-Gene_or_gene_product
may	O
contribute	O
to	O
the	O
inactivation	O
of	O
APC	B-Complex
/	I-Complex
C	I-Complex
(	I-Complex
Cdh1	I-Complex
)	I-Complex
.	O

Cdh1p	B-Gene_or_gene_product
is	O
a	O
substrate	O
-	O
specific	O
subunit	O
of	O
the	O
anaphase	B-Complex
-	I-Complex
promoting	I-Complex
complex	I-Complex
(	O
APC	B-Complex
/	I-Complex
C	I-Complex
)	O
,	O
which	O
functions	O
as	O
an	O
E3	O
ubiquitin	B-Gene_or_gene_product
ligase	O
to	O
degrade	O
the	O
mitotic	O
cyclin	B-Gene_or_gene_product
Clb2p	I-Gene_or_gene_product
and	O
other	O
substrates	O
during	O
the	O
G	O
(	O
1	O
)	O
phase	O
of	O
the	O
cell	O
cycle	O
.	O

Cdh1p	B-Gene_or_gene_product
is	O
phosphorylated	O
and	O
thereby	O
inactivated	O
at	O
the	O
G	O
(	O
1	O
)	O
/	O
S	O
transition	O
predominantly	O
by	O
Cdc28p	B-Complex
-	I-Complex
Clb5p	I-Complex
.	O

Here	O
we	O
show	O
that	O
Cdh1p	B-Gene_or_gene_product
is	O
nuclear	B-Cellular_component
during	O
the	O
G	O
(	O
1	O
)	O
phase	O
of	O
the	O
cell	O
cycle	O
,	O
but	O
redistributes	O
to	O
the	O
cytoplasm	B-Cellular_component
between	O
S	O
phase	O
and	O
the	O
end	O
of	O
mitosis	O
.	O

Nuclear	B-Cellular_component
export	O
of	O
Cdh1p	B-Gene_or_gene_product
is	O
regulated	O
by	O
phosphorylation	O
and	O
requires	O
active	O
Cdc28p	B-Gene_or_gene_product
kinase	O
.	O

Cdh1p	B-Gene_or_gene_product
binds	O
to	O
the	O
importin	O
Pse1p	B-Gene_or_gene_product
and	O
the	O
exportin	O
Msn5p	B-Gene_or_gene_product
,	O
which	O
is	O
necessary	O
and	O
sufficient	O
to	O
promote	O
efficient	O
export	O
of	O
Cdh1p	B-Gene_or_gene_product
in	O
vivo	O
.	O

Although	O
msn5delta	B-Gene_or_gene_product
cells	O
are	O
viable	O
,	O
they	O
are	O
sensitive	O
to	O
Cdh1p	B-Gene_or_gene_product
overexpression	O
.	O

Likewise	O
,	O
a	O
mutant	O
form	O
of	O
Cdh1p	B-Gene_or_gene_product
,	O
which	O
is	O
constitutively	O
nuclear	B-Cellular_component
,	O
prevents	O
accumulation	O
of	O
Clb2p	B-Gene_or_gene_product
and	O
leads	O
to	O
cell	O
cycle	O
arrest	O
when	O
overexpressed	O
in	O
wild	O
-	O
type	O
cells	O
.	O

Taken	O
together	O
,	O
these	O
results	O
suggest	O
that	O
phosphorylation	O
-	O
dependent	O
nuclear	B-Cellular_component
export	O
of	O
Cdh1p	B-Gene_or_gene_product
by	O
Msn5p	B-Gene_or_gene_product
contributes	O
to	O
efficient	O
inactivation	O
of	O
APC	B-Complex
/	I-Complex
C	I-Complex
(	I-Complex
Cdh1	I-Complex
)	I-Complex
.	O

Recruitment	O
of	O
NBS1	B-Gene_or_gene_product
into	O
PML	B-Gene_or_gene_product
oncogenic	O
domains	O
via	O
interaction	O
with	O
SP100	B-Gene_or_gene_product
protein	O
.	O

Nijmegen	O
breakage	O
syndrome	O
(	O
NBS	O
)	O
is	O
an	O
autosomal	O
recessive	O
disorder	O
characterized	O
by	O
microcephaly	O
,	O
chromosomal	B-Cellular_component
instability	O
,	O
radiation	O
sensitivity	O
,	O
and	O
an	O
increased	O
incidence	O
of	O
malignancies	O
.	O

NBS1	B-Gene_or_gene_product
,	O
the	O
protein	O
responsible	O
for	O
NBS	O
,	O
forms	O
a	O
complex	O
with	O
MRE11	B-Gene_or_gene_product
and	O
RAD50	B-Gene_or_gene_product
,	O
and	O
plays	O
a	O
vital	O
role	O
in	O
DNA	O
repair	O
,	O
cell	O
cycle	O
checkpoint	O
,	O
and	O
telomere	O
maintenance	O
.	O

Here	O
,	O
we	O
show	O
that	O
a	O
BRCA	B-Gene_or_gene_product
carboxyl	B-Simple_chemical
terminus	O
(	O
BRCT	O
)	O
domain	O
-	O
containing	O
region	O
of	O
NBS1	B-Gene_or_gene_product
interacts	O
with	O
a	O
nuclear	B-Cellular_component
dots	I-Cellular_component
-	O
associated	O
protein	O
,	O
SP100	B-Gene_or_gene_product
.	O

The	O
SP100	B-Gene_or_gene_product
and	O
NBS1	B-Gene_or_gene_product
proteins	O
co	O
-	O
localized	O
in	O
PODs	B-Cellular_component
and	O
APBs	B-Cellular_component
in	O
normal	O
human	O
fibroblast	O
MRC5	O
and	O
ALT	O
line	O
VA13	O
at	O
G2	O
phase	O
,	O
respectively	O
.	O

Introduction	O
of	O
PML	B-Gene_or_gene_product
and	O
SP100	B-Gene_or_gene_product
into	O
NT2	O
cells	O
,	O
which	O
express	O
no	O
detectable	O
amount	O
of	O
PML	B-Gene_or_gene_product
or	O
SP100	B-Gene_or_gene_product
proteins	O
,	O
resulted	O
in	O
localization	O
of	O
NBS1	B-Gene_or_gene_product
in	O
ectopically	O
expressed	O
PODs	B-Cellular_component
.	O

These	O
results	O
indicate	O
that	O
NBS1	B-Gene_or_gene_product
is	O
recruited	O
into	O
PODs	B-Cellular_component
via	O
interaction	O
with	O
SP100	B-Gene_or_gene_product
protein	O
.	O

Thus	O
,	O
interaction	O
between	O
the	O
NBS1	B-Gene_or_gene_product
and	O
SP100	B-Gene_or_gene_product
proteins	O
may	O
be	O
involved	O
in	O
genomic	O
stability	O
and	O
telomere	O
maintenance	O
.	O

Cyclin	B-Gene_or_gene_product
-	I-Gene_or_gene_product
dependent	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
:	O
linking	O
apoptosis	O
to	O
cell	O
cycle	O
and	O
mitotic	O
catastrophe	O
.	O

The	O
cyclin	B-Gene_or_gene_product
-	I-Gene_or_gene_product
dependent	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
1	I-Gene_or_gene_product
(	O
Cdk1	B-Gene_or_gene_product
)	O
,	O
formerly	O
called	O
Cdc2	B-Gene_or_gene_product
(	O
or	O
p34	B-Gene_or_gene_product
(	I-Gene_or_gene_product
Cdc2	I-Gene_or_gene_product
)	I-Gene_or_gene_product
)	O
,	O
interacts	O
with	O
cyclin	B-Gene_or_gene_product
B1	I-Gene_or_gene_product
to	O
form	O
an	O
active	O
heterodimer	O
.	O

The	O
activity	O
of	O
Cdk1	B-Gene_or_gene_product
is	O
subjected	O
to	O
a	O
complex	O
spatiotemporary	O
regulation	O
,	O
required	O
to	O
guarantee	O
its	O
scheduled	O
contribution	O
to	O
the	O
mitotic	O
prophase	O
and	O
metaphase	O
.	O

Moreover	O
,	O
the	O
activation	O
of	O
Cdk1	B-Gene_or_gene_product
may	O
be	O
required	O
for	O
apoptosis	O
induction	O
in	O
some	O
particular	O
pathways	O
of	O
cell	O
killing	O
.	O

This	O
applies	O
to	O
several	O
clinically	O
important	O
settings	O
,	O
for	O
instance	O
to	O
paclitaxel	B-Simple_chemical
-	O
induced	O
killing	O
of	O
breast	O
cancer	O
cells	O
,	O
in	O
which	O
the	O
ErbB2	B-Gene_or_gene_product
receptor	O
kinase	O
can	O
mediate	O
apoptosis	O
inhibition	O
through	O
inactivation	O
of	O
Cdk1	B-Gene_or_gene_product
.	O

The	O
activation	O
of	O
Cdk1	B-Gene_or_gene_product
participates	O
also	O
in	O
HIV	O
-	O
1	O
-	O
induced	O
apoptosis	O
,	O
upstream	O
of	O
the	O
p53	B-Gene_or_gene_product
-	O
dependent	O
mitochondrial	B-Cellular_component
permeabilization	O
step	O
.	O

An	O
unscheduled	O
Cdk1	B-Gene_or_gene_product
activation	O
may	O
contribute	O
to	O
neuronal	O
apoptosis	O
occurring	O
in	O
neurodegenerative	O
diseases	O
.	O

Finally	O
,	O
the	O
premature	O
activation	O
of	O
Cdk1	B-Gene_or_gene_product
can	O
lead	O
to	O
mitotic	O
catastrophe	O
,	O
for	O
instance	O
after	O
irradiation	O
-	O
induced	O
DNA	O
damage	O
.	O

Thus	O
,	O
a	O
cell	O
type	O
-	O
specific	O
modulation	O
of	O
Cdk1	B-Gene_or_gene_product
might	O
be	O
taken	O
advantage	O
of	O
for	O
the	O
therapeutic	O
correction	O
of	O
pathogenic	O
imbalances	O
in	O
apoptosis	O
control	O
.	O

betaTrCP	B-Gene_or_gene_product
-	O
mediated	O
proteolysis	O
of	O
NF	B-Complex
-	I-Complex
kappaB1	I-Complex
p105	B-Gene_or_gene_product
requires	O
phosphorylation	O
of	O
p105	B-Gene_or_gene_product
serines	B-Simple_chemical
927	I-Simple_chemical
and	O
932	B-Simple_chemical
.	O

NF	B-Complex
-	I-Complex
kappaB1	I-Complex
p105	B-Gene_or_gene_product
functions	O
both	O
as	O
a	O
precursor	O
of	O
NF	B-Complex
-	I-Complex
kappaB1	I-Complex
p50	B-Gene_or_gene_product
and	O
as	O
a	O
cytoplasmic	B-Cellular_component
inhibitor	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
.	O

Following	O
the	O
stimulation	O
of	O
cells	O
with	O
tumor	B-Gene_or_gene_product
necrosis	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
(	O
TNF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
)	O
,	O
the	O
IkappaB	B-Complex
kinase	I-Complex
(	O
IKK	B-Complex
)	O
complex	O
rapidly	O
phosphorylates	O
NF	B-Complex
-	I-Complex
kappaB1	I-Complex
p105	B-Gene_or_gene_product
on	O
serine	B-Simple_chemical
927	I-Simple_chemical
in	O
the	O
PEST	O
region	O
.	O

This	O
phosphorylation	O
is	O
essential	O
for	O
TNF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
to	O
trigger	O
p105	B-Gene_or_gene_product
degradation	O
,	O
which	O
releases	O
the	O
associated	O
Rel	B-Gene_or_gene_product
/	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
subunits	O
to	O
translocate	O
into	O
the	O
nucleus	B-Cellular_component
and	O
regulate	O
target	O
gene	O
transcription	O
.	O

Serine	B-Simple_chemical
927	I-Simple_chemical
resides	O
in	O
a	O
conserved	O
motif	O
(	O
Asp	B-Simple_chemical
-	O
Ser	B-Simple_chemical
(	I-Simple_chemical
927	I-Simple_chemical
)	I-Simple_chemical
-	O
Gly	B-Simple_chemical
-	O
Val	B-Simple_chemical
-	O
Glu	B-Simple_chemical
-	O
Thr	B-Simple_chemical
-	O
Ser	B-Simple_chemical
(	I-Simple_chemical
932	I-Simple_chemical
)	I-Simple_chemical
)	O
homologous	O
to	O
the	O
IKK	B-Complex
target	O
sequence	O
in	O
IkappaBalpha	B-Gene_or_gene_product
.	O

In	O
this	O
study	O
,	O
TNF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
-	O
induced	O
p105	B-Gene_or_gene_product
proteolysis	O
was	O
revealed	O
to	O
additionally	O
require	O
the	O
phosphorylation	O
of	O
serine	B-Simple_chemical
932	I-Simple_chemical
.	O

Experiments	O
with	O
IKK1	B-Gene_or_gene_product
(	O
-	O
/	O
-	O
)	O
and	O
IKK2	B-Gene_or_gene_product
(	O
-	O
/	O
-	O
)	O
double	O
knockout	O
embryonic	O
fibroblasts	O
demonstrate	O
that	O
the	O
IKK	B-Complex
complex	O
is	O
essential	O
for	O
TNF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
to	O
stimulate	O
phosphorylation	O
on	O
p105	B-Gene_or_gene_product
serines	B-Simple_chemical
927	I-Simple_chemical
and	O
932	B-Simple_chemical
.	O

Furthermore	O
,	O
purified	O
IKK1	B-Gene_or_gene_product
and	O
IKK2	B-Gene_or_gene_product
can	O
each	O
phosphorylate	O
a	O
glutathione	B-Gene_or_gene_product
S	I-Gene_or_gene_product
-	I-Gene_or_gene_product
transferase	I-Gene_or_gene_product
-	I-Gene_or_gene_product
p105	I-Gene_or_gene_product
(	O
758	O
-	O
967	O
)	O
fusion	O
protein	O
on	O
both	O
regulatory	O
serines	O
in	O
vitro	O
.	O

IKK	B-Complex
-	O
mediated	O
p105	B-Gene_or_gene_product
phosphorylation	O
generates	O
a	O
binding	O
site	O
for	O
betaTrCP	B-Gene_or_gene_product
,	O
the	O
receptor	O
subunit	O
of	O
an	O
SCF	O
-	O
type	O
ubiquitin	B-Gene_or_gene_product
E3	O
ligase	O
,	O
and	O
depletion	O
of	O
betaTrCP	B-Gene_or_gene_product
by	O
RNA	O
interference	O
blocks	O
TNF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
-	O
induced	O
p105	B-Gene_or_gene_product
ubiquitination	O
and	O
proteolysis	O
.	O

Phosphopeptide	O
competition	O
experiments	O
indicate	O
that	O
betaTrCP	B-Gene_or_gene_product
binds	O
p105	B-Gene_or_gene_product
more	O
effectively	O
when	O
both	O
serines	B-Simple_chemical
927	I-Simple_chemical
and	O
932	B-Simple_chemical
are	O
phosphorylated	O
.	O

Interestingly	O
,	O
however	O
,	O
betaTrCP	B-Gene_or_gene_product
affinity	O
for	O
the	O
IKK	B-Complex
-	O
phosphorylated	O
sequence	O
on	O
p105	B-Gene_or_gene_product
is	O
substantially	O
lower	O
than	O
that	O
on	O
IkappaBalpha	B-Gene_or_gene_product
.	O

Thus	O
,	O
it	O
appears	O
that	O
reduced	O
p105	B-Gene_or_gene_product
recruitment	O
of	O
betaTrCP	B-Gene_or_gene_product
and	O
subsequent	O
ubiquitination	O
may	O
contribute	O
to	O
delayed	O
p105	B-Gene_or_gene_product
proteolysis	O
after	O
TNF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
stimulation	O
relative	O
to	O
that	O
for	O
IkappaBalpha	B-Gene_or_gene_product
.	O

Regulation	O
of	O
lymphoid	B-Gene_or_gene_product
enhancer	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
1	I-Gene_or_gene_product
/	I-Gene_or_gene_product
T	I-Gene_or_gene_product
-	I-Gene_or_gene_product
cell	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
by	O
mitogen	B-Gene_or_gene_product
-	I-Gene_or_gene_product
activated	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
-	O
related	O
Nemo	B-Gene_or_gene_product
-	I-Gene_or_gene_product
like	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
-	O
dependent	O
phosphorylation	O
in	O
Wnt	B-Gene_or_gene_product
/	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
signaling	O
.	O

The	O
Wnt	B-Gene_or_gene_product
/	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
signaling	O
pathway	O
regulates	O
many	O
developmental	O
processes	O
by	O
modulating	O
gene	O
expression	O
.	O

Wnt	B-Gene_or_gene_product
signaling	O
induces	O
the	O
stabilization	O
of	O
cytosolic	B-Cellular_component
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
,	O
which	O
then	O
associates	O
with	O
lymphoid	B-Gene_or_gene_product
enhancer	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
and	O
T	B-Gene_or_gene_product
-	I-Gene_or_gene_product
cell	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
(	O
LEF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
/	O
TCF	B-Gene_or_gene_product
)	O
to	O
form	O
a	O
transcription	O
complex	O
that	O
activates	O
Wnt	B-Gene_or_gene_product
target	O
genes	O
.	O

Previously	O
,	O
we	O
have	O
shown	O
that	O
a	O
specific	O
mitogen	B-Gene_or_gene_product
-	I-Gene_or_gene_product
activated	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
(	I-Gene_or_gene_product
MAP	I-Gene_or_gene_product
)	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
pathway	O
involving	O
the	O
MAP	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
TAK1	I-Gene_or_gene_product
and	O
MAP	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
-	O
related	O
Nemo	B-Gene_or_gene_product
-	I-Gene_or_gene_product
like	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
(	O
NLK	B-Gene_or_gene_product
)	O
suppresses	O
Wnt	B-Gene_or_gene_product
signaling	O
.	O

In	O
this	O
study	O
,	O
we	O
investigated	O
the	O
relationships	O
among	O
NLK	B-Gene_or_gene_product
,	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
,	O
and	O
LEF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
/	I-Gene_or_gene_product
TCF	I-Gene_or_gene_product
.	O

We	O
found	O
that	O
NLK	B-Gene_or_gene_product
interacts	O
directly	O
with	O
LEF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
/	I-Gene_or_gene_product
TCF	I-Gene_or_gene_product
and	O
indirectly	O
with	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
via	O
LEF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
/	I-Gene_or_gene_product
TCF	I-Gene_or_gene_product
to	O
form	O
a	O
complex	O
.	O

NLK	B-Gene_or_gene_product
phosphorylates	O
LEF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
/	I-Gene_or_gene_product
TCF	I-Gene_or_gene_product
on	O
two	O
serine	B-Simple_chemical
/	O
threonine	B-Simple_chemical
residues	O
located	O
in	O
its	O
central	O
region	O
.	O

Mutation	O
of	O
both	O
residues	O
to	O
alanine	B-Simple_chemical
enhanced	O
LEF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
transcriptional	O
activity	O
and	O
rendered	O
it	O
resistant	O
to	O
inhibition	O
by	O
NLK	B-Gene_or_gene_product
.	O

Phosphorylation	O
of	O
TCF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
4	I-Gene_or_gene_product
by	O
NLK	B-Gene_or_gene_product
inhibited	O
DNA	O
binding	O
by	O
the	O
beta	B-Complex
-	I-Complex
catenin	I-Complex
-	I-Complex
TCF	I-Complex
-	I-Complex
4	I-Complex
complex	O
.	O

However	O
,	O
this	O
inhibition	O
was	O
abrogated	O
when	O
a	O
mutant	O
form	O
of	O
TCF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
4	I-Gene_or_gene_product
was	O
used	O
in	O
which	O
both	O
threonines	B-Simple_chemical
were	O
replaced	O
with	O
valines	B-Simple_chemical
.	O

These	O
results	O
suggest	O
that	O
NLK	B-Gene_or_gene_product
phosphorylation	O
on	O
these	O
sites	O
contributes	O
to	O
the	O
down	O
-	O
regulation	O
of	O
LEF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
/	I-Gene_or_gene_product
TCF	I-Gene_or_gene_product
transcriptional	O
activity	O
.	O

Tetrameric	O
oligomerization	O
of	O
IkappaB	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
gamma	I-Gene_or_gene_product
(	O
IKKgamma	B-Gene_or_gene_product
)	O
is	O
obligatory	O
for	O
IKK	B-Complex
complex	O
activity	O
and	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
activation	O
.	O

The	O
IkappaB	B-Complex
kinase	I-Complex
(	O
IKK	B-Complex
)	O
complex	O
mediates	O
activation	O
of	O
transcription	O
factor	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
by	O
phosphorylation	O
of	O
IkappaB	B-Gene_or_gene_product
proteins	O
.	O

Its	O
catalytic	O
subunits	O
,	O
IKKalpha	B-Gene_or_gene_product
and	O
IKKbeta	B-Gene_or_gene_product
,	O
require	O
association	O
with	O
the	O
regulatory	O
IKKgamma	B-Gene_or_gene_product
(	O
NEMO	B-Gene_or_gene_product
)	O
component	O
to	O
gain	O
full	O
basal	O
and	O
inducible	O
kinase	O
activity	O
.	O

However	O
,	O
the	O
oligomeric	O
composition	O
of	O
the	O
IKK	B-Complex
complex	O
and	O
its	O
regulation	O
by	O
IKKgamma	B-Gene_or_gene_product
are	O
poorly	O
understood	O
.	O

We	O
show	O
here	O
that	O
IKKgamma	B-Gene_or_gene_product
predominantly	O
forms	O
tetramers	O
and	O
interacts	O
with	O
IKKalpha	B-Gene_or_gene_product
or	O
IKKbeta	B-Gene_or_gene_product
in	O
this	O
state	O
.	O

We	O
propose	O
that	O
tetramerization	O
is	O
accomplished	O
by	O
a	O
prerequisite	O
dimerization	O
through	O
a	O
C	O
-	O
terminal	O
coiled	O
-	O
coil	O
minimal	O
oligomerization	O
domain	O
(	O
MOD	O
)	O
.	O

This	O
is	O
followed	O
by	O
dimerization	O
of	O
the	O
dimers	O
with	O
their	O
N	O
-	O
terminal	O
sequences	O
.	O

Tetrameric	O
IKKgamma	B-Complex
sequesters	O
four	O
kinase	O
molecules	O
,	O
yielding	O
a	O
gamma	B-Gene_or_gene_product
(	O
4	O
)	O
(	O
alpha	B-Gene_or_gene_product
/	O
beta	B-Gene_or_gene_product
)	O
(	O
4	O
)	O
stoichiometry	O
.	O

Deletion	O
of	O
the	O
MOD	O
leads	O
to	O
loss	O
of	O
tetramerization	O
and	O
of	O
phosphorylation	O
of	O
IKKbeta	B-Gene_or_gene_product
and	O
IKKgamma	B-Gene_or_gene_product
,	O
although	O
the	O
kinase	O
can	O
still	O
interact	O
with	O
the	O
resultant	O
IKKgamma	B-Gene_or_gene_product
monomers	O
and	O
dimers	O
.	O

Likewise	O
,	O
MOD	O
-	O
mediated	O
IKKgamma	B-Gene_or_gene_product
tetramerization	O
is	O
required	O
to	O
enhance	O
IKKbeta	B-Gene_or_gene_product
kinase	O
activity	O
when	O
overexpressed	O
in	O
293	O
cells	O
and	O
to	O
reconstitute	O
a	O
lipopolysaccharide	B-Simple_chemical
-	O
responsive	O
IKK	B-Complex
complex	O
in	O
pre	O
-	O
B	O
cells	O
.	O

These	O
data	O
thus	O
suggest	O
that	O
IKKgamma	B-Gene_or_gene_product
tetramerization	O
enforces	O
a	O
spatial	O
positioning	O
of	O
two	O
kinase	O
dimers	O
to	O
facilitate	O
transautophosphorylation	O
and	O
activation	O
.	O

Biotin	B-Simple_chemical
uptake	O
by	O
human	O
intestinal	O
and	O
liver	O
epithelial	O
cells	O
:	O
role	O
of	O
the	O
SMVT	B-Gene_or_gene_product
system	O
.	O

It	O
has	O
been	O
well	O
established	O
that	O
human	O
intestinal	O
and	O
liver	O
epithelial	O
cells	O
transport	O
biotin	B-Simple_chemical
via	O
an	O
Na	B-Simple_chemical
+	I-Simple_chemical
-	O
dependent	O
carrier	O
-	O
mediated	O
mechanism	O
.	O

The	O
sodium	B-Gene_or_gene_product
-	I-Gene_or_gene_product
dependent	I-Gene_or_gene_product
multivitamin	I-Gene_or_gene_product
transport	I-Gene_or_gene_product
(	O
SMVT	B-Gene_or_gene_product
)	O
,	O
a	O
biotin	B-Simple_chemical
transporter	O
,	O
is	O
expressed	O
in	O
both	O
cell	O
types	O
.	O

However	O
,	O
the	O
relative	O
contribution	O
of	O
SMVT	B-Gene_or_gene_product
toward	O
total	O
carrier	O
-	O
mediated	O
uptake	O
of	O
physiological	O
(	O
nanomolar	O
)	O
concentrations	O
of	O
biotin	B-Simple_chemical
by	O
these	O
cells	O
is	O
not	O
clear	O
.	O

Addressing	O
this	O
issue	O
is	O
important	O
,	O
especially	O
in	O
light	O
of	O
the	O
recent	O
identification	O
of	O
a	O
second	O
human	O
high	O
-	O
affinity	O
biotin	B-Simple_chemical
uptake	O
mechanism	O
that	O
operates	O
at	O
the	O
nanomolar	O
range	O
.	O

Hence	O
,	O
we	O
employed	O
a	O
physiological	O
approach	O
of	O
characterizing	O
biotin	B-Simple_chemical
uptake	O
by	O
human	O
-	O
derived	O
intestinal	O
Caco	O
-	O
2	O
and	O
HepG2	O
cells	O
at	O
the	O
nanomolar	O
concentration	O
range	O
.	O

We	O
also	O
employed	O
a	O
molecular	O
biology	O
approach	O
of	O
selectively	O
silencing	O
the	O
endogenous	O
SMVT	B-Gene_or_gene_product
of	O
these	O
cells	O
with	O
specific	O
small	O
interfering	O
RNAs	O
(	O
siRNAs	O
)	O
,	O
then	O
examining	O
carrier	O
-	O
mediated	O
biotin	B-Simple_chemical
uptake	O
.	O

The	O
results	O
showed	O
that	O
in	O
both	O
Caco	O
-	O
2	O
and	O
HepG2	O
cells	O
,	O
the	O
initial	O
rate	O
of	O
biotin	B-Simple_chemical
uptake	O
as	O
a	O
function	O
of	O
concentration	O
over	O
the	O
range	O
of	O
0	O
.	O
1	O
to	O
50	O
nM	O
to	O
be	O
linear	O
.	O

Furthermore	O
,	O
we	O
found	O
that	O
the	O
addition	O
of	O
100	O
nM	O
unlabeled	O
biotin	B-Simple_chemical
,	O
desthiobiotin	B-Simple_chemical
,	O
or	O
pantothenic	B-Simple_chemical
acid	I-Simple_chemical
to	O
the	O
incubation	O
medium	O
had	O
no	O
effect	O
on	O
the	O
uptake	O
of	O
2	O
.	O
6	O
nM	O
[	B-Simple_chemical
3H	I-Simple_chemical
]	I-Simple_chemical
biotin	I-Simple_chemical
.	O

Pretreatment	O
of	O
Caco	O
-	O
2	O
and	O
HepG2	O
cells	O
with	O
SMVT	B-Gene_or_gene_product
specific	O
siRNAs	O
substantially	O
reduced	O
SMVT	B-Gene_or_gene_product
mRNA	O
and	O
protein	O
levels	O
.	O

In	O
addition	O
,	O
carrier	O
-	O
mediated	O
[	B-Simple_chemical
3H	I-Simple_chemical
]	I-Simple_chemical
biotin	I-Simple_chemical
(	O
2	O
.	O
6	O
nM	O
)	O
uptake	O
by	O
Caco	O
-	O
2	O
and	O
HepG2	O
cells	O
was	O
severely	O
(	O
P	O
0	O
.	O
01	O
)	O
inhibited	O
by	O
the	O
siRNAs	O
pretreatment	O
.	O

These	O
results	O
demonstrate	O
that	O
the	O
recently	O
described	O
human	O
high	O
-	O
affinity	O
biotin	B-Simple_chemical
uptake	O
system	O
is	O
not	O
functional	O
in	O
intestinal	O
and	O
liver	O
epithelial	O
cells	O
.	O

In	O
addition	O
,	O
the	O
results	O
provide	O
strong	O
evidence	O
that	O
SMVT	B-Gene_or_gene_product
is	O
the	O
major	O
(	O
if	O
not	O
the	O
only	O
)	O
biotin	B-Simple_chemical
uptake	O
system	O
that	O
operates	O
in	O
these	O
cells	O
.	O

Identification	O
of	O
E	O
-	O
box	O
factor	O
TFE3	B-Gene_or_gene_product
as	O
a	O
functional	O
partner	O
for	O
the	O
E2F3	B-Gene_or_gene_product
transcription	O
factor	O
.	O

Various	O
studies	O
have	O
demonstrated	O
a	O
role	O
for	O
E2F	B-Gene_or_gene_product
proteins	O
in	O
the	O
control	O
of	O
transcription	O
of	O
genes	O
involved	O
in	O
DNA	O
replication	O
,	O
cell	O
cycle	O
progression	O
,	O
and	O
cell	O
fate	O
determination	O
.	O

Although	O
it	O
is	O
clear	O
that	O
the	O
functions	O
of	O
the	O
E2F	B-Gene_or_gene_product
proteins	O
overlap	O
,	O
there	O
is	O
also	O
evidence	O
for	O
specific	O
roles	O
for	O
individual	O
E2F	B-Gene_or_gene_product
proteins	O
in	O
the	O
control	O
of	O
apoptosis	O
and	O
cell	O
proliferation	O
.	O

Investigating	O
protein	O
interactions	O
that	O
might	O
provide	O
a	O
mechanistic	O
basis	O
for	O
the	O
specificity	O
of	O
E2F	B-Gene_or_gene_product
function	O
,	O
we	O
identified	O
the	O
E	O
-	O
box	O
binding	O
factor	O
TFE3	B-Gene_or_gene_product
as	O
an	O
E2F3	B-Gene_or_gene_product
-	O
specific	O
partner	O
.	O

We	O
also	O
show	O
that	O
this	O
interaction	O
is	O
dependent	O
on	O
the	O
marked	O
box	O
domain	O
of	O
E2F3	B-Gene_or_gene_product
.	O

We	O
provide	O
evidence	O
for	O
a	O
role	O
for	O
TFE3	B-Gene_or_gene_product
in	O
the	O
synergistic	O
activation	O
of	O
the	O
p68	B-Gene_or_gene_product
subunit	O
gene	O
of	O
DNA	B-Complex
polymerase	I-Complex
alpha	I-Complex
together	O
with	O
E2F3	B-Gene_or_gene_product
,	O
again	O
dependent	O
on	O
the	O
E2F3	B-Gene_or_gene_product
marked	O
box	O
domain	O
.	O

Chromatin	B-Cellular_component
immunoprecipitation	O
assays	O
showed	O
that	O
TFE3	B-Gene_or_gene_product
and	O
E2F3	B-Gene_or_gene_product
were	O
bound	O
to	O
the	O
p68	B-Gene_or_gene_product
promoter	O
in	O
vivo	O
and	O
that	O
the	O
interaction	O
of	O
either	O
E2F3	B-Gene_or_gene_product
or	O
TFE3	B-Gene_or_gene_product
with	O
the	O
promoter	O
was	O
facilitated	O
by	O
the	O
presence	O
of	O
both	O
proteins	O
.	O

In	O
contrast	O
,	O
neither	O
E2F1	B-Gene_or_gene_product
nor	O
E2F2	B-Gene_or_gene_product
interacted	O
with	O
the	O
p68	B-Gene_or_gene_product
promoter	O
under	O
these	O
conditions	O
.	O

We	O
propose	O
that	O
the	O
physical	O
interaction	O
of	O
TFE3	B-Gene_or_gene_product
and	O
E2F3	B-Gene_or_gene_product
facilitates	O
transcriptional	O
activation	O
of	O
the	O
p68	B-Gene_or_gene_product
gene	O
and	O
provides	O
strong	O
evidence	O
for	O
the	O
specificity	O
of	O
E2F	B-Gene_or_gene_product
function	O
.	O

A	O
subset	O
of	O
ATM	B-Gene_or_gene_product
-	O
and	O
ATR	B-Gene_or_gene_product
-	O
dependent	O
phosphorylation	O
events	O
requires	O
the	O
BRCA1	B-Gene_or_gene_product
protein	O
.	O

BRCA1	B-Gene_or_gene_product
is	O
a	O
central	O
component	O
of	O
the	O
DNA	O
damage	O
response	O
mechanism	O
and	O
defects	O
in	O
BRCA1	B-Gene_or_gene_product
confer	O
sensitivity	O
to	O
a	O
broad	O
range	O
of	O
DNA	O
damaging	O
agents	O
.	O

BRCA1	B-Gene_or_gene_product
is	O
required	O
for	O
homologous	O
recombination	O
and	O
DNA	O
damage	O
-	O
induced	O
S	O
and	O
G	O
(	O
2	O
)	O
/	O
M	O
phase	O
arrest	O
.	O

We	O
show	O
here	O
that	O
BRCA1	B-Gene_or_gene_product
is	O
required	O
for	O
ATM	B-Gene_or_gene_product
-	O
and	O
ATR	B-Gene_or_gene_product
-	O
dependent	O
phosphorylation	O
of	O
p53	B-Gene_or_gene_product
,	O
c	B-Gene_or_gene_product
-	I-Gene_or_gene_product
Jun	I-Gene_or_gene_product
,	O
Nbs1	B-Gene_or_gene_product
and	O
Chk2	B-Gene_or_gene_product
following	O
exposure	O
to	O
ionizing	O
or	O
ultraviolet	O
radiation	O
,	O
respectively	O
,	O
and	O
is	O
also	O
required	O
for	O
ATM	B-Gene_or_gene_product
phosphorylation	O
of	O
CtIP	B-Gene_or_gene_product
.	O

In	O
contrast	O
,	O
DNA	O
damage	O
-	O
induced	O
phosphorylation	O
of	O
the	O
histone	B-Gene_or_gene_product
variant	O
H2AX	B-Gene_or_gene_product
is	O
independent	O
of	O
BRCA1	B-Gene_or_gene_product
.	O

We	O
also	O
show	O
that	O
the	O
presence	O
of	O
BRCA1	B-Gene_or_gene_product
is	O
dispensable	O
for	O
DNA	O
damage	O
-	O
induced	O
phosphorylation	O
of	O
Rad9	B-Gene_or_gene_product
,	O
Hus1	B-Gene_or_gene_product
and	O
Rad17	B-Gene_or_gene_product
,	O
and	O
for	O
the	O
relocalization	O
of	O
Rad9	B-Gene_or_gene_product
and	O
Hus1	B-Gene_or_gene_product
.	O

We	O
propose	O
that	O
BRCA1	B-Gene_or_gene_product
facilitates	O
the	O
ability	O
of	O
ATM	B-Gene_or_gene_product
and	O
ATR	B-Gene_or_gene_product
to	O
phosphorylate	O
downstream	O
substrates	O
that	O
directly	O
influence	O
cell	O
cycle	O
checkpoint	O
arrest	O
and	O
apoptosis	O
,	O
but	O
that	O
BRCA1	B-Gene_or_gene_product
is	O
dispensable	O
for	O
the	O
phosphorylation	O
of	O
DNA	O
-	O
associated	O
ATM	B-Gene_or_gene_product
and	O
ATR	B-Gene_or_gene_product
substrates	O
.	O

Phosphorylation	O
of	O
p90	B-Gene_or_gene_product
ribosomal	I-Gene_or_gene_product
S6	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
(	O
RSK	B-Gene_or_gene_product
)	O
regulates	O
extracellular	B-Gene_or_gene_product
signal	I-Gene_or_gene_product
-	I-Gene_or_gene_product
regulated	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
docking	O
and	O
RSK	B-Gene_or_gene_product
activity	O
.	O

Stimulation	O
of	O
the	O
Ras	B-Gene_or_gene_product
/	O
extracellular	B-Gene_or_gene_product
signal	I-Gene_or_gene_product
-	I-Gene_or_gene_product
regulated	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
(	O
ERK	B-Gene_or_gene_product
)	O
pathway	O
can	O
modulate	O
cell	O
growth	O
,	O
proliferation	O
,	O
survival	O
,	O
and	O
motility	O
.	O

The	O
p90	B-Gene_or_gene_product
ribosomal	I-Gene_or_gene_product
S6	I-Gene_or_gene_product
kinases	I-Gene_or_gene_product
(	O
RSKs	B-Gene_or_gene_product
)	O
comprise	O
a	O
family	O
of	O
serine	B-Simple_chemical
/	O
threonine	B-Simple_chemical
kinases	O
that	O
lie	O
at	O
the	O
terminus	O
of	O
the	O
ERK	B-Gene_or_gene_product
pathway	O
.	O

Efficient	O
RSK	B-Gene_or_gene_product
activation	O
by	O
ERK	B-Gene_or_gene_product
requires	O
its	O
interaction	O
through	O
a	O
docking	O
site	O
located	O
near	O
the	O
C	O
terminus	O
of	O
RSK	B-Gene_or_gene_product
,	O
but	O
the	O
regulation	O
of	O
this	O
interaction	O
remains	O
unknown	O
.	O

In	O
this	O
report	O
we	O
show	O
that	O
RSK1	B-Gene_or_gene_product
and	O
ERK1	B-Gene_or_gene_product
/	I-Gene_or_gene_product
2	I-Gene_or_gene_product
form	O
a	O
complex	O
in	O
quiescent	O
HEK293	O
cells	O
that	O
transiently	O
dissociates	O
upon	O
mitogen	O
stimulation	O
.	O

Complex	O
dissociation	O
requires	O
phosphorylation	O
of	O
RSK1	B-Gene_or_gene_product
serine	B-Simple_chemical
749	I-Simple_chemical
,	O
which	O
is	O
a	O
mitogen	O
-	O
regulated	O
phosphorylation	O
site	O
located	O
near	O
the	O
ERK	B-Gene_or_gene_product
docking	O
site	O
.	O

Using	O
recombinant	O
RSK1	B-Gene_or_gene_product
proteins	O
,	O
we	O
find	O
that	O
serine	B-Simple_chemical
749	I-Simple_chemical
is	O
phosphorylated	O
by	O
the	O
N	O
-	O
terminal	O
kinase	O
domain	O
of	O
RSK1	B-Gene_or_gene_product
in	O
vitro	O
,	O
suggesting	O
that	O
ERK1	B-Gene_or_gene_product
/	I-Gene_or_gene_product
2	I-Gene_or_gene_product
dissociation	O
is	O
mediated	O
through	O
RSK1	B-Gene_or_gene_product
autophosphorylation	O
of	O
this	O
residue	O
.	O

Consistent	O
with	O
this	O
hypothesis	O
,	O
we	O
find	O
that	O
inactivating	O
mutations	O
in	O
the	O
RSK1	B-Gene_or_gene_product
kinase	O
domains	O
disrupted	O
the	O
mitogen	O
-	O
regulated	O
dissociation	O
of	O
ERK1	B-Gene_or_gene_product
/	I-Gene_or_gene_product
2	I-Gene_or_gene_product
in	O
vivo	O
.	O

Analysis	O
of	O
different	O
RSK	B-Gene_or_gene_product
isoforms	O
revealed	O
that	O
RSK1	B-Gene_or_gene_product
and	O
RSK2	B-Gene_or_gene_product
readily	O
dissociate	O
from	O
ERK1	B-Gene_or_gene_product
/	I-Gene_or_gene_product
2	I-Gene_or_gene_product
following	O
mitogen	O
stimulation	O
but	O
that	O
RSK3	B-Gene_or_gene_product
remains	O
associated	O
with	O
active	O
ERK1	B-Gene_or_gene_product
/	I-Gene_or_gene_product
2	I-Gene_or_gene_product
.	O

RSK	B-Gene_or_gene_product
activity	O
assays	O
revealed	O
that	O
RSK3	B-Gene_or_gene_product
also	O
remains	O
active	O
longer	O
than	O
RSK1	B-Gene_or_gene_product
and	O
RSK2	B-Gene_or_gene_product
,	O
suggesting	O
that	O
prolonged	O
ERK	B-Gene_or_gene_product
association	O
increased	O
the	O
duration	O
of	O
RSK3	B-Gene_or_gene_product
activation	O
.	O

These	O
results	O
provide	O
new	O
evidence	O
for	O
the	O
regulated	O
nature	O
of	O
ERK	B-Gene_or_gene_product
docking	O
interactions	O
and	O
reveal	O
important	O
differences	O
among	O
the	O
closely	O
related	O
RSK	B-Gene_or_gene_product
family	O
members	O
.	O

E2F6	B-Gene_or_gene_product
negatively	O
regulates	O
BRCA1	B-Gene_or_gene_product
in	O
human	O
cancer	O
cells	O
without	O
methylation	O
of	O
histone	B-Gene_or_gene_product
H3	I-Gene_or_gene_product
on	O
lysine	B-Simple_chemical
9	I-Simple_chemical
.	O

E2F6	B-Gene_or_gene_product
contains	O
a	O
DNA	O
binding	O
domain	O
that	O
is	O
very	O
similar	O
to	O
that	O
of	O
the	O
other	O
members	O
of	O
the	O
E2F	B-Gene_or_gene_product
family	O
of	O
transcriptional	O
regulators	O
.	O

However	O
,	O
E2F6	B-Gene_or_gene_product
cannot	O
bind	O
to	O
all	O
promoters	O
that	O
contain	O
consensus	O
E2F	B-Gene_or_gene_product
-	O
binding	O
sites	O
.	O

Therefore	O
,	O
we	O
used	O
a	O
combination	O
of	O
chromatin	B-Cellular_component
immunoprecipitation	O
and	O
genomic	O
microarrays	O
to	O
identify	O
promoters	O
bound	O
by	O
E2F6	B-Gene_or_gene_product
in	O
human	O
cells	O
.	O

Although	O
most	O
of	O
the	O
identified	O
promoters	O
were	O
bound	O
by	O
multiple	O
E2F	B-Gene_or_gene_product
family	O
members	O
,	O
one	O
promoter	O
was	O
bound	O
only	O
by	O
E2F6	B-Gene_or_gene_product
.	O

To	O
determine	O
which	O
of	O
the	O
newly	O
identified	O
promoters	O
were	O
regulated	O
by	O
E2F6	B-Gene_or_gene_product
,	O
we	O
reduced	O
the	O
level	O
of	O
E2F6	B-Gene_or_gene_product
by	O
using	O
RNA	O
interference	O
technology	O
.	O

We	O
found	O
that	O
mRNA	O
transcribed	O
from	O
promoters	O
bound	O
by	O
E2F6	B-Gene_or_gene_product
was	O
increased	O
after	O
reduction	O
of	O
the	O
amount	O
of	O
E2F6	B-Gene_or_gene_product
protein	O
in	O
the	O
cell	O
.	O

Interestingly	O
,	O
many	O
of	O
the	O
E2F6	B-Gene_or_gene_product
-	O
regulated	O
genes	O
encoded	O
functions	O
involved	O
in	O
tumor	O
suppression	O
and	O
the	O
maintenance	O
of	O
chromatin	B-Cellular_component
structure	O
.	O

Specifically	O
,	O
our	O
results	O
suggest	O
that	O
E2F6	B-Gene_or_gene_product
represses	O
transcription	O
of	O
the	O
brca1	B-Gene_or_gene_product
,	O
ctip	B-Gene_or_gene_product
,	O
art27	B-Gene_or_gene_product
,	O
hp1alpha	B-Gene_or_gene_product
,	O
and	O
the	O
rbap48	B-Gene_or_gene_product
genes	O
.	O

E2F6	B-Gene_or_gene_product
has	O
been	O
postulated	O
to	O
mediate	O
transcriptional	O
repression	O
by	O
recruiting	O
a	O
histone	B-Gene_or_gene_product
H3	I-Gene_or_gene_product
methyltransferase	I-Gene_or_gene_product
to	O
the	O
DNA	O
.	O

However	O
,	O
we	O
found	O
that	O
the	O
E2F6	B-Gene_or_gene_product
-	O
regulated	O
promoters	O
did	O
not	O
contain	O
histone	B-Gene_or_gene_product
H3	I-Gene_or_gene_product
methylated	O
at	O
lysine	B-Simple_chemical
9	I-Simple_chemical
.	O

To	O
determine	O
the	O
mechanism	O
by	O
which	O
E2F6	B-Gene_or_gene_product
regulates	O
transcription	O
,	O
we	O
performed	O
chromatin	B-Cellular_component
immunoprecipitation	O
before	O
and	O
after	O
the	O
introduction	O
of	O
small	O
inhibitory	O
ribonucleic	O
acids	O
specific	O
to	O
E2F6	B-Gene_or_gene_product
.	O

We	O
found	O
that	O
depletion	O
of	O
E2F6	B-Gene_or_gene_product
resulted	O
in	O
the	O
recruitment	O
of	O
E2F1	B-Gene_or_gene_product
to	O
the	O
target	O
promoters	O
.	O

In	O
summary	O
,	O
we	O
have	O
identified	O
48	O
endogenous	O
target	O
genes	O
of	O
E2F6	B-Gene_or_gene_product
and	O
have	O
shown	O
that	O
E2F6	B-Gene_or_gene_product
can	O
repress	O
target	O
promoters	O
in	O
a	O
manner	O
that	O
does	O
not	O
require	O
histone	B-Gene_or_gene_product
H3	I-Gene_or_gene_product
methylation	O
at	O
lysine	B-Simple_chemical
9	I-Simple_chemical
.	O

SIGIRR	B-Gene_or_gene_product
,	O
a	O
negative	O
regulator	O
of	O
Toll	B-Gene_or_gene_product
-	I-Gene_or_gene_product
like	I-Gene_or_gene_product
receptor	I-Gene_or_gene_product
-	I-Gene_or_gene_product
interleukin	I-Gene_or_gene_product
1	I-Gene_or_gene_product
receptor	I-Gene_or_gene_product
signaling	O
.	O

The	O
Toll	B-Gene_or_gene_product
-	I-Gene_or_gene_product
like	I-Gene_or_gene_product
receptor	I-Gene_or_gene_product
-	I-Gene_or_gene_product
interleukin	I-Gene_or_gene_product
1	I-Gene_or_gene_product
receptor	I-Gene_or_gene_product
signaling	O
(	O
TLR	B-Gene_or_gene_product
-	I-Gene_or_gene_product
IL	I-Gene_or_gene_product
-	I-Gene_or_gene_product
1R	I-Gene_or_gene_product
)	O
receptor	O
superfamily	O
is	O
important	O
in	O
differentially	O
recognizing	O
pathogen	O
products	O
and	O
eliciting	O
appropriate	O
immune	O
responses	O
.	O

These	O
receptors	O
alter	O
gene	O
expression	O
,	O
mainly	O
through	O
the	O
activation	O
of	O
nuclear	B-Complex
factor	I-Complex
-	I-Complex
kappaB	I-Complex
and	O
activating	B-Complex
protein	I-Complex
1	I-Complex
.	O

SIGIRR	B-Gene_or_gene_product
(	O
single	B-Gene_or_gene_product
immunoglobulin	I-Gene_or_gene_product
IL	I-Gene_or_gene_product
-	I-Gene_or_gene_product
1R	I-Gene_or_gene_product
-	I-Gene_or_gene_product
related	I-Gene_or_gene_product
molecule	I-Gene_or_gene_product
)	O
,	O
a	O
member	O
of	O
this	O
family	O
that	O
does	O
not	O
activate	O
these	O
factors	O
,	O
instead	O
negatively	O
modulates	O
immune	O
responses	O
.	O

Inflammation	O
is	O
enhanced	O
in	O
SIGIRR	B-Gene_or_gene_product
-	O
deficient	O
mice	O
,	O
as	O
shown	O
by	O
their	O
enhanced	O
chemokine	O
induction	O
after	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
injection	O
and	O
reduced	O
threshold	O
for	O
lethal	O
endotoxin	O
challenge	O
.	O

Cells	O
from	O
SIGIRR	B-Gene_or_gene_product
-	O
deficient	O
mice	O
showed	O
enhanced	O
activation	O
in	O
response	O
to	O
either	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
or	O
certain	O
Toll	B-Gene_or_gene_product
ligands	O
.	O

Finally	O
,	O
biochemical	O
analysis	O
indicated	O
that	O
SIGIRR	B-Gene_or_gene_product
binds	O
to	O
the	O
TLR	B-Gene_or_gene_product
-	I-Gene_or_gene_product
IL	I-Gene_or_gene_product
-	I-Gene_or_gene_product
1R	I-Gene_or_gene_product
signaling	O
components	O
in	O
a	O
ligand	O
-	O
dependent	O
way	O
.	O

Our	O
data	O
show	O
that	O
SIGIRR	B-Gene_or_gene_product
functions	O
as	O
a	O
biologically	O
important	O
modulator	O
of	O
TLR	B-Gene_or_gene_product
-	I-Gene_or_gene_product
IL	I-Gene_or_gene_product
-	I-Gene_or_gene_product
1R	I-Gene_or_gene_product
signaling	O
.	O

Molecular	O
cloning	O
and	O
characterization	O
of	O
a	O
novel	O
Rel	B-Gene_or_gene_product
/	I-Gene_or_gene_product
NF	I-Gene_or_gene_product
-	I-Gene_or_gene_product
kappa	I-Gene_or_gene_product
B	I-Gene_or_gene_product
family	O
member	O
displaying	O
structural	O
and	O
functional	O
homology	O
to	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
p50	B-Gene_or_gene_product
/	O
p105	B-Gene_or_gene_product
.	O

The	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
transcription	O
factor	O
has	O
been	O
implicated	O
in	O
the	O
inducible	O
expression	O
of	O
many	O
genes	O
,	O
including	O
inflammatory	O
,	O
immune	O
,	O
and	O
acute	O
-	O
phase	O
response	O
genes	O
.	O

NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
consists	O
of	O
two	O
subunits	O
,	O
50K	O
and	O
65K	O
polypeptides	O
.	O

The	O
genes	O
encoding	O
p50	B-Gene_or_gene_product
and	O
p65	B-Gene_or_gene_product
have	O
sequence	O
similarities	O
with	O
the	O
c	B-Gene_or_gene_product
-	I-Gene_or_gene_product
rel	I-Gene_or_gene_product
proto	O
-	O
oncogene	O
and	O
the	O
Drosophila	O
maternal	O
effect	O
gene	O
dorsal	O
.	O

We	O
describe	O
the	O
cloning	O
and	O
characterization	O
of	O
a	O
novel	O
rel	B-Gene_or_gene_product
-	O
related	O
gene	O
encoding	O
a	O
98K	O
product	O
that	O
shares	O
extensive	O
homology	O
with	O
the	O
p105	B-Gene_or_gene_product
precursor	O
of	O
the	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
p50	B-Gene_or_gene_product
protein	O
,	O
containing	O
both	O
a	O
Rel	B-Gene_or_gene_product
homology	O
and	O
SWI6	B-Gene_or_gene_product
/	O
ankyrin	B-Gene_or_gene_product
repeat	O
domain	O
.	O

We	O
demonstrate	O
that	O
p98	B-Gene_or_gene_product
is	O
proteolytically	O
processed	O
in	O
vivo	O
to	O
generate	O
a	O
55K	O
polypeptide	O
,	O
which	O
binds	O
to	O
kappa	B-Complex
B	I-Complex
sites	O
.	O

p55	B-Gene_or_gene_product
is	O
capable	O
of	O
forming	O
heterocomplexes	O
with	O
other	O
Rel	B-Gene_or_gene_product
/	I-Gene_or_gene_product
NF	I-Gene_or_gene_product
-	I-Gene_or_gene_product
kappa	I-Gene_or_gene_product
B	I-Gene_or_gene_product
family	O
members	O
,	O
which	O
can	O
bind	O
to	O
kappa	B-Complex
B	I-Complex
motifs	O
in	O
vitro	O
,	O
and	O
stimulate	O
transcription	O
of	O
reporter	O
genes	O
containing	O
these	O
cis	O
-	O
elements	O
in	O
vivo	O
.	O

The	O
identification	O
of	O
a	O
homolog	O
for	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
p50	B-Gene_or_gene_product
/	O
p105	B-Gene_or_gene_product
,	O
termed	O
p55	B-Gene_or_gene_product
/	O
p98	B-Gene_or_gene_product
,	O
gives	O
further	O
support	O
to	O
the	O
idea	O
that	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
is	O
a	O
collection	O
of	O
structurally	O
related	O
complexes	O
of	O
which	O
contribute	O
to	O
the	O
pleiotropic	O
regulatory	O
processes	O
originally	O
assigned	O
to	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
.	O

Ser	B-Simple_chemical
-	I-Simple_chemical
64	I-Simple_chemical
and	O
Ser	B-Simple_chemical
-	I-Simple_chemical
111	I-Simple_chemical
in	O
PHAS	B-Gene_or_gene_product
-	I-Gene_or_gene_product
I	I-Gene_or_gene_product
are	O
dispensable	O
for	O
insulin	B-Gene_or_gene_product
-	O
stimulated	O
dissociation	O
from	O
eIF4E	B-Gene_or_gene_product
.	O

Insulin	B-Gene_or_gene_product
stimulates	O
phosphorylation	O
of	O
multiple	O
sites	O
in	O
the	O
eIF4E	B-Gene_or_gene_product
-	O
binding	O
protein	O
,	O
PHAS	B-Gene_or_gene_product
-	I-Gene_or_gene_product
I	I-Gene_or_gene_product
,	O
leading	O
to	O
dissociation	O
of	O
the	O
PHAS	B-Complex
-	I-Complex
I	I-Complex
.	I-Complex
eIF4E	I-Complex
complex	O
and	O
to	O
an	O
increase	O
in	O
cap	O
-	O
dependent	O
translation	O
.	O

The	O
Ser	B-Simple_chemical
-	I-Simple_chemical
64	I-Simple_chemical
and	O
Ser	B-Simple_chemical
-	I-Simple_chemical
111	I-Simple_chemical
sites	O
have	O
been	O
proposed	O
to	O
have	O
key	O
roles	O
in	O
controlling	O
the	O
association	O
of	O
PHAS	B-Gene_or_gene_product
-	I-Gene_or_gene_product
I	I-Gene_or_gene_product
and	O
eIF4E	B-Gene_or_gene_product
.	O

To	O
determine	O
whether	O
the	O
effects	O
of	O
insulin	B-Gene_or_gene_product
require	O
these	O
sites	O
,	O
we	O
assessed	O
the	O
control	O
of	O
PHAS	B-Gene_or_gene_product
-	I-Gene_or_gene_product
I	I-Gene_or_gene_product
proteins	O
having	O
Ala	B-Simple_chemical
-	I-Simple_chemical
64	I-Simple_chemical
or	O
Ala	B-Simple_chemical
-	I-Simple_chemical
111	I-Simple_chemical
mutations	O
.	O

The	O
results	O
indicate	O
that	O
phosphorylation	O
of	O
neither	O
site	O
is	O
required	O
for	O
insulin	B-Gene_or_gene_product
to	O
promote	O
release	O
of	O
PHAS	B-Gene_or_gene_product
-	I-Gene_or_gene_product
I	I-Gene_or_gene_product
from	O
eIF4E	B-Gene_or_gene_product
.	O

Also	O
,	O
the	O
mutation	O
of	O
Ser	B-Simple_chemical
-	I-Simple_chemical
111	I-Simple_chemical
,	O
which	O
has	O
been	O
proposed	O
to	O
serve	O
as	O
a	O
necessary	O
priming	O
site	O
for	O
the	O
phosphorylation	O
of	O
other	O
sites	O
in	O
PHAS	B-Gene_or_gene_product
-	I-Gene_or_gene_product
I	I-Gene_or_gene_product
,	O
did	O
not	O
affect	O
the	O
phosphorylation	O
of	O
Thr	B-Simple_chemical
-	I-Simple_chemical
36	I-Simple_chemical
/	O
45	B-Simple_chemical
,	O
Ser	B-Simple_chemical
-	I-Simple_chemical
64	I-Simple_chemical
,	O
or	O
Thr	B-Simple_chemical
-	I-Simple_chemical
69	I-Simple_chemical
.	O

Insulin	B-Gene_or_gene_product
promoted	O
the	O
release	O
of	O
eIF4E	B-Gene_or_gene_product
from	O
PHAS	B-Gene_or_gene_product
-	I-Gene_or_gene_product
II	I-Gene_or_gene_product
,	O
a	O
PHAS	B-Gene_or_gene_product
isoform	O
that	O
lacks	O
the	O
Ser	B-Simple_chemical
-	I-Simple_chemical
111	I-Simple_chemical
site	O
,	O
but	O
it	O
was	O
without	O
effect	O
on	O
the	O
amount	O
of	O
eIF4E	B-Gene_or_gene_product
bound	O
to	O
the	O
third	O
isoform	O
,	O
PHAS	B-Gene_or_gene_product
-	I-Gene_or_gene_product
III	I-Gene_or_gene_product
.	O

The	O
results	O
demonstrate	O
that	O
contrary	O
to	O
widely	O
accepted	O
models	O
,	O
Ser	B-Simple_chemical
-	I-Simple_chemical
64	I-Simple_chemical
and	O
Ser	B-Simple_chemical
-	I-Simple_chemical
111	I-Simple_chemical
are	O
not	O
required	O
for	O
the	O
control	O
of	O
PHAS	B-Gene_or_gene_product
-	I-Gene_or_gene_product
I	I-Gene_or_gene_product
binding	O
to	O
eIF4E	B-Gene_or_gene_product
in	O
cells	O
,	O
implicating	O
phosphorylation	O
of	O
the	O
Thr	B-Simple_chemical
sites	O
in	O
dissociation	O
of	O
the	O
PHAS	B-Complex
-	I-Complex
I	I-Complex
.	I-Complex
eIF4E	I-Complex
complex	O
.	O

The	O
findings	O
also	O
indicate	O
that	O
PHAS	B-Gene_or_gene_product
-	I-Gene_or_gene_product
II	I-Gene_or_gene_product
,	O
but	O
not	O
PHAS	B-Gene_or_gene_product
-	I-Gene_or_gene_product
III	I-Gene_or_gene_product
,	O
contributes	O
to	O
the	O
control	O
of	O
protein	O
synthesis	O
by	O
insulin	B-Gene_or_gene_product
.	O

p73	B-Gene_or_gene_product
tumor	O
-	O
suppressor	O
activity	O
is	O
impaired	O
in	O
human	O
thyroid	O
cancer	O
.	O

The	O
p73	B-Gene_or_gene_product
protein	O
is	O
a	O
member	O
of	O
the	O
p53	B-Gene_or_gene_product
family	O
and	O
,	O
like	O
p53	B-Gene_or_gene_product
,	O
can	O
induce	O
cell	O
-	O
cycle	O
arrest	O
and	O
apoptosis	O
in	O
response	O
to	O
DNA	O
damage	O
.	O

Because	O
the	O
loss	O
of	O
p53	B-Gene_or_gene_product
function	O
is	O
responsible	O
for	O
the	O
progression	O
of	O
well	O
-	O
differentiated	O
thyroid	O
cancer	O
to	O
more	O
aggressive	O
phenotypes	O
,	O
we	O
hypothesized	O
that	O
p73	B-Gene_or_gene_product
might	O
also	O
be	O
involved	O
in	O
thyroid	O
carcinogenesis	O
.	O

We	O
find	O
that	O
normal	O
thyrocites	O
do	O
not	O
express	O
p73	B-Gene_or_gene_product
,	O
whereas	O
most	O
thyroid	O
malignancies	O
are	O
positive	O
for	O
p73	B-Gene_or_gene_product
expression	O
.	O

However	O
,	O
the	O
p73	B-Gene_or_gene_product
protein	O
of	O
thyroid	O
cancer	O
cells	O
is	O
unresponsive	O
to	O
DNA	O
-	O
damaging	O
agents	O
,	O
failing	O
to	O
elicit	O
a	O
block	O
of	O
the	O
cell	O
cycle	O
or	O
an	O
apoptotic	O
response	O
.	O

Notably	O
,	O
overexpression	O
of	O
transcriptionally	O
active	O
p73	B-Gene_or_gene_product
in	O
thyroid	O
cancer	O
lines	O
can	O
arrest	O
the	O
cell	O
cycle	O
but	O
is	O
still	O
unable	O
to	O
induce	O
cell	O
death	O
.	O

The	O
loss	O
of	O
p73	B-Gene_or_gene_product
biological	O
activity	O
in	O
neoplastic	O
thyroid	O
cells	O
is	O
partly	O
explained	O
by	O
its	O
interaction	O
with	O
transcriptionally	O
inactive	O
variants	O
of	O
p73	B-Gene_or_gene_product
(	O
DeltaNp73	B-Gene_or_gene_product
)	O
and	O
with	O
mutant	O
p53	B-Gene_or_gene_product
.	O

Our	O
findings	O
suggest	O
that	O
the	O
functional	O
impairment	O
of	O
p73	B-Gene_or_gene_product
could	O
be	O
involved	O
in	O
the	O
development	O
of	O
thyroid	O
malignancies	O
,	O
defining	O
p73	B-Gene_or_gene_product
as	O
a	O
potential	O
therapeutic	O
target	O
for	O
thyroid	O
cancer	O
.	O

Recruitment	O
of	O
Cdc28	B-Gene_or_gene_product
by	O
Whi3	B-Gene_or_gene_product
restricts	O
nuclear	B-Cellular_component
accumulation	O
of	O
the	O
G1	O
cyclin	B-Complex
-	I-Complex
Cdk	I-Complex
complex	O
to	O
late	O
G1	O
.	O

The	O
G1	O
cyclin	B-Gene_or_gene_product
Cln3	I-Gene_or_gene_product
is	O
a	O
key	O
activator	O
of	O
cell	O
-	O
cycle	O
entry	O
in	O
budding	O
yeast	O
.	O

Here	O
we	O
show	O
that	O
Whi3	B-Gene_or_gene_product
,	O
a	O
negative	O
G1	O
regulator	O
of	O
Cln3	B-Gene_or_gene_product
,	O
interacts	O
in	O
vivo	O
with	O
the	O
cyclin	B-Gene_or_gene_product
-	I-Gene_or_gene_product
dependent	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
Cdc28	I-Gene_or_gene_product
and	O
regulates	O
its	O
localization	O
in	O
the	O
cell	O
.	O

Efficient	O
interaction	O
with	O
Cdc28	B-Gene_or_gene_product
depends	O
on	O
an	O
N	O
-	O
terminal	O
domain	O
of	O
Whi3	B-Gene_or_gene_product
that	O
is	O
also	O
required	O
for	O
cytoplasmic	B-Cellular_component
localization	O
of	O
Cdc28	B-Gene_or_gene_product
,	O
and	O
for	O
proper	O
regulation	O
of	O
G1	O
length	O
and	O
filamentous	O
growth	O
.	O

On	O
the	O
other	O
hand	O
,	O
nuclear	B-Cellular_component
accumulation	O
of	O
Cdc28	B-Gene_or_gene_product
requires	O
the	O
nuclear	B-Cellular_component
localization	O
signal	O
of	O
Cln3	B-Gene_or_gene_product
,	O
which	O
is	O
also	O
found	O
in	O
Whi3	B-Complex
complexes	I-Complex
.	O

Both	O
Cln3	B-Gene_or_gene_product
and	O
Cdc28	B-Gene_or_gene_product
are	O
mainly	O
cytoplasmic	B-Cellular_component
during	O
early	O
G1	O
,	O
and	O
become	O
nuclear	B-Cellular_component
in	O
late	O
G1	O
.	O

However	O
,	O
Whi3	B-Gene_or_gene_product
-	O
deficient	O
cells	O
show	O
a	O
distinct	O
nuclear	B-Cellular_component
accumulation	O
of	O
Cln3	B-Gene_or_gene_product
and	O
Cdc28	B-Gene_or_gene_product
already	O
in	O
early	O
G1	O
.	O

We	O
propose	O
that	O
Whi3	B-Gene_or_gene_product
constitutes	O
a	O
cytoplasmic	B-Cellular_component
retention	O
device	O
for	O
Cln3	B-Complex
-	I-Complex
Cdc28	I-Complex
complexes	O
,	O
thus	O
defining	O
a	O
key	O
G1	O
event	O
in	O
yeast	O
cells	O
.	O

Regulation	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
signaling	O
by	O
Pin1	B-Gene_or_gene_product
-	O
dependent	O
prolyl	B-Simple_chemical
isomerization	O
and	O
ubiquitin	B-Gene_or_gene_product
-	O
mediated	O
proteolysis	O
of	O
p65	B-Gene_or_gene_product
/	O
RelA	B-Gene_or_gene_product
.	O

The	O
transcription	O
factor	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
is	O
activated	O
by	O
the	O
degradation	O
of	O
its	O
inhibitor	O
IkappaBalpha	B-Gene_or_gene_product
,	O
resulting	O
in	O
its	O
nuclear	B-Cellular_component
translocation	O
.	O

However	O
,	O
the	O
mechanism	O
by	O
which	O
nuclear	B-Cellular_component
NF	B-Complex
-	I-Complex
kappaB	I-Complex
is	O
subsequently	O
regulated	O
is	O
not	O
clear	O
.	O

Here	O
we	O
demonstrate	O
that	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
function	O
is	O
regulated	O
by	O
Pin1	B-Gene_or_gene_product
-	O
mediated	O
prolyl	B-Simple_chemical
isomerization	O
and	O
ubiquitin	B-Gene_or_gene_product
-	O
mediated	O
proteolysis	O
of	O
its	O
p65	B-Gene_or_gene_product
/	O
RelA	B-Gene_or_gene_product
subunit	O
.	O

Upon	O
cytokine	O
treatment	O
,	O
Pin1	B-Gene_or_gene_product
binds	O
to	O
the	O
pThr254	B-Simple_chemical
-	O
Pro	O
motif	O
in	O
p65	B-Gene_or_gene_product
and	O
inhibits	O
p65	B-Gene_or_gene_product
binding	O
to	O
IkappaBalpha	B-Gene_or_gene_product
,	O
resulting	O
in	O
increased	O
nuclear	B-Cellular_component
accumulation	O
and	O
protein	O
stability	O
of	O
p65	B-Gene_or_gene_product
and	O
enhanced	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
activity	O
.	O

Significantly	O
,	O
Pin1	B-Gene_or_gene_product
-	O
deficient	O
mice	O
and	O
cells	O
are	O
refractory	O
to	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
activation	O
by	O
cytokine	O
signals	O
.	O

Moreover	O
,	O
the	O
stability	O
of	O
p65	B-Gene_or_gene_product
is	O
controlled	O
by	O
ubiquitin	B-Gene_or_gene_product
-	O
mediated	O
proteolysis	O
,	O
facilitated	O
by	O
a	O
cytokine	O
signal	O
inhibitor	O
,	O
SOCS	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
,	O
acting	O
as	O
a	O
ubiquitin	B-Gene_or_gene_product
ligase	O
.	O

These	O
findings	O
uncover	O
two	O
important	O
mechanisms	O
of	O
regulating	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
signaling	O
and	O
offer	O
new	O
insight	O
into	O
the	O
pathogenesis	O
and	O
treatment	O
of	O
some	O
human	O
diseases	O
such	O
as	O
cancers	O
.	O

Structures	O
of	O
human	O
purine	B-Gene_or_gene_product
nucleoside	I-Gene_or_gene_product
phosphorylase	I-Gene_or_gene_product
complexed	O
with	O
inosine	B-Simple_chemical
and	O
ddI	B-Simple_chemical
.	O

Human	O
purine	B-Gene_or_gene_product
nucleoside	I-Gene_or_gene_product
phosphorylase	I-Gene_or_gene_product
(	O
PNP	B-Gene_or_gene_product
)	O
is	O
a	O
ubiquitous	O
enzyme	O
which	O
plays	O
a	O
key	O
role	O
in	O
the	O
purine	B-Simple_chemical
salvage	O
pathway	O
,	O
and	O
PNP	B-Gene_or_gene_product
deficiency	O
in	O
humans	O
leads	O
to	O
an	O
impairment	O
of	O
T	O
-	O
cell	O
function	O
,	O
usually	O
with	O
no	O
apparent	O
effect	O
on	O
B	O
-	O
cell	O
function	O
.	O

PNP	B-Gene_or_gene_product
is	O
highly	O
specific	O
for	O
6	B-Simple_chemical
-	I-Simple_chemical
oxopurine	I-Simple_chemical
nucleosides	I-Simple_chemical
and	O
exhibits	O
negligible	O
activity	O
for	O
6	B-Simple_chemical
-	I-Simple_chemical
aminopurine	I-Simple_chemical
nucleosides	I-Simple_chemical
.	O

The	O
catalytic	O
efficiency	O
for	O
inosine	B-Simple_chemical
is	O
350	O
,	O
000	O
-	O
fold	O
greater	O
than	O
for	O
adenosine	B-Simple_chemical
.	O

Adenine	B-Simple_chemical
nucleosides	I-Simple_chemical
and	O
nucleotides	B-Simple_chemical
are	O
deaminated	O
by	O
adenosine	B-Gene_or_gene_product
deaminase	I-Gene_or_gene_product
and	O
AMP	B-Gene_or_gene_product
deaminase	I-Gene_or_gene_product
to	O
their	O
corresponding	O
inosine	B-Simple_chemical
derivatives	O
which	O
,	O
in	O
turn	O
,	O
may	O
be	O
further	O
degraded	O
.	O

Here	O
we	O
report	O
the	O
crystal	O
structures	O
of	O
human	O
PNP	B-Gene_or_gene_product
in	O
complex	O
with	O
inosine	B-Simple_chemical
and	O
2	B-Simple_chemical
(	I-Simple_chemical
'	I-Simple_chemical
)	I-Simple_chemical
,	I-Simple_chemical
3	I-Simple_chemical
(	I-Simple_chemical
'	I-Simple_chemical
)	I-Simple_chemical
-	I-Simple_chemical
dideoxyinosine	I-Simple_chemical
,	O
refined	O
to	O
2	O
.	O
8A	O
resolution	O
using	O
synchrotron	O
radiation	O
.	O

The	O
present	O
structures	O
provide	O
explanation	O
for	O
ligand	O
binding	O
,	O
refine	O
the	O
purine	B-Simple_chemical
-	O
binding	O
site	O
,	O
and	O
can	O
be	O
used	O
for	O
future	O
inhibitor	O
design	O
.	O

Loss	O
of	O
the	O
tumor	O
suppressor	O
PML	B-Gene_or_gene_product
in	O
human	O
cancers	O
of	O
multiple	O
histologic	O
origins	O
.	O

BACKGROUND	O
:	O
The	O
PML	B-Gene_or_gene_product
gene	O
is	O
fused	O
to	O
the	O
RARalpha	B-Gene_or_gene_product
gene	O
in	O
the	O
vast	O
majority	O
of	O
acute	O
promyelocytic	O
leukemias	O
(	O
APL	O
)	O
and	O
has	O
been	O
implicated	O
in	O
the	O
control	O
of	O
key	O
tumor	O
-	O
suppressive	O
pathways	O
.	O

However	O
,	O
its	O
role	O
in	O
the	O
pathogenesis	O
of	O
human	O
cancers	O
other	O
than	O
APL	O
is	O
still	O
unclear	O
.	O

We	O
therefore	O
assessed	O
the	O
status	O
and	O
expression	O
of	O
the	O
PML	B-Gene_or_gene_product
gene	O
in	O
solid	O
tumors	O
of	O
multiple	O
histologic	O
origins	O
.	O

METHODS	O
:	O
We	O
created	O
tumor	O
tissue	O
microarrays	O
(	O
TTMs	O
)	O
with	O
samples	O
from	O
patients	O
with	O
colon	O
adenocarcinoma	O
(	O
n	O
=	O
109	O
)	O
,	O
lung	O
carcinoma	O
(	O
n	O
=	O
19	O
)	O
,	O
prostate	O
adenocarcinoma	O
(	O
n	O
=	O
36	O
)	O
,	O
breast	O
carcinoma	O
(	O
n	O
=	O
38	O
)	O
,	O
central	O
nervous	O
system	O
(	O
CNS	O
)	O
tumors	O
(	O
n	O
=	O
51	O
)	O
,	O
germ	O
cell	O
tumors	O
(	O
n	O
=	O
60	O
)	O
,	O
thyroid	O
carcinoma	O
(	O
n	O
=	O
32	O
)	O
,	O
adrenal	O
cortical	O
carcinoma	O
(	O
n	O
=	O
12	O
)	O
,	O
and	O
non	O
-	O
Hodgkin	O
'	O
s	O
lymphoma	O
(	O
n	O
=	O
251	O
)	O
and	O
from	O
normal	O
tissue	O
corresponding	O
to	O
each	O
histotype	O
and	O
analyzed	O
PML	B-Gene_or_gene_product
protein	O
and	O
mRNA	O
expression	O
by	O
immunohistochemistry	O
and	O
in	O
situ	O
hybridization	O
,	O
respectively	O
.	O

Tumor	O
cell	O
lines	O
(	O
n	O
=	O
64	O
)	O
of	O
various	O
histologic	O
origins	O
were	O
analyzed	O
for	O
PML	B-Gene_or_gene_product
protein	O
and	O
mRNA	O
expression	O
by	O
immunofluorescence	O
and	O
northern	O
blotting	O
,	O
respectively	O
.	O

DNA	O
from	O
microdissected	O
tumor	O
samples	O
and	O
cell	O
lines	O
was	O
analyzed	O
for	O
PML	B-Gene_or_gene_product
mutations	O
and	O
loss	O
of	O
heterozygosity	O
(	O
LOH	O
)	O
.	O

For	O
some	O
tumor	O
types	O
,	O
the	O
association	O
between	O
PML	B-Gene_or_gene_product
expression	O
and	O
tumor	O
stage	O
and	O
grade	O
was	O
analyzed	O
.	O

Statistical	O
tests	O
were	O
two	O
-	O
sided	O
.	O

RESULTS	O
:	O
All	O
normal	O
tissues	O
expressed	O
PML	B-Gene_or_gene_product
protein	O
.	O

PML	B-Gene_or_gene_product
protein	O
expression	O
was	O
reduced	O
or	O
abolished	O
in	O
prostate	O
adenocarcinomas	O
(	O
63	O
%	O
[	O
95	O
%	O
confidence	O
interval	O
[	O
CI	O
]	O
=	O
48	O
%	O
to	O
78	O
%	O
]	O
and	O
28	O
%	O
[	O
95	O
%	O
CI	O
=	O
13	O
%	O
to	O
43	O
%	O
]	O
,	O
respectively	O
)	O
,	O
colon	O
adenocarcinomas	O
(	O
31	O
%	O
[	O
95	O
%	O
CI	O
=	O
22	O
%	O
to	O
40	O
%	O
]	O
and	O
17	O
%	O
[	O
95	O
%	O
CI	O
=	O
10	O
%	O
to	O
24	O
%	O
]	O
)	O
,	O
breast	O
carcinomas	O
(	O
21	O
%	O
[	O
95	O
%	O
CI	O
=	O
8	O
%	O
to	O
34	O
%	O
]	O
and	O
31	O
%	O
[	O
95	O
%	O
CI	O
=	O
16	O
%	O
to	O
46	O
%	O
]	O
)	O
,	O
lung	O
carcinomas	O
(	O
36	O
%	O
[	O
95	O
%	O
CI	O
=	O
15	O
%	O
to	O
57	O
%	O
]	O
and	O
21	O
%	O
[	O
95	O
%	O
=	O
3	O
%	O
to	O
39	O
%	O
]	O
)	O
,	O
lymphomas	O
(	O
14	O
%	O
[	O
95	O
%	O
CI	O
=	O
10	O
%	O
to	O
18	O
%	O
]	O
and	O
69	O
%	O
[	O
95	O
%	O
CI	O
=	O
63	O
%	O
to	O
75	O
%	O
]	O
)	O
,	O
CNS	O
tumors	O
(	O
24	O
%	O
[	O
95	O
%	O
CI	O
=	O
13	O
%	O
to	O
35	O
%	O
]	O
and	O
49	O
%	O
[	O
95	O
%	O
CI	O
=	O
36	O
%	O
to	O
62	O
%	O
]	O
)	O
,	O
and	O
germ	O
cell	O
tumors	O
(	O
36	O
%	O
[	O
95	O
%	O
CI	O
=	O
24	O
%	O
to	O
48	O
%	O
]	O
and	O
48	O
%	O
[	O
95	O
%	O
CI	O
=	O
36	O
%	O
to	O
60	O
%	O
]	O
)	O
but	O
not	O
in	O
thyroid	O
or	O
adrenal	O
carcinomas	O
.	O

Loss	O
of	O
PML	B-Gene_or_gene_product
protein	O
expression	O
was	O
associated	O
with	O
tumor	O
progression	O
in	O
prostate	O
cancer	O
(	O
the	O
progression	O
from	O
prostatic	O
intraepithelial	O
neoplasia	O
to	O
invasive	O
carcinoma	O
was	O
associated	O
with	O
complete	O
PML	B-Gene_or_gene_product
loss	O
;	O
P	O
<	O
.	O
001	O
)	O
,	O
breast	O
cancer	O
(	O
complete	O
PML	B-Gene_or_gene_product
loss	O
was	O
associated	O
with	O
lymph	O
node	O
metastasis	O
;	O
P	O
=	O
.	O
01	O
)	O
,	O
and	O
CNS	O
tumors	O
(	O
complete	O
PML	B-Gene_or_gene_product
loss	O
was	O
associated	O
with	O
high	O
-	O
grade	O
tumors	O
;	O
P	O
=	O
.	O
003	O
)	O
.	O

PML	B-Gene_or_gene_product
mRNA	O
was	O
expressed	O
in	O
all	O
tumor	O
and	O
cell	O
line	O
samples	O
.	O

The	O
PML	B-Gene_or_gene_product
gene	O
was	O
rarely	O
mutated	O
and	O
was	O
not	O
subject	O
to	O
LOH	O
.	O

CONCLUSIONS	O
:	O
PML	B-Gene_or_gene_product
protein	O
expression	O
is	O
frequently	O
lost	O
in	O
human	O
cancers	O
of	O
various	O
histologic	O
origins	O
,	O
and	O
its	O
loss	O
associates	O
with	O
tumor	O
grade	O
and	O
progression	O
in	O
some	O
tumor	O
histotypes	O
.	O

mTOR	B-Gene_or_gene_product
-	O
dependent	O
activation	O
of	O
the	O
transcription	O
factor	O
TIF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
IA	I-Gene_or_gene_product
links	O
rRNA	O
synthesis	O
to	O
nutrient	O
availability	O
.	O

In	O
cycling	O
cells	O
,	O
transcription	O
of	O
ribosomal	B-Cellular_component
RNA	O
genes	O
by	O
RNA	B-Gene_or_gene_product
polymerase	I-Gene_or_gene_product
I	I-Gene_or_gene_product
(	O
Pol	B-Gene_or_gene_product
I	I-Gene_or_gene_product
)	O
is	O
tightly	O
coordinated	O
with	O
cell	O
growth	O
.	O

Here	O
,	O
we	O
show	O
that	O
the	O
mammalian	B-Gene_or_gene_product
target	I-Gene_or_gene_product
of	I-Gene_or_gene_product
rapamycin	I-Gene_or_gene_product
(	O
mTOR	B-Gene_or_gene_product
)	O
regulates	O
Pol	B-Gene_or_gene_product
I	I-Gene_or_gene_product
transcription	O
by	O
modulating	O
the	O
activity	O
of	O
TIF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
IA	I-Gene_or_gene_product
,	O
a	O
regulatory	O
factor	O
that	O
senses	O
nutrient	O
and	O
growth	O
-	O
factor	O
availability	O
.	O

Inhibition	O
of	O
mTOR	B-Gene_or_gene_product
signaling	O
by	O
rapamycin	B-Simple_chemical
inactivates	O
TIF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
IA	I-Gene_or_gene_product
and	O
impairs	O
transcription	O
-	O
initiation	O
complex	O
formation	O
.	O

Moreover	O
,	O
rapamycin	B-Simple_chemical
treatment	O
leads	O
to	O
translocation	O
of	O
TIF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
IA	I-Gene_or_gene_product
into	O
the	O
cytoplasm	B-Cellular_component
.	O

Rapamycin	B-Simple_chemical
-	O
mediated	O
inactivation	O
of	O
TIF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
IA	I-Gene_or_gene_product
is	O
caused	O
by	O
hypophosphorylation	O
of	O
Se	B-Simple_chemical
44	I-Simple_chemical
(	O
S44	B-Simple_chemical
)	O
and	O
hyperphosphorylation	O
of	O
Se	B-Simple_chemical
199	I-Simple_chemical
(	O
S199	B-Simple_chemical
)	O
.	O

Phosphorylation	O
at	O
these	O
sites	O
affects	O
TIF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
IA	I-Gene_or_gene_product
activity	O
in	O
opposite	O
ways	O
,	O
for	O
example	O
,	O
phosphorylation	O
of	O
S44	B-Simple_chemical
activates	O
and	O
S199	B-Simple_chemical
inactivates	O
TIF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
IA	I-Gene_or_gene_product
.	O

The	O
results	O
identify	O
a	O
new	O
target	O
formTOR	B-Gene_or_gene_product
-	O
signaling	O
pathways	O
and	O
elucidate	O
the	O
molecular	O
mechanism	O
underlying	O
mTOR	B-Gene_or_gene_product
-	O
dependent	O
regulation	O
of	O
RNA	O
synthesis	O
.	O

Coupling	O
morphogenesis	O
to	O
mitotic	O
entry	O
.	O

In	O
eukaryotes	O
,	O
cyclin	B-Gene_or_gene_product
B	I-Gene_or_gene_product
-	O
bound	O
cyclin	B-Gene_or_gene_product
-	I-Gene_or_gene_product
dependent	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
1	I-Gene_or_gene_product
promotes	O
mitotic	O
entry	O
but	O
is	O
held	O
in	O
check	O
,	O
in	O
part	O
,	O
by	O
Wee1	B-Gene_or_gene_product
protein	O
kinase	O
.	O

Timely	O
mitotic	O
entry	O
in	O
budding	O
yeast	O
requires	O
inactivation	O
of	O
Swe1	B-Gene_or_gene_product
(	O
Wee1	B-Gene_or_gene_product
ortholog	O
)	O
.	O

Perturbations	O
of	O
the	O
septin	B-Gene_or_gene_product
collar	B-Cellular_component
at	O
the	O
bud	B-Cellular_component
neck	I-Cellular_component
lead	O
to	O
Swe1	B-Gene_or_gene_product
stabilization	O
,	O
delaying	O
the	O
G	O
(	O
2	O
)	O
/	O
M	O
transition	O
.	O

Swe1	B-Gene_or_gene_product
is	O
recruited	O
to	O
the	O
neck	B-Cellular_component
and	O
hyperphosphorylated	O
before	O
ubiquitin	B-Gene_or_gene_product
-	O
mediated	O
degradation	O
.	O

Hsl1	B-Gene_or_gene_product
kinase	O
(	O
Nim1	B-Gene_or_gene_product
ortholog	O
)	O
,	O
a	O
negative	O
regulator	O
of	O
Wee1	B-Gene_or_gene_product
,	O
is	O
required	O
for	O
efficient	O
Swe1	B-Gene_or_gene_product
localization	O
at	O
the	O
neck	B-Cellular_component
but	O
seems	O
not	O
to	O
phosphorylate	O
Swe1	B-Gene_or_gene_product
.	O

Here	O
,	O
we	O
show	O
that	O
two	O
other	O
kinases	O
targeted	O
sequentially	O
to	O
the	O
neck	B-Cellular_component
,	O
Cla4	B-Gene_or_gene_product
/	I-Gene_or_gene_product
PAK	I-Gene_or_gene_product
and	O
Cdc5	B-Gene_or_gene_product
/	I-Gene_or_gene_product
Polo	I-Gene_or_gene_product
,	O
are	O
responsible	O
for	O
stepwise	O
phosphorylation	O
and	O
down	O
-	O
regulation	O
of	O
Swe1	B-Gene_or_gene_product
.	O

This	O
mechanism	O
links	O
assembly	O
of	O
a	O
cellular	B-Cellular_component
structure	I-Cellular_component
to	O
passage	O
into	O
mitosis	O
.	O

Bruton	B-Gene_or_gene_product
'	I-Gene_or_gene_product
s	I-Gene_or_gene_product
tyrosine	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
(	O
Btk	B-Gene_or_gene_product
)	O
-	O
the	O
critical	O
tyrosine	B-Simple_chemical
kinase	O
in	O
LPS	B-Simple_chemical
signalling	O
?	O

The	O
discovery	O
of	O
the	O
Toll	B-Gene_or_gene_product
-	I-Gene_or_gene_product
like	I-Gene_or_gene_product
receptors	I-Gene_or_gene_product
(	O
TLRs	B-Gene_or_gene_product
)	O
has	O
revolutionised	O
the	O
field	O
of	O
innate	O
immunity	O
.	O

One	O
unresolved	O
question	O
regarding	O
LPS	B-Simple_chemical
signalling	O
is	O
whether	O
there	O
is	O
a	O
role	O
for	O
tyrosine	B-Simple_chemical
kinases	O
downstream	O
of	O
the	O
LPS	B-Simple_chemical
receptor	O
.	O

Studies	O
in	O
mice	O
deficient	O
in	O
Bruton	B-Gene_or_gene_product
'	I-Gene_or_gene_product
s	I-Gene_or_gene_product
tyrosine	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
have	O
previously	O
shown	O
that	O
they	O
are	O
defective	O
in	O
their	O
responses	O
to	O
LPS	B-Simple_chemical
.	O

Further	O
investigation	O
into	O
the	O
role	O
of	O
Btk	B-Gene_or_gene_product
in	O
LPS	B-Simple_chemical
signalling	O
has	O
directly	O
implicated	O
Btk	B-Gene_or_gene_product
downstream	O
of	O
TLR4	B-Gene_or_gene_product
,	O
both	O
with	O
respect	O
to	O
p38	B-Gene_or_gene_product
MAPK	I-Gene_or_gene_product
activation	O
and	O
activation	O
of	O
the	O
transcription	O
factor	O
NFkappaB	B-Complex
.	O

In	O
fact	O
Btk	B-Gene_or_gene_product
is	O
activated	O
by	O
LPS	B-Simple_chemical
and	O
has	O
been	O
shown	O
to	O
directly	O
bind	O
TLR4	B-Gene_or_gene_product
and	O
the	O
key	O
proximal	O
signalling	O
proteins	O
involved	O
in	O
LPS	B-Simple_chemical
-	O
induced	O
NFkappaB	B-Complex
activation	O
,	O
MyD88	B-Gene_or_gene_product
,	O
Mal	B-Gene_or_gene_product
and	O
IRAK	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
.	O

These	O
recent	O
findings	O
point	O
to	O
a	O
direct	O
role	O
for	O
Btk	B-Gene_or_gene_product
in	O
LPS	B-Simple_chemical
signal	O
transduction	O
and	O
raise	O
interesting	O
questions	O
regarding	O
the	O
mode	O
of	O
activation	O
of	O
Btk	B-Gene_or_gene_product
following	O
LPS	B-Simple_chemical
stimulation	O
and	O
the	O
precise	O
nature	O
of	O
the	O
pathways	O
activated	O
downstream	O
of	O
Btk	B-Gene_or_gene_product
.	O

A	O
better	O
understanding	O
of	O
how	O
Btk	B-Gene_or_gene_product
functions	O
in	O
LPS	B-Simple_chemical
signalling	O
will	O
have	O
important	O
implications	O
for	O
inflammatory	O
and	O
autoimmune	O
disorders	O
and	O
therapies	O
thereof	O
.	O

Molecular	O
cloning	O
and	O
characterization	O
of	O
a	O
human	O
multisubstrate	O
specific	O
nucleotide	B-Simple_chemical
-	I-Simple_chemical
sugar	I-Simple_chemical
transporter	O
homologous	O
to	O
Drosophila	O
fringe	B-Gene_or_gene_product
connection	I-Gene_or_gene_product
.	O

Nucleotide	O
-	O
sugar	O
transporters	O
are	O
crucial	O
components	O
in	O
the	O
synthesis	O
of	O
glycoconjugates	O
.	O

We	O
identified	O
a	O
novel	O
human	O
nucleotide	B-Simple_chemical
-	I-Simple_chemical
sugar	I-Simple_chemical
transporter	O
gene	O
,	O
hfrc1	B-Gene_or_gene_product
,	O
which	O
is	O
homologous	O
to	O
Drosophila	O
melanogaster	O
fringe	B-Gene_or_gene_product
connection	I-Gene_or_gene_product
,	O
Caenorhabditis	O
elegans	O
sqv	B-Gene_or_gene_product
-	I-Gene_or_gene_product
7	I-Gene_or_gene_product
,	O
and	O
human	O
UGTrel7	B-Gene_or_gene_product
.	O

HFRC1	B-Gene_or_gene_product
was	O
localized	O
within	O
the	O
Golgi	B-Cellular_component
apparatus	I-Cellular_component
following	O
its	O
transient	O
expression	O
in	O
HCT116	O
cells	O
.	O

In	O
human	O
tissues	O
,	O
hfrc1	B-Gene_or_gene_product
and	O
UGTrel7	B-Gene_or_gene_product
exhibited	O
similar	O
tissue	O
distributions	O
,	O
although	O
hfrc1	B-Gene_or_gene_product
transcripts	O
showed	O
a	O
10	O
times	O
greater	O
abundance	O
than	O
those	O
of	O
UGTrel7	B-Gene_or_gene_product
.	O

The	O
heterologous	O
expression	O
of	O
HFRC1	B-Gene_or_gene_product
in	O
the	O
yeast	O
revealed	O
the	O
multisubstrate	O
specific	O
transport	O
activity	O
of	O
HFRC1	B-Gene_or_gene_product
(	O
for	O
UDP	B-Simple_chemical
-	I-Simple_chemical
N	I-Simple_chemical
-	I-Simple_chemical
acetylglucosamine	I-Simple_chemical
(	O
UDP	B-Simple_chemical
-	I-Simple_chemical
GlcNAc	I-Simple_chemical
)	O
,	O
UDP	B-Simple_chemical
-	I-Simple_chemical
glucose	I-Simple_chemical
(	O
UDP	B-Simple_chemical
-	I-Simple_chemical
Glc	I-Simple_chemical
)	O
,	O
and	O
GDP	B-Simple_chemical
-	I-Simple_chemical
mannose	I-Simple_chemical
(	O
GDP	B-Simple_chemical
-	I-Simple_chemical
Man	I-Simple_chemical
)	O
,	O
with	O
apparent	O
K	O
(	O
m	O
)	O
values	O
of	O
8	O
.	O
0	O
,	O
2	O
.	O
1	O
,	O
and	O
0	O
.	O
14	O
microm	O
,	O
respectively	O
)	O
.	O

In	O
the	O
mammalian	O
cells	O
,	O
HFRC1	B-Gene_or_gene_product
transported	O
UDP	B-Simple_chemical
-	I-Simple_chemical
GlcNAc	I-Simple_chemical
and	O
UDP	B-Simple_chemical
-	I-Simple_chemical
Glc	I-Simple_chemical
,	O
but	O
not	O
GDP	B-Simple_chemical
-	I-Simple_chemical
Man	I-Simple_chemical
.	O

Overexpression	O
of	O
the	O
hfrc1	B-Gene_or_gene_product
gene	O
in	O
HCT116	O
cells	O
modulated	O
the	O
cell	B-Cellular_component
surface	I-Cellular_component
heparan	B-Simple_chemical
sulfate	I-Simple_chemical
expression	O
status	O
.	O

These	O
results	O
suggest	O
that	O
HFRC1	B-Gene_or_gene_product
takes	O
part	O
in	O
the	O
synthesis	O
of	O
heparan	B-Simple_chemical
sulfate	I-Simple_chemical
by	O
regulating	O
the	O
level	O
of	O
UDP	B-Simple_chemical
-	I-Simple_chemical
GlcNAc	I-Simple_chemical
,	O
a	O
donor	O
substrate	O
for	O
the	O
heparan	B-Simple_chemical
sulfate	I-Simple_chemical
synthases	O
.	O

Hierarchical	O
requirement	O
of	O
SWI	B-Complex
/	I-Complex
SNF	I-Complex
in	O
retinoblastoma	B-Gene_or_gene_product
tumor	O
suppressor	O
-	O
mediated	O
repression	O
of	O
Plk1	B-Gene_or_gene_product
.	O

Plk1	B-Gene_or_gene_product
(	O
Polo	B-Gene_or_gene_product
-	I-Gene_or_gene_product
like	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
1	I-Gene_or_gene_product
)	O
is	O
a	O
critical	O
regulator	O
of	O
cell	O
cycle	O
progression	O
that	O
harbors	O
oncogenic	O
activity	O
and	O
exhibits	O
aberrant	O
expression	O
in	O
multiple	O
tumors	O
.	O

However	O
,	O
the	O
mechanism	O
through	O
which	O
Plk1	B-Gene_or_gene_product
expression	O
is	O
regulated	O
has	O
not	O
been	O
extensively	O
studied	O
.	O

Here	O
we	O
demonstrate	O
that	O
Plk1	B-Gene_or_gene_product
is	O
a	O
target	O
of	O
the	O
retinoblastoma	B-Gene_or_gene_product
tumor	O
suppressor	O
(	O
RB	B-Gene_or_gene_product
)	O
pathway	O
.	O

Activation	O
of	O
RB	B-Gene_or_gene_product
and	O
related	O
pocket	O
proteins	O
p107	B-Gene_or_gene_product
/	O
p130	B-Gene_or_gene_product
mediate	O
attenuation	O
of	O
Plk1	B-Gene_or_gene_product
.	O

Conversely	O
,	O
RB	B-Gene_or_gene_product
loss	O
deregulates	O
the	O
control	O
of	O
Plk1	B-Gene_or_gene_product
expression	O
.	O

RB	B-Gene_or_gene_product
pathway	O
activation	O
resulted	O
in	O
the	O
repression	O
of	O
Plk1	B-Gene_or_gene_product
promoter	O
activity	O
,	O
and	O
this	O
action	O
was	O
dependent	O
on	O
the	O
SWI	B-Complex
/	I-Complex
SNF	I-Complex
chromatin	B-Cellular_component
remodeling	O
complex	O
.	O

Although	O
SWI	B-Complex
/	I-Complex
SNF	I-Complex
subunits	O
are	O
lost	O
during	O
tumorigenesis	O
and	O
cooperate	O
with	O
RB	B-Gene_or_gene_product
for	O
transcriptional	O
repression	O
,	O
the	O
mechanism	O
through	O
which	O
SWI	B-Complex
/	I-Complex
SNF	I-Complex
impinges	O
on	O
RB	B-Gene_or_gene_product
action	O
is	O
unresolved	O
.	O

Therefore	O
,	O
we	O
delineated	O
the	O
requirement	O
of	O
SWI	B-Complex
/	I-Complex
SNF	I-Complex
for	O
three	O
critical	O
facets	O
of	O
Plk1	B-Gene_or_gene_product
promoter	O
regulation	O
:	O
transcription	O
factor	O
binding	O
,	O
corepressor	O
binding	O
,	O
and	O
histone	B-Gene_or_gene_product
modification	O
.	O

We	O
find	O
that	O
E2F4	B-Gene_or_gene_product
and	O
pocket	O
protein	O
association	O
with	O
the	O
Plk1	B-Gene_or_gene_product
promoter	O
is	O
independent	O
of	O
SWI	B-Complex
/	I-Complex
SNF	I-Complex
.	O

However	O
,	O
these	O
analyses	O
revealed	O
that	O
SWI	B-Complex
/	I-Complex
SNF	I-Complex
is	O
required	O
for	O
histone	B-Gene_or_gene_product
deacetylation	O
of	O
the	O
Plk1	B-Gene_or_gene_product
promoter	O
.	O

The	O
importance	O
of	O
SWI	B-Complex
/	I-Complex
SNF	I-Complex
-	O
dependent	O
histone	B-Gene_or_gene_product
deacetylation	O
of	O
the	O
Plk1	B-Gene_or_gene_product
promoter	O
was	O
evident	O
,	O
because	O
blockade	O
of	O
this	O
event	O
restored	O
Plk1	B-Gene_or_gene_product
expression	O
in	O
the	O
presence	O
of	O
active	O
RB	B-Gene_or_gene_product
.	O

In	O
summary	O
,	O
these	O
data	O
demonstrate	O
that	O
Plk1	B-Gene_or_gene_product
is	O
a	O
target	O
of	O
the	O
RB	B-Gene_or_gene_product
pathway	O
.	O

Moreover	O
,	O
these	O
findings	O
demonstrate	O
a	O
hierarchical	O
role	O
for	O
SWI	B-Complex
/	I-Complex
SNF	I-Complex
in	O
the	O
control	O
of	O
promoter	O
activity	O
through	O
histone	B-Gene_or_gene_product
modification	O
.	O

Human	O
recombinant	O
thiamine	B-Gene_or_gene_product
triphosphatase	I-Gene_or_gene_product
:	O
purification	O
,	O
secondary	O
structure	O
and	O
catalytic	O
properties	O
.	O

Thiamine	B-Simple_chemical
triphosphate	I-Simple_chemical
(	O
ThTP	B-Simple_chemical
)	O
is	O
found	O
in	O
most	O
living	O
organisms	O
and	O
it	O
may	O
act	O
as	O
a	O
phosphate	B-Simple_chemical
donor	O
for	O
protein	O
phosphorylation	O
.	O

We	O
have	O
recently	O
cloned	O
the	O
cDNA	O
coding	O
for	O
a	O
highly	O
specific	O
mammalian	O
25	O
kDa	O
thiamine	B-Gene_or_gene_product
triphosphatase	I-Gene_or_gene_product
(	O
ThTPase	B-Gene_or_gene_product
;	O
EC	B-Gene_or_gene_product
3	I-Gene_or_gene_product
.	I-Gene_or_gene_product
6	I-Gene_or_gene_product
.	I-Gene_or_gene_product
1	I-Gene_or_gene_product
.	I-Gene_or_gene_product
28	I-Gene_or_gene_product
)	O
.	O

As	O
the	O
enzyme	O
has	O
a	O
high	O
catalytic	O
efficiency	O
and	O
no	O
sequence	O
homology	O
with	O
known	O
phosphohydrolases	O
,	O
it	O
was	O
worth	O
investigating	O
its	O
structure	O
and	O
catalytic	O
properties	O
.	O

For	O
this	O
purpose	O
,	O
we	O
expressed	O
the	O
untagged	O
recombinant	O
human	O
ThTPase	B-Gene_or_gene_product
(	O
hThTPase	B-Gene_or_gene_product
)	O
in	O
E	O
.	O
coli	O
,	O
produced	O
the	O
protein	O
on	O
a	O
large	O
scale	O
and	O
purified	O
it	O
to	O
homogeneity	O
.	O

Its	O
kinetic	O
properties	O
were	O
similar	O
to	O
those	O
of	O
the	O
genuine	O
human	O
enzyme	O
,	O
indicating	O
that	O
the	O
recombinant	O
hThTPase	B-Gene_or_gene_product
is	O
completely	O
functional	O
.	O

Mg2	B-Simple_chemical
+	I-Simple_chemical
ions	O
were	O
required	O
for	O
activity	O
and	O
Ca2	B-Simple_chemical
+	I-Simple_chemical
inhibited	O
the	O
enzyme	O
by	O
competition	O
with	O
Mg2	B-Simple_chemical
+	I-Simple_chemical
.	O

With	O
ATP	B-Simple_chemical
as	O
substrate	O
,	O
the	O
catalytic	O
efficiency	O
was	O
10	O
(	O
-	O
4	O
)	O
-	O
fold	O
lower	O
than	O
with	O
ThTP	B-Simple_chemical
,	O
confirming	O
the	O
nearly	O
absolute	O
specificity	O
of	O
the	O
25	O
kDa	O
ThTPase	B-Gene_or_gene_product
for	O
ThTP	B-Simple_chemical
.	O

The	O
activity	O
was	O
maximum	O
at	O
pH	O
8	O
.	O
5	O
and	O
very	O
low	O
at	O
pH	O
6	O
.	O
0	O
.	O

Zn2	B-Simple_chemical
+	I-Simple_chemical
ions	O
were	O
inhibitory	O
at	O
micromolar	O
concentrations	O
at	O
pH	O
8	O
.	O
0	O
but	O
activated	O
at	O
pH	O
6	O
.	O
0	O
.	O

Kinetic	O
analysis	O
suggests	O
an	O
activator	O
site	O
for	O
Mg2	B-Simple_chemical
+	I-Simple_chemical
and	O
a	O
separate	O
regulatory	O
site	O
for	O
Zn2	B-Simple_chemical
+	I-Simple_chemical
.	O

The	O
effects	O
of	O
group	O
-	O
specific	O
reagents	O
such	O
as	O
Woodward	O
'	O
s	O
reagent	B-Simple_chemical
K	I-Simple_chemical
and	O
diethylpyrocarbonate	B-Simple_chemical
suggest	O
that	O
at	O
least	O
one	O
carboxyl	O
group	O
in	O
the	O
active	O
site	O
is	O
essential	O
for	O
catalysis	O
,	O
while	O
a	O
positively	O
charged	O
amino	O
group	O
may	O
be	O
involved	O
in	O
substrate	O
binding	O
.	O

The	O
secondary	O
structure	O
of	O
the	O
enzyme	O
,	O
as	O
determined	O
by	O
Fourier	O
-	O
transform	O
infrared	O
spectroscopy	O
,	O
was	O
predominantly	O
beta	O
-	O
sheet	O
and	O
alpha	O
-	O
helix	O
.	O

beta	B-Simple_chemical
-	I-Simple_chemical
D	I-Simple_chemical
-	I-Simple_chemical
Glucoside	I-Simple_chemical
suppresses	O
tumor	B-Gene_or_gene_product
necrosis	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
-	O
induced	O
activation	O
of	O
nuclear	B-Cellular_component
transcription	O
factor	O
kappaB	B-Complex
but	O
potentiates	O
apoptosis	O
.	O

Mangiferin	B-Simple_chemical
,	O
a	O
natural	O
polyphenol	B-Simple_chemical
is	O
known	O
to	O
exhibit	O
anti	O
-	O
inflammatory	O
,	O
antioxidant	O
,	O
and	O
antiviral	O
effects	O
.	O

However	O
the	O
molecular	O
mechanism	O
underlying	O
these	O
effects	O
has	O
not	O
been	O
well	O
characterized	O
.	O

Because	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
plays	O
an	O
important	O
role	O
in	O
these	O
processes	O
,	O
it	O
is	O
possible	O
that	O
mangiferin	B-Simple_chemical
modulates	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
activation	O
.	O

Our	O
results	O
show	O
that	O
mangiferin	B-Simple_chemical
blocks	O
tumor	B-Gene_or_gene_product
necrosis	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
(	O
TNF	B-Gene_or_gene_product
)	O
-	O
induced	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
activation	O
and	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
-	O
dependent	O
genes	O
like	O
ICAM1	B-Gene_or_gene_product
and	O
COX2	B-Gene_or_gene_product
.	O

The	O
effect	O
was	O
mediated	O
through	O
inhibition	O
of	O
IKK	B-Complex
activation	O
and	O
subsequent	O
blocking	O
of	O
phosphorylation	O
and	O
degradation	O
of	O
IkappaBalpha	B-Gene_or_gene_product
.	O

In	O
addition	O
,	O
mangiferin	B-Simple_chemical
inhibits	O
TNF	B-Gene_or_gene_product
-	O
induced	O
p65	B-Gene_or_gene_product
phosphorylation	O
as	O
well	O
as	O
translocation	O
to	O
the	O
nucleus	B-Cellular_component
and	O
also	O
inhibits	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
activation	O
induced	O
by	O
other	O
inflammatory	O
agents	O
like	O
PMA	B-Simple_chemical
,	O
ceramide	B-Simple_chemical
,	O
and	O
SA	B-Simple_chemical
-	I-Simple_chemical
LPS	I-Simple_chemical
.	O

Mangiferin	B-Simple_chemical
,	O
similar	O
to	O
the	O
other	O
known	O
antioxidants	O
,	O
NAC	B-Simple_chemical
and	O
PDTC	B-Simple_chemical
,	O
inhibits	O
TNF	B-Gene_or_gene_product
-	O
induced	O
reactive	B-Simple_chemical
oxygen	I-Simple_chemical
intermediate	I-Simple_chemical
(	O
ROI	B-Simple_chemical
)	O
generation	O
.	O

Since	O
intracellular	B-Cellular_component
glutathione	B-Simple_chemical
(	O
GSH	B-Simple_chemical
)	O
levels	O
are	O
known	O
to	O
modulate	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
levels	O
,	O
we	O
measured	O
the	O
levels	O
of	O
GSH	B-Simple_chemical
.	O

Mangiferin	B-Simple_chemical
enhances	O
glutathione	B-Simple_chemical
level	O
by	O
almost	O
2	O
-	O
fold	O
more	O
than	O
other	O
anti	O
-	O
oxidants	O
,	O
and	O
at	O
the	O
same	O
time	O
it	O
decreases	O
the	O
levels	O
of	O
GSSG	B-Simple_chemical
and	O
increases	O
the	O
activity	O
of	O
catalase	B-Gene_or_gene_product
.	O

Depletion	O
of	O
GSH	B-Simple_chemical
by	O
buthionine	B-Simple_chemical
sulfoximine	I-Simple_chemical
led	O
to	O
a	O
significant	O
reversal	O
of	O
mangiferin	B-Simple_chemical
effect	O
.	O

Hence	O
mangiferin	B-Simple_chemical
with	O
its	O
ability	O
to	O
inhibit	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
and	O
increase	O
the	O
intracellular	B-Cellular_component
GSH	B-Simple_chemical
levels	O
may	O
prove	O
to	O
be	O
a	O
potent	O
drug	O
for	O
anti	O
-	O
inflammatory	O
and	O
antioxidant	O
therapy	O
.	O

Mangiferin	B-Simple_chemical
-	O
mediated	O
down	O
-	O
regulation	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
also	O
potentiates	O
chemotherapeutic	O
agent	O
-	O
mediated	O
cell	O
death	O
,	O
suggesting	O
a	O
role	O
in	O
combination	O
therapy	O
for	O
cancer	O
.	O

Endogenous	O
ligands	O
of	O
Toll	B-Gene_or_gene_product
-	I-Gene_or_gene_product
like	I-Gene_or_gene_product
receptors	I-Gene_or_gene_product
.	O

Extensive	O
work	O
has	O
suggested	O
that	O
a	O
number	O
of	O
endogenous	O
molecules	O
such	O
as	O
heat	B-Gene_or_gene_product
shock	I-Gene_or_gene_product
proteins	I-Gene_or_gene_product
(	O
hsp	B-Gene_or_gene_product
)	O
may	O
be	O
potent	O
activators	O
of	O
the	O
innate	O
immune	O
system	O
capable	O
of	O
inducing	O
proinflammatory	O
cytokine	O
production	O
by	O
the	O
monocyte	O
-	O
macrophage	O
system	O
and	O
the	O
activation	O
and	O
maturation	O
of	O
dendritic	O
cells	O
.	O

The	O
cytokine	O
-	O
like	O
effects	O
of	O
these	O
endogenous	O
molecules	O
are	O
mediated	O
via	O
the	O
Toll	B-Gene_or_gene_product
-	I-Gene_or_gene_product
like	I-Gene_or_gene_product
receptor	I-Gene_or_gene_product
(	O
TLR	B-Gene_or_gene_product
)	O
signal	O
-	O
transduction	O
pathways	O
in	O
a	O
manner	O
similar	O
to	O
lipopolysaccharide	B-Simple_chemical
(	O
LPS	B-Simple_chemical
;	O
via	O
TLR4	B-Gene_or_gene_product
)	O
and	O
bacterial	O
lipoproteins	O
(	O
via	O
TLR2	B-Gene_or_gene_product
)	O
.	O

However	O
,	O
recent	O
evidence	O
suggests	O
that	O
the	O
reported	O
cytokine	O
effects	O
of	O
hsp	B-Gene_or_gene_product
may	O
be	O
a	O
result	O
of	O
the	O
contaminating	O
LPS	B-Simple_chemical
and	O
LPS	B-Simple_chemical
-	O
associated	O
molecules	O
.	O

The	O
reasons	O
for	O
previous	O
failure	O
to	O
recognize	O
the	O
contaminant	O
(	O
s	O
)	O
being	O
responsible	O
for	O
the	O
putative	O
TLR	B-Gene_or_gene_product
ligands	O
of	O
hsp	B-Gene_or_gene_product
include	O
failure	O
to	O
use	O
highly	O
purified	O
hsp	B-Gene_or_gene_product
free	O
of	O
LPS	B-Simple_chemical
contamination	O
;	O
failure	O
to	O
recognize	O
the	O
heat	O
sensitivity	O
of	O
LPS	B-Simple_chemical
;	O
and	O
failure	O
to	O
consider	O
contaminant	O
(	O
s	O
)	O
other	O
than	O
LPS	B-Simple_chemical
.	O

Whether	O
other	O
reported	O
putative	O
endogenous	O
ligands	O
of	O
TLR2	B-Gene_or_gene_product
and	O
TLR4	B-Gene_or_gene_product
are	O
a	O
result	O
of	O
contamination	O
of	O
pathogen	O
-	O
associated	O
molecular	O
patterns	O
is	O
not	O
clear	O
.	O

It	O
is	O
essential	O
that	O
efforts	O
should	O
be	O
directed	O
to	O
conclusively	O
determine	O
whether	O
the	O
reported	O
putative	O
endogenous	O
ligands	O
of	O
TLRs	B-Gene_or_gene_product
are	O
a	O
result	O
of	O
the	O
endogenous	O
molecules	O
or	O
of	O
contaminant	O
(	O
s	O
)	O
,	O
before	O
exploring	O
further	O
the	O
implication	O
and	O
therapeutic	O
potential	O
of	O
these	O
putative	O
TLR	B-Gene_or_gene_product
ligands	O
.	O

Targeted	O
proteomic	O
study	O
of	O
the	O
cyclin	B-Complex
-	I-Complex
Cdk	I-Complex
module	O
.	O

The	O
cell	O
division	O
cycle	O
of	O
the	O
yeast	O
S	O
.	O
cerevisiae	O
is	O
driven	O
by	O
one	O
Cdk	B-Gene_or_gene_product
(	O
cyclin	B-Gene_or_gene_product
-	I-Gene_or_gene_product
dependent	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
)	O
,	O
which	O
becomes	O
active	O
when	O
bound	O
to	O
one	O
of	O
nine	O
cyclin	B-Complex
subunits	O
.	O

Elucidation	O
of	O
Cdk	B-Gene_or_gene_product
substrates	O
and	O
other	O
Cdk	B-Gene_or_gene_product
-	O
associated	O
proteins	O
is	O
essential	O
for	O
a	O
full	O
understanding	O
of	O
the	O
cell	O
cycle	O
.	O

Here	O
,	O
we	O
report	O
the	O
results	O
of	O
a	O
targeted	O
proteomics	O
study	O
using	O
affinity	O
purification	O
coupled	O
to	O
mass	O
spectrometry	O
.	O

Our	O
study	O
identified	O
numerous	O
proteins	O
in	O
association	O
with	O
particular	O
cyclin	B-Complex
-	I-Complex
Cdk	I-Complex
complexes	O
.	O

These	O
included	O
phosphorylation	O
substrates	O
,	O
ubiquitination	O
-	O
degradation	O
proteins	O
,	O
adaptors	O
,	O
and	O
inhibitors	O
.	O

Some	O
associations	O
were	O
previously	O
known	O
,	O
and	O
for	O
others	O
,	O
we	O
confirmed	O
their	O
specificity	O
and	O
biological	O
relevance	O
.	O

Using	O
a	O
hypothesis	O
-	O
driven	O
mass	O
spectrometric	O
approach	O
,	O
we	O
also	O
mapped	O
in	O
vivo	O
phosphorylation	O
at	O
Cdk	B-Gene_or_gene_product
consensus	O
motif	O
-	O
containing	O
peptides	O
within	O
several	O
cyclin	B-Complex
-	O
associated	O
candidate	O
Cdk	B-Gene_or_gene_product
substrates	O
.	O

Our	O
results	O
demonstrate	O
that	O
this	O
approach	O
can	O
be	O
used	O
to	O
detect	O
a	O
host	O
of	O
transient	O
and	O
dynamic	O
protein	O
associations	O
within	O
a	O
biological	O
module	O
.	O

Genotoxic	O
stress	O
induces	O
expression	O
of	O
E2F4	B-Gene_or_gene_product
,	O
leading	O
to	O
its	O
association	O
with	O
p130	B-Gene_or_gene_product
in	O
prostate	O
carcinoma	O
cells	O
.	O

The	O
retinoblastoma	B-Gene_or_gene_product
(	O
pRb	B-Gene_or_gene_product
)	O
,	O
p107	B-Gene_or_gene_product
,	O
and	O
p130	B-Gene_or_gene_product
pocket	O
proteins	O
bind	O
to	O
the	O
E2F	B-Gene_or_gene_product
transcription	O
factors	O
to	O
control	O
gene	O
expression	O
.	O

E2F4	B-Gene_or_gene_product
protein	O
levels	O
increased	O
and	O
accumulated	O
in	O
the	O
nuclei	B-Cellular_component
of	O
prostate	O
carcinoma	O
cells	O
subjected	O
to	O
ionizing	O
radiation	O
(	O
IR	O
)	O
.	O

The	O
IR	O
-	O
induced	O
increase	O
of	O
E2F4	B-Gene_or_gene_product
levels	O
led	O
to	O
an	O
increase	O
in	O
E2F4	B-Gene_or_gene_product
binding	O
to	O
p130	B-Gene_or_gene_product
but	O
had	O
no	O
effect	O
on	O
E2F4	B-Complex
/	I-Complex
p107	I-Complex
or	O
E2F5	B-Complex
/	I-Complex
p130	I-Complex
complexes	O
.	O

The	O
increase	O
in	O
E2F4	B-Complex
/	I-Complex
p130	I-Complex
association	O
after	O
IR	O
was	O
observed	O
in	O
prostate	O
carcinoma	O
cells	O
regardless	O
of	O
their	O
sensitivity	O
to	O
androgens	O
,	O
but	O
not	O
in	O
breast	O
carcinoma	O
cells	O
.	O

E2F4	B-Complex
/	I-Complex
p130	I-Complex
complex	O
formation	O
was	O
dependent	O
on	O
dissociation	O
of	O
p130	B-Gene_or_gene_product
from	O
cyclin	B-Gene_or_gene_product
-	I-Gene_or_gene_product
dependent	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
2	I-Gene_or_gene_product
and	O
p130	B-Gene_or_gene_product
dephosphorylation	O
.	O

Disruption	O
of	O
E2F4	B-Gene_or_gene_product
through	O
small	O
interfering	O
RNA	O
prevented	O
p130	B-Gene_or_gene_product
/	O
E2F4	B-Gene_or_gene_product
complex	O
formation	O
and	O
sensitized	O
cells	O
to	O
IR	O
-	O
induced	O
apoptosis	O
,	O
leading	O
to	O
caspase	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3	I-Gene_or_gene_product
activation	O
,	O
cleavage	O
of	O
its	O
substrate	O
,	O
poly	O
(	O
ADP	O
-	O
ribose	O
)	O
polymerase	O
,	O
and	O
nuclear	B-Cellular_component
condensation	O
.	O

The	O
E2F4	B-Complex
/	I-Complex
p130	I-Complex
pocket	O
protein	O
complex	O
emerges	O
as	O
a	O
new	O
target	O
of	O
radiation	O
in	O
prostate	O
carcinoma	O
cells	O
.	O

Regulation	O
of	O
Toll	B-Gene_or_gene_product
/	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
-	I-Gene_or_gene_product
receptor	I-Gene_or_gene_product
-	O
mediated	O
gene	O
expression	O
by	O
the	O
inducible	O
nuclear	B-Cellular_component
protein	O
IkappaBzeta	B-Gene_or_gene_product
.	O

Toll	B-Gene_or_gene_product
-	I-Gene_or_gene_product
like	I-Gene_or_gene_product
receptors	I-Gene_or_gene_product
(	O
TLRs	B-Gene_or_gene_product
)	O
recognize	O
microbial	O
components	O
and	O
trigger	O
the	O
inflammatory	O
and	O
immune	O
responses	O
against	O
pathogens	O
.	O

IkappaBzeta	B-Gene_or_gene_product
(	O
also	O
known	O
as	O
MAIL	B-Gene_or_gene_product
and	O
INAP	B-Gene_or_gene_product
)	O
is	O
an	O
ankyrin	O
-	O
repeat	O
-	O
containing	O
nuclear	B-Cellular_component
protein	O
that	O
is	O
highly	O
homologous	O
to	O
the	O
IkappaB	B-Gene_or_gene_product
family	O
member	O
Bcl	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3	I-Gene_or_gene_product
(	O
refs	O
1	O
-	O
6	O
)	O
.	O

Transcription	O
of	O
IkappaBzeta	B-Gene_or_gene_product
is	O
rapidly	O
induced	O
by	O
stimulation	O
with	O
TLR	B-Gene_or_gene_product
ligands	O
and	O
interleukin	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
(	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
)	O
.	O

Here	O
we	O
show	O
that	O
IkappaBzeta	B-Gene_or_gene_product
is	O
indispensable	O
for	O
the	O
expression	O
of	O
a	O
subset	O
of	O
genes	O
activated	O
in	O
TLR	B-Gene_or_gene_product
/	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1R	I-Gene_or_gene_product
signalling	O
pathways	O
.	O

IkappaBzeta	B-Gene_or_gene_product
-	O
deficient	O
cells	O
show	O
severe	O
impairment	O
of	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
6	I-Gene_or_gene_product
production	O
in	O
response	O
to	O
a	O
variety	O
of	O
TLR	B-Gene_or_gene_product
ligands	O
as	O
well	O
as	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
,	O
but	O
not	O
in	O
response	O
to	O
tumour	B-Gene_or_gene_product
-	I-Gene_or_gene_product
necrosis	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
.	O

Endogenous	O
IkappaBzeta	B-Gene_or_gene_product
specifically	O
associates	O
with	O
the	O
p50	B-Gene_or_gene_product
subunit	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
,	O
and	O
is	O
recruited	O
to	O
the	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
binding	O
site	O
of	O
the	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
6	I-Gene_or_gene_product
promoter	O
on	O
stimulation	O
.	O

Moreover	O
,	O
NF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
kappaB1	I-Gene_or_gene_product
/	O
p50	B-Gene_or_gene_product
-	O
deficient	O
mice	O
show	O
responses	O
to	O
TLR	B-Gene_or_gene_product
/	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1R	I-Gene_or_gene_product
ligands	O
similar	O
to	O
those	O
of	O
IkappaBzeta	B-Gene_or_gene_product
-	O
deficient	O
mice	O
.	O

Endotoxin	O
-	O
induced	O
expression	O
of	O
other	O
genes	O
such	O
as	O
Il12b	B-Gene_or_gene_product
and	O
Csf2	B-Gene_or_gene_product
is	O
also	O
abrogated	O
in	O
IkappaBzeta	B-Gene_or_gene_product
-	O
deficient	O
macrophages	O
.	O

Given	O
that	O
the	O
lipopolysaccharide	B-Simple_chemical
-	O
induced	O
transcription	O
of	O
IkappaBzeta	B-Gene_or_gene_product
occurs	O
earlier	O
than	O
transcription	O
of	O
these	O
genes	O
,	O
some	O
TLR	B-Gene_or_gene_product
/	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1R	I-Gene_or_gene_product
-	O
mediated	O
responses	O
may	O
be	O
regulated	O
in	O
a	O
gene	O
expression	O
process	O
of	O
at	O
least	O
two	O
steps	O
that	O
requires	O
inducible	O
IkappaBzeta	B-Gene_or_gene_product
.	O

Phosphorylation	O
by	O
cyclin	B-Complex
B	I-Complex
-	I-Complex
Cdk	I-Complex
underlies	O
release	O
of	O
mitotic	O
exit	O
activator	O
Cdc14	B-Gene_or_gene_product
from	O
the	O
nucleolus	B-Cellular_component
.	O

Budding	O
yeast	O
protein	O
phosphatase	O
Cdc14	B-Gene_or_gene_product
is	O
sequestered	O
in	O
the	O
nucleolus	B-Cellular_component
in	O
an	O
inactive	O
state	O
during	O
interphase	O
by	O
the	O
anchor	O
protein	O
Net1	B-Gene_or_gene_product
.	O

Upon	O
entry	O
into	O
anaphase	O
,	O
the	O
Cdc14	B-Gene_or_gene_product
early	O
anaphase	O
release	O
(	O
FEAR	O
)	O
network	O
initiates	O
dispersal	O
of	O
active	O
Cdc14	B-Gene_or_gene_product
throughout	O
the	O
cell	O
.	O

We	O
report	O
that	O
the	O
FEARnetwork	O
promotes	O
phosphorylation	O
of	O
Net1	B-Gene_or_gene_product
by	O
cyclin	B-Gene_or_gene_product
-	I-Gene_or_gene_product
dependent	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
(	O
Cdk	B-Gene_or_gene_product
)	O
complexed	O
with	O
cyclin	B-Gene_or_gene_product
B1	I-Gene_or_gene_product
or	O
cyclin	B-Gene_or_gene_product
B2	I-Gene_or_gene_product
.	O

These	O
phosphorylations	O
appear	O
to	O
be	O
required	O
for	O
FEAR	O
and	O
sustain	O
the	O
proper	O
timing	O
of	O
late	O
mitotic	O
events	O
.	O

Thus	O
,	O
a	O
regulatory	O
circuit	O
exists	O
to	O
ensure	O
that	O
the	O
arbiter	O
of	O
the	O
mitotic	O
state	O
,	O
Cdk	B-Gene_or_gene_product
,	O
sets	O
in	O
motion	O
events	O
that	O
culminate	O
in	O
exit	O
from	O
mitosis	O
.	O

Transcription	O
of	O
dbpA	B-Gene_or_gene_product
,	O
a	O
Y	O
box	O
binding	O
protein	O
,	O
is	O
positively	O
regulated	O
by	O
E2F1	B-Gene_or_gene_product
:	O
implications	O
in	O
hepatocarcinogenesis	O
.	O

Human	O
hepatocellular	O
carcinoma	O
is	O
one	O
of	O
the	O
most	O
common	O
cancers	O
in	O
the	O
world	O
.	O

We	O
previously	O
showed	O
that	O
dbpA	B-Gene_or_gene_product
,	O
a	O
member	O
of	O
the	O
Y	O
box	O
family	O
of	O
proteins	O
,	O
could	O
accelerate	O
the	O
process	O
of	O
inflammation	O
-	O
induced	O
hepatocarcinogenesis	O
,	O
and	O
that	O
dbpA	B-Gene_or_gene_product
is	O
more	O
abundantly	O
expressed	O
in	O
hepatocellular	O
carcinoma	O
than	O
in	O
non	O
-	O
tumorous	O
tissue	O
.	O

In	O
this	O
study	O
,	O
to	O
clarify	O
the	O
mechanism	O
by	O
which	O
expression	O
of	O
dbpA	B-Gene_or_gene_product
is	O
enhanced	O
in	O
the	O
proliferative	O
state	O
,	O
we	O
examined	O
the	O
transcriptional	O
activity	O
of	O
the	O
dbpA	B-Gene_or_gene_product
promoter	O
region	O
.	O

We	O
focused	O
on	O
the	O
sequence	O
5	O
'	O
-	O
TTTGGGGC	O
-	O
3	O
'	O
(	O
-	O
8	O
to	O
-	O
1	O
in	O
the	O
promoter	O
region	O
)	O
resembling	O
the	O
E2F	B-Gene_or_gene_product
binding	O
site	O
(	O
one	O
base	O
mismatch	O
,	O
TFSEARCH	O
score	O
86	O
.	O
2	O
)	O
.	O

By	O
overexpressing	O
E2F1	B-Gene_or_gene_product
in	O
Huh	O
-	O
7	O
cells	O
,	O
transcriptional	O
activity	O
of	O
dbpA	B-Gene_or_gene_product
was	O
significantly	O
increased	O
,	O
and	O
this	O
increase	O
was	O
abolished	O
by	O
mutating	O
or	O
deleting	O
this	O
sequence	O
.	O

Thus	O
,	O
expression	O
of	O
dbpA	B-Gene_or_gene_product
was	O
positively	O
regulated	O
by	O
E2F1	B-Gene_or_gene_product
,	O
suggesting	O
that	O
one	O
of	O
the	O
effects	O
of	O
E2F1	B-Gene_or_gene_product
on	O
cell	O
proliferation	O
might	O
be	O
mediated	O
by	O
dbpA	B-Gene_or_gene_product
at	O
the	O
carcinogenesis	O
step	O
.	O

Upstream	O
and	O
downstream	O
of	O
mTOR	B-Gene_or_gene_product
.	O

The	O
evolutionarily	O
conserved	O
checkpoint	O
protein	O
kinase	O
,	O
TOR	B-Gene_or_gene_product
(	O
target	B-Gene_or_gene_product
of	I-Gene_or_gene_product
rapamycin	I-Gene_or_gene_product
)	O
,	O
has	O
emerged	O
as	O
a	O
major	O
effector	O
of	O
cell	O
growth	O
and	O
proliferation	O
via	O
the	O
regulation	O
of	O
protein	O
synthesis	O
.	O

Work	O
in	O
the	O
last	O
decade	O
clearly	O
demonstrates	O
that	O
TOR	B-Gene_or_gene_product
controls	O
protein	O
synthesis	O
through	O
a	O
stunning	O
number	O
of	O
downstream	O
targets	O
.	O

Some	O
of	O
the	O
targets	O
are	O
phosphorylated	O
directly	O
by	O
TOR	B-Gene_or_gene_product
,	O
but	O
many	O
are	O
phosphorylated	O
indirectly	O
.	O

In	O
this	O
review	O
,	O
we	O
summarize	O
some	O
recent	O
developments	O
in	O
this	O
fast	O
-	O
evolving	O
field	O
.	O

We	O
describe	O
both	O
the	O
upstream	O
components	O
of	O
the	O
signaling	O
pathway	O
(	O
s	O
)	O
that	O
activates	O
mammalian	B-Gene_or_gene_product
TOR	I-Gene_or_gene_product
(	O
mTOR	B-Gene_or_gene_product
)	O
and	O
the	O
downstream	O
targets	O
that	O
affect	O
protein	O
synthesis	O
.	O

We	O
also	O
summarize	O
the	O
roles	O
of	O
mTOR	B-Gene_or_gene_product
in	O
the	O
control	O
of	O
cell	O
growth	O
and	O
proliferation	O
,	O
as	O
well	O
as	O
its	O
relevance	O
to	O
cancer	O
and	O
synaptic	B-Cellular_component
plasticity	O
.	O

Phosphorylation	O
-	O
dependent	O
binding	O
of	O
mitotic	O
cyclins	B-Complex
to	O
Cdc6	B-Gene_or_gene_product
contributes	O
to	O
DNA	O
replication	O
control	O
.	O

Cyclin	B-Gene_or_gene_product
-	I-Gene_or_gene_product
dependent	I-Gene_or_gene_product
kinases	I-Gene_or_gene_product
(	O
CDKs	B-Gene_or_gene_product
)	O
limit	O
the	O
activation	O
of	O
DNA	O
replication	O
origins	O
to	O
once	O
per	O
cell	O
cycle	O
by	O
preventing	O
the	O
assembly	O
of	O
pre	B-Complex
-	I-Complex
replicative	I-Complex
complexes	I-Complex
(	O
pre	B-Complex
-	I-Complex
RCs	I-Complex
)	O
during	O
S	O
,	O
G2	O
and	O
M	O
phases	O
of	O
the	O
cell	O
cycle	O
in	O
the	O
budding	O
yeast	O
Saccharomyces	O
cerevisiae	O
.	O

CDKs	B-Gene_or_gene_product
inhibit	O
each	O
pre	B-Complex
-	I-Complex
RC	I-Complex
component	O
(	O
ORC	B-Gene_or_gene_product
,	O
Cdc6	B-Gene_or_gene_product
,	O
Cdt1	B-Complex
/	I-Complex
Mcm2	I-Complex
-	I-Complex
7	I-Complex
)	O
by	O
different	O
mechanisms	O
.	O

We	O
show	O
here	O
that	O
the	O
mitotic	O
CDK	B-Gene_or_gene_product
,	O
Clb2	B-Complex
/	I-Complex
Cdc28	I-Complex
,	O
binds	O
tightly	O
to	O
an	O
amino	O
-	O
terminal	O
domain	O
(	O
NTD	O
)	O
of	O
Cdc6	B-Gene_or_gene_product
,	O
and	O
that	O
Cdc6	B-Gene_or_gene_product
in	O
this	O
complex	O
is	O
unable	O
to	O
assemble	O
pre	B-Complex
-	I-Complex
RCs	I-Complex
.	O

We	O
present	O
evidence	O
indicating	O
that	O
this	O
Clb2	B-Gene_or_gene_product
-	O
dependent	O
mechanism	O
contributes	O
to	O
preventing	O
re	O
-	O
replication	O
in	O
vivo	O
.	O

CDK	B-Gene_or_gene_product
interaction	O
with	O
the	O
NTD	O
of	O
Cdc6	B-Gene_or_gene_product
is	O
mediated	O
by	O
the	O
cyclin	B-Complex
subunit	O
Clb2	B-Gene_or_gene_product
,	O
and	O
could	O
be	O
reconstituted	O
with	O
recombinant	O
Clb2	B-Gene_or_gene_product
protein	O
and	O
synthetic	O
NTD	O
peptides	O
.	O

Tight	O
Clb2	B-Gene_or_gene_product
binding	O
occurred	O
only	O
when	O
the	O
NTD	O
was	O
phosphorylated	O
on	O
CDK	B-Gene_or_gene_product
consensus	O
sites	O
.	O

Human	O
CDKs	B-Gene_or_gene_product
containing	O
cyclins	B-Gene_or_gene_product
A	I-Gene_or_gene_product
,	O
B	B-Gene_or_gene_product
and	O
E	B-Gene_or_gene_product
also	O
bound	O
specifically	O
to	O
phospho	O
-	O
NTD	O
peptides	O
.	O

We	O
propose	O
that	O
direct	O
binding	O
of	O
cyclins	B-Complex
to	O
phosphopeptide	O
motifs	O
may	O
be	O
a	O
widespread	O
phenomenon	O
contributing	O
to	O
the	O
targeting	O
of	O
CDKs	B-Gene_or_gene_product
to	O
substrates	O
.	O

Piceatannol	B-Simple_chemical
prevents	O
lipopolysaccharide	B-Simple_chemical
(	O
LPS	B-Simple_chemical
)	O
-	O
induced	O
nitric	B-Simple_chemical
oxide	I-Simple_chemical
(	O
NO	B-Simple_chemical
)	O
production	O
and	O
nuclear	B-Complex
factor	I-Complex
(	I-Complex
NF	I-Complex
)	I-Complex
-	I-Complex
kappaB	I-Complex
activation	O
by	O
inhibiting	O
IkappaB	B-Complex
kinase	I-Complex
(	O
IKK	B-Complex
)	O
.	O

The	O
effect	O
of	O
piceatannol	B-Simple_chemical
on	O
lipopolysaccharide	B-Simple_chemical
(	O
LPS	B-Simple_chemical
)	O
-	O
induced	O
nitric	B-Simple_chemical
oxide	I-Simple_chemical
(	O
NO	B-Simple_chemical
)	O
production	O
was	O
examined	O
.	O

Piceatannol	B-Simple_chemical
significantly	O
inhibited	O
NO	B-Simple_chemical
production	O
in	O
LPS	B-Simple_chemical
-	O
stimulated	O
RAW	O
264	O
.	O
7	O
cells	O
.	O

The	O
inhibition	O
was	O
due	O
to	O
the	O
reduced	O
expression	O
of	O
an	O
inducible	O
isoform	O
of	O
NO	B-Gene_or_gene_product
synthase	I-Gene_or_gene_product
(	O
iNOS	B-Gene_or_gene_product
)	O
.	O

The	O
inhibitory	O
effect	O
of	O
piceatannol	B-Simple_chemical
was	O
mediated	O
by	O
down	O
-	O
regulation	O
of	O
LPS	B-Simple_chemical
-	O
induced	O
nuclear	B-Complex
factor	I-Complex
(	I-Complex
NF	I-Complex
)	I-Complex
-	I-Complex
kappaB	I-Complex
activation	O
,	O
but	O
not	O
by	O
its	O
cytotoxic	O
action	O
.	O

Piceatannol	B-Simple_chemical
inhibited	O
IkappaB	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
(	I-Gene_or_gene_product
IKK	I-Gene_or_gene_product
)	I-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
and	O
beta	B-Gene_or_gene_product
phosphorylation	O
,	O
and	O
subsequently	O
IkappaB	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
phosphorylation	O
in	O
LPS	B-Simple_chemical
-	O
stimulated	O
RAW	O
264	O
.	O
7	O
cells	O
.	O

On	O
the	O
other	O
hand	O
,	O
piceatannol	B-Simple_chemical
did	O
not	O
affect	O
activation	O
of	O
mitogen	B-Gene_or_gene_product
-	I-Gene_or_gene_product
activated	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
(	I-Gene_or_gene_product
MAP	I-Gene_or_gene_product
)	I-Gene_or_gene_product
kinases	I-Gene_or_gene_product
including	O
extracellular	B-Gene_or_gene_product
signal	I-Gene_or_gene_product
regulated	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
1	I-Gene_or_gene_product
/	I-Gene_or_gene_product
2	I-Gene_or_gene_product
(	O
Erk1	B-Gene_or_gene_product
/	I-Gene_or_gene_product
2	I-Gene_or_gene_product
)	O
,	O
p38	B-Gene_or_gene_product
and	O
stress	B-Gene_or_gene_product
-	I-Gene_or_gene_product
activated	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
/	I-Gene_or_gene_product
c	I-Gene_or_gene_product
-	I-Gene_or_gene_product
Jun	I-Gene_or_gene_product
NH2	I-Gene_or_gene_product
-	I-Gene_or_gene_product
terminal	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
(	O
SAPK	B-Gene_or_gene_product
/	I-Gene_or_gene_product
JNK	I-Gene_or_gene_product
)	O
.	O

Piceatannol	B-Simple_chemical
inhibited	O
the	O
phosphorylation	O
of	O
Akt	B-Gene_or_gene_product
and	O
Raf	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
molecules	O
,	O
which	O
regulated	O
the	O
activation	O
of	O
IKK	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
and	O
beta	B-Gene_or_gene_product
phosphorylation	O
.	O

The	O
detailed	O
mechanism	O
of	O
the	O
inhibition	O
of	O
LPS	B-Simple_chemical
-	O
induced	O
NO	B-Simple_chemical
production	O
by	O
piceatannol	B-Simple_chemical
is	O
discussed	O
.	O

Novel	O
roles	O
of	O
TLR3	B-Gene_or_gene_product
tyrosine	B-Simple_chemical
phosphorylation	O
and	O
PI3	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
in	O
double	O
-	O
stranded	O
RNA	O
signaling	O
.	O

Double	O
-	O
stranded	O
RNA	O
(	O
dsRNA	O
)	O
,	O
a	O
frequent	O
byproduct	O
of	O
virus	O
infection	O
,	O
is	O
recognized	O
by	O
Toll	B-Gene_or_gene_product
-	I-Gene_or_gene_product
like	I-Gene_or_gene_product
receptor	I-Gene_or_gene_product
3	I-Gene_or_gene_product
(	O
TLR3	B-Gene_or_gene_product
)	O
to	O
mediate	O
innate	O
immune	O
response	O
to	O
virus	O
infection	O
.	O

TLR3	B-Gene_or_gene_product
signaling	O
activates	O
the	O
transcription	O
factor	O
IRF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3	I-Gene_or_gene_product
by	O
its	O
Ser	B-Simple_chemical
/	O
Thr	B-Simple_chemical
phosphorylation	O
,	O
accompanied	O
by	O
its	O
dimerization	O
and	O
nuclear	B-Cellular_component
translocation	O
.	O

It	O
has	O
been	O
reported	O
that	O
the	O
Ser	B-Simple_chemical
/	O
Thr	B-Simple_chemical
kinase	O
TBK	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
is	O
essential	O
for	O
TLR3	B-Gene_or_gene_product
-	O
mediated	O
activation	O
and	O
phosphorylation	O
of	O
IRF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3	I-Gene_or_gene_product
.	O

Here	O
we	O
report	O
that	O
dsRNA	O
-	O
activated	O
phosphorylation	O
of	O
two	O
specific	O
tyrosine	B-Simple_chemical
residues	O
of	O
TLR3	B-Gene_or_gene_product
is	O
essential	O
for	O
initiating	O
two	O
distinct	O
signaling	O
pathways	O
.	O

One	O
involves	O
activation	O
of	O
TBK	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
and	O
the	O
other	O
recruits	O
and	O
activates	O
PI3	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
and	O
the	O
downstream	O
kinase	O
,	O
Akt	B-Gene_or_gene_product
,	O
leading	O
to	O
full	O
phosphorylation	O
and	O
activation	O
of	O
IRF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3	I-Gene_or_gene_product
.	O

When	O
PI3	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
is	O
not	O
recruited	O
to	O
TLR3	B-Gene_or_gene_product
or	O
its	O
activity	O
is	O
blocked	O
,	O
IRF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3	I-Gene_or_gene_product
is	O
only	O
partially	O
phosphorylated	O
and	O
fails	O
to	O
bind	O
the	O
promoter	O
of	O
the	O
target	O
gene	O
in	O
dsRNA	O
-	O
treated	O
cells	O
.	O

Thus	O
,	O
the	O
PI3K	B-Gene_or_gene_product
-	O
Akt	B-Gene_or_gene_product
pathway	O
plays	O
an	O
essential	O
role	O
in	O
TLR3	B-Gene_or_gene_product
-	O
mediated	O
gene	O
induction	O
.	O

Cross	O
-	O
talk	O
between	O
pRb	B-Gene_or_gene_product
/	O
E2F	B-Gene_or_gene_product
and	O
Wnt	B-Gene_or_gene_product
/	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
pathways	O
:	O
E2F1	B-Gene_or_gene_product
induces	O
axin2	B-Gene_or_gene_product
leading	O
to	O
repression	O
of	O
Wnt	B-Gene_or_gene_product
signalling	O
and	O
to	O
increased	O
cell	O
death	O
.	O

The	O
pRb	B-Gene_or_gene_product
/	O
E2F	B-Gene_or_gene_product
and	O
Wnt	B-Gene_or_gene_product
/	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
pathways	O
are	O
two	O
of	O
the	O
most	O
frequently	O
deregulated	O
pathways	O
in	O
human	O
cancers	O
.	O

In	O
this	O
study	O
,	O
we	O
show	O
that	O
E2F1	B-Gene_or_gene_product
up	O
-	O
regulates	O
the	O
expression	O
of	O
axin2	B-Gene_or_gene_product
.	O

Further	O
,	O
we	O
show	O
that	O
axin2	B-Gene_or_gene_product
can	O
repress	O
Wnt	B-Gene_or_gene_product
signalling	O
leading	O
to	O
reduced	O
cell	O
growth	O
and	O
increased	O
cell	O
death	O
.	O

This	O
represents	O
cross	O
-	O
talk	O
between	O
major	O
pathways	O
involved	O
in	O
the	O
formation	O
of	O
tumours	O
.	O

We	O
use	O
our	O
data	O
to	O
suggest	O
a	O
novel	O
mechanism	O
for	O
tumour	O
suppression	O
.	O

PIG	B-Gene_or_gene_product
-	I-Gene_or_gene_product
V	I-Gene_or_gene_product
involved	O
in	O
transferring	O
the	O
second	O
mannose	B-Simple_chemical
in	O
glycosylphosphatidylinositol	B-Simple_chemical
.	O

Glycosylphosphatidylinositol	B-Simple_chemical
(	O
GPI	B-Simple_chemical
)	O
is	O
a	O
glycolipid	O
that	O
anchors	O
many	O
proteins	O
to	O
the	O
eukaryotic	O
cell	B-Cellular_component
surface	I-Cellular_component
.	O

The	O
biosynthetic	O
pathway	O
of	O
GPI	B-Simple_chemical
is	O
mediated	O
by	O
sequential	O
additions	O
of	O
sugars	B-Simple_chemical
and	O
other	O
components	O
to	O
phosphatidylinositol	B-Simple_chemical
.	O

Four	O
mannoses	B-Simple_chemical
in	O
the	O
GPI	B-Simple_chemical
are	O
transferred	O
from	O
dolichol	B-Simple_chemical
-	I-Simple_chemical
phosphate	I-Simple_chemical
-	I-Simple_chemical
mannose	I-Simple_chemical
(	O
Dol	B-Simple_chemical
-	I-Simple_chemical
P	I-Simple_chemical
-	I-Simple_chemical
Man	I-Simple_chemical
)	O
and	O
are	O
linked	O
through	O
different	O
glycosidic	O
linkages	O
.	O

Therefore	O
,	O
four	O
Dol	B-Simple_chemical
-	I-Simple_chemical
P	I-Simple_chemical
-	I-Simple_chemical
Man	I-Simple_chemical
-	O
dependent	O
mannosyltransferases	B-Gene_or_gene_product
,	O
GPI	B-Gene_or_gene_product
-	I-Gene_or_gene_product
MT	I-Gene_or_gene_product
-	I-Gene_or_gene_product
I	I-Gene_or_gene_product
,	O
-	B-Gene_or_gene_product
MT	I-Gene_or_gene_product
-	I-Gene_or_gene_product
II	I-Gene_or_gene_product
,	O
-	B-Gene_or_gene_product
MT	I-Gene_or_gene_product
-	I-Gene_or_gene_product
III	I-Gene_or_gene_product
,	O
and	O
-	B-Gene_or_gene_product
MT	I-Gene_or_gene_product
-	I-Gene_or_gene_product
IV	I-Gene_or_gene_product
for	O
the	O
first	O
,	O
second	O
,	O
third	O
,	O
and	O
fourth	O
mannoses	B-Simple_chemical
,	O
respectively	O
,	O
are	O
required	O
for	O
generation	O
of	O
GPI	B-Simple_chemical
.	O

GPI	B-Gene_or_gene_product
-	I-Gene_or_gene_product
MT	I-Gene_or_gene_product
-	I-Gene_or_gene_product
I	I-Gene_or_gene_product
(	O
PIG	B-Gene_or_gene_product
-	I-Gene_or_gene_product
M	I-Gene_or_gene_product
)	O
,	O
GPI	B-Gene_or_gene_product
-	I-Gene_or_gene_product
MT	I-Gene_or_gene_product
-	I-Gene_or_gene_product
III	I-Gene_or_gene_product
(	O
PIG	B-Gene_or_gene_product
-	I-Gene_or_gene_product
B	I-Gene_or_gene_product
)	O
,	O
and	O
GPI	B-Gene_or_gene_product
-	I-Gene_or_gene_product
MT	I-Gene_or_gene_product
-	I-Gene_or_gene_product
IV	I-Gene_or_gene_product
(	O
SMP3	B-Gene_or_gene_product
)	O
were	O
previously	O
reported	O
,	O
but	O
GPI	B-Gene_or_gene_product
-	I-Gene_or_gene_product
MT	I-Gene_or_gene_product
-	I-Gene_or_gene_product
II	I-Gene_or_gene_product
remains	O
to	O
be	O
identified	O
.	O

Here	O
we	O
report	O
the	O
cloning	O
of	O
PIG	B-Gene_or_gene_product
-	I-Gene_or_gene_product
V	I-Gene_or_gene_product
involved	O
in	O
transferring	O
the	O
second	O
mannose	B-Simple_chemical
in	O
the	O
GPI	B-Simple_chemical
anchor	O
.	O

Human	O
PIG	B-Gene_or_gene_product
-	I-Gene_or_gene_product
V	I-Gene_or_gene_product
encodes	O
a	O
493	O
-	O
amino	O
acid	O
,	O
endoplasmic	B-Cellular_component
reticulum	I-Cellular_component
(	O
ER	B-Cellular_component
)	O
resident	O
protein	O
with	O
eight	O
putative	O
transmembrane	B-Cellular_component
regions	O
.	O

Saccharomyces	O
cerevisiae	O
protein	O
encoded	O
in	O
open	O
reading	O
frame	O
YBR004c	O
,	O
which	O
we	O
termed	O
GPI18	B-Gene_or_gene_product
,	O
has	O
25	O
%	O
amino	O
acid	O
identity	O
to	O
human	O
PIG	B-Gene_or_gene_product
-	I-Gene_or_gene_product
V	I-Gene_or_gene_product
.	O

Viability	O
of	O
the	O
yeast	O
gpi18	O
deletion	O
mutant	O
was	O
restored	O
by	O
human	O
PIG	B-Gene_or_gene_product
-	I-Gene_or_gene_product
V	I-Gene_or_gene_product
cDNA	O
.	O

PIG	B-Gene_or_gene_product
-	I-Gene_or_gene_product
V	I-Gene_or_gene_product
has	O
two	O
functionally	O
important	O
conserved	O
regions	O
facing	O
the	O
ER	B-Cellular_component
lumen	I-Cellular_component
.	O

Taken	O
together	O
,	O
we	O
suggest	O
that	O
PIG	B-Gene_or_gene_product
-	I-Gene_or_gene_product
V	I-Gene_or_gene_product
is	O
the	O
second	O
mannosyltransferase	B-Gene_or_gene_product
in	O
GPI	B-Simple_chemical
anchor	O
biosynthesis	O
.	O

Identification	O
of	O
a	O
novel	O
class	O
of	O
target	O
genes	O
and	O
a	O
novel	O
type	O
of	O
binding	O
sequence	O
of	O
heat	B-Gene_or_gene_product
shock	I-Gene_or_gene_product
transcription	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
in	O
Saccharomyces	O
cerevisiae	O
.	O

In	O
response	O
to	O
hyperthermia	O
,	O
heat	B-Gene_or_gene_product
shock	I-Gene_or_gene_product
transcription	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
(	O
HSF	B-Gene_or_gene_product
)	O
activates	O
transcription	O
of	O
a	O
set	O
of	O
genes	O
encoding	O
heat	B-Gene_or_gene_product
shock	I-Gene_or_gene_product
proteins	I-Gene_or_gene_product
(	O
HSPs	B-Gene_or_gene_product
)	O
.	O

The	O
promoter	O
regions	O
of	O
HSP	B-Gene_or_gene_product
genes	O
contain	O
the	O
HSF	B-Gene_or_gene_product
binding	O
sequence	O
called	O
the	O
heat	O
shock	O
element	O
(	O
HSE	O
)	O
,	O
which	O
consists	O
of	O
contiguous	O
inverted	O
repeats	O
of	O
the	O
sequence	O
5	O
'	O
-	O
nGAAn	O
-	O
3	O
'	O
(	O
where	O
n	O
is	O
any	O
nucleotide	O
)	O
.	O

We	O
have	O
constructed	O
an	O
hsf1	B-Gene_or_gene_product
mutant	O
of	O
Saccharomyces	O
cerevisiae	O
and	O
analyzed	O
genome	O
-	O
wide	O
changes	O
in	O
heat	O
shock	O
response	O
in	O
the	O
mutant	O
cells	O
.	O

The	O
results	O
have	O
revealed	O
that	O
Hsf1	B-Gene_or_gene_product
is	O
necessary	O
for	O
heat	O
-	O
induced	O
transcription	O
of	O
not	O
only	O
HSP	B-Gene_or_gene_product
but	O
also	O
genes	O
encoding	O
proteins	O
involved	O
in	O
diverse	O
cellular	O
processes	O
such	O
as	O
protein	O
degradation	O
,	O
detoxification	O
,	O
energy	O
generation	O
,	O
carbohydrate	B-Simple_chemical
metabolism	O
,	O
and	O
maintenance	O
of	O
cell	B-Cellular_component
wall	I-Cellular_component
integrity	O
.	O

Approximately	O
half	O
of	O
the	O
Hsf1	B-Gene_or_gene_product
-	O
regulated	O
genes	O
lacked	O
the	O
typical	O
HSE	O
in	O
their	O
promoter	O
regions	O
.	O

Instead	O
,	O
several	O
of	O
these	O
genes	O
have	O
a	O
novel	O
Hsf1	B-Gene_or_gene_product
binding	O
sequence	O
that	O
contains	O
three	O
direct	O
repeats	O
of	O
nTTCn	O
(	O
or	O
nGAAn	O
)	O
interrupted	O
by	O
5	O
bp	O
.	O

The	O
number	O
and	O
spacing	O
of	O
the	O
repeating	O
units	O
are	O
critical	O
determinants	O
for	O
heat	O
-	O
induced	O
transcription	O
as	O
well	O
as	O
for	O
recognition	O
by	O
Hsf1	B-Gene_or_gene_product
.	O

In	O
the	O
yeast	O
genome	O
,	O
the	O
presence	O
of	O
the	O
sequence	O
is	O
enriched	O
in	O
Hsf1	B-Gene_or_gene_product
-	O
regulated	O
genes	O
,	O
suggesting	O
that	O
it	O
is	O
generally	O
used	O
as	O
an	O
HSE	O
in	O
the	O
Hsf1	B-Gene_or_gene_product
regulon	O
.	O

NF	B-Complex
-	I-Complex
{	I-Complex
kappa	I-Complex
}	I-Complex
B	I-Complex
is	O
transported	O
into	O
the	O
nucleus	B-Cellular_component
by	O
importin	B-Gene_or_gene_product
{	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
}	I-Gene_or_gene_product
3	I-Gene_or_gene_product
and	O
importin	B-Gene_or_gene_product
{	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
}	I-Gene_or_gene_product
4	I-Gene_or_gene_product
.	O

NF	B-Complex
-	I-Complex
kappaB	I-Complex
transcription	O
factors	O
are	O
retained	O
in	O
the	O
cytoplasm	B-Cellular_component
in	O
an	O
inactive	O
form	O
until	O
they	O
are	O
activated	O
and	O
rapidly	O
imported	O
into	O
the	O
nucleus	B-Cellular_component
.	O

We	O
identified	O
importin	B-Gene_or_gene_product
alpha3	I-Gene_or_gene_product
and	O
importin	B-Gene_or_gene_product
alpha4	I-Gene_or_gene_product
as	O
the	O
main	O
importin	B-Gene_or_gene_product
alpha	I-Gene_or_gene_product
isoforms	O
mediating	O
TNF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
-	O
stimulated	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
p50	I-Complex
/	I-Complex
p65	I-Complex
heterodimer	O
translocation	O
into	O
the	O
nucleus	B-Cellular_component
.	O

Importin	B-Gene_or_gene_product
alpha3	I-Gene_or_gene_product
and	O
alpha4	B-Gene_or_gene_product
are	O
close	O
relatives	O
in	O
the	O
human	O
importin	B-Gene_or_gene_product
alpha	I-Gene_or_gene_product
family	O
.	O

We	O
show	O
that	O
importin	B-Gene_or_gene_product
alpha3	I-Gene_or_gene_product
isoform	O
also	O
mediates	O
nuclear	B-Cellular_component
import	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
p50	I-Complex
homodimer	O
in	O
nonstimulated	O
cells	O
.	O

Importin	B-Gene_or_gene_product
alpha3	I-Gene_or_gene_product
is	O
shown	O
to	O
directly	O
bind	O
to	O
previously	O
characterized	O
nuclear	B-Cellular_component
localization	O
signals	O
(	O
NLSs	O
)	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
p50	B-Gene_or_gene_product
and	O
p65	B-Gene_or_gene_product
proteins	O
.	O

Importin	B-Gene_or_gene_product
alpha	I-Gene_or_gene_product
molecules	O
are	O
known	O
to	O
have	O
armadillo	O
repeats	O
that	O
constitute	O
the	O
N	O
-	O
terminal	O
and	O
C	O
-	O
terminal	O
NLS	O
binding	O
sites	O
.	O

We	O
demonstrate	O
by	O
site	O
-	O
directed	O
mutagenesis	O
that	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
p50	B-Gene_or_gene_product
binds	O
to	O
the	O
N	O
-	O
terminal	O
and	O
p65	B-Gene_or_gene_product
to	O
the	O
C	O
-	O
terminal	O
NLS	O
binding	O
site	O
of	O
importin	B-Gene_or_gene_product
alpha3	I-Gene_or_gene_product
.	O

In	O
vitro	O
competition	O
experiments	O
and	O
analysis	O
of	O
cellular	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
suggest	O
that	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
binds	O
to	O
importin	B-Gene_or_gene_product
alpha	I-Gene_or_gene_product
only	O
when	O
it	O
is	O
free	O
of	O
IkappaBalpha	B-Gene_or_gene_product
.	O

The	O
present	O
study	O
demonstrates	O
that	O
the	O
nuclear	B-Cellular_component
import	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
is	O
a	O
highly	O
regulated	O
process	O
mediated	O
by	O
a	O
subset	O
of	O
importin	B-Gene_or_gene_product
alpha	I-Gene_or_gene_product
molecules	O
.	O

Regulation	O
of	O
neuron	O
survival	O
and	O
death	O
by	O
p130	B-Gene_or_gene_product
and	O
associated	O
chromatin	B-Cellular_component
modifiers	O
.	O

E2F	B-Gene_or_gene_product
-	O
mediated	O
gene	O
repression	O
plays	O
a	O
key	O
role	O
in	O
regulation	O
of	O
neuron	O
survival	O
and	O
death	O
.	O

However	O
,	O
the	O
key	O
molecules	O
involved	O
in	O
such	O
regulation	O
and	O
the	O
mechanisms	O
by	O
which	O
they	O
respond	O
to	O
apoptotic	O
stimuli	O
are	O
largely	O
unknown	O
.	O

Here	O
we	O
show	O
that	O
p130	B-Gene_or_gene_product
is	O
the	O
predominant	O
Rb	B-Gene_or_gene_product
family	O
member	O
associated	O
with	O
E2F	B-Gene_or_gene_product
in	O
neurons	O
,	O
that	O
its	O
major	O
partner	O
for	O
repression	O
of	O
pro	O
-	O
apoptotic	O
genes	O
is	O
E2F4	B-Gene_or_gene_product
,	O
and	O
that	O
the	O
p130	B-Complex
-	I-Complex
E2F4	I-Complex
complex	O
recruits	O
the	O
chromatin	B-Cellular_component
modifiers	O
HDAC1	B-Gene_or_gene_product
and	O
Suv39H1	B-Gene_or_gene_product
to	O
promote	O
gene	O
silencing	O
and	O
neuron	O
survival	O
.	O

Apoptotic	O
stimuli	O
induce	O
neuron	O
death	O
by	O
sequentially	O
causing	O
p130	B-Gene_or_gene_product
hyperphosphorylation	O
,	O
dissociation	O
of	O
p130	B-Complex
-	I-Complex
E2F4	I-Complex
-	I-Complex
Suv39H1	I-Complex
-	I-Complex
HDAC	I-Complex
complexes	O
,	O
altered	O
modification	O
of	O
H3	B-Gene_or_gene_product
histone	I-Gene_or_gene_product
and	O
gene	O
derepression	O
.	O

Experimental	O
suppression	O
of	O
such	O
events	O
blocks	O
neuron	O
death	O
while	O
interference	O
with	O
the	O
synthesis	O
of	O
E2F4	B-Gene_or_gene_product
or	O
p130	B-Gene_or_gene_product
,	O
or	O
with	O
the	O
interaction	O
of	O
E2F4	B-Complex
-	I-Complex
p130	I-Complex
with	O
chromatin	B-Cellular_component
modifiers	O
,	O
induces	O
neuron	O
death	O
.	O

Thus	O
,	O
neuron	O
survival	O
and	O
death	O
are	O
dependent	O
on	O
the	O
integrity	O
of	O
E2F4	B-Complex
-	I-Complex
p130	I-Complex
-	I-Complex
HDAC	I-Complex
/	I-Complex
Suv39H1	I-Complex
complexes	O
.	O

Production	O
of	O
matrix	B-Gene_or_gene_product
metalloproteinase	I-Gene_or_gene_product
-	I-Gene_or_gene_product
9	I-Gene_or_gene_product
by	O
activated	O
human	O
monocytes	O
involves	O
a	O
phosphatidylinositol	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
/	O
Akt	B-Gene_or_gene_product
/	O
IKKalpha	B-Gene_or_gene_product
/	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
pathway	O
.	O

Matrix	B-Gene_or_gene_product
metalloproteinase	I-Gene_or_gene_product
-	I-Gene_or_gene_product
9	I-Gene_or_gene_product
(	O
MMP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
9	I-Gene_or_gene_product
)	O
is	O
considered	O
to	O
be	O
an	O
important	O
component	O
in	O
the	O
progression	O
of	O
inflammation	O
.	O

Monocytes	O
/	O
macrophages	O
are	O
prominent	O
at	O
inflammation	O
sites	O
,	O
and	O
activation	O
of	O
these	O
cells	O
by	O
stimulants	O
,	O
such	O
as	O
lipopolysaccharide	B-Simple_chemical
(	O
LPS	B-Simple_chemical
)	O
or	O
tumor	B-Gene_or_gene_product
necrosis	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
and	O
granulocyte	B-Gene_or_gene_product
macrophage	I-Gene_or_gene_product
-	I-Gene_or_gene_product
colony	I-Gene_or_gene_product
stimulating	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
,	O
leads	O
to	O
the	O
production	O
of	O
significant	O
amounts	O
of	O
MMP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
9	I-Gene_or_gene_product
.	O

Here	O
,	O
we	O
show	O
that	O
LPS	B-Simple_chemical
stimulation	O
of	O
monocytes	O
results	O
in	O
MMP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
9	I-Gene_or_gene_product
production	O
through	O
a	O
phosphatidylinositol	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
(	O
PI	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3K	I-Gene_or_gene_product
)	O
/	O
Akt	B-Gene_or_gene_product
/	O
inhibitor	B-Gene_or_gene_product
of	I-Gene_or_gene_product
kappaB	I-Gene_or_gene_product
(	I-Gene_or_gene_product
IkappaB	I-Gene_or_gene_product
)	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
(	O
IKKalpha	B-Gene_or_gene_product
)	O
/	O
nuclear	B-Complex
factor	I-Complex
(	I-Complex
NF	I-Complex
)	I-Complex
-	I-Complex
kappaB	I-Complex
pathway	O
.	O

This	O
new	O
role	O
for	O
Akt	B-Gene_or_gene_product
in	O
signaling	O
leading	O
to	O
MMP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
9	I-Gene_or_gene_product
production	O
was	O
demonstrated	O
by	O
inhibitor	O
and	O
immunoprecipitation	O
studies	O
.	O

LY294002	B-Simple_chemical
or	O
wortmannin	B-Simple_chemical
,	O
inhibitors	O
of	O
PI	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3K	I-Gene_or_gene_product
,	O
suppressed	O
LPS	B-Simple_chemical
-	O
induced	O
Akt	B-Gene_or_gene_product
activity	O
and	O
MMP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
9	I-Gene_or_gene_product
production	O
.	O

Evidence	O
for	O
the	O
participation	O
of	O
Akt	B-Gene_or_gene_product
in	O
monocyte	O
MMP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
9	I-Gene_or_gene_product
synthesis	O
was	O
demonstrated	O
by	O
the	O
inhibition	O
of	O
MMP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
9	I-Gene_or_gene_product
by	O
SH	B-Simple_chemical
-	I-Simple_chemical
5	I-Simple_chemical
,	O
a	O
specific	O
inhibitor	O
of	O
Akt	B-Gene_or_gene_product
.	O

The	O
mechanism	O
by	O
which	O
Akt	B-Gene_or_gene_product
regulates	O
MMP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
9	I-Gene_or_gene_product
is	O
through	O
the	O
activation	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
,	O
as	O
shown	O
by	O
coimmunoprecipitation	O
of	O
the	O
phosphorylated	O
form	O
of	O
IKKalpha	B-Gene_or_gene_product
and	O
Akt	B-Gene_or_gene_product
as	O
well	O
as	O
the	O
SH	B-Simple_chemical
-	I-Simple_chemical
5	I-Simple_chemical
suppression	O
of	O
the	O
dissociation	O
of	O
IkappaB	B-Gene_or_gene_product
from	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
and	O
the	O
activation	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
p65	B-Gene_or_gene_product
.	O

The	O
role	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
in	O
regulation	O
of	O
MMP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
9	I-Gene_or_gene_product
was	O
demonstrated	O
further	O
by	O
the	O
inhibition	O
of	O
MMP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
9	I-Gene_or_gene_product
production	O
by	O
proteasome	O
inhibitors	O
,	O
lactacystin	B-Simple_chemical
and	O
MG	B-Simple_chemical
-	I-Simple_chemical
132	I-Simple_chemical
,	O
which	O
prevented	O
the	O
ubiquitination	O
and	O
dissociation	O
of	O
IkappaB	B-Gene_or_gene_product
from	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
.	O

This	O
is	O
the	O
first	O
demonstration	O
that	O
Akt	B-Gene_or_gene_product
is	O
involved	O
in	O
the	O
signaling	O
pathway	O
leading	O
to	O
the	O
production	O
of	O
monocyte	O
MMP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
9	I-Gene_or_gene_product
and	O
provides	O
an	O
additional	O
approach	O
in	O
the	O
regulation	O
of	O
this	O
enzyme	O
in	O
human	O
primary	O
monocytes	O
.	O

Phosphorylation	O
and	O
functional	O
inactivation	O
of	O
TSC2	B-Gene_or_gene_product
by	O
Erk	B-Gene_or_gene_product
implications	O
for	O
tuberous	O
sclerosis	O
and	O
cancer	O
pathogenesis	O
.	O

Tuberous	O
sclerosis	O
(	O
TSC	O
)	O
is	O
a	O
tumor	O
syndrome	O
caused	O
by	O
mutation	O
in	O
TSC1	B-Gene_or_gene_product
or	O
TSC2	B-Gene_or_gene_product
genes	O
.	O

TSC	O
tumorigenesis	O
is	O
not	O
always	O
accompanied	O
by	O
loss	O
of	O
heterozygosity	O
(	O
LOH	O
)	O
.	O

Recently	O
,	O
extracellular	B-Gene_or_gene_product
signal	I-Gene_or_gene_product
-	I-Gene_or_gene_product
regulated	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
(	O
Erk	B-Gene_or_gene_product
)	O
has	O
been	O
found	O
activated	O
in	O
TSC	O
lesions	O
lacking	O
TSC1	B-Gene_or_gene_product
or	O
TSC2	B-Gene_or_gene_product
LOH	O
.	O

Here	O
,	O
we	O
show	O
that	O
Erk	B-Gene_or_gene_product
may	O
play	O
a	O
critical	O
role	O
in	O
TSC	O
progression	O
through	O
posttranslational	O
inactivation	O
of	O
TSC2	B-Gene_or_gene_product
.	O

Erk	B-Gene_or_gene_product
-	O
dependent	O
phosphorylation	O
leads	O
to	O
TSC1	B-Complex
-	I-Complex
TSC2	I-Complex
dissociation	O
and	O
markedly	O
impairs	O
TSC2	B-Gene_or_gene_product
ability	O
to	O
inhibit	O
mTOR	B-Gene_or_gene_product
signaling	O
,	O
cell	O
proliferation	O
,	O
and	O
oncogenic	O
transformation	O
.	O

Importantly	O
,	O
expression	O
of	O
an	O
Erk	B-Gene_or_gene_product
nonphosphorylatable	O
TSC2	B-Gene_or_gene_product
mutant	O
in	O
TSC2	B-Gene_or_gene_product
+	O
/	O
-	O
tumor	O
cells	O
where	O
Erk	B-Gene_or_gene_product
is	O
constitutively	O
activated	O
blocks	O
tumorigenecity	O
in	O
vivo	O
,	O
while	O
wild	O
-	O
type	O
TSC2	B-Gene_or_gene_product
is	O
ineffective	O
.	O

Our	O
findings	O
position	O
the	O
Ras	B-Gene_or_gene_product
/	O
MAPK	B-Gene_or_gene_product
pathway	O
upstream	O
of	O
the	O
TSC	B-Complex
complex	I-Complex
and	O
suggest	O
that	O
Erk	B-Gene_or_gene_product
may	O
modulate	O
mTOR	B-Gene_or_gene_product
signaling	O
and	O
contribute	O
to	O
disease	O
progression	O
through	O
phosphorylation	O
and	O
inactivation	O
of	O
TSC2	B-Gene_or_gene_product
.	O

H19	B-Gene_or_gene_product
mRNA	O
-	O
like	O
noncoding	O
RNA	O
promotes	O
breast	O
cancer	O
cell	O
proliferation	O
through	O
positive	O
control	O
by	O
E2F1	B-Gene_or_gene_product
.	O

The	O
imprinted	O
H19	B-Gene_or_gene_product
gene	O
has	O
riboregulatory	O
functions	O
.	O

We	O
show	O
here	O
that	O
H19	B-Gene_or_gene_product
transcription	O
is	O
up	O
-	O
regulated	O
during	O
the	O
S	O
-	O
phase	O
of	O
growth	O
-	O
stimulated	O
cells	O
and	O
that	O
the	O
H19	B-Gene_or_gene_product
promoter	O
is	O
activated	O
by	O
E2F1	B-Gene_or_gene_product
in	O
breast	O
cancer	O
cells	O
.	O

H19	B-Gene_or_gene_product
repression	O
by	O
pRb	B-Gene_or_gene_product
and	O
E2F6	B-Gene_or_gene_product
confirms	O
the	O
E2F1	B-Gene_or_gene_product
-	O
dependent	O
control	O
of	O
the	O
H19	B-Gene_or_gene_product
promoter	O
.	O

Consistently	O
,	O
we	O
demonstrate	O
by	O
chromatin	B-Cellular_component
immunoprecipitation	O
assays	O
that	O
endogenous	O
E2F1	B-Gene_or_gene_product
is	O
recruited	O
to	O
the	O
H19	B-Gene_or_gene_product
promoter	O
in	O
vivo	O
.	O

The	O
functionality	O
of	O
E2F	B-Gene_or_gene_product
promoter	O
sites	O
was	O
further	O
confirmed	O
by	O
gel	O
shift	O
and	O
mutagenesis	O
experiments	O
,	O
revealing	O
that	O
these	O
sites	O
are	O
required	O
for	O
binding	O
and	O
promoter	O
response	O
to	O
E2F1	B-Gene_or_gene_product
exogenous	O
expression	O
and	O
serum	O
stimulation	O
.	O

Furthermore	O
,	O
we	O
show	O
that	O
H19	B-Gene_or_gene_product
overexpression	O
confers	O
a	O
growth	O
advantage	O
on	O
breast	O
cancer	O
cells	O
released	O
from	O
growth	O
arrest	O
as	O
well	O
as	O
in	O
asynchronously	O
growing	O
cells	O
.	O

The	O
H19	B-Gene_or_gene_product
knockdown	O
by	O
small	O
interfering	O
RNA	O
duplexes	O
impedes	O
S	O
-	O
phase	O
entry	O
in	O
both	O
wild	O
-	O
type	O
and	O
stably	O
H19	O
-	O
transfected	O
cells	O
.	O

Based	O
on	O
these	O
findings	O
,	O
we	O
conclude	O
that	O
the	O
H19	B-Gene_or_gene_product
RNA	O
is	O
actively	O
linked	O
to	O
E2F1	B-Gene_or_gene_product
to	O
promote	O
cell	O
cycle	O
progression	O
of	O
breast	O
cancer	O
cells	O
.	O

This	O
clearly	O
supports	O
the	O
H19	B-Gene_or_gene_product
oncogenic	O
function	O
in	O
breast	O
tumor	O
genesis	O
.	O

Mip1	B-Gene_or_gene_product
,	O
an	O
MEKK2	B-Gene_or_gene_product
-	O
interacting	O
protein	O
,	O
controls	O
MEKK2	B-Gene_or_gene_product
dimerization	O
and	O
activation	O
.	O

Mitogen	B-Gene_or_gene_product
-	I-Gene_or_gene_product
activated	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
(	O
MAPK	B-Gene_or_gene_product
)	O
cascades	O
are	O
central	O
components	O
of	O
the	O
intracellular	B-Cellular_component
signaling	O
networks	O
used	O
by	O
eukaryotic	O
cells	O
to	O
respond	O
to	O
a	O
wide	O
spectrum	O
of	O
extracellular	B-Cellular_component
stimuli	O
.	O

An	O
MAPK	B-Gene_or_gene_product
is	O
activated	O
by	O
an	O
MAPK	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
,	O
which	O
in	O
turn	O
is	O
activated	O
by	O
an	O
MAPK	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
(	O
MAP3K	B-Gene_or_gene_product
)	O
.	O

However	O
,	O
little	O
is	O
known	O
about	O
the	O
molecular	O
aspects	O
of	O
the	O
regulation	O
and	O
activation	O
of	O
large	O
numbers	O
of	O
MAP3Ks	B-Gene_or_gene_product
that	O
are	O
crucial	O
in	O
relaying	O
upstream	O
receptor	O
-	O
mediated	O
signals	O
through	O
the	O
MAPK	B-Gene_or_gene_product
cascades	O
to	O
induce	O
various	O
physiological	O
responses	O
.	O

In	O
this	O
study	O
,	O
we	O
identified	O
a	O
novel	O
MEKK2	B-Gene_or_gene_product
-	O
interacting	O
protein	O
,	O
Mip1	B-Gene_or_gene_product
,	O
that	O
regulates	O
MEKK2	B-Gene_or_gene_product
dimerization	O
and	O
activation	O
by	O
forming	O
a	O
complex	O
with	O
inactive	O
and	O
nonphosphorylated	O
MEKK2	B-Gene_or_gene_product
.	O

In	O
particular	O
,	O
Mip1	B-Gene_or_gene_product
prevented	O
MEKK2	B-Gene_or_gene_product
activation	O
by	O
blocking	O
MEKK2	B-Gene_or_gene_product
dimer	O
formation	O
,	O
which	O
in	O
turn	O
blocked	O
JNKK2	B-Gene_or_gene_product
,	O
c	B-Gene_or_gene_product
-	I-Gene_or_gene_product
Jun	I-Gene_or_gene_product
N	I-Gene_or_gene_product
-	I-Gene_or_gene_product
terminal	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
1	I-Gene_or_gene_product
(	O
JNK1	B-Gene_or_gene_product
)	O
,	O
extracellular	B-Gene_or_gene_product
signal	I-Gene_or_gene_product
-	I-Gene_or_gene_product
regulated	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
5	I-Gene_or_gene_product
,	O
and	O
AP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
reporter	O
gene	O
activation	O
by	O
MEKK2	B-Gene_or_gene_product
.	O

Furthermore	O
,	O
we	O
found	O
that	O
the	O
endogenous	O
Mip1	B-Complex
-	I-Complex
MEKK2	I-Complex
complex	O
was	O
dissociated	O
transiently	O
following	O
epidermal	O
growth	O
factor	O
stimulation	O
.	O

In	O
contrast	O
,	O
the	O
knockdown	O
of	O
Mip1	B-Gene_or_gene_product
expression	O
by	O
siRNA	O
augmented	O
the	O
MEKK2	B-Gene_or_gene_product
-	O
mediated	O
JNK	B-Gene_or_gene_product
and	O
AP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
reporter	O
activation	O
.	O

Together	O
,	O
our	O
data	O
suggest	O
a	O
novel	O
model	O
for	O
MEKK2	B-Gene_or_gene_product
regulation	O
and	O
activation	O
.	O

PTEN	B-Gene_or_gene_product
represses	O
RNA	B-Gene_or_gene_product
Polymerase	I-Gene_or_gene_product
I	I-Gene_or_gene_product
transcription	O
by	O
disrupting	O
the	O
SL1	B-Complex
complex	O
.	O

PTEN	B-Gene_or_gene_product
is	O
a	O
tumor	O
suppressor	O
whose	O
function	O
is	O
frequently	O
lost	O
in	O
human	O
cancer	O
.	O

It	O
possesses	O
a	O
lipid	O
phosphatase	O
activity	O
that	O
represses	O
the	O
activation	O
of	O
PI3	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
/	O
Akt	B-Gene_or_gene_product
signaling	O
,	O
leading	O
to	O
decreased	O
cell	O
growth	O
,	O
proliferation	O
,	O
and	O
survival	O
.	O

The	O
potential	O
for	O
PTEN	B-Gene_or_gene_product
to	O
regulate	O
transcription	O
of	O
the	O
large	O
rRNAs	O
by	O
RNA	B-Gene_or_gene_product
polymerase	I-Gene_or_gene_product
I	I-Gene_or_gene_product
(	O
RNA	B-Gene_or_gene_product
Pol	I-Gene_or_gene_product
I	I-Gene_or_gene_product
)	O
was	O
investigated	O
.	O

As	O
increased	O
synthesis	O
of	O
rRNAs	O
is	O
a	O
hallmark	O
of	O
neoplastic	O
transformation	O
,	O
the	O
ability	O
of	O
PTEN	B-Gene_or_gene_product
to	O
control	O
the	O
transcription	O
of	O
rRNAs	O
might	O
be	O
crucial	O
for	O
its	O
tumor	O
suppressor	O
function	O
.	O

The	O
expression	O
of	O
PTEN	B-Gene_or_gene_product
in	O
PTEN	B-Gene_or_gene_product
-	O
deficient	O
cells	O
represses	O
RNA	B-Gene_or_gene_product
Pol	I-Gene_or_gene_product
I	I-Gene_or_gene_product
transcription	O
,	O
while	O
decreasing	O
PTEN	B-Gene_or_gene_product
expression	O
enhances	O
transcription	O
.	O

PTEN	B-Gene_or_gene_product
-	O
mediated	O
repression	O
requires	O
its	O
lipid	O
phosphatase	O
activity	O
and	O
is	O
independent	O
of	O
the	O
p53	B-Gene_or_gene_product
status	O
of	O
the	O
cell	O
.	O

This	O
event	O
can	O
be	O
uncoupled	O
from	O
PTEN	B-Gene_or_gene_product
'	O
s	O
ability	O
to	O
regulate	O
the	O
cell	O
cycle	O
.	O

RNA	B-Gene_or_gene_product
Pol	I-Gene_or_gene_product
I	I-Gene_or_gene_product
is	O
regulated	O
through	O
PI3	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
/	O
Akt	B-Gene_or_gene_product
/	O
mammalian	B-Gene_or_gene_product
target	I-Gene_or_gene_product
of	I-Gene_or_gene_product
rapamycin	I-Gene_or_gene_product
/	O
S6	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
,	O
and	O
the	O
expression	O
of	O
constitutively	O
activated	O
S6	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
is	O
able	O
to	O
abrogate	O
transcription	O
repression	O
by	O
PTEN	B-Gene_or_gene_product
.	O

No	O
change	O
in	O
the	O
expression	O
of	O
the	O
RNA	B-Gene_or_gene_product
Pol	I-Gene_or_gene_product
I	I-Gene_or_gene_product
transcription	O
components	O
,	O
upstream	B-Gene_or_gene_product
binding	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
or	O
SL1	B-Gene_or_gene_product
,	O
was	O
observed	O
upon	O
PTEN	B-Gene_or_gene_product
expression	O
.	O

However	O
,	O
chromatin	B-Cellular_component
immunoprecipitation	O
assays	O
demonstrate	O
that	O
PTEN	B-Gene_or_gene_product
differentially	O
reduces	O
the	O
occupancy	O
of	O
the	O
SL1	B-Gene_or_gene_product
subunits	O
on	O
the	O
rRNA	O
gene	O
promoter	O
.	O

Furthermore	O
,	O
PTEN	B-Gene_or_gene_product
induces	O
dissociation	O
of	O
the	O
SL1	B-Gene_or_gene_product
subunits	O
.	O

Together	O
,	O
these	O
results	O
demonstrate	O
that	O
PTEN	B-Gene_or_gene_product
represses	O
RNA	B-Gene_or_gene_product
Pol	I-Gene_or_gene_product
I	I-Gene_or_gene_product
transcription	O
through	O
a	O
novel	O
mechanism	O
that	O
involves	O
disruption	O
of	O
the	O
SL1	B-Complex
complex	O
.	O

Pocket	B-Complex
protein	I-Complex
complexes	I-Complex
are	O
recruited	O
to	O
distinct	O
targets	O
in	O
quiescent	O
and	O
proliferating	O
cells	O
.	O

Biochemical	O
and	O
genetic	O
studies	O
have	O
determined	O
that	O
retinoblastoma	B-Gene_or_gene_product
protein	O
(	O
pRB	B-Gene_or_gene_product
)	O
tumor	O
suppressor	O
family	O
members	O
have	O
overlapping	O
functions	O
.	O

However	O
,	O
these	O
studies	O
have	O
largely	O
failed	O
to	O
distinguish	O
functional	O
differences	O
between	O
the	O
highly	O
related	O
p107	B-Gene_or_gene_product
and	O
p130	B-Gene_or_gene_product
proteins	O
.	O

Moreover	O
,	O
most	O
studies	O
pertaining	O
to	O
the	O
pRB	B-Gene_or_gene_product
family	O
and	O
its	O
principal	O
target	O
,	O
the	O
E2F	B-Gene_or_gene_product
transcription	O
factor	O
,	O
have	O
focused	O
on	O
cells	O
that	O
have	O
reinitiated	O
a	O
cell	O
cycle	O
from	O
quiescence	O
,	O
although	O
recent	O
studies	O
suggest	O
that	O
cycling	O
cells	O
exhibit	O
layers	O
of	O
regulation	O
distinct	O
from	O
mitogenically	O
stimulated	O
cells	O
.	O

Using	O
genome	O
-	O
wide	O
chromatin	B-Cellular_component
immunoprecipitation	O
,	O
we	O
show	O
that	O
there	O
are	O
distinct	O
classes	O
of	O
genes	O
directly	O
regulated	O
by	O
unique	O
combinations	O
of	O
E2F4	B-Gene_or_gene_product
,	O
p107	B-Gene_or_gene_product
,	O
and	O
p130	B-Gene_or_gene_product
,	O
including	O
a	O
group	O
of	O
genes	O
specifically	O
regulated	O
in	O
cycling	O
cells	O
.	O

These	O
groups	O
exhibit	O
both	O
distinct	O
histone	B-Gene_or_gene_product
acetylation	O
signatures	O
and	O
patterns	O
of	O
mammalian	O
Sin3B	B-Gene_or_gene_product
corepressor	O
recruitment	O
.	O

Our	O
findings	O
suggest	O
that	O
cell	O
cycle	O
-	O
dependent	O
repression	O
results	O
from	O
recruitment	O
of	O
an	O
unexpected	O
array	O
of	O
diverse	O
complexes	O
and	O
reveals	O
specific	O
differences	O
between	O
transcriptional	O
regulation	O
in	O
cycling	O
and	O
quiescent	O
cells	O
.	O

In	O
addition	O
,	O
factor	O
location	O
analyses	O
have	O
,	O
for	O
the	O
first	O
time	O
,	O
allowed	O
the	O
identification	O
of	O
novel	O
and	O
specific	O
targets	O
of	O
the	O
highly	O
related	O
transcriptional	O
regulators	O
p107	B-Gene_or_gene_product
and	O
p130	B-Gene_or_gene_product
,	O
suggesting	O
new	O
and	O
distinct	O
regulatory	O
networks	O
engaged	O
by	O
each	O
protein	O
in	O
continuously	O
cycling	O
cells	O
.	O

CsgA	B-Gene_or_gene_product
is	O
a	O
pathogen	O
-	O
associated	O
molecular	O
pattern	O
of	O
Salmonella	O
enterica	O
serotype	O
Typhimurium	O
that	O
is	O
recognized	O
by	O
Toll	B-Gene_or_gene_product
-	I-Gene_or_gene_product
like	I-Gene_or_gene_product
receptor	I-Gene_or_gene_product
2	I-Gene_or_gene_product
.	O

Knowledge	O
about	O
the	O
origin	O
and	O
identity	O
of	O
the	O
microbial	O
products	O
recognized	O
by	O
the	O
innate	O
immune	O
system	O
is	O
important	O
for	O
understanding	O
the	O
pathogenesis	O
of	O
inflammatory	O
diseases	O
.	O

We	O
investigated	O
the	O
potential	O
role	O
of	O
Salmonella	O
enterica	O
serotype	O
Typhimurium	O
fimbriae	B-Cellular_component
as	O
pathogen	O
-	O
associated	O
molecular	O
patterns	O
(	O
PAMPs	O
)	O
that	O
may	O
stimulate	O
innate	O
pathways	O
of	O
inflammation	O
.	O

We	O
screened	O
a	O
panel	O
of	O
11	O
mutants	O
,	O
each	O
carrying	O
a	O
deletion	O
of	O
a	O
different	O
fimbrial	O
operon	O
,	O
for	O
their	O
enteropathogenicity	O
using	O
the	O
calf	O
model	O
of	O
human	O
gastroenteritis	O
.	O

One	O
mutant	O
(	O
csgBA	O
)	O
was	O
attenuated	O
in	O
its	O
ability	O
to	O
elicit	O
fluid	O
accumulation	O
and	O
GROalpha	B-Gene_or_gene_product
mRNA	O
expression	O
in	O
bovine	O
ligated	O
ileal	O
loops	O
.	O

The	O
mechanism	O
by	O
which	O
thin	B-Cellular_component
curled	I-Cellular_component
fimbriae	I-Cellular_component
encoded	O
by	O
the	O
csg	B-Gene_or_gene_product
genes	O
contribute	O
to	O
inflammation	O
was	O
further	O
investigated	O
using	O
tissue	O
culture	O
.	O

The	O
S	O
.	O

Typhimurium	O
csgBA	O
mutant	O
induced	O
significantly	O
less	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
8	I-Gene_or_gene_product
production	O
than	O
the	O
wild	O
type	O
in	O
human	O
macrophage	O
-	O
like	O
cells	O
.	O

Purified	O
thin	B-Cellular_component
curled	I-Cellular_component
fimbriae	I-Cellular_component
induced	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
8	I-Gene_or_gene_product
expression	O
in	O
human	O
embryonic	O
kidney	O
(	O
HEK293	O
)	O
cells	O
transfected	O
with	O
Toll	B-Complex
-	I-Complex
like	I-Complex
receptor	I-Complex
(	I-Complex
TLR	I-Complex
)	I-Complex
2	I-Complex
/	I-Complex
CD14	I-Complex
but	O
not	O
in	O
cells	O
transfected	O
with	O
TLR5	B-Gene_or_gene_product
,	O
TLR4	B-Complex
/	I-Complex
MD2	I-Complex
/	I-Complex
CD14	I-Complex
or	O
TLR11	B-Gene_or_gene_product
.	O

Fusion	O
proteins	O
between	O
the	O
major	O
fimbrial	O
subunit	O
of	O
thin	B-Cellular_component
curled	I-Cellular_component
fimbriae	I-Cellular_component
(	O
CsgA	B-Gene_or_gene_product
)	O
and	O
glutathione	B-Gene_or_gene_product
-	I-Gene_or_gene_product
S	I-Gene_or_gene_product
-	I-Gene_or_gene_product
transferase	I-Gene_or_gene_product
(	O
GST	B-Gene_or_gene_product
)	O
elicited	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
8	I-Gene_or_gene_product
production	O
in	O
HEK293	O
cells	O
transfected	O
with	O
TLR2	B-Complex
/	I-Complex
CD14	I-Complex
.	O

Proteinase	B-Gene_or_gene_product
K	I-Gene_or_gene_product
treatment	O
abrogated	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
8	I-Gene_or_gene_product
production	O
elicited	O
in	O
these	O
cells	O
by	O
GST	B-Gene_or_gene_product
-	I-Gene_or_gene_product
CsgA	I-Gene_or_gene_product
,	O
but	O
not	O
by	O
synthetic	O
lipoprotein	O
.	O

GST	B-Gene_or_gene_product
-	I-Gene_or_gene_product
CsgA	I-Gene_or_gene_product
elicited	O
more	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
6	I-Gene_or_gene_product
production	O
than	O
GST	B-Gene_or_gene_product
in	O
bone	O
marrow	O
-	O
derived	O
macrophages	O
from	O
TLR2	B-Gene_or_gene_product
+	O
/	O
+	O
mice	O
,	O
while	O
there	O
was	O
no	O
difference	O
in	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
6	I-Gene_or_gene_product
secretion	O
between	O
GST	B-Gene_or_gene_product
-	I-Gene_or_gene_product
CsgA	I-Gene_or_gene_product
and	O
GST	B-Gene_or_gene_product
in	O
macrophages	O
from	O
TLR2	B-Gene_or_gene_product
-	O
/	O
-	O
mice	O
.	O

These	O
data	O
suggested	O
that	O
CsgA	B-Gene_or_gene_product
is	O
a	O
PAMP	O
that	O
is	O
recognized	O
by	O
TLR2	B-Gene_or_gene_product
.	O

Cdc28	B-Gene_or_gene_product
-	O
dependent	O
regulation	O
of	O
the	O
Cdc5	B-Gene_or_gene_product
/	O
Polo	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
.	O

Polo	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
is	O
activated	O
as	O
cells	O
enter	O
mitosis	O
and	O
plays	O
a	O
central	O
role	O
in	O
coordinating	O
diverse	O
mitotic	O
events	O
,	O
yet	O
the	O
mechanisms	O
leading	O
to	O
activation	O
of	O
Polo	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
are	O
poorly	O
understood	O
.	O

Work	O
in	O
Xenopus	O
meiotic	O
cell	O
cycles	O
has	O
suggested	O
that	O
Polo	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
functions	O
in	O
a	O
pathway	O
that	O
helps	O
trigger	O
activation	O
of	O
Cdk1	B-Gene_or_gene_product
.	O

However	O
,	O
studies	O
in	O
other	O
organisms	O
have	O
suggested	O
that	O
activation	O
of	O
Polo	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
is	O
dependent	O
upon	O
Cdk1	B-Gene_or_gene_product
and	O
therefore	O
occurs	O
downstream	O
of	O
Cdk1	B-Gene_or_gene_product
activation	O
.	O

In	O
this	O
study	O
,	O
we	O
have	O
investigated	O
the	O
role	O
of	O
Cdk1	B-Gene_or_gene_product
in	O
the	O
activation	O
of	O
budding	O
yeast	O
Polo	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
.	O

The	O
budding	O
yeast	O
homologs	O
of	O
Cdk1	B-Gene_or_gene_product
and	O
Polo	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
are	O
referred	O
to	O
as	O
Cdc28	B-Gene_or_gene_product
and	O
Cdc5	B-Gene_or_gene_product
.	O

We	O
show	O
that	O
signaling	O
from	O
Cdc28	B-Gene_or_gene_product
is	O
required	O
to	O
maintain	O
Cdc5	B-Gene_or_gene_product
activity	O
in	O
vivo	O
.	O

Furthermore	O
,	O
purified	O
Cdc28	B-Gene_or_gene_product
associated	O
with	O
the	O
mitotic	O
cyclin	B-Gene_or_gene_product
Clb2	I-Gene_or_gene_product
is	O
sufficient	O
to	O
activate	O
purified	O
Cdc5	B-Gene_or_gene_product
in	O
vitro	O
.	O

A	O
single	O
Cdc28	B-Gene_or_gene_product
consensus	O
phosphorylation	O
site	O
found	O
at	O
threonine	B-Simple_chemical
242	I-Simple_chemical
in	O
the	O
activation	O
loop	O
segment	O
of	O
Cdc5	B-Gene_or_gene_product
is	O
required	O
for	O
Cdc5	B-Gene_or_gene_product
function	O
in	O
vivo	O
and	O
for	O
kinase	O
activity	O
in	O
vitro	O
,	O
whereas	O
four	O
other	O
Cdc28	B-Gene_or_gene_product
consensus	O
sites	O
are	O
dispensable	O
.	O

Analysis	O
of	O
Cdc5	B-Gene_or_gene_product
phosphorylation	O
by	O
mass	O
spectrometry	O
indicates	O
that	O
threonine	B-Simple_chemical
242	I-Simple_chemical
is	O
phosphorylated	O
in	O
vivo	O
.	O

These	O
results	O
suggest	O
that	O
Cdc28	B-Gene_or_gene_product
activates	O
Cdc5	B-Gene_or_gene_product
via	O
phosphorylation	O
of	O
threonine	B-Simple_chemical
242	I-Simple_chemical
.	O

GC	O
-	O
selective	O
DNA	O
-	O
binding	O
antibiotic	O
,	O
mithramycin	B-Simple_chemical
A	I-Simple_chemical
,	O
reveals	O
multiple	O
points	O
of	O
control	O
in	O
the	O
regulation	O
of	O
Hdm2	B-Gene_or_gene_product
protein	O
synthesis	O
.	O

The	O
primary	O
role	O
of	O
the	O
Hdm2	B-Gene_or_gene_product
/	O
Mdm2	B-Gene_or_gene_product
oncoprotein	O
is	O
to	O
regulate	O
the	O
levels	O
and	O
activity	O
of	O
the	O
transcription	O
factor	O
p53	B-Gene_or_gene_product
.	O

Hdm2	B-Gene_or_gene_product
synthesis	O
is	O
itself	O
tightly	O
controlled	O
and	O
,	O
as	O
demonstrated	O
by	O
a	O
recently	O
described	O
SNP	O
(	O
SNP309	O
)	O
in	O
the	O
hdm2	B-Gene_or_gene_product
-	O
P2	O
promoter	O
,	O
minor	O
variations	O
in	O
Hdm2	B-Gene_or_gene_product
expression	O
have	O
phenotypic	O
consequences	O
on	O
radiation	O
sensitivity	O
and	O
cancer	O
predisposition	O
.	O

To	O
further	O
define	O
mechanisms	O
regulating	O
Hdm2	B-Gene_or_gene_product
expression	O
,	O
we	O
have	O
investigated	O
the	O
effects	O
of	O
the	O
GC	O
-	O
selective	O
DNA	O
-	O
binding	O
drug	O
,	O
Mithramycin	B-Simple_chemical
A	I-Simple_chemical
(	O
MA	B-Simple_chemical
)	O
on	O
hdm2	B-Gene_or_gene_product
mRNA	O
transcription	O
,	O
trafficking	O
,	O
and	O
translation	O
.	O

Firstly	O
we	O
show	O
that	O
the	O
constitutive	O
hdm2	B-Gene_or_gene_product
-	O
P1	O
promoter	O
is	O
inhibited	O
by	O
MA	B-Simple_chemical
.	O

We	O
define	O
,	O
for	O
the	O
first	O
time	O
,	O
the	O
minimal	O
sequence	O
elements	O
that	O
are	O
required	O
for	O
P1	O
-	O
promoter	O
activity	O
and	O
identify	O
those	O
which	O
confer	O
MA	B-Simple_chemical
sensitivity	O
.	O

Secondly	O
,	O
MA	B-Simple_chemical
induces	O
p53	B-Gene_or_gene_product
-	O
dependent	O
transcription	O
from	O
the	O
hdm2	B-Gene_or_gene_product
-	O
P2	O
promoter	O
.	O

Thirdly	O
,	O
and	O
critically	O
,	O
MA	B-Simple_chemical
also	O
inhibits	O
Hdm2	B-Gene_or_gene_product
synthesis	O
at	O
the	O
post	O
-	O
transcriptional	O
level	O
,	O
with	O
negative	O
effects	O
on	O
hdm2	B-Gene_or_gene_product
mRNA	O
nuclear	B-Cellular_component
export	O
and	O
translation	O
.	O

This	O
study	O
highlights	O
the	O
complex	O
interplay	O
between	O
the	O
pathways	O
that	O
regulate	O
Hdm2	B-Gene_or_gene_product
protein	O
synthesis	O
in	O
cancer	O
cells	O
,	O
and	O
furthermore	O
emphasizes	O
the	O
export	O
of	O
hdm2	B-Gene_or_gene_product
mRNA	O
from	O
the	O
nucleus	B-Cellular_component
to	O
the	O
cytoplasm	B-Cellular_component
as	O
a	O
key	O
point	O
of	O
control	O
in	O
this	O
process	O
.	O

ASK	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
(	O
apoptosis	B-Gene_or_gene_product
signal	I-Gene_or_gene_product
-	I-Gene_or_gene_product
regulating	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
1	I-Gene_or_gene_product
)	O
is	O
a	O
direct	O
E2F	B-Gene_or_gene_product
target	O
gene	O
.	O

In	O
the	O
present	O
study	O
,	O
we	O
show	O
that	O
E2Fs	B-Gene_or_gene_product
(	O
E2	B-Gene_or_gene_product
promoter	I-Gene_or_gene_product
-	I-Gene_or_gene_product
binding	I-Gene_or_gene_product
factors	I-Gene_or_gene_product
)	O
regulate	O
the	O
expression	O
of	O
ASK	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
(	O
apoptosis	B-Gene_or_gene_product
signal	I-Gene_or_gene_product
-	I-Gene_or_gene_product
regulating	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
1	I-Gene_or_gene_product
)	O
,	O
which	O
encodes	O
a	O
mitogen	B-Gene_or_gene_product
-	I-Gene_or_gene_product
activated	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
,	O
also	O
known	O
as	O
MAP3K5	B-Gene_or_gene_product
.	O

Its	O
mRNA	O
expression	O
is	O
cell	O
-	O
cycle	O
-	O
regulated	O
in	O
human	O
T98G	O
cells	O
released	O
from	O
serum	O
starvation	O
.	O

Moreover	O
,	O
overexpression	O
and	O
RNA	O
interference	O
experiments	O
support	O
the	O
requirement	O
of	O
endogenous	O
E2F	B-Complex
/	I-Complex
DP	I-Complex
(	O
E2F	B-Complex
dimerization	I-Complex
partner	I-Complex
)	O
activity	O
for	O
ASK	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
expression	O
.	O

Characterization	O
of	O
the	O
human	O
ASK	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
promoter	O
demonstrates	O
that	O
the	O
-	O
95	O
/	O
+	O
11	O
region	O
is	O
critical	O
for	O
E2F	B-Gene_or_gene_product
-	O
mediated	O
up	O
-	O
regulation	O
.	O

Chromatin	B-Cellular_component
immunoprecipitation	O
assays	O
show	O
that	O
E2F1	B-Gene_or_gene_product
-	O
E2F4	B-Gene_or_gene_product
are	O
bound	O
in	O
vivo	O
to	O
the	O
ASK	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
promoter	O
in	O
cycling	O
cells	O
,	O
probably	O
through	O
a	O
non	O
-	O
consensus	O
E2F	B-Gene_or_gene_product
-	O
binding	O
site	O
located	O
12	O
bp	O
upstream	O
of	O
the	O
transcription	O
start	O
site	O
.	O

Mutation	O
of	O
this	O
site	O
completely	O
abolishes	O
the	O
ASK	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
promoter	O
response	O
to	O
E2Fs	B-Gene_or_gene_product
as	O
well	O
as	O
the	O
E2F1	B-Gene_or_gene_product
binding	O
in	O
electrophoretic	O
mobility	O
-	O
shift	O
experiments	O
.	O

Our	O
results	O
indicate	O
that	O
E2Fs	B-Gene_or_gene_product
modulate	O
the	O
expression	O
of	O
ASK	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
and	O
suggest	O
that	O
some	O
of	O
the	O
cellular	O
functions	O
of	O
ASK	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
may	O
be	O
under	O
the	O
control	O
of	O
E2F	B-Gene_or_gene_product
transcription	O
factors	O
.	O

Moreover	O
,	O
the	O
up	O
-	O
regulation	O
of	O
ASK	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
may	O
also	O
favour	O
the	O
p53	B-Gene_or_gene_product
-	O
independent	O
E2F1	B-Gene_or_gene_product
apoptotic	O
activity	O
.	O

Glc7	B-Gene_or_gene_product
/	O
protein	B-Gene_or_gene_product
phosphatase	I-Gene_or_gene_product
1	I-Gene_or_gene_product
regulatory	I-Gene_or_gene_product
subunits	I-Gene_or_gene_product
can	O
oppose	O
the	O
Ipl1	B-Gene_or_gene_product
/	O
aurora	B-Gene_or_gene_product
protein	O
kinase	O
by	O
redistributing	O
Glc7	B-Gene_or_gene_product
.	O

Faithful	O
chromosome	B-Cellular_component
segregation	O
depends	O
on	O
the	O
opposing	O
activities	O
of	O
the	O
budding	O
yeast	O
Glc7	B-Gene_or_gene_product
/	O
PP1	B-Gene_or_gene_product
protein	O
phosphatase	O
and	O
Ipl1	B-Gene_or_gene_product
/	O
Aurora	B-Gene_or_gene_product
protein	O
kinase	O
.	O

We	O
explored	O
the	O
relationship	O
between	O
Glc7	B-Gene_or_gene_product
and	O
Ipl1	B-Gene_or_gene_product
and	O
found	O
that	O
the	O
phosphorylation	O
of	O
the	O
Ipl1	B-Gene_or_gene_product
substrate	O
,	O
Dam1	B-Gene_or_gene_product
,	O
was	O
altered	O
by	O
decreased	O
Glc7	B-Gene_or_gene_product
activity	O
,	O
whereas	O
Ipl1	B-Gene_or_gene_product
levels	O
,	O
localization	O
,	O
and	O
kinase	O
activity	O
were	O
not	O
.	O

These	O
data	O
strongly	O
suggest	O
that	O
Glc7	B-Gene_or_gene_product
ensures	O
accurate	O
chromosome	B-Cellular_component
segregation	O
by	O
dephosphorylating	O
Ipl1	B-Gene_or_gene_product
targets	O
rather	O
than	O
regulating	O
the	O
Ipl1	B-Gene_or_gene_product
kinase	O
.	O

To	O
identify	O
potential	O
Glc7	B-Gene_or_gene_product
and	O
Ipl1	B-Gene_or_gene_product
substrates	O
,	O
we	O
isolated	O
ipl1	B-Gene_or_gene_product
-	I-Gene_or_gene_product
321	I-Gene_or_gene_product
dosage	O
suppressors	O
.	O

Seven	O
genes	O
(	O
SDS22	B-Gene_or_gene_product
,	O
BUD14	B-Gene_or_gene_product
,	O
GIP3	B-Gene_or_gene_product
,	O
GIP4	B-Gene_or_gene_product
,	O
SOL1	B-Gene_or_gene_product
,	O
SOL2	B-Gene_or_gene_product
,	O
and	O
PEX31	B-Gene_or_gene_product
)	O
encode	O
newly	O
identified	O
ipl1	B-Gene_or_gene_product
dosage	O
suppressors	O
,	O
and	O
all	O
10	O
suppressors	O
encode	O
proteins	O
that	O
physically	O
interact	O
with	O
Glc7	B-Gene_or_gene_product
.	O

The	O
overexpression	O
of	O
the	O
Gip3	B-Gene_or_gene_product
and	O
Gip4	B-Gene_or_gene_product
suppressors	O
altered	O
Glc7	B-Gene_or_gene_product
localization	O
,	O
indicating	O
they	O
are	O
previously	O
unidentified	O
Glc7	B-Gene_or_gene_product
regulatory	O
subunits	O
.	O

In	O
addition	O
,	O
the	O
overexpression	O
of	O
Gip3	B-Gene_or_gene_product
and	O
Gip4	B-Gene_or_gene_product
from	O
the	O
galactose	B-Simple_chemical
promoter	O
restored	O
Dam1	B-Gene_or_gene_product
phosphorylation	O
in	O
ipl1	B-Gene_or_gene_product
-	I-Gene_or_gene_product
321	I-Gene_or_gene_product
mutant	O
cells	O
and	O
caused	O
wild	O
-	O
type	O
cells	O
to	O
arrest	O
in	O
metaphase	O
with	O
unsegregated	O
chromosomes	B-Cellular_component
,	O
suggesting	O
that	O
Gip3	B-Gene_or_gene_product
and	O
Gip4	B-Gene_or_gene_product
overexpression	O
impairs	O
Glc7	B-Gene_or_gene_product
'	O
s	O
mitotic	O
functions	O
.	O

We	O
therefore	O
propose	O
that	O
the	O
overexpression	O
of	O
Glc7	B-Gene_or_gene_product
regulatory	O
subunits	O
can	O
titrate	O
Glc7	B-Gene_or_gene_product
away	O
from	O
relevant	O
Ipl1	B-Gene_or_gene_product
targets	O
and	O
thereby	O
suppress	O
ipl1	B-Gene_or_gene_product
-	I-Gene_or_gene_product
321	I-Gene_or_gene_product
cells	O
by	O
restoring	O
the	O
balance	O
of	O
phosphatase	O
/	O
kinase	O
activity	O
.	O

Detection	O
of	O
catalase	B-Gene_or_gene_product
in	O
rat	O
heart	O
mitochondria	B-Cellular_component
.	O

The	O
presence	O
of	O
heme	B-Simple_chemical
-	O
containing	O
catalase	B-Gene_or_gene_product
in	O
rat	O
heart	O
mitochondria	B-Cellular_component
(	O
20	O
+	O
/	O
-	O
5	O
units	O
/	O
mg	O
)	O
was	O
demonstrated	O
by	O
biochemical	O
and	O
immunocytochemical	O
analysis	O
.	O

Intact	O
rat	O
heart	O
mitochondria	B-Cellular_component
efficiently	O
consumed	O
exogenously	O
added	O
H2O2	B-Simple_chemical
.	O

The	O
rate	O
of	O
H2O2	B-Simple_chemical
consumption	O
was	O
not	O
influenced	O
by	O
succinate	B-Simple_chemical
,	O
glutamate	B-Simple_chemical
/	O
malate	B-Simple_chemical
,	O
or	O
N	B-Simple_chemical
-	I-Simple_chemical
ethylmaleimide	I-Simple_chemical
but	O
was	O
significantly	O
inhibited	O
by	O
cyanide	B-Simple_chemical
.	O

Hydrogen	B-Simple_chemical
peroxide	I-Simple_chemical
decomposition	O
by	O
mitochondria	B-Cellular_component
yielded	O
molecular	O
oxygen	B-Simple_chemical
in	O
a	O
2	O
:	O
1	O
stoichiometry	O
,	O
consistent	O
with	O
a	O
catalytic	O
mechanism	O
.	O

Mitochondrial	B-Cellular_component
fractionation	O
studies	O
and	O
quantitative	O
electron	O
microscopic	O
immunocytochemistry	O
revealed	O
that	O
most	O
catalase	B-Gene_or_gene_product
was	O
matrix	B-Cellular_component
-	O
associated	O
.	O

Electrophoretic	O
analysis	O
and	O
Western	O
blotting	O
of	O
the	O
mitochondrial	B-Cellular_component
matrix	I-Cellular_component
fraction	O
indicated	O
the	O
presence	O
of	O
a	O
protein	O
with	O
similar	O
electrophoretic	O
mobility	O
to	O
bovine	O
and	O
rat	O
liver	O
catalase	B-Gene_or_gene_product
and	O
immunoreactive	O
to	O
anti	O
-	O
catalase	B-Gene_or_gene_product
antibody	O
.	O

Myocardial	O
tissue	O
has	O
a	O
lower	O
catalase	O
-	O
specific	O
activity	O
and	O
a	O
greater	O
mitochondrial	B-Cellular_component
H2O2	B-Simple_chemical
production	O
/	O
g	O
of	O
tissue	O
than	O
most	O
organs	O
.	O

Thus	O
catalase	B-Gene_or_gene_product
,	O
representing	O
0	O
.	O
025	O
%	O
of	O
heart	O
mitochondrial	B-Cellular_component
protein	O
,	O
is	O
important	O
for	O
detoxifying	O
mitochondrial	B-Cellular_component
derived	O
H2O2	B-Simple_chemical
and	O
represents	O
a	O
key	O
antioxidant	O
defense	O
mechanism	O
for	O
myocardial	O
tissue	O
.	O

Nuclear	B-Cellular_component
export	O
of	O
S6K1	B-Gene_or_gene_product
II	I-Gene_or_gene_product
is	O
regulated	O
by	O
protein	B-Complex
kinase	I-Complex
CK2	I-Complex
phosphorylation	O
at	O
Ser	B-Simple_chemical
-	I-Simple_chemical
17	I-Simple_chemical
.	O

Ribosomal	B-Cellular_component
S6	B-Gene_or_gene_product
kinases	I-Gene_or_gene_product
(	O
S6Ks	B-Gene_or_gene_product
)	O
are	O
principal	O
players	O
in	O
the	O
regulation	O
of	O
cell	O
growth	O
and	O
energy	O
metabolism	O
.	O

Signaling	O
via	O
phosphatidylinositol	B-Gene_or_gene_product
3	I-Gene_or_gene_product
-	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
and	O
mammalian	B-Gene_or_gene_product
target	I-Gene_or_gene_product
of	I-Gene_or_gene_product
rapamycin	I-Gene_or_gene_product
pathways	O
mediates	O
the	O
activation	O
of	O
S6K	B-Gene_or_gene_product
in	O
response	O
to	O
various	O
mitogenic	O
stimuli	O
.	O

The	O
family	O
of	O
S6Ks	B-Gene_or_gene_product
consists	O
of	O
two	O
forms	O
,	O
S6K1	B-Gene_or_gene_product
and	O
-	B-Gene_or_gene_product
2	I-Gene_or_gene_product
,	O
that	O
have	O
cytoplasmic	B-Cellular_component
and	O
nuclear	B-Cellular_component
splicing	O
variants	O
,	O
S6K1	B-Gene_or_gene_product
II	I-Gene_or_gene_product
and	O
S6K1	B-Gene_or_gene_product
I	I-Gene_or_gene_product
,	O
respectively	O
.	O

Nuclear	B-Cellular_component
-	O
cytoplasmic	B-Cellular_component
shuttling	O
of	O
both	O
isoforms	O
induced	O
by	O
mitogenic	O
stimuli	O
has	O
been	O
reported	O
recently	O
.	O

Here	O
we	O
present	O
the	O
identification	O
of	O
protein	B-Complex
kinase	I-Complex
CK2	I-Complex
(	O
CK2	B-Complex
)	O
as	O
a	O
novel	O
binding	O
and	O
regulatory	O
partner	O
for	O
S6K1	B-Gene_or_gene_product
II	I-Gene_or_gene_product
.	O

The	O
interaction	O
between	O
S6K1	B-Gene_or_gene_product
II	I-Gene_or_gene_product
and	O
CK2beta	B-Gene_or_gene_product
regulatory	O
subunit	O
was	O
initially	O
identified	O
in	O
a	O
yeast	O
two	O
-	O
hybrid	O
screen	O
and	O
further	O
confirmed	O
by	O
co	O
-	O
immunoprecipitation	O
of	O
transiently	O
expressed	O
and	O
endogenous	O
proteins	O
.	O

The	O
interaction	O
between	O
S6K1	B-Gene_or_gene_product
II	I-Gene_or_gene_product
and	O
CK2	B-Complex
was	O
found	O
to	O
occur	O
in	O
serum	O
-	O
starved	O
and	O
serum	O
-	O
stimulated	O
cells	O
.	O

In	O
addition	O
,	O
we	O
found	O
that	O
S6K1	B-Gene_or_gene_product
II	I-Gene_or_gene_product
is	O
a	O
substrate	O
for	O
CK2	B-Complex
.	O

The	O
localization	O
of	O
the	O
CK2	B-Complex
phosphorylation	O
site	O
was	O
narrowed	O
down	O
to	O
Ser	B-Simple_chemical
-	I-Simple_chemical
17	I-Simple_chemical
in	O
S6K1	B-Gene_or_gene_product
II	I-Gene_or_gene_product
.	O

Mutational	O
analysis	O
and	O
the	O
use	O
of	O
phosphospecific	O
antibody	O
indicate	O
that	O
Ser	B-Simple_chemical
-	I-Simple_chemical
17	I-Simple_chemical
is	O
a	O
major	O
in	O
vitro	O
and	O
in	O
vivo	O
phosphorylation	O
site	O
for	O
CK2	B-Complex
.	O

Functional	O
studies	O
reveal	O
that	O
,	O
in	O
contrast	O
to	O
the	O
wild	O
type	O
kinase	O
,	O
the	O
phosphorylation	O
-	O
mimicking	O
mutant	O
of	O
S6K1	B-Gene_or_gene_product
II	I-Gene_or_gene_product
(	O
S17E	B-Gene_or_gene_product
)	O
retains	O
its	O
cytoplasmic	B-Cellular_component
localization	O
in	O
serum	O
-	O
stimulated	O
cells	O
.	O

Treatment	O
of	O
cells	O
with	O
the	O
nuclear	B-Cellular_component
export	O
inhibitor	O
leptomycin	B-Simple_chemical
B	I-Simple_chemical
revealed	O
that	O
the	O
S17E	B-Gene_or_gene_product
mutant	O
accumulates	O
in	O
the	O
nucleus	B-Cellular_component
to	O
the	O
same	O
extent	O
as	O
S6K1	B-Gene_or_gene_product
II	I-Gene_or_gene_product
wild	O
type	O
.	O

These	O
results	O
indicate	O
that	O
nuclear	B-Cellular_component
import	O
of	O
the	O
S17E	B-Gene_or_gene_product
mutant	O
is	O
not	O
affected	O
,	O
although	O
the	O
export	O
is	O
significantly	O
enhanced	O
.	O

We	O
also	O
provide	O
evidence	O
that	O
nuclear	B-Cellular_component
export	O
of	O
S6K1	B-Gene_or_gene_product
is	O
mediated	O
by	O
a	O
CRM1	B-Gene_or_gene_product
-	O
dependent	O
mechanism	O
.	O

Taken	O
together	O
,	O
this	O
study	O
establishes	O
a	O
functional	O
link	O
between	O
S6K1	B-Gene_or_gene_product
II	I-Gene_or_gene_product
and	O
CK2	B-Complex
signaling	O
,	O
which	O
involves	O
the	O
regulation	O
of	O
S6K1	B-Gene_or_gene_product
II	I-Gene_or_gene_product
nuclear	B-Cellular_component
export	O
by	O
CK2	B-Complex
-	O
mediated	O
phosphorylation	O
of	O
Ser	B-Simple_chemical
-	I-Simple_chemical
17	I-Simple_chemical
.	O

Invariant	O
Leu	B-Simple_chemical
preceding	O
turn	O
motif	O
phosphorylation	O
site	O
controls	O
the	O
interaction	O
of	O
protein	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
C	I-Gene_or_gene_product
with	O
Hsp70	B-Gene_or_gene_product
.	O

Heat	O
shock	O
proteins	O
play	O
important	O
roles	O
in	O
regulating	O
signal	O
transduction	O
in	O
cells	O
by	O
associating	O
with	O
,	O
and	O
stabilizing	O
,	O
diverse	O
signaling	O
molecules	O
,	O
including	O
protein	O
kinases	O
.	O

Previously	O
,	O
we	O
have	O
shown	O
that	O
heat	O
shock	O
protein	O
Hsp70	B-Gene_or_gene_product
associates	O
with	O
protein	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
C	I-Gene_or_gene_product
(	O
PKC	B-Gene_or_gene_product
)	O
via	O
an	O
interaction	O
that	O
is	O
triggered	O
by	O
dephosphorylation	O
at	O
the	O
turn	O
phosphorylation	O
motif	O
.	O

Here	O
we	O
have	O
identified	O
an	O
invariant	O
residue	O
in	O
the	O
carboxyl	O
terminus	O
of	O
PKC	B-Gene_or_gene_product
that	O
mediates	O
the	O
binding	O
to	O
Hsp70	B-Gene_or_gene_product
.	O

Specifically	O
,	O
we	O
show	O
that	O
Hsp70	B-Gene_or_gene_product
binds	O
to	O
Leu	B-Simple_chemical
(	O
Leu	B-Simple_chemical
-	I-Simple_chemical
640	I-Simple_chemical
)	O
immediately	O
preceding	O
the	O
conserved	O
turn	O
motif	O
autophosphorylation	O
site	O
(	O
Thr	B-Simple_chemical
-	I-Simple_chemical
641	I-Simple_chemical
)	O
in	O
PKC	B-Gene_or_gene_product
betaII	I-Gene_or_gene_product
.	O

Co	O
-	O
immunoprecipitation	O
experiments	O
reveal	O
that	O
mutation	O
of	O
Leu	B-Simple_chemical
-	I-Simple_chemical
640	I-Simple_chemical
to	O
Gly	B-Simple_chemical
decreases	O
the	O
interaction	O
of	O
Hsp70	B-Gene_or_gene_product
with	O
PKC	B-Gene_or_gene_product
betaII	I-Gene_or_gene_product
.	O

This	O
weakened	O
interaction	O
between	O
Hsp70	B-Gene_or_gene_product
and	O
the	O
mutant	O
PKCs	B-Gene_or_gene_product
results	O
in	O
accumulation	O
of	O
dephosphorylated	O
PKC	B-Gene_or_gene_product
in	O
the	O
detergent	O
-	O
insoluble	O
fraction	O
of	O
cells	O
.	O

In	O
addition	O
,	O
the	O
Hsp70	B-Gene_or_gene_product
-	O
binding	O
mutant	O
is	O
considerably	O
more	O
sensitive	O
to	O
down	O
-	O
regulation	O
compared	O
with	O
WT	O
PKC	B-Gene_or_gene_product
:	O
disruption	O
of	O
Hsp70	B-Gene_or_gene_product
binding	O
leads	O
to	O
accelerated	O
dephosphorylation	O
and	O
enhanced	O
ubiquitination	O
of	O
mutant	O
PKC	B-Gene_or_gene_product
upon	O
phorbol	B-Simple_chemical
ester	I-Simple_chemical
treatment	O
.	O

Last	O
,	O
pulse	O
-	O
chase	O
experiments	O
demonstrate	O
that	O
Hsp70	B-Gene_or_gene_product
preferentially	O
binds	O
the	O
species	O
of	O
mature	O
PKC	B-Gene_or_gene_product
that	O
has	O
become	O
dephosphorylated	O
compared	O
with	O
the	O
newly	O
synthesized	O
protein	O
that	O
has	O
yet	O
to	O
be	O
phosphorylated	O
.	O

Thus	O
,	O
Hsp70	B-Gene_or_gene_product
binds	O
a	O
hydrophobic	O
residue	O
preceding	O
the	O
turn	O
motif	O
,	O
protecting	O
PKC	B-Gene_or_gene_product
from	O
down	O
-	O
regulation	O
and	O
sustaining	O
the	O
signaling	O
lifetime	O
of	O
the	O
kinase	O
.	O

Acm1	B-Gene_or_gene_product
is	O
a	O
negative	O
regulator	O
of	O
the	O
CDH1	B-Gene_or_gene_product
-	O
dependent	O
anaphase	B-Complex
-	I-Complex
promoting	I-Complex
complex	I-Complex
/	I-Complex
cyclosome	I-Complex
in	O
budding	O
yeast	O
.	O

Cdh1	B-Gene_or_gene_product
is	O
a	O
coactivator	O
of	O
the	O
anaphase	B-Complex
-	I-Complex
promoting	I-Complex
complex	I-Complex
/	I-Complex
cyclosome	I-Complex
(	O
APC	B-Complex
/	I-Complex
C	I-Complex
)	O
and	O
contributes	O
to	O
mitotic	O
exit	O
and	O
G1	O
maintenance	O
by	O
facilitating	O
the	O
polyubiquitination	O
and	O
subsequent	O
proteolysis	O
of	O
specific	O
substrates	O
.	O

Here	O
,	O
we	O
report	O
that	O
budding	O
yeast	O
Cdh1	B-Gene_or_gene_product
is	O
a	O
component	O
of	O
a	O
cell	O
cycle	O
-	O
regulated	O
complex	O
that	O
includes	O
the	O
14	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3	I-Gene_or_gene_product
-	I-Gene_or_gene_product
3	I-Gene_or_gene_product
homologs	O
Bmh1	B-Gene_or_gene_product
and	O
Bmh2	B-Gene_or_gene_product
and	O
a	O
previously	O
uncharacterized	O
protein	O
,	O
which	O
we	O
name	O
Acm1	B-Gene_or_gene_product
(	O
APC	B-Gene_or_gene_product
/	I-Gene_or_gene_product
CCdh1	I-Gene_or_gene_product
modulator	I-Gene_or_gene_product
1	I-Gene_or_gene_product
)	O
.	O

Association	O
of	O
Cdh1	B-Gene_or_gene_product
with	O
Bmh1	B-Gene_or_gene_product
and	O
Bmh2	B-Gene_or_gene_product
requires	O
Acm1	B-Gene_or_gene_product
,	O
and	O
the	O
Acm1	B-Gene_or_gene_product
protein	O
is	O
cell	O
cycle	O
regulated	O
,	O
appearing	O
late	O
in	O
G1	O
and	O
disappearing	O
in	O
late	O
M	O
.	O

In	O
acm1Delta	B-Gene_or_gene_product
strains	O
,	O
Cdh1	B-Gene_or_gene_product
localization	O
to	O
the	O
bud	B-Cellular_component
neck	I-Cellular_component
and	O
association	O
with	O
two	O
substrates	O
,	O
Clb2	B-Gene_or_gene_product
and	O
Hsl1	B-Gene_or_gene_product
,	O
were	O
strongly	O
enhanced	O
.	O

Several	O
lines	O
of	O
evidence	O
suggest	O
that	O
Acm1	B-Gene_or_gene_product
can	O
suppress	O
APC	B-Complex
/	I-Complex
CCdh1	I-Complex
-	O
mediated	O
proteolysis	O
of	O
mitotic	O
cyclins	B-Gene_or_gene_product
.	O

First	O
,	O
overexpression	O
of	O
Acm1	B-Gene_or_gene_product
fully	O
restored	O
viability	O
to	O
cells	O
expressing	O
toxic	O
levels	O
of	O
Cdh1	B-Gene_or_gene_product
or	O
a	O
constitutively	O
active	O
Cdh1	B-Gene_or_gene_product
mutant	O
lacking	O
inhibitory	O
phosphorylation	O
sites	O
.	O

Second	O
,	O
overexpression	O
of	O
Acm1	B-Gene_or_gene_product
was	O
toxic	O
in	O
sic1Delta	B-Gene_or_gene_product
cells	O
.	O

Third	O
,	O
ACM1	B-Gene_or_gene_product
deletion	O
exacerbated	O
a	O
low	O
-	O
penetrance	O
elongated	O
-	O
bud	B-Cellular_component
phenotype	O
caused	O
by	O
modest	O
overexpression	O
of	O
Cdh1	B-Gene_or_gene_product
.	O

This	O
bud	B-Cellular_component
elongation	O
was	O
independent	O
of	O
the	O
morphogenesis	O
checkpoint	O
,	O
and	O
the	O
combination	O
of	O
acm1Delta	B-Gene_or_gene_product
and	O
hsl1Delta	B-Gene_or_gene_product
resulted	O
in	O
a	O
dramatic	O
enhancement	O
of	O
bud	B-Cellular_component
elongation	O
and	O
G2	O
/	O
M	O
delay	O
.	O

Effects	O
on	O
bud	B-Cellular_component
elongation	O
were	O
attenuated	O
when	O
Cdh1	B-Gene_or_gene_product
was	O
replaced	O
with	O
a	O
mutant	O
lacking	O
the	O
C	O
-	O
terminal	O
IR	O
dipeptide	O
,	O
suggesting	O
that	O
APC	B-Complex
/	I-Complex
C	I-Complex
-	O
dependent	O
proteolysis	O
is	O
required	O
for	O
this	O
phenotype	O
.	O

We	O
propose	O
that	O
Acm1	B-Gene_or_gene_product
and	O
Bmh1	B-Gene_or_gene_product
/	O
Bmh2	B-Gene_or_gene_product
constitute	O
a	O
specialized	O
inhibitor	O
of	O
APC	B-Complex
/	I-Complex
CCdh1	I-Complex
.	O

Teratogen	O
-	O
induced	O
activation	O
of	O
p53	B-Gene_or_gene_product
in	O
early	O
postimplantation	O
mouse	O
embryos	O
.	O

Hyperthermia	O
(	O
HS	O
)	O
and	O
4	B-Simple_chemical
-	I-Simple_chemical
hydroperoxycyclophosphamide	I-Simple_chemical
(	O
4CP	B-Simple_chemical
)	O
activate	O
the	O
mitochondrial	B-Cellular_component
apoptotic	O
pathway	O
in	O
day	O
9	O
mouse	O
embryos	O
.	O

Previous	O
microarray	O
analyses	O
Microarray	O
analyses	O
revealed	O
that	O
several	O
p53	B-Gene_or_gene_product
target	O
genes	O
are	O
upregulated	O
after	O
exposure	O
to	O
HS	O
or	O
4CP	B-Simple_chemical
,	O
suggesting	O
a	O
role	O
for	O
p53	B-Gene_or_gene_product
in	O
teratogen	O
-	O
induced	O
apoptosis	O
.	O

To	O
explore	O
the	O
role	O
of	O
p53	B-Gene_or_gene_product
,	O
we	O
assessed	O
the	O
activation	O
of	O
p53	B-Gene_or_gene_product
in	O
day	O
9	O
mouse	O
embryos	O
exposed	O
to	O
HS	O
or	O
4CP	B-Simple_chemical
in	O
vitro	O
.	O

Both	O
teratogens	O
induced	O
the	O
accumulation	O
of	O
p53	B-Gene_or_gene_product
and	O
phosphorylation	O
of	O
p53	B-Gene_or_gene_product
at	O
ser	B-Simple_chemical
-	I-Simple_chemical
15	I-Simple_chemical
,	O
two	O
hallmarks	O
of	O
p53	B-Gene_or_gene_product
activation	O
.	O

HS	O
and	O
4CP	B-Simple_chemical
also	O
induced	O
an	O
increase	O
in	O
Noxa	B-Gene_or_gene_product
and	O
Puma	B-Gene_or_gene_product
mRNAs	O
,	O
transcripts	O
of	O
two	O
known	O
proapoptotic	O
p53	B-Gene_or_gene_product
target	O
genes	O
;	O
however	O
,	O
these	O
two	O
teratogens	O
did	O
not	O
induce	O
significant	O
increases	O
in	O
NOXA	B-Gene_or_gene_product
and	O
PUMA	B-Gene_or_gene_product
proteins	O
,	O
suggesting	O
that	O
p53	B-Gene_or_gene_product
does	O
not	O
activate	O
the	O
mitochondrial	B-Cellular_component
apoptotic	O
pathway	O
by	O
transcriptionally	O
upregulating	O
the	O
expression	O
of	O
NOXA	B-Gene_or_gene_product
and	O
PUMA	B-Gene_or_gene_product
proteins	O
.	O

HS	O
and	O
4CP	B-Simple_chemical
also	O
induced	O
the	O
expression	O
of	O
p21	B-Gene_or_gene_product
mRNA	O
and	O
protein	O
,	O
suggesting	O
a	O
role	O
for	O
p53	B-Gene_or_gene_product
in	O
teratogen	O
-	O
induced	O
cell	O
cycle	O
arrest	O
.	O

Previously	O
,	O
we	O
also	O
showed	O
that	O
HS	O
and	O
4CP	B-Simple_chemical
activate	O
the	O
apoptotic	O
pathway	O
in	O
the	O
embryo	O
proper	O
(	O
head	O
and	O
trunk	O
)	O
but	O
not	O
in	O
the	O
heart	O
.	O

We	O
now	O
show	O
that	O
HS	O
and	O
4CP	B-Simple_chemical
induce	O
a	O
robust	O
activation	O
of	O
p53	B-Gene_or_gene_product
in	O
the	O
embryo	O
proper	O
but	O
an	O
attenuated	O
induction	O
in	O
the	O
heart	O
.	O

HS	O
and	O
4CP	B-Simple_chemical
induce	O
the	O
expression	O
of	O
p21	B-Gene_or_gene_product
protein	O
in	O
majority	O
of	O
the	O
cells	O
in	O
the	O
embryo	O
;	O
however	O
,	O
expression	O
of	O
NOXA	B-Gene_or_gene_product
and	O
PUMA	B-Gene_or_gene_product
proteins	O
were	O
not	O
significantly	O
induced	O
in	O
heads	O
,	O
hearts	O
,	O
or	O
trunks	O
of	O
day	O
9	O
embryos	O
.	O

Overall	O
,	O
our	O
results	O
suggest	O
that	O
p53	B-Gene_or_gene_product
may	O
play	O
a	O
transcription	O
-	O
dependent	O
role	O
in	O
teratogen	O
-	O
induced	O
cell	O
cycle	O
arrest	O
but	O
a	O
transcription	O
-	O
independent	O
role	O
in	O
teratogen	O
-	O
induced	O
apoptosis	O
in	O
day	O
9	O
mouse	O
embryos	O
exposed	O
to	O
HS	O
or	O
4CP	B-Simple_chemical
.	O

Identification	O
of	O
novel	O
phosphorylation	O
sites	O
in	O
MSK1	B-Gene_or_gene_product
by	O
precursor	O
ion	O
scanning	O
MS	O
.	O

MSK1	B-Gene_or_gene_product
(	O
mitogen	B-Gene_or_gene_product
-	I-Gene_or_gene_product
and	I-Gene_or_gene_product
stress	I-Gene_or_gene_product
-	I-Gene_or_gene_product
activated	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
1	I-Gene_or_gene_product
)	O
is	O
a	O
dual	O
kinase	O
domain	O
protein	O
that	O
acts	O
downstream	O
of	O
the	O
ERK1	B-Gene_or_gene_product
/	I-Gene_or_gene_product
2	I-Gene_or_gene_product
(	O
extracellular	B-Gene_or_gene_product
-	I-Gene_or_gene_product
signal	I-Gene_or_gene_product
-	I-Gene_or_gene_product
regulated	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
1	I-Gene_or_gene_product
/	I-Gene_or_gene_product
2	I-Gene_or_gene_product
)	O
and	O
p38	B-Gene_or_gene_product
MAPK	I-Gene_or_gene_product
(	O
mitogen	B-Gene_or_gene_product
-	I-Gene_or_gene_product
activated	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
)	O
signalling	O
pathways	O
in	O
cells	O
.	O

MSK1	B-Gene_or_gene_product
,	O
and	O
its	O
related	O
isoform	O
MSK2	B-Gene_or_gene_product
,	O
phosphorylate	O
the	O
transcription	O
factors	O
CREB	B-Gene_or_gene_product
(	O
cAMP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
response	I-Gene_or_gene_product
-	I-Gene_or_gene_product
element	I-Gene_or_gene_product
-	I-Gene_or_gene_product
binding	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
)	O
and	O
ATF1	B-Gene_or_gene_product
(	O
activating	B-Gene_or_gene_product
transcription	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
1	I-Gene_or_gene_product
)	O
,	O
and	O
the	O
chromatin	B-Cellular_component
proteins	O
histone	B-Gene_or_gene_product
H3	I-Gene_or_gene_product
and	O
HMGN1	B-Gene_or_gene_product
(	O
high	B-Gene_or_gene_product
-	I-Gene_or_gene_product
mobility	I-Gene_or_gene_product
-	I-Gene_or_gene_product
group	I-Gene_or_gene_product
nucleosomal	I-Gene_or_gene_product
-	I-Gene_or_gene_product
binding	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
1	I-Gene_or_gene_product
)	O
in	O
response	O
to	O
either	O
mitogenic	O
stimulation	O
or	O
cellular	O
stress	O
.	O

MSK1	B-Gene_or_gene_product
activity	O
is	O
tightly	O
regulated	O
in	O
cells	O
,	O
and	O
activation	O
requires	O
the	O
phosphorylation	O
of	O
MSK1	B-Gene_or_gene_product
by	O
either	O
ERK1	B-Gene_or_gene_product
/	I-Gene_or_gene_product
2	I-Gene_or_gene_product
or	O
p38a	B-Gene_or_gene_product
.	O

This	O
results	O
in	O
activation	O
of	O
the	O
C	O
-	O
terminal	O
kinase	O
domain	O
,	O
which	O
then	O
phosphorylates	O
further	O
sites	O
in	O
MSK1	B-Gene_or_gene_product
,	O
leading	O
to	O
the	O
activation	O
of	O
the	O
N	O
-	O
terminal	O
kinase	O
domain	O
and	O
phosphorylation	O
of	O
substrates	O
.	O

Here	O
,	O
we	O
use	O
precursor	O
ion	O
scanning	O
MS	O
to	O
identify	O
five	O
previously	O
unknown	O
sites	O
in	O
MSK1	B-Gene_or_gene_product
:	O
Thr630	B-Simple_chemical
,	O
Ser647	B-Simple_chemical
,	O
Ser657	B-Simple_chemical
,	O
Ser695	B-Simple_chemical
and	O
Thr700	B-Simple_chemical
.	O

One	O
of	O
these	O
sites	O
,	O
Thr700	B-Simple_chemical
,	O
was	O
found	O
to	O
be	O
a	O
third	O
site	O
in	O
MSK1	B-Gene_or_gene_product
phosphorylated	O
by	O
the	O
upstream	O
kinases	O
ERK1	B-Gene_or_gene_product
/	I-Gene_or_gene_product
2	I-Gene_or_gene_product
and	O
p38a	B-Gene_or_gene_product
.	O

Mutation	O
of	O
Thr700	B-Simple_chemical
resulted	O
in	O
an	O
increased	O
basal	O
activity	O
of	O
MSK1	B-Gene_or_gene_product
,	O
but	O
this	O
could	O
be	O
further	O
increased	O
by	O
stimulation	O
with	O
PMA	O
or	O
UV	O
-	O
C	O
radiation	O
.	O

Surprisingly	O
,	O
however	O
,	O
mutation	O
of	O
Thr700	B-Simple_chemical
resulted	O
in	O
a	O
dramatic	O
loss	O
of	O
Thr581	B-Simple_chemical
phosphorylation	O
,	O
a	O
site	O
essential	O
for	O
activity	O
.	O

Mutation	O
of	O
Thr700	B-Simple_chemical
and	O
Thr581	B-Simple_chemical
to	O
an	O
alanine	B-Simple_chemical
residue	O
resulted	O
in	O
an	O
inactive	O
kinase	O
,	O
while	O
mutation	O
of	O
both	O
sites	O
to	O
an	O
aspartic	B-Simple_chemical
acid	I-Simple_chemical
residue	O
resulted	O
in	O
a	O
kinase	O
with	O
a	O
significant	O
basal	O
activity	O
that	O
could	O
not	O
be	O
further	O
stimulated	O
.	O

Together	O
these	O
results	O
are	O
consistent	O
with	O
a	O
mechanism	O
by	O
which	O
Thr700	B-Simple_chemical
phosphorylation	O
relieves	O
the	O
inhibition	O
of	O
MSK1	B-Gene_or_gene_product
by	O
a	O
C	O
-	O
terminal	O
autoinhibitory	O
helix	O
and	O
helps	O
induce	O
a	O
conformational	O
shift	O
that	O
protects	O
Thr581	B-Simple_chemical
from	O
dephosphorylation	O
.	O

The	O
Rtt109	B-Complex
-	I-Complex
Vps75	I-Complex
histone	B-Gene_or_gene_product
acetyltransferase	I-Gene_or_gene_product
complex	O
acetylates	O
non	O
-	O
nucleosomal	O
histone	B-Gene_or_gene_product
H3	I-Gene_or_gene_product
.	O

Acetylation	O
of	O
lysine	B-Simple_chemical
56	I-Simple_chemical
of	O
histone	B-Gene_or_gene_product
H3	I-Gene_or_gene_product
(	O
H3	B-Gene_or_gene_product
-	O
Lys	B-Simple_chemical
-	I-Simple_chemical
56	I-Simple_chemical
)	O
occurs	O
in	O
S	O
phase	O
and	O
disappears	O
during	O
G	O
(	O
2	O
)	O
/	O
M	O
phase	O
of	O
the	O
cell	O
cycle	O
.	O

However	O
,	O
it	O
is	O
not	O
clear	O
how	O
this	O
modification	O
is	O
regulated	O
during	O
the	O
progression	O
of	O
the	O
cell	O
cycle	O
.	O

We	O
and	O
others	O
have	O
shown	O
that	O
the	O
histone	B-Gene_or_gene_product
acetyltransferase	I-Gene_or_gene_product
(	O
HAT	B-Gene_or_gene_product
)	O
Rtt109	B-Gene_or_gene_product
is	O
the	O
primary	O
HAT	B-Gene_or_gene_product
responsible	O
for	O
acetylating	O
H3	B-Gene_or_gene_product
-	O
Lys	B-Simple_chemical
-	I-Simple_chemical
56	I-Simple_chemical
in	O
budding	O
yeast	O
.	O

Here	O
we	O
show	O
that	O
Rtt109	B-Gene_or_gene_product
forms	O
a	O
complex	O
with	O
Vps75	B-Gene_or_gene_product
and	O
that	O
both	O
recombinant	O
Rtt109	B-Complex
-	I-Complex
Vps75	I-Complex
complexes	O
and	O
native	O
complexes	O
purified	O
from	O
yeast	O
cells	O
acetylate	O
H3	B-Gene_or_gene_product
present	O
in	O
H3	B-Gene_or_gene_product
/	O
H4	B-Gene_or_gene_product
/	O
H2A	B-Gene_or_gene_product
/	O
H2B	B-Gene_or_gene_product
core	O
histones	B-Gene_or_gene_product
but	O
not	O
other	O
histones	B-Gene_or_gene_product
.	O

In	O
addition	O
,	O
both	O
recombinant	O
and	O
native	O
Rtt109	B-Complex
-	I-Complex
Vps75	I-Complex
HAT	B-Gene_or_gene_product
complexes	O
exhibited	O
no	O
detectable	O
activity	O
toward	O
nucleosomal	B-Cellular_component
H3	B-Gene_or_gene_product
,	O
suggesting	O
that	O
H3	B-Gene_or_gene_product
-	O
Lys	B-Simple_chemical
-	I-Simple_chemical
56	I-Simple_chemical
acetylation	O
is	O
at	O
least	O
in	O
part	O
regulated	O
by	O
the	O
inability	O
of	O
Rtt109	B-Complex
-	I-Complex
Vps75	I-Complex
complexes	O
to	O
acetylate	O
nucleosomal	B-Cellular_component
H3	B-Gene_or_gene_product
during	O
G	O
(	O
2	O
)	O
/	O
M	O
phase	O
of	O
the	O
cell	O
cycle	O
.	O

Further	O
,	O
Rtt109	B-Gene_or_gene_product
bound	O
mutant	O
H3	B-Complex
/	I-Complex
H4	I-Complex
tetramers	O
composed	O
of	O
histones	B-Gene_or_gene_product
lacking	O
their	O
N	O
-	O
terminal	O
tail	O
domains	O
less	O
efficiently	O
than	O
wild	O
-	O
type	O
H3	B-Complex
/	I-Complex
H4	I-Complex
tetramers	O
,	O
and	O
Rtt109	B-Complex
-	I-Complex
Vps75	I-Complex
complexes	O
displayed	O
reduced	O
HAT	B-Gene_or_gene_product
activity	O
toward	O
these	O
mutant	O
H3	B-Complex
/	I-Complex
H4	I-Complex
tetramers	O
.	O

Thus	O
,	O
the	O
N	O
termini	O
of	O
H3	B-Complex
/	I-Complex
H4	I-Complex
tetramers	O
are	O
required	O
for	O
efficient	O
acetylation	O
of	O
H3	B-Gene_or_gene_product
by	O
the	O
Rtt109	B-Complex
-	I-Complex
Vps75	I-Complex
complex	O
.	O

Taken	O
together	O
,	O
these	O
studies	O
provide	O
insights	O
into	O
how	O
H3	B-Gene_or_gene_product
-	O
Lys	B-Simple_chemical
-	I-Simple_chemical
56	I-Simple_chemical
acetylation	O
is	O
regulated	O
during	O
the	O
cell	O
cycle	O
.	O

The	O
Aurora	B-Gene_or_gene_product
kinase	O
Ipl1	B-Gene_or_gene_product
maintains	O
the	O
centromeric	B-Cellular_component
localization	O
of	O
PP2A	B-Complex
to	O
protect	O
cohesin	B-Complex
during	O
meiosis	O
.	O

Homologue	O
segregation	O
during	O
the	O
first	O
meiotic	O
division	O
requires	O
the	O
proper	O
spatial	O
regulation	O
of	O
sister	B-Cellular_component
chromatid	I-Cellular_component
cohesion	O
and	O
its	O
dissolution	O
along	O
chromosome	B-Cellular_component
arms	I-Cellular_component
,	O
but	O
its	O
protection	O
at	O
centromeric	B-Cellular_component
regions	I-Cellular_component
.	O

This	O
protection	O
requires	O
the	O
conserved	O
MEI	B-Gene_or_gene_product
-	I-Gene_or_gene_product
S332	I-Gene_or_gene_product
/	O
Sgo1	B-Gene_or_gene_product
proteins	O
that	O
localize	O
to	O
centromeric	B-Cellular_component
regions	I-Cellular_component
and	O
also	O
recruit	O
the	O
PP2A	B-Complex
phosphatase	O
by	O
binding	O
its	O
regulatory	O
subunit	O
,	O
Rts1	B-Gene_or_gene_product
.	O

Centromeric	B-Cellular_component
Rts1	B-Gene_or_gene_product
/	O
PP2A	B-Complex
then	O
locally	O
prevents	O
cohesion	O
dissolution	O
possibly	O
by	O
dephosphorylating	O
the	O
protein	O
complex	O
cohesin	B-Complex
.	O

We	O
show	O
that	O
Aurora	B-Gene_or_gene_product
B	I-Gene_or_gene_product
kinase	O
in	O
Saccharomyces	O
cerevisiae	O
(	O
Ipl1	B-Gene_or_gene_product
)	O
is	O
also	O
essential	O
for	O
the	O
protection	O
of	O
meiotic	O
centromeric	B-Cellular_component
cohesion	O
.	O

Coupled	O
with	O
a	O
previous	O
study	O
in	O
Drosophila	O
melanogaster	O
,	O
this	O
meiotic	O
function	O
of	O
Aurora	B-Gene_or_gene_product
B	I-Gene_or_gene_product
kinase	O
appears	O
to	O
be	O
conserved	O
among	O
eukaryotes	O
.	O

Furthermore	O
,	O
we	O
show	O
that	O
Sgo1	B-Gene_or_gene_product
recruits	O
Ipl1	B-Gene_or_gene_product
to	O
centromeric	B-Cellular_component
regions	I-Cellular_component
.	O

In	O
the	O
absence	O
of	O
Ipl1	B-Gene_or_gene_product
,	O
Rts1	B-Gene_or_gene_product
can	O
initially	O
bind	O
to	O
centromeric	B-Cellular_component
regions	I-Cellular_component
but	O
disappears	O
from	O
these	O
regions	B-Cellular_component
after	O
anaphase	O
I	O
onset	O
.	O

We	O
suggest	O
that	O
centromeric	B-Cellular_component
Ipl1	B-Gene_or_gene_product
ensures	O
the	O
continued	O
centromeric	B-Cellular_component
presence	O
of	O
active	O
Rts1	B-Gene_or_gene_product
/	O
PP2A	B-Complex
,	O
which	O
in	O
turn	O
locally	O
protects	O
cohesin	B-Complex
and	O
cohesion	O
.	O

PRAS40	B-Gene_or_gene_product
is	O
an	O
insulin	B-Gene_or_gene_product
-	O
regulated	O
inhibitor	O
of	O
the	O
mTORC1	B-Complex
protein	O
kinase	O
.	O

The	O
heterotrimeric	O
mTORC1	B-Complex
protein	O
kinase	O
nucleates	O
a	O
signaling	O
network	O
that	O
promotes	O
cell	O
growth	O
in	O
response	O
to	O
insulin	B-Gene_or_gene_product
and	O
becomes	O
constitutively	O
active	O
in	O
cells	O
missing	O
the	O
TSC1	B-Gene_or_gene_product
or	O
TSC2	B-Gene_or_gene_product
tumor	O
suppressors	O
.	O

Insulin	B-Gene_or_gene_product
stimulates	O
the	O
phosphorylation	O
of	O
S6K1	B-Gene_or_gene_product
,	O
an	O
mTORC1	B-Complex
substrate	O
,	O
but	O
it	O
is	O
not	O
known	O
how	O
mTORC1	B-Complex
kinase	O
activity	O
is	O
regulated	O
.	O

We	O
identify	O
PRAS40	B-Gene_or_gene_product
as	O
a	O
raptor	B-Gene_or_gene_product
-	O
interacting	O
protein	O
that	O
binds	O
to	O
mTORC1	B-Complex
in	O
insulin	B-Gene_or_gene_product
-	O
deprived	O
cells	O
and	O
whose	O
in	O
vitro	O
interaction	O
with	O
mTORC1	B-Complex
is	O
disrupted	O
by	O
high	O
salt	B-Simple_chemical
concentrations	O
.	O

PRAS40	B-Gene_or_gene_product
inhibits	O
cell	O
growth	O
,	O
S6K1	B-Gene_or_gene_product
phosphorylation	O
,	O
and	O
rheb	B-Gene_or_gene_product
-	O
induced	O
activation	O
of	O
the	O
mTORC1	B-Complex
pathway	O
,	O
and	O
in	O
vitro	O
it	O
prevents	O
the	O
great	O
increase	O
in	O
mTORC1	B-Complex
kinase	O
activity	O
induced	O
by	O
rheb1	B-Complex
-	I-Complex
GTP	I-Complex
.	O

Insulin	B-Gene_or_gene_product
stimulates	O
Akt	B-Gene_or_gene_product
/	O
PKB	B-Gene_or_gene_product
-	O
mediated	O
phosphorylation	O
of	O
PRAS40	B-Gene_or_gene_product
,	O
which	O
prevents	O
its	O
inhibition	O
of	O
mTORC1	B-Complex
in	O
cells	O
and	O
in	O
vitro	O
.	O

We	O
propose	O
that	O
the	O
relative	O
strengths	O
of	O
the	O
rheb	B-Gene_or_gene_product
-	O
and	O
PRAS40	B-Gene_or_gene_product
-	O
mediated	O
inputs	O
to	O
mTORC1	B-Complex
set	O
overall	O
pathway	O
activity	O
and	O
that	O
insulin	B-Gene_or_gene_product
activates	O
mTORC1	B-Complex
through	O
the	O
coordinated	O
regulation	O
of	O
both	O
.	O

Beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
arrestin	I-Gene_or_gene_product
is	O
a	O
necessary	O
component	O
of	O
Wnt	B-Gene_or_gene_product
/	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
signaling	O
in	O
vitro	O
and	O
in	O
vivo	O
.	O

The	O
Wnt	B-Gene_or_gene_product
/	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
signaling	O
pathway	O
is	O
crucial	O
for	O
proper	O
embryonic	O
development	O
and	O
tissue	O
homeostasis	O
.	O

The	O
phosphoprotein	O
dishevelled	B-Gene_or_gene_product
(	O
Dvl	B-Gene_or_gene_product
)	O
is	O
an	O
integral	O
part	O
of	O
Wnt	B-Gene_or_gene_product
signaling	O
and	O
has	O
recently	O
been	O
shown	O
to	O
interact	O
with	O
the	O
multifunctional	O
scaffolding	O
protein	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
arrestin	I-Gene_or_gene_product
.	O

Using	O
Dvl	B-Gene_or_gene_product
deletion	O
constructs	O
,	O
we	O
found	O
that	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
arrestin	I-Gene_or_gene_product
binds	O
a	O
region	O
N	O
-	O
terminal	O
of	O
the	O
PDZ	O
domain	O
of	O
Dvl	B-Gene_or_gene_product
,	O
which	O
contains	O
casein	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
1	I-Gene_or_gene_product
(	O
CK1	B-Gene_or_gene_product
)	O
phosphorylation	O
sites	O
.	O

Inhibition	O
of	O
Wnt	B-Gene_or_gene_product
signaling	O
by	O
CK1	B-Gene_or_gene_product
inhibitors	O
reduced	O
the	O
binding	O
of	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
arrestin	I-Gene_or_gene_product
to	O
Dvl	B-Gene_or_gene_product
.	O

Moreover	O
,	O
mouse	O
embryonic	O
fibroblasts	O
lacking	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
arrestins	I-Gene_or_gene_product
were	O
able	O
to	O
phosphorylate	O
LRP6	B-Gene_or_gene_product
in	O
response	O
to	O
Wnt	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3a	I-Gene_or_gene_product
but	O
decreased	O
the	O
activation	O
of	O
Dvl	B-Gene_or_gene_product
and	O
blocked	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
signaling	O
.	O

In	O
addition	O
,	O
we	O
found	O
that	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
arrestin	I-Gene_or_gene_product
can	O
bind	O
axin	B-Gene_or_gene_product
and	O
forms	O
a	O
trimeric	O
complex	O
with	O
axin	B-Gene_or_gene_product
and	O
Dvl	B-Gene_or_gene_product
.	O

Furthermore	O
,	O
treatment	O
of	O
Xenopus	O
laevis	O
embryos	O
with	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
arrestin	I-Gene_or_gene_product
morpholinos	O
reduced	O
the	O
activation	O
of	O
endogenous	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
,	O
decreased	O
the	O
expression	O
of	O
the	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
target	O
gene	O
,	O
Xnr3	B-Gene_or_gene_product
,	O
and	O
blocked	O
axis	O
duplication	O
induced	O
by	O
X	B-Gene_or_gene_product
-	I-Gene_or_gene_product
Wnt	I-Gene_or_gene_product
-	I-Gene_or_gene_product
8	I-Gene_or_gene_product
,	O
CK1epsilon	B-Gene_or_gene_product
,	O
or	O
DshDeltaDEP	B-Gene_or_gene_product
,	O
but	O
not	O
by	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
.	O

Thus	O
,	O
our	O
results	O
identify	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
arrestin	I-Gene_or_gene_product
as	O
a	O
necessary	O
component	O
for	O
Wnt	B-Gene_or_gene_product
/	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
signaling	O
,	O
linking	O
Dvl	B-Gene_or_gene_product
and	O
axin	B-Gene_or_gene_product
,	O
and	O
open	O
a	O
vast	O
array	O
of	O
signaling	O
avenues	O
and	O
possibilities	O
for	O
cross	O
-	O
talk	O
with	O
other	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
arrestin	I-Gene_or_gene_product
-	O
dependent	O
signaling	O
pathways	O
.	O

Mammalian	O
DET1	B-Gene_or_gene_product
regulates	O
Cul4A	B-Gene_or_gene_product
activity	O
and	O
forms	O
stable	O
complexes	O
with	O
E2	O
ubiquitin	B-Gene_or_gene_product
-	I-Gene_or_gene_product
conjugating	I-Gene_or_gene_product
enzymes	I-Gene_or_gene_product
.	O

DET1	B-Gene_or_gene_product
(	O
de	B-Gene_or_gene_product
-	I-Gene_or_gene_product
etiolated	I-Gene_or_gene_product
1	I-Gene_or_gene_product
)	O
is	O
an	O
essential	O
negative	O
regulator	O
of	O
plant	O
light	O
responses	O
,	O
and	O
it	O
is	O
a	O
component	O
of	O
the	O
Arabidopsis	O
thaliana	O
CDD	B-Complex
complex	O
containing	O
DDB1	B-Gene_or_gene_product
and	O
COP10	B-Gene_or_gene_product
ubiquitin	I-Gene_or_gene_product
E2	I-Gene_or_gene_product
variant	I-Gene_or_gene_product
.	O

Human	O
DET1	B-Gene_or_gene_product
has	O
recently	O
been	O
isolated	O
as	O
one	O
of	O
the	O
DDB1	B-Gene_or_gene_product
-	O
and	O
Cul4A	B-Gene_or_gene_product
-	O
associated	O
factors	O
,	O
along	O
with	O
an	O
array	O
of	O
WD40	O
-	O
containing	O
substrate	O
receptors	O
of	O
the	O
Cul4A	B-Complex
-	I-Complex
DDB1	I-Complex
ubiquitin	B-Gene_or_gene_product
ligase	O
.	O

However	O
,	O
DET1	B-Gene_or_gene_product
differs	O
from	O
conventional	O
substrate	O
receptors	O
of	O
cullin	B-Gene_or_gene_product
E3	O
ligases	O
in	O
both	O
biochemical	O
behavior	O
and	O
activity	O
.	O

Here	O
we	O
report	O
that	O
mammalian	O
DET1	B-Gene_or_gene_product
forms	O
stable	O
DDD	B-Complex
-	I-Complex
E2	I-Complex
complexes	O
,	O
consisting	O
of	O
DDB1	B-Gene_or_gene_product
,	O
DDA1	B-Gene_or_gene_product
(	O
DET1	B-Gene_or_gene_product
,	I-Gene_or_gene_product
DDB1	I-Gene_or_gene_product
associated	I-Gene_or_gene_product
1	I-Gene_or_gene_product
)	O
,	O
and	O
a	O
member	O
of	O
the	O
UBE2E	B-Gene_or_gene_product
group	O
of	O
canonical	O
ubiquitin	B-Gene_or_gene_product
-	I-Gene_or_gene_product
conjugating	I-Gene_or_gene_product
enzymes	I-Gene_or_gene_product
.	O

DDD	B-Complex
-	I-Complex
E2	I-Complex
complexes	O
interact	O
with	O
multiple	O
ubiquitin	B-Gene_or_gene_product
E3	I-Gene_or_gene_product
ligases	I-Gene_or_gene_product
.	O

We	O
show	O
that	O
the	O
E2	B-Gene_or_gene_product
component	I-Gene_or_gene_product
cannot	O
maintain	O
the	O
ubiquitin	B-Gene_or_gene_product
thioester	O
linkage	O
once	O
bound	O
to	O
the	O
DDD	B-Complex
core	O
,	O
rendering	O
mammalian	O
DDD	B-Complex
-	I-Complex
E2	I-Complex
equivalent	O
to	O
the	O
Arabidopsis	O
CDD	B-Complex
complex	O
.	O

While	O
free	O
UBE2E	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3	I-Gene_or_gene_product
is	O
active	O
and	O
able	O
to	O
enhance	O
UbcH5	B-Gene_or_gene_product
/	O
Cul4A	B-Gene_or_gene_product
activity	O
,	O
the	O
DDD	B-Complex
core	O
specifically	O
inhibits	O
Cul4A	B-Gene_or_gene_product
-	O
dependent	O
polyubiquitin	B-Gene_or_gene_product
chain	O
assembly	O
in	O
vitro	O
.	O

Overexpression	O
of	O
DET1	B-Gene_or_gene_product
inhibits	O
UV	O
-	O
induced	O
CDT1	B-Gene_or_gene_product
degradation	O
in	O
cultured	O
cells	O
.	O

These	O
findings	O
demonstrate	O
that	O
the	O
conserved	O
DET1	B-Complex
complex	I-Complex
modulates	O
Cul4A	B-Gene_or_gene_product
functions	O
by	O
a	O
novel	O
mechanism	O
.	O

Cdc34	B-Gene_or_gene_product
C	O
-	O
terminal	O
tail	O
phosphorylation	O
regulates	O
Skp1	B-Complex
/	I-Complex
cullin	I-Complex
/	I-Complex
F	I-Complex
-	I-Complex
box	I-Complex
(	O
SCF	B-Complex
)	O
-	O
mediated	O
ubiquitination	O
and	O
cell	O
cycle	O
progression	O
.	O

The	O
ubiquitin	B-Gene_or_gene_product
-	O
conjugating	O
enzyme	O
Cdc34	B-Gene_or_gene_product
(	O
cell	B-Gene_or_gene_product
division	I-Gene_or_gene_product
cycle	I-Gene_or_gene_product
34	I-Gene_or_gene_product
)	O
plays	O
an	O
essential	O
role	O
in	O
promoting	O
the	O
G1	O
-	O
S	O
-	O
phase	O
transition	O
of	O
the	O
eukaryotic	O
cell	O
cycle	O
and	O
is	O
phosphorylated	O
in	O
vivo	O
.	O

In	O
the	O
present	O
study	O
,	O
we	O
investigated	O
if	O
phosphorylation	O
regulates	O
Cdc34	B-Gene_or_gene_product
function	O
.	O

We	O
mapped	O
the	O
in	O
vivo	O
phosphorylation	O
sites	O
on	O
budding	O
yeast	O
Cdc34	B-Gene_or_gene_product
(	O
yCdc34	B-Gene_or_gene_product
;	O
Ser207	B-Simple_chemical
and	O
Ser216	B-Simple_chemical
)	O
and	O
human	O
Cdc34	B-Gene_or_gene_product
(	O
hCdc34	B-Gene_or_gene_product
Ser203	B-Simple_chemical
,	O
Ser222	B-Simple_chemical
and	O
Ser231	B-Simple_chemical
)	O
to	O
serine	B-Simple_chemical
residues	O
in	O
the	O
acidic	O
tail	O
domain	O
,	O
a	O
region	O
that	O
is	O
critical	O
for	O
Cdc34	B-Gene_or_gene_product
'	O
s	O
cell	O
cycle	O
function	O
.	O

CK2	B-Gene_or_gene_product
(	O
protein	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
CK2	I-Gene_or_gene_product
)	O
phosphorylates	O
both	O
yCdc34	B-Gene_or_gene_product
and	O
hCdc34	B-Gene_or_gene_product
on	O
these	O
sites	O
in	O
vitro	O
.	O

CK2	B-Gene_or_gene_product
-	O
mediated	O
phosphorylation	O
increased	O
yCdc34	B-Gene_or_gene_product
ubiquitination	O
activity	O
towards	O
the	O
yeast	O
Saccharomyces	O
cerevisiae	O
Sic1	B-Gene_or_gene_product
in	O
vitro	O
,	O
when	O
assayed	O
in	O
the	O
presence	O
of	O
its	O
cognate	O
SCFCdc4	B-Complex
E3	O
ligase	O
[	O
where	O
SCF	B-Complex
is	O
Skp1	B-Complex
(	I-Complex
S	I-Complex
-	I-Complex
phase	I-Complex
kinase	I-Complex
-	I-Complex
associated	I-Complex
protein	I-Complex
1	I-Complex
)	I-Complex
/	I-Complex
cullin	I-Complex
/	I-Complex
F	I-Complex
-	I-Complex
box	I-Complex
]	O
.	O

Similarly	O
,	O
mutation	O
of	O
the	O
yCdc34	B-Gene_or_gene_product
phosphorylation	O
sites	O
to	O
alanine	B-Simple_chemical
,	O
aspartate	B-Simple_chemical
or	O
glutamate	B-Simple_chemical
residues	O
altered	O
Cdc34	B-Gene_or_gene_product
-	O
SCFCdc4	B-Complex
-	O
mediated	O
Sic1	B-Gene_or_gene_product
ubiquitination	O
activity	O
.	O

Similar	O
results	O
were	O
obtained	O
when	O
yCdc34	B-Gene_or_gene_product
'	I-Gene_or_gene_product
s	I-Gene_or_gene_product
ubiquitination	O
activity	O
was	O
assayed	O
in	O
the	O
absence	O
of	O
SCFCdc4	B-Complex
,	O
indicating	O
that	O
phosphorylation	O
regulates	O
the	O
intrinsic	O
catalytic	O
activity	O
of	O
Cdc34	B-Gene_or_gene_product
.	O

To	O
evaluate	O
the	O
in	O
vivo	O
consequences	O
of	O
altered	O
Cdc34	B-Gene_or_gene_product
activity	O
,	O
wild	O
-	O
type	O
yCdc34	B-Gene_or_gene_product
and	O
the	O
phosphosite	O
mutants	O
were	O
introduced	O
into	O
an	O
S	O
.	O
cerevisiae	O
cdc34	B-Gene_or_gene_product
deletion	O
strain	O
and	O
,	O
following	O
synchronization	O
in	O
G1	O
-	O
phase	O
,	O
progression	O
through	O
the	O
cell	O
cycle	O
was	O
monitored	O
.	O

Consistent	O
with	O
the	O
increased	O
ubiquitination	O
activity	O
in	O
vitro	O
,	O
cells	O
expressing	O
the	O
phosphosite	O
mutants	O
with	O
higher	O
catalytic	O
activity	O
exhibited	O
accelerated	O
cell	O
cycle	O
progression	O
and	O
Sic1	B-Gene_or_gene_product
degradation	O
.	O

These	O
studies	O
demonstrate	O
that	O
CK2	B-Gene_or_gene_product
-	O
mediated	O
phosphorylation	O
of	O
Cdc34	B-Gene_or_gene_product
on	O
the	O
acidic	O
tail	O
domain	O
stimulates	O
Cdc34	B-Gene_or_gene_product
-	O
SCFCdc4	B-Complex
ubiquitination	O
activity	O
and	O
cell	O
cycle	O
progression	O
.	O

PRAS40	B-Gene_or_gene_product
regulates	O
mTORC1	B-Complex
kinase	O
activity	O
by	O
functioning	O
as	O
a	O
direct	O
inhibitor	O
of	O
substrate	O
binding	O
.	O

Mammalian	B-Gene_or_gene_product
target	I-Gene_or_gene_product
of	I-Gene_or_gene_product
rapamycin	I-Gene_or_gene_product
(	O
mTOR	B-Gene_or_gene_product
)	O
functions	O
in	O
two	O
distinct	O
signaling	O
complexes	O
,	O
mTORC1	B-Complex
and	O
mTORC2	B-Complex
.	O

In	O
response	O
to	O
insulin	B-Gene_or_gene_product
and	O
nutrients	O
,	O
mTORC1	B-Complex
,	O
consisting	O
of	O
mTOR	B-Gene_or_gene_product
,	O
raptor	B-Gene_or_gene_product
(	O
regulatory	B-Gene_or_gene_product
-	I-Gene_or_gene_product
associated	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
of	I-Gene_or_gene_product
mTOR	I-Gene_or_gene_product
)	O
,	O
and	O
mLST8	B-Gene_or_gene_product
,	O
is	O
activated	O
and	O
phosphorylates	O
eukaryotic	B-Gene_or_gene_product
initiation	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
4E	I-Gene_or_gene_product
-	I-Gene_or_gene_product
binding	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
(	O
4EBP	B-Gene_or_gene_product
)	O
and	O
p70	B-Gene_or_gene_product
S6	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
to	O
promote	O
protein	O
synthesis	O
and	O
cell	O
size	O
.	O

Previously	O
we	O
found	O
that	O
activation	O
of	O
mTOR	B-Gene_or_gene_product
kinase	O
in	O
response	O
to	O
insulin	B-Gene_or_gene_product
was	O
associated	O
with	O
increased	O
4EBP1	B-Gene_or_gene_product
binding	O
to	O
raptor	B-Gene_or_gene_product
.	O

Here	O
we	O
identify	O
prolinerich	B-Gene_or_gene_product
Akt	I-Gene_or_gene_product
substrate	I-Gene_or_gene_product
40	I-Gene_or_gene_product
(	O
PRAS40	B-Gene_or_gene_product
)	O
as	O
a	O
binding	O
partner	O
for	O
mTORC1	B-Complex
.	O

A	O
putative	O
TOR	B-Gene_or_gene_product
signaling	O
motif	O
,	O
FVMDE	O
,	O
is	O
identified	O
in	O
PRAS40	B-Gene_or_gene_product
and	O
shown	O
to	O
be	O
required	O
for	O
interaction	O
with	O
raptor	B-Gene_or_gene_product
.	O

Insulin	B-Gene_or_gene_product
stimulation	O
markedly	O
decreases	O
the	O
level	O
of	O
PRAS40	B-Gene_or_gene_product
bound	O
by	O
mTORC1	B-Complex
.	O

Recombinant	O
PRAS40	B-Gene_or_gene_product
inhibits	O
mTORC1	B-Complex
kinase	O
activity	O
in	O
vivo	O
and	O
in	O
vitro	O
,	O
and	O
this	O
inhibition	O
depends	O
on	O
PRAS40	B-Gene_or_gene_product
association	O
with	O
raptor	B-Gene_or_gene_product
.	O

Furthermore	O
,	O
decreasing	O
PRAS40	B-Gene_or_gene_product
expression	O
by	O
short	O
hairpin	O
RNA	O
enhances	O
4E	B-Gene_or_gene_product
-	I-Gene_or_gene_product
BP1	I-Gene_or_gene_product
binding	O
to	O
raptor	B-Gene_or_gene_product
,	O
and	O
recombinant	O
PRAS40	B-Gene_or_gene_product
competes	O
with	O
4E	B-Gene_or_gene_product
-	I-Gene_or_gene_product
BP1	I-Gene_or_gene_product
binding	O
to	O
raptor	B-Gene_or_gene_product
.	O

We	O
,	O
therefore	O
,	O
propose	O
that	O
PRAS40	B-Gene_or_gene_product
regulates	O
mTORC1	B-Complex
kinase	O
activity	O
by	O
functioning	O
as	O
a	O
direct	O
inhibitor	O
of	O
substrate	O
binding	O
.	O

Acute	O
alcohol	B-Simple_chemical
exposure	O
exerts	O
anti	O
-	O
inflammatory	O
effects	O
by	O
inhibiting	O
IkappaB	B-Complex
kinase	I-Complex
activity	O
and	O
p65	B-Gene_or_gene_product
phosphorylation	O
in	O
human	O
monocytes	O
.	O

Acute	O
alcohol	B-Simple_chemical
use	O
is	O
associated	O
with	O
impaired	O
immune	O
responses	O
and	O
decreased	O
proinflammatory	O
cytokine	O
production	O
.	O

Our	O
earlier	O
studies	O
have	O
shown	O
that	O
acute	O
alcohol	B-Simple_chemical
intake	O
inhibits	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
DNA	O
binding	O
in	O
an	O
IkappaBalpha	B-Gene_or_gene_product
-	O
independent	O
manner	O
.	O

We	O
report	O
using	O
human	O
peripheral	O
blood	O
monocytes	O
and	O
Chinese	O
hamster	O
ovary	O
cells	O
transfected	O
with	O
CD14	B-Gene_or_gene_product
cells	O
that	O
acute	O
alcohol	B-Simple_chemical
treatment	O
in	O
vitro	O
exerts	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
inhibition	O
by	O
disrupting	O
phosphorylation	O
of	O
p65	B-Gene_or_gene_product
.	O

Immunoprecipitation	O
of	O
p65	B-Gene_or_gene_product
and	O
IkappaBalpha	B-Gene_or_gene_product
revealed	O
that	O
acute	O
alcohol	B-Simple_chemical
exposure	O
for	O
1	O
h	O
decreased	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
-	I-Complex
IkappaBalpha	I-Complex
complexes	O
in	O
the	O
cytoplasm	B-Cellular_component
.	O

Phosphorylation	O
of	O
p65	B-Gene_or_gene_product
at	O
Ser	B-Simple_chemical
(	I-Simple_chemical
536	I-Simple_chemical
)	I-Simple_chemical
is	O
mediated	O
by	O
IkappaB	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
(	I-Gene_or_gene_product
IKK	I-Gene_or_gene_product
)	I-Gene_or_gene_product
beta	I-Gene_or_gene_product
and	O
is	O
required	O
for	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
-	O
dependent	O
cellular	O
responses	O
.	O

We	O
show	O
that	O
acute	O
alcohol	B-Simple_chemical
treatment	O
decreased	O
LPS	B-Simple_chemical
-	O
induced	O
IKKalpha	B-Gene_or_gene_product
and	O
IKKbeta	B-Gene_or_gene_product
activity	O
resulting	O
in	O
decreased	O
phosphorylation	O
of	O
p65	B-Gene_or_gene_product
at	O
Ser	B-Simple_chemical
(	I-Simple_chemical
536	I-Simple_chemical
)	I-Simple_chemical
.	O

Furthermore	O
,	O
nuclear	B-Cellular_component
expression	O
of	O
IKKalpha	B-Gene_or_gene_product
increased	O
after	O
alcohol	B-Simple_chemical
treatment	O
,	O
which	O
may	O
contribute	O
to	O
inhibition	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
.	O

Decreased	O
phosphorylation	O
of	O
nuclear	B-Cellular_component
p65	B-Gene_or_gene_product
at	O
Ser	B-Simple_chemical
(	I-Simple_chemical
276	I-Simple_chemical
)	I-Simple_chemical
was	O
likely	O
not	O
due	O
to	O
alcohol	B-Simple_chemical
-	O
induced	O
inhibition	O
of	O
protein	B-Complex
kinase	I-Complex
A	I-Complex
and	O
mitogen	B-Gene_or_gene_product
-	I-Gene_or_gene_product
and	I-Gene_or_gene_product
stress	I-Gene_or_gene_product
-	I-Gene_or_gene_product
activated	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
activity	O
.	O

Although	O
decreased	O
IkappaBalpha	B-Gene_or_gene_product
phosphorylation	O
after	O
acute	O
alcohol	B-Simple_chemical
treatment	O
was	O
attributable	O
to	O
reduced	O
IKKbeta	B-Gene_or_gene_product
activity	O
,	O
degradation	O
of	O
IkappaBalpha	B-Gene_or_gene_product
during	O
alcohol	B-Simple_chemical
exposure	O
was	O
IKKbeta	B-Gene_or_gene_product
-	O
independent	O
.	O

Alcohol	B-Simple_chemical
-	O
induced	O
degradation	O
of	O
IkappaBalpha	B-Gene_or_gene_product
in	O
the	O
presence	O
of	O
a	O
26S	B-Cellular_component
proteasome	I-Cellular_component
inhibitor	O
suggested	O
proteasome	B-Cellular_component
-	O
independent	O
IkappaBalpha	B-Gene_or_gene_product
degradation	O
.	O

Collectively	O
,	O
our	O
studies	O
suggest	O
that	O
acute	O
alcohol	B-Simple_chemical
exposure	O
modulates	O
IkappaBalpha	B-Gene_or_gene_product
-	O
independent	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
activity	O
primarily	O
by	O
affecting	O
phosphorylation	O
of	O
p65	B-Gene_or_gene_product
.	O

These	O
findings	O
further	O
implicate	O
an	O
important	O
role	O
for	O
IKKbeta	B-Gene_or_gene_product
in	O
the	O
acute	O
effects	O
of	O
alcohol	B-Simple_chemical
in	O
immune	O
cells	O
.	O

Differential	O
susceptibility	O
of	O
yeast	O
S	O
and	O
M	O
phase	O
CDK	B-Gene_or_gene_product
complexes	O
to	O
inhibitory	O
tyrosine	B-Simple_chemical
phosphorylation	O
.	O

BACKGROUND	O
:	O
Several	O
checkpoint	O
pathways	O
employ	O
Wee1	B-Gene_or_gene_product
-	O
mediated	O
inhibitory	O
tyrosine	B-Simple_chemical
phosphorylation	O
of	O
cyclin	B-Gene_or_gene_product
-	I-Gene_or_gene_product
dependent	I-Gene_or_gene_product
kinases	I-Gene_or_gene_product
(	O
CDKs	B-Gene_or_gene_product
)	O
to	O
restrain	O
cell	O
-	O
cycle	O
progression	O
.	O

Whereas	O
in	O
vertebrates	O
this	O
strategy	O
can	O
delay	O
both	O
DNA	O
replication	O
and	O
mitosis	O
,	O
in	O
yeast	O
cells	O
only	O
mitosis	O
is	O
delayed	O
.	O

This	O
is	O
particularly	O
surprising	O
because	O
yeasts	O
,	O
unlike	O
vertebrates	O
,	O
employ	O
a	O
single	O
family	O
of	O
cyclins	B-Gene_or_gene_product
(	I-Gene_or_gene_product
B	I-Gene_or_gene_product
type	I-Gene_or_gene_product
)	I-Gene_or_gene_product
and	O
the	O
same	O
CDK	B-Gene_or_gene_product
to	O
promote	O
both	O
S	O
phase	O
and	O
mitosis	O
.	O

The	O
G2	O
-	O
specific	O
arrest	O
could	O
be	O
explained	O
in	O
two	O
fundamentally	O
different	O
ways	O
:	O
tyrosine	B-Simple_chemical
phosphorylation	O
of	O
cyclin	B-Complex
/	I-Complex
CDK	I-Complex
complexes	O
could	O
leave	O
sufficient	O
residual	O
activity	O
to	O
promote	O
S	O
phase	O
,	O
or	O
S	O
phase	O
-	O
promoting	O
cyclin	B-Complex
/	I-Complex
CDK	I-Complex
complexes	O
could	O
somehow	O
be	O
protected	O
from	O
checkpoint	O
-	O
induced	O
tyrosine	B-Simple_chemical
phosphorylation	O
.	O

RESULTS	O
:	O
We	O
demonstrate	O
that	O
in	O
Saccharomyces	O
cerevisiae	O
,	O
several	O
cyclin	B-Complex
/	I-Complex
CDK	I-Complex
complexes	O
are	O
protected	O
from	O
inhibitory	O
tyrosine	B-Simple_chemical
phosphorylation	O
,	O
allowing	O
Clb5	B-Gene_or_gene_product
,	I-Gene_or_gene_product
6p	I-Gene_or_gene_product
to	O
promote	O
DNA	O
replication	O
and	O
Clb3	B-Gene_or_gene_product
,	I-Gene_or_gene_product
4p	I-Gene_or_gene_product
to	O
promote	O
spindle	B-Cellular_component
assembly	O
,	O
even	O
under	O
checkpoint	O
-	O
inducing	O
conditions	O
that	O
block	O
nuclear	B-Cellular_component
division	O
.	O

In	O
vivo	O
,	O
S	O
phase	O
-	O
promoting	O
Clb5p	B-Complex
/	I-Complex
Cdc28p	I-Complex
complexes	O
were	O
phosphorylated	O
more	O
slowly	O
and	O
dephosphorylated	O
more	O
effectively	O
than	O
were	O
mitosis	O
-	O
promoting	O
Clb2p	B-Complex
/	I-Complex
Cdc28p	I-Complex
complexes	O
.	O

Moreover	O
,	O
we	O
show	O
that	O
the	O
CDK	B-Gene_or_gene_product
inhibitor	I-Gene_or_gene_product
(	O
CKI	B-Gene_or_gene_product
)	O
Sic1p	B-Gene_or_gene_product
protects	O
bound	O
Clb5p	B-Complex
/	I-Complex
Cdc28p	I-Complex
complexes	O
from	O
tyrosine	B-Simple_chemical
phosphorylation	O
,	O
allowing	O
the	O
accumulation	O
of	O
unphosphorylated	O
complexes	O
that	O
are	O
unleashed	O
when	O
Sic1p	B-Gene_or_gene_product
is	O
degraded	O
to	O
promote	O
S	O
phase	O
.	O

The	O
vertebrate	O
CKI	B-Gene_or_gene_product
p27	B-Gene_or_gene_product
(	O
Kip1	B-Gene_or_gene_product
)	O
similarly	O
protects	O
Cyclin	B-Complex
A	I-Complex
/	I-Complex
Cdk2	I-Complex
complexes	O
from	O
Wee1	B-Gene_or_gene_product
,	O
suggesting	O
that	O
the	O
antagonism	O
between	O
CKIs	B-Gene_or_gene_product
and	O
Wee1	B-Gene_or_gene_product
is	O
evolutionarily	O
conserved	O
.	O

CONCLUSIONS	O
:	O
In	O
yeast	O
cells	O
,	O
the	O
combination	O
of	O
CKI	B-Gene_or_gene_product
binding	O
and	O
preferential	O
phosphorylation	O
/	O
dephosphorylation	O
of	O
different	O
B	B-Complex
cyclin	I-Complex
/	I-Complex
CDK	I-Complex
complexes	O
renders	O
S	O
phase	O
progression	O
immune	O
from	O
checkpoints	O
acting	O
via	O
CDK	B-Gene_or_gene_product
tyrosine	B-Simple_chemical
phosphorylation	O
.	O

Adiponectin	B-Gene_or_gene_product
enhances	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
6	I-Gene_or_gene_product
production	O
in	O
human	O
synovial	O
fibroblast	O
via	O
an	O
AdipoR1	B-Gene_or_gene_product
receptor	O
,	O
AMPK	B-Complex
,	O
p38	B-Gene_or_gene_product
,	O
and	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
pathway	O
.	O

Articular	O
adipose	O
tissue	O
is	O
a	O
ubiquitous	O
component	O
of	O
human	O
joints	O
,	O
and	O
adiponectin	B-Gene_or_gene_product
is	O
a	O
protein	O
hormone	O
secreted	O
predominantly	O
by	O
differentiated	O
adipocytes	O
and	O
involved	O
in	O
energy	O
homeostasis	O
.	O

We	O
investigated	O
the	O
signaling	O
pathway	O
involved	O
in	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
6	I-Gene_or_gene_product
production	O
caused	O
by	O
adiponectin	B-Gene_or_gene_product
in	O
both	O
rheumatoid	O
arthritis	O
synovial	O
fibroblasts	O
and	O
osteoarthritis	O
synovial	O
fibroblasts	O
.	O

Rheumatoid	O
arthritis	O
synovial	O
fibroblasts	O
and	O
osteoarthritis	O
synovial	O
fibroblasts	O
expressed	O
the	O
AdipoR1	B-Gene_or_gene_product
and	O
AdipoR2	B-Gene_or_gene_product
isoforms	O
of	O
the	O
adiponectin	B-Gene_or_gene_product
receptor	O
.	O

Adiponectin	B-Gene_or_gene_product
caused	O
concentration	O
-	O
and	O
time	O
-	O
dependent	O
increases	O
in	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
6	I-Gene_or_gene_product
production	O
.	O

Adiponectin	B-Gene_or_gene_product
-	O
mediated	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
6	I-Gene_or_gene_product
production	O
was	O
attenuated	O
by	O
AdipoR1	B-Gene_or_gene_product
and	O
5	B-Gene_or_gene_product
'	I-Gene_or_gene_product
-	I-Gene_or_gene_product
AMP	I-Gene_or_gene_product
-	I-Gene_or_gene_product
activated	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
(	I-Gene_or_gene_product
AMPK	I-Gene_or_gene_product
)	I-Gene_or_gene_product
alpha1	I-Gene_or_gene_product
small	O
interference	O
RNA	O
.	O

Pretreatment	O
with	O
AMPK	B-Complex
inhibitor	O
(	O
araA	B-Gene_or_gene_product
and	O
compound	B-Simple_chemical
C	I-Simple_chemical
)	O
,	O
p38	B-Gene_or_gene_product
inhibitor	O
(	O
SB203580	B-Simple_chemical
)	O
,	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
inhibitor	O
,	O
IkappaB	B-Gene_or_gene_product
protease	O
inhibitor	O
,	O
and	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
inhibitor	O
peptide	O
also	O
inhibited	O
the	O
potentiating	O
action	O
of	O
adiponectin	B-Gene_or_gene_product
.	O

Adiponectin	B-Gene_or_gene_product
increased	O
the	O
kinase	O
activity	O
and	O
phosphorylation	O
of	O
AMPK	B-Complex
and	O
p38	B-Gene_or_gene_product
.	O

Stimulation	O
of	O
synovial	O
fibroblasts	O
with	O
adiponectin	B-Gene_or_gene_product
activated	O
IkappaB	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
/	O
beta	B-Gene_or_gene_product
(	O
IKK	B-Gene_or_gene_product
alpha	I-Gene_or_gene_product
/	O
beta	B-Gene_or_gene_product
)	O
,	O
IkappaBalpha	B-Gene_or_gene_product
phosphorylation	O
,	O
IkappaBalpha	B-Gene_or_gene_product
degradation	O
,	O
p65	B-Gene_or_gene_product
phosphorylation	O
at	O
Ser	B-Simple_chemical
(	I-Simple_chemical
276	I-Simple_chemical
)	I-Simple_chemical
,	O
p65	B-Gene_or_gene_product
and	O
p50	B-Gene_or_gene_product
translocation	O
from	O
the	O
cytosol	B-Cellular_component
to	O
the	O
nucleus	B-Cellular_component
,	O
and	O
kappaB	B-Complex
-	O
luciferase	O
activity	O
.	O

Adiponectin	B-Gene_or_gene_product
-	O
mediated	O
an	O
increase	O
of	O
IKK	B-Gene_or_gene_product
alpha	I-Gene_or_gene_product
/	O
beta	B-Gene_or_gene_product
activity	O
,	O
kappaB	B-Complex
-	O
luciferase	O
activity	O
,	O
and	O
p65	B-Gene_or_gene_product
and	O
p50	B-Gene_or_gene_product
binding	O
to	O
the	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
element	O
and	O
was	O
inhibited	O
by	O
compound	B-Simple_chemical
C	I-Simple_chemical
,	O
SB203580	B-Simple_chemical
and	O
AdipoR1	B-Gene_or_gene_product
small	O
interference	O
RNA	O
.	O

Our	O
results	O
suggest	O
that	O
adiponectin	B-Gene_or_gene_product
increased	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
6	I-Gene_or_gene_product
production	O
in	O
synovial	O
fibroblasts	O
via	O
the	O
AdipoR1	B-Gene_or_gene_product
receptor	O
/	O
AMPK	B-Complex
/	O
p38	B-Gene_or_gene_product
/	O
IKKalphabeta	B-Gene_or_gene_product
and	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
signaling	O
pathway	O
.	O

S6K1	B-Gene_or_gene_product
-	O
mediated	O
disassembly	O
of	O
mitochondrial	B-Cellular_component
URI	B-Complex
/	I-Complex
PP1gamma	I-Complex
complexes	O
activates	O
a	O
negative	O
feedback	O
program	O
that	O
counters	O
S6K1	B-Gene_or_gene_product
survival	O
signaling	O
.	O

S6	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
1	I-Gene_or_gene_product
(	O
S6K1	B-Gene_or_gene_product
)	O
acts	O
to	O
integrate	O
nutrient	O
and	O
growth	O
factor	O
signals	O
to	O
promote	O
cell	O
growth	O
but	O
also	O
cell	O
survival	O
as	O
a	O
mitochondria	B-Cellular_component
-	O
tethered	O
protein	O
kinase	O
that	O
phosphorylates	O
and	O
inactivates	O
the	O
proapoptotic	O
molecule	O
BAD	B-Gene_or_gene_product
.	O

Here	O
we	O
report	O
that	O
the	O
prefoldin	O
chaperone	O
URI	B-Gene_or_gene_product
represents	O
a	O
mitochondrial	B-Cellular_component
substrate	O
of	O
S6K1	B-Gene_or_gene_product
.	O

In	O
growth	O
factor	O
-	O
deprived	O
or	O
rapamycin	B-Simple_chemical
-	O
treated	O
cells	O
,	O
URI	B-Gene_or_gene_product
forms	O
stable	O
complexes	O
with	O
protein	B-Gene_or_gene_product
phosphatase	I-Gene_or_gene_product
(	I-Gene_or_gene_product
PP	I-Gene_or_gene_product
)	I-Gene_or_gene_product
1gamma	I-Gene_or_gene_product
at	O
mitochondria	B-Cellular_component
,	O
thereby	O
inhibiting	O
the	O
activity	O
of	O
the	O
bound	O
enzyme	O
.	O

Growth	O
factor	O
stimulation	O
induces	O
disassembly	O
of	O
URI	B-Complex
/	I-Complex
PP1gamma	I-Complex
complexes	O
through	O
S6K1	B-Gene_or_gene_product
-	O
mediated	O
phosphorylation	O
of	O
URI	B-Gene_or_gene_product
at	O
serine	B-Simple_chemical
371	I-Simple_chemical
.	O

This	O
activates	O
a	O
PP1gamma	B-Gene_or_gene_product
-	O
dependent	O
negative	O
feedback	O
program	O
that	O
decreases	O
S6K1	B-Gene_or_gene_product
activity	O
and	O
BAD	B-Gene_or_gene_product
phosphorylation	O
,	O
thereby	O
altering	O
the	O
threshold	O
for	O
apoptosis	O
.	O

These	O
findings	O
establish	O
URI	B-Gene_or_gene_product
and	O
PP1gamma	B-Gene_or_gene_product
as	O
integral	O
components	O
of	O
an	O
S6K1	B-Gene_or_gene_product
-	O
regulated	O
mitochondrial	B-Cellular_component
pathway	O
dedicated	O
,	O
in	O
part	O
,	O
to	O
oppose	O
sustained	O
S6K1	B-Gene_or_gene_product
survival	O
signaling	O
and	O
to	O
ensure	O
that	O
the	O
mitochondrial	B-Cellular_component
threshold	O
for	O
apoptosis	O
is	O
set	O
in	O
accord	O
with	O
nutrient	O
and	O
growth	O
factor	O
availability	O
.	O

PRAS40	B-Gene_or_gene_product
and	O
PRR5	B-Gene_or_gene_product
-	I-Gene_or_gene_product
like	I-Gene_or_gene_product
protein	O
are	O
new	O
mTOR	B-Gene_or_gene_product
interactors	O
that	O
regulate	O
apoptosis	O
.	O

TOR	B-Gene_or_gene_product
(	O
Target	B-Gene_or_gene_product
of	I-Gene_or_gene_product
Rapamycin	I-Gene_or_gene_product
)	O
is	O
a	O
highly	O
conserved	O
protein	O
kinase	O
and	O
a	O
central	O
controller	O
of	O
cell	O
growth	O
.	O

TOR	B-Gene_or_gene_product
is	O
found	O
in	O
two	O
functionally	O
and	O
structurally	O
distinct	O
multiprotein	O
complexes	O
termed	O
TOR	B-Complex
complex	I-Complex
1	I-Complex
(	O
TORC1	B-Complex
)	O
and	O
TOR	B-Complex
complex	I-Complex
2	I-Complex
(	O
TORC2	B-Complex
)	O
.	O

In	O
the	O
present	O
study	O
,	O
we	O
developed	O
a	O
two	O
-	O
dimensional	O
liquid	O
chromatography	O
tandem	O
mass	O
spectrometry	O
(	O
2D	O
LC	O
-	O
MS	O
/	O
MS	O
)	O
based	O
proteomic	O
strategy	O
to	O
identify	O
new	O
mammalian	B-Gene_or_gene_product
TOR	I-Gene_or_gene_product
(	O
mTOR	B-Gene_or_gene_product
)	O
binding	O
proteins	O
.	O

We	O
report	O
the	O
identification	O
of	O
Proline	B-Gene_or_gene_product
-	I-Gene_or_gene_product
rich	I-Gene_or_gene_product
Akt	I-Gene_or_gene_product
substrate	I-Gene_or_gene_product
(	O
PRAS40	B-Gene_or_gene_product
)	O
and	O
the	O
hypothetical	O
protein	O
Q6MZQ0	B-Gene_or_gene_product
/	O
FLJ14213	B-Gene_or_gene_product
/	O
CAE45978	B-Gene_or_gene_product
as	O
new	O
mTOR	B-Gene_or_gene_product
binding	O
proteins	O
.	O

PRAS40	B-Gene_or_gene_product
binds	O
mTORC1	B-Complex
via	O
Raptor	B-Gene_or_gene_product
,	O
and	O
is	O
an	O
mTOR	B-Gene_or_gene_product
phosphorylation	O
substrate	O
.	O

PRAS40	B-Gene_or_gene_product
inhibits	O
mTORC1	B-Complex
autophosphorylation	O
and	O
mTORC1	B-Complex
kinase	O
activity	O
toward	O
eIF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
4E	I-Gene_or_gene_product
binding	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
(	O
4E	B-Gene_or_gene_product
-	I-Gene_or_gene_product
BP	I-Gene_or_gene_product
)	O
and	O
PRAS40	B-Gene_or_gene_product
itself	O
.	O

HeLa	O
cells	O
in	O
which	O
PRAS40	B-Gene_or_gene_product
was	O
knocked	O
down	O
were	O
protected	O
against	O
induction	O
of	O
apoptosis	O
by	O
TNFalpha	B-Gene_or_gene_product
and	O
cycloheximide	B-Simple_chemical
.	O

Rapamycin	B-Simple_chemical
failed	O
to	O
mimic	O
the	O
pro	O
-	O
apoptotic	O
effect	O
of	O
PRAS40	B-Gene_or_gene_product
,	O
suggesting	O
that	O
PRAS40	B-Gene_or_gene_product
mediates	O
apoptosis	O
independently	O
of	O
its	O
inhibitory	O
effect	O
on	O
mTORC1	B-Complex
.	O

Q6MZQ0	B-Gene_or_gene_product
is	O
structurally	O
similar	O
to	O
proline	B-Gene_or_gene_product
rich	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
5	I-Gene_or_gene_product
(	O
PRR5	B-Gene_or_gene_product
)	O
and	O
was	O
therefore	O
named	O
PRR5	B-Gene_or_gene_product
-	I-Gene_or_gene_product
Like	I-Gene_or_gene_product
(	O
PRR5L	B-Gene_or_gene_product
)	O
.	O

PRR5L	B-Gene_or_gene_product
binds	O
specifically	O
to	O
mTORC2	B-Complex
,	O
via	O
Rictor	B-Gene_or_gene_product
and	O
/	O
or	O
SIN1	B-Gene_or_gene_product
.	O

Unlike	O
other	O
mTORC2	B-Complex
members	O
,	O
PRR5L	B-Gene_or_gene_product
is	O
not	O
required	O
for	O
mTORC2	B-Complex
integrity	O
or	O
kinase	O
activity	O
,	O
but	O
dissociates	O
from	O
mTORC2	B-Complex
upon	O
knock	O
down	O
of	O
tuberous	B-Gene_or_gene_product
sclerosis	I-Gene_or_gene_product
complex	I-Gene_or_gene_product
1	I-Gene_or_gene_product
(	O
TSC1	B-Gene_or_gene_product
)	O
and	O
TSC2	B-Gene_or_gene_product
.	O

Hyperactivation	O
of	O
mTOR	B-Gene_or_gene_product
by	O
TSC1	B-Gene_or_gene_product
/	O
2	B-Gene_or_gene_product
knock	O
down	O
enhanced	O
apoptosis	O
whereas	O
PRR5L	B-Gene_or_gene_product
knock	O
down	O
reduced	O
apoptosis	O
.	O

PRR5L	B-Gene_or_gene_product
knock	O
down	O
reduced	O
apoptosis	O
also	O
in	O
mTORC2	B-Complex
deficient	O
cells	O
.	O

The	O
above	O
suggests	O
that	O
mTORC2	B-Complex
-	O
dissociated	O
PRR5L	B-Gene_or_gene_product
may	O
promote	O
apoptosis	O
when	O
mTOR	B-Gene_or_gene_product
is	O
hyperactive	O
.	O

Thus	O
,	O
PRAS40	B-Gene_or_gene_product
and	O
PRR5L	B-Gene_or_gene_product
are	O
novel	O
mTOR	B-Gene_or_gene_product
-	O
associated	O
proteins	O
that	O
control	O
the	O
balance	O
between	O
cell	O
growth	O
and	O
cell	O
death	O
.	O

Bub1	B-Gene_or_gene_product
kinase	O
targets	O
Sgo1	B-Gene_or_gene_product
to	O
ensure	O
efficient	O
chromosome	B-Cellular_component
biorientation	O
in	O
budding	O
yeast	O
mitosis	O
.	O

During	O
cell	O
division	O
all	O
chromosomes	B-Cellular_component
must	O
be	O
segregated	O
accurately	O
to	O
each	O
daughter	O
cell	O
.	O

Errors	O
in	O
this	O
process	O
give	O
rise	O
to	O
aneuploidy	O
,	O
which	O
leads	O
to	O
birth	O
defects	O
and	O
is	O
implicated	O
in	O
cancer	O
progression	O
.	O

The	O
spindle	B-Cellular_component
checkpoint	O
is	O
a	O
surveillance	O
mechanism	O
that	O
ensures	O
high	O
fidelity	O
of	O
chromosome	B-Cellular_component
segregation	O
by	O
inhibiting	O
anaphase	O
until	O
all	O
kinetochores	B-Cellular_component
have	O
established	O
bipolar	O
attachments	O
to	O
spindle	B-Cellular_component
microtubules	I-Cellular_component
.	O

Bub1	B-Gene_or_gene_product
kinase	O
is	O
a	O
core	O
component	O
of	O
the	O
spindle	B-Cellular_component
checkpoint	O
,	O
and	O
cells	O
lacking	O
Bub1	B-Gene_or_gene_product
fail	O
to	O
arrest	O
in	O
response	O
to	O
microtubule	B-Cellular_component
drugs	O
and	O
precociously	O
segregate	O
their	O
DNA	O
.	O

The	O
mitotic	O
role	O
(	O
s	O
)	O
of	O
Bub1	B-Gene_or_gene_product
kinase	O
activity	O
remain	O
elusive	O
,	O
and	O
it	O
is	O
controversial	O
whether	O
this	O
C	O
-	O
terminal	O
domain	O
of	O
Bub1p	B-Gene_or_gene_product
is	O
required	O
for	O
spindle	B-Cellular_component
checkpoint	O
arrest	O
.	O

Here	O
we	O
make	O
a	O
detailed	O
analysis	O
of	O
budding	O
yeast	O
cells	O
lacking	O
the	O
kinase	O
domain	O
(	O
bub1DeltaK	B-Gene_or_gene_product
)	O
.	O

We	O
show	O
that	O
despite	O
being	O
able	O
to	O
arrest	O
in	O
response	O
to	O
microtubule	B-Cellular_component
depolymerisation	O
and	O
kinetochore	B-Cellular_component
-	O
microtubule	B-Cellular_component
attachment	O
defects	O
,	O
bub1DeltaK	B-Gene_or_gene_product
cells	O
are	O
sensitive	O
to	O
microtubule	B-Cellular_component
drugs	O
.	O

This	O
is	O
because	O
bub1DeltaK	B-Gene_or_gene_product
cells	O
display	O
significant	O
chromosome	B-Cellular_component
mis	O
-	O
segregation	O
upon	O
release	O
from	O
nocodazole	B-Simple_chemical
arrest	O
.	O

bub1DeltaK	B-Gene_or_gene_product
cells	O
mislocalise	O
Sgo1p	B-Gene_or_gene_product
,	O
and	O
we	O
demonstrate	O
that	O
both	O
the	O
Bub1	B-Gene_or_gene_product
kinase	O
domain	O
and	O
Sgo1p	B-Gene_or_gene_product
are	O
required	O
for	O
accurate	O
chromosome	B-Cellular_component
biorientation	O
after	O
nocodazole	B-Simple_chemical
treatment	O
.	O

We	O
propose	O
that	O
Bub1	B-Gene_or_gene_product
kinase	O
and	O
Sgo1p	B-Gene_or_gene_product
act	O
together	O
to	O
ensure	O
efficient	O
biorientation	O
of	O
sister	B-Cellular_component
chromatids	I-Cellular_component
during	O
mitosis	O
.	O

Shugoshin	B-Gene_or_gene_product
promotes	O
sister	O
kinetochore	B-Cellular_component
biorientation	O
in	O
Saccharomyces	O
cerevisiae	O
.	O

Chromosome	B-Cellular_component
segregation	O
must	O
be	O
executed	O
accurately	O
during	O
both	O
mitotic	O
and	O
meiotic	O
cell	O
divisions	O
.	O

Sgo1	B-Gene_or_gene_product
plays	O
a	O
key	O
role	O
in	O
ensuring	O
faithful	O
chromosome	B-Cellular_component
segregation	O
in	O
at	O
least	O
two	O
ways	O
.	O

During	O
meiosis	O
this	O
protein	O
regulates	O
the	O
removal	O
of	O
cohesins	B-Complex
,	O
the	O
proteins	O
that	O
hold	O
sister	O
chromatids	B-Cellular_component
together	O
,	O
from	O
chromosomes	B-Cellular_component
.	O

During	O
mitosis	O
,	O
Sgo1	B-Gene_or_gene_product
is	O
required	O
for	O
sensing	O
the	O
absence	O
of	O
tension	O
caused	O
by	O
sister	O
kinetochores	B-Cellular_component
not	O
being	O
attached	O
to	O
microtubules	B-Cellular_component
emanating	O
from	O
opposite	O
poles	O
.	O

Here	O
we	O
describe	O
a	O
differential	O
requirement	O
for	O
Sgo1	B-Gene_or_gene_product
in	O
the	O
segregation	O
of	O
homologous	O
chromosomes	B-Cellular_component
and	O
sister	O
chromatids	B-Cellular_component
.	O

Sgo1	B-Gene_or_gene_product
plays	O
only	O
a	O
minor	O
role	O
in	O
segregating	O
homologous	O
chromosomes	B-Cellular_component
at	O
meiosis	O
I	O
.	O

In	O
contrast	O
,	O
Sgo1	B-Gene_or_gene_product
is	O
important	O
to	O
bias	O
sister	O
kinetochores	B-Cellular_component
toward	O
biorientation	O
.	O

We	O
suggest	O
that	O
Sgo1	B-Gene_or_gene_product
acts	O
at	O
sister	O
kinetochores	B-Cellular_component
to	O
promote	O
their	O
biorientation	O
.	O

Suppressive	O
effects	O
of	O
flavonoid	B-Simple_chemical
fisetin	I-Simple_chemical
on	O
lipopolysaccharide	B-Simple_chemical
-	O
induced	O
microglial	O
activation	O
and	O
neurotoxicity	O
.	O

Microglia	O
are	O
innate	O
immune	O
cells	O
in	O
the	O
central	O
nervous	O
system	O
.	O

Activation	O
of	O
microglia	O
plays	O
an	O
important	O
role	O
in	O
the	O
processes	O
of	O
several	O
neurodegenerative	O
diseases	O
including	O
Alzheimer	O
'	O
s	O
disease	O
,	O
Parkinson	O
'	O
s	O
disease	O
,	O
and	O
HIV	O
dementia	O
.	O

Activated	O
microglia	O
can	O
produce	O
various	O
proinflammatory	O
cytokines	O
and	O
nitric	B-Simple_chemical
oxide	I-Simple_chemical
(	O
NO	B-Simple_chemical
)	O
,	O
which	O
may	O
exert	O
neurotoxic	O
effects	O
.	O

Inhibition	O
of	O
microglia	O
activation	O
may	O
alleviate	O
neurodegeneration	O
under	O
these	O
conditions	O
.	O

To	O
search	O
for	O
the	O
novel	O
therapeutic	O
agents	O
against	O
neuroinflammatory	O
diseases	O
,	O
we	O
have	O
screened	O
a	O
series	O
of	O
flavonoid	B-Simple_chemical
compounds	O
using	O
a	O
cell	O
-	O
based	O
assay	O
.	O

Our	O
studies	O
showed	O
that	O
fisetin	B-Simple_chemical
markedly	O
suppressed	O
the	O
production	O
of	O
tumor	B-Gene_or_gene_product
necrosis	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
(	I-Gene_or_gene_product
TNF	I-Gene_or_gene_product
)	I-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
,	O
NO	B-Simple_chemical
,	O
and	O
prostaglandin	B-Simple_chemical
(	I-Simple_chemical
PG	I-Simple_chemical
)	I-Simple_chemical
E2	I-Simple_chemical
in	O
lipopolysaccharide	B-Simple_chemical
(	O
LPS	B-Simple_chemical
)	O
-	O
stimulated	O
BV	O
-	O
2	O
microglia	O
cells	O
or	O
primary	O
microglia	O
cultures	O
.	O

Fisetin	B-Simple_chemical
also	O
inhibited	O
the	O
gene	O
expression	O
of	O
TNF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
,	O
interleukin	B-Gene_or_gene_product
(	I-Gene_or_gene_product
IL	I-Gene_or_gene_product
)	I-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
beta	I-Gene_or_gene_product
,	O
cyclooxygenase	B-Gene_or_gene_product
(	O
COX	B-Gene_or_gene_product
-	I-Gene_or_gene_product
2	I-Gene_or_gene_product
)	O
and	O
inducible	O
nitric	B-Gene_or_gene_product
oxide	I-Gene_or_gene_product
synthase	I-Gene_or_gene_product
(	O
iNOS	B-Gene_or_gene_product
)	O
at	O
both	O
mRNA	O
and	O
protein	O
levels	O
.	O

Fisetin	B-Simple_chemical
significantly	O
suppressed	O
I	B-Gene_or_gene_product
kappa	I-Gene_or_gene_product
B	I-Gene_or_gene_product
degradation	O
,	O
nuclear	B-Cellular_component
translocation	O
of	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
,	O
and	O
phosphorylation	O
of	O
p38	B-Gene_or_gene_product
mitogen	I-Gene_or_gene_product
-	I-Gene_or_gene_product
activated	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
(	O
MAPKs	B-Gene_or_gene_product
)	O
in	O
the	O
LPS	B-Simple_chemical
-	O
stimulated	O
BV	O
-	O
2	O
microglia	O
cells	O
.	O

In	O
addition	O
,	O
fisetin	B-Simple_chemical
reduced	O
cytotoxicity	O
of	O
LPS	B-Simple_chemical
-	O
stimulated	O
microglia	O
toward	O
B35	O
neuroblastoma	O
cells	O
in	O
a	O
co	O
-	O
culture	O
system	O
.	O

These	O
results	O
indicate	O
that	O
fisetin	B-Simple_chemical
has	O
a	O
strong	O
anti	O
-	O
inflammatory	O
activity	O
in	O
brain	O
microglia	O
,	O
and	O
could	O
be	O
a	O
potential	O
therapeutic	O
agent	O
for	O
the	O
treatment	O
of	O
neuroinflammatory	O
diseases	O
.	O

Dephosphorylation	O
and	O
activation	O
of	O
a	O
p34cdc2	B-Complex
/	I-Complex
cyclin	I-Complex
B	I-Complex
complex	O
in	O
vitro	O
by	O
human	O
CDC25	B-Gene_or_gene_product
protein	O
.	O

Oocytes	O
arrested	O
in	O
the	O
G2	O
phase	O
of	O
the	O
cell	O
cycle	O
contain	O
a	O
p34cdc2	B-Complex
/	I-Complex
cyclin	I-Complex
B	I-Complex
complex	O
which	O
is	O
kept	O
in	O
an	O
inactive	O
form	O
by	O
phosphorylation	O
of	O
its	O
p34cdc2	B-Gene_or_gene_product
subunit	O
on	O
tyrosine	B-Simple_chemical
,	O
threonine	B-Simple_chemical
and	O
perhaps	O
serine	B-Simple_chemical
residues	O
.	O

The	O
phosphatase	O
(	O
s	O
)	O
involved	O
in	O
p34cdc2	B-Gene_or_gene_product
dephosphorylation	O
is	O
unknown	O
,	O
but	O
the	O
product	O
of	O
the	O
fission	O
yeast	O
cdc25	B-Gene_or_gene_product
+	O
gene	O
,	O
and	O
its	O
homologues	O
in	O
budding	O
yeast	O
and	O
Drosophila	O
are	O
probably	O
positive	O
regulators	O
of	O
the	O
transition	O
from	O
G2	O
to	O
M	O
phase	O
.	O

We	O
have	O
purified	O
the	O
inactive	O
p34cdc2	B-Complex
/	I-Complex
cyclin	I-Complex
B	I-Complex
complex	O
from	O
G2	O
-	O
arrested	O
starfish	O
oocytes	O
.	O

Addition	O
of	O
the	O
purified	O
bacterially	O
expressed	O
product	O
of	O
the	O
human	O
homologue	O
of	O
the	O
fission	O
yeast	O
cdc25	B-Gene_or_gene_product
+	O
gene	O
(	O
p54CDC25H	B-Gene_or_gene_product
)	O
triggers	O
p34cdc2	B-Gene_or_gene_product
dephosphorylation	O
and	O
activates	O
H1	B-Gene_or_gene_product
histone	I-Gene_or_gene_product
kinase	O
activity	O
in	O
this	O
preparation	O
.	O

We	O
propose	O
that	O
the	O
cdc25	B-Gene_or_gene_product
+	O
gene	O
product	O
directly	O
activates	O
the	O
p34cdc2	B-Complex
-	I-Complex
cyclin	I-Complex
B	I-Complex
complex	O
.	O

Transcriptional	O
activation	O
of	O
CLN1	B-Gene_or_gene_product
,	O
CLN2	B-Gene_or_gene_product
,	O
and	O
a	O
putative	O
new	O
G1	O
cyclin	B-Gene_or_gene_product
(	O
HCS26	B-Gene_or_gene_product
)	O
by	O
SWI4	B-Gene_or_gene_product
,	O
a	O
positive	O
regulator	O
of	O
G1	O
-	O
specific	O
transcription	O
.	O

SWI4	B-Gene_or_gene_product
of	O
budding	O
yeast	O
codes	O
for	O
a	O
component	O
of	O
a	O
transcription	O
factor	O
(	O
cell	B-Gene_or_gene_product
cycle	I-Gene_or_gene_product
box	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
,	O
or	O
CCBF	B-Gene_or_gene_product
)	O
necessary	O
for	O
G1	O
-	O
specific	O
expression	O
of	O
HO	B-Gene_or_gene_product
.	O

We	O
show	O
that	O
SWI4	B-Gene_or_gene_product
is	O
essential	O
for	O
haploid	O
cell	O
viability	O
at	O
high	O
temperature	O
and	O
in	O
a	O
/	O
alpha	O
cells	O
at	O
all	O
temperatures	O
:	O
SWI4	B-Gene_or_gene_product
-	O
deficient	O
cells	O
arrest	O
as	O
large	O
unbudded	O
cells	O
.	O

Eight	O
high	O
copy	O
number	O
plasmids	O
were	O
identified	O
that	O
allow	O
swi4	B-Gene_or_gene_product
-	O
strains	O
to	O
grow	O
under	O
nonpermissive	O
conditions	O
.	O

Two	O
carry	O
G1	O
cyclin	B-Gene_or_gene_product
genes	O
,	O
CLN1	B-Gene_or_gene_product
and	O
CLN2	B-Gene_or_gene_product
;	O
another	O
carries	O
HCS26	B-Gene_or_gene_product
,	O
coding	O
for	O
a	O
putative	O
cyclin	B-Gene_or_gene_product
,	O
a	O
/	O
alpha	O
swi4	B-Gene_or_gene_product
-	O
mutants	O
exhibit	O
3	O
-	O
to	O
20	O
-	O
fold	O
reductions	O
in	O
the	O
levels	O
of	O
CLN1	B-Gene_or_gene_product
,	O
CLN2	B-Gene_or_gene_product
,	O
and	O
HCS26	B-Gene_or_gene_product
transcripts	O
.	O

The	O
requirement	O
of	O
SWI4	B-Gene_or_gene_product
for	O
transcription	O
appears	O
to	O
be	O
direct	O
:	O
each	O
gene	O
contains	O
sites	O
similar	O
to	O
the	O
CCBF	B-Gene_or_gene_product
-	O
binding	O
site	O
;	O
CCBF	B-Gene_or_gene_product
binds	O
to	O
the	O
upstream	O
region	O
of	O
HCS26	B-Gene_or_gene_product
.	O

We	O
propose	O
that	O
SWI4	B-Gene_or_gene_product
participates	O
in	O
a	O
positive	O
feedback	O
loop	O
by	O
which	O
CLN1	B-Gene_or_gene_product
,	O
CLN2	B-Gene_or_gene_product
,	O
and	O
possibly	O
HCS26	B-Gene_or_gene_product
promote	O
their	O
own	O
transcription	O
in	O
G1	O
.	O

The	O
Cdk	B-Gene_or_gene_product
inhibitor	O
p27	B-Gene_or_gene_product
in	O
human	O
cancer	O
:	O
prognostic	O
potential	O
and	O
relevance	O
to	O
anticancer	O
therapy	O
.	O

The	O
cyclin	B-Gene_or_gene_product
-	I-Gene_or_gene_product
dependent	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
(	O
Cdk	B-Gene_or_gene_product
)	O
inhibitor	O
p27	B-Gene_or_gene_product
(	O
also	O
known	O
as	O
KIP1	B-Gene_or_gene_product
)	O
regulates	O
cell	O
proliferation	O
,	O
cell	O
motility	O
and	O
apoptosis	O
.	O

Interestingly	O
,	O
the	O
protein	O
can	O
exert	O
both	O
positive	O
and	O
negative	O
functions	O
on	O
these	O
processes	O
.	O

Diverse	O
post	O
-	O
translational	O
modifications	O
determine	O
the	O
physiological	O
role	O
of	O
p27	B-Gene_or_gene_product
.	O

Phosphorylation	O
regulates	O
p27	B-Gene_or_gene_product
binding	O
to	O
and	O
inhibition	O
of	O
cyclin	B-Complex
-	I-Complex
Cdk	I-Complex
complexes	O
,	O
its	O
localization	O
and	O
its	O
ubiquitin	B-Gene_or_gene_product
-	O
mediated	O
proteolysis	O
.	O

In	O
cancers	O
,	O
p27	B-Gene_or_gene_product
is	O
inactivated	O
through	O
impaired	O
synthesis	O
,	O
accelerated	O
degradation	O
and	O
by	O
mislocalization	O
.	O

Moreover	O
,	O
studies	O
in	O
several	O
tumour	O
types	O
indicate	O
that	O
p27	B-Gene_or_gene_product
expression	O
levels	O
have	O
both	O
prognostic	O
and	O
therapeutic	O
implications	O
.	O

Regulation	O
of	O
proline	B-Gene_or_gene_product
-	I-Gene_or_gene_product
rich	I-Gene_or_gene_product
Akt	I-Gene_or_gene_product
substrate	I-Gene_or_gene_product
of	I-Gene_or_gene_product
40	I-Gene_or_gene_product
kDa	I-Gene_or_gene_product
(	O
PRAS40	B-Gene_or_gene_product
)	O
function	O
by	O
mammalian	B-Complex
target	I-Complex
of	I-Complex
rapamycin	I-Complex
complex	I-Complex
1	I-Complex
(	O
mTORC1	B-Complex
)	O
-	O
mediated	O
phosphorylation	O
.	O

The	O
rapamycin	B-Simple_chemical
-	O
sensitive	O
mammalian	B-Complex
target	I-Complex
of	I-Complex
rapamycin	I-Complex
(	I-Complex
mTOR	I-Complex
)	I-Complex
complex	I-Complex
1	I-Complex
(	O
mTORC1	B-Complex
)	O
contains	O
mTOR	B-Gene_or_gene_product
,	O
raptor	B-Gene_or_gene_product
,	O
mLST8	B-Gene_or_gene_product
,	O
and	O
PRAS40	B-Gene_or_gene_product
(	O
proline	B-Gene_or_gene_product
-	I-Gene_or_gene_product
rich	I-Gene_or_gene_product
Akt	I-Gene_or_gene_product
substrate	I-Gene_or_gene_product
of	I-Gene_or_gene_product
40	I-Gene_or_gene_product
kDa	I-Gene_or_gene_product
)	O
.	O

PRAS40	B-Gene_or_gene_product
functions	O
as	O
a	O
negative	O
regulator	O
when	O
bound	O
to	O
mTORC1	B-Complex
,	O
and	O
it	O
dissociates	O
from	O
mTORC1	B-Complex
in	O
response	O
to	O
insulin	B-Gene_or_gene_product
.	O

PRAS40	B-Gene_or_gene_product
has	O
been	O
demonstrated	O
to	O
be	O
a	O
substrate	O
of	O
mTORC1	B-Complex
,	O
and	O
one	O
phosphorylation	O
site	O
,	O
Ser	B-Simple_chemical
-	I-Simple_chemical
183	I-Simple_chemical
,	O
has	O
been	O
identified	O
.	O

In	O
this	O
study	O
,	O
we	O
used	O
two	O
-	O
dimensional	O
phosphopeptide	O
mapping	O
in	O
conjunction	O
with	O
mutational	O
analysis	O
to	O
show	O
that	O
in	O
addition	O
to	O
Ser	B-Simple_chemical
-	I-Simple_chemical
183	I-Simple_chemical
,	O
mTORC1	B-Complex
also	O
phosphorylates	O
Ser	B-Simple_chemical
-	I-Simple_chemical
212	I-Simple_chemical
and	O
Ser	B-Simple_chemical
-	I-Simple_chemical
221	I-Simple_chemical
in	O
PRAS40	B-Gene_or_gene_product
when	O
assayed	O
in	O
vitro	O
.	O

Mutation	O
of	O
all	O
three	O
residues	O
to	O
Ala	B-Simple_chemical
markedly	O
reduces	O
mTORC1	B-Complex
-	O
mediated	O
phosphorylation	O
of	O
PRAS40	B-Gene_or_gene_product
in	O
vitro	O
.	O

All	O
three	O
sites	O
were	O
confirmed	O
to	O
be	O
phosphorylated	O
in	O
vivo	O
by	O
[	B-Simple_chemical
(	I-Simple_chemical
32	I-Simple_chemical
)	I-Simple_chemical
P	I-Simple_chemical
]	I-Simple_chemical
orthophosphate	I-Simple_chemical
labeling	O
and	O
peptide	O
mapping	O
.	O

Phosphorylation	O
of	O
Ser	B-Simple_chemical
-	I-Simple_chemical
221	I-Simple_chemical
and	O
Ser	B-Simple_chemical
-	I-Simple_chemical
183	I-Simple_chemical
but	O
not	O
Ser	B-Simple_chemical
-	I-Simple_chemical
212	I-Simple_chemical
is	O
sensitive	O
to	O
rapamycin	B-Simple_chemical
treatment	O
.	O

Furthermore	O
,	O
we	O
demonstrate	O
that	O
mutation	O
of	O
Ser	B-Simple_chemical
-	I-Simple_chemical
221	I-Simple_chemical
to	O
Ala	B-Simple_chemical
reduces	O
the	O
interaction	O
with	O
14	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3	I-Gene_or_gene_product
-	I-Gene_or_gene_product
3	I-Gene_or_gene_product
to	O
the	O
same	O
extent	O
as	O
mutation	O
of	O
Thr	B-Simple_chemical
-	I-Simple_chemical
246	I-Simple_chemical
,	O
the	O
Akt	B-Gene_or_gene_product
/	O
protein	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
B	I-Gene_or_gene_product
-	O
phosphorylated	O
site	O
.	O

We	O
also	O
find	O
that	O
mutation	O
of	O
Ser	B-Simple_chemical
-	I-Simple_chemical
221	I-Simple_chemical
to	O
Ala	B-Simple_chemical
increases	O
the	O
inhibitory	O
activity	O
of	O
PRAS40	B-Gene_or_gene_product
toward	O
mTORC1	B-Complex
.	O

We	O
propose	O
that	O
after	O
mTORC1	B-Complex
kinase	O
activation	O
by	O
upstream	O
regulators	O
,	O
PRAS40	B-Gene_or_gene_product
is	O
phosphorylated	O
directly	O
by	O
mTOR	B-Gene_or_gene_product
,	O
thus	O
contributing	O
to	O
the	O
relief	O
of	O
PRAS40	B-Gene_or_gene_product
-	O
mediated	O
substrate	O
competition	O
.	O

WD40	O
protein	O
FBW5	B-Gene_or_gene_product
promotes	O
ubiquitination	O
of	O
tumor	O
suppressor	O
TSC2	B-Gene_or_gene_product
by	O
DDB1	B-Complex
-	I-Complex
CUL4	I-Complex
-	I-Complex
ROC1	I-Complex
ligase	O
.	O

Tuberous	O
sclerosis	O
(	O
TSC	O
)	O
is	O
an	O
autosomal	O
dominant	O
disease	O
characterized	O
by	O
hamartoma	O
formation	O
in	O
various	O
organs	O
and	O
is	O
caused	O
by	O
mutations	O
targeting	O
either	O
the	O
TSC1	B-Gene_or_gene_product
or	O
TSC2	B-Gene_or_gene_product
genes	O
.	O

TSC1	B-Gene_or_gene_product
and	O
TSC2	B-Gene_or_gene_product
proteins	O
form	O
a	O
functionally	O
interdependent	O
dimeric	O
complex	O
.	O

Phosphorylation	O
of	O
either	O
TSC	B-Complex
subunit	O
by	O
different	O
kinases	O
regulates	O
the	O
function	O
of	O
TSC	B-Complex
and	O
represents	O
a	O
major	O
mechanism	O
to	O
integrate	O
various	O
signals	O
into	O
a	O
centralized	O
cell	O
growth	O
pathway	O
.	O

The	O
majority	O
of	O
disease	O
-	O
associated	O
mutations	O
targeting	O
either	O
TSC1	B-Gene_or_gene_product
or	O
TSC2	B-Gene_or_gene_product
results	O
in	O
a	O
substantial	O
decrease	O
in	O
protein	O
level	O
,	O
suggesting	O
that	O
protein	O
turnover	O
also	O
plays	O
a	O
critical	O
role	O
in	O
TSC	B-Complex
regulation	O
.	O

Here	O
we	O
report	O
that	O
TSC2	B-Gene_or_gene_product
protein	O
binds	O
to	O
FBW5	B-Gene_or_gene_product
,	O
a	O
DDB1	B-Gene_or_gene_product
-	I-Gene_or_gene_product
binding	I-Gene_or_gene_product
WD40	I-Gene_or_gene_product
(	O
DWD	B-Gene_or_gene_product
)	O
protein	O
,	O
and	O
is	O
recruited	O
by	O
FBW5	B-Gene_or_gene_product
to	O
the	O
DDB1	B-Complex
-	I-Complex
CUL4	I-Complex
-	I-Complex
ROC1	I-Complex
E3	O
ubiquitin	B-Gene_or_gene_product
ligase	O
.	O

Overexpression	O
of	O
FBW5	B-Gene_or_gene_product
or	O
CUL4A	B-Gene_or_gene_product
promotes	O
TSC2	B-Gene_or_gene_product
protein	O
degradation	O
,	O
and	O
this	O
is	O
abrogated	O
by	O
the	O
coexpression	O
of	O
TSC1	B-Gene_or_gene_product
.	O

Conversely	O
,	O
depletion	O
of	O
FBW5	B-Gene_or_gene_product
,	O
DDB1	B-Gene_or_gene_product
,	O
or	O
CUL4A	B-Gene_or_gene_product
/	O
B	B-Gene_or_gene_product
stabilizes	O
TSC2	B-Gene_or_gene_product
.	O

Ddb1	B-Gene_or_gene_product
or	O
Cul4	B-Gene_or_gene_product
mutations	O
in	O
Drosophila	O
result	O
in	O
Gigas	B-Gene_or_gene_product
/	O
TSC2	B-Gene_or_gene_product
protein	O
accumulation	O
and	O
cause	O
growth	O
defects	O
that	O
can	O
be	O
partially	O
rescued	O
by	O
Gigas	B-Gene_or_gene_product
/	O
Tsc2	B-Gene_or_gene_product
reduction	O
.	O

These	O
results	O
indicate	O
that	O
FBW5	B-Complex
-	I-Complex
DDB1	I-Complex
-	I-Complex
CUL4	I-Complex
-	I-Complex
ROC1	I-Complex
is	O
an	O
E3	O
ubiquitin	B-Gene_or_gene_product
ligase	O
regulating	O
TSC2	B-Gene_or_gene_product
protein	O
stability	O
and	O
TSC	B-Complex
complex	O
turnover	O
.	O

Activation	O
of	O
TLR	B-Gene_or_gene_product
-	I-Gene_or_gene_product
4	I-Gene_or_gene_product
and	O
liver	O
injury	O
via	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
in	O
rat	O
with	O
acute	O
cholangitis	O
.	O

BACKGROUND	O
:	O
Toll	B-Gene_or_gene_product
-	I-Gene_or_gene_product
like	I-Gene_or_gene_product
receptors	I-Gene_or_gene_product
(	O
TLRs	B-Gene_or_gene_product
)	O
are	O
a	O
family	O
of	O
type	O
1	O
transmembrane	O
receptors	O
,	O
which	O
can	O
recognize	O
different	O
pathogen	O
-	O
associated	O
molecular	O
patterns	O
.	O

Among	O
them	O
,	O
TLR	B-Gene_or_gene_product
-	I-Gene_or_gene_product
4	I-Gene_or_gene_product
is	O
specific	O
to	O
lipopolysaccharide	B-Simple_chemical
.	O

It	O
transfers	O
the	O
infection	O
signal	O
into	O
the	O
cell	O
and	O
promotes	O
the	O
translocation	O
of	O
nuclear	B-Complex
factor	I-Complex
kappa	I-Complex
B	I-Complex
(	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
)	O
to	O
the	O
nucleus	B-Cellular_component
and	O
the	O
subsequent	O
transcriptional	O
activation	O
of	O
genes	O
encoding	O
pro	O
-	O
and	O
anti	O
-	O
inflammatory	O
cytokines	O
and	O
chemokines	O
.	O

Acute	O
cholangitis	O
(	O
AC	O
)	O
is	O
a	O
common	O
biliary	O
tract	O
infection	O
in	O
oriental	O
countries	O
,	O
and	O
often	O
leads	O
to	O
liver	O
injury	O
.	O

The	O
activation	O
of	O
TLR	B-Gene_or_gene_product
-	I-Gene_or_gene_product
4	I-Gene_or_gene_product
and	O
its	O
significance	O
in	O
liver	O
injury	O
in	O
rats	O
with	O
AC	O
remain	O
unclear	O
.	O

METHODS	O
:	O
Rat	O
models	O
of	O
AC	O
(	O
biliary	O
tract	O
obstruction	O
+	O
E	O
.	O
coli	O
injection	O
,	O
n	O
=	O
36	O
)	O
and	O
control	O
models	O
(	O
biliary	O
tract	O
obstruction	O
+	O
saline	O
,	O
n	O
=	O
18	O
)	O
were	O
made	O
.	O

Liver	O
tissue	O
injury	O
was	O
investigated	O
by	O
pathological	O
examination	O
.	O

The	O
levels	O
of	O
serum	O
TNF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
and	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
10	I-Gene_or_gene_product
were	O
measured	O
by	O
enzyme	O
-	O
linked	O
immunosorbent	O
assay	O
,	O
and	O
the	O
expressions	O
of	O
TLR	B-Gene_or_gene_product
-	I-Gene_or_gene_product
4	I-Gene_or_gene_product
,	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
mRNAs	O
and	O
proteins	O
in	O
the	O
liver	O
were	O
detected	O
by	O
RT	O
-	O
PCR	O
,	O
immunohistochemical	O
staining	O
and	O
Western	O
blotting	O
,	O
respectively	O
.	O

RESULTS	O
:	O
Severe	O
liver	O
tissue	O
injury	O
in	O
rats	O
with	O
AC	O
was	O
evident	O
as	O
shown	O
by	O
pathological	O
examination	O
.	O

TLR	B-Gene_or_gene_product
-	I-Gene_or_gene_product
4	I-Gene_or_gene_product
and	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
were	O
strongly	O
expressed	O
in	O
the	O
cytoplasm	B-Cellular_component
of	O
hepatocytes	O
in	O
the	O
AC	O
group	O
.	O

They	O
were	O
negative	O
or	O
slightly	O
positive	O
in	O
the	O
control	O
group	O
.	O

TLR	B-Gene_or_gene_product
-	I-Gene_or_gene_product
4	I-Gene_or_gene_product
mRNA	O
and	O
protein	O
in	O
the	O
liver	O
of	O
rats	O
with	O
AC	O
increased	O
1	O
hour	O
after	O
biliary	O
tract	O
ligation	O
and	O
E	O
.	O
coli	O
injection	O
,	O
and	O
peaked	O
at	O
6	O
hours	O
after	O
surgery	O
.	O

Twenty	O
-	O
four	O
hours	O
later	O
,	O
they	O
began	O
to	O
decrease	O
.	O

The	O
expression	O
of	O
TLR	B-Gene_or_gene_product
-	I-Gene_or_gene_product
4	I-Gene_or_gene_product
was	O
paralleled	O
by	O
that	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
in	O
the	O
liver	O
and	O
TNF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
in	O
serum	O
.	O

CONCLUSION	O
:	O
The	O
higher	O
expression	O
of	O
TLR	B-Gene_or_gene_product
-	I-Gene_or_gene_product
4	I-Gene_or_gene_product
in	O
the	O
liver	O
of	O
rats	O
with	O
AC	O
may	O
be	O
involved	O
in	O
liver	O
injury	O
through	O
the	O
activation	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
and	O
release	O
of	O
cytokines	O
such	O
as	O
TNF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
.	O

5	B-Simple_chemical
-	I-Simple_chemical
Aza	I-Simple_chemical
-	I-Simple_chemical
2	I-Simple_chemical
'	I-Simple_chemical
-	I-Simple_chemical
deoxycytidine	I-Simple_chemical
restores	O
proapoptotic	O
function	O
of	O
p53	B-Gene_or_gene_product
in	O
cancer	O
cells	O
resistant	O
to	O
p53	B-Gene_or_gene_product
-	O
induced	O
apoptosis	O
.	O

The	O
expression	O
of	O
p53	B-Gene_or_gene_product
-	O
target	O
genes	O
encoding	O
the	O
proapoptotic	O
factor	O
Noxa	B-Gene_or_gene_product
,	O
but	O
not	O
PUMA	B-Gene_or_gene_product
,	O
was	O
not	O
induced	O
by	O
p53	B-Gene_or_gene_product
in	O
HCT116	O
and	O
SW480	O
cells	O
,	O
which	O
show	O
resistance	O
to	O
apoptosis	O
in	O
response	O
to	O
p53	B-Gene_or_gene_product
overexpression	O
.	O

The	O
lack	O
of	O
p53	B-Gene_or_gene_product
inducibility	O
of	O
Noxa	B-Gene_or_gene_product
was	O
restored	O
by	O
treatment	O
with	O
the	O
DNA	B-Gene_or_gene_product
methyltransferase	I-Gene_or_gene_product
inhibitor	O
5	B-Simple_chemical
-	I-Simple_chemical
Aza	I-Simple_chemical
-	I-Simple_chemical
2	I-Simple_chemical
'	I-Simple_chemical
-	I-Simple_chemical
deoxycytidine	I-Simple_chemical
(	O
5	B-Simple_chemical
-	I-Simple_chemical
aza	I-Simple_chemical
-	I-Simple_chemical
CdR	I-Simple_chemical
)	O
.	O

Furthermore	O
,	O
p53	B-Gene_or_gene_product
induced	O
apoptosis	O
in	O
HCT116	O
and	O
SW480	O
cells	O
treated	O
with	O
5	B-Simple_chemical
-	I-Simple_chemical
aza	I-Simple_chemical
-	I-Simple_chemical
CdR	I-Simple_chemical
.	O

Moreover	O
,	O
the	O
inhibition	O
of	O
Noxa	B-Gene_or_gene_product
expression	O
by	O
RNAi	O
in	O
5	B-Simple_chemical
-	I-Simple_chemical
aza	I-Simple_chemical
-	I-Simple_chemical
CdR	I-Simple_chemical
-	O
treated	O
HCT116	O
cells	O
resulted	O
in	O
the	O
partial	O
inhibition	O
of	O
p53	B-Gene_or_gene_product
-	O
induced	O
apoptosis	O
.	O

These	O
results	O
suggest	O
that	O
epigenetic	O
cancer	O
therapy	O
is	O
possible	O
for	O
some	O
cancers	O
in	O
combination	O
with	O
forced	O
p53	B-Gene_or_gene_product
activation	O
.	O

Regulation	O
of	O
Bax	B-Gene_or_gene_product
activation	O
and	O
apoptotic	O
response	O
to	O
UV	O
irradiation	O
by	O
p53	B-Gene_or_gene_product
transcription	O
-	O
dependent	O
and	O
-	O
independent	O
pathways	O
.	O

The	O
Trp53	B-Gene_or_gene_product
tumor	O
suppressor	O
gene	O
product	O
(	O
p53	B-Gene_or_gene_product
)	O
functions	O
in	O
the	O
nucleus	B-Cellular_component
to	O
regulate	O
proapoptotic	O
genes	O
,	O
whereas	O
cytoplasmic	B-Cellular_component
p53	B-Gene_or_gene_product
directly	O
activates	O
proapoptotic	O
Bcl	B-Gene_or_gene_product
-	I-Gene_or_gene_product
2	I-Gene_or_gene_product
proteins	O
to	O
permeabilize	O
mitochondria	B-Cellular_component
and	O
initiate	O
apoptosis	O
.	O

Here	O
,	O
we	O
demonstrate	O
that	O
both	O
p53	B-Gene_or_gene_product
transcription	O
-	O
dependent	O
and	O
-	O
independent	O
pathways	O
contribute	O
to	O
UV	O
-	O
induced	O
apoptosis	O
.	O

First	O
we	O
show	O
that	O
Pifithrin	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
,	O
a	O
small	O
molecule	O
inhibitor	O
of	O
p53	B-Gene_or_gene_product
transcriptional	O
activity	O
,	O
delays	O
Bax	B-Gene_or_gene_product
translocation	O
and	O
cell	O
death	O
by	O
UV	O
irradiation	O
.	O

Then	O
using	O
CHX	B-Simple_chemical
(	O
cycloheximide	B-Simple_chemical
)	O
to	O
prevent	O
new	O
protein	O
expression	O
in	O
response	O
to	O
p53	B-Gene_or_gene_product
,	O
we	O
also	O
find	O
that	O
Bax	B-Gene_or_gene_product
translocation	O
and	O
cell	O
death	O
by	O
UV	O
irradiation	O
are	O
delayed	O
.	O

Furthermore	O
we	O
find	O
that	O
overexpression	O
of	O
Bcl	B-Gene_or_gene_product
-	I-Gene_or_gene_product
x	I-Gene_or_gene_product
(	I-Gene_or_gene_product
L	I-Gene_or_gene_product
)	I-Gene_or_gene_product
,	O
an	O
inhibitor	O
of	O
cytoplasmic	B-Cellular_component
p53	B-Gene_or_gene_product
after	O
UV	O
irradiation	O
,	O
prevents	O
cell	O
death	O
.	O

Finally	O
,	O
we	O
observe	O
that	O
Pifithrin	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
and	O
CHX	B-Simple_chemical
effectively	O
inhibit	O
PUMA	B-Gene_or_gene_product
expression	O
by	O
UV	O
irradiation	O
.	O

Taken	O
together	O
,	O
these	O
data	O
indicate	O
that	O
the	O
nuclear	B-Cellular_component
p53	B-Gene_or_gene_product
promotes	O
PUMA	B-Gene_or_gene_product
expression	O
,	O
which	O
then	O
displaces	O
cytoplasmic	B-Cellular_component
p53	B-Gene_or_gene_product
from	O
Bcl	B-Gene_or_gene_product
-	I-Gene_or_gene_product
x	I-Gene_or_gene_product
(	I-Gene_or_gene_product
L	I-Gene_or_gene_product
)	I-Gene_or_gene_product
,	O
allowing	O
p53	B-Gene_or_gene_product
to	O
induce	O
mitochondrial	B-Cellular_component
permeabilization	O
,	O
thereby	O
triggering	O
apoptosis	O
.	O

Identification	O
of	O
genes	O
,	O
including	O
the	O
gene	O
encoding	O
p27Kip1	B-Gene_or_gene_product
,	O
regulated	O
by	O
serine	B-Simple_chemical
276	I-Simple_chemical
phosphorylation	O
of	O
the	O
p65	B-Gene_or_gene_product
subunit	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
.	O

Phosphorylation	O
of	O
the	O
p65	B-Gene_or_gene_product
subunit	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
is	O
required	O
for	O
its	O
transcriptional	O
activity	O
.	O

Recent	O
reports	O
show	O
that	O
phosphorylation	O
of	O
p65	B-Gene_or_gene_product
at	O
serine	B-Simple_chemical
276	I-Simple_chemical
regulates	O
only	O
a	O
subset	O
of	O
genes	O
,	O
such	O
as	O
those	O
encoding	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
6	I-Gene_or_gene_product
,	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
8	I-Gene_or_gene_product
,	O
Gro	B-Gene_or_gene_product
-	I-Gene_or_gene_product
beta	I-Gene_or_gene_product
,	O
and	O
ICAM	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
.	O

In	O
order	O
to	O
identify	O
additional	O
genes	O
regulated	O
by	O
serine	B-Simple_chemical
276	I-Simple_chemical
phosphorylation	O
,	O
HepG2	O
hepatoma	O
cells	O
were	O
infected	O
with	O
adenoviruses	O
encoding	O
either	O
wild	O
-	O
type	O
p65	B-Gene_or_gene_product
or	O
the	O
S276A	B-Gene_or_gene_product
mutant	O
of	O
p65	B-Gene_or_gene_product
,	O
followed	O
by	O
DNA	O
microarray	O
analysis	O
.	O

The	O
results	O
show	O
that	O
mutation	O
of	O
serine	B-Simple_chemical
276	I-Simple_chemical
affected	O
the	O
expression	O
of	O
several	O
genes	O
that	O
encode	O
proteins	O
involved	O
in	O
cell	O
cycle	O
regulation	O
,	O
signal	O
transduction	O
,	O
transcription	O
,	O
and	O
metabolism	O
.	O

Notably	O
,	O
expression	O
of	O
S276A	B-Gene_or_gene_product
increased	O
the	O
mRNA	O
and	O
protein	O
level	O
of	O
p27	B-Gene_or_gene_product
,	O
a	O
cell	O
cycle	O
inhibitory	O
protein	O
,	O
which	O
led	O
to	O
an	O
increased	O
association	O
of	O
p27	B-Gene_or_gene_product
with	O
cdk2	B-Gene_or_gene_product
,	O
and	O
inhibition	O
of	O
cdk2	B-Gene_or_gene_product
activity	O
.	O

Furthermore	O
,	O
while	O
wild	O
-	O
type	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
is	O
known	O
to	O
increase	O
cell	O
proliferation	O
in	O
a	O
number	O
of	O
different	O
cancer	O
cell	O
lines	O
,	O
our	O
data	O
shows	O
that	O
S276A	B-Gene_or_gene_product
inhibited	O
cell	O
proliferation	O
.	O

Evidence	O
is	O
mounting	O
that	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
plays	O
a	O
pivotal	O
role	O
in	O
oncogenesis	O
.	O

Therapeutic	O
agents	O
that	O
regulate	O
the	O
phosphorylation	O
of	O
serine	B-Simple_chemical
276	I-Simple_chemical
and	O
p27	B-Gene_or_gene_product
gene	O
expression	O
,	O
therefore	O
,	O
may	O
be	O
useful	O
as	O
anti	O
-	O
cancer	O
agents	O
in	O
the	O
future	O
.	O

Analysis	O
of	O
nucleocytoplasmic	B-Cellular_component
shuttling	O
of	O
NF	B-Complex
kappa	I-Complex
B	I-Complex
proteins	O
in	O
human	O
leukocytes	O
.	O

Controlled	O
nucleocytoplasmic	B-Cellular_component
localization	O
regulates	O
activity	O
of	O
NF	B-Complex
kappa	I-Complex
B	I-Complex
as	O
well	O
as	O
other	O
transcription	O
factors	O
.	O

Analysis	O
of	O
the	O
nucleocytoplasmic	B-Cellular_component
protein	O
shuttling	O
has	O
been	O
greatly	O
facilitated	O
by	O
the	O
use	O
of	O
leptomycin	B-Simple_chemical
B	I-Simple_chemical
(	O
LMB	B-Simple_chemical
)	O
,	O
an	O
inhibitor	O
of	O
CRM1	B-Gene_or_gene_product
-	O
dependent	O
nuclear	B-Cellular_component
export	O
.	O

The	O
authors	O
have	O
previously	O
shown	O
that	O
LMB	B-Simple_chemical
inhibits	O
NF	B-Complex
kappa	I-Complex
B	I-Complex
activity	O
in	O
human	O
neutrophils	O
by	O
increasing	O
the	O
nuclear	B-Cellular_component
accumulation	O
of	O
NF	B-Complex
kappa	I-Complex
B	I-Complex
inhibitor	O
,	O
I	B-Gene_or_gene_product
kappa	I-Gene_or_gene_product
B	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
.	O

In	O
this	O
chapter	O
,	O
the	O
authors	O
describe	O
a	O
protocol	O
that	O
uses	O
LMB	B-Simple_chemical
to	O
study	O
the	O
nucleocytoplasmic	B-Cellular_component
shuttling	O
of	O
I	B-Gene_or_gene_product
kappa	I-Gene_or_gene_product
B	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
in	O
human	O
macrophage	O
-	O
like	O
U937	O
cells	O
,	O
thus	O
inhibiting	O
NF	B-Complex
kappa	I-Complex
B	I-Complex
activity	O
.	O

This	O
protocol	O
should	O
be	O
readily	O
adaptable	O
to	O
analyze	O
the	O
nucleocytoplasmic	B-Cellular_component
shuttling	O
of	O
other	O
proteins	O
in	O
human	O
leukocytes	O
.	O

Wnt	B-Gene_or_gene_product
pathway	O
,	O
an	O
essential	O
role	O
in	O
bone	O
regeneration	O
.	O

Fracture	O
repair	O
is	O
a	O
complex	O
regenerative	O
process	O
initiated	O
in	O
response	O
to	O
injury	O
,	O
resulting	O
in	O
optimal	O
restoration	O
of	O
skeletal	O
function	O
.	O

Although	O
histology	O
characteristics	O
at	O
various	O
phases	O
of	O
fracture	O
repair	O
are	O
clear	O
and	O
well	O
established	O
,	O
much	O
remains	O
to	O
be	O
understood	O
about	O
the	O
process	O
of	O
bone	O
healing	O
,	O
particularly	O
at	O
the	O
molecular	O
signaling	O
level	O
.	O

During	O
the	O
past	O
decade	O
,	O
secreted	O
signaling	O
molecules	O
of	O
the	O
Wnt	B-Gene_or_gene_product
family	O
have	O
been	O
widely	O
investigated	O
and	O
found	O
to	O
play	O
a	O
central	O
role	O
in	O
controlling	O
embryonic	O
development	O
processes	O
.	O

Wnt	B-Gene_or_gene_product
signaling	O
pathway	O
also	O
plays	O
a	O
pivotal	O
role	O
in	O
the	O
regulation	O
of	O
bone	O
mass	O
.	O

Recent	O
published	O
data	O
reveal	O
that	O
Wnt	B-Gene_or_gene_product
signaling	O
pathway	O
is	O
activated	O
during	O
postnatal	O
bone	O
regenerative	O
events	O
,	O
such	O
as	O
ectopic	O
endochondral	O
bone	O
formation	O
and	O
fracture	O
repair	O
.	O

Dysregulation	O
of	O
this	O
pathway	O
greatly	O
inhibits	O
bone	O
formation	O
and	O
healing	O
process	O
.	O

Interestingly	O
,	O
activation	O
of	O
Wnt	B-Gene_or_gene_product
pathway	O
has	O
potential	O
to	O
improve	O
bone	O
healing	O
,	O
but	O
only	O
utilized	O
after	O
mesenchymal	O
cells	O
have	O
become	O
committed	O
to	O
the	O
osteoblast	O
lineage	O
.	O

These	O
advances	O
suggest	O
an	O
essential	O
role	O
of	O
Wnt	B-Gene_or_gene_product
pathway	O
in	O
bone	O
regeneration	O
.	O

Biomechanical	O
thresholds	O
regulate	O
inflammation	O
through	O
the	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
pathway	O
:	O
experiments	O
and	O
modeling	O
.	O

BACKGROUND	O
:	O
During	O
normal	O
physical	O
activities	O
cartilage	O
experiences	O
dynamic	O
compressive	O
forces	O
that	O
are	O
essential	O
to	O
maintain	O
cartilage	O
integrity	O
.	O

However	O
,	O
at	O
non	O
-	O
physiologic	O
levels	O
these	O
signals	O
can	O
induce	O
inflammation	O
and	O
initiate	O
cartilage	O
destruction	O
.	O

Here	O
,	O
by	O
examining	O
the	O
pro	O
-	O
inflammatory	O
signaling	O
networks	O
,	O
we	O
developed	O
a	O
mathematical	O
model	O
to	O
show	O
the	O
magnitude	O
-	O
dependent	O
regulation	O
of	O
chondrocytic	O
responses	O
by	O
compressive	O
forces	O
.	O

METHODOLOGY	O
/	O
PRINCIPAL	O
FINDINGS	O
:	O
Chondrocytic	O
cells	O
grown	O
in	O
3	O
-	O
D	O
scaffolds	O
were	O
subjected	O
to	O
various	O
magnitudes	O
of	O
dynamic	O
compressive	O
strain	O
(	O
DCS	O
)	O
,	O
and	O
the	O
regulation	O
of	O
pro	O
-	O
inflammatory	O
gene	O
expression	O
via	O
activation	O
of	O
nuclear	B-Complex
factor	I-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
(	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
)	O
signaling	O
cascade	O
examined	O
.	O

Experimental	O
evidences	O
provide	O
the	O
existence	O
of	O
a	O
threshold	O
in	O
the	O
magnitude	O
of	O
DCS	O
that	O
regulates	O
the	O
mRNA	O
expression	O
of	O
nitric	B-Gene_or_gene_product
oxide	I-Gene_or_gene_product
synthase	I-Gene_or_gene_product
(	O
NOS2	B-Gene_or_gene_product
)	O
,	O
an	O
inducible	O
pro	O
-	O
inflammatory	O
enzyme	O
.	O

Interestingly	O
,	O
below	O
this	O
threshold	O
,	O
DCS	O
inhibits	O
the	O
interleukin	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1beta	I-Gene_or_gene_product
(	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1beta	I-Gene_or_gene_product
)	O
-	O
induced	O
pro	O
-	O
inflammatory	O
gene	O
expression	O
,	O
with	O
the	O
degree	O
of	O
suppression	O
depending	O
on	O
the	O
magnitude	O
of	O
DCS	O
.	O

This	O
suppression	O
of	O
NOS2	B-Gene_or_gene_product
by	O
DCS	O
correlates	O
with	O
the	O
attenuation	O
of	O
the	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
signaling	O
pathway	O
as	O
measured	O
by	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1beta	I-Gene_or_gene_product
-	O
induced	O
phosphorylation	O
of	O
the	O
inhibitor	O
of	O
kappa	B-Gene_or_gene_product
B	I-Gene_or_gene_product
(	I-Gene_or_gene_product
IkappaB	I-Gene_or_gene_product
)	I-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
,	O
degradation	O
of	O
IkappaB	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
and	O
IkappaB	B-Gene_or_gene_product
-	I-Gene_or_gene_product
beta	I-Gene_or_gene_product
,	O
and	O
subsequent	O
nuclear	B-Cellular_component
translocation	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
p65	B-Gene_or_gene_product
.	O

A	O
mathematical	O
model	O
developed	O
to	O
understand	O
the	O
complex	O
dynamics	O
of	O
the	O
system	O
predicts	O
two	O
thresholds	O
in	O
the	O
magnitudes	O
of	O
DCS	O
,	O
one	O
for	O
the	O
inhibition	O
of	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1beta	I-Gene_or_gene_product
-	O
induced	O
expression	O
of	O
NOS2	B-Gene_or_gene_product
by	O
DCS	O
at	O
low	O
magnitudes	O
,	O
and	O
second	O
for	O
the	O
DCS	O
-	O
induced	O
expression	O
of	O
NOS2	B-Gene_or_gene_product
at	O
higher	O
magnitudes	O
.	O

CONCLUSIONS	O
/	O
SIGNIFICANCE	O
:	O
Experimental	O
and	O
computational	O
results	O
indicate	O
that	O
biomechanical	O
signals	O
suppress	O
and	O
induce	O
inflammation	O
at	O
critical	O
thresholds	O
through	O
activation	O
/	O
suppression	O
of	O
the	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
signaling	O
pathway	O
.	O

These	O
thresholds	O
arise	O
due	O
to	O
the	O
bistable	O
behavior	O
of	O
the	O
networks	O
originating	O
from	O
the	O
positive	O
feedback	O
loop	O
between	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
and	O
its	O
target	O
genes	O
.	O

These	O
findings	O
lay	O
initial	O
groundwork	O
for	O
the	O
identification	O
of	O
the	O
thresholds	O
in	O
physical	O
activities	O
that	O
can	O
differentiate	O
its	O
favorable	O
actions	O
from	O
its	O
unfavorable	O
consequences	O
on	O
joints	O
.	O

The	O
isolation	O
,	O
total	O
synthesis	O
and	O
structure	O
elucidation	O
of	O
chlorofusin	B-Simple_chemical
,	O
a	O
natural	O
product	O
inhibitor	O
of	O
the	O
p53	B-Gene_or_gene_product
-	O
mDM2	B-Gene_or_gene_product
protein	O
-	O
protein	O
interaction	O
.	O

Inhibitors	O
of	O
key	O
protein	O
-	O
protein	O
interactions	O
are	O
emerging	O
as	O
exciting	O
therapeutic	O
targets	O
for	O
the	O
treatment	O
of	O
cancer	O
.	O

One	O
such	O
interaction	O
between	O
MDM2	B-Gene_or_gene_product
(	O
HDM2	B-Gene_or_gene_product
)	O
and	O
p53	B-Gene_or_gene_product
,	O
that	O
silences	O
the	O
tumour	O
suppression	O
activities	O
of	O
p53	B-Gene_or_gene_product
,	O
was	O
found	O
to	O
be	O
inhibited	O
by	O
the	O
recently	O
isolated	O
natural	O
product	O
chlorofusin	B-Simple_chemical
.	O

Synthetic	O
studies	O
on	O
this	O
complex	O
natural	O
product	O
summarized	O
herein	O
have	O
served	O
to	O
reassign	O
its	O
chromophore	O
relative	O
stereochemistry	O
,	O
assign	O
its	O
absolute	O
stereochemistry	O
,	O
and	O
provided	O
access	O
to	O
a	O
series	O
of	O
key	O
analogues	O
and	O
partial	O
structures	O
for	O
biological	O
evaluation	O
.	O

Degradation	O
of	O
phosphorylated	O
p53	B-Gene_or_gene_product
by	O
viral	O
protein	O
-	O
ECS	B-Complex
E3	O
ligase	O
complex	O
.	O

p53	B-Gene_or_gene_product
-	O
signaling	O
is	O
modulated	O
by	O
viruses	O
to	O
establish	O
a	O
host	O
cellular	O
environment	O
advantageous	O
for	O
their	O
propagation	O
.	O

The	O
Epstein	O
-	O
Barr	O
virus	O
(	O
EBV	O
)	O
lytic	O
program	O
induces	O
phosphorylation	O
of	O
p53	B-Gene_or_gene_product
,	O
which	O
prevents	O
interaction	O
with	O
MDM2	B-Gene_or_gene_product
.	O

Here	O
,	O
we	O
show	O
that	O
induction	O
of	O
EBV	O
lytic	O
program	O
leads	O
to	O
degradation	O
of	O
p53	B-Gene_or_gene_product
via	O
an	O
ubiquitin	B-Gene_or_gene_product
-	O
proteasome	O
pathway	O
independent	O
of	O
MDM2	B-Gene_or_gene_product
.	O

The	O
BZLF1	B-Gene_or_gene_product
protein	O
directly	O
functions	O
as	O
an	O
adaptor	O
component	O
of	O
the	O
ECS	B-Complex
(	O
Elongin	B-Complex
B	I-Complex
/	I-Complex
C	I-Complex
-	I-Complex
Cul2	I-Complex
/	I-Complex
5	I-Complex
-	I-Complex
SOCS	I-Complex
-	I-Complex
box	I-Complex
protein	I-Complex
)	O
ubiquitin	B-Gene_or_gene_product
ligase	O
complex	O
targeting	O
p53	B-Gene_or_gene_product
for	O
degradation	O
.	O

Intringuingly	O
,	O
C	O
-	O
terminal	O
phosphorylation	O
of	O
p53	B-Gene_or_gene_product
resulting	O
from	O
activated	O
DNA	O
damage	O
response	O
by	O
viral	O
lytic	O
replication	O
enhances	O
its	O
binding	O
to	O
BZLF1	B-Gene_or_gene_product
protein	O
.	O

Purified	O
BZLF1	B-Gene_or_gene_product
protein	O
-	O
associated	O
ECS	B-Complex
could	O
be	O
shown	O
to	O
catalyze	O
ubiquitination	O
of	O
phospho	O
-	O
mimetic	O
p53	B-Gene_or_gene_product
more	O
efficiently	O
than	O
the	O
wild	O
-	O
type	O
in	O
vitro	O
.	O

The	O
compensation	O
of	O
p53	B-Gene_or_gene_product
at	O
middle	O
and	O
late	O
stages	O
of	O
the	O
lytic	O
infection	O
inhibits	O
viral	O
DNA	O
replication	O
and	O
production	O
during	O
lytic	O
infection	O
,	O
suggesting	O
that	O
the	O
degradation	O
of	O
p53	B-Gene_or_gene_product
is	O
required	O
for	O
efficient	O
viral	O
propagation	O
.	O

Taken	O
together	O
,	O
these	O
findings	O
demonstrate	O
a	O
role	O
for	O
the	O
BZLF1	B-Gene_or_gene_product
protein	O
-	O
associated	O
ECS	B-Complex
ligase	O
complex	O
in	O
regulation	O
of	O
p53	B-Gene_or_gene_product
phosphorylated	O
by	O
activated	O
DNA	O
damage	O
signaling	O
during	O
viral	O
lytic	O
infection	O
.	O

Interaction	O
with	O
vascular	O
endothelium	O
enhances	O
survival	O
in	O
primary	O
chronic	O
lymphocytic	O
leukemia	O
cells	O
via	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
activation	O
and	O
de	O
novo	O
gene	O
transcription	O
.	O

Chronic	O
lymphocytic	O
leukemia	O
(	O
CLL	O
)	O
cells	O
rapidly	O
undergo	O
apoptosis	O
in	O
vitro	O
,	O
suggesting	O
that	O
the	O
in	O
vivo	O
microenvironment	O
provides	O
crucial	O
antiapoptotic	O
signals	O
.	O

Overexpression	O
of	O
the	O
antiapoptotic	O
proteins	O
Bcl	B-Gene_or_gene_product
-	I-Gene_or_gene_product
2	I-Gene_or_gene_product
and	O
Mcl	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
is	O
a	O
hallmark	O
of	O
CLL	O
,	O
and	O
their	O
expression	O
is	O
further	O
enhanced	O
in	O
the	O
lymphoid	O
tissues	O
.	O

However	O
,	O
the	O
high	O
levels	O
of	O
Mcl	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
found	O
in	O
peripheral	O
blood	O
samples	O
,	O
coupled	O
with	O
its	O
short	O
half	O
-	O
life	O
,	O
led	O
us	O
to	O
hypothesize	O
that	O
it	O
must	O
be	O
actively	O
maintained	O
in	O
the	O
peripheral	O
circulation	O
.	O

Coculture	O
of	O
CLL	O
cells	O
with	O
human	O
vascular	O
endothelial	O
cells	O
significantly	O
enhanced	O
tumor	O
cell	O
survival	O
,	O
an	O
effect	O
that	O
was	O
not	O
observed	O
with	O
normal	O
B	O
cells	O
.	O

This	O
was	O
associated	O
with	O
elevated	O
levels	O
of	O
the	O
antiapoptotic	O
proteins	O
Bcl	B-Gene_or_gene_product
-	I-Gene_or_gene_product
2	I-Gene_or_gene_product
,	O
Mcl	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
,	O
and	O
Bcl	B-Gene_or_gene_product
-	I-Gene_or_gene_product
X	I-Gene_or_gene_product
(	I-Gene_or_gene_product
L	I-Gene_or_gene_product
)	I-Gene_or_gene_product
and	O
marked	O
increased	O
expression	O
of	O
CD38	B-Gene_or_gene_product
and	O
CD49d	B-Gene_or_gene_product
,	O
both	O
of	O
which	O
are	O
associated	O
with	O
clinically	O
aggressive	O
disease	O
.	O

Because	O
CD38	B-Gene_or_gene_product
,	O
CD49d	B-Gene_or_gene_product
,	O
and	O
some	O
Bcl	B-Gene_or_gene_product
-	I-Gene_or_gene_product
2	I-Gene_or_gene_product
family	O
genes	O
are	O
transcriptional	O
targets	O
for	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
,	O
we	O
assessed	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
activation	O
following	O
coculture	O
with	O
endothelial	O
cells	O
.	O

DNA	O
binding	O
of	O
the	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
subunit	O
Rel	B-Gene_or_gene_product
A	I-Gene_or_gene_product
was	O
significantly	O
increased	O
and	O
strongly	O
correlated	O
with	O
changes	O
in	O
transcription	O
of	O
CD38	B-Gene_or_gene_product
,	O
CD49d	B-Gene_or_gene_product
,	O
BCL2	B-Gene_or_gene_product
,	O
MCL1	B-Gene_or_gene_product
,	O
and	O
BCLXL	B-Gene_or_gene_product
,	O
effects	O
that	O
were	O
reversed	O
by	O
a	O
peptide	O
inhibitor	O
of	O
Rel	B-Gene_or_gene_product
A	I-Gene_or_gene_product
.	O

These	O
effects	O
were	O
not	O
observed	O
following	O
coculture	O
with	O
nonendothelial	O
cell	O
lines	O
.	O

Therefore	O
,	O
CLL	O
cells	O
receive	O
specific	O
survival	O
signals	O
following	O
interaction	O
with	O
endothelial	O
cells	O
mediated	O
through	O
the	O
activation	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
and	O
the	O
induction	O
of	O
downstream	O
target	O
genes	O
.	O

This	O
type	O
of	O
interaction	O
in	O
the	O
peripheral	O
vasculature	O
may	O
explain	O
the	O
constitutive	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
activation	O
and	O
the	O
overexpression	O
of	O
Bcl	B-Gene_or_gene_product
-	I-Gene_or_gene_product
2	I-Gene_or_gene_product
family	O
proteins	O
commonly	O
seen	O
in	O
this	O
disease	O
.	O

Interaction	O
of	O
the	O
TNFR	B-Gene_or_gene_product
-	I-Gene_or_gene_product
receptor	I-Gene_or_gene_product
associated	O
factor	O
TRAF1	B-Gene_or_gene_product
with	O
I	B-Gene_or_gene_product
-	I-Gene_or_gene_product
kappa	I-Gene_or_gene_product
B	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
-	I-Gene_or_gene_product
2	I-Gene_or_gene_product
and	O
TRAF2	B-Gene_or_gene_product
indicates	O
a	O
regulatory	O
function	O
for	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
signaling	O
.	O

BACKGROUND	O
:	O
I	B-Gene_or_gene_product
-	I-Gene_or_gene_product
kappa	I-Gene_or_gene_product
B	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
2	I-Gene_or_gene_product
(	O
IKK2	B-Gene_or_gene_product
or	O
IKK	B-Gene_or_gene_product
-	I-Gene_or_gene_product
beta	I-Gene_or_gene_product
)	O
is	O
one	O
of	O
the	O
most	O
crucial	O
signaling	O
kinases	O
for	O
activation	O
of	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
,	O
a	O
transcription	O
factor	O
that	O
is	O
important	O
for	O
inflammation	O
,	O
cell	O
survival	O
and	O
differentiation	O
.	O

Since	O
many	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
activating	O
pathways	O
converge	O
at	O
the	O
level	O
of	O
IKK2	B-Gene_or_gene_product
,	O
molecular	O
interactions	O
of	O
this	O
kinase	O
are	O
pivotal	O
for	O
regulation	O
of	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
signaling	O
.	O

METHODOLOGY	O
/	O
PRINCIPAL	O
FINDINGS	O
:	O
We	O
searched	O
for	O
proteins	O
interacting	O
with	O
IKK2	B-Gene_or_gene_product
using	O
the	O
C	O
-	O
terminal	O
part	O
(	O
amino	B-Simple_chemical
acids	I-Simple_chemical
466	I-Simple_chemical
-	O
756	B-Simple_chemical
)	O
as	O
bait	O
in	O
a	O
yeast	O
two	O
-	O
hybrid	O
system	O
and	O
identified	O
the	O
N	O
-	O
terminal	O
part	O
(	O
amino	B-Simple_chemical
acids	I-Simple_chemical
1	I-Simple_chemical
-	O
228	B-Simple_chemical
)	O
of	O
the	O
TNF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
receptor	I-Gene_or_gene_product
associated	O
factor	O
TRAF1	B-Gene_or_gene_product
as	O
putative	O
interaction	O
partner	O
.	O

The	O
interaction	O
was	O
confirmed	O
in	O
human	O
cells	O
by	O
mammalian	O
two	O
-	O
hybrid	O
and	O
coimmunoprecipitation	O
experiments	O
.	O

The	O
IKK2	B-Gene_or_gene_product
/	O
TRAF1	B-Gene_or_gene_product
interaction	O
seemed	O
weaker	O
than	O
the	O
interaction	O
between	O
TRAF1	B-Gene_or_gene_product
and	O
TRAF2	B-Gene_or_gene_product
,	O
an	O
important	O
activating	O
adapter	O
molecule	O
of	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
signaling	O
.	O

Reporter	O
gene	O
and	O
kinase	O
assays	O
using	O
ectopic	O
expression	O
of	O
TRAF1	B-Gene_or_gene_product
indicated	O
that	O
it	O
can	O
both	O
activate	O
and	O
inhibit	O
IKK2	B-Gene_or_gene_product
and	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
.	O

Co	O
-	O
expression	O
of	O
fluorescently	O
tagged	O
TRAF1	B-Gene_or_gene_product
and	O
TRAF2	B-Gene_or_gene_product
at	O
different	O
ratios	O
implied	O
that	O
TRAF1	B-Gene_or_gene_product
can	O
affect	O
clustering	O
and	O
presumably	O
the	O
activating	O
function	O
of	O
TRAF2	B-Gene_or_gene_product
in	O
a	O
dose	O
dependent	O
manner	O
.	O

CONCLUSIONS	O
/	O
SIGNIFICANCE	O
:	O
The	O
observation	O
that	O
TRAF1	B-Gene_or_gene_product
can	O
either	O
activate	O
or	O
inhibit	O
the	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
pathway	O
and	O
the	O
fact	O
that	O
it	O
influences	O
the	O
oligomerization	O
of	O
TRAF2	B-Gene_or_gene_product
indicates	O
that	O
relative	O
levels	O
of	O
IKK2	B-Gene_or_gene_product
,	O
TRAF1	B-Gene_or_gene_product
and	O
TRAF2	B-Gene_or_gene_product
may	O
be	O
important	O
for	O
regulation	O
of	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
activity	O
.	O

Since	O
TRAF1	B-Gene_or_gene_product
is	O
an	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
induced	O
gene	O
,	O
it	O
might	O
act	O
as	O
a	O
feedback	O
effector	O
molecule	O
.	O

Human	O
adipose	O
tissue	O
-	O
derived	O
mesenchymal	O
stem	O
cells	O
facilitate	O
the	O
immunosuppressive	O
effect	O
of	O
cyclosporin	B-Simple_chemical
A	I-Simple_chemical
on	O
T	O
lymphocytes	O
through	O
Jagged	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
-	O
mediated	O
inhibition	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
signaling	O
.	O

OBJECTIVE	O
:	O
Cyclosporine	B-Simple_chemical
A	I-Simple_chemical
(	O
CsA	B-Simple_chemical
)	O
,	O
known	O
as	O
an	O
effective	O
immunosuppressive	O
agent	O
,	O
is	O
widely	O
used	O
in	O
clinical	O
fields	O
.	O

Mesenchymal	O
stem	O
cells	O
may	O
exert	O
immunomodulatory	O
effects	O
on	O
the	O
immune	O
system	O
,	O
but	O
the	O
exact	O
mechanisms	O
underlying	O
them	O
remain	O
controversial	O
.	O

Here	O
we	O
investigated	O
whether	O
human	O
adipose	O
tissue	O
-	O
derived	O
mesenchymal	O
stem	O
cells	O
(	O
AMSCs	O
)	O
facilitate	O
in	O
vitro	O
the	O
immunomodulatory	O
effects	O
of	O
CsA	B-Simple_chemical
and	O
we	O
explored	O
the	O
molecule	O
mechanisms	O
that	O
may	O
be	O
involved	O
.	O

MATERIALS	O
AND	O
METHODS	O
:	O
Proliferation	O
of	O
T	O
lymphocytes	O
was	O
measured	O
by	O
uptake	O
of	O
(	B-Simple_chemical
3	I-Simple_chemical
)	I-Simple_chemical
H	I-Simple_chemical
-	I-Simple_chemical
thymidine	I-Simple_chemical
.	O

Transcription	O
and	O
production	O
of	O
interleukin	B-Gene_or_gene_product
-	I-Gene_or_gene_product
2	I-Gene_or_gene_product
and	O
interferon	B-Gene_or_gene_product
-	I-Gene_or_gene_product
gamma	I-Gene_or_gene_product
were	O
evaluated	O
by	O
real	O
-	O
time	O
quantitative	O
polymerase	O
chain	O
reaction	O
,	O
reverse	O
transcription	O
polymerase	O
chain	O
reaction	O
,	O
and	O
enzyme	O
-	O
linked	O
immunosorbent	O
assay	O
.	O

Nuclear	B-Complex
factor	I-Complex
-	I-Complex
kappaB	I-Complex
(	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
)	O
was	O
assayed	O
by	O
Western	O
blotting	O
and	O
electrophoretic	O
mobility	O
shift	O
assay	O
.	O

Expression	O
of	O
Jagged	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
,	O
Jagged	B-Gene_or_gene_product
-	I-Gene_or_gene_product
2	I-Gene_or_gene_product
,	O
and	O
Delta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
of	O
AMSCs	O
were	O
surveyed	O
by	O
flow	O
cytometric	O
analysis	O
and	O
Western	O
blotting	O
.	O

RESULTS	O
:	O
The	O
combination	O
of	O
moderate	O
-	O
dose	O
AMSCs	O
and	O
low	O
-	O
dose	O
CsA	B-Simple_chemical
was	O
significantly	O
more	O
powerful	O
than	O
moderate	O
-	O
dose	O
AMSCs	O
or	O
large	O
-	O
dose	O
CsA	B-Simple_chemical
alone	O
in	O
suppressing	O
transcription	O
and	O
production	O
of	O
interleukin	B-Gene_or_gene_product
-	I-Gene_or_gene_product
2	I-Gene_or_gene_product
and	O
interferon	B-Gene_or_gene_product
-	I-Gene_or_gene_product
gamma	I-Gene_or_gene_product
,	O
activation	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
,	O
and	O
proliferation	O
of	O
T	O
lymphocytes	O
.	O

In	O
addition	O
,	O
AMSCs	O
expressed	O
a	O
high	O
level	O
of	O
Jagged	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
,	O
which	O
induced	O
activation	O
of	O
Notch	B-Gene_or_gene_product
signaling	O
in	O
T	O
lymphocytes	O
,	O
thus	O
reducing	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
activity	O
.	O

Anti	O
-	O
Jagged	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
neutralizing	O
antibody	O
and	O
N	B-Simple_chemical
[	I-Simple_chemical
N	I-Simple_chemical
-	I-Simple_chemical
(	I-Simple_chemical
3	I-Simple_chemical
,	I-Simple_chemical
5	I-Simple_chemical
-	I-Simple_chemical
difluorophenacetyl	I-Simple_chemical
-	I-Simple_chemical
L	I-Simple_chemical
-	I-Simple_chemical
alanyl	I-Simple_chemical
)	I-Simple_chemical
]	I-Simple_chemical
-	I-Simple_chemical
S	I-Simple_chemical
-	I-Simple_chemical
phenylglycine	I-Simple_chemical
t	I-Simple_chemical
-	I-Simple_chemical
butyl	I-Simple_chemical
ester	I-Simple_chemical
could	O
reverse	O
this	O
trend	O
.	O

CONCLUSIONS	O
:	O
Human	O
AMSCs	O
facilitate	O
the	O
immunosuppressive	O
effect	O
of	O
CsA	B-Simple_chemical
on	O
T	O
lymphocytes	O
through	O
Jagged	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
/	O
Notch	B-Gene_or_gene_product
-	O
related	O
inhibition	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
signaling	O
.	O

The	O
combination	O
of	O
AMSCs	O
and	O
CsA	B-Simple_chemical
represents	O
a	O
rationale	O
therapeutic	O
approach	O
aimed	O
to	O
prevent	O
adverse	O
effects	O
of	O
CsA	B-Simple_chemical
while	O
maintaining	O
its	O
adequate	O
immunosuppressive	O
effect	O
.	O

Expression	O
of	O
Jagged	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
on	O
AMSCs	O
may	O
provide	O
an	O
effective	O
mechanism	O
for	O
the	O
immunomodulatory	O
activity	O
of	O
AMSCs	O
via	O
direct	O
cell	O
-	O
cell	O
interaction	O
.	O

Identification	O
of	O
calcium	B-Gene_or_gene_product
-	I-Gene_or_gene_product
activated	I-Gene_or_gene_product
neutral	I-Gene_or_gene_product
protease	I-Gene_or_gene_product
as	O
a	O
processing	O
enzyme	O
of	O
human	B-Gene_or_gene_product
interleukin	I-Gene_or_gene_product
1	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
.	O

We	O
describe	O
here	O
the	O
involvement	O
of	O
calcium	B-Gene_or_gene_product
-	I-Gene_or_gene_product
activated	I-Gene_or_gene_product
neutral	I-Gene_or_gene_product
protease	I-Gene_or_gene_product
(	O
CANP	B-Gene_or_gene_product
or	O
calpain	B-Gene_or_gene_product
,	O
EC	B-Gene_or_gene_product
3	I-Gene_or_gene_product
.	I-Gene_or_gene_product
4	I-Gene_or_gene_product
.	I-Gene_or_gene_product
22	I-Gene_or_gene_product
.	I-Gene_or_gene_product
17	I-Gene_or_gene_product
)	O
in	O
calcium	B-Simple_chemical
-	O
dependent	O
proteolytic	O
processing	O
of	O
the	O
precursor	O
of	O
human	B-Gene_or_gene_product
interleukin	I-Gene_or_gene_product
1	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
(	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
)	O
into	O
mature	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
.	O

Calcium	B-Simple_chemical
ionophore	O
ionomycin	B-Simple_chemical
enhanced	O
proteolytic	O
processing	O
of	O
pre	O
-	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
and	O
the	O
release	O
of	O
mature	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
either	O
from	O
lipopolysaccharide	B-Simple_chemical
(	O
LPS	B-Simple_chemical
)	O
-	O
activated	O
human	O
adherent	O
mononuclear	O
cells	O
or	O
from	O
a	O
human	O
bladder	O
carcinoma	O
cell	O
line	O
(	O
HTB9	O
5637	O
)	O
that	O
constitutively	O
produces	O
human	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
and	O
-	B-Gene_or_gene_product
beta	I-Gene_or_gene_product
.	O

The	O
proteolytic	O
processing	O
of	O
pre	O
-	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
was	O
completely	O
inhibited	O
by	O
EGTA	B-Simple_chemical
.	O

Similar	O
calcium	B-Simple_chemical
-	O
dependent	O
proteolytic	O
processing	O
of	O
pre	O
-	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
was	O
also	O
observed	O
with	O
lysates	O
of	O
either	O
LPS	B-Simple_chemical
-	O
activated	O
human	O
adherent	O
mononuclear	O
cells	O
or	O
HTB9	O
5637	O
cells	O
.	O

Since	O
the	O
optimal	O
pH	O
for	O
processing	O
was	O
between	O
7	O
and	O
8	O
,	O
and	O
E	B-Simple_chemical
-	I-Simple_chemical
64	I-Simple_chemical
(	O
a	O
cysteine	B-Simple_chemical
protease	O
inhibitor	O
)	O
and	O
leupeptin	B-Simple_chemical
(	O
a	O
serine	B-Simple_chemical
and	O
cysteine	B-Simple_chemical
protease	O
inhibitor	O
)	O
both	O
inhibited	O
this	O
processing	O
by	O
cell	O
lysates	O
,	O
we	O
hypothesized	O
that	O
a	O
calcium	B-Gene_or_gene_product
-	I-Gene_or_gene_product
activated	I-Gene_or_gene_product
neutral	I-Gene_or_gene_product
protease	I-Gene_or_gene_product
,	O
CANP	B-Gene_or_gene_product
,	O
might	O
be	O
responsible	O
for	O
this	O
processing	O
.	O

This	O
hypothesis	O
was	O
supported	O
by	O
data	O
showing	O
that	O
the	O
specific	O
CANP	B-Gene_or_gene_product
inhibitor	O
peptide	O
inhibited	O
this	O
proteolysis	O
in	O
cell	O
lysates	O
in	O
a	O
dose	O
-	O
dependent	O
fashion	O
(	O
IC50	O
=	O
0	O
.	O
05	O
microM	O
)	O
and	O
that	O
treatment	O
of	O
pre	O
-	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
with	O
purified	O
CANP	B-Gene_or_gene_product
yielded	O
the	O
17	O
-	O
kDa	O
mature	O
form	O
of	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
,	O
which	O
has	O
an	O
amino	O
terminus	O
identical	O
with	O
that	O
reported	O
for	O
mature	O
human	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
.	O

Taken	O
together	O
,	O
these	O
findings	O
indicate	O
that	O
calcium	B-Simple_chemical
-	O
dependent	O
proteolytic	O
processing	O
of	O
pre	O
-	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
is	O
selectively	O
mediated	O
by	O
CANP	B-Gene_or_gene_product
.	O

NF	B-Complex
-	I-Complex
kappaB	I-Complex
suppresses	O
HIF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1alpha	I-Gene_or_gene_product
response	O
by	O
competing	O
for	O
P300	B-Gene_or_gene_product
binding	O
.	O

Hypoxia	O
has	O
emerged	O
as	O
a	O
key	O
determinant	O
of	O
osteogenesis	O
.	O

HIF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1alpha	I-Gene_or_gene_product
is	O
the	O
transcription	O
factor	O
mediating	O
hypoxia	O
responses	O
that	O
include	O
induction	O
of	O
VEGF	B-Gene_or_gene_product
and	O
related	O
bone	O
induction	O
.	O

Inflammatory	O
signals	O
antagonize	O
bone	O
repair	O
via	O
the	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
pathway	O
.	O

The	O
present	O
investigation	O
explored	O
the	O
functional	O
relationship	O
of	O
hypoxia	O
(	O
HIF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1alpha	I-Gene_or_gene_product
function	O
)	O
and	O
inflammatory	O
signaling	O
(	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
)	O
in	O
stem	O
like	O
and	O
osteoprogenitor	O
cell	O
lines	O
.	O

The	O
potential	O
interaction	O
between	O
HIF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1alpha	I-Gene_or_gene_product
and	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
signaling	O
was	O
explored	O
by	O
co	O
-	O
transfection	O
studies	O
in	O
hFOB	O
with	O
p65	B-Gene_or_gene_product
,	O
HIF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1alpha	I-Gene_or_gene_product
and	O
9x	B-Gene_or_gene_product
-	I-Gene_or_gene_product
HRE	I-Gene_or_gene_product
-	I-Gene_or_gene_product
luc	I-Gene_or_gene_product
or	O
HIF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1alpha	I-Gene_or_gene_product
target	O
genes	O
reporter	O
plasmids	O
.	O

Nuclear	B-Cellular_component
cross	O
-	O
talk	O
was	O
directly	O
tested	O
using	O
the	O
mammalian	O
Gal4	B-Gene_or_gene_product
/	I-Gene_or_gene_product
VP16	I-Gene_or_gene_product
two	O
-	O
hybrid	O
,	O
and	O
confirmed	O
by	O
co	O
-	O
immunoprecipitation	O
/	O
western	O
blotting	O
assays	O
.	O

The	O
results	O
show	O
that	O
inflammatory	O
stimulation	O
(	O
TNF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
treatment	O
)	O
causes	O
a	O
marked	O
inhibition	O
of	O
HIF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1alpha	I-Gene_or_gene_product
function	O
at	O
the	O
HRE	O
in	O
all	O
cell	O
lines	O
studied	O
.	O

Also	O
,	O
co	O
-	O
transfection	O
with	O
p65	B-Gene_or_gene_product
expression	O
vector	O
leads	O
to	O
reduced	O
hVEGFp	B-Gene_or_gene_product
transcription	O
after	O
DFO	O
-	O
induced	O
hypoxia	O
.	O

However	O
,	O
TNF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
treatment	O
had	O
little	O
effect	O
on	O
HIF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1alpha	I-Gene_or_gene_product
mRNA	O
levels	O
.	O

The	O
functional	O
interaction	O
of	O
Gal4	B-Gene_or_gene_product
-	I-Gene_or_gene_product
HIF	I-Gene_or_gene_product
-	I-Gene_or_gene_product
1alpha	I-Gene_or_gene_product
and	O
VP16	B-Gene_or_gene_product
-	I-Gene_or_gene_product
p300	I-Gene_or_gene_product
fusion	O
proteins	O
is	O
effectively	O
blocked	O
by	O
expression	O
of	O
p65	B-Gene_or_gene_product
in	O
a	O
dose	O
dependent	O
manner	O
.	O

It	O
was	O
concluded	O
that	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
-	O
mediated	O
inflammatory	O
signaling	O
is	O
able	O
to	O
block	O
HIF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1alpha	I-Gene_or_gene_product
transactivation	O
at	O
HRE	O
-	O
encoding	O
genes	O
by	O
direct	O
competition	O
for	O
p300	B-Gene_or_gene_product
binding	O
at	O
the	O
promoter	O
.	O

Inflammation	O
may	O
influence	O
the	O
stem	O
cell	O
niche	O
and	O
tissue	O
regeneration	O
by	O
influencing	O
cellular	O
responses	O
to	O
hypoxia	O
.	O

ZAS3	B-Gene_or_gene_product
represses	O
NFkappaB	B-Complex
-	O
dependent	O
transcription	O
by	O
direct	O
competition	O
for	O
DNA	O
binding	O
.	O

NFkappaB	B-Complex
and	O
ZAS3	B-Gene_or_gene_product
are	O
transcription	O
factors	O
that	O
control	O
important	O
cellular	O
processes	O
including	O
immunity	O
,	O
cell	O
survival	O
and	O
apoptosis	O
.	O

Although	O
both	O
proteins	O
bind	O
the	O
kappaB	B-Complex
-	O
motif	O
,	O
they	O
produce	O
opposite	O
physiological	O
consequences	O
;	O
NFkappaB	B-Complex
activates	O
transcription	O
,	O
promotes	O
cell	O
growth	O
and	O
is	O
often	O
found	O
to	O
be	O
constitutively	O
expressed	O
in	O
cancer	O
cells	O
,	O
while	O
ZAS3	B-Gene_or_gene_product
generally	O
represses	O
transcription	O
,	O
inhibits	O
cell	O
proliferation	O
and	O
is	O
downregulated	O
in	O
some	O
cancers	O
.	O

Here	O
,	O
we	O
show	O
that	O
ZAS3	B-Gene_or_gene_product
inhibits	O
NFkappaB	B-Complex
-	O
dependent	O
transcription	O
by	O
competing	O
with	O
NFkappaB	B-Complex
for	O
the	O
kappaB	B-Complex
-	O
motif	O
.	O

Transient	O
transfection	O
studies	O
show	O
that	O
N	O
-	O
terminal	O
645	O
amino	O
acids	O
is	O
sufficient	O
to	O
repress	O
transcription	O
activated	O
by	O
NFkappaB	B-Complex
,	O
and	O
that	O
the	O
identical	O
region	O
also	O
possesses	O
intrinsic	O
repression	O
activity	O
to	O
inhibit	O
basal	O
transcription	O
from	O
a	O
promoter	O
.	O

Finally	O
,	O
in	O
vitro	O
DNA	O
-	O
protein	O
interaction	O
analysis	O
shows	O
that	O
ZAS3	B-Gene_or_gene_product
is	O
able	O
to	O
displace	O
NFkappaB	B-Complex
by	O
competing	O
with	O
NFkappaB	B-Complex
for	O
the	O
kappaB	B-Complex
-	O
motif	O
.	O

It	O
is	O
conceivable	O
that	O
ZAS3	B-Gene_or_gene_product
has	O
therapeutic	O
potential	O
for	O
controlling	O
aberrant	O
activation	O
of	O
NFkappaB	B-Complex
in	O
various	O
diseases	O
.	O

The	O
interplay	O
between	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
and	O
E2F1	B-Gene_or_gene_product
coordinately	O
regulates	O
inflammation	O
and	O
metabolism	O
in	O
human	O
cardiac	O
cells	O
.	O

Pyruvate	B-Gene_or_gene_product
dehydrogenase	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
4	I-Gene_or_gene_product
(	O
PDK4	B-Gene_or_gene_product
)	O
inhibition	O
by	O
nuclear	B-Complex
factor	I-Complex
-	I-Complex
kappaB	I-Complex
(	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
)	O
is	O
related	O
to	O
a	O
shift	O
towards	O
increased	O
glycolysis	O
during	O
cardiac	O
pathological	O
processes	O
such	O
as	O
cardiac	O
hypertrophy	O
and	O
heart	O
failure	O
.	O

The	O
transcription	O
factors	O
estrogen	B-Gene_or_gene_product
-	I-Gene_or_gene_product
related	I-Gene_or_gene_product
receptor	I-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
(	O
ERRalpha	B-Gene_or_gene_product
)	O
and	O
peroxisome	B-Gene_or_gene_product
proliferator	I-Gene_or_gene_product
-	I-Gene_or_gene_product
activated	I-Gene_or_gene_product
receptor	I-Gene_or_gene_product
(	O
PPAR	B-Gene_or_gene_product
)	O
regulate	O
PDK4	B-Gene_or_gene_product
expression	O
through	O
the	O
potent	O
transcriptional	O
coactivator	O
PPARgamma	B-Gene_or_gene_product
coactivator	I-Gene_or_gene_product
-	I-Gene_or_gene_product
1alpha	I-Gene_or_gene_product
(	O
PGC	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1alpha	I-Gene_or_gene_product
)	O
.	O

NF	B-Complex
-	I-Complex
kappaB	I-Complex
activation	O
in	O
AC16	O
cardiac	O
cells	O
inhibit	O
ERRalpha	B-Gene_or_gene_product
and	O
PPARbeta	B-Gene_or_gene_product
/	O
delta	B-Gene_or_gene_product
transcriptional	O
activity	O
,	O
resulting	O
in	O
reduced	O
PGC	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1alpha	I-Gene_or_gene_product
and	O
PDK4	B-Gene_or_gene_product
expression	O
,	O
and	O
an	O
enhanced	O
glucose	B-Simple_chemical
oxidation	O
rate	O
.	O

However	O
,	O
addition	O
of	O
the	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
inhibitor	O
parthenolide	B-Simple_chemical
to	O
these	O
cells	O
prevents	O
the	O
downregulation	O
of	O
PDK4	B-Gene_or_gene_product
expression	O
but	O
not	O
ERRalpha	B-Gene_or_gene_product
and	O
PPARbeta	B-Gene_or_gene_product
/	O
delta	B-Gene_or_gene_product
DNA	O
binding	O
activity	O
,	O
thus	O
suggesting	O
that	O
additional	O
transcription	O
factors	O
are	O
regulating	O
PDK4	B-Gene_or_gene_product
.	O

Interestingly	O
,	O
a	O
recent	O
study	O
has	O
demonstrated	O
that	O
the	O
transcription	O
factor	O
E2F1	B-Gene_or_gene_product
,	O
which	O
is	O
crucial	O
for	O
cell	O
cycle	O
control	O
,	O
may	O
regulate	O
PDK4	B-Gene_or_gene_product
expression	O
.	O

Given	O
that	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
may	O
antagonize	O
the	O
transcriptional	O
activity	O
of	O
E2F1	B-Gene_or_gene_product
in	O
cardiac	O
myocytes	O
,	O
we	O
sought	O
to	O
study	O
whether	O
inflammatory	O
processes	O
driven	O
by	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
can	O
downregulate	O
PDK4	B-Gene_or_gene_product
expression	O
in	O
human	O
cardiac	O
AC16	O
cells	O
through	O
E2F1	B-Gene_or_gene_product
inhibition	O
.	O

Protein	O
coimmunoprecipitation	O
indicated	O
that	O
PDK4	B-Gene_or_gene_product
downregulation	O
entailed	O
enhanced	O
physical	O
interaction	O
between	O
the	O
p65	B-Gene_or_gene_product
subunit	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
and	O
E2F1	B-Gene_or_gene_product
.	O

Chromatin	B-Cellular_component
immunoprecipitation	O
analyses	O
demonstrated	O
that	O
p65	B-Gene_or_gene_product
translocation	O
into	O
the	O
nucleus	B-Cellular_component
prevented	O
the	O
recruitment	O
of	O
E2F1	B-Gene_or_gene_product
to	O
the	O
PDK4	B-Gene_or_gene_product
promoter	O
and	O
its	O
subsequent	O
E2F1	B-Gene_or_gene_product
-	O
dependent	O
gene	O
transcription	O
.	O

Interestingly	O
,	O
the	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
inhibitor	O
parthenolide	B-Simple_chemical
prevented	O
the	O
inhibition	O
of	O
E2F1	B-Gene_or_gene_product
,	O
while	O
E2F1	B-Gene_or_gene_product
overexpression	O
reduced	O
interleukin	B-Gene_or_gene_product
expression	O
in	O
stimulated	O
cardiac	O
cells	O
.	O

Based	O
on	O
these	O
findings	O
,	O
we	O
propose	O
that	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
acts	O
as	O
a	O
molecular	O
switch	O
that	O
regulates	O
E2F1	B-Gene_or_gene_product
-	O
dependent	O
PDK4	B-Gene_or_gene_product
gene	O
transcription	O
.	O

Heteroarylketones	B-Simple_chemical
inhibit	O
astroglial	O
interleukin	B-Gene_or_gene_product
-	I-Gene_or_gene_product
6	I-Gene_or_gene_product
expression	O
via	O
a	O
STAT3	B-Gene_or_gene_product
/	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
signaling	O
pathway	O
.	O

BACKGROUND	O
:	O
Elevated	O
brain	O
levels	O
of	O
the	O
pleiotropic	O
cytokine	O
interleukin	B-Gene_or_gene_product
-	I-Gene_or_gene_product
6	I-Gene_or_gene_product
,	O
which	O
is	O
mainly	O
secreted	O
from	O
activated	O
local	O
astrocytes	O
,	O
contribute	O
to	O
pathological	O
events	O
including	O
neuroinflammation	O
and	O
neurodegeneration	O
.	O

Thus	O
,	O
inhibition	O
of	O
pathological	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
6	I-Gene_or_gene_product
expression	O
provides	O
a	O
rationale	O
strategy	O
for	O
targeting	O
the	O
onset	O
or	O
further	O
progression	O
of	O
neurological	O
disorders	O
including	O
Alzheimer	O
'	O
s	O
disease	O
,	O
multiple	O
sclerosis	O
,	O
Parkinson	O
'	O
s	O
disease	O
and	O
traumatic	O
brain	O
injury	O
.	O

The	O
purpose	O
of	O
this	O
study	O
was	O
to	O
identify	O
and	O
to	O
characterize	O
new	O
potent	O
inhibitors	O
of	O
astrocytic	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
6	I-Gene_or_gene_product
expression	O
for	O
further	O
therapeutic	O
development	O
of	O
novel	O
anti	O
-	O
inflammatory	O
and	O
neuroprotective	O
drugs	O
.	O

METHODS	O
:	O
Oncostatin	B-Gene_or_gene_product
M	I-Gene_or_gene_product
(	O
OSM	B-Gene_or_gene_product
)	O
-	O
treated	O
human	O
glioma	O
U343	O
cells	O
were	O
used	O
as	O
model	O
for	O
induction	O
of	O
astrocytic	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
6	I-Gene_or_gene_product
expression	O
.	O

This	O
model	O
was	O
characterized	O
by	O
immunoblotting	O
,	O
siRNA	O
technique	O
,	O
ELISA	O
and	O
qRT	O
-	O
PCR	O
and	O
used	O
to	O
screen	O
low	O
molecular	O
weight	O
compound	O
libraries	O
for	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
6	I-Gene_or_gene_product
-	O
lowering	O
effects	O
.	O

To	O
validate	O
bioactive	O
compounds	O
identified	O
from	O
library	O
screens	O
,	O
bacterial	O
lipopolysaccharide	O
was	O
used	O
to	O
induce	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
6	I-Gene_or_gene_product
expression	O
in	O
cultivated	O
primary	O
astrocytes	O
and	O
in	O
mice	O
in	O
vivo	O
.	O

To	O
dissect	O
underlying	O
molecular	O
mechanisms	O
,	O
protein	O
extracts	O
from	O
OSM	B-Gene_or_gene_product
-	O
treated	O
U343	O
cells	O
were	O
analyzed	O
by	O
phospho	O
-	O
specific	O
immunoblotting	O
and	O
immunocytochemistry	O
as	O
well	O
as	O
by	O
co	O
-	O
immunoprecipitation	O
.	O

RESULTS	O
:	O
OSM	B-Gene_or_gene_product
-	O
treatment	O
(	O
100	O
ng	O
/	O
ml	O
;	O
24	O
h	O
)	O
led	O
to	O
30	O
-	O
fold	O
increase	O
of	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
6	I-Gene_or_gene_product
secretion	O
from	O
U343	O
cells	O
.	O

The	O
temporal	O
profile	O
of	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
6	I-Gene_or_gene_product
mRNA	O
induction	O
displayed	O
a	O
biphasic	O
induction	O
pattern	O
with	O
peak	O
synthesis	O
at	O
1	O
h	O
(	O
6	O
.	O
5	O
-	O
fold	O
)	O
and	O
16	O
h	O
(	O
5	O
.	O
5	O
-	O
fold	O
)	O
post	O
stimulation	O
.	O

IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
6	I-Gene_or_gene_product
protein	O
release	O
did	O
not	O
show	O
that	O
biphasic	O
pattern	O
and	O
was	O
detected	O
as	O
early	O
as	O
3	O
h	O
post	O
stimulation	O
reaching	O
a	O
maximum	O
at	O
24	O
h	O
.	O

The	O
screen	O
of	O
compound	O
libraries	O
identified	O
a	O
set	O
of	O
heteroarylketones	B-Simple_chemical
(	O
HAKs	B-Simple_chemical
)	O
as	O
potent	O
inhibitors	O
of	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
6	I-Gene_or_gene_product
secretion	O
.	O

HAK	B-Simple_chemical
compounds	O
affected	O
the	O
second	O
peak	O
in	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
6	I-Gene_or_gene_product
mRNA	O
synthesis	O
,	O
whereas	O
the	O
first	O
peak	O
was	O
insensitive	O
to	O
HAK	B-Simple_chemical
treatment	O
.	O

HAK	B-Simple_chemical
compounds	O
also	O
suppressed	O
lipopolysaccharide	O
-	O
induced	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
6	I-Gene_or_gene_product
expression	O
in	O
primary	O
murine	O
astrocytes	O
as	O
well	O
as	O
in	O
brain	O
and	O
plasma	O
samples	O
from	O
lipopolysaccharide	O
-	O
treated	O
mice	O
.	O

Finally	O
,	O
HAK	B-Simple_chemical
compounds	O
were	O
demonstrated	O
to	O
specifically	O
suppress	O
the	O
OSM	B-Gene_or_gene_product
-	O
induced	O
phosphorylation	O
of	O
STAT3	B-Gene_or_gene_product
at	O
serine	B-Simple_chemical
727	I-Simple_chemical
and	O
the	O
physical	O
interaction	O
of	O
pSTAT3S727	B-Gene_or_gene_product
with	O
p65	B-Gene_or_gene_product
.	O

CONCLUSION	O
:	O
Heteroarylketone	B-Simple_chemical
compounds	O
are	O
potent	O
inhibitors	O
of	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
6	I-Gene_or_gene_product
expression	O
in	O
vitro	O
and	O
in	O
vivo	O
and	O
may	O
represent	O
a	O
new	O
class	O
of	O
potent	O
anti	O
-	O
inflammatory	O
and	O
neuroprotective	O
drugs	O
.	O

The	O
Ran	B-Gene_or_gene_product
-	O
binding	O
protein	O
RanBPM	B-Gene_or_gene_product
can	O
depress	O
the	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
pathway	O
by	O
interacting	O
with	O
TRAF6	B-Gene_or_gene_product
.	O

Ran	B-Gene_or_gene_product
-	I-Gene_or_gene_product
binding	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
in	I-Gene_or_gene_product
microtubule	I-Gene_or_gene_product
-	I-Gene_or_gene_product
organizing	I-Gene_or_gene_product
center	I-Gene_or_gene_product
(	O
RanBPM	B-Gene_or_gene_product
)	O
has	O
been	O
reported	O
to	O
interact	O
with	O
the	O
neurotrophin	B-Gene_or_gene_product
receptors	O
p75NTR	B-Gene_or_gene_product
and	O
TrkA	B-Gene_or_gene_product
,	O
meanwhile	O
p75NTR	B-Gene_or_gene_product
and	O
TrkA	B-Gene_or_gene_product
can	O
also	O
interact	O
with	O
TRAF6	B-Gene_or_gene_product
.	O

Whether	O
RanBPM	B-Gene_or_gene_product
interacts	O
directly	O
with	O
TRAF6	B-Gene_or_gene_product
has	O
not	O
yet	O
been	O
established	O
.	O

In	O
this	O
study	O
,	O
using	O
a	O
yeast	O
two	O
-	O
hybrid	O
system	O
and	O
glutathione	B-Gene_or_gene_product
-	I-Gene_or_gene_product
S	I-Gene_or_gene_product
:	I-Gene_or_gene_product
-	I-Gene_or_gene_product
transferase	I-Gene_or_gene_product
pull	O
-	O
down	O
assays	O
,	O
we	O
determined	O
that	O
RanBPM	B-Gene_or_gene_product
binds	O
to	O
the	O
TRAF6	B-Gene_or_gene_product
C	O
-	O
terminus	O
through	O
its	O
SPRY	O
motif	O
.	O

Complex	O
formation	O
between	O
overexpressed	O
RanBPM	B-Gene_or_gene_product
and	O
TRAF6	B-Gene_or_gene_product
was	O
also	O
confirmed	O
with	O
a	O
co	O
-	O
immunoprecipitation	O
assay	O
,	O
laser	O
scanning	O
confocal	O
and	O
fluorescence	O
resonance	O
energy	O
transfer	O
.	O

Additional	O
co	O
-	O
immunoprecipitation	O
experiments	O
verified	O
that	O
endogenous	O
RanBPM	B-Gene_or_gene_product
and	O
TRAF6	B-Gene_or_gene_product
interact	O
in	O
several	O
mammalian	O
cell	O
lines	O
.	O

A	O
series	O
of	O
experiments	O
revealed	O
that	O
RanBPM	B-Gene_or_gene_product
influences	O
TRAF6	B-Gene_or_gene_product
ubiquitination	O
and	O
the	O
TRAF6	B-Gene_or_gene_product
-	O
triggered	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
signaling	O
pathway	O
through	O
RanBPM	B-Gene_or_gene_product
'	O
s	O
interaction	O
with	O
TRAF6	B-Gene_or_gene_product
.	O

These	O
data	O
suggest	O
that	O
RanBPM	B-Gene_or_gene_product
participates	O
in	O
gene	O
transcription	O
by	O
binding	O
to	O
TRAF6	B-Gene_or_gene_product
.	O

Involvement	O
of	O
JNK	B-Gene_or_gene_product
and	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
pathways	O
in	O
lipopolysaccharide	B-Simple_chemical
(	O
LPS	B-Simple_chemical
)	O
-	O
induced	O
BAG3	B-Gene_or_gene_product
expression	O
in	O
human	O
monocytic	O
cells	O
.	O

Lipopolysaccharide	B-Simple_chemical
(	O
LPS	B-Simple_chemical
)	O
is	O
an	O
outer	B-Cellular_component
-	I-Cellular_component
membrane	I-Cellular_component
glycolipid	B-Simple_chemical
component	O
of	O
Gram	O
-	O
negative	O
bacteria	O
known	O
for	O
its	O
fervent	O
ability	O
to	O
activate	O
monocytic	O
cells	O
and	O
for	O
its	O
potent	O
proinflammatory	O
capabilities	O
.	O

Bcl	B-Gene_or_gene_product
-	I-Gene_or_gene_product
2	I-Gene_or_gene_product
-	I-Gene_or_gene_product
associated	I-Gene_or_gene_product
athanogene	I-Gene_or_gene_product
3	I-Gene_or_gene_product
(	O
BAG3	B-Gene_or_gene_product
)	O
is	O
a	O
survival	O
protein	O
that	O
has	O
been	O
shown	O
to	O
be	O
stimulated	O
during	O
cell	O
response	O
to	O
stressful	O
conditions	O
,	O
such	O
as	O
exposure	O
to	O
high	O
temperature	O
,	O
heavy	O
metals	O
,	O
proteasome	O
inhibition	O
,	O
and	O
human	O
immunodeficiency	O
virus	O
1	O
(	O
HIV	O
-	O
1	O
)	O
infection	O
.	O

In	O
addition	O
,	O
BAG3	B-Gene_or_gene_product
regulates	O
replication	O
of	O
Varicella	O
-	O
Zoster	O
Virus	O
(	O
VZV	O
)	O
and	O
Herpes	O
Simplex	O
Virus	O
(	O
HSV	O
)	O
replication	O
,	O
suggesting	O
that	O
BAG3	B-Gene_or_gene_product
could	O
participate	O
in	O
the	O
host	O
response	O
to	O
infection	O
.	O

In	O
the	O
current	O
study	O
,	O
we	O
found	O
that	O
LPS	B-Simple_chemical
increased	O
the	O
expression	O
of	O
BAG3	B-Gene_or_gene_product
in	O
a	O
dose	O
-	O
and	O
time	O
-	O
dependent	O
manner	O
.	O

Actinomycin	B-Simple_chemical
D	I-Simple_chemical
completely	O
blocked	O
the	O
LPS	B-Simple_chemical
-	O
induced	O
BAG3	B-Gene_or_gene_product
accumulation	O
,	O
as	O
well	O
as	O
LPS	B-Simple_chemical
activated	O
the	O
proximal	O
promoter	O
of	O
BAG3	B-Gene_or_gene_product
gene	O
,	O
supported	O
that	O
the	O
induction	O
by	O
LPS	B-Simple_chemical
occurred	O
at	O
the	O
level	O
of	O
gene	O
transcription	O
.	O

LPS	B-Simple_chemical
-	O
induced	O
BAG3	B-Gene_or_gene_product
expression	O
was	O
blocked	O
by	O
JNK	B-Gene_or_gene_product
or	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
inhibition	O
,	O
suggesting	O
that	O
JNK	B-Gene_or_gene_product
and	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
pathways	O
participated	O
in	O
BAG3	B-Gene_or_gene_product
induction	O
by	O
LPS	B-Simple_chemical
.	O

In	O
addition	O
,	O
we	O
also	O
found	O
that	O
induction	O
of	O
BAG3	B-Gene_or_gene_product
was	O
implicated	O
in	O
monocytic	O
cell	O
adhesion	O
to	O
extracellular	B-Cellular_component
matrix	I-Cellular_component
induced	O
by	O
LPS	B-Simple_chemical
.	O

Overall	O
,	O
the	O
data	O
support	O
that	O
BAG3	B-Gene_or_gene_product
is	O
induced	O
by	O
LPS	B-Simple_chemical
via	O
JNK	B-Gene_or_gene_product
and	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
-	O
dependent	O
signals	O
,	O
and	O
involved	O
in	O
monocytic	O
cell	O
-	O
extracellular	B-Cellular_component
matrix	I-Cellular_component
interaction	O
,	O
suggesting	O
that	O
BAG3	B-Gene_or_gene_product
may	O
have	O
a	O
role	O
in	O
the	O
host	O
response	O
to	O
LPS	B-Simple_chemical
stimulation	O
.	O

p53	B-Gene_or_gene_product
mutants	O
induce	O
transcription	O
of	O
NF	B-Complex
-	I-Complex
kappaB2	I-Complex
in	O
H1299	O
cells	O
through	O
CBP	B-Gene_or_gene_product
and	O
STAT	B-Gene_or_gene_product
binding	O
on	O
the	O
NF	B-Complex
-	I-Complex
kappaB2	I-Complex
promoter	O
and	O
gain	O
of	O
function	O
activity	O
.	O

Cancer	O
cells	O
with	O
p53	B-Gene_or_gene_product
mutations	O
,	O
in	O
general	O
,	O
grow	O
more	O
aggressively	O
than	O
those	O
with	O
wild	O
-	O
type	O
p53	B-Gene_or_gene_product
and	O
show	O
"	O
gain	O
of	O
function	O
"	O
(	O
GOF	O
)	O
phenotypes	O
such	O
as	O
increased	O
growth	O
rate	O
,	O
enhanced	O
resistance	O
to	O
chemotherapeutic	O
drugs	O
,	O
increased	O
cell	O
motility	O
and	O
tumorigenicity	O
;	O
although	O
the	O
mechanism	O
for	O
this	O
function	O
remains	O
unknown	O
.	O

In	O
this	O
communication	O
we	O
report	O
that	O
p53	B-Gene_or_gene_product
-	O
mediated	O
NF	B-Complex
-	I-Complex
kappaB2	I-Complex
up	O
-	O
regulation	O
significantly	O
contributes	O
to	O
the	O
aggressive	O
oncogenic	O
behavior	O
of	O
cancer	O
cells	O
.	O

Lowering	O
the	O
level	O
of	O
mutant	O
p53	B-Gene_or_gene_product
in	O
a	O
number	O
of	O
cancer	O
cell	O
lines	O
resulted	O
in	O
a	O
loss	O
of	O
GOF	O
phenotypes	O
directly	O
implicating	O
p53	B-Gene_or_gene_product
mutants	O
in	O
the	O
process	O
.	O

RNAi	O
against	O
NF	B-Complex
-	I-Complex
kappaB2	I-Complex
in	O
naturally	O
occurring	O
cancer	O
cell	O
lines	O
also	O
lowers	O
GOF	O
activities	O
.	O

In	O
H1299	O
cells	O
expressing	O
mutant	O
p53	B-Gene_or_gene_product
,	O
chromatin	B-Cellular_component
immunoprecipitation	O
(	O
ChIP	O
)	O
assays	O
indicate	O
that	O
mutant	O
p53	B-Gene_or_gene_product
induces	O
histone	B-Gene_or_gene_product
acetylation	O
at	O
specific	O
sites	O
on	O
the	O
regulatory	O
regions	O
of	O
its	O
target	O
genes	O
.	O

ChIP	O
assays	O
using	O
antibodies	O
against	O
transcription	O
factors	O
putatively	O
capable	O
of	O
interacting	O
with	O
the	O
NF	B-Complex
-	I-Complex
kappaB2	I-Complex
promoter	O
show	O
increased	O
interaction	O
of	O
CBP	B-Gene_or_gene_product
and	O
STAT2	B-Gene_or_gene_product
in	O
the	O
presence	O
of	O
mutant	O
p53	B-Gene_or_gene_product
.	O

Thus	O
,	O
we	O
propose	O
that	O
in	O
H1299	O
cells	O
,	O
mutant	O
p53	B-Gene_or_gene_product
elevates	O
expression	O
of	O
genes	O
capable	O
of	O
enhancing	O
cell	O
proliferation	O
,	O
motility	O
,	O
and	O
tumorigenicity	O
by	O
inducing	O
acetylation	O
of	O
histones	O
via	O
recruitment	O
of	O
CBP	B-Gene_or_gene_product
and	O
STAT2	B-Gene_or_gene_product
on	O
the	O
promoters	O
causing	O
CBP	B-Gene_or_gene_product
-	O
mediated	O
histone	B-Gene_or_gene_product
acetylation	O
.	O

New	O
molecular	O
bridge	O
between	O
RelA	B-Gene_or_gene_product
/	O
p65	B-Gene_or_gene_product
and	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
target	O
genes	O
via	O
histone	B-Gene_or_gene_product
acetyltransferase	I-Gene_or_gene_product
TIP60	B-Gene_or_gene_product
cofactor	O
.	O

The	O
nuclear	B-Complex
factor	I-Complex
-	I-Complex
kappaB	I-Complex
(	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
)	O
family	O
is	O
involved	O
in	O
the	O
expressions	O
of	O
numerous	O
genes	O
,	O
in	O
development	O
,	O
apoptosis	O
,	O
inflammatory	O
responses	O
,	O
and	O
oncogenesis	O
.	O

In	O
this	O
study	O
we	O
identified	O
four	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
target	O
genes	O
that	O
are	O
modulated	O
by	O
TIP60	B-Gene_or_gene_product
.	O

We	O
also	O
found	O
that	O
TIP60	B-Gene_or_gene_product
interacts	O
with	O
the	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
RelA	B-Gene_or_gene_product
/	O
p65	B-Gene_or_gene_product
subunit	O
and	O
increases	O
its	O
transcriptional	O
activity	O
through	O
protein	O
-	O
protein	O
interaction	O
.	O

Although	O
TIP60	B-Gene_or_gene_product
binds	O
with	O
RelA	B-Gene_or_gene_product
/	O
p65	B-Gene_or_gene_product
using	O
its	O
histone	B-Gene_or_gene_product
acetyltransferase	I-Gene_or_gene_product
domain	O
,	O
TIP60	B-Gene_or_gene_product
does	O
not	O
directly	O
acetylate	O
RelA	B-Gene_or_gene_product
/	O
p65	B-Gene_or_gene_product
.	O

However	O
,	O
TIP60	B-Gene_or_gene_product
maintained	O
acetylated	O
Lys	B-Simple_chemical
-	I-Simple_chemical
310	I-Simple_chemical
RelA	B-Gene_or_gene_product
/	O
p65	B-Gene_or_gene_product
levels	O
in	O
the	O
TNF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
-	O
dependent	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
signaling	O
pathway	O
.	O

In	O
chromatin	B-Cellular_component
immunoprecipitation	O
assay	O
,	O
TIP60	B-Gene_or_gene_product
was	O
primarily	O
recruited	O
to	O
the	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
6	I-Gene_or_gene_product
,	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
8	I-Gene_or_gene_product
,	O
C	B-Gene_or_gene_product
-	I-Gene_or_gene_product
IAP1	I-Gene_or_gene_product
,	O
and	O
XIAP	B-Gene_or_gene_product
promoters	O
in	O
TNF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
stimulation	O
followed	O
by	O
acetylation	O
of	O
histones	B-Gene_or_gene_product
H3	I-Gene_or_gene_product
and	O
H4	B-Gene_or_gene_product
.	O

Chromatin	B-Cellular_component
remodeling	O
by	O
TIP60	B-Gene_or_gene_product
involved	O
the	O
sequential	O
recruitment	O
of	O
acetyl	B-Simple_chemical
-	I-Simple_chemical
Lys	I-Simple_chemical
-	I-Simple_chemical
310	I-Simple_chemical
RelA	B-Gene_or_gene_product
/	O
p65	B-Gene_or_gene_product
to	O
its	O
target	O
gene	O
promoters	O
.	O

Furthermore	O
,	O
we	O
showed	O
that	O
up	O
-	O
regulated	O
TIP60	B-Gene_or_gene_product
expression	O
was	O
correlated	O
with	O
acetyl	B-Simple_chemical
-	I-Simple_chemical
Lys	I-Simple_chemical
-	I-Simple_chemical
310	I-Simple_chemical
RelA	B-Gene_or_gene_product
/	O
p65	B-Gene_or_gene_product
expressions	O
in	O
hepatocarcinoma	O
tissues	O
.	O

Taken	O
together	O
these	O
results	O
suggest	O
that	O
TIP60	B-Gene_or_gene_product
is	O
involved	O
in	O
the	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
pathway	O
through	O
protein	O
interaction	O
with	O
RelA	B-Gene_or_gene_product
/	O
p65	B-Gene_or_gene_product
and	O
that	O
it	O
modulates	O
the	O
transcriptional	O
activity	O
of	O
RelA	B-Gene_or_gene_product
/	O
p65	B-Gene_or_gene_product
in	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
-	O
dependent	O
gene	O
expression	O
.	O

Interaction	O
with	O
CREB	B-Gene_or_gene_product
binding	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
modulates	O
the	O
activities	O
of	O
Nrf2	B-Gene_or_gene_product
and	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
in	O
cystic	O
fibrosis	O
airway	O
epithelial	O
cells	O
.	O

Cystic	O
fibrosis	O
(	O
CF	O
)	O
is	O
characterized	O
by	O
inflammatory	O
lung	O
disease	O
that	O
significantly	O
contributes	O
to	O
morbidity	O
and	O
mortality	O
.	O

Airway	O
epithelial	O
cells	O
play	O
a	O
role	O
in	O
the	O
inflammatory	O
signaling	O
in	O
CF	O
and	O
have	O
been	O
reported	O
to	O
exhibit	O
a	O
number	O
of	O
dysfunctions	O
in	O
signaling	O
cascades	O
that	O
modulate	O
inflammation	O
.	O

Previously	O
,	O
we	O
reported	O
that	O
the	O
activity	O
of	O
nuclear	B-Gene_or_gene_product
factor	I-Gene_or_gene_product
erythroid	I-Gene_or_gene_product
-	I-Gene_or_gene_product
derived	I-Gene_or_gene_product
-	I-Gene_or_gene_product
like	I-Gene_or_gene_product
2	I-Gene_or_gene_product
(	O
Nrf2	B-Gene_or_gene_product
)	O
,	O
a	O
transcription	O
factor	O
that	O
regulates	O
antioxidant	O
and	O
cytoprotective	O
protein	O
expression	O
,	O
is	O
diminished	O
in	O
CF	O
epithelia	O
(	O
7	O
)	O
.	O

In	O
this	O
report	O
,	O
we	O
examined	O
the	O
mechanism	O
of	O
Nrf2	B-Gene_or_gene_product
dysregulation	O
in	O
vitro	O
in	O
human	O
airway	O
epithelial	O
cell	O
lines	O
and	O
primary	O
cells	O
and	O
in	O
vivo	O
in	O
nasal	O
epithelia	O
excised	O
from	O
DeltaF508	O
CF	O
mutant	O
mice	O
.	O

We	O
found	O
that	O
cAMP	B-Simple_chemical
-	O
mediated	O
signaling	O
markedly	O
reduces	O
Nrf2	B-Gene_or_gene_product
activity	O
in	O
CF	O
vs	O
.	O
non	O
-	O
CF	O
cells	O
.	O

Rp	B-Simple_chemical
-	I-Simple_chemical
cAMPS	I-Simple_chemical
,	O
a	O
cAMP	B-Simple_chemical
competitor	O
,	O
significantly	O
corrected	O
Nrf2	B-Gene_or_gene_product
activity	O
in	O
CF	O
cells	O
,	O
predominantly	O
by	O
increasing	O
the	O
nuclear	B-Cellular_component
accumulation	O
of	O
the	O
transcription	O
factor	O
.	O

Furthermore	O
,	O
we	O
found	O
that	O
Rp	B-Simple_chemical
-	I-Simple_chemical
cAMPS	I-Simple_chemical
significantly	O
decreased	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
activation	O
following	O
inflammatory	O
stimulation	O
of	O
CF	O
cells	O
.	O

Further	O
investigation	O
revealed	O
that	O
Nrf2	B-Gene_or_gene_product
and	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
compete	O
for	O
the	O
transcriptional	O
coactivator	O
cAMP	B-Gene_or_gene_product
responsive	I-Gene_or_gene_product
element	I-Gene_or_gene_product
-	I-Gene_or_gene_product
binding	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
(	I-Gene_or_gene_product
CREB	I-Gene_or_gene_product
)	I-Gene_or_gene_product
binding	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
(	O
CBP	B-Gene_or_gene_product
)	O
and	O
that	O
Rp	B-Simple_chemical
-	I-Simple_chemical
cAMPS	I-Simple_chemical
shifts	O
CBP	B-Gene_or_gene_product
association	O
in	O
favor	O
of	O
Nrf2	B-Gene_or_gene_product
.	O

Thus	O
our	O
findings	O
provide	O
a	O
link	O
between	O
feedback	O
to	O
CF	O
transmembrane	O
regulator	O
dysfunction	O
and	O
dysregulation	O
of	O
an	O
inflammatory	O
signaling	O
pathway	O
that	O
modulates	O
the	O
coordinated	O
activities	O
of	O
Nrf2	B-Gene_or_gene_product
and	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
.	O

Furthermore	O
,	O
our	O
studies	O
suggest	O
that	O
strategies	O
that	O
shift	O
CBP	B-Gene_or_gene_product
association	O
away	O
from	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
and	O
toward	O
Nrf2	B-Gene_or_gene_product
could	O
have	O
potential	O
therapeutic	O
efficacy	O
for	O
reducing	O
inflammation	O
in	O
patients	O
with	O
CF	O
.	O

E3	B-Gene_or_gene_product
-	I-Gene_or_gene_product
14	I-Gene_or_gene_product
.	I-Gene_or_gene_product
7K	I-Gene_or_gene_product
is	O
recruited	O
to	O
TNF	B-Complex
-	I-Complex
receptor	I-Complex
1	I-Complex
and	O
blocks	O
TNF	B-Gene_or_gene_product
cytolysis	O
independent	O
from	O
interaction	O
with	O
optineurin	B-Gene_or_gene_product
.	O

Escape	O
from	O
the	O
host	O
immune	O
system	O
is	O
essential	O
for	O
intracellular	B-Cellular_component
pathogens	O
.	O

The	O
adenoviral	O
protein	O
E3	B-Gene_or_gene_product
-	I-Gene_or_gene_product
14	I-Gene_or_gene_product
.	I-Gene_or_gene_product
7K	I-Gene_or_gene_product
(	O
14	B-Gene_or_gene_product
.	I-Gene_or_gene_product
7K	I-Gene_or_gene_product
)	O
is	O
known	O
as	O
a	O
general	O
inhibitor	O
of	O
tumor	B-Gene_or_gene_product
necrosis	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
(	O
TNF	B-Gene_or_gene_product
)	O
-	O
induced	O
apoptosis	O
.	O

It	O
efficiently	O
blocks	O
TNF	B-Complex
-	I-Complex
receptor	I-Complex
1	I-Complex
(	O
TNFR1	B-Complex
)	O
internalization	O
but	O
the	O
underlying	O
molecular	O
mechanism	O
still	O
remains	O
elusive	O
.	O

Direct	O
interaction	O
of	O
14	B-Gene_or_gene_product
.	I-Gene_or_gene_product
7K	I-Gene_or_gene_product
and	O
/	O
or	O
associated	O
proteins	O
with	O
the	O
TNFR1	B-Complex
complex	O
has	O
been	O
discussed	O
although	O
to	O
date	O
not	O
proven	O
.	O

In	O
our	O
study	O
,	O
we	O
provide	O
for	O
the	O
first	O
time	O
evidence	O
for	O
recruitment	O
of	O
14	B-Gene_or_gene_product
.	I-Gene_or_gene_product
7K	I-Gene_or_gene_product
and	O
the	O
14	B-Gene_or_gene_product
.	I-Gene_or_gene_product
7K	I-Gene_or_gene_product
interacting	O
protein	O
optineurin	B-Gene_or_gene_product
to	O
TNFR1	B-Complex
.	O

Various	O
functions	O
have	O
been	O
implicated	O
for	O
optineurin	B-Gene_or_gene_product
such	O
as	O
regulation	O
of	O
receptor	O
endocytosis	O
,	O
vesicle	B-Cellular_component
trafficking	O
,	O
regulation	O
of	O
the	O
nuclear	B-Complex
factor	I-Complex
kappaB	I-Complex
(	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
)	O
pathway	O
and	O
antiviral	O
signaling	O
.	O

We	O
therefore	O
hypothesized	O
that	O
binding	O
of	O
optineurin	B-Gene_or_gene_product
to	O
14	B-Gene_or_gene_product
.	I-Gene_or_gene_product
7K	I-Gene_or_gene_product
and	O
recruitment	O
of	O
both	O
proteins	O
to	O
the	O
TNFR1	B-Complex
complex	O
is	O
essential	O
for	O
protection	O
against	O
TNF	B-Gene_or_gene_product
-	O
induced	O
cytotoxic	O
effects	O
.	O

To	O
precisely	O
dissect	O
the	O
individual	O
role	O
of	O
14	B-Gene_or_gene_product
.	I-Gene_or_gene_product
7K	I-Gene_or_gene_product
and	O
optineurin	B-Gene_or_gene_product
,	O
we	O
generated	O
and	O
characterized	O
a	O
14	B-Gene_or_gene_product
.	I-Gene_or_gene_product
7K	I-Gene_or_gene_product
mutant	O
that	O
does	O
not	O
confer	O
TNF	B-Gene_or_gene_product
-	O
resistance	O
but	O
is	O
still	O
able	O
to	O
interact	O
with	O
optineurin	B-Gene_or_gene_product
.	O

In	O
H1299	O
and	O
KB	O
cells	O
expressing	O
14	B-Gene_or_gene_product
.	I-Gene_or_gene_product
7K	I-Gene_or_gene_product
wild	O
-	O
type	O
protein	O
,	O
neither	O
decrease	O
in	O
cell	O
viability	O
nor	O
cleavage	O
of	O
caspases	O
was	O
observed	O
upon	O
stimulation	O
with	O
TNF	B-Gene_or_gene_product
.	O

In	O
sharp	O
contrast	O
,	O
cells	O
expressing	O
the	O
non	O
-	O
protective	O
mutant	O
of	O
14	B-Gene_or_gene_product
.	I-Gene_or_gene_product
7K	I-Gene_or_gene_product
displayed	O
reduced	O
viability	O
and	O
cleavage	O
of	O
initiator	O
and	O
effector	O
caspases	O
upon	O
TNF	B-Gene_or_gene_product
treatment	O
,	O
indicating	O
ongoing	O
apoptotic	O
cell	O
death	O
.	O

Knockdown	O
of	O
optineurin	B-Gene_or_gene_product
in	O
14	B-Gene_or_gene_product
.	I-Gene_or_gene_product
7K	I-Gene_or_gene_product
expressing	O
cells	O
did	O
not	O
alter	O
the	O
protective	O
effect	O
as	O
measured	O
by	O
cell	O
viability	O
and	O
caspase	B-Gene_or_gene_product
activation	O
.	O

Taken	O
together	O
,	O
we	O
conclude	O
that	O
optineurin	B-Gene_or_gene_product
despite	O
its	O
substantial	O
role	O
in	O
vesicular	O
trafficking	O
,	O
endocytosis	O
of	O
cell	B-Cellular_component
surface	I-Cellular_component
receptors	O
and	O
recruitment	O
to	O
the	O
TNFR1	B-Complex
complex	O
is	O
dispensable	O
for	O
the	O
14	B-Gene_or_gene_product
.	I-Gene_or_gene_product
7K	I-Gene_or_gene_product
-	O
mediated	O
protection	O
against	O
TNF	B-Gene_or_gene_product
-	O
induced	O
apoptosis	O
.	O

HTLV	O
-	O
1	O
tax	B-Gene_or_gene_product
-	O
induced	O
rapid	O
senescence	O
is	O
driven	O
by	O
the	O
transcriptional	O
activity	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
and	O
depends	O
on	O
chronically	O
activated	O
IKKalpha	B-Gene_or_gene_product
and	O
p65	B-Gene_or_gene_product
/	O
RelA	B-Gene_or_gene_product
.	O

The	O
HTLV	O
-	O
1	O
oncoprotein	O
Tax	B-Gene_or_gene_product
is	O
a	O
potent	O
activator	O
of	O
classical	O
and	O
alternative	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
pathways	O
and	O
is	O
thought	O
to	O
promote	O
cell	O
proliferation	O
and	O
transformation	O
via	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
activation	O
.	O

We	O
showed	O
recently	O
that	O
hyperactivation	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
by	O
Tax	B-Gene_or_gene_product
triggers	O
a	O
cellular	O
senescence	O
response	O
(	O
H	O
.	O
Zhi	O
et	O
al	O
.	O
,	O
PLoS	O
Pathog	O
.	O
7	O
:	O
e1002025	O
,	O
2011	O
)	O
.	O

Inhibition	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
activation	O
by	O
expression	O
of	O
I	B-Gene_or_gene_product
-	I-Gene_or_gene_product
kappaBalpha	I-Gene_or_gene_product
superrepressor	O
or	O
by	O
small	O
hairpin	O
RNA	O
(	O
shRNA	O
)	O
-	O
mediated	O
knockdown	O
of	O
p65	B-Gene_or_gene_product
/	O
RelA	B-Gene_or_gene_product
rescues	O
cells	O
from	O
Tax	B-Gene_or_gene_product
-	O
induced	O
rapid	O
senescence	O
(	O
Tax	B-Gene_or_gene_product
-	O
IRS	O
)	O
.	O

Here	O
we	O
demonstrate	O
that	O
Tax	B-Gene_or_gene_product
-	O
IRS	O
is	O
driven	O
by	O
the	O
transcriptional	O
activity	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
.	O

Knockdown	O
of	O
IKKgamma	B-Gene_or_gene_product
,	O
the	O
primary	O
Tax	B-Gene_or_gene_product
target	O
,	O
by	O
shRNAs	O
abrogated	O
Tax	B-Gene_or_gene_product
-	O
mediated	O
activation	O
of	O
both	O
classical	O
and	O
alternative	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
pathways	O
and	O
rendered	O
knockdown	O
cells	O
resistant	O
to	O
Tax	B-Gene_or_gene_product
-	O
IRS	O
.	O

Consistent	O
with	O
a	O
critical	O
role	O
of	O
IKKalpha	B-Gene_or_gene_product
in	O
the	O
transcriptional	O
activity	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
,	O
IKKalpha	B-Gene_or_gene_product
deficiency	O
drastically	O
decreased	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
trans	O
-	O
activation	O
by	O
Tax	B-Gene_or_gene_product
,	O
although	O
it	O
only	O
modestly	O
reduced	O
Tax	B-Gene_or_gene_product
-	O
mediated	O
I	B-Gene_or_gene_product
-	I-Gene_or_gene_product
kappaBalpha	I-Gene_or_gene_product
degradation	O
and	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
nuclear	B-Cellular_component
localization	O
.	O

In	O
contrast	O
,	O
although	O
IKKbeta	B-Gene_or_gene_product
knockdown	O
attenuated	O
Tax	B-Gene_or_gene_product
-	O
induced	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
transcriptional	O
activation	O
,	O
the	O
residual	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
activation	O
in	O
IKKbeta	B-Gene_or_gene_product
-	O
deficient	O
cells	O
was	O
sufficient	O
to	O
trigger	O
Tax	B-Gene_or_gene_product
-	O
IRS	O
.	O

Importantly	O
,	O
the	O
phenotypes	O
of	O
NIK	B-Gene_or_gene_product
and	O
TAK1	B-Gene_or_gene_product
knockdown	O
were	O
similar	O
to	O
those	O
of	O
IKKalpha	B-Gene_or_gene_product
and	O
IKKbeta	B-Gene_or_gene_product
knockdown	O
,	O
respectively	O
.	O

Finally	O
,	O
double	O
knockdown	O
of	O
RelB	B-Gene_or_gene_product
and	O
p100	B-Gene_or_gene_product
had	O
a	O
minor	O
effect	O
on	O
senescence	O
induction	O
by	O
Tax	B-Gene_or_gene_product
.	O

These	O
data	O
suggest	O
that	O
Tax	B-Gene_or_gene_product
,	O
through	O
its	O
interaction	O
with	O
IKKgamma	B-Gene_or_gene_product
,	O
helps	O
recruit	O
NIK	B-Gene_or_gene_product
and	O
TAK1	B-Gene_or_gene_product
for	O
IKKalpha	B-Gene_or_gene_product
and	O
IKKbeta	B-Gene_or_gene_product
activation	O
,	O
respectively	O
.	O

In	O
the	O
presence	O
of	O
Tax	B-Gene_or_gene_product
,	O
the	O
delineation	O
between	O
the	O
classical	O
and	O
alternative	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
pathways	O
becomes	O
obscured	O
.	O

The	O
senescence	O
checkpoint	O
triggered	O
by	O
Tax	B-Gene_or_gene_product
is	O
driven	O
by	O
the	O
transcriptional	O
activity	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
,	O
which	O
depends	O
on	O
activated	O
IKKalpha	B-Gene_or_gene_product
and	O
p65	B-Gene_or_gene_product
/	O
RelA	B-Gene_or_gene_product
.	O

Propylene	B-Simple_chemical
glycol	I-Simple_chemical
ingestion	O
causes	O
D	O
-	O
lactic	O
acidosis	O
.	O

Propylene	B-Simple_chemical
glycol	I-Simple_chemical
(	O
1	B-Simple_chemical
,	I-Simple_chemical
2	I-Simple_chemical
-	I-Simple_chemical
propanediol	I-Simple_chemical
)	O
is	O
a	O
solvent	O
in	O
numerous	O
pharmaceuticals	O
and	O
a	O
major	O
preservative	O
and	O
source	O
of	O
carbohydrates	B-Simple_chemical
in	O
processed	O
foods	O
.	O

In	O
mammals	O
,	O
propylene	B-Simple_chemical
glycol	I-Simple_chemical
is	O
metabolized	O
similar	O
to	O
ethanol	B-Simple_chemical
,	O
proceeding	O
via	O
hepatic	O
alcohol	B-Gene_or_gene_product
and	I-Gene_or_gene_product
aldehyde	I-Gene_or_gene_product
dehydrogenases	I-Gene_or_gene_product
to	O
lactate	B-Simple_chemical
,	O
which	O
can	O
then	O
enter	O
gluconeogenesis	O
.	O

We	O
observed	O
that	O
cats	O
ingesting	O
1	O
.	O
6	O
gm	O
of	O
propylene	B-Simple_chemical
glycol	I-Simple_chemical
/	O
kg	O
body	O
weight	O
/	O
day	O
developed	O
increased	O
anion	B-Simple_chemical
gap	O
.	O

To	O
investigate	O
this	O
further	O
,	O
we	O
measured	O
D	B-Simple_chemical
-	I-Simple_chemical
and	O
L	B-Simple_chemical
-	I-Simple_chemical
lactate	I-Simple_chemical
concentrations	O
in	O
these	O
cats	O
;	O
we	O
also	O
measured	O
D	B-Simple_chemical
-	I-Simple_chemical
lactate	I-Simple_chemical
in	O
cats	O
ingesting	O
high	O
doses	O
of	O
propylene	B-Simple_chemical
glycol	I-Simple_chemical
(	O
8	O
.	O
0	O
gm	O
/	O
kg	O
)	O
.	O

While	O
L	B-Simple_chemical
-	I-Simple_chemical
lactate	I-Simple_chemical
actually	O
decreased	O
throughout	O
the	O
35	O
-	O
day	O
course	O
of	O
propylene	B-Simple_chemical
glycol	I-Simple_chemical
feeding	O
,	O
D	B-Simple_chemical
-	I-Simple_chemical
lactate	I-Simple_chemical
levels	O
were	O
significantly	O
increased	O
on	O
a	O
dose	O
-	O
dependent	O
basis	O
and	O
correlated	O
positively	O
with	O
anion	B-Simple_chemical
gap	O
.	O

In	O
cats	O
ingesting	O
the	O
high	O
dose	O
of	O
propylene	B-Simple_chemical
glycol	I-Simple_chemical
,	O
D	B-Simple_chemical
-	I-Simple_chemical
lactate	I-Simple_chemical
concentrations	O
were	O
as	O
high	O
as	O
7	O
mmol	O
/	O
liter	O
,	O
levels	O
associated	O
with	O
encephalopathy	O
in	O
humans	O
.	O

Indeed	O
,	O
this	O
group	O
of	O
cats	O
developed	O
depression	O
and	O
ataxia	O
,	O
consistent	O
with	O
intoxication	O
by	O
D	B-Simple_chemical
-	I-Simple_chemical
lactate	I-Simple_chemical
.	O

These	O
findings	O
are	O
significant	O
not	O
only	O
for	O
animals	O
ingesting	O
diets	O
which	O
contain	O
propylene	B-Simple_chemical
glycol	I-Simple_chemical
,	O
but	O
for	O
humans	O
who	O
receive	O
propylene	B-Simple_chemical
glycol	I-Simple_chemical
-	O
containing	O
medications	O
.	O

Visfatin	B-Gene_or_gene_product
induces	O
stromal	B-Gene_or_gene_product
cell	I-Gene_or_gene_product
-	I-Gene_or_gene_product
derived	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
expression	O
by	O
beta1	B-Gene_or_gene_product
integrin	I-Gene_or_gene_product
signaling	O
in	O
colorectal	O
cancer	O
cells	O
.	O

Obesity	O
has	O
been	O
shown	O
to	O
be	O
associated	O
with	O
the	O
risk	O
of	O
colorectal	O
cancer	O
(	O
CRC	O
)	O
.	O

Adipokines	O
produced	O
by	O
the	O
adipose	O
tissue	O
are	O
linked	O
to	O
some	O
malignancies	O
,	O
including	O
CRC	O
.	O

Visfatin	B-Gene_or_gene_product
is	O
an	O
adipokine	O
shown	O
to	O
be	O
a	O
biomarker	O
of	O
CRC	O
malignant	O
potential	O
.	O

In	O
addition	O
,	O
the	O
stromal	B-Gene_or_gene_product
cell	I-Gene_or_gene_product
-	I-Gene_or_gene_product
derived	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
(	O
SDF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
)	O
has	O
been	O
reported	O
to	O
play	O
a	O
role	O
in	O
CRC	O
progression	O
.	O

Although	O
the	O
relationship	O
between	O
visfatin	B-Gene_or_gene_product
and	O
CRC	O
has	O
been	O
established	O
,	O
the	O
underlying	O
mechanism	O
has	O
not	O
been	O
clarified	O
.	O

We	O
investigated	O
the	O
molecular	O
mechanism	O
governing	O
the	O
interaction	O
between	O
visfatin	B-Gene_or_gene_product
stimulation	O
and	O
SDF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
expression	O
in	O
human	O
CRC	O
cell	O
lines	O
.	O

We	O
found	O
that	O
visfatin	B-Gene_or_gene_product
stimulation	O
led	O
to	O
an	O
increase	O
in	O
the	O
expression	O
and	O
secretion	O
of	O
SDF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
in	O
CRC	O
DLD	O
-	O
1	O
and	O
SW48	O
cells	O
.	O

Experiments	O
involving	O
specific	O
inhibitors	O
and	O
small	O
interfering	O
RNA	O
demonstrated	O
that	O
the	O
activation	O
of	O
ERK	B-Gene_or_gene_product
and	O
p38	B-Gene_or_gene_product
mitogen	B-Gene_or_gene_product
-	I-Gene_or_gene_product
activated	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
(	O
MAPK	B-Gene_or_gene_product
)	O
pathways	O
are	O
critical	O
for	O
visfatin	B-Gene_or_gene_product
-	O
induced	O
SDF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
expression	O
.	O

Analysis	O
of	O
transcription	O
factor	O
binding	O
using	O
ELISA	O
and	O
luciferase	B-Gene_or_gene_product
reporter	O
assays	O
revealed	O
that	O
visfatin	B-Gene_or_gene_product
increased	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
-	O
and	O
AP	B-Complex
-	I-Complex
1	I-Complex
-	O
DNA	O
-	O
binding	O
activities	O
in	O
DLD	O
-	O
1	O
cells	O
.	O

Inhibition	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
and	O
AP	B-Complex
-	I-Complex
1	I-Complex
activation	O
blocked	O
the	O
visfatin	B-Gene_or_gene_product
-	O
induced	O
expression	O
and	O
activity	O
of	O
the	O
SDF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
promoter	O
.	O

The	O
effect	O
of	O
visfatin	B-Gene_or_gene_product
on	O
DLD	O
-	O
1	O
signaling	O
and	O
SDF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
expression	O
was	O
mediated	O
by	O
beta1	B-Gene_or_gene_product
integrin	I-Gene_or_gene_product
.	O

In	O
summary	O
,	O
these	O
findings	O
provide	O
novel	O
insights	O
pertaining	O
to	O
the	O
pathophysiological	O
role	O
of	O
visfatin	B-Gene_or_gene_product
in	O
CRC	O
.	O

J	O
.	O

Cell	O
.	O

Physiol	O
.	O

(	O
c	O
)	O
2012	O
Wiley	O
Periodicals	O
,	O
Inc	O
.	O

Functional	O
properties	O
of	O
phosphorylated	O
elongation	B-Gene_or_gene_product
factor	I-Gene_or_gene_product
2	I-Gene_or_gene_product
.	O

The	O
effect	O
of	O
phosphorylation	O
on	O
the	O
functional	O
activity	O
of	O
eukaryotic	B-Gene_or_gene_product
elongation	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
2	I-Gene_or_gene_product
(	O
eEF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
2	I-Gene_or_gene_product
)	O
was	O
studied	O
using	O
a	O
purified	O
phosphorylated	O
factor	O
.	O

The	O
modified	O
factor	O
was	O
unable	O
to	O
stimulate	O
protein	O
synthesis	O
in	O
an	O
eEF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
2	I-Gene_or_gene_product
-	O
dependent	O
rabbit	O
reticulocyte	O
lysate	O
.	O

The	O
functional	O
alteration	O
was	O
further	O
analyzed	O
by	O
measuring	O
the	O
effects	O
of	O
phosphorylation	O
on	O
the	O
ability	O
of	O
the	O
factor	O
to	O
catalyse	O
the	O
ribosome	B-Cellular_component
-	O
dependent	O
hydrolysis	O
of	O
GTP	B-Simple_chemical
.	O

Kinetic	O
analysis	O
showed	O
that	O
both	O
phosphorylated	O
and	O
unmodified	O
factor	O
was	O
able	O
to	O
hydrolyse	O
GTP	B-Simple_chemical
with	O
approximately	O
the	O
same	O
maximum	O
rate	O
,	O
indicating	O
that	O
the	O
rate	O
of	O
nucleotide	O
exchange	O
was	O
not	O
impaired	O
by	O
the	O
modification	O
.	O

However	O
,	O
the	O
phosphorylated	O
factor	O
showed	O
a	O
marked	O
reduction	O
in	O
the	O
second	O
-	O
order	O
rate	O
constant	O
,	O
suggesting	O
that	O
the	O
phosphorylation	O
interfered	O
with	O
ribosome	B-Complex
.	I-Complex
eEF	I-Complex
-	I-Complex
2	I-Complex
complex	O
formation	O
by	O
reducing	O
the	O
affinity	O
of	O
eEF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
2	I-Gene_or_gene_product
for	O
the	O
ribosome	B-Cellular_component
.	O

This	O
assumption	O
was	O
confirmed	O
by	O
direct	O
measurements	O
of	O
the	O
dissociation	O
constants	O
for	O
the	O
ribosomal	B-Cellular_component
complexes	O
containing	O
unmodified	O
and	O
phosphorylated	O
eEF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
2	I-Gene_or_gene_product
.	O

Human	O
gastric	B-Gene_or_gene_product
lipase	I-Gene_or_gene_product
.	O

The	O
N	O
-	O
terminal	O
tetrapeptide	O
is	O
essential	O
for	O
lipid	B-Simple_chemical
binding	O
and	O
lipase	B-Gene_or_gene_product
activity	O
.	O

Human	O
gastric	B-Gene_or_gene_product
lipase	I-Gene_or_gene_product
subjected	O
to	O
limited	O
tryptic	O
proteolysis	O
lost	O
its	O
ability	O
to	O
hydrolyze	O
emulsified	O
long	O
-	O
chain	O
triacylglycerol	B-Simple_chemical
.	O

Activity	O
against	O
a	O
water	B-Simple_chemical
-	O
soluble	O
substrate	O
was	O
however	O
retained	O
,	O
indicating	O
that	O
proteolysis	O
did	O
not	O
affect	O
the	O
active	O
site	O
.	O

Sequence	O
analysis	O
revealed	O
that	O
trypsin	B-Gene_or_gene_product
specifically	O
cleaved	O
the	O
linkage	O
between	O
lysine	B-Simple_chemical
-	I-Simple_chemical
4	I-Simple_chemical
and	O
leucine	B-Simple_chemical
-	I-Simple_chemical
5	I-Simple_chemical
.	O

This	O
cleavage	O
rendered	O
the	O
enzyme	O
unable	O
to	O
bind	O
to	O
emulsified	O
triacylglycerol	B-Simple_chemical
particles	O
,	O
e	O
.	O
g	O
.	O
human	O
milk	O
fat	B-Simple_chemical
globules	I-Simple_chemical
.	O

We	O
suggest	O
that	O
the	O
N	O
-	O
terminal	O
tetrapeptide	O
,	O
in	O
particular	O
lysine	B-Simple_chemical
-	I-Simple_chemical
4	I-Simple_chemical
,	O
is	O
essential	O
for	O
the	O
binding	O
of	O
human	O
gastric	B-Gene_or_gene_product
lipase	I-Gene_or_gene_product
to	O
lipid	B-Simple_chemical
/	O
water	B-Simple_chemical
interfaces	O
,	O
and	O
hence	O
,	O
for	O
its	O
physiological	O
function	O
.	O

Genetic	O
evidence	O
for	O
a	O
common	O
enzyme	O
catalyzing	O
the	O
second	O
step	O
in	O
the	O
degradation	O
of	O
proline	B-Simple_chemical
and	O
hydroxyproline	B-Simple_chemical
.	O

The	O
initial	O
step	O
in	O
the	O
degradation	O
pathways	O
of	O
proline	B-Simple_chemical
and	O
hydroxyproline	B-Simple_chemical
is	O
catalyzed	O
by	O
proline	B-Gene_or_gene_product
oxidase	I-Gene_or_gene_product
and	O
hydroxyproline	B-Gene_or_gene_product
oxidase	I-Gene_or_gene_product
,	O
yielding	O
delta	B-Simple_chemical
1	I-Simple_chemical
-	I-Simple_chemical
pyrroline	I-Simple_chemical
-	I-Simple_chemical
5	I-Simple_chemical
-	I-Simple_chemical
carboxylate	I-Simple_chemical
and	O
delta	B-Simple_chemical
1	I-Simple_chemical
-	I-Simple_chemical
pyrroline	I-Simple_chemical
-	I-Simple_chemical
3	I-Simple_chemical
-	I-Simple_chemical
hydroxy	I-Simple_chemical
-	I-Simple_chemical
5	I-Simple_chemical
-	I-Simple_chemical
carboxylate	I-Simple_chemical
,	O
respectively	O
.	O

The	O
second	O
step	O
is	O
the	O
oxidation	O
of	O
delta	B-Simple_chemical
1	I-Simple_chemical
-	I-Simple_chemical
pyrroline	I-Simple_chemical
-	I-Simple_chemical
5	I-Simple_chemical
-	I-Simple_chemical
carboxylate	I-Simple_chemical
to	O
glutamate	B-Simple_chemical
and	O
delta	B-Simple_chemical
1	I-Simple_chemical
-	I-Simple_chemical
pyrroline	I-Simple_chemical
-	I-Simple_chemical
3	I-Simple_chemical
-	I-Simple_chemical
hydroxy	I-Simple_chemical
-	I-Simple_chemical
5	I-Simple_chemical
-	I-Simple_chemical
carboxylate	I-Simple_chemical
to	O
gamma	B-Simple_chemical
-	I-Simple_chemical
hydroxy	I-Simple_chemical
-	I-Simple_chemical
glutamate	I-Simple_chemical
.	O

To	O
determine	O
if	O
this	O
second	O
step	O
in	O
the	O
degradation	O
of	O
proline	B-Simple_chemical
and	O
hydroxyproline	B-Simple_chemical
is	O
catalyzed	O
by	O
a	O
common	O
or	O
by	O
separate	O
enzyme	O
(	O
s	O
)	O
,	O
we	O
developed	O
a	O
radioisotopic	O
assay	O
for	O
delta	B-Gene_or_gene_product
1	I-Gene_or_gene_product
-	I-Gene_or_gene_product
pyrroline	I-Gene_or_gene_product
-	I-Gene_or_gene_product
3	I-Gene_or_gene_product
-	I-Gene_or_gene_product
hydroxy	I-Gene_or_gene_product
-	I-Gene_or_gene_product
5	I-Gene_or_gene_product
-	I-Gene_or_gene_product
carboxylate	I-Gene_or_gene_product
dehydrogenase	I-Gene_or_gene_product
activity	O
.	O

We	O
then	O
compared	O
delta1	B-Gene_or_gene_product
-	I-Gene_or_gene_product
pyrroline	I-Gene_or_gene_product
-	I-Gene_or_gene_product
3	I-Gene_or_gene_product
-	I-Gene_or_gene_product
hydroxy	I-Gene_or_gene_product
-	I-Gene_or_gene_product
5	I-Gene_or_gene_product
-	I-Gene_or_gene_product
carboxylate	I-Gene_or_gene_product
dehydrogenase	I-Gene_or_gene_product
activity	O
with	O
that	O
of	O
delta	B-Gene_or_gene_product
1	I-Gene_or_gene_product
-	I-Gene_or_gene_product
pyrroline	I-Gene_or_gene_product
-	I-Gene_or_gene_product
5	I-Gene_or_gene_product
-	I-Gene_or_gene_product
carboxylate	I-Gene_or_gene_product
dehydrogenase	I-Gene_or_gene_product
in	O
fibroblasts	O
and	O
leukocytes	O
from	O
type	O
II	O
hyperprolinemia	O
patients	O
,	O
heterozygotes	O
,	O
and	O
controls	O
.	O

We	O
found	O
that	O
cells	O
from	O
type	O
II	O
hyperprolinemia	O
patients	O
were	O
deficient	O
in	O
both	O
dehydrogenase	O
activities	O
.	O

Furthermore	O
,	O
these	O
activities	O
were	O
highly	O
correlated	O
over	O
the	O
range	O
found	O
in	O
the	O
normals	O
,	O
heterozygotes	O
,	O
and	O
patients	O
.	O

We	O
conclude	O
from	O
these	O
data	O
that	O
a	O
common	O
delta	B-Gene_or_gene_product
1	I-Gene_or_gene_product
-	I-Gene_or_gene_product
pyrroline	I-Gene_or_gene_product
-	I-Gene_or_gene_product
5	I-Gene_or_gene_product
-	I-Gene_or_gene_product
carboxylate	I-Gene_or_gene_product
dehydrogenase	I-Gene_or_gene_product
catalyzes	O
the	O
oxidation	O
of	O
both	O
delta	B-Simple_chemical
1	I-Simple_chemical
-	I-Simple_chemical
pyrroline	I-Simple_chemical
-	I-Simple_chemical
5	I-Simple_chemical
-	I-Simple_chemical
carboxylate	I-Simple_chemical
and	O
delta	B-Simple_chemical
1	I-Simple_chemical
-	I-Simple_chemical
pyrroline	I-Simple_chemical
-	I-Simple_chemical
3	I-Simple_chemical
-	I-Simple_chemical
hydroxy	I-Simple_chemical
-	I-Simple_chemical
5	I-Simple_chemical
-	I-Simple_chemical
carboxylate	I-Simple_chemical
,	O
and	O
that	O
this	O
activity	O
is	O
deficient	O
in	O
type	O
II	O
hyperprolinemia	O
.	O

The	O
isolation	O
and	O
characterization	O
of	O
cDNA	O
encoding	O
human	O
and	O
rat	O
brain	O
inositol	B-Gene_or_gene_product
polyphosphate	I-Gene_or_gene_product
4	I-Gene_or_gene_product
-	I-Gene_or_gene_product
phosphatase	I-Gene_or_gene_product
.	O

Inositol	B-Gene_or_gene_product
polyphosphate	I-Gene_or_gene_product
4	I-Gene_or_gene_product
-	I-Gene_or_gene_product
phosphatase	I-Gene_or_gene_product
,	O
an	O
enzyme	O
of	O
the	O
inositol	B-Simple_chemical
phosphate	I-Simple_chemical
signaling	O
pathway	O
,	O
catalyzes	O
the	O
hydrolysis	O
of	O
the	O
4	O
-	O
position	O
phosphate	B-Simple_chemical
of	O
inositol	B-Simple_chemical
3	I-Simple_chemical
,	I-Simple_chemical
4	I-Simple_chemical
-	I-Simple_chemical
bisphosphate	I-Simple_chemical
,	O
inositol	B-Simple_chemical
1	I-Simple_chemical
,	I-Simple_chemical
3	I-Simple_chemical
,	I-Simple_chemical
4	I-Simple_chemical
-	I-Simple_chemical
trisphosphate	I-Simple_chemical
,	O
and	O
phosphatidylinositol	B-Simple_chemical
3	I-Simple_chemical
,	I-Simple_chemical
4	I-Simple_chemical
-	I-Simple_chemical
bisphosphate	I-Simple_chemical
.	O

The	O
amino	O
acid	O
sequences	O
of	O
tryptic	O
and	O
CNBr	O
peptides	O
of	O
the	O
enzyme	O
isolated	O
from	O
rat	O
brain	O
were	O
determined	O
.	O

Degenerate	O
oligonucleotide	O
primers	O
based	O
on	O
this	O
sequence	O
were	O
used	O
to	O
amplify	O
a	O
74	O
-	O
base	O
pair	O
polymerase	O
chain	O
reaction	O
product	O
.	O

This	O
product	O
was	O
used	O
to	O
isolate	O
a	O
5607	O
-	O
base	O
pair	O
composite	O
cDNA	O
,	O
which	O
had	O
an	O
open	O
reading	O
frame	O
encoding	O
a	O
protein	O
with	O
939	O
amino	O
acids	O
with	O
a	O
predicted	O
molecular	O
mass	O
of	O
105	O
,	O
588	O
Da	O
.	O

The	O
rat	O
brain	O
polymerase	O
chain	O
reaction	O
product	O
was	O
used	O
as	O
a	O
probe	O
to	O
isolate	O
a	O
human	O
brain	O
cDNA	O
that	O
predicts	O
a	O
protein	O
with	O
938	O
amino	O
acids	O
and	O
a	O
molecular	O
mass	O
of	O
105	O
,	O
710	O
Da	O
.	O

Remarkably	O
,	O
the	O
human	O
and	O
rat	O
proteins	O
were	O
97	O
%	O
identical	O
.	O

Recombinant	O
rat	O
protein	O
expressed	O
in	O
Escherichia	O
coli	O
catalyzed	O
the	O
hydrolysis	O
of	O
all	O
three	O
substrates	O
of	O
the	O
4	B-Gene_or_gene_product
-	I-Gene_or_gene_product
phosphatase	I-Gene_or_gene_product
.	O

Northern	O
blot	O
hybridization	O
indicates	O
that	O
the	O
4	B-Gene_or_gene_product
-	I-Gene_or_gene_product
phosphatase	I-Gene_or_gene_product
is	O
widely	O
expressed	O
in	O
rat	O
tissues	O
with	O
the	O
highest	O
levels	O
of	O
expression	O
occurring	O
in	O
brain	O
,	O
heart	O
,	O
and	O
skeletal	O
muscle	O
.	O

Polyclonal	O
antiserum	O
directed	O
against	O
the	O
carboxyl	O
terminus	O
of	O
the	O
4	B-Gene_or_gene_product
-	I-Gene_or_gene_product
phosphatase	I-Gene_or_gene_product
immunoprecipitated	O
>	O
95	O
%	O
of	O
the	O
4	B-Gene_or_gene_product
-	I-Gene_or_gene_product
phosphatase	I-Gene_or_gene_product
activity	O
in	O
crude	O
homogenates	O
of	O
rat	O
brain	O
,	O
heart	O
,	O
skeletal	O
muscle	O
,	O
and	O
spleen	O
,	O
suggesting	O
that	O
this	O
enzyme	O
accounts	O
for	O
the	O
4	B-Simple_chemical
-	I-Simple_chemical
phosphate	I-Simple_chemical
activity	O
present	O
in	O
rat	O
tissues	O
.	O

This	O
antiserum	O
also	O
immunoprecipitated	O
the	O
4	B-Gene_or_gene_product
-	I-Gene_or_gene_product
phosphatase	I-Gene_or_gene_product
from	O
human	O
platelet	O
sonicates	O
.	O

The	O
sequence	O
of	O
phosphatidylinositol	B-Gene_or_gene_product
-	I-Gene_or_gene_product
4	I-Gene_or_gene_product
-	I-Gene_or_gene_product
phosphate	I-Gene_or_gene_product
5	I-Gene_or_gene_product
-	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
defines	O
a	O
novel	O
family	O
of	O
lipid	B-Simple_chemical
kinases	O
.	O

Phosphatidylinositol	B-Simple_chemical
4	I-Simple_chemical
,	I-Simple_chemical
5	I-Simple_chemical
-	I-Simple_chemical
bisphosphate	I-Simple_chemical
(	O
PtdIns	B-Simple_chemical
(	I-Simple_chemical
4	I-Simple_chemical
,	I-Simple_chemical
5	I-Simple_chemical
)	I-Simple_chemical
P2	I-Simple_chemical
)	O
occupies	O
an	O
essential	O
position	O
in	O
the	O
phosphoinositide	B-Simple_chemical
signal	O
transduction	O
cascades	O
as	O
the	O
precursor	O
to	O
second	O
messengers	O
and	O
is	O
thought	O
to	O
regulate	O
many	O
cellular	O
proteins	O
directly	O
.	O

The	O
final	O
step	O
in	O
the	O
synthesis	O
of	O
PtdIns	B-Simple_chemical
(	I-Simple_chemical
4	I-Simple_chemical
,	I-Simple_chemical
5	I-Simple_chemical
)	I-Simple_chemical
P2	I-Simple_chemical
is	O
the	O
phosphorylation	O
of	O
PtdIns	B-Simple_chemical
(	I-Simple_chemical
4	I-Simple_chemical
)	I-Simple_chemical
P	I-Simple_chemical
-	I-Simple_chemical
by	O
PtdIns	B-Gene_or_gene_product
(	I-Gene_or_gene_product
4	I-Gene_or_gene_product
)	I-Gene_or_gene_product
P	I-Gene_or_gene_product
5	I-Gene_or_gene_product
-	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
(	O
PIP5K	B-Gene_or_gene_product
)	O
.	O

Using	O
peptide	O
sequences	O
from	O
a	O
purified	O
PIP5K	B-Gene_or_gene_product
,	O
a	O
cDNA	O
for	O
a	O
human	O
placental	O
PIP5K	B-Gene_or_gene_product
was	O
isolated	O
and	O
sequenced	O
.	O

Expression	O
of	O
this	O
cDNA	O
in	O
Escherichia	O
coli	O
produced	O
an	O
active	O
PIP5K	B-Gene_or_gene_product
.	O

Surprisingly	O
,	O
the	O
sequence	O
of	O
this	O
PIP5K	B-Gene_or_gene_product
has	O
no	O
homology	O
to	O
known	O
PtdIns	B-Gene_or_gene_product
kinases	I-Gene_or_gene_product
or	O
protein	O
kinases	O
.	O

However	O
,	O
the	O
PIP5K	B-Gene_or_gene_product
is	O
homologous	O
to	O
the	O
Saccharomyces	O
cerevisiae	O
proteins	O
Fab1p	B-Gene_or_gene_product
and	O
Mss4p	B-Gene_or_gene_product
.	O

Growth	O
suppression	O
by	O
p18	B-Gene_or_gene_product
,	O
a	O
p16INK4	B-Gene_or_gene_product
/	O
MTS1	B-Gene_or_gene_product
-	O
and	O
p14INK4B	B-Gene_or_gene_product
/	O
MTS2	B-Gene_or_gene_product
-	O
related	O
CDK6	B-Gene_or_gene_product
inhibitor	O
,	O
correlates	O
with	O
wild	O
-	O
type	O
pRb	B-Gene_or_gene_product
function	O
.	O

The	O
D	B-Gene_or_gene_product
-	I-Gene_or_gene_product
type	I-Gene_or_gene_product
cyclin	I-Gene_or_gene_product
-	I-Gene_or_gene_product
dependent	I-Gene_or_gene_product
kinases	I-Gene_or_gene_product
CDK4	B-Gene_or_gene_product
and	O
CDK6	B-Gene_or_gene_product
are	O
complexed	O
with	O
many	O
small	O
cellular	O
proteins	O
(	O
p14	B-Gene_or_gene_product
,	O
p15	B-Gene_or_gene_product
,	O
p16	B-Gene_or_gene_product
,	O
p18	B-Gene_or_gene_product
,	O
and	O
p20	B-Gene_or_gene_product
)	O
.	O

We	O
have	O
isolated	O
cDNA	O
sequences	O
corresponding	O
to	O
the	O
MTS2	B-Gene_or_gene_product
genomic	O
fragment	O
that	O
encodes	O
the	O
CDK4	B-Gene_or_gene_product
-	O
and	O
CDK6	B-Gene_or_gene_product
-	O
associated	O
p14	B-Gene_or_gene_product
protein	O
.	O

By	O
use	O
of	O
a	O
yeast	O
interaction	O
screen	O
to	O
search	O
for	O
CDK6	B-Gene_or_gene_product
-	O
interacting	O
proteins	O
,	O
we	O
have	O
also	O
identified	O
an	O
18	O
-	O
kD	O
human	O
protein	O
,	O
p18	B-Gene_or_gene_product
,	O
that	O
is	O
a	O
homolog	O
of	O
the	O
cyclin	B-Complex
D	I-Complex
-	I-Complex
CDK4	I-Complex
inhibitors	O
p16	B-Gene_or_gene_product
(	O
INK4A	B-Gene_or_gene_product
/	O
MTS1	B-Gene_or_gene_product
)	O
and	O
p14	B-Gene_or_gene_product
(	O
MTS2	B-Gene_or_gene_product
/	O
INK4B	B-Gene_or_gene_product
)	O
.	O

Both	O
in	O
vivo	O
and	O
in	O
vitro	O
,	O
p18	B-Gene_or_gene_product
interacts	O
strongly	O
with	O
CDK6	B-Gene_or_gene_product
,	O
weakly	O
with	O
CDK4	B-Gene_or_gene_product
,	O
and	O
exhibits	O
no	O
detectable	O
interaction	O
with	O
the	O
other	O
known	O
CDKs	B-Gene_or_gene_product
.	O

Recombinant	O
p18	B-Gene_or_gene_product
inhibits	O
the	O
kinase	O
activity	O
of	O
cyclin	B-Complex
D	I-Complex
-	I-Complex
CDK6	I-Complex
.	O

Distinct	O
from	O
the	O
p21	B-Gene_or_gene_product
/	O
p27	B-Gene_or_gene_product
family	O
of	O
CDK	B-Gene_or_gene_product
inhibitors	O
that	O
form	O
ternary	O
complexes	O
with	O
cyclin	B-Complex
-	I-Complex
CDKs	I-Complex
,	O
only	O
binary	O
complexes	O
of	O
p14	B-Gene_or_gene_product
,	O
p16	B-Gene_or_gene_product
,	O
and	O
p18	B-Gene_or_gene_product
were	O
found	O
in	O
association	O
with	O
CDK4	B-Gene_or_gene_product
and	O
/	O
or	O
CDK6	B-Gene_or_gene_product
.	O

Ectopic	O
expression	O
of	O
p18	B-Gene_or_gene_product
or	O
p16	B-Gene_or_gene_product
suppresses	O
cell	O
growth	O
with	O
a	O
correlated	O
dependence	O
on	O
endogenous	O
wild	O
-	O
type	O
pRb	B-Gene_or_gene_product
.	O

Reconstitution	O
of	O
a	O
yeast	O
protein	O
kinase	O
cascade	O
in	O
vitro	O
:	O
activation	O
of	O
the	O
yeast	O
MEK	B-Gene_or_gene_product
homologue	O
STE7	B-Gene_or_gene_product
by	O
STE11	B-Gene_or_gene_product
.	O

The	O
mating	O
-	O
factor	O
response	O
pathway	O
of	O
Saccharomyces	O
cerevisiae	O
employs	O
a	O
set	O
of	O
protein	O
kinase	O
similar	O
to	O
kinases	O
that	O
function	O
in	O
signal	O
transduction	O
pathways	O
of	O
metazoans	O
.	O

We	O
have	O
purified	O
the	O
yeast	O
protein	O
kinases	O
encoded	O
by	O
STE11	B-Gene_or_gene_product
,	O
STE7	B-Gene_or_gene_product
,	O
and	O
FUS3	B-Gene_or_gene_product
as	O
fusions	O
to	O
glutathione	B-Gene_or_gene_product
S	I-Gene_or_gene_product
-	I-Gene_or_gene_product
transferase	I-Gene_or_gene_product
(	O
GST	B-Gene_or_gene_product
)	O
and	O
reconstituted	O
a	O
kinase	O
cascade	O
in	O
which	O
STE11	B-Gene_or_gene_product
phosphorylates	O
and	O
activates	O
STE7	B-Gene_or_gene_product
,	O
which	O
in	O
turn	O
phosphorylates	O
the	O
mitogen	B-Gene_or_gene_product
-	I-Gene_or_gene_product
activated	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
FUS3	B-Gene_or_gene_product
.	O

GST	B-Gene_or_gene_product
-	I-Gene_or_gene_product
STE11	I-Gene_or_gene_product
is	O
active	O
even	O
when	O
purified	O
from	O
cells	O
that	O
have	O
not	O
been	O
treated	O
with	O
alpha	O
-	O
factor	O
.	O

This	O
observation	O
raises	O
the	O
possibility	O
that	O
STE11	B-Gene_or_gene_product
activity	O
is	O
governed	O
by	O
an	O
inhibitor	O
which	O
is	O
regulated	O
by	O
pheromone	O
.	O

We	O
also	O
identify	O
a	O
STE11	B-Gene_or_gene_product
-	O
dependent	O
phosphorylation	O
site	O
in	O
STE7	B-Gene_or_gene_product
which	O
is	O
required	O
for	O
activity	O
of	O
STE7	B-Gene_or_gene_product
.	O

Conservation	O
of	O
this	O
site	O
in	O
the	O
mammalian	O
STE7	B-Gene_or_gene_product
homologue	O
MEK	B-Gene_or_gene_product
and	O
other	O
STE7	B-Gene_or_gene_product
relatives	O
suggests	O
that	O
this	O
may	O
be	O
a	O
regulatory	O
phosphorylation	O
site	O
in	O
all	O
MAP	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
kinases	I-Gene_or_gene_product
.	O

Cyclic	B-Complex
AMP	I-Complex
-	I-Complex
dependent	I-Complex
protein	I-Complex
kinase	I-Complex
phosphorylates	O
rabbit	O
reticulocyte	O
elongation	B-Gene_or_gene_product
factor	I-Gene_or_gene_product
-	I-Gene_or_gene_product
2	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
and	O
induces	O
calcium	B-Simple_chemical
-	O
independent	O
activity	O
.	O

The	O
catalytic	O
subunit	O
of	O
cyclic	B-Complex
AMP	I-Complex
-	I-Complex
dependent	I-Complex
protein	I-Complex
kinase	I-Complex
(	O
PKA	B-Complex
)	O
phosphorylated	O
purified	O
calcium	B-Simple_chemical
/	O
calmodulin	B-Gene_or_gene_product
-	O
dependent	O
eukaryotic	B-Gene_or_gene_product
elongation	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
-	I-Gene_or_gene_product
2	I-Gene_or_gene_product
(	I-Gene_or_gene_product
eEF	I-Gene_or_gene_product
-	I-Gene_or_gene_product
2	I-Gene_or_gene_product
)	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
,	O
isolated	O
from	O
rabbit	O
reticulocyte	O
lysates	O
.	O

It	O
maximally	O
incorporated	O
about	O
1	O
mol	O
of	O
phosphate	B-Simple_chemical
/	O
mol	O
of	O
eEF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
2	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
.	O

The	O
Km	O
of	O
eEF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
2	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
for	O
PKA	B-Complex
was	O
calculated	O
to	O
be	O
7	O
microM	O
.	O

Phosphorylation	O
of	O
eEF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
2	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
by	O
PKA	B-Complex
induced	O
calcium	B-Simple_chemical
-	O
independent	O
activity	O
which	O
amounted	O
to	O
40	O
-	O
50	O
%	O
of	O
the	O
total	O
activity	O
measured	O
in	O
the	O
presence	O
of	O
calcium	B-Simple_chemical
.	O

Furthermore	O
,	O
the	O
level	O
of	O
calcium	B-Simple_chemical
-	O
independent	O
activity	O
induced	O
by	O
phosphorylation	O
by	O
PKA	B-Complex
was	O
similar	O
to	O
that	O
induced	O
by	O
the	O
calcium	O
-	O
stimulated	O
autophosphorylation	O
of	O
eEF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
2	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
.	O

Phosphopeptide	O
mapping	O
of	O
eEF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
2	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
labelled	O
by	O
autophosphorylation	O
and	O
by	O
PKA	B-Complex
revealed	O
a	O
number	O
of	O
common	O
phosphopeptides	O
.	O

This	O
suggests	O
that	O
PKA	B-Complex
may	O
phosphorylate	O
the	O
same	O
site	O
(	O
s	O
)	O
which	O
are	O
phosphorylated	O
autocatalytically	O
and	O
which	O
are	O
responsible	O
for	O
the	O
induction	O
of	O
calcium	B-Simple_chemical
-	O
independent	O
activity	O
.	O

The	O
possible	O
implications	O
these	O
findings	O
have	O
for	O
the	O
control	O
of	O
translation	O
are	O
discussed	O
.	O

A	O
dominant	O
negative	O
protein	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
C	I-Gene_or_gene_product
zeta	I-Gene_or_gene_product
subspecies	O
blocks	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
activation	O
.	O

Nuclear	B-Complex
factor	I-Complex
kappa	I-Complex
B	I-Complex
(	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
)	O
plays	O
a	O
critical	O
role	O
in	O
the	O
regulation	O
of	O
a	O
number	O
of	O
genes	O
.	O

NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
is	O
a	O
heterodimer	O
of	O
50	O
-	O
and	O
65	O
-	O
kDa	O
subunits	O
sequestered	O
in	O
the	O
cytoplasm	B-Cellular_component
complexed	O
to	O
inhibitory	O
protein	O
I	B-Gene_or_gene_product
kappa	I-Gene_or_gene_product
B	I-Gene_or_gene_product
.	O

Following	O
stimulation	O
of	O
cells	O
,	O
I	B-Gene_or_gene_product
kappa	I-Gene_or_gene_product
B	I-Gene_or_gene_product
dissociates	O
from	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
,	O
allowing	O
its	O
translocation	O
to	O
the	O
nucleus	B-Cellular_component
,	O
where	O
it	O
carries	O
out	O
the	O
transactivation	O
function	O
.	O

The	O
precise	O
mechanism	O
controlling	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
activation	O
and	O
the	O
involvement	O
of	O
members	O
of	O
the	O
protein	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
C	I-Gene_or_gene_product
(	O
PKC	B-Gene_or_gene_product
)	O
family	O
of	O
isotypes	O
have	O
previously	O
been	O
investigated	O
.	O

It	O
was	O
found	O
that	O
phorbol	B-Simple_chemical
myristate	I-Simple_chemical
acetate	I-Simple_chemical
,	O
(	O
PMA	B-Simple_chemical
)	O
which	O
is	O
a	O
potent	O
stimulant	O
of	O
phorbol	B-Simple_chemical
ester	I-Simple_chemical
-	O
sensitive	O
PKC	B-Gene_or_gene_product
isotypes	O
,	O
activates	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
.	O

However	O
,	O
the	O
role	O
of	O
PMA	B-Simple_chemical
-	O
sensitive	O
PKCs	B-Gene_or_gene_product
in	O
vivo	O
is	O
not	O
as	O
apparent	O
.	O

It	O
has	O
recently	O
been	O
demonstrated	O
in	O
the	O
model	O
system	O
of	O
Xenopus	O
laevis	O
oocytes	O
that	O
the	O
PMA	B-Simple_chemical
-	O
insensitive	O
PKC	B-Gene_or_gene_product
isotype	O
,	O
zeta	B-Gene_or_gene_product
PKC	I-Gene_or_gene_product
,	O
is	O
a	O
required	O
step	O
in	O
the	O
activation	O
of	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
in	O
response	O
to	O
ras	B-Gene_or_gene_product
p21	I-Gene_or_gene_product
.	O

We	O
demonstrate	O
here	O
that	O
overexpression	O
of	O
zeta	B-Gene_or_gene_product
PKC	I-Gene_or_gene_product
is	O
by	O
itself	O
sufficient	O
to	O
stimulate	O
a	O
permanent	O
translocation	O
of	O
functionally	O
active	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
into	O
the	O
nucleus	B-Cellular_component
of	O
NIH	O
3T3	O
fibroblasts	O
and	O
that	O
transfection	O
of	O
a	O
kinase	O
-	O
defective	O
dominant	O
negative	O
mutant	O
of	O
zeta	B-Gene_or_gene_product
PKC	I-Gene_or_gene_product
dramatically	O
inhibits	O
the	O
kappa	B-Complex
B	I-Complex
-	O
dependent	O
transactivation	O
of	O
a	O
chloramphenicol	B-Complex
acetyltransferase	I-Complex
reporter	O
plasmid	O
in	O
NIH	O
3T3	O
fibroblasts	O
.	O

All	O
these	O
results	O
support	O
the	O
notion	O
that	O
zeta	B-Gene_or_gene_product
PKC	I-Gene_or_gene_product
plays	O
a	O
decisive	O
role	O
in	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
regulation	O
in	O
mammalian	O
cells	O
.	O

The	O
yeast	O
Cln3	B-Gene_or_gene_product
protein	O
is	O
an	O
unstable	O
activator	O
of	O
Cdc28	B-Gene_or_gene_product
.	O

The	O
Cln3	B-Gene_or_gene_product
cyclin	B-Gene_or_gene_product
homolog	O
of	O
Saccharomyces	O
cerevisiae	O
functions	O
to	O
promote	O
cell	O
cycle	O
START	O
for	O
only	O
a	O
short	O
time	O
following	O
its	O
synthesis	O
.	O

Cln3	B-Gene_or_gene_product
protein	O
is	O
highly	O
unstable	O
and	O
is	O
stabilized	O
by	O
C	O
-	O
terminal	O
truncation	O
.	O

Cln3	B-Gene_or_gene_product
binds	O
to	O
Cdc28	B-Gene_or_gene_product
,	O
a	O
protein	O
kinase	O
catalytic	O
subunit	O
essential	O
for	O
cell	O
cycle	O
START	O
,	O
and	O
Cln3	B-Gene_or_gene_product
instability	O
requires	O
Cdc28	B-Gene_or_gene_product
activity	O
.	O

The	O
long	O
functional	O
lifetime	O
and	O
the	O
hyperactivity	O
of	O
C	O
-	O
terminally	O
truncated	O
Cln3	B-Gene_or_gene_product
(	O
Cln3	B-Gene_or_gene_product
-	I-Gene_or_gene_product
2	I-Gene_or_gene_product
)	O
relative	O
to	O
those	O
of	O
full	O
-	O
length	O
Cln3	B-Gene_or_gene_product
are	O
affected	O
by	O
mutations	O
in	O
CDC28	B-Gene_or_gene_product
:	O
the	O
functional	O
lifetime	O
of	O
Cln3	B-Gene_or_gene_product
-	I-Gene_or_gene_product
2	I-Gene_or_gene_product
is	O
drastically	O
reduced	O
by	O
the	O
cdc28	B-Gene_or_gene_product
-	O
13	O
mutation	O
at	O
the	O
permissive	O
temperature	O
,	O
and	O
the	O
cdc28	B-Gene_or_gene_product
-	O
4	O
mutation	O
at	O
the	O
permissive	O
temperature	O
completely	O
blocks	O
the	O
function	O
of	O
Cln3	B-Gene_or_gene_product
-	I-Gene_or_gene_product
2	I-Gene_or_gene_product
while	O
only	O
partially	O
reducing	O
the	O
function	O
of	O
full	O
-	O
length	O
Cln3	B-Gene_or_gene_product
.	O

Thus	O
,	O
sequences	O
in	O
the	O
C	O
-	O
terminal	O
third	O
of	O
Cln3	B-Gene_or_gene_product
might	O
help	O
stabilize	O
functional	O
Cdc28	B-Gene_or_gene_product
-	O
Cln3	B-Gene_or_gene_product
association	O
,	O
as	O
well	O
as	O
decreasing	O
the	O
lifetime	O
of	O
the	O
Cln3	B-Gene_or_gene_product
protein	O
.	O

These	O
and	O
other	O
results	O
strongly	O
support	O
the	O
idea	O
that	O
Cln	B-Gene_or_gene_product
proteins	O
function	O
to	O
activate	O
Cdc28	B-Gene_or_gene_product
at	O
START	O
.	O

Rac	B-Gene_or_gene_product
GTPase	O
interacts	O
specifically	O
with	O
phosphatidylinositol	B-Gene_or_gene_product
3	I-Gene_or_gene_product
-	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
.	O

The	O
Rac	B-Gene_or_gene_product
GTP	B-Simple_chemical
-	O
binding	O
proteins	O
are	O
members	O
of	O
the	O
Rho	B-Gene_or_gene_product
family	O
and	O
regulate	O
growth	O
factor	O
-	O
stimulated	O
actin	B-Gene_or_gene_product
assembly	O
in	O
a	O
variety	O
of	O
cells	O
.	O

The	O
formation	O
of	O
phosphorylated	O
inositol	B-Simple_chemical
lipids	I-Simple_chemical
has	O
been	O
implicated	O
in	O
control	O
of	O
the	O
processes	O
initiating	O
and	O
regulating	O
such	O
actin	B-Gene_or_gene_product
polymerization	O
.	O

Associations	O
of	O
Rho	B-Gene_or_gene_product
family	O
GTP	B-Simple_chemical
-	O
binding	O
proteins	O
with	O
enzymes	O
involved	O
in	O
lipid	B-Simple_chemical
metabolism	O
have	O
been	O
described	O
.	O

Here	O
we	O
demonstrate	O
a	O
direct	O
and	O
specific	O
interaction	O
of	O
Rac	B-Gene_or_gene_product
proteins	O
with	O
phosphatidylinositol	B-Gene_or_gene_product
(	I-Gene_or_gene_product
PI	I-Gene_or_gene_product
)	I-Gene_or_gene_product
3	I-Gene_or_gene_product
-	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
.	O

This	O
interaction	O
is	O
dependent	O
upon	O
Rac	B-Gene_or_gene_product
being	O
in	O
a	O
GTP	B-Simple_chemical
-	O
bound	O
state	O
and	O
requires	O
an	O
intact	O
Rac	B-Gene_or_gene_product
effector	O
domain	O
.	O

In	O
contrast	O
,	O
direct	O
binding	O
of	O
RhoA	B-Gene_or_gene_product
to	O
PI	B-Gene_or_gene_product
3	I-Gene_or_gene_product
-	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
could	O
not	O
be	O
detected	O
.	O

Rac	B-Complex
-	I-Complex
GTP	I-Complex
also	O
bound	O
to	O
PI	B-Gene_or_gene_product
3	I-Gene_or_gene_product
-	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
in	O
Swiss	O
3T3	O
fibroblast	O
and	O
human	O
neutrophil	O
lysates	O
,	O
and	O
increased	O
PI	B-Gene_or_gene_product
3	I-Gene_or_gene_product
-	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
activity	O
became	O
associated	O
with	O
Rac	B-Complex
-	I-Complex
GTP	I-Complex
in	O
platelet	O
-	O
derived	O
growth	O
factor	O
-	O
stimulated	O
cells	O
.	O

Interaction	O
of	O
Rac	B-Complex
-	I-Complex
GTP	I-Complex
with	O
PI	B-Gene_or_gene_product
3	I-Gene_or_gene_product
-	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
in	O
vitro	O
stimulated	O
the	O
activity	O
of	O
the	O
enzyme	O
by	O
2	O
-	O
9	O
-	O
fold	O
.	O

A	O
specific	O
interaction	O
of	O
active	O
Rac	B-Gene_or_gene_product
with	O
PI	B-Gene_or_gene_product
3	I-Gene_or_gene_product
-	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
might	O
be	O
important	O
in	O
regulation	O
of	O
the	O
actin	B-Gene_or_gene_product
cytoskeleton	B-Cellular_component
.	O

Wild	O
type	O
and	O
mutant	O
human	O
heart	O
(	B-Gene_or_gene_product
R	I-Gene_or_gene_product
)	I-Gene_or_gene_product
-	I-Gene_or_gene_product
3	I-Gene_or_gene_product
-	I-Gene_or_gene_product
hydroxybutyrate	I-Gene_or_gene_product
dehydrogenase	I-Gene_or_gene_product
expressed	O
in	O
insect	O
cells	O
.	O

(	B-Gene_or_gene_product
R	I-Gene_or_gene_product
)	I-Gene_or_gene_product
-	I-Gene_or_gene_product
3	I-Gene_or_gene_product
-	I-Gene_or_gene_product
Hydroxybutyrate	I-Gene_or_gene_product
dehydrogenase	I-Gene_or_gene_product
(	O
BDH	B-Gene_or_gene_product
)	O
is	O
a	O
lipid	B-Simple_chemical
-	O
requiring	O
mitochondrial	B-Cellular_component
enzyme	O
with	O
a	O
specific	O
requirement	O
of	O
phosphatidylcholine	B-Simple_chemical
(	O
PC	B-Simple_chemical
)	O
for	O
function	O
.	O

PC	B-Simple_chemical
is	O
an	O
allosteric	O
activator	O
that	O
enhances	O
NAD	B-Simple_chemical
(	I-Simple_chemical
H	I-Simple_chemical
)	I-Simple_chemical
binding	O
to	O
BDH	B-Gene_or_gene_product
.	O

The	O
enzyme	O
serves	O
as	O
a	O
paradigm	O
to	O
study	O
specific	O
lipid	B-Simple_chemical
-	O
protein	O
interactions	O
in	O
membranes	B-Cellular_component
.	O

Analysis	O
of	O
the	O
primary	O
sequence	O
of	O
BDH	B-Gene_or_gene_product
,	O
as	O
determined	O
by	O
molecular	O
cloning	O
,	O
predicts	O
that	O
lipid	B-Simple_chemical
binding	O
and	O
substrate	O
specificity	O
are	O
contributed	O
by	O
the	O
C	O
-	O
terminal	O
third	O
of	O
the	O
protein	O
[	O
Marks	O
,	O
A	O
.	O
R	O
.	O
,	O
McIntyre	O
,	O
J	O
.	O
O	O
.	O
,	O
Duncan	O
,	O
T	O
.	O
M	O
.	O
,	O
Erdjument	O
-	O
Bromage	O
,	O
H	O
.	O
,	O
Tempst	O
,	O
P	O
.	O
,	O
&	O
Fleischer	O
,	O
S	O
.	O
(	O
1992	O
)	O
J	O
.	O
Biol	O
.	O
Chem	O
.	O
267	O
,	O
15459	O
-	O
15463	O
]	O
.	O

The	O
mature	O
form	O
of	O
human	O
heart	O
BDH	B-Gene_or_gene_product
has	O
now	O
been	O
expressed	O
in	O
catalytically	O
active	O
form	O
in	O
insect	O
cells	O
(	O
Sf9	O
,	O
Spodoptera	O
frugiperda	O
)	O
transfected	O
with	O
BDH	B-Gene_or_gene_product
-	O
cDNA	O
in	O
baculovirus	O
.	O

Endogenous	O
PC	B-Simple_chemical
in	O
the	O
insect	O
cells	O
fulfills	O
the	O
lipid	B-Simple_chemical
requirement	O
for	O
the	O
expressed	O
BDH	B-Gene_or_gene_product
since	O
enzymatic	O
activity	O
is	O
lost	O
upon	O
digestion	O
with	O
phospholipase	B-Gene_or_gene_product
A2	I-Gene_or_gene_product
and	O
restored	O
selectively	O
by	O
reconstitution	O
with	O
PC	B-Simple_chemical
vesicles	O
.	O

The	O
K	O
(	O
m	O
)	O
s	O
for	O
NAD	B-Simple_chemical
+	I-Simple_chemical
and	O
(	B-Simple_chemical
R	I-Simple_chemical
)	I-Simple_chemical
-	I-Simple_chemical
3	I-Simple_chemical
-	I-Simple_chemical
hydroxybutyrate	I-Simple_chemical
(	O
R	B-Simple_chemical
-	I-Simple_chemical
HOB	I-Simple_chemical
)	O
of	O
expressed	O
BDH	B-Gene_or_gene_product
are	O
similar	O
to	O
those	O
for	O
bovine	O
heart	O
or	O
rat	O
liver	O
BDH	B-Gene_or_gene_product
in	O
mitochondria	B-Cellular_component
.	O

Replacing	O
Cys242	B-Simple_chemical
(	O
the	O
only	O
cysteine	B-Simple_chemical
in	O
the	O
C	O
-	O
terminal	O
domain	O
)	O
with	O
serine	B-Simple_chemical
by	O
site	O
-	O
directed	O
mutagenesis	O
resulted	O
in	O
a	O
10	O
-	O
fold	O
increase	O
in	O
K	O
(	O
m	O
)	O
for	O
R	B-Simple_chemical
-	I-Simple_chemical
HOB	I-Simple_chemical
with	O
no	O
change	O
in	O
the	O
K	O
(	O
m	O
)	O
for	O
NAD	B-Simple_chemical
+	I-Simple_chemical
,	O
indicating	O
a	O
role	O
for	O
Cys242	B-Simple_chemical
in	O
substrate	O
binding	O
.	O

Carboxypeptidase	B-Gene_or_gene_product
cleavage	O
studies	O
had	O
indicated	O
a	O
requirement	O
of	O
the	O
C	O
-	O
terminal	O
for	O
catalysis	O
and	O
a	O
role	O
in	O
lipid	B-Simple_chemical
binding	O
[	O
Adami	O
,	O
P	O
.	O
,	O
Duncan	O
,	O
T	O
.	O
M	O
.	O
,	O
McIntyre	O
,	O
J	O
.	O
O	O
.	O
,	O
Carter	O
,	O
C	O
.	O
E	O
.	O
,	O
Fu	O
,	O
C	O
.	O
,	O
Melin	O
,	O
M	O
.	O
,	O
Latruffe	O
,	O
N	O
.	O
,	O
&	O
Fleischer	O
,	O
S	O
.	O
(	O
1993	O
)	O
Biochem	O
J	O
.	O
292	O
,	O
863	O
-	O
872	O
]	O
.	O

We	O
now	O
show	O
that	O
deletion	O
of	O
twelve	O
C	O
-	O
terminal	O
amino	O
acids	O
to	O
form	O
a	O
truncated	O
BDH	B-Gene_or_gene_product
mutant	O
results	O
in	O
loss	O
of	O
enzymic	O
function	O
.	O

The	O
expression	O
in	O
Sf	O
9	O
cells	O
of	O
the	O
constitutively	O
active	O
full	O
-	O
length	O
mature	O
form	O
of	O
human	O
heart	O
BDH	B-Gene_or_gene_product
and	O
the	O
first	O
expression	O
and	O
characterization	O
of	O
BDH	B-Gene_or_gene_product
mutants	O
validate	O
this	O
system	O
for	O
structure	O
-	O
function	O
studies	O
of	O
BDH	B-Gene_or_gene_product
.	O

Nuclear	O
localization	O
of	O
DP	B-Gene_or_gene_product
and	O
E2F	B-Gene_or_gene_product
transcription	O
factors	O
by	O
heterodimeric	O
partners	O
and	O
retinoblastoma	B-Gene_or_gene_product
protein	O
family	O
members	O
.	O

E2F	B-Gene_or_gene_product
is	O
a	O
family	O
of	O
transcription	O
factors	O
implicated	O
in	O
the	O
regulation	O
of	O
genes	O
required	O
for	O
progression	O
through	O
G1	O
and	O
entry	O
into	O
the	O
S	O
phase	O
.	O

The	O
transcriptionally	O
active	O
forms	O
of	O
E2F	B-Gene_or_gene_product
are	O
heterodimers	O
composed	O
of	O
one	O
polypeptide	O
encoded	O
by	O
the	O
E2F	B-Gene_or_gene_product
gene	O
family	O
and	O
one	O
polypeptide	O
encoded	O
by	O
the	O
DP	B-Gene_or_gene_product
gene	O
family	O
.	O

The	O
transcriptional	O
activity	O
of	O
E2F	B-Complex
/	I-Complex
DP	I-Complex
heterodimers	O
is	O
influenced	O
by	O
association	O
with	O
the	O
members	O
of	O
the	O
retinoblastoma	B-Gene_or_gene_product
tumor	O
suppressor	O
protein	O
family	O
(	O
pRb	B-Gene_or_gene_product
,	O
p107	B-Gene_or_gene_product
,	O
and	O
p130	B-Gene_or_gene_product
)	O
.	O

Here	O
the	O
intracellular	B-Cellular_component
distribution	O
of	O
E2F	B-Gene_or_gene_product
and	O
DP	B-Gene_or_gene_product
proteins	O
was	O
investigated	O
in	O
transiently	O
transfected	O
Chinese	O
hamster	O
and	O
human	O
cells	O
.	O

In	O
transfected	O
cells	O
,	O
DP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
did	O
not	O
accumulate	O
in	O
the	O
nucleus	B-Cellular_component
unless	O
it	O
was	O
coexpressed	O
with	O
the	O
heterodimeric	O
partners	O
E2F	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
,	O
E2F	B-Gene_or_gene_product
-	I-Gene_or_gene_product
2	I-Gene_or_gene_product
,	O
or	O
E2F	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3	I-Gene_or_gene_product
.	O

Domain	O
mapping	O
experiments	O
showed	O
that	O
regions	O
of	O
E2F	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
and	O
DP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
that	O
are	O
required	O
for	O
stable	O
association	O
of	O
the	O
two	O
proteins	O
were	O
also	O
required	O
for	O
nuclear	B-Cellular_component
localization	O
of	O
DP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
.	O

Unlike	O
E2F	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
,	O
-	B-Gene_or_gene_product
2	I-Gene_or_gene_product
,	O
and	O
-	B-Gene_or_gene_product
3	I-Gene_or_gene_product
,	O
E2F	B-Gene_or_gene_product
-	I-Gene_or_gene_product
4	I-Gene_or_gene_product
did	O
not	O
accumulate	O
in	O
the	O
nucleus	B-Cellular_component
unless	O
it	O
was	O
coexpressed	O
with	O
DP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
2	I-Gene_or_gene_product
,	O
p107	B-Gene_or_gene_product
and	O
p130	B-Gene_or_gene_product
,	O
but	O
not	O
pRb	B-Gene_or_gene_product
,	O
stimulated	O
nuclear	B-Cellular_component
localization	O
of	O
E2F	B-Gene_or_gene_product
-	I-Gene_or_gene_product
4	I-Gene_or_gene_product
,	O
either	O
alone	O
or	O
in	O
combination	O
with	O
DP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
2	I-Gene_or_gene_product
.	O

These	O
results	O
indicate	O
that	O
DP	B-Gene_or_gene_product
proteins	O
preferentially	O
associate	O
with	O
specific	O
E2F	B-Gene_or_gene_product
partners	O
,	O
and	O
suggest	O
that	O
the	O
ability	O
of	O
specific	O
E2F	B-Complex
/	I-Complex
DP	I-Complex
heterodimers	O
to	O
localize	O
in	O
the	O
nucleus	B-Cellular_component
contributes	O
to	O
the	O
regulation	O
of	O
E2F	B-Gene_or_gene_product
activity	O
.	O

Basal	O
phosphorylation	O
of	O
the	O
PEST	O
domain	O
in	O
the	O
I	B-Gene_or_gene_product
(	I-Gene_or_gene_product
kappa	I-Gene_or_gene_product
)	I-Gene_or_gene_product
B	I-Gene_or_gene_product
(	I-Gene_or_gene_product
beta	I-Gene_or_gene_product
)	I-Gene_or_gene_product
regulates	O
its	O
functional	O
interaction	O
with	O
the	O
c	B-Gene_or_gene_product
-	I-Gene_or_gene_product
rel	I-Gene_or_gene_product
proto	O
-	O
oncogene	O
product	O
.	O

The	O
product	O
of	O
the	O
c	B-Gene_or_gene_product
-	I-Gene_or_gene_product
rel	I-Gene_or_gene_product
proto	O
-	O
oncogene	O
(	O
c	B-Gene_or_gene_product
-	I-Gene_or_gene_product
Rel	I-Gene_or_gene_product
)	O
belongs	O
to	O
the	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
/	O
Rel	B-Gene_or_gene_product
family	O
of	O
polypeptides	O
and	O
has	O
been	O
implicated	O
in	O
the	O
transcriptional	O
control	O
of	O
cell	O
proliferation	O
and	O
immune	O
function	O
.	O

In	O
human	O
T	O
lymphocytes	O
,	O
c	B-Gene_or_gene_product
-	I-Gene_or_gene_product
Rel	I-Gene_or_gene_product
is	O
sequestered	O
in	O
the	O
cytoplasmic	B-Cellular_component
compartment	I-Cellular_component
by	O
constitutively	O
phosphorylated	O
inhibitors	O
,	O
including	O
I	B-Gene_or_gene_product
(	I-Gene_or_gene_product
kappa	I-Gene_or_gene_product
)	I-Gene_or_gene_product
B	I-Gene_or_gene_product
(	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
)	I-Gene_or_gene_product
and	O
I	B-Gene_or_gene_product
(	I-Gene_or_gene_product
kappa	I-Gene_or_gene_product
)	I-Gene_or_gene_product
B	I-Gene_or_gene_product
(	I-Gene_or_gene_product
beta	I-Gene_or_gene_product
)	I-Gene_or_gene_product
.	O

Studies	O
with	O
bacterially	O
expressed	O
forms	O
of	O
these	O
inhibitory	O
proteins	O
revealed	O
that	O
unphosphorylated	O
I	B-Gene_or_gene_product
(	I-Gene_or_gene_product
kappa	I-Gene_or_gene_product
)	I-Gene_or_gene_product
B	I-Gene_or_gene_product
(	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
)	I-Gene_or_gene_product
but	O
not	O
I	B-Gene_or_gene_product
(	I-Gene_or_gene_product
kappa	I-Gene_or_gene_product
)	I-Gene_or_gene_product
B	I-Gene_or_gene_product
(	I-Gene_or_gene_product
beta	I-Gene_or_gene_product
)	I-Gene_or_gene_product
assembles	O
with	O
c	B-Gene_or_gene_product
-	I-Gene_or_gene_product
Rel	I-Gene_or_gene_product
and	O
inhibits	O
its	O
DNA	O
binding	O
activity	O
.	O

Furthermore	O
,	O
latent	O
I	B-Complex
(	I-Complex
kappa	I-Complex
)	I-Complex
B	I-Complex
(	I-Complex
beta	I-Complex
)	I-Complex
-	I-Complex
c	I-Complex
-	I-Complex
Rel	I-Complex
complexes	O
derived	O
from	O
mammalian	O
cells	O
were	O
sensitive	O
to	O
phosphatase	B-Simple_chemical
treatment	O
,	O
whereas	O
I	B-Complex
(	I-Complex
kappa	I-Complex
)	I-Complex
B	I-Complex
(	I-Complex
alpha	I-Complex
)	I-Complex
-	I-Complex
c	I-Complex
-	I-Complex
Rel	I-Complex
complexes	O
were	O
resistant	O
.	O

We	O
have	O
identified	O
a	O
constitutive	O
protein	O
kinase	O
in	O
unstimulated	O
T	O
cells	O
that	O
associates	O
with	O
and	O
phosphorylates	O
I	B-Gene_or_gene_product
(	I-Gene_or_gene_product
kappa	I-Gene_or_gene_product
)	I-Gene_or_gene_product
B	I-Gene_or_gene_product
(	I-Gene_or_gene_product
beta	I-Gene_or_gene_product
)	I-Gene_or_gene_product
in	O
vitro	O
.	O

The	O
substrate	O
specificity	O
,	O
electrophoretic	O
mobility	O
,	O
and	O
antigenic	O
properties	O
of	O
this	O
I	B-Gene_or_gene_product
(	I-Gene_or_gene_product
kappa	I-Gene_or_gene_product
)	I-Gene_or_gene_product
B	I-Gene_or_gene_product
(	I-Gene_or_gene_product
beta	I-Gene_or_gene_product
)	I-Gene_or_gene_product
-	I-Gene_or_gene_product
associated	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
(	O
BAK	B-Gene_or_gene_product
)	O
suggest	O
identity	O
with	O
casein	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
II	I-Gene_or_gene_product
(	O
CKII	B-Gene_or_gene_product
)	O
,	O
an	O
enzyme	O
known	O
to	O
mediate	O
basal	O
phosphorylation	O
of	O
I	B-Gene_or_gene_product
(	I-Gene_or_gene_product
kappa	I-Gene_or_gene_product
)	I-Gene_or_gene_product
B	I-Gene_or_gene_product
(	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
)	I-Gene_or_gene_product
.	O

Phosphorylation	O
of	O
recombinant	O
I	B-Gene_or_gene_product
(	I-Gene_or_gene_product
kappa	I-Gene_or_gene_product
)	I-Gene_or_gene_product
B	I-Gene_or_gene_product
(	I-Gene_or_gene_product
beta	I-Gene_or_gene_product
)	I-Gene_or_gene_product
by	O
either	O
BAK	B-Gene_or_gene_product
or	O
CKII	B-Gene_or_gene_product
restored	O
the	O
capacity	O
of	O
this	O
inhibitor	O
to	O
antagonize	O
the	O
DNA	O
binding	O
activity	O
of	O
c	B-Gene_or_gene_product
-	I-Gene_or_gene_product
Rel	I-Gene_or_gene_product
.	O

Peptide	O
mapping	O
and	O
mutational	O
analyses	O
localized	O
the	O
bulk	O
of	O
the	O
basal	O
phosphorylation	O
sites	O
in	O
I	B-Gene_or_gene_product
(	I-Gene_or_gene_product
kappa	I-Gene_or_gene_product
)	I-Gene_or_gene_product
B	I-Gene_or_gene_product
(	I-Gene_or_gene_product
beta	I-Gene_or_gene_product
)	I-Gene_or_gene_product
to	O
the	O
C	O
-	O
terminal	O
PEST	O
domain	O
,	O
which	O
contains	O
two	O
potential	O
acceptors	O
for	O
CKII	B-Gene_or_gene_product
-	O
mediated	O
phosphoryl	O
group	O
transfer	O
(	O
Ser	B-Simple_chemical
-	I-Simple_chemical
313	I-Simple_chemical
and	O
Ser	B-Simple_chemical
-	I-Simple_chemical
315	I-Simple_chemical
)	O
.	O

Point	O
mutations	O
introduced	O
into	O
the	O
full	O
-	O
length	O
inhibitor	O
at	O
Ser	B-Simple_chemical
-	I-Simple_chemical
313	I-Simple_chemical
and	O
Ser	B-Simple_chemical
-	I-Simple_chemical
315	I-Simple_chemical
led	O
to	O
a	O
significant	O
reduction	O
in	O
the	O
phosphorylation	O
of	O
I	B-Gene_or_gene_product
(	I-Gene_or_gene_product
kappa	I-Gene_or_gene_product
)	I-Gene_or_gene_product
B	I-Gene_or_gene_product
(	I-Gene_or_gene_product
beta	I-Gene_or_gene_product
)	I-Gene_or_gene_product
and	O
severely	O
impaired	O
its	O
c	B-Gene_or_gene_product
-	I-Gene_or_gene_product
Rel	I-Gene_or_gene_product
inhibitory	O
function	O
in	O
vivo	O
.	O

Taken	O
together	O
,	O
these	O
findings	O
strongly	O
suggest	O
that	O
basal	O
phosphorylation	O
of	O
the	O
PEST	O
domain	O
of	O
I	B-Gene_or_gene_product
(	I-Gene_or_gene_product
kappa	I-Gene_or_gene_product
)	I-Gene_or_gene_product
B	I-Gene_or_gene_product
(	I-Gene_or_gene_product
beta	I-Gene_or_gene_product
)	I-Gene_or_gene_product
at	O
consensus	O
CKII	B-Gene_or_gene_product
sites	O
is	O
required	O
for	O
the	O
efficient	O
formation	O
of	O
latent	O
I	B-Complex
(	I-Complex
kappa	I-Complex
)	I-Complex
B	I-Complex
(	I-Complex
beta	I-Complex
)	I-Complex
-	I-Complex
c	I-Complex
-	I-Complex
Rel	I-Complex
complexes	O
.	O

Molecular	O
cloning	O
and	O
characterization	O
of	O
a	O
novel	O
protein	O
kinase	O
with	O
a	O
catalytic	O
domain	O
homologous	O
to	O
mitogen	B-Gene_or_gene_product
-	I-Gene_or_gene_product
activated	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
.	O

Mitogen	B-Gene_or_gene_product
-	I-Gene_or_gene_product
activated	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
(	O
MAPK	B-Gene_or_gene_product
)	O
signaling	O
cascades	O
include	O
MAPK	B-Gene_or_gene_product
or	O
extracellular	B-Gene_or_gene_product
signal	I-Gene_or_gene_product
-	I-Gene_or_gene_product
regulated	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
(	O
ERK	B-Gene_or_gene_product
)	O
,	O
MAPK	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
(	O
MKK	B-Gene_or_gene_product
or	O
MEK	B-Gene_or_gene_product
)	O
,	O
and	O
MAPK	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
(	O
MAPKKK	B-Gene_or_gene_product
or	O
MEKK	B-Gene_or_gene_product
)	O
.	O

MAPKK	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
/	O
MEKK	B-Gene_or_gene_product
phosphorylates	O
and	O
activates	O
its	O
downstream	O
protein	O
kinase	O
,	O
MAPK	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
/	O
MEK	B-Gene_or_gene_product
,	O
which	O
in	O
turn	O
activates	O
MAPK	B-Gene_or_gene_product
.	O

We	O
report	O
herein	O
the	O
isolation	O
of	O
a	O
cDNA	O
encoding	O
a	O
novel	O
protein	O
kinase	O
designated	O
MAPKKK5	B-Gene_or_gene_product
from	O
a	O
human	O
macrophage	O
library	O
.	O

The	O
nucleotide	O
sequence	O
predicts	O
that	O
MAPKKK5	B-Gene_or_gene_product
encodes	O
an	O
open	O
reading	O
frame	O
of	O
1374	O
amino	O
acids	O
with	O
all	O
11	O
kinase	O
subdomains	O
.	O

The	O
putative	O
catalytic	O
domain	O
of	O
MAPKKK5	B-Gene_or_gene_product
shows	O
significant	O
sequence	O
homology	O
to	O
the	O
kinase	O
domains	O
of	O
the	O
MAPKKK	B-Gene_or_gene_product
/	O
MEKK	B-Gene_or_gene_product
level	O
protein	O
kinases	O
from	O
mouse	O
MEKK2	B-Gene_or_gene_product
and	O
-	B-Gene_or_gene_product
3	I-Gene_or_gene_product
,	O
Drosophila	O
melanogaster	O
PK92B	B-Gene_or_gene_product
,	O
Saccharomyces	O
cerevisiae	O
STE11	B-Gene_or_gene_product
,	O
and	O
Schizosaccharomyces	O
pombe	O
BYR2	B-Gene_or_gene_product
.	O

Northern	O
blot	O
analysis	O
showed	O
that	O
MAPKKK5	B-Gene_or_gene_product
transcript	O
is	O
abundantly	O
expressed	O
in	O
human	O
heart	O
and	O
pancreas	O
.	O

When	O
transiently	O
expressed	O
in	O
COS	O
and	O
293	O
cells	O
,	O
MAPKKK5	B-Gene_or_gene_product
markedly	O
activated	O
c	B-Gene_or_gene_product
-	I-Gene_or_gene_product
Jun	I-Gene_or_gene_product
N	I-Gene_or_gene_product
-	I-Gene_or_gene_product
terminal	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
or	O
stress	B-Gene_or_gene_product
-	I-Gene_or_gene_product
activated	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
,	O
but	O
not	O
MAPK	B-Gene_or_gene_product
/	O
ERK	B-Gene_or_gene_product
.	O

Furthermore	O
,	O
MAPKKK5	B-Gene_or_gene_product
that	O
was	O
immunoprecipitated	O
from	O
transfected	O
293	O
cells	O
was	O
able	O
to	O
phosphorylate	O
and	O
activate	O
MKK4	B-Gene_or_gene_product
in	O
vitro	O
,	O
suggesting	O
that	O
MAPKKK5	B-Gene_or_gene_product
may	O
be	O
an	O
upstream	O
activator	O
of	O
MKK4	B-Gene_or_gene_product
in	O
the	O
c	B-Gene_or_gene_product
-	I-Gene_or_gene_product
Jun	I-Gene_or_gene_product
N	I-Gene_or_gene_product
-	I-Gene_or_gene_product
terminal	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
pathway	O
.	O

Thyroid	O
Na	B-Gene_or_gene_product
+	I-Gene_or_gene_product
/	I-Gene_or_gene_product
I	I-Gene_or_gene_product
-	I-Gene_or_gene_product
symporter	I-Gene_or_gene_product
.	O

Mechanism	O
,	O
stoichiometry	O
,	O
and	O
specificity	O
.	O

The	O
rat	O
thyroid	O
Na	B-Gene_or_gene_product
+	I-Gene_or_gene_product
/	I-Gene_or_gene_product
I	I-Gene_or_gene_product
-	I-Gene_or_gene_product
symporter	I-Gene_or_gene_product
(	O
NIS	B-Gene_or_gene_product
)	O
was	O
expressed	O
in	O
Xenopus	O
laevis	O
oocytes	O
and	O
characterized	O
using	O
electrophysiological	O
,	O
tracer	O
uptake	O
,	O
and	O
electron	O
microscopic	O
methods	O
.	O

NIS	B-Gene_or_gene_product
activity	O
was	O
found	O
to	O
be	O
electrogenic	O
and	O
Na	B-Simple_chemical
+	I-Simple_chemical
-	O
dependent	O
(	O
Na	B-Simple_chemical
+	I-Simple_chemical
>	O
Li	B-Simple_chemical
+	I-Simple_chemical
>	O
H	B-Simple_chemical
+	I-Simple_chemical
)	O
.	O

The	O
apparent	O
affinity	O
constants	O
for	O
Na	B-Simple_chemical
+	I-Simple_chemical
and	O
I	B-Simple_chemical
-	I-Simple_chemical
were	O
28	O
+	O
/	O
-	O
3	O
mM	O
and	O
33	O
+	O
/	O
-	O
9	O
microM	O
,	O
respectively	O
.	O

Stoichiometry	O
of	O
Na	B-Simple_chemical
+	I-Simple_chemical
/	O
anion	B-Simple_chemical
cotransport	O
was	O
2	O
:	O
1	O
.	O

NIS	B-Gene_or_gene_product
was	O
capable	O
of	O
transporting	O
a	O
wide	O
variety	O
of	O
anions	B-Simple_chemical
(	O
I	B-Simple_chemical
-	I-Simple_chemical
,	O
ClO3	B-Simple_chemical
-	I-Simple_chemical
,	O
SCN	B-Simple_chemical
-	I-Simple_chemical
,	O
SeCN	B-Simple_chemical
-	I-Simple_chemical
,	O
NO3	B-Simple_chemical
-	I-Simple_chemical
,	O
Br	B-Simple_chemical
-	I-Simple_chemical
,	O
BF4	B-Simple_chemical
-	I-Simple_chemical
,	O
IO4	B-Simple_chemical
-	I-Simple_chemical
,	O
BrO3	B-Simple_chemical
-	I-Simple_chemical
,	O
but	O
perchlorate	B-Simple_chemical
(	O
ClO4	B-Simple_chemical
-	I-Simple_chemical
)	O
was	O
not	O
transported	O
.	O

In	O
the	O
absence	O
of	O
anion	B-Simple_chemical
substrate	O
,	O
NIS	B-Gene_or_gene_product
exhibited	O
a	O
Na	B-Simple_chemical
+	I-Simple_chemical
-	O
dependent	O
leak	O
current	O
(	O
approximately	O
35	O
%	O
of	O
maximum	O
substrate	O
-	O
induced	O
current	O
)	O
with	O
an	O
apparent	O
Na	B-Simple_chemical
+	I-Simple_chemical
affinity	O
of	O
74	O
+	O
/	O
-	O
14	O
mM	O
and	O
a	O
Hill	O
coefficient	O
(	O
n	O
)	O
of	O
1	O
.	O

In	O
response	O
to	O
step	O
voltage	O
changes	O
,	O
NIS	B-Gene_or_gene_product
exhibited	O
current	O
transients	O
that	O
relaxed	O
with	O
a	O
time	O
constant	O
of	O
8	O
-	O
14	O
ms	O
.	O

Presteady	O
-	O
state	O
charge	O
movements	O
(	O
integral	O
of	O
the	O
current	O
transients	O
)	O
versus	O
voltage	O
relations	O
obey	O
a	O
Boltzmann	O
relation	O
.	O

The	O
voltage	O
for	O
half	O
-	O
maximal	O
charge	O
translocation	O
(	O
V0	O
.	O
5	O
)	O
was	O
-	O
15	O
+	O
/	O
-	O
3	O
mV	O
,	O
and	O
the	O
apparent	O
valence	O
of	O
the	O
movable	O
charge	O
was	O
1	O
.	O

Total	O
charge	O
was	O
insensitive	O
to	O
[	B-Simple_chemical
Na	I-Simple_chemical
+	I-Simple_chemical
]	I-Simple_chemical
o	I-Simple_chemical
,	O
but	O
V0	O
.	O
5	O
shifted	O
to	O
more	O
negative	O
potentials	O
as	O
[	B-Simple_chemical
Na	I-Simple_chemical
+	I-Simple_chemical
]	I-Simple_chemical
o	I-Simple_chemical
was	O
reduced	O
.	O

NIS	B-Gene_or_gene_product
charge	O
movements	O
are	O
attributed	O
to	O
the	O
conformational	O
changes	O
of	O
the	O
empty	O
transporter	O
within	O
the	O
membrane	B-Cellular_component
electric	O
field	O
.	O

The	O
turnover	O
rate	O
of	O
NIS	B-Gene_or_gene_product
was	O
>	O
/	O
=	O
22	O
s	O
-	O
1	O
in	O
the	O
Na	B-Simple_chemical
+	I-Simple_chemical
uniport	O
mode	O
and	O
>	O
/	O
=	O
36	O
s	O
-	O
1	O
in	O
the	O
Na	B-Simple_chemical
+	I-Simple_chemical
/	O
I	B-Simple_chemical
-	I-Simple_chemical
cotransport	O
mode	O
.	O

Transporter	O
density	O
in	O
the	O
plasma	B-Cellular_component
membrane	I-Cellular_component
was	O
determined	O
using	O
freeze	O
-	O
fracture	O
electron	O
microscopy	O
.	O

Expression	O
of	O
NIS	B-Gene_or_gene_product
in	O
oocytes	O
led	O
to	O
a	O
approximately	O
2	O
.	O

5	O
-	O
fold	O
increase	O
in	O
the	O
density	O
of	O
plasma	B-Cellular_component
membrane	I-Cellular_component
protoplasmic	I-Cellular_component
face	I-Cellular_component
intramembrane	B-Cellular_component
particles	O
.	O

On	O
the	O
basis	O
of	O
the	O
kinetic	O
results	O
,	O
we	O
propose	O
an	O
ordered	O
simultaneous	O
transport	O
mechanism	O
in	O
which	O
the	O
binding	O
of	O
Na	B-Simple_chemical
+	I-Simple_chemical
to	O
NIS	B-Gene_or_gene_product
occurs	O
first	O
.	O

MyD88	B-Gene_or_gene_product
:	O
an	O
adapter	O
that	O
recruits	O
IRAK	B-Gene_or_gene_product
to	O
the	O
IL	B-Complex
-	I-Complex
1	I-Complex
receptor	I-Complex
complex	O
.	O

IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
is	O
a	O
proinflammatory	O
cytokine	O
that	O
signals	O
through	O
a	O
receptor	O
complex	O
of	O
two	O
different	O
transmembrane	B-Cellular_component
chains	O
to	O
generate	O
multiple	O
cellular	O
responses	O
,	O
including	O
activation	O
of	O
the	O
transcription	O
factor	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
.	O

Here	O
we	O
show	O
that	O
MyD88	B-Gene_or_gene_product
,	O
a	O
previously	O
described	O
protein	O
of	O
unknown	O
function	O
,	O
is	O
recruited	O
to	O
the	O
IL	B-Complex
-	I-Complex
1	I-Complex
receptor	I-Complex
complex	O
following	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
stimulation	O
.	O

MyD88	B-Gene_or_gene_product
binds	O
to	O
both	O
IRAK	B-Gene_or_gene_product
(	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
receptor	I-Gene_or_gene_product
-	I-Gene_or_gene_product
associated	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
)	O
and	O
the	O
heterocomplex	O
(	O
the	O
signaling	O
complex	O
)	O
of	O
the	O
two	O
receptor	O
chains	O
and	O
thereby	O
mediates	O
the	O
association	O
of	O
IRAK	B-Gene_or_gene_product
with	O
the	O
receptor	O
.	O

Ectopic	O
expression	O
of	O
MyD88	B-Gene_or_gene_product
or	O
its	O
death	O
domain	O
-	O
containing	O
N	O
-	O
terminus	O
activates	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
.	O

The	O
C	O
-	O
terminus	O
of	O
MyD88	B-Gene_or_gene_product
interacts	O
with	O
the	O
IL	B-Complex
-	I-Complex
1	I-Complex
receptor	I-Complex
and	O
blocks	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
activation	O
induced	O
by	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
,	O
but	O
not	O
by	O
TNF	B-Gene_or_gene_product
.	O

Thus	O
,	O
MyD88	B-Gene_or_gene_product
plays	O
the	O
same	O
role	O
in	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
signaling	O
as	O
TRADD	B-Gene_or_gene_product
and	O
Tube	B-Gene_or_gene_product
do	O
in	O
TNF	B-Gene_or_gene_product
and	O
Toll	B-Gene_or_gene_product
pathways	O
,	O
respectively	O
:	O
it	O
couples	O
a	O
serine	B-Simple_chemical
/	O
threonine	B-Simple_chemical
protein	O
kinase	O
to	O
the	O
receptor	O
complex	O
.	O

The	O
characterization	O
of	O
butyrate	B-Simple_chemical
transport	O
across	O
pig	O
and	O
human	O
colonic	B-Cellular_component
luminal	I-Cellular_component
membrane	I-Cellular_component
.	O

1	O
.	O

Luminal	B-Cellular_component
membrane	I-Cellular_component
vesicles	I-Cellular_component
(	O
LMV	B-Cellular_component
)	O
were	O
isolated	O
from	O
human	O
and	O
pig	O
colonic	O
tissues	O
.	O

They	O
were	O
characterized	O
in	O
terms	O
of	O
purity	O
and	O
ability	O
to	O
transport	O
[	B-Simple_chemical
14C	I-Simple_chemical
]	I-Simple_chemical
butyrate	I-Simple_chemical
.	O

2	O
.	O

The	O
activity	O
of	O
cysteine	B-Simple_chemical
-	O
sensitive	O
alkaline	B-Gene_or_gene_product
phosphatase	I-Gene_or_gene_product
,	O
and	O
the	O
abundance	O
of	O
villin	B-Gene_or_gene_product
,	O
NHE2	B-Gene_or_gene_product
and	O
NHE3	B-Gene_or_gene_product
proteins	O
,	O
markers	O
of	O
the	O
colonic	B-Cellular_component
luminal	I-Cellular_component
membrane	I-Cellular_component
,	O
were	O
significantly	O
enriched	O
in	O
the	O
LMV	B-Cellular_component
compared	O
with	O
the	O
original	O
cellular	O
homogenate	O
.	O

The	O
LMV	B-Cellular_component
were	O
free	O
from	O
contamination	O
by	O
other	O
cellular	B-Cellular_component
organelles	I-Cellular_component
and	O
basolateral	B-Cellular_component
membranes	I-Cellular_component
,	O
as	O
revealed	O
by	O
the	O
negligible	O
presence	O
of	O
either	O
specific	O
marker	O
enzyme	O
activity	O
or	O
characteristic	O
immunogenic	O
protein	O
.	O

3	O
.	O

The	O
transport	O
of	O
butyrate	B-Simple_chemical
into	O
the	O
luminal	B-Cellular_component
membrane	I-Cellular_component
vesicles	I-Cellular_component
was	O
enhanced	O
5	O
-	O
fold	O
at	O
pH	O
5	O
.	O
5	O
compared	O
with	O
pH	O
8	O
.	O
0	O
.	O

Butyrate	B-Simple_chemical
transport	O
was	O
temperature	O
dependent	O
,	O
and	O
was	O
stimulated	O
in	O
the	O
presence	O
of	O
an	O
outward	O
-	O
directed	O
anion	B-Simple_chemical
gradient	O
in	O
the	O
order	O
of	O
butyrate	B-Simple_chemical
>	O
bicarbonate	B-Simple_chemical
>	O
propionate	B-Simple_chemical
>	O
chloride	B-Simple_chemical
.	O

Kinetic	O
analysis	O
of	O
increasing	O
substrate	O
concentration	O
showed	O
saturation	O
kinetics	O
with	O
an	O
apparent	O
Km	O
value	O
of	O
14	O
.	O
8	O
+	O
/	O
-	O
3	O
.	O
6	O
mM	O
and	O
a	O
Vmax	O
of	O
54	O
+	O
/	O
-	O
14	O
nmol	O
min	O
-	O
1	O
(	O
mg	O
protein	O
)	O
-	O
1	O
.	O

4	O
.	O

Butyrate	B-Simple_chemical
transport	O
was	O
significantly	O
reduced	O
in	O
the	O
presence	O
of	O
short	B-Simple_chemical
chain	I-Simple_chemical
fatty	I-Simple_chemical
acids	I-Simple_chemical
(	O
SCFA	B-Simple_chemical
)	O
,	O
acetate	B-Simple_chemical
,	O
propionate	B-Simple_chemical
and	O
other	O
monocarboxylates	B-Simple_chemical
(	O
pyruvate	B-Simple_chemical
and	O
L	B-Simple_chemical
-	I-Simple_chemical
lactate	I-Simple_chemical
)	O
.	O

Butyrate	B-Simple_chemical
uptake	O
was	O
inhibited	O
by	O
several	O
cysteine	B-Simple_chemical
group	O
modifying	O
reagents	O
such	O
as	O
p	B-Simple_chemical
-	I-Simple_chemical
chloromercuribenzosulphonic	I-Simple_chemical
acid	I-Simple_chemical
(	O
pCMBS	B-Simple_chemical
)	O
,	O
p	B-Simple_chemical
-	I-Simple_chemical
chloromercuribenzoate	I-Simple_chemical
(	O
pCMB	B-Simple_chemical
)	O
,	O
mersalyl	B-Simple_chemical
acid	I-Simple_chemical
and	O
HgCl2	B-Simple_chemical
,	O
but	O
not	O
by	O
the	O
stilbene	B-Simple_chemical
anion	I-Simple_chemical
exchange	O
inhibitors	O
,	O
4	B-Simple_chemical
,	I-Simple_chemical
4	I-Simple_chemical
'	I-Simple_chemical
-	I-Simple_chemical
diisothiocyanostilbene	I-Simple_chemical
-	I-Simple_chemical
2	I-Simple_chemical
,	I-Simple_chemical
2	I-Simple_chemical
'	I-Simple_chemical
-	I-Simple_chemical
disulphonate	I-Simple_chemical
(	O
DIDS	B-Simple_chemical
)	O
and	O
4	B-Simple_chemical
,	I-Simple_chemical
4	I-Simple_chemical
'	I-Simple_chemical
-	I-Simple_chemical
dinitrostilbene	I-Simple_chemical
-	I-Simple_chemical
2	I-Simple_chemical
,	I-Simple_chemical
2	I-Simple_chemical
'	I-Simple_chemical
-	I-Simple_chemical
disulphonate	I-Simple_chemical
(	O
SITS	B-Simple_chemical
)	O
.	O

5	O
.	O

The	O
described	O
properties	O
of	O
butyrate	B-Simple_chemical
transport	O
across	O
the	O
luminal	B-Cellular_component
pole	I-Cellular_component
of	O
the	O
colon	O
suggest	O
the	O
involvement	O
of	O
a	O
carrier	O
protein	O
,	O
in	O
the	O
form	O
of	O
a	O
pH	O
-	O
activated	O
anion	B-Simple_chemical
exchange	O
process	O
.	O

The	O
transporter	O
is	O
distinct	O
from	O
the	O
erythrocyte	O
band	O
-	O
3	O
type	O
anion	B-Simple_chemical
exchanger	O
and	O
may	O
belong	O
to	O
the	O
monocarboxylate	B-Gene_or_gene_product
-	I-Gene_or_gene_product
type	I-Gene_or_gene_product
transport	I-Gene_or_gene_product
proteins	I-Gene_or_gene_product
(	O
MCT1	B-Gene_or_gene_product
)	O
.	O

Cloning	O
and	O
functional	O
expression	O
of	O
a	O
cDNA	O
encoding	O
a	O
mammalian	O
sodium	B-Simple_chemical
-	O
dependent	O
vitamin	B-Simple_chemical
transporter	O
mediating	O
the	O
uptake	O
of	O
pantothenate	B-Simple_chemical
,	O
biotin	B-Simple_chemical
,	O
and	O
lipoate	B-Simple_chemical
.	O

Previous	O
studies	O
have	O
shown	O
that	O
a	O
Na	B-Simple_chemical
+	I-Simple_chemical
-	O
dependent	O
transport	O
system	O
is	O
responsible	O
for	O
the	O
transplacental	O
transfer	O
of	O
the	O
vitamins	B-Simple_chemical
pantothenate	B-Simple_chemical
and	O
biotin	B-Simple_chemical
and	O
the	O
essential	O
metabolite	O
lipoate	B-Simple_chemical
.	O

We	O
now	O
report	O
the	O
isolation	O
of	O
a	O
rat	O
placental	O
cDNA	O
encoding	O
a	O
transport	O
protein	O
responsible	O
for	O
this	O
function	O
.	O

The	O
cloned	O
cDNA	O
,	O
when	O
expressed	O
in	O
HeLa	O
cells	O
,	O
induces	O
Na	B-Simple_chemical
+	I-Simple_chemical
-	O
dependent	O
pantothenate	B-Simple_chemical
and	O
biotin	B-Simple_chemical
transport	O
activities	O
.	O

The	O
transporter	O
is	O
specific	O
for	O
pantothenate	B-Simple_chemical
,	O
biotin	B-Simple_chemical
,	O
and	O
lipoate	B-Simple_chemical
.	O

The	O
Michaelis	O
-	O
Menten	O
constant	O
(	O
Kt	O
)	O
for	O
the	O
transport	O
of	O
pantothenate	B-Simple_chemical
and	O
biotin	B-Simple_chemical
in	O
cDNA	O
-	O
transfected	O
cells	O
is	O
4	O
.	O
9	O
+	O
/	O
-	O
1	O
.	O
1	O
and	O
15	O
.	O
1	O
+	O
/	O
-	O
1	O
.	O
2	O
microM	O
,	O
respectively	O
.	O

The	O
transport	O
of	O
both	O
vitamins	B-Simple_chemical
in	O
cDNA	O
-	O
transfected	O
cells	O
is	O
inhibited	O
by	O
lipoate	B-Simple_chemical
with	O
an	O
inhibition	O
constant	O
(	O
Ki	O
)	O
of	O
approximately	O
5	O
microM	O
.	O

The	O
nucleotide	O
sequence	O
of	O
the	O
cDNA	O
(	O
sodium	B-Gene_or_gene_product
-	I-Gene_or_gene_product
dependent	I-Gene_or_gene_product
multivitamin	I-Gene_or_gene_product
transporter	I-Gene_or_gene_product
(	O
SMVT	B-Gene_or_gene_product
)	O
)	O
predicts	O
a	O
protein	O
of	O
68	O
.	O
6	O
kDa	O
with	O
634	O
amino	O
acids	O
and	O
12	O
potential	O
transmembrane	O
domains	O
.	O

Protein	O
data	O
base	O
search	O
indicates	O
significant	O
sequence	O
similarity	O
between	O
SMVT	B-Gene_or_gene_product
and	O
known	O
members	O
of	O
the	O
Na	B-Gene_or_gene_product
+	I-Gene_or_gene_product
-	I-Gene_or_gene_product
dependent	I-Gene_or_gene_product
glucose	I-Gene_or_gene_product
transporter	I-Gene_or_gene_product
family	O
.	O

Northern	O
blot	O
analysis	O
shows	O
that	O
SMVT	B-Gene_or_gene_product
transcripts	O
are	O
present	O
in	O
all	O
of	O
the	O
tissues	O
that	O
were	O
tested	O
.	O

The	O
size	O
of	O
the	O
principal	O
transcript	O
is	O
3	O
.	O
2	O
kilobases	O
.	O

SMVT	B-Gene_or_gene_product
represents	O
the	O
first	O
Na	B-Simple_chemical
+	I-Simple_chemical
-	O
dependent	O
vitamin	B-Simple_chemical
transporter	O
to	O
be	O
cloned	O
from	O
a	O
mammalian	O
tissue	O
.	O

p53	B-Gene_or_gene_product
-	O
dependent	O
and	O
-	O
independent	O
regulation	O
of	O
the	O
death	B-Gene_or_gene_product
receptor	I-Gene_or_gene_product
KILLER	B-Gene_or_gene_product
/	O
DR5	B-Gene_or_gene_product
gene	O
expression	O
in	O
response	O
to	O
genotoxic	O
stress	O
and	O
tumor	B-Gene_or_gene_product
necrosis	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
.	O

The	O
death	B-Gene_or_gene_product
receptor	I-Gene_or_gene_product
(	O
DR	B-Gene_or_gene_product
)	O
KILLER	B-Gene_or_gene_product
/	O
DR5	B-Gene_or_gene_product
gene	O
has	O
recently	O
been	O
identified	O
as	O
a	O
doxorubicin	B-Simple_chemical
-	O
regulated	O
transcript	O
that	O
was	O
also	O
induced	O
by	O
exogenous	O
wild	O
-	O
type	O
p53	B-Gene_or_gene_product
in	O
p53	B-Gene_or_gene_product
-	O
negative	O
cells	O
.	O

KILLER	B-Gene_or_gene_product
/	O
DR5	B-Gene_or_gene_product
gene	O
encodes	O
a	O
DR	B-Gene_or_gene_product
containing	O
cell	B-Cellular_component
surface	I-Cellular_component
protein	O
that	O
is	O
highly	O
homologous	O
to	O
DR4	B-Gene_or_gene_product
,	O
another	O
DR	B-Gene_or_gene_product
of	O
the	O
tumor	B-Gene_or_gene_product
necrosis	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
(	I-Gene_or_gene_product
TNF	I-Gene_or_gene_product
)	I-Gene_or_gene_product
receptor	I-Gene_or_gene_product
family	O
.	O

Both	O
DR4	B-Gene_or_gene_product
and	O
KILLER	B-Gene_or_gene_product
/	O
DR5	B-Gene_or_gene_product
independently	O
bind	O
to	O
their	O
specific	O
ligand	O
TRAIL	B-Gene_or_gene_product
and	O
engage	O
the	O
caspase	B-Gene_or_gene_product
cascade	O
to	O
induce	O
apoptosis	O
.	O

TRID	B-Gene_or_gene_product
(	O
also	O
known	O
as	O
TRAIL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
R3	I-Gene_or_gene_product
)	O
is	O
an	O
antiapoptotic	O
decoy	O
receptor	O
that	O
lacks	O
the	O
cytoplasmic	B-Cellular_component
death	O
domain	O
and	O
competes	O
with	O
KILLER	B-Gene_or_gene_product
/	O
DR5	B-Gene_or_gene_product
and	O
DR4	B-Gene_or_gene_product
for	O
binding	O
to	O
TRAIL	B-Gene_or_gene_product
.	O

In	O
this	O
study	O
,	O
we	O
demonstrate	O
that	O
the	O
DR	B-Gene_or_gene_product
KILLER	I-Gene_or_gene_product
/	O
DR5	B-Gene_or_gene_product
gene	O
is	O
regulated	O
in	O
a	O
p53	B-Gene_or_gene_product
-	O
dependent	O
and	O
-	O
independent	O
manner	O
during	O
genotoxic	O
and	O
nongenotoxic	O
stress	O
-	O
induced	O
apoptosis	O
.	O

Just	O
like	O
other	O
p53	B-Gene_or_gene_product
-	O
regulated	O
genes	O
,	O
ionizing	O
radiation	O
induction	O
of	O
KILLER	B-Gene_or_gene_product
/	O
DR5	B-Gene_or_gene_product
occurs	O
in	O
p53	B-Gene_or_gene_product
wild	O
-	O
type	O
cells	O
,	O
whereas	O
methyl	B-Simple_chemical
methanesulfonate	I-Simple_chemical
regulation	O
of	O
KILLER	B-Gene_or_gene_product
/	O
DR5	B-Gene_or_gene_product
occurs	O
in	O
a	O
p53	B-Gene_or_gene_product
-	O
dependent	O
and	O
-	O
independent	O
manner	O
.	O

However	O
,	O
unlike	O
other	O
p53	B-Gene_or_gene_product
-	O
regulated	O
genes	O
,	O
KILLER	B-Gene_or_gene_product
/	O
DR5	B-Gene_or_gene_product
is	O
not	O
regulated	O
following	O
UV	O
irradiation	O
.	O

TNF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
,	O
a	O
nongenotoxic	O
cytokine	O
,	O
also	O
induced	O
the	O
expression	O
of	O
KILLER	B-Gene_or_gene_product
/	O
DR5	B-Gene_or_gene_product
in	O
a	O
number	O
of	O
cancer	O
cell	O
lines	O
,	O
irrespective	O
of	O
p53	B-Gene_or_gene_product
status	O
.	O

TNF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
did	O
not	O
alter	O
the	O
KILLER	B-Gene_or_gene_product
/	O
DR5	B-Gene_or_gene_product
mRNA	O
stability	O
,	O
suggesting	O
that	O
the	O
TNF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
regulation	O
of	O
KILLER	B-Gene_or_gene_product
/	O
DRS	B-Gene_or_gene_product
expression	O
appears	O
transcriptional	O
.	O

We	O
also	O
provide	O
evidence	O
that	O
KILLER	B-Gene_or_gene_product
/	O
DR5	B-Gene_or_gene_product
is	O
regulated	O
in	O
a	O
trigger	O
and	O
cell	O
type	O
-	O
specific	O
manner	O
and	O
that	O
its	O
induction	O
by	O
TNF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
,	O
p53	B-Gene_or_gene_product
,	O
or	O
DNA	O
damage	O
is	O
not	O
the	O
consequence	O
of	O
apoptosis	O
induced	O
by	O
these	O
agents	O
.	O

Unlike	O
KILLER	B-Gene_or_gene_product
/	O
DR5	B-Gene_or_gene_product
,	O
none	O
of	O
the	O
other	O
KILLER	B-Gene_or_gene_product
/	O
DR5	B-Gene_or_gene_product
family	O
members	O
,	O
including	O
DR4	B-Gene_or_gene_product
,	O
TRID	B-Gene_or_gene_product
,	O
or	O
the	O
ligand	O
TRAIL	B-Gene_or_gene_product
,	O
displayed	O
genotoxic	O
stress	O
or	O
TNF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
regulation	O
in	O
a	O
p53	B-Gene_or_gene_product
transcription	O
-	O
dependent	O
manner	O
.	O

Thus	O
,	O
KILLER	B-Gene_or_gene_product
/	O
DR5	B-Gene_or_gene_product
appears	O
a	O
bona	O
fide	O
downstream	O
target	O
of	O
p53	B-Gene_or_gene_product
that	O
is	O
also	O
regulated	O
in	O
a	O
cell	O
type	O
-	O
specific	O
,	O
trigger	O
-	O
dependent	O
,	O
and	O
p53	B-Gene_or_gene_product
-	O
independent	O
manner	O
.	O

The	O
eukaryotic	O
UDP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
N	I-Gene_or_gene_product
-	I-Gene_or_gene_product
acetylglucosamine	I-Gene_or_gene_product
pyrophosphorylases	I-Gene_or_gene_product
.	O

Gene	O
cloning	O
,	O
protein	O
expression	O
,	O
and	O
catalytic	O
mechanism	O
.	O

A	O
search	O
of	O
the	O
yeast	O
data	O
base	O
for	O
a	O
protein	O
homologous	O
to	O
Escherichia	O
coli	O
UDP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
N	I-Gene_or_gene_product
-	I-Gene_or_gene_product
acetylglucosamine	I-Gene_or_gene_product
pyrophosphorylase	I-Gene_or_gene_product
yielded	O
UAP1	B-Gene_or_gene_product
(	O
UDP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
N	I-Gene_or_gene_product
-	I-Gene_or_gene_product
acetylglucosamine	I-Gene_or_gene_product
pyrophosphorylase	I-Gene_or_gene_product
)	O
,	O
the	O
Saccharomyces	O
cerevisiae	O
gene	O
for	O
UDP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
N	I-Gene_or_gene_product
-	I-Gene_or_gene_product
acetylglucosamine	I-Gene_or_gene_product
pyrophosphorylase	I-Gene_or_gene_product
.	O

The	O
Candida	O
albicans	O
and	O
human	O
homologs	O
were	O
also	O
cloned	O
by	O
screening	O
a	O
C	O
.	O
albicans	O
genomic	O
library	O
and	O
a	O
human	O
testis	O
cDNA	O
library	O
,	O
respectively	O
.	O

Sequence	O
analysis	O
revealed	O
that	O
the	O
human	O
UAP1	B-Gene_or_gene_product
cDNA	O
was	O
identical	O
to	O
previously	O
reported	O
AGX1	B-Gene_or_gene_product
.	O

A	O
null	O
mutation	O
of	O
the	O
S	O
.	O
cerevisiae	O
UAP1	B-Gene_or_gene_product
(	O
ScUAP1	B-Gene_or_gene_product
)	O
gene	O
was	O
lethal	O
,	O
and	O
when	O
expressed	O
under	O
the	O
control	O
of	O
ScUAP1	B-Gene_or_gene_product
promoter	O
,	O
both	O
C	O
.	O
albicans	O
and	O
Homo	O
sapiens	O
UAP1	B-Gene_or_gene_product
(	O
CaUAP1	B-Gene_or_gene_product
and	O
HsUAP1	B-Gene_or_gene_product
)	O
rescued	O
the	O
ScUAP1	B-Gene_or_gene_product
-	O
deficient	O
S	O
.	O
cerevisiae	O
cells	O
.	O

All	O
the	O
recombinant	O
ScUap1p	B-Gene_or_gene_product
,	O
CaUap1p	B-Gene_or_gene_product
,	O
and	O
HsUap1p	B-Gene_or_gene_product
possessed	O
UDP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
N	I-Gene_or_gene_product
-	I-Gene_or_gene_product
acetylglucosamine	I-Gene_or_gene_product
pyrophosphorylase	I-Gene_or_gene_product
activities	O
in	O
vitro	O
.	O

The	O
yeast	O
Uap1p	B-Gene_or_gene_product
utilized	O
N	B-Simple_chemical
-	I-Simple_chemical
acetylglucosamine	I-Simple_chemical
-	I-Simple_chemical
1	I-Simple_chemical
-	I-Simple_chemical
phosphate	I-Simple_chemical
as	O
the	O
substrate	O
,	O
and	O
together	O
with	O
Agm1p	B-Gene_or_gene_product
,	O
it	O
produced	O
UDP	B-Simple_chemical
-	I-Simple_chemical
N	I-Simple_chemical
-	I-Simple_chemical
acetylglucosamine	I-Simple_chemical
from	O
N	B-Simple_chemical
-	I-Simple_chemical
acetylglucosamine	I-Simple_chemical
-	I-Simple_chemical
6	I-Simple_chemical
-	I-Simple_chemical
phosphate	I-Simple_chemical
.	O

These	O
results	O
demonstrate	O
that	O
the	O
UAP1	B-Gene_or_gene_product
genes	O
indeed	O
specify	O
eukaryotic	O
UDP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
GlcNAc	I-Gene_or_gene_product
pyrophosphorylase	I-Gene_or_gene_product
and	O
that	O
phosphomutase	O
reaction	O
precedes	O
uridyltransfer	O
.	O

Sequence	O
comparison	O
with	O
other	O
UDP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
sugar	I-Gene_or_gene_product
pyrophosphorylases	I-Gene_or_gene_product
revealed	O
that	O
amino	O
acid	O
residues	O
,	O
Gly112	B-Simple_chemical
,	O
Gly114	B-Simple_chemical
,	O
Thr115	B-Simple_chemical
,	O
Arg116	B-Simple_chemical
,	O
Pro122	B-Simple_chemical
,	O
and	O
Lys123	B-Simple_chemical
of	O
ScUap1p	B-Gene_or_gene_product
are	O
highly	O
conserved	O
in	O
UDP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
sugar	I-Gene_or_gene_product
pyrophosphorylases	I-Gene_or_gene_product
reported	O
to	O
date	O
.	O

Among	O
these	O
amino	O
acids	O
,	O
alanine	B-Simple_chemical
substitution	O
for	O
Gly112	B-Simple_chemical
,	O
Arg116	B-Simple_chemical
,	O
or	O
Lys123	B-Simple_chemical
severely	O
diminished	O
the	O
activity	O
,	O
suggesting	O
that	O
Gly112	B-Simple_chemical
,	O
Arg116	B-Simple_chemical
,	O
or	O
Lys123	B-Simple_chemical
are	O
possible	O
catalytic	O
residues	O
of	O
the	O
enzyme	O
.	O

Control	O
of	O
cyclin	B-Gene_or_gene_product
B1	I-Gene_or_gene_product
localization	O
through	O
regulated	O
binding	O
of	O
the	O
nuclear	B-Cellular_component
export	O
factor	O
CRM1	B-Gene_or_gene_product
.	O

Activation	O
of	O
the	O
Cyclin	B-Complex
B	I-Complex
/	I-Complex
Cdc2	I-Complex
kinase	O
complex	O
triggers	O
entry	O
into	O
mitosis	O
in	O
all	O
eukaryotic	O
cells	O
.	O

Cyclin	B-Gene_or_gene_product
B1	I-Gene_or_gene_product
localization	O
changes	O
dramatically	O
during	O
the	O
cell	O
cycle	O
,	O
precipitously	O
transiting	O
from	O
the	O
cytoplasm	B-Cellular_component
to	O
the	O
nucleus	B-Cellular_component
at	O
the	O
beginning	O
of	O
mitosis	O
.	O

Presumably	O
,	O
this	O
relocalization	O
promotes	O
the	O
phosphorylation	O
of	O
nuclear	B-Cellular_component
targets	O
critical	O
for	O
chromatin	B-Cellular_component
condensation	O
and	O
nuclear	B-Cellular_component
envelope	I-Cellular_component
breakdown	O
.	O

We	O
show	O
here	O
that	O
the	O
previously	O
characterized	O
cytoplasmic	B-Cellular_component
retention	O
sequence	O
of	O
Cyclin	B-Gene_or_gene_product
B1	I-Gene_or_gene_product
,	O
responsible	O
for	O
its	O
interphase	O
cytoplasmic	B-Cellular_component
localization	O
,	O
is	O
actually	O
an	O
autonomous	O
nuclear	B-Cellular_component
export	O
sequence	O
,	O
capable	O
of	O
directing	O
nuclear	B-Cellular_component
export	O
of	O
a	O
heterologous	O
protein	O
,	O
and	O
able	O
to	O
bind	O
specifically	O
to	O
the	O
recently	O
identified	O
export	O
mediator	O
,	O
CRM1	B-Gene_or_gene_product
.	O

We	O
propose	O
that	O
the	O
observed	O
cytoplasmic	B-Cellular_component
localization	O
of	O
Cyclin	B-Gene_or_gene_product
B1	I-Gene_or_gene_product
during	O
interphase	O
reflects	O
the	O
equilibrium	O
between	O
ongoing	O
nuclear	B-Cellular_component
import	O
and	O
rapid	O
CRM1	B-Gene_or_gene_product
-	O
mediated	O
export	O
.	O

In	O
support	O
of	O
this	O
hypothesis	O
,	O
we	O
found	O
that	O
treatment	O
of	O
cells	O
with	O
leptomycin	B-Simple_chemical
B	I-Simple_chemical
,	O
which	O
disrupted	O
Cyclin	B-Gene_or_gene_product
B1	I-Gene_or_gene_product
-	O
CRM1	B-Gene_or_gene_product
interactions	O
,	O
led	O
to	O
a	O
marked	O
nuclear	B-Cellular_component
accumulation	O
of	O
Cyclin	B-Gene_or_gene_product
B1	I-Gene_or_gene_product
.	O

In	O
mitosis	O
,	O
Cyclin	B-Gene_or_gene_product
B1	I-Gene_or_gene_product
undergoes	O
phosphorylation	O
at	O
several	O
sites	O
,	O
a	O
subset	O
of	O
which	O
have	O
been	O
proposed	O
to	O
play	O
a	O
role	O
in	O
Cyclin	B-Gene_or_gene_product
B1	I-Gene_or_gene_product
accumulation	O
in	O
the	O
nucleus	B-Cellular_component
.	O

Both	O
CRM1	B-Gene_or_gene_product
binding	O
and	O
the	O
ability	O
to	O
direct	O
nuclear	B-Cellular_component
export	O
were	O
affected	O
by	O
mutation	O
of	O
these	O
phosphorylation	O
sites	O
;	O
thus	O
,	O
we	O
propose	O
that	O
Cyclin	B-Gene_or_gene_product
B1	I-Gene_or_gene_product
phosphorylation	O
at	O
the	O
G2	O
/	O
M	O
transition	O
prevents	O
its	O
interaction	O
with	O
CRM1	B-Gene_or_gene_product
,	O
thereby	O
reducing	O
nuclear	B-Cellular_component
export	O
and	O
facilitating	O
nuclear	B-Cellular_component
accumulation	O
.	O

Interleukin	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
.	O

Interleukin	B-Gene_or_gene_product
1	I-Gene_or_gene_product
(	O
IL1	B-Gene_or_gene_product
)	O
is	O
a	O
primary	O
regulator	O
of	O
inflammatory	O
and	O
immune	O
responses	O
.	O

Via	O
its	O
type	B-Gene_or_gene_product
I	I-Gene_or_gene_product
receptor	I-Gene_or_gene_product
it	O
activates	O
specific	O
protein	O
kinases	O
,	O
including	O
the	O
NF	B-Gene_or_gene_product
kappa	I-Gene_or_gene_product
B	I-Gene_or_gene_product
inducing	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
(	O
NIK	B-Gene_or_gene_product
)	O
and	O
three	O
distinct	O
mitogen	B-Gene_or_gene_product
-	I-Gene_or_gene_product
activated	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
(	I-Gene_or_gene_product
MAP	I-Gene_or_gene_product
)	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
cascades	O
.	O

These	O
modulate	O
a	O
number	O
of	O
transcription	O
factors	O
including	O
NF	B-Complex
kappa	I-Complex
B	I-Complex
,	O
AP1	B-Complex
and	O
CREB	B-Gene_or_gene_product
each	O
of	O
which	O
regulate	O
a	O
plethora	O
of	O
immediate	O
early	O
genes	O
central	O
to	O
the	O
inflammatory	O
response	O
.	O

Phase	O
I	O
clinical	O
trials	O
of	O
the	O
soluble	O
type	B-Gene_or_gene_product
I	I-Gene_or_gene_product
receptor	I-Gene_or_gene_product
and	O
IRAP	B-Gene_or_gene_product
indicate	O
that	O
these	O
have	O
potential	O
anti	O
-	O
inflammatory	O
effects	O
.	O

Cell	O
cycle	O
regulation	O
of	O
the	O
Saccharomyces	O
cerevisiae	O
polo	B-Gene_or_gene_product
-	O
like	O
kinase	O
cdc5p	B-Gene_or_gene_product
.	O

Progression	O
through	O
and	O
completion	O
of	O
mitosis	O
require	O
the	O
actions	O
of	O
the	O
evolutionarily	O
conserved	O
Polo	B-Gene_or_gene_product
kinase	O
.	O

We	O
have	O
determined	O
that	O
the	O
levels	O
of	O
Cdc5p	B-Gene_or_gene_product
,	O
a	O
Saccharomyces	O
cerevisiae	O
member	O
of	O
the	O
Polo	B-Gene_or_gene_product
family	O
of	O
mitotic	O
kinases	O
,	O
are	O
cell	O
cycle	O
regulated	O
.	O

Cdc5p	B-Gene_or_gene_product
accumulates	O
in	O
the	O
nuclei	B-Cellular_component
of	O
G2	O
/	O
M	O
-	O
phase	O
cells	O
,	O
and	O
its	O
levels	O
decline	O
dramatically	O
as	O
cells	O
progress	O
through	O
anaphase	O
and	O
begin	O
telophase	O
.	O

We	O
report	O
that	O
Cdc5p	B-Gene_or_gene_product
levels	O
are	O
sensitive	O
to	O
mutations	O
in	O
key	O
components	O
of	O
the	O
anaphase	B-Complex
-	I-Complex
promoting	I-Complex
complex	I-Complex
(	O
APC	B-Complex
)	O
.	O

We	O
have	O
determined	O
that	O
Cdc5p	B-Gene_or_gene_product
-	O
associated	O
kinase	O
activity	O
is	O
restricted	O
to	O
G2	O
/	O
M	O
and	O
that	O
this	O
activity	O
is	O
posttranslationally	O
regulated	O
.	O

These	O
results	O
further	O
link	O
the	O
actions	O
of	O
the	O
APC	B-Complex
to	O
the	O
completion	O
of	O
mitosis	O
and	O
suggest	O
possible	O
roles	O
for	O
Cdc5p	B-Gene_or_gene_product
during	O
progression	O
through	O
and	O
completion	O
of	O
mitosis	O
.	O

The	O
potential	O
tumor	O
suppressor	O
p73	B-Gene_or_gene_product
differentially	O
regulates	O
cellular	O
p53	B-Gene_or_gene_product
target	O
genes	O
.	O

p73	B-Gene_or_gene_product
,	O
a	O
potential	O
tumor	O
suppressor	O
,	O
is	O
a	O
p53	B-Gene_or_gene_product
homologue	O
.	O

Transient	O
over	O
expression	O
of	O
p73	B-Gene_or_gene_product
in	O
cells	O
can	O
induce	O
apoptosis	O
and	O
p21	B-Gene_or_gene_product
,	O
a	O
cellular	O
p53	B-Gene_or_gene_product
target	O
gene	O
primarily	O
responsible	O
for	O
p53	B-Gene_or_gene_product
-	O
dependent	O
cell	O
cycle	O
arrest	O
.	O

To	O
further	O
characterize	O
the	O
role	O
of	O
p73	B-Gene_or_gene_product
in	O
tumor	O
suppression	O
,	O
we	O
established	O
several	O
groups	O
of	O
cell	O
lines	O
that	O
inducibly	O
express	O
p73	B-Gene_or_gene_product
under	O
a	O
tetracycline	B-Simple_chemical
-	O
regulated	O
promoter	O
.	O

By	O
using	O
these	O
cell	O
lines	O
,	O
we	O
found	O
that	O
p73	B-Gene_or_gene_product
can	O
induce	O
both	O
cell	O
cycle	O
arrest	O
and	O
apoptosis	O
.	O

We	O
also	O
found	O
that	O
p73	B-Gene_or_gene_product
can	O
activate	O
some	O
but	O
not	O
all	O
of	O
the	O
previously	O
identified	O
p53	B-Gene_or_gene_product
cellular	O
target	O
genes	O
.	O

Furthermore	O
,	O
we	O
found	O
that	O
the	O
transcriptional	O
activities	O
of	O
p53	B-Gene_or_gene_product
,	O
p73	B-Gene_or_gene_product
alpha	I-Gene_or_gene_product
,	O
and	O
p73	B-Gene_or_gene_product
beta	I-Gene_or_gene_product
to	O
induce	O
their	O
common	O
cellular	O
target	O
genes	O
differ	O
among	O
one	O
another	O
.	O

These	O
results	O
suggest	O
that	O
p73	B-Gene_or_gene_product
is	O
both	O
similar	O
to	O
and	O
different	O
from	O
p53	B-Gene_or_gene_product
in	O
their	O
signaling	O
pathways	O
leading	O
to	O
tumor	O
suppression	O
.	O

Linkage	O
of	O
ATM	B-Gene_or_gene_product
to	O
cell	O
cycle	O
regulation	O
by	O
the	O
Chk2	B-Gene_or_gene_product
protein	O
kinase	O
.	O

In	O
response	O
to	O
DNA	O
damage	O
and	O
replication	O
blocks	O
,	O
cells	O
prevent	O
cell	O
cycle	O
progression	O
through	O
the	O
control	O
of	O
critical	O
cell	O
cycle	O
regulators	O
.	O

We	O
identified	O
Chk2	B-Gene_or_gene_product
,	O
the	O
mammalian	O
homolog	O
of	O
the	O
Saccharomyces	O
cerevisiae	O
Rad53	B-Gene_or_gene_product
and	O
Schizosaccharomyces	O
pombe	O
Cds1	B-Gene_or_gene_product
protein	O
kinases	O
required	O
for	O
the	O
DNA	O
damage	O
and	O
replication	O
checkpoints	O
.	O

Chk2	B-Gene_or_gene_product
was	O
rapidly	O
phosphorylated	O
and	O
activated	O
in	O
response	O
to	O
replication	O
blocks	O
and	O
DNA	O
damage	O
;	O
the	O
response	O
to	O
DNA	O
damage	O
occurred	O
in	O
an	O
ataxia	B-Gene_or_gene_product
telangiectasia	I-Gene_or_gene_product
mutated	I-Gene_or_gene_product
(	O
ATM	B-Gene_or_gene_product
)	O
-	O
dependent	O
manner	O
.	O

In	O
vitro	O
,	O
Chk2	B-Gene_or_gene_product
phosphorylated	O
Cdc25C	B-Gene_or_gene_product
on	O
serine	B-Simple_chemical
-	I-Simple_chemical
216	I-Simple_chemical
,	O
a	O
site	O
known	O
to	O
be	O
involved	O
in	O
negative	O
regulation	O
of	O
Cdc25C	B-Gene_or_gene_product
.	O

This	O
is	O
the	O
same	O
site	O
phosphorylated	O
by	O
the	O
protein	O
kinase	O
Chk1	B-Gene_or_gene_product
,	O
which	O
suggests	O
that	O
,	O
in	O
response	O
to	O
DNA	O
damage	O
and	O
DNA	O
replicational	O
stress	O
,	O
Chk1	B-Gene_or_gene_product
and	O
Chk2	B-Gene_or_gene_product
may	O
phosphorylate	O
Cdc25C	B-Gene_or_gene_product
to	O
prevent	O
entry	O
into	O
mitosis	O
.	O

ATM	B-Gene_or_gene_product
associates	O
with	O
and	O
phosphorylates	O
p53	B-Gene_or_gene_product
:	O
mapping	O
the	O
region	O
of	O
interaction	O
.	O

The	O
human	O
genetic	O
disorder	O
ataxia	O
-	O
telangiectasia	O
(	O
AT	O
)	O
is	O
characterized	O
by	O
immunodeficiency	O
,	O
progressive	O
cerebellar	O
ataxia	O
,	O
radiosensitivity	O
,	O
cell	O
cycle	O
checkpoint	O
defects	O
and	O
cancer	O
predisposition	O
.	O

The	O
gene	O
mutated	O
in	O
this	O
syndrome	O
,	O
ATM	B-Gene_or_gene_product
(	O
for	O
AT	B-Gene_or_gene_product
mutated	I-Gene_or_gene_product
)	O
,	O
encodes	O
a	O
protein	O
containing	O
a	O
phosphatidyl	B-Gene_or_gene_product
-	I-Gene_or_gene_product
inositol	I-Gene_or_gene_product
3	I-Gene_or_gene_product
-	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
(	O
PI	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
)	O
-	O
like	O
domain	O
.	O

ATM	B-Gene_or_gene_product
also	O
contains	O
a	O
proline	B-Simple_chemical
-	O
rich	O
region	O
and	O
a	O
leucine	B-Simple_chemical
zipper	O
,	O
both	O
of	O
which	O
implicate	O
this	O
protein	O
in	O
signal	O
transduction	O
.	O

The	O
proline	B-Simple_chemical
-	O
rich	O
region	O
has	O
been	O
shown	O
to	O
bind	O
to	O
the	O
SH3	O
domain	O
of	O
c	B-Gene_or_gene_product
-	I-Gene_or_gene_product
Abl	I-Gene_or_gene_product
,	O
which	O
facilitates	O
its	O
phosphorylation	O
and	O
activation	O
by	O
ATM	B-Gene_or_gene_product
.	O

Previous	O
results	O
have	O
demonstrated	O
that	O
AT	O
cells	O
are	O
defective	O
in	O
the	O
G1	O
/	O
S	O
checkpoint	O
activated	O
after	O
radiation	O
damage	O
and	O
that	O
this	O
defect	O
is	O
attributable	O
to	O
a	O
defective	O
p53	B-Gene_or_gene_product
signal	O
transduction	O
pathway	O
.	O

We	O
report	O
here	O
direct	O
interaction	O
between	O
ATM	B-Gene_or_gene_product
and	O
p53	B-Gene_or_gene_product
involving	O
two	O
regions	O
in	O
ATM	B-Gene_or_gene_product
,	O
one	O
at	O
the	O
amino	O
terminus	O
and	O
the	O
other	O
at	O
the	O
carboxy	O
terminus	O
,	O
corresponding	O
to	O
the	O
PI	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
domain	O
.	O

Recombinant	O
ATM	B-Gene_or_gene_product
protein	O
phosphorylates	O
p53	B-Gene_or_gene_product
on	O
serine	B-Simple_chemical
15	I-Simple_chemical
near	O
the	O
N	O
terminus	O
.	O

Furthermore	O
,	O
ectopic	O
expression	O
of	O
ATM	B-Gene_or_gene_product
in	O
AT	O
cells	O
restores	O
normal	O
ionizing	O
radiation	O
(	O
IR	O
)	O
-	O
induced	O
phosphorylation	O
of	O
p53	B-Gene_or_gene_product
,	O
whereas	O
expression	O
of	O
ATM	B-Gene_or_gene_product
antisense	O
RNA	O
in	O
control	O
cells	O
abrogates	O
the	O
rapid	O
IR	O
-	O
induced	O
phosphorylation	O
of	O
p53	B-Gene_or_gene_product
on	O
serine	B-Simple_chemical
15	I-Simple_chemical
.	O

These	O
results	O
demonstrate	O
that	O
ATM	B-Gene_or_gene_product
can	O
bind	O
p53	B-Gene_or_gene_product
directly	O
and	O
is	O
responsible	O
for	O
its	O
serine	B-Simple_chemical
15	I-Simple_chemical
phosphorylation	O
,	O
thereby	O
contributing	O
to	O
the	O
activation	O
and	O
stabilization	O
of	O
p53	B-Gene_or_gene_product
during	O
the	O
IR	O
-	O
induced	O
DNA	O
damage	O
response	O
.	O

The	O
metabolism	O
of	O
6	B-Simple_chemical
-	I-Simple_chemical
deoxyhexoses	I-Simple_chemical
in	O
bacterial	O
and	O
animal	O
cells	O
.	O

L	B-Simple_chemical
-	I-Simple_chemical
fucose	I-Simple_chemical
and	O
L	B-Simple_chemical
-	I-Simple_chemical
rhamnose	I-Simple_chemical
are	O
two	O
6	B-Simple_chemical
-	I-Simple_chemical
deoxyhexoses	I-Simple_chemical
naturally	O
occurring	O
in	O
several	O
complex	O
carbohydrates	B-Simple_chemical
.	O

In	O
prokaryotes	O
both	O
of	O
them	O
are	O
found	O
in	O
polysaccharides	B-Simple_chemical
of	O
the	O
cell	B-Cellular_component
wall	I-Cellular_component
,	O
while	O
in	O
animals	O
only	O
L	B-Simple_chemical
-	I-Simple_chemical
fucose	I-Simple_chemical
has	O
been	O
described	O
,	O
which	O
mainly	O
participates	O
to	O
the	O
structure	O
of	O
glycoconjugates	O
,	O
either	O
in	O
the	O
cell	B-Cellular_component
membrane	I-Cellular_component
or	O
secreted	O
in	O
biological	O
fluids	O
,	O
such	O
as	O
ABH	O
blood	O
groups	O
and	O
Lewis	O
system	O
antigens	O
.	O

L	B-Simple_chemical
-	I-Simple_chemical
fucose	I-Simple_chemical
and	O
L	B-Simple_chemical
-	I-Simple_chemical
rhamnose	I-Simple_chemical
are	O
synthesized	O
by	O
two	O
de	O
novo	O
biosynthetic	O
pathways	O
starting	O
from	O
GDP	B-Simple_chemical
-	I-Simple_chemical
D	I-Simple_chemical
-	I-Simple_chemical
mannose	I-Simple_chemical
and	O
dTDP	B-Simple_chemical
-	I-Simple_chemical
D	I-Simple_chemical
-	I-Simple_chemical
glucose	I-Simple_chemical
,	O
respectively	O
,	O
which	O
share	O
several	O
common	O
features	O
.	O

The	O
first	O
step	O
for	O
both	O
pathways	O
is	O
a	O
dehydration	O
reaction	O
catalyzed	O
by	O
specific	O
nucleotide	B-Gene_or_gene_product
-	I-Gene_or_gene_product
sugar	I-Gene_or_gene_product
dehydratases	I-Gene_or_gene_product
.	O

This	O
leads	O
to	O
the	O
formation	O
of	O
unstable	O
4	B-Simple_chemical
-	I-Simple_chemical
keto	I-Simple_chemical
-	I-Simple_chemical
6	I-Simple_chemical
-	I-Simple_chemical
deoxy	I-Simple_chemical
intermediates	O
,	O
which	O
undergo	O
a	O
subsequent	O
epimerization	O
reaction	O
responsible	O
for	O
the	O
change	O
from	O
D	O
-	O
to	O
L	O
-	O
conformation	O
,	O
and	O
then	O
a	O
NADPH	B-Simple_chemical
-	O
dependent	O
reduction	O
of	O
the	O
4	O
-	O
keto	O
group	O
,	O
with	O
the	O
consequent	O
formation	O
of	O
either	O
GDP	B-Simple_chemical
-	I-Simple_chemical
L	I-Simple_chemical
-	I-Simple_chemical
fucose	I-Simple_chemical
or	O
dTDP	B-Simple_chemical
-	I-Simple_chemical
L	I-Simple_chemical
-	I-Simple_chemical
rhamnose	I-Simple_chemical
.	O

These	O
compounds	O
are	O
then	O
the	O
substrates	O
of	O
specific	O
glycosyltransferases	B-Gene_or_gene_product
which	O
are	O
responsible	O
for	O
insertion	O
of	O
either	O
L	B-Simple_chemical
-	I-Simple_chemical
fucose	I-Simple_chemical
or	O
L	B-Simple_chemical
-	I-Simple_chemical
rhamnose	I-Simple_chemical
in	O
the	O
corresponding	O
glycoconjugates	O
.	O

The	O
enzyme	O
involved	O
in	O
the	O
first	O
step	O
of	O
GDP	B-Simple_chemical
-	I-Simple_chemical
L	I-Simple_chemical
-	I-Simple_chemical
fucose	I-Simple_chemical
biosynthesis	O
in	O
E	O
.	O
coli	O
,	O
i	O
.	O
e	O
.	O
,	O
GDP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
D	I-Gene_or_gene_product
-	I-Gene_or_gene_product
mannose	I-Gene_or_gene_product
4	I-Gene_or_gene_product
,	I-Gene_or_gene_product
6	I-Gene_or_gene_product
dehydratase	I-Gene_or_gene_product
,	O
has	O
been	O
recently	O
expressed	O
as	O
recombinant	O
protein	O
and	O
characterized	O
in	O
our	O
laboratory	O
.	O

We	O
have	O
also	O
cloned	O
and	O
fully	O
characterized	O
a	O
human	O
protein	O
,	O
formerly	O
named	O
FX	B-Gene_or_gene_product
,	O
and	O
an	O
E	O
.	O
coli	O
protein	O
,	O
WcaG	B-Gene_or_gene_product
,	O
which	O
display	O
both	O
the	O
epimerase	O
and	O
the	O
reductase	O
activities	O
,	O
thus	O
indicating	O
that	O
only	O
two	O
enzymes	O
are	O
required	O
for	O
GDP	B-Simple_chemical
-	I-Simple_chemical
L	I-Simple_chemical
-	I-Simple_chemical
fucose	I-Simple_chemical
production	O
.	O

Fucosylated	O
complex	O
glycoconjugates	O
at	O
the	O
cell	B-Cellular_component
surface	I-Cellular_component
can	O
then	O
be	O
recognized	O
by	O
specific	O
counter	O
-	O
receptors	O
in	O
interacting	O
cells	O
,	O
these	O
mechanisms	O
initiating	O
important	O
processes	O
including	O
inflammation	O
and	O
metastasis	O
.	O

The	O
second	O
pathway	O
starting	O
from	O
dTDP	B-Simple_chemical
-	I-Simple_chemical
D	I-Simple_chemical
-	I-Simple_chemical
glucose	I-Simple_chemical
leads	O
to	O
the	O
synthesis	O
of	O
antibiotic	O
glycosides	B-Simple_chemical
or	O
,	O
alternatively	O
,	O
to	O
the	O
production	O
of	O
dTDP	B-Simple_chemical
-	I-Simple_chemical
L	I-Simple_chemical
-	I-Simple_chemical
rhamnose	I-Simple_chemical
.	O

While	O
several	O
sets	O
of	O
data	O
are	O
available	O
on	O
the	O
first	O
enzyme	O
of	O
the	O
pathway	O
,	O
i	O
.	O
e	O
.	O
,	O
dTDP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
D	I-Gene_or_gene_product
-	I-Gene_or_gene_product
glucose	I-Gene_or_gene_product
dehydratase	I-Gene_or_gene_product
,	O
the	O
enzymes	O
involved	O
in	O
the	O
following	O
steps	O
still	O
need	O
to	O
be	O
identified	O
and	O
characterized	O
.	O

