Regulation	O
of	O
Saccharomyces	O
cerevisiae	O
kinetochores	B-Cellular_component
by	O
the	O
type	B-Gene_or_gene_product
1	I-Gene_or_gene_product
phosphatase	I-Gene_or_gene_product
Glc7p	B-Gene_or_gene_product
.	O

We	O
have	O
investigated	O
the	O
role	O
of	O
protein	O
phosphorylation	O
in	O
regulation	O
of	O
Saccharomyces	O
cerevisiae	O
kinetochores	B-Cellular_component
.	O

By	O
use	O
of	O
phosphatase	O
inhibitors	O
and	O
a	O
type	B-Gene_or_gene_product
1	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
phosphatase	I-Gene_or_gene_product
mutant	O
(	O
glc7	B-Gene_or_gene_product
-	I-Gene_or_gene_product
10	I-Gene_or_gene_product
)	O
,	O
we	O
show	O
that	O
the	O
microtubule	B-Cellular_component
binding	O
activity	O
,	O
but	O
not	O
the	O
centromeric	B-Cellular_component
DNA	O
-	O
binding	O
activity	O
,	O
of	O
the	O
kinetochore	B-Cellular_component
complex	O
is	O
regulated	O
by	O
a	O
balance	O
between	O
a	O
protein	O
kinase	O
and	O
the	O
type	B-Gene_or_gene_product
1	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
phosphatase	I-Gene_or_gene_product
(	O
PP1	B-Gene_or_gene_product
)	O
encoded	O
by	O
the	O
GLC7	O
gene	O
.	O

glc7	B-Gene_or_gene_product
-	I-Gene_or_gene_product
10	I-Gene_or_gene_product
mutant	O
cells	O
exhibit	O
low	O
kinetochore	B-Cellular_component
-	O
microtubule	B-Cellular_component
binding	O
activity	O
in	O
vitro	O
and	O
a	O
high	O
frequency	O
of	O
chromosome	B-Cellular_component
loss	O
in	O
vivo	O
.	O

Specifically	O
,	O
the	O
Ndc10p	B-Gene_or_gene_product
component	O
of	O
the	O
centromere	O
DNA	O
-	O
binding	O
CBF3	B-Complex
complex	O
is	O
altered	O
by	O
the	O
glc7	B-Gene_or_gene_product
-	I-Gene_or_gene_product
10	I-Gene_or_gene_product
mutation	O
;	O
Ndc10p	B-Gene_or_gene_product
is	O
hyperphosphorylated	O
in	O
glc7	B-Gene_or_gene_product
-	I-Gene_or_gene_product
10	I-Gene_or_gene_product
extracts	O
.	O

Furthermore	O
,	O
addition	O
of	O
recombinant	O
Ndc10p	B-Gene_or_gene_product
reconstitutes	O
the	O
microtubule	B-Cellular_component
-	O
binding	O
activity	O
of	O
a	O
glc7	B-Gene_or_gene_product
-	I-Gene_or_gene_product
10	I-Gene_or_gene_product
extract	O
to	O
wild	O
-	O
type	O
levels	O
.	O

Finally	O
,	O
the	O
glc7	B-Gene_or_gene_product
-	I-Gene_or_gene_product
10	I-Gene_or_gene_product
-	O
induced	O
mitotic	O
arrest	O
is	O
abolished	O
in	O
spindle	B-Cellular_component
checkpoint	O
mutants	O
,	O
suggesting	O
that	O
defects	O
in	O
kinetochore	B-Cellular_component
-	O
microtubule	B-Cellular_component
interactions	O
caused	O
by	O
hyperphosphorylation	O
of	O
kinetochore	B-Cellular_component
proteins	O
activate	O
the	O
spindle	B-Cellular_component
checkpoint	O
.	O

Human	O
placental	O
Na	B-Gene_or_gene_product
+	I-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

Cloning	O
,	O
functional	O
expression	O
,	O
gene	O
structure	O
,	O
and	O
chromosomal	B-Cellular_component
localization	O
.	O

We	O
have	O
cloned	O
the	O
human	O
Na	B-Gene_or_gene_product
+	I-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
which	O
transports	O
the	O
water	O
-	O
soluble	O
vitamins	O
pantothenate	B-Simple_chemical
,	O
biotin	B-Simple_chemical
,	O
and	O
lipoate	B-Simple_chemical
,	O
from	O
a	O
placental	O
choriocarcinoma	O
cell	O
line	O
(	O
JAR	O
)	O
.	O

The	O
cDNA	O
codes	O
for	O
a	O
protein	O
of	O
635	O
amino	O
acids	O
with	O
12	O
transmembrane	B-Cellular_component
domains	O
and	O
4	O
putative	O
sites	O
for	O
N	O
-	O
linked	O
glycosylation	O
.	O

The	O
human	O
SMVT	B-Gene_or_gene_product
exhibits	O
a	O
high	O
degree	O
of	O
homology	O
(	O
84	O
%	O
identity	O
and	O
89	O
%	O
similarity	O
)	O
to	O
the	O
rat	O
counterpart	O
.	O

When	O
expressed	O
in	O
HRPE	O
cells	O
,	O
the	O
cDNA	O
-	O
induced	O
transport	O
process	O
is	O
obligatorily	O
dependent	O
on	O
Na	B-Simple_chemical
+	I-Simple_chemical
and	O
accepts	O
pantothenate	B-Simple_chemical
,	O
biotin	B-Simple_chemical
,	O
and	O
lipoate	B-Simple_chemical
as	O
substrates	O
.	O

The	O
relationship	O
between	O
the	O
cDNA	O
-	O
specific	O
uptake	O
rate	O
of	O
pantothenate	B-Simple_chemical
or	O
biotin	B-Simple_chemical
and	O
Na	B-Simple_chemical
+	I-Simple_chemical
concentration	O
is	O
sigmoidal	O
with	O
a	O
Na	B-Simple_chemical
+	I-Simple_chemical
:	O
vitamin	O
stoichiometry	O
of	O
2	O
:	O
1	O
.	O

The	O
human	O
SMVT	B-Gene_or_gene_product
,	O
when	O
expressed	O
in	O
Xenopus	O
laevis	O
oocytes	O
,	O
induces	O
inward	O
currents	O
in	O
the	O
presence	O
of	O
pantothenate	B-Simple_chemical
,	O
biotin	B-Simple_chemical
,	O
and	O
lipoate	B-Simple_chemical
in	O
a	O
Na	B-Simple_chemical
+	I-Simple_chemical
-	O
,	O
concentration	O
-	O
,	O
and	O
potential	O
-	O
dependent	O
manner	O
.	O

We	O
also	O
report	O
here	O
on	O
the	O
structural	O
organization	O
and	O
chromosomal	B-Cellular_component
localization	O
of	O
the	O
human	O
SMVT	B-Gene_or_gene_product
gene	O
.	O

The	O
SMVT	B-Gene_or_gene_product
gene	O
is	O
approximately	O
14	O
kilobase	O
pairs	O
in	O
length	O
and	O
consists	O
of	O
17	O
exons	O
.	O

The	O
SMVT	B-Gene_or_gene_product
gene	O
is	O
located	O
on	O
chromosome	B-Cellular_component
2p23	I-Cellular_component
as	O
evidenced	O
by	O
somatic	O
cell	O
hybrid	O
analysis	O
and	O
fluorescence	O
in	O
situ	O
hybridization	O
.	O

Aldose	B-Gene_or_gene_product
reductase	I-Gene_or_gene_product
,	O
a	O
key	O
enzyme	O
in	O
the	O
oxidative	O
deamination	O
of	O
norepinephrine	B-Simple_chemical
in	O
rats	O
.	O

The	O
sympathoneural	O
neurotransmitter	O
norepinephrine	B-Simple_chemical
(	O
NE	B-Simple_chemical
)	O
is	O
deaminated	O
to	O
3	B-Simple_chemical
,	I-Simple_chemical
4	I-Simple_chemical
-	I-Simple_chemical
dihydroxymandelaldehyde	I-Simple_chemical
(	O
DHMAL	B-Simple_chemical
)	O
and	O
subsequently	O
converted	O
to	O
either	O
3	B-Simple_chemical
,	I-Simple_chemical
4	I-Simple_chemical
-	I-Simple_chemical
dihydroxymandelic	I-Simple_chemical
acid	I-Simple_chemical
(	O
DHMA	B-Simple_chemical
)	O
or	O
3	B-Simple_chemical
,	I-Simple_chemical
4	I-Simple_chemical
-	I-Simple_chemical
dihydroxyphenylglycol	I-Simple_chemical
(	O
DHPG	B-Simple_chemical
)	O
.	O

In	O
this	O
study	O
,	O
we	O
investigated	O
the	O
relative	O
importance	O
of	O
aldose	B-Gene_or_gene_product
reductase	I-Gene_or_gene_product
versus	O
aldehyde	B-Gene_or_gene_product
reductase	I-Gene_or_gene_product
in	O
the	O
formation	O
of	O
DHPG	B-Simple_chemical
from	O
DHMAL	B-Simple_chemical
.	O

The	O
in	O
vitro	O
incubation	O
of	O
NE	B-Simple_chemical
with	O
aldose	B-Gene_or_gene_product
reductase	I-Gene_or_gene_product
in	O
the	O
presence	O
of	O
monoamine	B-Gene_or_gene_product
oxidase	I-Gene_or_gene_product
(	O
MAO	B-Gene_or_gene_product
)	O
resulted	O
in	O
the	O
formation	O
of	O
DHPG	B-Simple_chemical
,	O
which	O
was	O
confirmed	O
by	O
mass	O
spectrometry	O
.	O

Although	O
aldehyde	B-Gene_or_gene_product
reductase	I-Gene_or_gene_product
also	O
generated	O
DHPG	B-Simple_chemical
,	O
its	O
activity	O
was	O
much	O
lower	O
than	O
that	O
of	O
aldose	B-Gene_or_gene_product
reductase	I-Gene_or_gene_product
.	O

With	O
northern	O
blotting	O
,	O
the	O
expression	O
of	O
both	O
aldose	B-Gene_or_gene_product
reductase	I-Gene_or_gene_product
and	O
aldehyde	B-Gene_or_gene_product
reductase	I-Gene_or_gene_product
was	O
detected	O
in	O
rat	O
superior	O
cervical	O
ganglia	O
.	O

However	O
,	O
with	O
western	O
blotting	O
,	O
only	O
aldose	B-Gene_or_gene_product
reductase	I-Gene_or_gene_product
was	O
immunologically	O
detectable	O
.	O

Treatment	O
of	O
rats	O
with	O
aldose	B-Gene_or_gene_product
reductase	I-Gene_or_gene_product
inhibitors	O
for	O
3	O
days	O
increased	O
the	O
plasma	O
level	O
of	O
DHMA	B-Simple_chemical
.	O

There	O
was	O
no	O
correlation	O
between	O
the	O
selectivity	O
of	O
inhibitors	O
and	O
effects	O
on	O
NE	B-Simple_chemical
metabolite	O
levels	O
.	O

A	O
significant	O
decrease	O
in	O
DHPG	B-Simple_chemical
,	O
however	O
,	O
was	O
obtained	O
only	O
with	O
an	O
extremely	O
high	O
dose	O
(	O
9	O
mg	O
/	O
kg	O
/	O
day	O
)	O
of	O
the	O
nonselective	O
inhibitor	O
AL	B-Simple_chemical
1576	I-Simple_chemical
.	O

The	O
present	O
study	O
confirmed	O
that	O
aldose	B-Gene_or_gene_product
reductase	I-Gene_or_gene_product
generates	O
DHPG	B-Simple_chemical
from	O
NE	B-Simple_chemical
in	O
the	O
presence	O
of	O
MAO	B-Gene_or_gene_product
.	O

In	O
rat	O
sympathetic	O
neurons	O
,	O
aldose	B-Gene_or_gene_product
reductase	I-Gene_or_gene_product
appears	O
to	O
be	O
more	O
important	O
than	O
aldehyde	B-Gene_or_gene_product
reductase	I-Gene_or_gene_product
for	O
the	O
formation	O
of	O
DHPG	B-Simple_chemical
.	O

However	O
,	O
when	O
aldose	B-Gene_or_gene_product
reductase	I-Gene_or_gene_product
is	O
inhibited	O
,	O
it	O
appears	O
that	O
aldehyde	B-Gene_or_gene_product
reductase	I-Gene_or_gene_product
can	O
compensate	O
for	O
the	O
conversion	O
of	O
DHMAL	B-Simple_chemical
to	O
DHPG	B-Simple_chemical
,	O
indicating	O
redundancy	O
in	O
the	O
reduction	O
pathway	O
.	O

Phosphorylation	O
of	O
deoxycytidine	B-Simple_chemical
analog	O
monophosphates	B-Simple_chemical
by	O
UMP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
CMP	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
:	O
molecular	O
characterization	O
of	O
the	O
human	O
enzyme	O
.	O

Phosphorylation	O
of	O
deoxycytidine	B-Simple_chemical
analogs	O
by	O
cellular	O
enzymes	O
is	O
a	O
prerequisite	O
for	O
the	O
activity	O
of	O
these	O
compounds	O
.	O

We	O
have	O
investigated	O
the	O
kinetic	O
parameters	O
for	O
the	O
phosphorylation	O
of	O
1	B-Simple_chemical
-	I-Simple_chemical
beta	I-Simple_chemical
-	I-Simple_chemical
D	I-Simple_chemical
-	I-Simple_chemical
arabinofuranosylcytosine	I-Simple_chemical
(	O
araC	B-Simple_chemical
)	O
and	O
2	B-Simple_chemical
'	I-Simple_chemical
,	I-Simple_chemical
2	I-Simple_chemical
'	I-Simple_chemical
-	I-Simple_chemical
difluorodeoxycytidine	I-Simple_chemical
(	O
dFdC	B-Simple_chemical
)	O
to	O
their	O
diphosphate	B-Simple_chemical
forms	O
catalyzed	O
by	O
human	O
UMP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
CMP	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
.	O

We	O
cloned	O
the	O
cDNA	O
of	O
this	O
enzyme	O
to	O
enable	O
characterization	O
of	O
the	O
recombinant	O
protein	O
,	O
determine	O
its	O
expression	O
in	O
different	O
tissues	O
,	O
and	O
determine	O
the	O
chromosome	B-Cellular_component
location	O
of	O
the	O
gene	O
.	O

We	O
showed	O
that	O
the	O
recombinant	O
UMP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
CMP	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
phosphorylated	O
CMP	B-Simple_chemical
,	O
dCMP	B-Simple_chemical
,	O
and	O
UMP	B-Simple_chemical
with	O
highest	O
efficiency	O
and	O
dUMP	B-Simple_chemical
,	O
AMP	B-Simple_chemical
,	O
and	O
dAMP	B-Simple_chemical
with	O
lower	O
efficiency	O
.	O

The	O
monophosphates	B-Simple_chemical
of	O
araC	B-Simple_chemical
and	O
dFdC	B-Simple_chemical
were	O
shown	O
to	O
be	O
phosphorylated	O
with	O
similar	O
efficiency	O
as	O
dCMP	B-Simple_chemical
and	O
CMP	B-Simple_chemical
.	O

We	O
further	O
showed	O
,	O
in	O
a	O
combined	O
enzymatic	O
assay	O
,	O
that	O
human	O
deoxycytidine	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
and	O
UMP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
CMP	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
together	O
phosphorylated	O
araC	B-Simple_chemical
,	O
dFdC	B-Simple_chemical
,	O
and	O
2	B-Simple_chemical
'	I-Simple_chemical
,	I-Simple_chemical
3	I-Simple_chemical
'	I-Simple_chemical
-	I-Simple_chemical
dideoxycytidine	I-Simple_chemical
to	O
their	O
diphosphate	B-Simple_chemical
forms	O
.	O

Northern	O
blot	O
analysis	O
showed	O
that	O
the	O
UMP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
CMP	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
mRNA	O
was	O
ubiquitously	O
present	O
in	O
human	O
tissues	O
as	O
a	O
3	O
.	O
9	O
-	O
kb	O
transcript	O
with	O
highest	O
levels	O
in	O
pancreas	O
,	O
skeletal	O
muscle	O
,	O
and	O
liver	O
.	O

The	O
human	O
UMP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
CMP	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
gene	O
was	O
localized	O
to	O
chromosome	B-Cellular_component
1p34	I-Cellular_component
.	I-Cellular_component
1	I-Cellular_component
-	I-Cellular_component
1p33	I-Cellular_component
by	O
radiation	O
hybrid	O
analysis	O
.	O

We	O
further	O
expressed	O
the	O
UMP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
CMP	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
as	O
a	O
fusion	O
protein	O
to	O
the	O
green	B-Gene_or_gene_product
fluorescent	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
in	O
Chinese	O
hamster	O
ovary	O
cells	O
,	O
and	O
showed	O
that	O
the	O
fusion	O
protein	O
was	O
located	O
in	O
the	O
cytosol	B-Cellular_component
and	O
nucleus	B-Cellular_component
.	O

Hsl7	B-Gene_or_gene_product
localizes	O
to	O
a	O
septin	B-Gene_or_gene_product
ring	O
and	O
serves	O
as	O
an	O
adapter	O
in	O
a	O
regulatory	O
pathway	O
that	O
relieves	O
tyrosine	B-Simple_chemical
phosphorylation	O
of	O
Cdc28	B-Gene_or_gene_product
protein	O
kinase	O
in	O
Saccharomyces	O
cerevisiae	O
.	O

Successful	O
mitosis	O
requires	O
faithful	O
DNA	O
replication	O
,	O
spindle	B-Cellular_component
assembly	O
,	O
chromosome	B-Cellular_component
segregation	O
,	O
and	O
cell	O
division	O
.	O

In	O
the	O
budding	O
yeast	O
Saccharomyces	O
cerevisiae	O
,	O
the	O
G	O
(	O
2	O
)	O
-	O
to	O
-	O
M	O
transition	O
requires	O
activation	O
of	O
Clb	O
-	O
bound	O
forms	O
of	O
the	O
protein	O
kinase	O
,	O
Cdc28	B-Gene_or_gene_product
.	O

These	O
complexes	O
are	O
held	O
in	O
an	O
inactive	O
state	O
via	O
phosphorylation	O
of	O
Tyr19	B-Simple_chemical
in	O
the	O
ATP	B-Simple_chemical
-	O
binding	O
loop	O
of	O
Cdc28	B-Gene_or_gene_product
by	O
the	O
Swe1	B-Gene_or_gene_product
protein	O
kinase	O
.	O

The	O
HSL1	B-Gene_or_gene_product
and	O
HSL7	B-Gene_or_gene_product
gene	O
products	O
act	O
as	O
negative	O
regulators	O
of	O
Swe1	B-Gene_or_gene_product
.	O

Hsl1	B-Gene_or_gene_product
is	O
a	O
large	O
(	O
1	O
,	O
518	O
-	O
residue	O
)	O
protein	O
kinase	O
with	O
an	O
N	O
-	O
terminal	O
catalytic	O
domain	O
and	O
a	O
very	O
long	O
C	O
-	O
terminal	O
extension	O
.	O

Hsl1	B-Gene_or_gene_product
localizes	O
to	O
the	O
incipient	O
site	O
of	O
cytokinesis	O
in	O
the	O
bud	B-Cellular_component
neck	I-Cellular_component
in	O
a	O
septin	B-Gene_or_gene_product
-	O
dependent	O
manner	O
;	O
however	O
,	O
the	O
function	O
of	O
Hsl7	B-Gene_or_gene_product
was	O
not	O
previously	O
known	O
.	O

Using	O
both	O
indirect	O
immunofluorescence	O
with	O
anti	O
-	O
Hsl7	B-Gene_or_gene_product
antibodies	O
and	O
a	O
fusion	O
of	O
Hsl7	B-Gene_or_gene_product
to	O
green	O
fluorescent	O
protein	O
,	O
we	O
found	O
that	O
Hsl7	B-Gene_or_gene_product
also	O
localizes	O
to	O
the	O
bud	B-Cellular_component
neck	I-Cellular_component
,	O
congruent	O
with	O
the	O
septin	B-Gene_or_gene_product
ring	O
that	O
faces	O
the	O
daughter	O
cell	O
.	O

Both	O
Swe1	B-Gene_or_gene_product
and	O
a	O
segment	O
of	O
the	O
C	O
terminus	O
of	O
Hsl1	B-Gene_or_gene_product
(	O
which	O
has	O
no	O
sequence	O
counterpart	O
in	O
two	O
Hsl1	B-Gene_or_gene_product
-	O
related	O
protein	O
kinases	O
,	O
Gin4	B-Gene_or_gene_product
and	O
Kcc4	B-Gene_or_gene_product
)	O
were	O
identified	O
as	O
gene	O
products	O
that	O
interact	O
with	O
Hsl7	B-Gene_or_gene_product
in	O
a	O
two	O
-	O
hybrid	O
screen	O
of	O
a	O
random	O
S	O
.	O
cerevisiae	O
cDNA	O
library	O
.	O

Hsl7	B-Gene_or_gene_product
plus	O
Swe1	B-Gene_or_gene_product
and	O
Hsl7	B-Gene_or_gene_product
plus	O
Hsl1	B-Gene_or_gene_product
can	O
be	O
coimmunoprecipitated	O
from	O
extracts	O
of	O
cells	O
overexpressing	O
these	O
proteins	O
,	O
confirming	O
that	O
Hsl7	B-Gene_or_gene_product
physically	O
associates	O
with	O
both	O
partners	O
.	O

Also	O
consistent	O
with	O
the	O
two	O
-	O
hybrid	O
results	O
,	O
Hsl7	B-Gene_or_gene_product
coimmunoprecipitates	O
with	O
full	O
-	O
length	O
Hsl1	B-Gene_or_gene_product
less	O
efficiently	O
than	O
with	O
a	O
C	O
-	O
terminal	O
fragment	O
of	O
Hsl1	B-Gene_or_gene_product
.	O

Moreover	O
,	O
Hsl7	B-Gene_or_gene_product
does	O
not	O
localize	O
to	O
the	O
bud	B-Cellular_component
neck	I-Cellular_component
in	O
an	O
hsl1Delta	B-Gene_or_gene_product
mutant	O
,	O
whereas	O
Hsl1	B-Gene_or_gene_product
is	O
localized	O
normally	O
in	O
an	O
hsl7Delta	B-Gene_or_gene_product
mutant	O
.	O

Phosphorylation	O
and	O
ubiquitinylation	O
of	O
Swe1	B-Gene_or_gene_product
,	O
preludes	O
to	O
its	O
destruction	O
,	O
are	O
severely	O
reduced	O
in	O
cells	O
lacking	O
either	O
Hsl1	B-Gene_or_gene_product
or	O
Hsl7	B-Gene_or_gene_product
(	O
or	O
both	O
)	O
,	O
as	O
judged	O
by	O
an	O
electrophoretic	O
mobility	O
shift	O
assay	O
.	O

Collectively	O
,	O
these	O
data	O
suggest	O
that	O
formation	O
of	O
the	O
septin	B-Gene_or_gene_product
rings	O
provides	O
sites	O
for	O
docking	O
Hsl1	B-Gene_or_gene_product
,	O
exposing	O
its	O
C	O
terminus	O
and	O
thereby	O
permitting	O
recruitment	O
of	O
Hsl7	B-Gene_or_gene_product
.	O

Hsl7	B-Gene_or_gene_product
,	O
in	O
turn	O
,	O
presents	O
its	O
cargo	O
of	O
bound	O
Swe1	B-Gene_or_gene_product
,	O
allowing	O
phosphorylation	O
by	O
Hsl1	B-Gene_or_gene_product
.	O

Thus	O
,	O
Hsl1	B-Gene_or_gene_product
and	O
Hsl7	B-Gene_or_gene_product
promote	O
proper	O
timing	O
of	O
cell	O
cycle	O
progression	O
by	O
coupling	O
septin	B-Gene_or_gene_product
ring	O
assembly	O
to	O
alleviation	O
of	O
Swe1	B-Gene_or_gene_product
-	O
dependent	O
inhibition	O
of	O
Cdc28	B-Gene_or_gene_product
.	O

Furthermore	O
,	O
like	O
septins	B-Gene_or_gene_product
and	O
Hsl1	B-Gene_or_gene_product
,	O
homologs	O
of	O
Hsl7	B-Gene_or_gene_product
are	O
found	O
in	O
fission	O
yeast	O
,	O
flies	O
,	O
worms	O
,	O
and	O
humans	O
,	O
suggesting	O
that	O
its	O
function	O
in	O
this	O
control	O
mechanism	O
may	O
be	O
conserved	O
in	O
all	O
eukaryotes	O
.	O

New	O
insights	O
into	O
the	O
regulation	O
of	O
protein	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
C	I-Gene_or_gene_product
and	O
novel	O
phorbol	B-Simple_chemical
ester	I-Simple_chemical
receptors	O
.	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
,	O
a	O
family	O
of	O
related	O
serine	B-Simple_chemical
-	O
threonine	B-Simple_chemical
kinases	O
,	O
is	O
a	O
key	O
player	O
in	O
the	O
cellular	O
responses	O
mediated	O
by	O
the	O
second	O
messenger	O
diacylglycerol	B-Simple_chemical
(	O
DAG	B-Simple_chemical
)	O
and	O
the	O
phorbol	B-Simple_chemical
ester	I-Simple_chemical
tumor	O
promoters	O
.	O

The	O
traditional	O
view	O
of	O
PKCs	B-Gene_or_gene_product
as	O
DAG	B-Simple_chemical
/	O
phospholipid	B-Simple_chemical
-	O
regulated	O
proteins	O
has	O
expanded	O
in	O
the	O
last	O
few	O
years	O
by	O
three	O
seminal	O
discoveries	O
.	O

First	O
,	O
PKC	B-Gene_or_gene_product
activity	O
and	O
maturation	O
is	O
controlled	O
by	O
autophosphorylation	O
and	O
transphosphorylation	O
mechanisms	O
,	O
which	O
includes	O
phosphorylation	O
of	O
PKC	B-Gene_or_gene_product
isozymes	O
by	O
phosphoinositide	B-Gene_or_gene_product
-	I-Gene_or_gene_product
dependent	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
kinases	I-Gene_or_gene_product
(	O
PDKs	B-Gene_or_gene_product
)	O
and	O
tyrosine	B-Simple_chemical
kinases	O
.	O

Second	O
,	O
PKC	B-Gene_or_gene_product
activity	O
and	O
localization	O
are	O
regulated	O
by	O
direct	O
interaction	O
with	O
different	O
types	O
of	O
interacting	O
proteins	O
.	O

Protein	O
-	O
protein	O
interactions	O
are	O
now	O
recognized	O
as	O
important	O
mechanisms	O
that	O
target	O
individual	O
PKCs	B-Gene_or_gene_product
to	O
different	O
intracellular	B-Cellular_component
compartments	I-Cellular_component
and	O
confer	O
selectivity	O
by	O
associating	O
individual	O
isozymes	O
with	O
specific	O
substrates	O
.	O

Last	O
,	O
the	O
discovery	O
of	O
novel	O
phorbol	B-Simple_chemical
ester	I-Simple_chemical
receptors	O
lacking	O
kinase	O
activity	O
allows	O
us	O
to	O
speculate	O
that	O
some	O
of	O
the	O
biological	O
responses	O
elicited	O
by	O
phorbol	B-Simple_chemical
esters	I-Simple_chemical
or	O
by	O
activation	O
of	O
receptors	O
coupled	O
to	O
elevation	O
in	O
DAG	B-Simple_chemical
levels	O
could	O
be	O
mediated	O
by	O
PKC	B-Gene_or_gene_product
-	O
independent	O
pathways	O
.	O

Differential	O
roles	O
of	O
TLR2	B-Gene_or_gene_product
and	O
TLR4	B-Gene_or_gene_product
in	O
recognition	O
of	O
gram	O
-	O
negative	O
and	O
gram	O
-	O
positive	O
bacterial	O
cell	B-Cellular_component
wall	I-Cellular_component
components	O
.	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
TLR	I-Gene_or_gene_product
)	I-Gene_or_gene_product
2	I-Gene_or_gene_product
and	O
TLR4	B-Gene_or_gene_product
are	O
implicated	O
in	O
the	O
recognition	O
of	O
various	O
bacterial	O
cell	B-Cellular_component
wall	I-Cellular_component
components	O
,	O
such	O
as	O
lipopolysaccharide	B-Simple_chemical
(	O
LPS	B-Simple_chemical
)	O
.	O

To	O
investigate	O
in	O
vivo	O
roles	O
of	O
TLR2	B-Gene_or_gene_product
,	O
we	O
generated	O
TLR2	B-Gene_or_gene_product
-	O
deficient	O
mice	O
.	O

In	O
contrast	O
to	O
LPS	B-Simple_chemical
unresponsiveness	O
in	O
TLR4	B-Gene_or_gene_product
-	O
deficient	O
mice	O
,	O
TLR2	B-Gene_or_gene_product
-	O
deficient	O
mice	O
responded	O
to	O
LPS	B-Simple_chemical
to	O
the	O
same	O
extent	O
as	O
wild	O
-	O
type	O
mice	O
.	O

TLR2	B-Gene_or_gene_product
-	O
deficient	O
macrophages	O
were	O
hyporesponsive	O
to	O
several	O
Gram	O
-	O
positive	O
bacterial	O
cell	B-Cellular_component
walls	I-Cellular_component
as	O
well	O
as	O
Staphylococcus	O
aureus	O
peptidoglycan	B-Simple_chemical
.	O

TLR4	B-Gene_or_gene_product
-	O
deficient	O
macrophages	O
lacked	O
the	O
response	O
to	O
Gram	O
-	O
positive	O
lipoteichoic	B-Simple_chemical
acids	I-Simple_chemical
.	O

These	O
results	O
demonstrate	O
that	O
TLR2	B-Gene_or_gene_product
and	O
TLR4	B-Gene_or_gene_product
recognize	O
different	O
bacterial	O
cell	B-Cellular_component
wall	I-Cellular_component
components	O
in	O
vivo	O
and	O
TLR2	B-Gene_or_gene_product
plays	O
a	O
major	O
role	O
in	O
Gram	O
-	O
positive	O
bacterial	O
recognition	O
.	O

Identification	O
of	O
a	O
central	O
phosphorylation	O
site	O
in	O
p21	B-Gene_or_gene_product
-	I-Gene_or_gene_product
activated	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
regulating	O
autoinhibition	O
and	O
kinase	O
activity	O
.	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
Pak	B-Gene_or_gene_product
)	O
/	O
Ste20	B-Gene_or_gene_product
kinases	O
are	O
regulated	O
in	O
vitro	O
and	O
in	O
vivo	O
by	O
the	O
small	O
GTP	B-Simple_chemical
-	O
binding	O
proteins	O
Rac	B-Gene_or_gene_product
and	O
Cdc42	B-Gene_or_gene_product
and	O
lipids	B-Simple_chemical
,	O
such	O
as	O
sphingosine	B-Simple_chemical
,	O
which	O
stimulate	O
autophosphorylation	O
and	O
phosphorylation	O
of	O
exogenous	O
substrates	O
.	O

The	O
mechanism	O
of	O
Pak	B-Gene_or_gene_product
activation	O
by	O
these	O
agents	O
remains	O
unclear	O
.	O

We	O
investigated	O
Pak	B-Gene_or_gene_product
kinase	O
activation	O
in	O
more	O
detail	O
to	O
gain	O
insight	O
into	O
the	O
interplay	O
between	O
the	O
GTPase	O
/	O
sphingosine	B-Simple_chemical
binding	O
,	O
an	O
intramolecular	O
inhibitory	O
interaction	O
,	O
and	O
autophosphorylation	O
.	O

We	O
present	O
biochemical	O
evidence	O
that	O
an	O
autoinhibitory	O
domain	O
(	O
ID	O
)	O
contained	O
within	O
amino	O
acid	O
residues	O
67	O
-	O
150	O
of	O
Pak1	B-Gene_or_gene_product
interacts	O
with	O
the	O
carboxyl	O
-	O
terminal	O
kinase	O
domain	O
and	O
that	O
this	O
interaction	O
is	O
regulated	O
in	O
a	O
GTPase	O
-	O
dependent	O
fashion	O
.	O

Cdc42	B-Gene_or_gene_product
-	O
and	O
sphingosine	B-Simple_chemical
-	O
stimulated	O
Pak1	B-Gene_or_gene_product
activity	O
can	O
be	O
inhibited	O
in	O
trans	O
by	O
recombinant	O
ID	O
peptide	O
,	O
indicating	O
similarities	O
in	O
their	O
mode	O
of	O
activation	O
.	O

However	O
,	O
Pak1	B-Gene_or_gene_product
,	O
which	O
was	O
autophosphorylated	O
in	O
response	O
to	O
either	O
GTPase	O
or	O
sphingosine	B-Simple_chemical
,	O
is	O
highly	O
active	O
and	O
is	O
insensitive	O
to	O
inhibition	O
by	O
the	O
ID	O
peptide	O
.	O

We	O
identified	O
phospho	O
-	O
acceptor	O
site	O
threonine	B-Simple_chemical
423	I-Simple_chemical
in	O
the	O
kinase	O
activation	O
loop	O
as	O
a	O
critical	O
determinant	O
for	O
the	O
sensitivity	O
to	O
autoinhibition	O
and	O
enzymatic	O
activity	O
.	O

Phosphorylation	O
studies	O
suggested	O
that	O
the	O
stimulatory	O
effect	O
of	O
both	O
GTPase	O
and	O
sphingosine	B-Simple_chemical
results	O
in	O
exposure	O
of	O
the	O
activation	O
loop	O
,	O
making	O
it	O
accessible	O
for	O
intermolecular	O
phosphorylation	O
.	O

Loss	O
of	O
imprinting	O
and	O
elevated	O
expression	O
of	O
wild	O
-	O
type	O
p73	B-Gene_or_gene_product
in	O
human	O
gastric	O
adenocarcinoma	O
.	O

The	O
p73	B-Gene_or_gene_product
gene	O
located	O
at	O
1p36	O
.	O
3	O
encodes	O
for	O
a	O
protein	O
with	O
significant	O
similarity	O
to	O
p53	B-Gene_or_gene_product
.	O

To	O
investigate	O
the	O
penetrance	O
of	O
p73	B-Gene_or_gene_product
in	O
gastric	O
carcinogenesis	O
,	O
we	O
analyzed	O
the	O
expression	O
,	O
allelotype	O
,	O
and	O
mutation	O
of	O
p73	B-Gene_or_gene_product
in	O
five	O
cell	O
lines	O
and	O
75	O
tissues	O
.	O

Although	O
extremely	O
low	O
levels	O
of	O
p73	B-Gene_or_gene_product
expression	O
were	O
observed	O
in	O
all	O
noncancerous	O
gastric	O
tissues	O
and	O
four	O
of	O
five	O
cell	O
lines	O
,	O
a	O
significant	O
elevation	O
of	O
p73	B-Gene_or_gene_product
was	O
detected	O
in	O
37	O
of	O
39	O
(	O
94	O
.	O
9	O
%	O
)	O
carcinoma	O
tissues	O
.	O

Furthermore	O
,	O
a	O
tumor	O
-	O
specific	O
increase	O
of	O
p73	B-Gene_or_gene_product
was	O
identified	O
in	O
14	O
of	O
16	O
(	O
87	O
.	O
5	O
%	O
)	O
matched	O
sets	O
.	O

Allelotyping	O
analysis	O
using	O
a	O
StyI	O
or	O
BanI	O
polymorphism	O
revealed	O
that	O
5	O
of	O
21	O
(	O
23	O
.	O
8	O
%	O
)	O
informative	O
carcinomas	O
,	O
but	O
none	O
of	O
19	O
noncancerous	O
cases	O
,	O
express	O
p73	B-Gene_or_gene_product
biallelically	O
,	O
suggesting	O
the	O
transcriptional	O
activation	O
of	O
a	O
silent	O
allele	O
in	O
a	O
subset	O
of	O
cancers	O
.	O

Whereas	O
the	O
transcription	O
of	O
an	O
active	O
allele	O
was	O
markedly	O
induced	O
by	O
serum	O
starvation	O
or	O
clump	O
formation	O
of	O
the	O
cells	O
,	O
treatment	O
with	O
5	B-Simple_chemical
-	I-Simple_chemical
aza	I-Simple_chemical
-	I-Simple_chemical
2	I-Simple_chemical
'	I-Simple_chemical
deoxycytidine	I-Simple_chemical
activated	O
a	O
silent	O
allele	O
with	O
a	O
subsequent	O
up	O
-	O
regulation	O
of	O
an	O
active	O
allele	O
,	O
supporting	O
the	O
genomic	O
imprinting	O
and	O
autoregulation	O
of	O
the	O
gene	O
.	O

Allelic	O
deletion	O
or	O
mutation	O
of	O
the	O
gene	O
was	O
not	O
found	O
,	O
and	O
no	O
association	O
of	O
p73	B-Gene_or_gene_product
expression	O
with	O
the	O
mutational	O
status	O
of	O
p53	B-Gene_or_gene_product
or	O
expression	O
of	O
p21Waf1	B-Gene_or_gene_product
was	O
recognized	O
.	O

Taken	O
together	O
,	O
this	O
study	O
argues	O
that	O
p73	B-Gene_or_gene_product
is	O
not	O
a	O
target	O
of	O
genetic	O
alteration	O
in	O
gastric	O
carcinogenesis	O
and	O
suggests	O
that	O
overexpression	O
of	O
p73	B-Gene_or_gene_product
might	O
be	O
triggered	O
by	O
physiological	O
stresses	O
accompanied	O
with	O
outgrowth	O
of	O
tumors	O
,	O
such	O
as	O
hypoxia	O
or	O
nutrient	O
deprivation	O
.	O

Activity	O
of	O
the	O
human	O
cytochrome	B-Gene_or_gene_product
c1	I-Gene_or_gene_product
promoter	O
is	O
modulated	O
by	O
E2F	B-Gene_or_gene_product
.	O

The	O
human	O
cytochrome	B-Gene_or_gene_product
c	I-Gene_or_gene_product
(	I-Gene_or_gene_product
1	I-Gene_or_gene_product
)	I-Gene_or_gene_product
promoter	O
is	O
strongly	O
activated	O
in	O
transfected	O
Drosophila	O
SL2	O
cells	O
expressing	O
exogenous	O
human	O
E2F1	B-Gene_or_gene_product
.	O

Transfection	O
-	O
deletion	O
experiments	O
,	O
DNase	B-Gene_or_gene_product
I	I-Gene_or_gene_product
protection	O
by	O
E2F1	B-Gene_or_gene_product
and	O
gel	O
mobility	O
-	O
shift	O
experiments	O
locate	O
E2F1	B-Gene_or_gene_product
activation	O
sites	O
to	O
two	O
regions	O
on	O
either	O
side	O
of	O
the	O
transcription	O
start	O
site	O
.	O

Deletion	O
of	O
either	O
region	O
prevents	O
E2F1	B-Gene_or_gene_product
activation	O
in	O
transfected	O
SL2	O
cells	O
,	O
suggesting	O
a	O
co	O
-	O
operative	O
interaction	O
between	O
them	O
.	O

E2F6	B-Gene_or_gene_product
,	O
a	O
member	O
of	O
the	O
E2F	B-Gene_or_gene_product
family	O
that	O
lacks	O
transactivation	O
domains	O
but	O
contains	O
specific	O
suppressor	O
domains	O
,	O
inhibits	O
cytochrome	B-Gene_or_gene_product
c	I-Gene_or_gene_product
(	I-Gene_or_gene_product
1	I-Gene_or_gene_product
)	I-Gene_or_gene_product
promoter	O
activity	O
when	O
co	O
-	O
transfected	O
into	O
HeLa	O
cells	O
,	O
indicating	O
that	O
the	O
E2F	B-Gene_or_gene_product
proteins	O
modulate	O
the	O
cytochrome	B-Gene_or_gene_product
c	I-Gene_or_gene_product
(	I-Gene_or_gene_product
1	I-Gene_or_gene_product
)	I-Gene_or_gene_product
promoter	O
in	O
mammalian	O
cells	O
.	O

However	O
,	O
E2F	B-Gene_or_gene_product
is	O
not	O
a	O
general	O
regulator	O
of	O
oxidative	O
phosphorylation	O
genes	O
since	O
three	O
additional	O
nuclear	B-Cellular_component
-	O
encoded	O
mitochondrial	B-Cellular_component
genes	O
were	O
unaffected	O
by	O
E2F1	B-Gene_or_gene_product
or	O
E2F6	B-Gene_or_gene_product
.	O

Nuclear	B-Cellular_component
-	O
specific	O
degradation	O
of	O
Far1	B-Gene_or_gene_product
is	O
controlled	O
by	O
the	O
localization	O
of	O
the	O
F	O
-	O
box	O
protein	O
Cdc4	B-Gene_or_gene_product
.	O

Far1	B-Gene_or_gene_product
is	O
a	O
bifunctional	O
protein	O
that	O
is	O
required	O
to	O
arrest	O
the	O
cell	O
cycle	O
and	O
establish	O
cell	O
polarity	O
during	O
yeast	O
mating	O
.	O

Here	O
we	O
show	O
that	O
SCF	B-Complex
(	I-Complex
Cdc4	I-Complex
)	I-Complex
ubiquitylates	O
Far1	B-Gene_or_gene_product
in	O
the	O
nucleus	B-Cellular_component
,	O
which	O
in	O
turn	O
targets	O
the	O
multi	O
-	O
ubiquitylated	O
protein	O
to	O
26S	B-Cellular_component
proteasomes	I-Cellular_component
most	O
likely	O
located	O
at	O
the	O
nuclear	B-Cellular_component
envelope	I-Cellular_component
.	O

In	O
response	O
to	O
mating	O
pheromones	O
,	O
a	O
fraction	O
of	O
Far1	B-Gene_or_gene_product
was	O
stabilized	O
after	O
its	O
export	O
into	O
the	O
cytoplasm	B-Cellular_component
by	O
Ste21	B-Gene_or_gene_product
/	O
Msn5	B-Gene_or_gene_product
.	O

Preventing	O
nuclear	B-Cellular_component
export	O
destabilized	O
Far1	B-Gene_or_gene_product
,	O
while	O
conversely	O
cytoplasmic	B-Cellular_component
Far1	B-Gene_or_gene_product
was	O
stabilized	O
,	O
although	O
the	O
protein	O
was	O
efficiently	O
phosphorylated	O
in	O
a	O
Cdc28	B-Complex
-	I-Complex
Cln	I-Complex
-	O
dependent	O
manner	O
.	O

The	O
core	O
SCF	B-Complex
subunits	O
Cdc53	B-Gene_or_gene_product
,	O
Hrt1	B-Gene_or_gene_product
and	O
Skp1	B-Gene_or_gene_product
were	O
distributed	O
in	O
the	O
nucleus	B-Cellular_component
and	O
the	O
cytoplasm	B-Cellular_component
,	O
whereas	O
the	O
F	O
-	O
box	O
protein	O
Cdc4	B-Gene_or_gene_product
was	O
exclusively	O
nuclear	B-Cellular_component
.	O

A	O
cytoplasmic	B-Cellular_component
form	O
of	O
Cdc4	B-Gene_or_gene_product
was	O
unable	O
to	O
complement	O
the	O
growth	O
defect	O
of	O
cdc4	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
cells	O
,	O
but	O
it	O
was	O
sufficient	O
to	O
degrade	O
Far1	B-Gene_or_gene_product
in	O
the	O
cytoplasm	B-Cellular_component
.	O

Our	O
results	O
illustrate	O
the	O
importance	O
of	O
subcellular	B-Cellular_component
localization	O
of	O
F	O
-	O
box	O
proteins	O
,	O
and	O
provide	O
an	O
example	O
of	O
how	O
an	O
extracellular	O
signal	O
regulates	O
protein	O
stability	O
at	O
the	O
level	O
of	O
substrate	O
localization	O
.	O

Evidence	O
for	O
an	O
accessory	O
protein	O
function	O
for	O
Toll	B-Gene_or_gene_product
-	I-Gene_or_gene_product
like	I-Gene_or_gene_product
receptor	I-Gene_or_gene_product
1	I-Gene_or_gene_product
in	O
anti	O
-	O
bacterial	O
responses	O
.	O

Members	O
of	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
family	O
are	O
components	O
of	O
the	O
mammalian	O
anti	O
-	O
microbial	O
response	O
,	O
signaling	O
with	O
a	O
domain	O
closely	O
related	O
to	O
that	O
of	O
IL	B-Complex
-	I-Complex
1	I-Complex
receptors	I-Complex
.	O

In	O
this	O
report	O
the	O
expression	O
and	O
function	O
of	O
TLR1	B-Gene_or_gene_product
,	O
a	O
TLR	B-Gene_or_gene_product
of	O
unknown	O
function	O
,	O
are	O
examined	O
.	O

TLR1	B-Gene_or_gene_product
is	O
expressed	O
by	O
monocytes	O
,	O
as	O
demonstrated	O
using	O
a	O
novel	O
mAb	O
.	O

Monocytes	O
also	O
express	O
TLR2	B-Gene_or_gene_product
.	O

TLR1	B-Gene_or_gene_product
transfection	O
of	O
HeLa	O
cells	O
,	O
which	O
express	O
neither	O
TLR1	B-Gene_or_gene_product
nor	O
TLR2	B-Gene_or_gene_product
,	O
was	O
not	O
sufficient	O
to	O
confer	O
responsiveness	O
to	O
several	O
microbial	O
extracts	O
.	O

However	O
,	O
cotransfection	O
of	O
TLR1	B-Gene_or_gene_product
and	O
TLR2	B-Gene_or_gene_product
resulted	O
in	O
enhanced	O
signaling	O
by	O
HeLa	O
cells	O
to	O
soluble	O
factors	O
released	O
from	O
Neisseria	O
meningitidis	O
relative	O
to	O
the	O
response	O
with	O
either	O
TLR	B-Gene_or_gene_product
alone	O
.	O

This	O
phenomenon	O
was	O
also	O
seen	O
with	O
high	O
concentrations	O
of	O
some	O
preparations	O
of	O
LPS	B-Simple_chemical
.	O

The	O
N	O
.	O
meningitidis	O
factors	O
recognized	O
by	O
TLR1	B-Gene_or_gene_product
/	O
TLR2	B-Gene_or_gene_product
were	O
not	O
released	O
by	O
N	O
.	O
meningitidis	O
mutant	O
in	O
the	O
LpxA	B-Gene_or_gene_product
gene	O
.	O

Although	O
LpxA	B-Gene_or_gene_product
is	O
required	O
for	O
LPS	B-Simple_chemical
biosynthesis	O
,	O
because	O
cooperation	O
between	O
TLR1	B-Gene_or_gene_product
and	O
TLR2	B-Gene_or_gene_product
was	O
not	O
seen	O
with	O
all	O
LPS	B-Simple_chemical
preparations	O
,	O
the	O
microbial	O
component	O
(	O
s	O
)	O
TLR1	B-Gene_or_gene_product
/	O
2	B-Gene_or_gene_product
recognizes	O
is	O
likely	O
to	O
be	O
a	O
complex	O
of	O
LPS	B-Simple_chemical
and	O
other	O
molecules	O
or	O
a	O
compound	O
metabolically	O
and	O
chemically	O
related	O
to	O
LPS	B-Simple_chemical
.	O

The	O
functional	O
IL	B-Complex
-	I-Complex
1R	I-Complex
consists	O
of	O
a	O
heterodimer	O
;	O
this	O
report	O
suggests	O
a	O
similar	O
mechanism	O
for	O
TLR1	B-Gene_or_gene_product
and	O
TLR2	B-Gene_or_gene_product
,	O
for	O
certain	O
agonists	O
.	O

These	O
data	O
further	O
suggest	O
that	O
mammalian	O
responsiveness	O
to	O
some	O
bacterial	O
products	O
may	O
be	O
mediated	O
by	O
combinations	O
of	O
TLRs	B-Gene_or_gene_product
,	O
suggesting	O
a	O
mechanism	O
for	O
diversifying	O
the	O
repertoire	O
of	O
Toll	B-Gene_or_gene_product
-	O
mediated	O
responses	O
.	O

PIG	B-Gene_or_gene_product
-	I-Gene_or_gene_product
M	I-Gene_or_gene_product
transfers	O
the	O
first	O
mannose	B-Simple_chemical
to	O
glycosylphosphatidylinositol	B-Simple_chemical
on	O
the	O
lumenal	B-Cellular_component
side	I-Cellular_component
of	O
the	O
ER	B-Cellular_component
.	O

Glycosylphosphatidylinositol	B-Simple_chemical
(	O
GPI	B-Simple_chemical
)	O
acts	O
as	O
a	O
membrane	B-Cellular_component
anchor	O
of	O
many	O
cell	B-Cellular_component
surface	I-Cellular_component
proteins	O
.	O

Its	O
structure	O
and	O
biosynthetic	O
pathway	O
are	O
generally	O
conserved	O
among	O
eukaryotic	O
organisms	O
,	O
with	O
a	O
number	O
of	O
differences	O
.	O

In	O
particular	O
,	O
mammalian	O
and	O
protozoan	O
mannosyltransferases	B-Gene_or_gene_product
needed	O
for	O
addition	O
of	O
the	O
first	O
mannose	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
have	O
different	O
substrate	O
specificities	O
and	O
are	O
targets	O
of	O
species	O
-	O
specific	O
inhibitors	O
of	O
GPI	B-Simple_chemical
biosynthesis	O
.	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
however	O
,	O
has	O
not	O
been	O
molecularly	O
characterized	O
.	O

Characterization	O
of	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
would	O
also	O
help	O
to	O
clarify	O
the	O
topology	O
of	O
GPI	B-Simple_chemical
biosynthesis	O
.	O

Here	O
,	O
we	O
report	O
a	O
human	O
cell	O
line	O
defective	O
in	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
and	O
the	O
gene	O
responsible	O
,	O
PIG	B-Gene_or_gene_product
-	I-Gene_or_gene_product
M	I-Gene_or_gene_product
.	O

PIG	B-Gene_or_gene_product
-	I-Gene_or_gene_product
M	I-Gene_or_gene_product
encodes	O
a	O
new	O
type	O
of	O
mannosyltransferase	B-Gene_or_gene_product
of	O
423	O
amino	O
acids	O
,	O
bearing	O
multiple	O
transmembrane	B-Cellular_component
domains	O
.	O

PIG	B-Gene_or_gene_product
-	I-Gene_or_gene_product
M	I-Gene_or_gene_product
has	O
a	O
functionally	O
important	O
DXD	O
motif	O
,	O
a	O
characteristic	O
of	O
many	O
glycosyltransferases	B-Gene_or_gene_product
,	O
within	O
a	O
domain	O
facing	O
the	O
lumen	B-Cellular_component
of	O
the	O
endoplasmic	B-Cellular_component
reticulum	I-Cellular_component
(	O
ER	B-Cellular_component
)	O
,	O
indicating	O
that	O
transfer	O
of	O
the	O
first	O
mannose	B-Simple_chemical
to	O
GPI	B-Simple_chemical
occurs	O
on	O
the	O
lumenal	B-Cellular_component
side	I-Cellular_component
of	O
the	O
ER	B-Cellular_component
membrane	I-Cellular_component
.	O

Human	O
neuron	O
-	O
committed	O
teratocarcinoma	O
NT2	O
cell	O
line	O
has	O
abnormal	O
ND10	B-Cellular_component
structures	I-Cellular_component
and	O
is	O
poorly	O
infected	O
by	O
herpes	O
simplex	O
virus	O
type	O
1	O
.	O

Herpes	O
simplex	O
virus	O
type	O
1	O
(	O
HSV	O
-	O
1	O
)	O
immediate	B-Gene_or_gene_product
-	I-Gene_or_gene_product
early	I-Gene_or_gene_product
regulatory	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
ICP0	I-Gene_or_gene_product
stimulates	O
the	O
initiation	O
of	O
lytic	O
infection	O
and	O
reactivation	O
from	O
quiescence	O
in	O
human	O
fibroblast	O
cells	O
.	O

These	O
functions	O
correlate	O
with	O
its	O
ability	O
to	O
localize	O
to	O
and	O
disrupt	O
centromeres	B-Cellular_component
and	O
specific	O
subnuclear	O
structures	O
known	O
as	O
ND10	B-Cellular_component
,	O
PML	B-Cellular_component
nuclear	I-Cellular_component
bodies	I-Cellular_component
,	O
or	O
promyelocytic	B-Cellular_component
oncogenic	I-Cellular_component
domains	I-Cellular_component
.	O

Since	O
the	O
natural	O
site	O
of	O
herpesvirus	O
latency	O
is	O
in	O
neurons	O
,	O
we	O
investigated	O
the	O
status	O
of	O
ND10	B-Cellular_component
and	O
centromeres	B-Cellular_component
in	O
uninfected	O
and	O
infected	O
human	O
cells	O
with	O
neuronal	O
characteristics	O
.	O

We	O
found	O
that	O
NT2	O
cells	O
,	O
a	O
neuronally	O
committed	O
human	O
teratocarcinoma	O
cell	O
line	O
,	O
have	O
abnormal	O
ND10	B-Cellular_component
characterized	O
by	O
low	O
expression	O
of	O
the	O
major	O
ND10	B-Cellular_component
component	O
PML	B-Gene_or_gene_product
and	O
no	O
detectable	O
expression	O
of	O
another	O
major	O
ND10	B-Cellular_component
antigen	O
,	O
Sp100	B-Gene_or_gene_product
.	O

In	O
addition	O
,	O
PML	B-Gene_or_gene_product
is	O
less	O
extensively	O
modified	O
by	O
the	O
ubiquitin	B-Gene_or_gene_product
-	O
like	O
protein	O
SUMO	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
in	O
NT2	O
cells	O
compared	O
to	O
fibroblasts	O
.	O

After	O
treatment	O
with	O
retinoic	B-Simple_chemical
acid	I-Simple_chemical
,	O
NT2	O
cells	O
differentiate	O
into	O
neuron	O
-	O
like	O
hNT	O
cells	O
which	O
express	O
very	O
high	O
levels	O
of	O
both	O
PML	B-Gene_or_gene_product
and	O
Sp100	B-Gene_or_gene_product
.	O

Infection	O
of	O
both	O
NT2	O
and	O
hNT	O
cells	O
by	O
HSV	O
-	O
1	O
was	O
poor	O
compared	O
to	O
human	O
fibroblasts	O
,	O
and	O
after	O
low	O
-	O
multiplicity	O
infection	O
yields	O
of	O
virus	O
were	O
reduced	O
by	O
2	O
to	O
3	O
orders	O
of	O
magnitude	O
.	O

ICP0	B-Gene_or_gene_product
-	O
deficient	O
mutants	O
were	O
also	O
disabled	O
in	O
the	O
neuron	O
-	O
related	O
cell	O
lines	O
,	O
and	O
cells	O
quiescently	O
infected	O
with	O
an	O
ICP0	B-Gene_or_gene_product
-	O
null	O
virus	O
could	O
be	O
established	O
.	O

These	O
results	O
correlated	O
with	O
less	O
-	O
efficient	O
disruption	O
of	O
ND10	B-Cellular_component
and	O
centromeres	B-Cellular_component
induced	O
by	O
ICP0	B-Gene_or_gene_product
in	O
NT2	O
and	O
hNT	O
cells	O
.	O

Furthermore	O
,	O
the	O
ability	O
of	O
ICP0	B-Gene_or_gene_product
to	O
activate	O
gene	O
expression	O
in	O
transfection	O
assays	O
in	O
NT2	O
cells	O
was	O
poor	O
compared	O
to	O
Vero	O
cells	O
.	O

These	O
results	O
suggest	O
that	O
a	O
contributory	O
factor	O
in	O
the	O
reduced	O
HSV	O
-	O
1	O
replication	O
in	O
the	O
neuron	O
-	O
related	O
cells	O
is	O
inefficient	O
ICP0	B-Gene_or_gene_product
function	O
;	O
it	O
is	O
possible	O
that	O
this	O
is	O
pertinent	O
to	O
the	O
establishment	O
of	O
latent	O
infection	O
in	O
neurons	O
in	O
vivo	O
.	O

The	O
SV40	B-Gene_or_gene_product
small	I-Gene_or_gene_product
t	I-Gene_or_gene_product
-	I-Gene_or_gene_product
antigen	I-Gene_or_gene_product
prevents	O
mammary	O
gland	O
differentiation	O
and	O
induces	O
breast	O
cancer	O
formation	O
in	O
transgenic	O
mice	O
;	O
truncated	O
large	B-Gene_or_gene_product
T	I-Gene_or_gene_product
-	I-Gene_or_gene_product
antigen	I-Gene_or_gene_product
molecules	O
harboring	O
the	O
intact	O
p53	B-Gene_or_gene_product
and	O
pRb	B-Gene_or_gene_product
binding	O
region	O
do	O
not	O
have	O
this	O
effect	O
.	O

We	O
report	O
here	O
for	O
the	O
first	O
time	O
,	O
that	O
the	O
SV40	B-Gene_or_gene_product
small	I-Gene_or_gene_product
t	I-Gene_or_gene_product
-	I-Gene_or_gene_product
antigen	I-Gene_or_gene_product
inhibits	O
mammary	O
gland	O
differentiation	O
during	O
mid	O
-	O
pregnancy	O
and	O
that	O
about	O
10	O
%	O
of	O
multiparous	O
WAP	B-Gene_or_gene_product
-	O
SVt	B-Gene_or_gene_product
transgenic	O
animals	O
develop	O
breast	O
tumors	O
with	O
latencies	O
ranging	O
from	O
10	O
-	O
17	O
months	O
.	O

Cyclin	B-Gene_or_gene_product
D1	I-Gene_or_gene_product
is	O
deregulated	O
and	O
over	O
expressed	O
in	O
the	O
small	B-Gene_or_gene_product
t	I-Gene_or_gene_product
-	I-Gene_or_gene_product
antigen	I-Gene_or_gene_product
positive	O
mammary	O
gland	O
epithelial	O
cells	O
(	O
ME	O
-	O
cells	O
)	O
and	O
in	O
the	O
breast	O
tumor	O
cells	O
.	O

SV40	B-Gene_or_gene_product
small	I-Gene_or_gene_product
t	I-Gene_or_gene_product
-	I-Gene_or_gene_product
antigen	I-Gene_or_gene_product
immortalized	O
ME	O
-	O
cells	O
(	O
t	O
-	O
ME	O
-	O
cells	O
)	O
exhibit	O
a	O
strong	O
intranuclear	O
cyclin	B-Gene_or_gene_product
D1	I-Gene_or_gene_product
staining	O
,	O
also	O
in	O
the	O
absence	O
of	O
external	O
growth	O
factors	O
and	O
the	O
cells	O
continue	O
to	O
divide	O
for	O
several	O
days	O
without	O
serum	O
.	O

In	O
addition	O
,	O
the	O
expression	O
rate	O
of	O
cyclin	B-Gene_or_gene_product
E	I-Gene_or_gene_product
and	O
p21	B-Gene_or_gene_product
(	O
Waf1	B-Gene_or_gene_product
)	O
but	O
not	O
of	O
p53	B-Gene_or_gene_product
is	O
increased	O
.	O

Coimmunoprecipitation	O
experiments	O
revealed	O
that	O
p21	B-Gene_or_gene_product
(	O
Waf1	B-Gene_or_gene_product
)	O
is	O
mainly	O
associated	O
with	O
the	O
cyclin	B-Complex
D	I-Complex
/	I-Complex
CDK4	I-Complex
but	O
not	O
with	O
the	O
cyclin	B-Complex
E	I-Complex
/	I-Complex
CDK2	I-Complex
complex	O
.	O

WAP	O
-	O
SVT	O
transgenic	O
animals	O
exhibit	O
an	O
almost	O
regular	O
mammary	O
gland	O
development	O
until	O
late	O
pregnancy	O
but	O
the	O
majority	O
of	O
the	O
ME	O
-	O
cells	O
are	O
eliminated	O
by	O
apoptosis	O
during	O
the	O
early	O
lactation	O
period	O
.	O

Tumor	O
formation	O
is	O
delayed	O
and	O
less	O
efficient	O
than	O
in	O
T	B-Gene_or_gene_product
/	O
t	B-Gene_or_gene_product
-	I-Gene_or_gene_product
antigen	I-Gene_or_gene_product
positive	O
animals	O
.	O

Sequestration	O
of	O
p53	B-Gene_or_gene_product
and	O
pRb	B-Gene_or_gene_product
by	O
the	O
N	O
-	O
terminal	O
truncated	O
T	B-Gene_or_gene_product
-	I-Gene_or_gene_product
antigen	I-Gene_or_gene_product
molecules	O
(	O
T1	B-Gene_or_gene_product
-	I-Gene_or_gene_product
antigen	I-Gene_or_gene_product
and	O
T2	B-Gene_or_gene_product
-	I-Gene_or_gene_product
antigen	I-Gene_or_gene_product
)	O
does	O
not	O
affect	O
mammary	O
gland	O
differentiation	O
and	O
the	O
transgenic	O
animals	O
(	O
WAP	B-Gene_or_gene_product
-	O
SVBst	B-Gene_or_gene_product
-	O
Bam	B-Gene_or_gene_product
)	O
do	O
not	O
develop	O
breast	O
tumors	O
.	O

TAK1	B-Gene_or_gene_product
is	O
a	O
ubiquitin	B-Gene_or_gene_product
-	O
dependent	O
kinase	O
of	O
MKK	B-Gene_or_gene_product
and	O
IKK	B-Complex
.	O

TRAF6	B-Gene_or_gene_product
is	O
a	O
signal	O
transducer	O
that	O
activates	O
IkappaB	B-Complex
kinase	I-Complex
(	O
IKK	B-Complex
)	O
and	O
Jun	B-Gene_or_gene_product
amino	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
in	O
response	O
to	O
pro	O
-	O
inflammatory	O
mediators	O
such	O
as	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
and	O
lipopolysaccharides	B-Simple_chemical
(	O
LPS	B-Simple_chemical
)	O
.	O

IKK	B-Complex
activation	O
by	O
TRAF6	B-Gene_or_gene_product
requires	O
two	O
intermediary	O
factors	O
,	O
TRAF6	B-Gene_or_gene_product
-	I-Gene_or_gene_product
regulated	I-Gene_or_gene_product
IKK	I-Gene_or_gene_product
activator	I-Gene_or_gene_product
1	I-Gene_or_gene_product
(	O
TRIKA1	B-Gene_or_gene_product
)	O
and	O
TRIKA2	B-Gene_or_gene_product
(	O
ref	O
.	O
5	O
)	O
.	O

TRIKA1	B-Complex
is	O
a	O
dimeric	O
ubiquitin	B-Gene_or_gene_product
-	O
conjugating	O
enzyme	O
complex	O
composed	O
of	O
Ubc13	B-Gene_or_gene_product
and	O
Uev1A	B-Gene_or_gene_product
(	O
or	O
the	O
functionally	O
equivalent	O
Mms2	B-Gene_or_gene_product
)	O
.	O

This	O
Ubc	B-Complex
complex	I-Complex
,	O
together	O
with	O
TRAF6	B-Gene_or_gene_product
,	O
catalyses	O
the	O
formation	O
of	O
a	O
Lys	B-Simple_chemical
63	I-Simple_chemical
(	O
K63	B-Simple_chemical
)	O
-	O
linked	O
polyubiquitin	B-Gene_or_gene_product
chain	O
that	O
mediates	O
IKK	B-Complex
activation	O
through	O
a	O
unique	O
proteasome	O
-	O
independent	O
mechanism	O
.	O

Here	O
we	O
report	O
the	O
purification	O
and	O
identification	O
of	O
TRIKA2	B-Complex
,	O
which	O
is	O
composed	O
of	O
TAK1	B-Gene_or_gene_product
,	O
TAB1	B-Gene_or_gene_product
and	O
TAB2	B-Gene_or_gene_product
,	O
a	O
protein	O
kinase	O
complex	O
previously	O
implicated	O
in	O
IKK	B-Complex
activation	O
through	O
an	O
unknown	O
mechanism	O
.	O

We	O
find	O
that	O
the	O
TAK1	B-Gene_or_gene_product
kinase	O
complex	O
phosphorylates	O
and	O
activates	O
IKK	B-Complex
in	O
a	O
manner	O
that	O
depends	O
on	O
TRAF6	B-Gene_or_gene_product
and	O
Ubc13	B-Complex
-	I-Complex
Uev1A	I-Complex
.	O

Moreover	O
,	O
the	O
activity	O
of	O
TAK1	B-Gene_or_gene_product
to	O
phosphorylate	O
MKK6	B-Gene_or_gene_product
,	O
which	O
activates	O
the	O
JNK	B-Gene_or_gene_product
-	O
p38	B-Gene_or_gene_product
kinase	O
pathway	O
,	O
is	O
directly	O
regulated	O
by	O
K63	B-Simple_chemical
-	O
linked	O
polyubiquitination	O
.	O

We	O
also	O
provide	O
evidence	O
that	O
TRAF6	B-Gene_or_gene_product
is	O
conjugated	O
by	O
the	O
K63	B-Simple_chemical
polyubiquitin	B-Gene_or_gene_product
chains	O
.	O

These	O
results	O
indicate	O
that	O
ubiquitination	O
has	O
an	O
important	O
regulatory	O
role	O
in	O
stress	O
response	O
pathways	O
,	O
including	O
those	O
of	O
IKK	B-Complex
and	O
JNK	B-Gene_or_gene_product
.	O

Rb	B-Gene_or_gene_product
targets	O
histone	B-Gene_or_gene_product
H3	I-Gene_or_gene_product
methylation	O
and	O
HP1	B-Gene_or_gene_product
to	O
promoters	O
.	O

In	O
eukaryotic	O
cells	O
the	O
histone	B-Gene_or_gene_product
methylase	O
SUV39H1	B-Gene_or_gene_product
and	O
the	O
methyl	B-Simple_chemical
-	I-Simple_chemical
lysine	I-Simple_chemical
binding	O
protein	O
HP1	B-Gene_or_gene_product
functionally	O
interact	O
to	O
repress	O
transcription	O
at	O
heterochromatic	O
sites	O
.	O

Lysine	B-Simple_chemical
9	I-Simple_chemical
of	O
histone	B-Gene_or_gene_product
H3	I-Gene_or_gene_product
is	O
methylated	O
by	O
SUV39H1	B-Gene_or_gene_product
(	O
ref	O
.	O
2	O
)	O
,	O
creating	O
a	O
binding	O
site	O
for	O
the	O
chromo	O
domain	O
of	O
HP1	B-Gene_or_gene_product
(	O
refs	O
3	O
,	O
4	O
)	O
.	O

Here	O
we	O
show	O
that	O
SUV39H1	B-Gene_or_gene_product
and	O
HP1	B-Gene_or_gene_product
are	O
both	O
involved	O
in	O
the	O
repressive	O
functions	O
of	O
the	O
retinoblastoma	B-Gene_or_gene_product
(	O
Rb	B-Gene_or_gene_product
)	O
protein	O
.	O

Rb	B-Gene_or_gene_product
associates	O
with	O
SUV39H1	B-Gene_or_gene_product
and	O
HP1	B-Gene_or_gene_product
in	O
vivo	O
by	O
means	O
of	O
its	O
pocket	O
domain	O
.	O

SUV39H1	B-Gene_or_gene_product
cooperates	O
with	O
Rb	B-Gene_or_gene_product
to	O
repress	O
the	O
cyclin	B-Gene_or_gene_product
E	I-Gene_or_gene_product
promoter	O
,	O
and	O
in	O
fibroblasts	O
that	O
are	O
disrupted	O
for	O
SUV39	B-Gene_or_gene_product
,	O
the	O
activity	O
of	O
the	O
cyclin	B-Gene_or_gene_product
E	I-Gene_or_gene_product
and	O
cyclin	B-Gene_or_gene_product
A2	I-Gene_or_gene_product
genes	O
are	O
specifically	O
elevated	O
.	O

Chromatin	B-Cellular_component
immunoprecipitations	O
show	O
that	O
Rb	B-Gene_or_gene_product
is	O
necessary	O
to	O
direct	O
methylation	O
of	O
histone	B-Gene_or_gene_product
H3	I-Gene_or_gene_product
,	O
and	O
is	O
necessary	O
for	O
binding	O
of	O
HP1	B-Gene_or_gene_product
to	O
the	O
cyclin	B-Gene_or_gene_product
E	I-Gene_or_gene_product
promoter	O
.	O

These	O
results	O
indicate	O
that	O
the	O
SUV39H1	B-Complex
-	I-Complex
HP1	I-Complex
complex	O
is	O
not	O
only	O
involved	O
in	O
heterochromatic	O
silencing	O
but	O
also	O
has	O
a	O
role	O
in	O
repression	O
of	O
euchromatic	O
genes	O
by	O
Rb	B-Gene_or_gene_product
and	O
perhaps	O
other	O
co	O
-	O
repressor	O
proteins	O
.	O

p70S6	B-Gene_or_gene_product
kinase	O
signals	O
cell	O
survival	O
as	O
well	O
as	O
growth	O
,	O
inactivating	O
the	O
pro	O
-	O
apoptotic	O
molecule	O
BAD	B-Gene_or_gene_product
.	O

Cytokines	O
often	O
deliver	O
simultaneous	O
,	O
yet	O
distinct	O
,	O
cell	O
growth	O
and	O
cell	O
survival	O
signals	O
.	O

The	O
70	B-Gene_or_gene_product
-	I-Gene_or_gene_product
kDa	I-Gene_or_gene_product
ribosomal	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
S6	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
(	O
p70S6K	B-Gene_or_gene_product
)	O
is	O
known	O
to	O
regulate	O
cell	O
growth	O
by	O
inducing	O
protein	O
synthesis	O
components	O
.	O

We	O
purified	O
membrane	B-Cellular_component
-	O
based	O
p70S6K	B-Gene_or_gene_product
as	O
a	O
kinase	O
responsible	O
for	O
site	O
-	O
specific	O
phosphorylation	O
of	O
BAD	B-Gene_or_gene_product
,	O
which	O
inactivates	O
this	O
proapoptotic	O
molecule	O
.	O

Rapamycin	B-Simple_chemical
inhibited	O
mitochondrial	O
-	O
based	O
p70S6K	B-Gene_or_gene_product
,	O
which	O
prevented	O
phosphorylation	O
of	O
Ser	B-Simple_chemical
-	I-Simple_chemical
136	I-Simple_chemical
on	O
BAD	B-Gene_or_gene_product
and	O
blocked	O
cell	O
survival	O
induced	O
by	O
insulin	B-Gene_or_gene_product
-	I-Gene_or_gene_product
like	I-Gene_or_gene_product
growth	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
1	I-Gene_or_gene_product
(	O
IGF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
)	O
.	O

Moreover	O
,	O
IGF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
-	O
induced	O
phosphorylation	O
of	O
BAD	B-Gene_or_gene_product
Ser	B-Simple_chemical
-	I-Simple_chemical
136	I-Simple_chemical
was	O
abolished	O
in	O
p70S6K	B-Gene_or_gene_product
-	O
deficient	O
cells	O
.	O

Thus	O
,	O
p70S6K	B-Gene_or_gene_product
is	O
itself	O
a	O
dual	O
pathway	O
kinase	O
,	O
signaling	O
cell	O
survival	O
as	O
well	O
as	O
growth	O
through	O
differential	O
substrates	O
which	O
include	O
mitochondrial	B-Cellular_component
BAD	B-Gene_or_gene_product
and	O
the	O
ribosomal	B-Cellular_component
subunit	O
S6	B-Gene_or_gene_product
,	O
respectively	O
.	O

Polyamine	O
depletion	O
induces	O
rapid	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
activation	O
in	O
IEC	O
-	O
6	O
cells	O
.	O

The	O
proliferation	O
of	O
the	O
rat	O
intestinal	O
mucosal	O
IEC	O
-	O
6	O
cell	O
line	O
requires	O
polyamines	O
,	O
whose	O
synthesis	O
is	O
catalyzed	O
by	O
the	O
enzyme	O
ornithine	B-Gene_or_gene_product
decarboxylase	I-Gene_or_gene_product
(	O
ODC	B-Gene_or_gene_product
)	O
.	O

ODC	B-Gene_or_gene_product
inhibition	O
leads	O
to	O
polyamine	O
depletion	O
,	O
as	O
well	O
as	O
inhibition	O
of	O
both	O
cell	O
proliferation	O
and	O
apoptosis	O
by	O
regulating	O
gene	O
expression	O
.	O

The	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
transcription	O
factor	O
regulates	O
genes	O
involved	O
in	O
apoptotic	O
,	O
immune	O
,	O
and	O
inflammatory	O
responses	O
.	O

In	O
the	O
present	O
study	O
we	O
tested	O
the	O
hypothesis	O
that	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
is	O
activated	O
following	O
ODC	B-Gene_or_gene_product
inhibition	O
.	O

We	O
found	O
that	O
the	O
inhibition	O
of	O
ODC	B-Gene_or_gene_product
by	O
alpha	B-Simple_chemical
-	I-Simple_chemical
difluoromethylornithine	I-Simple_chemical
(	O
DFMO	B-Simple_chemical
)	O
resulted	O
in	O
a	O
approximately	O
50	O
%	O
decrease	O
in	O
intracellular	B-Cellular_component
putrescine	B-Simple_chemical
levels	O
within	O
1	O
h	O
.	O

NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
is	O
activated	O
by	O
DFMO	B-Simple_chemical
through	O
the	O
degradation	O
of	O
the	O
inhibitory	O
protein	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
that	O
sequesters	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
in	O
the	O
cytoplasm	B-Cellular_component
.	O

The	O
DFMO	B-Simple_chemical
-	O
induced	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
complexes	O
contain	O
the	O
p65	B-Gene_or_gene_product
and	O
p50	B-Gene_or_gene_product
members	O
of	O
the	O
Rel	B-Gene_or_gene_product
protein	O
family	O
.	O

DFMO	B-Simple_chemical
-	O
induced	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
activation	O
was	O
accompanied	O
by	O
the	O
translocation	O
of	O
p65	B-Gene_or_gene_product
from	O
the	O
cytoplasm	B-Cellular_component
into	O
the	O
nucleus	B-Cellular_component
.	O

DFMO	B-Simple_chemical
selectively	O
inhibited	O
a	O
gene	O
reporter	O
construct	O
dependent	O
on	O
the	O
kappa	B-Complex
B	I-Complex
site	O
present	O
in	O
the	O
HLA	B-Gene_or_gene_product
-	I-Gene_or_gene_product
B7	I-Gene_or_gene_product
gene	O
.	O

In	O
contrast	O
,	O
DFMO	B-Simple_chemical
had	O
no	O
effect	O
on	O
a	O
gene	O
reporter	O
construct	O
dependent	O
on	O
the	O
kappa	B-Complex
B	I-Complex
site	O
present	O
in	O
the	O
interleukin	B-Gene_or_gene_product
-	I-Gene_or_gene_product
8	I-Gene_or_gene_product
gene	O
.	O

Thus	O
,	O
we	O
report	O
that	O
ODC	B-Gene_or_gene_product
inhibition	O
activates	O
the	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
transcription	O
factor	O
,	O
which	O
may	O
mediate	O
the	O
altered	O
physiological	O
state	O
of	O
intestinal	O
cells	O
that	O
occurs	O
following	O
polyamine	O
depletion	O
.	O

Nucleocytoplasmic	B-Cellular_component
shuttling	O
of	O
p130	B-Gene_or_gene_product
/	O
RBL2	B-Gene_or_gene_product
:	O
novel	O
regulatory	O
mechanism	O
.	O

The	O
retinoblastoma	O
-	O
related	O
pocket	O
proteins	O
pRb	B-Gene_or_gene_product
,	O
p107	B-Gene_or_gene_product
,	O
and	O
p130	B-Gene_or_gene_product
are	O
implicated	O
in	O
the	O
control	O
of	O
cell	O
proliferation	O
,	O
differentiation	O
,	O
and	O
transformation	O
.	O

The	O
function	O
of	O
pocket	O
proteins	O
is	O
in	O
part	O
mediated	O
by	O
their	O
ability	O
to	O
inhibit	O
specific	O
E2F	B-Gene_or_gene_product
transcription	O
factors	O
.	O

The	O
transcriptional	O
activity	O
of	O
E2Fs	B-Gene_or_gene_product
is	O
controlled	O
by	O
alteration	O
of	O
their	O
nucleocytoplasmic	B-Cellular_component
localization	O
during	O
the	O
cell	O
cycle	O
.	O

p130	B-Gene_or_gene_product
was	O
observed	O
to	O
shuttle	O
between	O
the	O
nucleus	B-Cellular_component
and	O
cytoplasm	B-Cellular_component
in	O
a	O
heterokaryon	O
fusion	O
assay	O
,	O
suggesting	O
the	O
presence	O
of	O
nuclear	B-Cellular_component
and	O
cytoplasmic	B-Cellular_component
localization	O
signals	O
.	O

Two	O
independent	O
nuclear	B-Cellular_component
localization	O
signals	O
(	O
NLS	O
)	O
that	O
could	O
target	O
reporter	O
proteins	O
to	O
the	O
nucleus	B-Cellular_component
in	O
transient	O
transfection	O
and	O
microinjection	O
experiments	O
were	O
identified	O
in	O
the	O
C	O
terminus	O
of	O
p130	B-Gene_or_gene_product
.	O

In	O
addition	O
to	O
the	O
C	O
-	O
terminal	O
NLS	O
,	O
the	O
intact	O
pocket	O
domain	O
of	O
p130	B-Gene_or_gene_product
itself	O
was	O
sufficient	O
for	O
nuclear	B-Cellular_component
translocation	O
.	O

Moreover	O
,	O
an	O
additional	O
functional	O
NLS	O
was	O
mapped	O
within	O
the	O
unique	O
Loop	O
region	O
of	O
p130	B-Gene_or_gene_product
.	O

An	O
N	O
-	O
terminal	O
domain	O
that	O
conferred	O
cytoplasmic	B-Cellular_component
localization	O
was	O
identified	O
.	O

Removal	O
of	O
the	O
entire	O
N	O
terminus	O
did	O
not	O
affect	O
the	O
ability	O
of	O
p130	B-Gene_or_gene_product
to	O
interact	O
with	O
E2F	B-Gene_or_gene_product
and	O
to	O
induce	O
growth	O
arrest	O
.	O

A	O
model	O
suggesting	O
that	O
the	O
activity	O
of	O
pRb	B-Gene_or_gene_product
family	O
members	O
can	O
be	O
regulated	O
by	O
intracellular	B-Cellular_component
trafficking	O
of	O
the	O
proteins	O
is	O
proposed	O
.	O

S	B-Gene_or_gene_product
-	I-Gene_or_gene_product
Cdk	I-Gene_or_gene_product
-	O
dependent	O
phosphorylation	O
of	O
Sld2	B-Gene_or_gene_product
essential	O
for	O
chromosomal	B-Cellular_component
DNA	O
replication	O
in	O
budding	O
yeast	O
.	O

Cyclin	B-Gene_or_gene_product
-	I-Gene_or_gene_product
dependent	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
kinases	I-Gene_or_gene_product
(	O
Cdks	B-Gene_or_gene_product
)	O
in	O
eukaryotic	O
cells	O
work	O
as	O
a	O
key	O
enzyme	O
at	O
various	O
points	O
in	O
the	O
cell	O
cycle	O
.	O

At	O
the	O
onset	O
of	O
S	O
phase	O
,	O
active	O
S	O
-	O
phase	O
Cdks	B-Gene_or_gene_product
(	O
S	B-Gene_or_gene_product
-	I-Gene_or_gene_product
Cdks	I-Gene_or_gene_product
)	O
are	O
essential	O
for	O
chromosomal	B-Cellular_component
DNA	O
replication	O
.	O

Although	O
several	O
replication	O
proteins	O
are	O
phosphorylated	O
in	O
a	O
Cdk	B-Gene_or_gene_product
-	O
dependent	O
manner	O
,	O
the	O
biological	O
effects	O
of	O
phosphorylation	O
of	O
these	O
proteins	O
on	O
the	O
activation	O
of	O
DNA	O
replication	O
have	O
not	O
been	O
elucidated	O
.	O

Here	O
we	O
show	O
that	O
Sld2	B-Gene_or_gene_product
(	O
ref	O
.	O
4	O
)	O
(	O
also	O
known	O
as	O
Drc1	B-Gene_or_gene_product
;	O
ref	O
.	O
5	O
)	O
,	O
one	O
of	O
the	O
replication	O
proteins	O
of	O
budding	O
yeast	O
(	O
Saccharomyces	O
cerevisiae	O
)	O
,	O
is	O
phosphorylated	O
in	O
S	O
phase	O
in	O
an	O
S	B-Gene_or_gene_product
-	I-Gene_or_gene_product
Cdk	I-Gene_or_gene_product
-	O
dependent	O
manner	O
,	O
and	O
mutant	O
Sld2	B-Gene_or_gene_product
lacking	O
all	O
the	O
preferred	O
Cdk	B-Gene_or_gene_product
phosphorylation	O
sites	O
(	O
All	B-Gene_or_gene_product
-	I-Gene_or_gene_product
A	I-Gene_or_gene_product
)	O
is	O
defective	O
in	O
chromosomal	B-Cellular_component
DNA	O
replication	O
.	O

Moreover	O
,	O
the	O
complex	O
that	O
contains	O
,	O
at	O
least	O
,	O
Sld2	B-Gene_or_gene_product
and	O
Dpb11	B-Gene_or_gene_product
(	O
ref	O
.	O
6	O
)	O
(	O
the	O
Sld2	B-Complex
-	I-Complex
Dpb11	I-Complex
complex	O
)	O
is	O
formed	O
predominantly	O
in	O
S	O
phase	O
;	O
the	O
All	B-Gene_or_gene_product
-	I-Gene_or_gene_product
A	I-Gene_or_gene_product
protein	O
is	O
defective	O
in	O
this	O
complex	O
formation	O
.	O

Because	O
this	O
complex	O
is	O
suggested	O
to	O
be	O
essential	O
for	O
chromosomal	B-Cellular_component
DNA	O
replication	O
,	O
it	O
seems	O
likely	O
that	O
S	B-Gene_or_gene_product
-	I-Gene_or_gene_product
Cdk	I-Gene_or_gene_product
positively	O
regulates	O
formation	O
of	O
the	O
Sld2	B-Complex
-	I-Complex
Dpb11	I-Complex
complex	O
and	O
,	O
consequently	O
,	O
chromosomal	B-Cellular_component
DNA	O
replication	O
.	O

Steps	O
in	O
assembly	O
of	O
silent	O
chromatin	B-Cellular_component
in	O
yeast	O
:	O
Sir3	B-Gene_or_gene_product
-	O
independent	O
binding	O
of	O
a	O
Sir2	B-Complex
/	I-Complex
Sir4	I-Complex
complex	O
to	O
silencers	O
and	O
role	O
for	O
Sir2	B-Gene_or_gene_product
-	O
dependent	O
deacetylation	O
.	O

Transcriptional	O
silencing	O
at	O
the	O
budding	O
yeast	O
silent	O
mating	O
type	O
(	O
HM	O
)	O
loci	O
and	O
telomeric	O
DNA	O
regions	O
requires	O
Sir2	B-Gene_or_gene_product
,	O
a	O
conserved	O
NAD	B-Simple_chemical
-	O
dependent	O
histone	B-Gene_or_gene_product
deacetylase	I-Gene_or_gene_product
,	O
Sir3	B-Gene_or_gene_product
,	O
Sir4	B-Gene_or_gene_product
,	O
histones	B-Gene_or_gene_product
H3	I-Gene_or_gene_product
and	O
H4	B-Gene_or_gene_product
,	O
and	O
several	O
DNA	O
-	O
binding	O
proteins	O
.	O

Silencing	O
at	O
the	O
yeast	O
ribosomal	O
DNA	O
(	O
rDNA	O
)	O
repeats	O
requires	O
a	O
complex	O
containing	O
Sir2	B-Gene_or_gene_product
,	O
Net1	B-Gene_or_gene_product
,	O
and	O
Cdc14	B-Gene_or_gene_product
.	O

Here	O
we	O
show	O
that	O
the	O
native	O
Sir2	B-Complex
/	I-Complex
Sir4	I-Complex
complex	O
is	O
composed	O
solely	O
of	O
Sir2	B-Gene_or_gene_product
and	O
Sir4	B-Gene_or_gene_product
and	O
that	O
native	O
Sir3	B-Gene_or_gene_product
is	O
not	O
associated	O
with	O
other	O
proteins	O
.	O

We	O
further	O
show	O
that	O
the	O
initial	O
binding	O
of	O
the	O
Sir2	B-Complex
/	I-Complex
Sir4	I-Complex
complex	O
to	O
DNA	O
sites	O
that	O
nucleate	O
silencing	O
,	O
accompanied	O
by	O
partial	O
Sir2	B-Gene_or_gene_product
-	O
dependent	O
histone	B-Gene_or_gene_product
deacetylation	O
,	O
occurs	O
independently	O
of	O
Sir3	B-Gene_or_gene_product
and	O
is	O
likely	O
to	O
be	O
the	O
first	O
step	O
in	O
assembly	O
of	O
silent	O
chromatin	B-Cellular_component
at	O
the	O
HM	O
loci	O
and	O
telomeres	O
.	O

The	O
enzymatic	O
activity	O
of	O
Sir2	B-Gene_or_gene_product
is	O
not	O
required	O
for	O
this	O
initial	O
binding	O
,	O
but	O
is	O
required	O
for	O
the	O
association	O
of	O
silencing	O
proteins	O
with	O
regions	O
distal	O
from	O
nucleation	O
sites	O
.	O

At	O
the	O
rDNA	O
repeats	O
,	O
we	O
show	O
that	O
histone	B-Gene_or_gene_product
H3	I-Gene_or_gene_product
and	O
H4	B-Gene_or_gene_product
tails	O
are	O
required	O
for	O
silencing	O
and	O
rDNA	O
-	O
associated	O
H4	B-Gene_or_gene_product
is	O
hypoacetylated	O
in	O
a	O
Sir2	B-Gene_or_gene_product
-	O
dependent	O
manner	O
.	O

However	O
,	O
the	O
binding	O
of	O
Sir2	B-Gene_or_gene_product
to	O
rDNA	O
is	O
independent	O
of	O
its	O
histone	B-Gene_or_gene_product
deacetylase	I-Gene_or_gene_product
activity	O
.	O

Together	O
,	O
these	O
results	O
support	O
a	O
stepwise	O
model	O
for	O
the	O
assembly	O
of	O
silent	O
chromatin	B-Cellular_component
domains	O
in	O
Saccharomyces	O
cerevisiae	O
.	O

Role	O
of	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
in	O
TNF	B-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
and	O
apoptosis	O
in	O
hepatocytes	O
.	O

Nuclear	B-Complex
factor	I-Complex
-	I-Complex
kappaB	I-Complex
(	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
)	O
prevents	O
hepatocytes	O
from	O
undergoing	O
apoptosis	O
during	O
development	O
and	O
liver	O
regeneration	O
.	O

Mice	O
with	O
inactivated	O
glycogen	B-Gene_or_gene_product
synthase	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
(	I-Gene_or_gene_product
GSK	I-Gene_or_gene_product
)	I-Gene_or_gene_product
-	I-Gene_or_gene_product
3beta	I-Gene_or_gene_product
die	O
from	O
hepatocyte	O
apoptosis	O
during	O
development	O
due	O
to	O
a	O
defect	O
in	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
activation	O
(	O
Hoeflich	O
KP	O
,	O
Luo	O
J	O
,	O
Rubie	O
EA	O
,	O
Tsao	O
MS	O
,	O
Jin	O
O	O
,	O
and	O
Woodgett	O
JR	O
.	O
Nature	O
406	O
:	O
86	O
-	O
90	O
,	O
2000	O
)	O
.	O

In	O
this	O
study	O
,	O
we	O
determined	O
the	O
role	O
of	O
GSK	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3	I-Gene_or_gene_product
in	O
TNF	B-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
and	O
cell	O
death	O
in	O
primary	O
hepatocytes	O
.	O

LiCl	B-Simple_chemical
,	O
an	O
established	O
inhibitor	O
of	O
GSK	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3	I-Gene_or_gene_product
,	O
sensitized	O
primary	O
rat	O
hepatocytes	O
toward	O
TNF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
-	O
mediated	O
apoptosis	O
resulting	O
in	O
90	O
%	O
cell	O
death	O
after	O
24	O
h	O
.	O

This	O
was	O
accompanied	O
by	O
increased	O
caspase	B-Gene_or_gene_product
8	I-Gene_or_gene_product
-	I-Gene_or_gene_product
like	I-Gene_or_gene_product
and	O
3	B-Gene_or_gene_product
-	I-Gene_or_gene_product
like	I-Gene_or_gene_product
activities	O
,	O
nuclear	B-Cellular_component
fragmentation	O
and	O
DNA	O
laddering	O
.	O

LiCl	B-Simple_chemical
treatment	O
had	O
no	O
effect	O
on	O
IkappaB	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
degradation	O
,	O
IkappaB	B-Complex
kinase	I-Complex
(	O
IKK	B-Complex
)	O
activity	O
,	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
binding	O
activity	O
,	O
and	O
p65	B-Gene_or_gene_product
nuclear	B-Cellular_component
import	O
and	O
export	O
,	O
but	O
decreased	O
transcription	O
of	O
the	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
-	O
dependent	O
inducible	O
nitric	B-Gene_or_gene_product
oxide	I-Gene_or_gene_product
synthase	I-Gene_or_gene_product
gene	O
and	O
a	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
-	O
driven	O
reporter	O
gene	O
.	O

The	O
p65	B-Gene_or_gene_product
sequence	O
revealed	O
four	O
potential	O
GSK	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3	I-Gene_or_gene_product
phosphorylation	O
sites	O
within	O
its	O
COOH	O
-	O
terminal	O
transactivation	O
domains	O
and	O
recombinant	O
GSK	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3beta	I-Gene_or_gene_product
phosphorylated	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
-	O
p65	B-Gene_or_gene_product
(	O
354	O
-	O
551	O
)	O
,	O
but	O
not	O
GST	B-Gene_or_gene_product
-	O
p65	B-Gene_or_gene_product
(	O
1	O
-	O
305	O
)	O
in	O
vitro	O
.	O

These	O
results	O
indicate	O
that	O
GSK	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3	I-Gene_or_gene_product
protects	O
hepatocytes	O
from	O
TNF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
-	O
induced	O
apoptosis	O
through	O
p65	B-Gene_or_gene_product
phosphorylation	O
and	O
upregulation	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
transactivation	O
.	O

An	O
E2F	B-Gene_or_gene_product
-	O
binding	O
site	O
mediates	O
the	O
activation	O
of	O
the	O
proliferative	O
isoform	O
of	O
6	B-Gene_or_gene_product
-	I-Gene_or_gene_product
phosphofructo	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
fructose	B-Gene_or_gene_product
-	I-Gene_or_gene_product
2	I-Gene_or_gene_product
,	I-Gene_or_gene_product
6	I-Gene_or_gene_product
-	I-Gene_or_gene_product
bisphosphatase	I-Gene_or_gene_product
by	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

In	O
the	O
present	O
study	O
,	O
we	O
demonstrate	O
that	O
E2F	B-Gene_or_gene_product
is	O
implicated	O
in	O
the	O
regulation	O
of	O
the	O
glycolytic	O
enzyme	O
6	B-Gene_or_gene_product
-	I-Gene_or_gene_product
phosphofructo	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
fructose	B-Gene_or_gene_product
-	I-Gene_or_gene_product
2	I-Gene_or_gene_product
,	I-Gene_or_gene_product
6	I-Gene_or_gene_product
-	I-Gene_or_gene_product
bisphosphatase	I-Gene_or_gene_product
(	O
6PF2K	B-Gene_or_gene_product
/	O
Fru	B-Gene_or_gene_product
-	I-Gene_or_gene_product
2	I-Gene_or_gene_product
,	I-Gene_or_gene_product
6	I-Gene_or_gene_product
-	I-Gene_or_gene_product
BPase	I-Gene_or_gene_product
)	O
during	O
cell	O
division	O
.	O

The	O
expression	O
of	O
this	O
enzyme	O
is	O
induced	O
during	O
the	O
G	O
(	O
1	O
)	O
/	O
S	O
transition	O
of	O
the	O
cell	O
cycle	O
.	O

We	O
identified	O
and	O
monitored	O
the	O
E2F	B-Complex
-	I-Complex
pocket	I-Complex
protein	I-Complex
complexes	O
that	O
bind	O
to	O
the	O
E2F	B-Gene_or_gene_product
site	O
of	O
the	O
F	O
-	O
type	O
promoter	O
during	O
cell	O
-	O
cycle	O
entry	O
,	O
and	O
we	O
analysed	O
their	O
contribution	O
to	O
the	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
PI	B-Gene_or_gene_product
3	I-Gene_or_gene_product
-	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
)	O
-	O
mediated	O
regulation	O
of	O
the	O
promoter	O
.	O

We	O
found	O
that	O
the	O
predominant	O
E2F	B-Complex
complex	I-Complex
bound	O
to	O
the	O
F	O
-	O
type	O
promoter	O
in	O
unstimulated	O
/	O
quiescent	O
cells	O
contains	O
E2F4	B-Gene_or_gene_product
,	O
DP1	B-Gene_or_gene_product
and	O
p130	B-Gene_or_gene_product
proteins	O
.	O

In	O
serum	O
-	O
stimulated	O
(	O
S	O
-	O
phase	O
)	O
cells	O
,	O
the	O
composition	O
of	O
the	O
complex	O
switched	O
to	O
E2F1	B-Gene_or_gene_product
/	I-Gene_or_gene_product
4	I-Gene_or_gene_product
,	O
DP1	B-Gene_or_gene_product
and	O
p107	B-Gene_or_gene_product
,	O
together	O
with	O
cyclin	B-Gene_or_gene_product
A	I-Gene_or_gene_product
and	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
.	O

Treatment	O
with	O
the	O
PI	B-Gene_or_gene_product
3	I-Gene_or_gene_product
-	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
specific	O
inhibitor	O
LY	B-Simple_chemical
294002	I-Simple_chemical
prevented	O
the	O
formation	O
of	O
the	O
S	O
-	O
phase	O
complex	O
,	O
suggesting	O
that	O
activation	O
of	O
the	O
PI	B-Gene_or_gene_product
3	I-Gene_or_gene_product
-	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
pathway	O
is	O
essential	O
for	O
the	O
formation	O
of	O
this	O
complex	O
.	O

Further	O
supporting	O
this	O
idea	O
,	O
we	O
obtained	O
results	O
showing	O
that	O
treatment	O
of	O
cycling	O
NIH	O
3T3	O
cells	O
with	O
either	O
wortmannin	B-Simple_chemical
or	O
LY	B-Simple_chemical
294002	I-Simple_chemical
induces	O
the	O
accumulation	O
of	O
the	O
transcriptionally	O
repressive	O
p130	B-Complex
-	I-Complex
E2F4	I-Complex
-	I-Complex
DP1	I-Complex
complex	O
.	O

Using	O
the	O
Rat	O
-	O
1	O
ER	B-Gene_or_gene_product
-	I-Gene_or_gene_product
E2F1	I-Gene_or_gene_product
cell	O
line	O
where	O
E2F1	B-Gene_or_gene_product
activity	O
can	O
be	O
conditionally	O
induced	O
,	O
we	O
demonstrated	O
that	O
E2F	B-Gene_or_gene_product
activity	O
is	O
involved	O
in	O
the	O
in	O
vivo	O
transcriptional	O
regulation	O
of	O
the	O
F	O
-	O
type	O
6PF2K	B-Gene_or_gene_product
/	O
Fru	B-Gene_or_gene_product
-	I-Gene_or_gene_product
2	I-Gene_or_gene_product
,	I-Gene_or_gene_product
6	I-Gene_or_gene_product
-	I-Gene_or_gene_product
BPase	I-Gene_or_gene_product
gene	O
.	O

Taken	O
together	O
,	O
our	O
results	O
show	O
that	O
the	O
F	O
-	O
type	O
6PF2K	B-Gene_or_gene_product
/	O
Fru	B-Gene_or_gene_product
-	I-Gene_or_gene_product
2	I-Gene_or_gene_product
,	I-Gene_or_gene_product
6	I-Gene_or_gene_product
-	I-Gene_or_gene_product
BPase	I-Gene_or_gene_product
is	O
a	O
genuine	O
E2F	B-Gene_or_gene_product
-	O
regulated	O
gene	O
,	O
and	O
that	O
its	O
regulation	O
by	O
the	O
PI	B-Gene_or_gene_product
3	I-Gene_or_gene_product
-	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
pathway	O
is	O
at	O
least	O
partially	O
mediated	O
through	O
the	O
E2F	B-Gene_or_gene_product
transcription	O
factor	O
.	O

Two	O
TOR	B-Gene_or_gene_product
complexes	O
,	O
only	O
one	O
of	O
which	O
is	O
rapamycin	B-Simple_chemical
sensitive	O
,	O
have	O
distinct	O
roles	O
in	O
cell	O
growth	O
control	O
.	O

The	O
target	B-Gene_or_gene_product
of	I-Gene_or_gene_product
rapamycin	I-Gene_or_gene_product
(	O
TOR	B-Gene_or_gene_product
)	O
proteins	O
in	O
Saccharomyces	O
cerevisiae	O
,	O
TOR1	B-Gene_or_gene_product
and	O
TOR2	B-Gene_or_gene_product
,	O
redundantly	O
regulate	O
growth	O
in	O
a	O
rapamycin	B-Simple_chemical
-	O
sensitive	O
manner	O
.	O

TOR2	B-Gene_or_gene_product
additionally	O
regulates	O
polarization	O
of	O
the	O
actin	B-Gene_or_gene_product
cytoskeleton	B-Cellular_component
in	O
a	O
rapamycin	B-Simple_chemical
-	O
insensitive	O
manner	O
.	O

We	O
describe	O
two	O
functionally	O
distinct	O
TOR	B-Gene_or_gene_product
complexes	O
.	O

TOR	B-Complex
Complex	I-Complex
1	I-Complex
(	O
TORC1	B-Complex
)	O
contains	O
TOR1	B-Gene_or_gene_product
or	O
TOR2	B-Gene_or_gene_product
,	O
KOG1	B-Gene_or_gene_product
(	O
YHR186c	B-Gene_or_gene_product
)	O
,	O
and	O
LST8	B-Gene_or_gene_product
.	O

TORC2	B-Complex
contains	O
TOR2	B-Gene_or_gene_product
,	O
AVO1	B-Gene_or_gene_product
(	O
YOL078w	B-Gene_or_gene_product
)	O
,	O
AVO2	B-Gene_or_gene_product
(	O
YMR068w	B-Gene_or_gene_product
)	O
,	O
AVO3	B-Gene_or_gene_product
(	O
YER093c	B-Gene_or_gene_product
)	O
,	O
and	O
LST8	B-Gene_or_gene_product
.	O

FKBP	B-Complex
-	I-Complex
rapamycin	I-Complex
binds	O
TORC1	B-Complex
,	O
and	O
TORC1	B-Complex
disruption	O
mimics	O
rapamycin	B-Simple_chemical
treatment	O
,	O
suggesting	O
that	O
TORC1	B-Complex
mediates	O
the	O
rapamycin	B-Simple_chemical
-	O
sensitive	O
,	O
TOR	B-Gene_or_gene_product
-	O
shared	O
pathway	O
.	O

FKBP	B-Complex
-	I-Complex
rapamycin	I-Complex
fails	O
to	O
bind	O
TORC2	B-Complex
,	O
and	O
TORC2	B-Complex
disruption	O
causes	O
an	O
actin	B-Gene_or_gene_product
defect	O
,	O
suggesting	O
that	O
TORC2	B-Complex
mediates	O
the	O
rapamycin	B-Simple_chemical
-	O
insensitive	O
,	O
TOR2	B-Gene_or_gene_product
-	O
unique	O
pathway	O
.	O

Thus	O
,	O
the	O
distinct	O
TOR	B-Gene_or_gene_product
complexes	O
account	O
for	O
the	O
diversity	O
,	O
specificity	O
,	O
and	O
selective	O
rapamycin	B-Simple_chemical
inhibition	O
of	O
TOR	B-Gene_or_gene_product
signaling	O
.	O

TORC1	B-Complex
and	O
possibly	O
TORC2	B-Complex
are	O
conserved	O
from	O
yeast	O
to	O
man	O
.	O

Summary	O
and	O
comparison	O
of	O
the	O
signaling	O
mechanisms	O
of	O
the	O
Toll	B-Gene_or_gene_product
/	I-Gene_or_gene_product
interleukin	I-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
receptor	I-Gene_or_gene_product
family	O
.	O

The	O
Toll	B-Gene_or_gene_product
/	I-Gene_or_gene_product
interleukin	I-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
(	I-Gene_or_gene_product
IL	I-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
TIR	B-Gene_or_gene_product
)	O
family	O
comprises	O
two	O
groups	O
of	O
transmembrane	B-Cellular_component
proteins	O
,	O
which	O
share	O
functional	O
and	O
structural	O
properties	O
.	O

The	O
members	O
of	O
the	O
IL	B-Complex
-	I-Complex
1	I-Complex
receptor	I-Complex
(	O
IL	B-Complex
-	I-Complex
1R	I-Complex
)	O
subfamily	O
are	O
characterized	O
by	O
three	O
extracellular	B-Cellular_component
immunoglobulin	B-Gene_or_gene_product
(	O
Ig	B-Gene_or_gene_product
)	O
-	O
like	O
domains	O
.	O

They	O
form	O
heterodimeric	O
signaling	O
receptor	O
complexes	O
consisting	O
of	O
receptor	O
and	O
accessory	O
proteins	O
.	O

The	O
members	O
of	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
subfamily	O
recognize	O
alarm	O
signals	O
that	O
can	O
be	O
derived	O
either	O
from	O
pathogens	O
or	O
the	O
host	O
itself	O
.	O

TLRs	B-Gene_or_gene_product
possess	O
leucine	B-Simple_chemical
-	O
rich	O
repeats	O
in	O
their	O
extracellular	B-Cellular_component
part	O
.	O

TLRs	B-Gene_or_gene_product
can	O
form	O
dimeric	O
receptor	O
complexes	O
consisting	O
of	O
two	O
different	O
TLRs	B-Gene_or_gene_product
or	O
homodimers	O
in	O
the	O
case	O
of	O
TLR4	B-Gene_or_gene_product
.	O

The	O
TLR4	B-Gene_or_gene_product
receptor	O
complex	O
requires	O
supportive	O
molecules	O
for	O
optimal	O
response	O
to	O
its	O
ligand	O
lipopolysaccharide	B-Simple_chemical
(	O
LPS	B-Simple_chemical
)	O
.	O

A	O
hallmark	O
of	O
the	O
TIR	B-Gene_or_gene_product
family	O
is	O
the	O
cytoplasmic	B-Cellular_component
TIR	B-Gene_or_gene_product
domain	O
that	O
is	O
indispensable	O
for	O
signal	O
transduction	O
.	O

The	O
TIR	B-Gene_or_gene_product
domain	O
serves	O
as	O
a	O
scaffold	O
for	O
a	O
series	O
of	O
protein	O
-	O
protein	O
interactions	O
which	O
result	O
in	O
the	O
activation	O
of	O
a	O
unique	O
signaling	O
module	O
consisting	O
of	O
MyD88	B-Gene_or_gene_product
,	O
interleukin	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
receptor	I-Gene_or_gene_product
associated	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
(	O
IRAK	B-Gene_or_gene_product
)	O
family	O
members	O
and	O
Tollip	B-Gene_or_gene_product
,	O
which	O
is	O
used	O
exclusively	O
by	O
TIR	B-Gene_or_gene_product
family	O
members	O
.	O

Subsequently	O
,	O
several	O
central	O
signaling	O
pathways	O
are	O
activated	O
in	O
parallel	O
,	O
the	O
activation	O
of	O
NFkappaB	B-Complex
being	O
the	O
most	O
prominent	O
event	O
of	O
the	O
inflammatory	O
response	O
.	O

Recent	O
developments	O
indicate	O
that	O
in	O
addition	O
to	O
the	O
common	O
signaling	O
module	O
MyD88	B-Complex
/	I-Complex
IRAK	I-Complex
/	I-Complex
Tollip	I-Complex
,	O
other	O
molecules	O
can	O
modulate	O
signaling	O
by	O
TLRs	B-Gene_or_gene_product
,	O
especially	O
of	O
TLR4	B-Gene_or_gene_product
,	O
resulting	O
in	O
differential	O
biological	O
answers	O
to	O
distinct	O
pathogenic	O
structures	O
.	O

Subtle	O
differences	O
in	O
TLR	B-Gene_or_gene_product
signaling	O
pathways	O
are	O
now	O
becoming	O
apparent	O
,	O
which	O
reveal	O
how	O
the	O
innate	O
immune	O
system	O
decides	O
at	O
a	O
very	O
early	O
stage	O
the	O
direction	O
in	O
which	O
the	O
adaptive	O
immune	O
response	O
will	O
develop	O
.	O

The	O
creation	O
of	O
pathogen	O
-	O
specific	O
mediator	O
environments	O
by	O
dendritic	O
cells	O
defines	O
whether	O
a	O
cellular	O
or	O
humoral	O
response	O
will	O
be	O
activated	O
in	O
response	O
to	O
the	O
pathogen	O
.	O

Cloning	O
and	O
expression	O
of	O
the	O
human	O
N	B-Gene_or_gene_product
-	I-Gene_or_gene_product
acetylglutamate	I-Gene_or_gene_product
synthase	I-Gene_or_gene_product
gene	O
.	O

N	B-Gene_or_gene_product
-	I-Gene_or_gene_product
acetylglutamate	I-Gene_or_gene_product
synthase	I-Gene_or_gene_product
(	O
NAGS	B-Gene_or_gene_product
,	O
E	B-Gene_or_gene_product
.	I-Gene_or_gene_product
C	I-Gene_or_gene_product
.	I-Gene_or_gene_product
2	I-Gene_or_gene_product
.	I-Gene_or_gene_product
3	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
)	O
is	O
a	O
mitochondrial	B-Cellular_component
enzyme	O
catalyzing	O
the	O
formation	O
of	O
N	B-Simple_chemical
-	I-Simple_chemical
acetylglutamate	I-Simple_chemical
(	O
NAG	B-Simple_chemical
)	O
,	O
an	O
essential	O
allosteric	O
activator	O
of	O
carbamylphosphate	B-Gene_or_gene_product
synthase	I-Gene_or_gene_product
I	I-Gene_or_gene_product
(	O
CPSI	B-Gene_or_gene_product
)	O
,	O
the	O
first	O
enzyme	O
of	O
the	O
urea	B-Simple_chemical
cycle	O
.	O

Patients	O
with	O
NAGS	B-Gene_or_gene_product
deficiency	O
develop	O
hyperammonemia	O
because	O
CPSI	B-Gene_or_gene_product
is	O
inactive	O
without	O
NAG	B-Simple_chemical
.	O

The	O
human	O
NAGS	B-Gene_or_gene_product
cDNA	O
was	O
isolated	O
from	O
a	O
liver	O
library	O
based	O
on	O
its	O
similarity	O
to	O
mouse	O
NAGS	B-Gene_or_gene_product
.	O

The	O
deduced	O
amino	O
acid	O
sequence	O
contains	O
an	O
N	O
-	O
terminal	O
putative	O
mitochondrial	B-Cellular_component
targeting	O
signal	O
of	O
49	O
amino	O
acids	O
(	O
63	O
%	O
identity	O
with	O
mouse	O
NAGS	B-Gene_or_gene_product
)	O
followed	O
by	O
a	O
"	O
variable	O
domain	O
"	O
of	O
45	O
amino	O
acids	O
(	O
35	O
%	O
identity	O
)	O
and	O
a	O
"	O
conserved	O
domain	O
"	O
of	O
440	O
amino	O
acids	O
(	O
92	O
%	O
identity	O
)	O
.	O

A	O
cDNA	O
sequence	O
containing	O
the	O
"	O
conserved	O
domain	O
"	O
complements	O
an	O
NAGS	B-Gene_or_gene_product
-	O
deficient	O
Escherichia	O
coli	O
strain	O
and	O
the	O
recombinant	O
protein	O
has	O
arginine	B-Simple_chemical
-	O
responsive	O
NAGS	B-Gene_or_gene_product
catalytic	O
activity	O
.	O

The	O
NAGS	B-Gene_or_gene_product
gene	O
is	O
expressed	O
in	O
the	O
liver	O
and	O
small	O
intestine	O
;	O
the	O
intestinal	O
transcript	O
is	O
smaller	O
in	O
size	O
than	O
liver	O
transcript	O
.	O

Osmium	B-Simple_chemical
(	I-Simple_chemical
VI	I-Simple_chemical
)	I-Simple_chemical
complexes	I-Simple_chemical
of	O
the	O
3	B-Simple_chemical
'	I-Simple_chemical
,	I-Simple_chemical
5	I-Simple_chemical
'	I-Simple_chemical
-	I-Simple_chemical
dinucleoside	I-Simple_chemical
monophosphates	I-Simple_chemical
,	O
ApU	B-Simple_chemical
and	O
UpA	B-Simple_chemical
.	O

The	O
dinucleoside	B-Simple_chemical
monophosphates	I-Simple_chemical
,	O
ApU	B-Simple_chemical
and	O
UpA	B-Simple_chemical
,	O
react	O
with	O
potassium	B-Simple_chemical
osmate	I-Simple_chemical
(	I-Simple_chemical
VI	I-Simple_chemical
)	I-Simple_chemical
and	O
2	B-Simple_chemical
,	I-Simple_chemical
2	I-Simple_chemical
'	I-Simple_chemical
-	I-Simple_chemical
bipyridyl	I-Simple_chemical
to	O
form	O
the	O
corresponding	O
oxo	B-Simple_chemical
-	I-Simple_chemical
osmium	I-Simple_chemical
(	I-Simple_chemical
VI	I-Simple_chemical
)	I-Simple_chemical
bipyridyl	I-Simple_chemical
sugar	I-Simple_chemical
ester	I-Simple_chemical
in	O
which	O
the	O
osmate	B-Simple_chemical
group	O
is	O
bonded	O
to	O
the	O
terminal	O
2	B-Simple_chemical
'	I-Simple_chemical
,	I-Simple_chemical
3	I-Simple_chemical
'	I-Simple_chemical
-	I-Simple_chemical
glycol	I-Simple_chemical
.	O

Osmium	B-Simple_chemical
(	I-Simple_chemical
VIII	I-Simple_chemical
)	I-Simple_chemical
tetroxide	I-Simple_chemical
and	O
2	B-Simple_chemical
,	I-Simple_chemical
2	I-Simple_chemical
'	I-Simple_chemical
-	I-Simple_chemical
bipyridyl	I-Simple_chemical
react	O
with	O
the	O
dinucleosides	O
to	O
form	O
the	O
corresponding	O
oxo	B-Simple_chemical
-	I-Simple_chemical
osmium	I-Simple_chemical
(	I-Simple_chemical
VI	I-Simple_chemical
)	I-Simple_chemical
bipyridyl	I-Simple_chemical
heterocyclic	I-Simple_chemical
esters	I-Simple_chemical
which	O
result	O
from	O
addition	O
of	O
the	O
tetroxide	B-Simple_chemical
to	O
the	O
5	O
,	O
6	O
-	O
double	O
bond	O
of	O
the	O
uracil	B-Simple_chemical
residue	O
.	O

Although	O
capable	O
of	O
transesterification	O
reactions	O
,	O
these	O
heterocyclic	B-Simple_chemical
esters	I-Simple_chemical
are	O
exceptionally	O
stable	O
toward	O
exchange	O
reactions	O
in	O
solution	O
.	O

No	O
apparent	O
exchange	O
was	O
observed	O
after	O
1	O
month	O
.	O

This	O
reaction	O
thus	O
seems	O
promising	O
for	O
single	O
-	O
site	O
osmium	B-Simple_chemical
labeling	O
in	O
polynucleotides	O
.	O

Transcriptional	O
activation	O
of	O
the	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
p65	B-Gene_or_gene_product
subunit	I-Gene_or_gene_product
by	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
(	O
MSK1	B-Gene_or_gene_product
)	O
.	O

Nuclear	B-Complex
factor	I-Complex
kappaB	I-Complex
(	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
)	O
is	O
one	O
of	O
the	O
key	O
regulators	O
of	O
transcription	O
of	O
a	O
variety	O
of	O
genes	O
involved	O
in	O
immune	O
and	O
inflammatory	O
responses	O
.	O

NF	B-Complex
-	I-Complex
kappaB	I-Complex
activity	O
has	O
long	O
been	O
thought	O
to	O
be	O
regulated	O
mainly	O
by	O
IkappaB	B-Gene_or_gene_product
family	O
members	O
,	O
which	O
keep	O
the	O
transcription	O
factor	O
complex	O
in	O
an	O
inactive	O
form	O
in	O
the	O
cytoplasm	B-Cellular_component
by	O
masking	O
the	O
nuclear	B-Cellular_component
localization	O
signal	O
.	O

Nowadays	O
,	O
the	O
importance	O
of	O
additional	O
mechanisms	O
controlling	O
the	O
nuclear	B-Cellular_component
transcription	O
potential	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
is	O
generally	O
accepted	O
.	O

We	O
show	O
that	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
inhibitors	O
SB203580	B-Simple_chemical
and	O
PD98059	B-Simple_chemical
or	O
U0126	B-Simple_chemical
,	O
as	O
well	O
as	O
a	O
potent	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
(	O
MSK1	B-Gene_or_gene_product
)	O
inhibitor	O
H89	B-Simple_chemical
,	O
counteract	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
mediated	O
stimulation	O
of	O
p65	B-Gene_or_gene_product
transactivation	O
capacity	O
.	O

Mutational	O
analysis	O
of	O
p65	B-Gene_or_gene_product
revealed	O
Ser276	B-Simple_chemical
as	O
a	O
target	O
for	O
phosphorylation	O
and	O
transactivation	O
in	O
response	O
to	O
TNF	B-Gene_or_gene_product
.	O

Moreover	O
,	O
we	O
identified	O
MSK1	B-Gene_or_gene_product
as	O
a	O
nuclear	B-Cellular_component
kinase	O
for	O
p65	B-Gene_or_gene_product
,	O
since	O
MSK1	B-Gene_or_gene_product
associates	O
with	O
p65	B-Gene_or_gene_product
in	O
a	O
stimulus	O
-	O
dependent	O
way	O
and	O
phosphorylates	O
p65	B-Gene_or_gene_product
at	O
Ser276	B-Simple_chemical
.	O

This	O
effect	O
represents	O
,	O
together	O
with	O
phosphorylation	O
of	O
nucleosome	O
components	O
such	O
as	O
histone	B-Gene_or_gene_product
H3	I-Gene_or_gene_product
,	O
an	O
essential	O
step	O
leading	O
to	O
selective	O
transcriptional	O
activation	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
-	O
dependent	O
gene	O
expression	O
.	O

Human	O
claspin	B-Gene_or_gene_product
is	O
required	O
for	O
replication	O
checkpoint	O
control	O
.	O

Claspin	B-Gene_or_gene_product
is	O
a	O
newly	O
identified	O
protein	O
that	O
regulates	O
Chk1	B-Gene_or_gene_product
activation	O
in	O
Xenopus	O
.	O

In	O
the	O
present	O
study	O
we	O
investigated	O
the	O
role	O
of	O
human	O
Claspin	B-Gene_or_gene_product
in	O
the	O
DNA	O
damage	O
/	O
replication	O
checkpoint	O
in	O
mammalian	O
cells	O
.	O

We	O
observed	O
that	O
human	O
Claspin	B-Gene_or_gene_product
is	O
a	O
cell	O
cycle	O
regulated	O
protein	O
that	O
peaks	O
at	O
S	O
/	O
G2	O
phase	O
.	O

Claspin	B-Gene_or_gene_product
localizes	O
in	O
the	O
nuclei	B-Cellular_component
,	O
but	O
it	O
only	O
associates	O
with	O
Chk1	B-Gene_or_gene_product
following	O
replication	O
stress	O
or	O
other	O
types	O
of	O
DNA	O
damage	O
.	O

In	O
addition	O
,	O
Claspin	B-Gene_or_gene_product
is	O
phosphorylated	O
in	O
response	O
to	O
replication	O
stress	O
,	O
and	O
this	O
phosphorylation	O
appears	O
to	O
be	O
required	O
for	O
its	O
association	O
with	O
Chk1	B-Gene_or_gene_product
.	O

Moreover	O
,	O
Claspin	B-Gene_or_gene_product
interacts	O
with	O
the	O
checkpoint	O
proteins	O
ATR	B-Gene_or_gene_product
and	O
Rad9	B-Gene_or_gene_product
.	O

Given	O
that	O
both	O
the	O
ATR	B-Gene_or_gene_product
and	O
Rad9	B-Complex
-	I-Complex
Rad1	I-Complex
-	I-Complex
Hus1	I-Complex
complexes	O
are	O
involved	O
in	O
Chk1	B-Gene_or_gene_product
activation	O
,	O
it	O
is	O
possible	O
that	O
Claspin	B-Gene_or_gene_product
works	O
as	O
an	O
adaptor	O
molecule	O
bringing	O
these	O
molecules	O
together	O
.	O

Using	O
small	O
interfering	O
RNA	O
technology	O
,	O
we	O
have	O
shown	O
that	O
down	O
-	O
regulation	O
of	O
Claspin	B-Gene_or_gene_product
expression	O
inhibits	O
Chk1	B-Gene_or_gene_product
activation	O
in	O
response	O
to	O
replication	O
stress	O
.	O

More	O
importantly	O
,	O
down	O
-	O
regulation	O
of	O
Claspin	B-Gene_or_gene_product
augments	O
the	O
premature	O
chromatin	B-Cellular_component
condensation	O
induced	O
by	O
hydroxyurea	B-Simple_chemical
,	O
inhibits	O
the	O
UV	O
-	O
induced	O
reduction	O
of	O
DNA	O
synthesis	O
,	O
and	O
decreases	O
cell	O
survival	O
.	O

Taken	O
together	O
,	O
these	O
data	O
imply	O
a	O
potentially	O
critical	O
role	O
for	O
Claspin	B-Gene_or_gene_product
in	O
replication	O
checkpoint	O
control	O
in	O
mammalian	O
cells	O
.	O

Cbl	B-Gene_or_gene_product
-	I-Gene_or_gene_product
b	I-Gene_or_gene_product
negatively	O
regulates	O
B	B-Gene_or_gene_product
cell	I-Gene_or_gene_product
antigen	I-Gene_or_gene_product
receptor	I-Gene_or_gene_product
signaling	O
in	O
mature	O
B	O
cells	O
through	O
ubiquitination	O
of	O
the	O
tyrosine	B-Simple_chemical
kinase	O
Syk	B-Gene_or_gene_product
.	O

Members	O
of	O
the	O
Cbl	B-Gene_or_gene_product
family	O
of	O
molecular	O
adaptors	O
play	O
key	O
roles	O
in	O
regulating	O
tyrosine	B-Simple_chemical
kinase	O
-	O
dependent	O
signaling	O
in	O
a	O
variety	O
of	O
cellular	O
systems	O
.	O

Here	O
we	O
provide	O
evidence	O
that	O
in	O
B	O
cells	O
Cbl	B-Gene_or_gene_product
-	I-Gene_or_gene_product
b	I-Gene_or_gene_product
functions	O
as	O
a	O
negative	O
regulator	O
of	O
B	B-Gene_or_gene_product
cell	I-Gene_or_gene_product
antigen	I-Gene_or_gene_product
receptor	I-Gene_or_gene_product
(	O
BCR	B-Gene_or_gene_product
)	O
signaling	O
during	O
the	O
normal	O
course	O
of	O
a	O
response	O
.	O

In	O
B	O
cells	O
from	O
Cbl	B-Gene_or_gene_product
-	O
b	O
-	O
deficient	O
mice	O
cross	O
-	O
linking	O
the	O
BCRs	B-Gene_or_gene_product
resulted	O
in	O
sustained	O
phosphorylation	O
of	O
Igalpha	B-Gene_or_gene_product
,	O
Syk	B-Gene_or_gene_product
,	O
and	O
phospholipase	B-Gene_or_gene_product
C	I-Gene_or_gene_product
(	I-Gene_or_gene_product
PLC	I-Gene_or_gene_product
)	I-Gene_or_gene_product
-	I-Gene_or_gene_product
gamma2	I-Gene_or_gene_product
,	O
leading	O
to	O
prolonged	O
Ca2	B-Simple_chemical
+	I-Simple_chemical
mobilization	O
,	O
and	O
increases	O
in	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
and	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
protein	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
(	O
JNK	B-Gene_or_gene_product
)	O
phosphorylation	O
and	O
surface	B-Cellular_component
expression	O
of	O
the	O
activation	O
marker	O
,	O
CD69	B-Gene_or_gene_product
.	O

Image	O
analysis	O
following	O
BCR	B-Gene_or_gene_product
cross	O
-	O
linking	O
showed	O
sustained	O
polarization	O
of	O
the	O
BCRs	B-Gene_or_gene_product
into	O
large	O
signaling	O
-	O
active	O
caps	O
associated	O
with	O
phosphorylated	O
Syk	B-Gene_or_gene_product
in	O
Cbl	B-Gene_or_gene_product
-	I-Gene_or_gene_product
b	I-Gene_or_gene_product
-	O
deficient	O
B	O
cells	O
in	O
contrast	O
to	O
the	O
BCRs	B-Gene_or_gene_product
in	O
Cbl	B-Gene_or_gene_product
-	I-Gene_or_gene_product
b	I-Gene_or_gene_product
-	O
expressing	O
B	O
cells	O
that	O
rapidly	O
proceeded	O
to	O
form	O
small	O
,	O
condensed	O
,	O
signaling	O
inactive	O
caps	O
.	O

Significantly	O
,	O
prolonged	O
phosphorylation	O
of	O
Syk	B-Gene_or_gene_product
correlated	O
with	O
reduced	O
ubiquitination	O
of	O
Syk	B-Gene_or_gene_product
indicating	O
that	O
Cbl	B-Gene_or_gene_product
-	I-Gene_or_gene_product
b	I-Gene_or_gene_product
negatively	O
regulates	O
BCR	B-Gene_or_gene_product
signaling	O
by	O
targeting	O
Syk	B-Gene_or_gene_product
for	O
ubiquitination	O
.	O

Proteolytic	O
signaling	O
by	O
TNFalpha	B-Gene_or_gene_product
:	O
caspase	B-Gene_or_gene_product
activation	O
and	O
IkappaB	B-Gene_or_gene_product
degradation	O
.	O

Following	O
binding	O
its	O
death	B-Gene_or_gene_product
receptor	I-Gene_or_gene_product
on	O
the	O
plasma	B-Cellular_component
membrane	I-Cellular_component
,	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
induces	O
the	O
receptor	O
trimerization	O
and	O
recruits	O
a	O
number	O
of	O
death	O
domain	O
-	O
containing	O
molecules	O
to	O
form	O
the	O
receptor	O
complex	O
.	O

The	O
complex	O
promotes	O
activation	O
of	O
downstream	O
caspase	B-Gene_or_gene_product
cascade	O
and	O
induces	O
degradation	O
of	O
IkappaBalpha	B-Gene_or_gene_product
.	O

Caspases	B-Gene_or_gene_product
are	O
activated	O
using	O
mechanisms	O
of	O
oligomeration	O
and	O
'	O
self	O
-	O
controlled	O
proteolysis	O
'	O
.	O

According	O
to	O
their	O
structures	O
and	O
functions	O
,	O
apoptosis	O
related	O
caspases	B-Gene_or_gene_product
can	O
be	O
divided	O
into	O
upstream	O
and	O
downstream	O
caspases	B-Gene_or_gene_product
.	O

In	O
general	O
,	O
upstream	O
caspases	B-Gene_or_gene_product
cleave	O
and	O
activate	O
downstream	O
caspases	B-Gene_or_gene_product
by	O
proteolysis	O
of	O
the	O
Asp	O
-	O
X	O
site	O
.	O

Activated	O
caspases	B-Gene_or_gene_product
then	O
cleaved	O
target	O
substrates	O
.	O

To	O
date	O
,	O
more	O
than	O
70	O
proteins	O
have	O
been	O
identified	O
to	O
be	O
substrates	O
of	O
caspases	B-Gene_or_gene_product
in	O
mammalian	O
cells	O
.	O

Caspases	B-Gene_or_gene_product
can	O
alter	O
the	O
function	O
of	O
their	O
target	O
proteins	O
by	O
destroying	O
structural	O
components	O
of	O
the	O
cytoskeleton	B-Cellular_component
and	O
nuclear	B-Cellular_component
scaffold	I-Cellular_component
or	O
by	O
removing	O
their	O
regulatory	O
domains	O
.	O

Activation	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
is	O
dependent	O
on	O
the	O
degradation	O
of	O
IkappaBalpha	B-Gene_or_gene_product
.	O

IkappaB	B-Complex
kinase	I-Complex
(	O
IKK	B-Complex
)	O
phosphorylates	O
IkappaBalpha	B-Gene_or_gene_product
at	O
the	O
residues	B-Simple_chemical
32	I-Simple_chemical
and	O
36	B-Simple_chemical
followed	O
by	O
polyubiquitination	O
at	O
lysine	B-Simple_chemical
21	I-Simple_chemical
and	O
22	B-Simple_chemical
and	O
subsequent	O
degradation	O
of	O
the	O
molecules	O
by	O
26S	B-Cellular_component
proteasome	I-Cellular_component
.	O

There	O
is	O
extensive	O
crosstalk	O
between	O
the	O
apoptotic	O
and	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
signaling	O
pathways	O
that	O
emanate	O
from	O
TNF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
R1	I-Gene_or_gene_product
.	O

On	O
the	O
one	O
hand	O
,	O
activation	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
can	O
inactivate	O
caspases	B-Gene_or_gene_product
;	O
on	O
the	O
other	O
hand	O
,	O
activated	O
caspases	B-Gene_or_gene_product
can	O
inhibit	O
the	O
activation	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
.	O

Both	O
processes	O
involve	O
in	O
proteolysis	O
.	O

This	O
crosstalk	O
may	O
be	O
important	O
for	O
maintaining	O
the	O
balance	O
between	O
the	O
two	O
pathways	O
and	O
for	O
determining	O
whether	O
a	O
cell	O
should	O
live	O
or	O
die	O
.	O

The	O
roles	O
of	O
APC	B-Gene_or_gene_product
and	O
Axin	B-Gene_or_gene_product
derived	O
from	O
experimental	O
and	O
theoretical	O
analysis	O
of	O
the	O
Wnt	B-Gene_or_gene_product
pathway	O
.	O

Wnt	B-Gene_or_gene_product
signaling	O
plays	O
an	O
important	O
role	O
in	O
both	O
oncogenesis	O
and	O
development	O
.	O

Activation	O
of	O
the	O
Wnt	B-Gene_or_gene_product
pathway	O
results	O
in	O
stabilization	O
of	O
the	O
transcriptional	O
coactivator	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
.	O

Recent	O
studies	O
have	O
demonstrated	O
that	O
axin	B-Gene_or_gene_product
,	O
which	O
coordinates	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
degradation	O
,	O
is	O
itself	O
degraded	O
.	O

Although	O
the	O
key	O
molecules	O
required	O
for	O
transducing	O
a	O
Wnt	B-Gene_or_gene_product
signal	O
have	O
been	O
identified	O
,	O
a	O
quantitative	O
understanding	O
of	O
this	O
pathway	O
has	O
been	O
lacking	O
.	O

We	O
have	O
developed	O
a	O
mathematical	O
model	O
for	O
the	O
canonical	O
Wnt	B-Gene_or_gene_product
pathway	O
that	O
describes	O
the	O
interactions	O
among	O
the	O
core	O
components	O
:	O
Wnt	B-Gene_or_gene_product
,	O
Frizzled	B-Gene_or_gene_product
,	O
Dishevelled	B-Gene_or_gene_product
,	O
GSK3beta	B-Gene_or_gene_product
,	O
APC	B-Gene_or_gene_product
,	O
axin	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
TCF	B-Gene_or_gene_product
.	O

Using	O
a	O
system	O
of	O
differential	O
equations	O
,	O
the	O
model	O
incorporates	O
the	O
kinetics	O
of	O
protein	O
-	O
protein	O
interactions	O
,	O
protein	O
synthesis	O
/	O
degradation	O
,	O
and	O
phosphorylation	O
/	O
dephosphorylation	O
.	O

We	O
initially	O
defined	O
a	O
reference	O
state	O
of	O
kinetic	O
,	O
thermodynamic	O
,	O
and	O
flux	O
data	O
from	O
experiments	O
using	O
Xenopus	O
extracts	O
.	O

Predictions	O
based	O
on	O
the	O
analysis	O
of	O
the	O
reference	O
state	O
were	O
used	O
iteratively	O
to	O
develop	O
a	O
more	O
refined	O
model	O
from	O
which	O
we	O
analyzed	O
the	O
effects	O
of	O
prolonged	O
and	O
transient	O
Wnt	B-Gene_or_gene_product
stimulation	O
on	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
and	O
axin	B-Gene_or_gene_product
turnover	O
.	O

We	O
predict	O
several	O
unusual	O
features	O
of	O
the	O
Wnt	B-Gene_or_gene_product
pathway	O
,	O
some	O
of	O
which	O
we	O
tested	O
experimentally	O
.	O

An	O
insight	O
from	O
our	O
model	O
,	O
which	O
we	O
confirmed	O
experimentally	O
,	O
is	O
that	O
the	O
two	O
scaffold	O
proteins	O
axin	B-Gene_or_gene_product
and	O
APC	B-Gene_or_gene_product
promote	O
the	O
formation	O
of	O
degradation	O
complexes	O
in	O
very	O
different	O
ways	O
.	O

We	O
can	O
also	O
explain	O
the	O
importance	O
of	O
axin	B-Gene_or_gene_product
degradation	O
in	O
amplifying	O
and	O
sharpening	O
the	O
Wnt	B-Gene_or_gene_product
signal	O
,	O
and	O
we	O
show	O
that	O
the	O
dependence	O
of	O
axin	B-Gene_or_gene_product
degradation	O
on	O
APC	B-Gene_or_gene_product
is	O
an	O
essential	O
part	O
of	O
an	O
unappreciated	O
regulatory	O
loop	O
that	O
prevents	O
the	O
accumulation	O
of	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
at	O
decreased	O
APC	B-Gene_or_gene_product
concentrations	O
.	O

By	O
applying	O
control	O
analysis	O
to	O
our	O
mathematical	O
model	O
,	O
we	O
demonstrate	O
the	O
modular	O
design	O
,	O
sensitivity	O
,	O
and	O
robustness	O
of	O
the	O
Wnt	B-Gene_or_gene_product
pathway	O
and	O
derive	O
an	O
explicit	O
expression	O
for	O
tumor	O
suppression	O
and	O
oncogenicity	O
.	O

Characterizing	O
the	O
role	O
of	O
MDM2	B-Gene_or_gene_product
in	O
diethylnitrosamine	B-Simple_chemical
induced	O
acute	O
liver	O
damage	O
and	O
development	O
of	O
pre	O
-	O
neoplastic	O
lesions	O
.	O

Pre	O
-	O
neoplastic	O
lesions	O
in	O
rodent	O
liver	O
often	O
express	O
high	O
levels	O
of	O
MDM2	B-Gene_or_gene_product
and	O
lack	O
a	O
p53	B-Gene_or_gene_product
response	O
to	O
DNA	O
damage	O
.	O

The	O
question	O
we	O
posed	O
was	O
whether	O
there	O
is	O
a	O
liver	O
-	O
specific	O
regulation	O
of	O
the	O
p53	B-Gene_or_gene_product
/	O
MDM2	B-Gene_or_gene_product
feedback	O
loop	O
and	O
if	O
it	O
can	O
be	O
related	O
to	O
the	O
development	O
of	O
pre	O
-	O
neoplastic	O
lesions	O
,	O
referred	O
to	O
as	O
enzyme	O
altered	O
foci	O
(	O
EAF	O
)	O
in	O
rats	O
.	O

Acute	O
responses	O
of	O
p53	B-Gene_or_gene_product
and	O
MDM2	B-Gene_or_gene_product
to	O
diethylnitrosamine	B-Simple_chemical
(	O
DEN	B-Simple_chemical
)	O
were	O
characterized	O
by	O
employing	O
immunohistochemistry	O
,	O
western	O
blotting	O
,	O
RT	O
-	O
PCR	O
and	O
in	O
situ	O
hybridization	O
.	O

A	O
single	O
dose	O
of	O
DEN	B-Simple_chemical
induced	O
a	O
centrilobular	O
p53	B-Gene_or_gene_product
response	O
that	O
peaked	O
at	O
24	O
h	O
.	O

It	O
was	O
associated	O
with	O
transcriptional	O
activation	O
of	O
MDM2	B-Gene_or_gene_product
and	O
signs	O
of	O
apoptosis	O
.	O

However	O
,	O
in	O
midzonal	O
hepatocytes	O
,	O
which	O
constitutively	O
expressed	O
high	O
levels	O
of	O
cytoplasmic	B-Cellular_component
MDM2	B-Gene_or_gene_product
,	O
there	O
was	O
a	O
rapid	O
-	O
onset	O
but	O
transient	O
p53	B-Gene_or_gene_product
response	O
.	O

It	O
was	O
terminated	O
at	O
24	O
h	O
and	O
there	O
were	O
no	O
signs	O
of	O
apoptosis	O
.	O

The	O
rapidly	O
declining	O
p53	B-Gene_or_gene_product
levels	O
in	O
midzonal	O
areas	O
was	O
preceded	O
by	O
a	O
transient	O
peak	O
in	O
MDM2	B-Gene_or_gene_product
mRNA	O
levels	O
at	O
6	O
h	O
.	O

Rats	O
pre	O
-	O
treated	O
with	O
repeated	O
low	O
or	O
high	O
weekly	O
doses	O
of	O
DEN	B-Simple_chemical
exhibited	O
EAF	O
and	O
these	O
lesions	O
expressed	O
high	O
levels	O
of	O
cytoplasmic	B-Cellular_component
MDM2	B-Gene_or_gene_product
.	O

Using	O
MDM2	B-Gene_or_gene_product
as	O
a	O
marker	O
for	O
EAF	O
gave	O
similar	O
results	O
as	O
using	O
glutathione	B-Gene_or_gene_product
transferase	I-Gene_or_gene_product
-	I-Gene_or_gene_product
P	I-Gene_or_gene_product
(	O
GST	B-Gene_or_gene_product
-	I-Gene_or_gene_product
P	I-Gene_or_gene_product
)	O
as	O
a	O
marker	O
.	O

Furthermore	O
,	O
small	O
EAF	O
,	O
elicited	O
by	O
low	O
doses	O
of	O
DEN	B-Simple_chemical
,	O
were	O
preferentially	O
localized	O
to	O
midzonal	O
areas	O
.	O

It	O
is	O
concluded	O
that	O
in	O
centrilobular	O
areas	O
DEN	B-Simple_chemical
-	O
induced	O
alterations	O
in	O
p53	B-Gene_or_gene_product
/	O
MDM2	B-Gene_or_gene_product
levels	O
are	O
compatible	O
with	O
a	O
previously	O
described	O
feedback	O
loop	O
.	O

An	O
attenuated	O
p53	B-Gene_or_gene_product
response	O
in	O
midzonal	O
hepatocytes	O
can	O
be	O
related	O
to	O
a	O
high	O
constitutive	O
expression	O
of	O
MDM2	B-Gene_or_gene_product
in	O
these	O
cells	O
.	O

The	O
localization	O
of	O
small	O
EAF	O
to	O
midzonal	O
areas	O
,	O
and	O
the	O
fact	O
that	O
EAF	O
cells	O
expressed	O
high	O
levels	O
of	O
MDM2	B-Gene_or_gene_product
,	O
indicates	O
that	O
MDM2	B-Gene_or_gene_product
expression	O
is	O
a	O
factor	O
governing	O
initiation	O
and	O
early	O
development	O
of	O
EAF	O
.	O

The	O
data	O
support	O
the	O
hypothesis	O
that	O
EAF	O
hepatocytes	O
are	O
initiated	O
via	O
epigenetic	O
mechanisms	O
.	O

Reciprocal	O
regulation	O
between	O
Slt2	B-Gene_or_gene_product
MAPK	B-Gene_or_gene_product
and	O
isoforms	O
of	O
Msg5	B-Gene_or_gene_product
dual	B-Gene_or_gene_product
-	I-Gene_or_gene_product
specificity	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
phosphatase	I-Gene_or_gene_product
modulates	O
the	O
yeast	O
cell	O
integrity	O
pathway	O
.	O

Dual	B-Gene_or_gene_product
-	I-Gene_or_gene_product
specificity	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
phosphatases	I-Gene_or_gene_product
(	O
DSPs	B-Gene_or_gene_product
)	O
are	O
involved	O
in	O
the	O
negative	O
regulation	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
kinases	I-Gene_or_gene_product
(	O
MAPKs	B-Gene_or_gene_product
)	O
by	O
dephosphorylating	O
both	O
threonine	B-Simple_chemical
-	O
and	O
tyrosine	B-Simple_chemical
-	O
conserved	O
residues	O
located	O
at	O
the	O
activation	O
loop	O
.	O

Here	O
we	O
show	O
that	O
Msg5	B-Gene_or_gene_product
DSP	B-Gene_or_gene_product
activity	O
is	O
essential	O
for	O
maintaining	O
a	O
low	O
level	O
of	O
signaling	O
through	O
the	O
cell	O
integrity	O
pathway	O
in	O
Saccharomyces	O
cerevisiae	O
.	O

Consistent	O
with	O
a	O
role	O
of	O
this	O
phosphatase	O
on	O
cell	B-Cellular_component
wall	I-Cellular_component
physiology	O
,	O
cells	O
lacking	O
Msg5	B-Gene_or_gene_product
displayed	O
an	O
increased	O
sensitivity	O
to	O
the	O
cell	B-Cellular_component
wall	I-Cellular_component
-	O
interfering	O
compound	O
Congo	B-Simple_chemical
Red	I-Simple_chemical
.	O

We	O
have	O
observed	O
that	O
the	O
N	O
-	O
terminal	O
non	O
-	O
catalytic	O
region	O
of	O
this	O
phosphatase	O
was	O
responsible	O
for	O
binding	O
to	O
the	O
kinase	O
domain	O
of	O
Slt2	B-Gene_or_gene_product
,	O
the	O
MAPK	B-Gene_or_gene_product
that	O
operates	O
in	O
this	O
pathway	O
.	O

In	O
vivo	O
and	O
in	O
vitro	O
experiments	O
revealed	O
that	O
both	O
proteins	O
act	O
on	O
each	O
other	O
.	O

Msg5	B-Gene_or_gene_product
bound	O
and	O
dephosphorylated	O
activated	O
Slt2	B-Gene_or_gene_product
.	O

Reciprocally	O
,	O
Slt2	B-Gene_or_gene_product
phosphorylated	O
Msg5	B-Gene_or_gene_product
as	O
a	O
consequence	O
of	O
the	O
activation	O
of	O
the	O
cell	O
integrity	O
pathway	O
.	O

In	O
addition	O
,	O
alternative	O
use	O
of	O
translation	O
initiation	O
sites	O
at	O
MSG5	B-Gene_or_gene_product
resulted	O
in	O
two	O
protein	O
forms	O
that	O
are	O
functional	O
on	O
Slt2	B-Gene_or_gene_product
and	O
became	O
equally	O
phosphorylated	O
following	O
activation	O
of	O
this	O
MAPK	B-Gene_or_gene_product
.	O

Under	O
activating	O
conditions	O
,	O
a	O
decrease	O
in	O
the	O
affinity	O
between	O
Msg5	B-Gene_or_gene_product
and	O
Slt2	B-Gene_or_gene_product
was	O
observed	O
,	O
leading	O
us	O
to	O
suggest	O
that	O
the	O
mechanism	O
by	O
which	O
Slt2	B-Gene_or_gene_product
controls	O
the	O
action	O
of	O
Msg5	B-Gene_or_gene_product
was	O
via	O
the	O
modulation	O
of	O
protein	O
-	O
protein	O
interactions	O
.	O

Our	O
results	O
indicate	O
the	O
existence	O
of	O
posttranscriptional	O
mechanisms	O
of	O
regulation	O
of	O
DSPs	B-Gene_or_gene_product
in	O
yeast	O
and	O
provide	O
new	O
insights	O
into	O
the	O
negative	O
control	O
of	O
the	O
cell	O
integrity	O
pathway	O
.	O

Role	O
of	O
vav1	B-Gene_or_gene_product
-	O
and	O
src	B-Gene_or_gene_product
-	O
related	O
tyrosine	B-Simple_chemical
kinases	O
in	O
macrophage	O
activation	O
by	O
CpG	O
DNA	O
.	O

Macrophage	O
activation	O
by	O
CpG	O
DNA	O
requires	O
toll	B-Gene_or_gene_product
-	I-Gene_or_gene_product
like	I-Gene_or_gene_product
receptor	I-Gene_or_gene_product
9	I-Gene_or_gene_product
and	O
the	O
adaptor	O
protein	O
MyD88	B-Gene_or_gene_product
.	O

Gram	O
-	O
negative	O
bacterial	O
lipopolysaccharide	B-Simple_chemical
also	O
activates	O
macrophages	O
via	O
a	O
toll	B-Gene_or_gene_product
-	I-Gene_or_gene_product
like	I-Gene_or_gene_product
receptor	I-Gene_or_gene_product
pathway	O
(	O
TLR	B-Gene_or_gene_product
-	I-Gene_or_gene_product
4	I-Gene_or_gene_product
)	O
,	O
but	O
we	O
and	O
others	O
have	O
reported	O
that	O
lipopolysaccharide	B-Simple_chemical
also	O
stimulates	O
tyrosine	B-Simple_chemical
phosphorylation	O
in	O
macrophages	O
.	O

Herein	O
we	O
report	O
that	O
exposure	O
of	O
RAW	O
264	O
.	O
7	O
murine	O
macrophages	O
to	O
CpG	O
DNA	O
(	O
but	O
not	O
non	O
-	O
CpG	O
DNA	O
)	O
provoked	O
the	O
rapid	O
tyrosine	B-Simple_chemical
phosphorylation	O
of	O
vav1	B-Gene_or_gene_product
.	O

PP1	B-Gene_or_gene_product
,	O
a	O
selective	O
inhibitor	O
of	O
src	B-Gene_or_gene_product
-	O
related	O
tyrosine	B-Simple_chemical
kinases	O
,	O
blocked	O
both	O
the	O
CpG	O
DNA	O
-	O
mediated	O
tyrosine	B-Simple_chemical
phosphorylation	O
of	O
vav1	B-Gene_or_gene_product
and	O
the	O
CpG	O
DNA	O
-	O
mediated	O
up	O
-	O
regulation	O
of	O
macrophage	O
tumor	B-Gene_or_gene_product
necrosis	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
secretion	O
and	O
inducible	O
nitric	B-Gene_or_gene_product
-	I-Gene_or_gene_product
oxide	I-Gene_or_gene_product
synthase	I-Gene_or_gene_product
protein	O
accumulation	O
.	O

Furthermore	O
,	O
we	O
found	O
that	O
the	O
inducible	O
expression	O
of	O
any	O
of	O
three	O
dominant	O
interfering	O
mutants	O
of	O
vav1	B-Gene_or_gene_product
(	O
a	O
truncated	O
protein	O
,	O
vavC	B-Gene_or_gene_product
;	O
a	O
form	O
containing	O
a	O
point	O
mutation	O
in	O
the	O
regulatory	O
tyrosine	B-Simple_chemical
residue	O
,	O
vavYF174	B-Gene_or_gene_product
;	O
and	O
a	O
form	O
with	O
an	O
in	O
-	O
frame	O
deletion	O
of	O
six	O
amino	O
acids	O
required	O
for	O
the	O
guanidine	B-Gene_or_gene_product
nucleotide	I-Gene_or_gene_product
exchange	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
(	O
GEF	B-Gene_or_gene_product
)	O
activity	O
of	O
vav1	B-Gene_or_gene_product
for	O
rac	B-Gene_or_gene_product
family	O
GTPases	O
,	O
vavGEFmt	B-Gene_or_gene_product
)	O
consistently	O
inhibited	O
CpG	O
DNA	O
-	O
mediated	O
up	O
-	O
regulation	O
of	O
tumor	B-Gene_or_gene_product
necrosis	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
secretion	O
and	O
inducible	O
nitric	B-Gene_or_gene_product
-	I-Gene_or_gene_product
oxide	I-Gene_or_gene_product
synthase	I-Gene_or_gene_product
protein	O
accumulation	O
in	O
RAW	O
-	O
TT10	O
macrophages	O
.	O

Finally	O
,	O
we	O
determined	O
that	O
CpG	O
DNA	O
-	O
mediated	O
up	O
-	O
regulation	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
activity	O
(	O
but	O
not	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
activation	O
)	O
was	O
inhibited	O
by	O
preincubation	O
with	O
PP1	B-Gene_or_gene_product
or	O
by	O
expression	O
of	O
the	O
truncated	O
vavC	B-Gene_or_gene_product
mutant	O
.	O

Taken	O
together	O
,	O
our	O
results	O
indicate	O
that	O
the	O
tyrosine	B-Simple_chemical
phosphorylation	O
of	O
vav1	B-Gene_or_gene_product
by	O
a	O
src	B-Gene_or_gene_product
-	O
related	O
tyrosine	B-Simple_chemical
kinase	O
or	O
kinases	O
plays	O
an	O
important	O
role	O
in	O
the	O
macrophage	O
response	O
to	O
CpG	O
DNA	O
.	O

E2F	B-Gene_or_gene_product
-	O
dependent	O
histone	B-Gene_or_gene_product
acetylation	O
and	O
recruitment	O
of	O
the	O
Tip60	B-Complex
acetyltransferase	I-Complex
complex	I-Complex
to	O
chromatin	B-Cellular_component
in	O
late	O
G1	O
.	O

E2F	B-Gene_or_gene_product
proteins	O
can	O
either	O
activate	O
or	O
repress	O
transcription	O
.	O

Following	O
mitogenic	O
stimulation	O
,	O
repressive	O
E2F4	B-Complex
-	I-Complex
p130	I-Complex
-	I-Complex
histone	I-Complex
deacetylase	I-Complex
complexes	I-Complex
dissociate	O
from	O
,	O
while	O
activating	O
species	O
(	O
E2F1	B-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
associate	O
with	O
,	O
target	O
promoters	O
.	O

Histones	B-Gene_or_gene_product
H3	I-Gene_or_gene_product
and	O
H4	B-Gene_or_gene_product
simultaneously	O
become	O
hyperacetylated	O
,	O
but	O
it	O
remains	O
unclear	O
whether	O
this	O
is	O
a	O
prerequisite	O
or	O
a	O
consequence	O
of	O
E2F	B-Gene_or_gene_product
binding	O
.	O

Here	O
,	O
we	O
show	O
that	O
activating	O
E2F	B-Gene_or_gene_product
species	O
are	O
required	O
for	O
hyperacetylation	O
of	O
target	O
chromatin	B-Cellular_component
in	O
human	O
cells	O
.	O

Overexpression	O
of	O
a	O
dominant	O
-	O
negative	O
(	O
DN	O
)	O
E2F1	B-Gene_or_gene_product
mutant	O
in	O
serum	O
-	O
stimulated	O
T98G	O
cells	O
blocked	O
all	O
E2F	B-Gene_or_gene_product
binding	O
,	O
H4	B-Gene_or_gene_product
acetylation	O
,	O
and	O
,	O
albeit	O
partially	O
,	O
H3	B-Gene_or_gene_product
acetylation	O
.	O

Target	O
gene	O
activation	O
and	O
S	O
-	O
phase	O
entry	O
were	O
also	O
blocked	O
by	O
DN	O
E2F1	B-Gene_or_gene_product
.	O

Conversely	O
,	O
ectopic	O
activation	O
of	O
E2F1	B-Gene_or_gene_product
rapidly	O
induced	O
H3	B-Gene_or_gene_product
and	O
H4	B-Gene_or_gene_product
acetylation	O
,	O
demonstrating	O
a	O
direct	O
role	O
for	O
E2F	B-Gene_or_gene_product
in	O
these	O
events	O
.	O

E2F1	B-Gene_or_gene_product
was	O
previously	O
shown	O
to	O
bind	O
the	O
histone	B-Gene_or_gene_product
acetyltransferases	I-Gene_or_gene_product
(	O
HATs	B-Gene_or_gene_product
)	O
p300	B-Gene_or_gene_product
/	O
CBP	B-Gene_or_gene_product
and	O
PCAF	B-Gene_or_gene_product
/	O
GCN5	B-Gene_or_gene_product
.	O

In	O
our	O
hands	O
,	O
ectopically	O
expressed	O
E2F1	B-Gene_or_gene_product
also	O
bound	O
the	O
unrelated	O
HAT	B-Gene_or_gene_product
Tip60	B-Gene_or_gene_product
and	O
induced	O
recruitment	O
of	O
five	O
subunits	O
of	O
the	O
Tip60	B-Complex
complex	I-Complex
(	O
Tip60	B-Gene_or_gene_product
,	O
TRRAP	B-Gene_or_gene_product
,	O
p400	B-Gene_or_gene_product
,	O
Tip48	B-Gene_or_gene_product
,	O
and	O
Tip49	B-Gene_or_gene_product
)	O
to	O
target	O
promoters	O
in	O
vivo	O
.	O

Moreover	O
,	O
E2F	B-Gene_or_gene_product
-	O
dependent	O
recruitment	O
of	O
Tip60	B-Gene_or_gene_product
to	O
chromatin	B-Cellular_component
occurred	O
in	O
late	O
G	O
(	O
1	O
)	O
following	O
serum	O
stimulation	O
.	O

We	O
speculate	O
that	O
the	O
activities	O
of	O
multiple	O
HAT	B-Complex
complexes	I-Complex
account	O
for	O
E2F	B-Gene_or_gene_product
-	O
dependent	O
acetylation	O
,	O
transcription	O
,	O
and	O
S	O
-	O
phase	O
entry	O
.	O

E2F1	B-Gene_or_gene_product
uses	O
the	O
ATM	B-Gene_or_gene_product
signaling	O
pathway	O
to	O
induce	O
p53	B-Gene_or_gene_product
and	O
Chk2	B-Gene_or_gene_product
phosphorylation	O
and	O
apoptosis	O
.	O

The	O
p53	B-Gene_or_gene_product
tumor	O
suppressor	O
protein	O
is	O
phosphorylated	O
and	O
activated	O
by	O
several	O
DNA	O
damage	O
-	O
inducible	O
kinases	O
,	O
such	O
as	O
ATM	B-Gene_or_gene_product
,	O
and	O
is	O
a	O
key	O
effector	O
of	O
the	O
DNA	O
damage	O
response	O
by	O
promoting	O
cell	O
cycle	O
arrest	O
or	O
apoptosis	O
.	O

Deregulation	O
of	O
the	O
Rb	B-Gene_or_gene_product
-	O
E2F1	B-Gene_or_gene_product
pathway	O
also	O
results	O
in	O
the	O
activation	O
of	O
p53	B-Gene_or_gene_product
and	O
the	O
promotion	O
of	O
apoptosis	O
,	O
and	O
this	O
contributes	O
to	O
the	O
suppression	O
of	O
tumor	O
development	O
.	O

Here	O
,	O
we	O
describe	O
a	O
novel	O
connection	O
between	O
E2F1	B-Gene_or_gene_product
and	O
the	O
ATM	B-Gene_or_gene_product
DNA	O
damage	O
response	O
pathway	O
.	O

In	O
primary	O
human	O
fibroblasts	O
lacking	O
functional	O
ATM	B-Gene_or_gene_product
,	O
the	O
ability	O
of	O
E2F1	B-Gene_or_gene_product
to	O
induce	O
the	O
phosphorylation	O
of	O
p53	B-Gene_or_gene_product
and	O
apoptosis	O
is	O
impaired	O
.	O

In	O
contrast	O
,	O
ATM	B-Gene_or_gene_product
status	O
has	O
no	O
effect	O
on	O
transcriptional	O
activation	O
of	O
target	O
genes	O
or	O
the	O
stimulation	O
of	O
DNA	O
synthesis	O
by	O
E2F1	B-Gene_or_gene_product
.	O

Cells	O
containing	O
mutant	O
Nijmegen	B-Gene_or_gene_product
breakage	I-Gene_or_gene_product
syndrome	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
(	O
NBS1	B-Gene_or_gene_product
)	O
,	O
a	O
component	O
of	O
the	O
Mre11	B-Complex
-	I-Complex
Rad50	I-Complex
DNA	O
repair	O
complex	O
,	O
also	O
have	O
attenuated	O
p53	B-Gene_or_gene_product
phosphorylation	O
and	O
apoptosis	O
in	O
response	O
to	O
E2F1	B-Gene_or_gene_product
expression	O
.	O

Moreover	O
,	O
E2F1	B-Gene_or_gene_product
induces	O
ATM	B-Gene_or_gene_product
-	O
and	O
NBS1	B-Gene_or_gene_product
-	O
dependent	O
phosphorylation	O
of	O
the	O
checkpoint	O
kinase	O
Chk2	B-Gene_or_gene_product
at	O
Thr68	B-Simple_chemical
,	O
a	O
phosphorylation	O
site	O
that	O
stimulates	O
Chk2	B-Gene_or_gene_product
activity	O
.	O

Delayed	O
gammaH2AX	B-Gene_or_gene_product
phosphorylation	O
and	O
absence	O
of	O
ATM	B-Gene_or_gene_product
autophosphorylation	O
at	O
Ser1981	B-Simple_chemical
suggest	O
that	O
E2F1	B-Gene_or_gene_product
stimulates	O
ATM	B-Gene_or_gene_product
through	O
a	O
unique	O
mechanism	O
that	O
is	O
distinct	O
from	O
agents	O
that	O
cause	O
DNA	O
double	O
-	O
strand	O
breaks	O
.	O

These	O
findings	O
identify	O
new	O
roles	O
for	O
several	O
DNA	O
damage	O
response	O
factors	O
by	O
demonstrating	O
that	O
they	O
also	O
participate	O
in	O
the	O
oncogenic	O
stress	O
signaling	O
pathway	O
between	O
E2F1	B-Gene_or_gene_product
and	O
p53	B-Gene_or_gene_product
.	O

Expression	O
of	O
MCM10	B-Gene_or_gene_product
and	O
TopBP1	B-Gene_or_gene_product
is	O
regulated	O
by	O
cell	O
proliferation	O
and	O
UV	O
irradiation	O
via	O
the	O
E2F	B-Gene_or_gene_product
transcription	O
factor	O
.	O

MCM10	B-Gene_or_gene_product
and	O
TopBP1	B-Gene_or_gene_product
function	O
in	O
the	O
initiation	O
of	O
DNA	O
replication	O
,	O
by	O
regulating	O
the	O
chromatin	B-Cellular_component
binding	O
of	O
the	O
DNA	B-Complex
polymerase	I-Complex
alpha	I-Complex
loading	O
factor	O
,	O
CDC45	B-Gene_or_gene_product
.	O

TopBP1	B-Gene_or_gene_product
is	O
also	O
known	O
as	O
a	O
DNA	O
damage	O
response	O
protein	O
.	O

In	O
this	O
study	O
,	O
we	O
showed	O
that	O
the	O
transcription	O
of	O
human	O
MCM10	B-Gene_or_gene_product
and	O
TopBP1	B-Gene_or_gene_product
is	O
activated	O
by	O
transcription	O
factors	O
E2F1	B-Gene_or_gene_product
-	O
3	B-Gene_or_gene_product
,	O
but	O
not	O
by	O
factors	O
E2F4	B-Gene_or_gene_product
-	O
7	B-Gene_or_gene_product
.	O

Analysis	O
of	O
various	O
MCM10	B-Gene_or_gene_product
and	O
TopBP1	B-Gene_or_gene_product
promoter	O
constructs	O
showed	O
that	O
an	O
E2F	B-Gene_or_gene_product
-	O
responsive	O
sequence	O
in	O
the	O
vicinity	O
of	O
the	O
transcription	O
initiation	O
site	O
is	O
necessary	O
for	O
the	O
E2F1	B-Gene_or_gene_product
-	O
induced	O
activation	O
of	O
MCM10	B-Gene_or_gene_product
and	O
TopBP1	B-Gene_or_gene_product
gene	O
transcription	O
,	O
which	O
is	O
further	O
suppressed	O
by	O
pRb	B-Gene_or_gene_product
.	O

The	O
promoter	O
activities	O
of	O
human	O
MCM10	B-Gene_or_gene_product
and	O
TopBP1	B-Gene_or_gene_product
were	O
demonstrated	O
to	O
be	O
growth	O
dependent	O
via	O
the	O
E2F	B-Gene_or_gene_product
-	O
responsive	O
sequence	O
.	O

Although	O
E2F1	B-Gene_or_gene_product
was	O
stabilized	O
by	O
ultraviolet	O
(	O
UV	O
)	O
irradiation	O
,	O
the	O
mRNA	O
expression	O
level	O
of	O
TopBP1	B-Gene_or_gene_product
was	O
suppressed	O
in	O
HCT116	O
human	O
diploid	O
colon	O
cancer	O
cells	O
.	O

We	O
showed	O
,	O
by	O
performing	O
chromatin	B-Cellular_component
immunoprecipitation	O
that	O
,	O
in	O
response	O
to	O
UV	O
irradiation	O
but	O
not	O
doxorubicin	B-Simple_chemical
treatment	O
,	O
E2F4	B-Gene_or_gene_product
accumulated	O
on	O
the	O
MCM10	B-Gene_or_gene_product
and	O
TopBP1	B-Gene_or_gene_product
promoters	O
.	O

Our	O
data	O
suggest	O
a	O
model	O
in	O
which	O
UV	O
irradiation	O
-	O
induced	O
DNA	O
damage	O
depends	O
,	O
at	O
least	O
in	O
part	O
,	O
on	O
the	O
accumulation	O
of	O
the	O
E2F4	B-Gene_or_gene_product
transcription	O
factor	O
on	O
the	O
MCM10	B-Gene_or_gene_product
and	O
TopBP1	B-Gene_or_gene_product
promoters	O
,	O
which	O
results	O
in	O
suppression	O
of	O
DNA	O
replication	O
.	O

Direct	O
binding	O
of	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
to	O
retinoblastoma	B-Gene_or_gene_product
protein	I-Gene_or_gene_product
:	O
novel	O
links	O
between	O
apoptotic	O
signaling	O
and	O
cell	O
cycle	O
machinery	O
.	O

The	O
retinoblastoma	B-Gene_or_gene_product
protein	I-Gene_or_gene_product
Rb	B-Gene_or_gene_product
has	O
antiproliferative	O
and	O
antiapoptotic	O
functions	O
.	O

Our	O
previous	O
studies	O
have	O
shown	O
that	O
certain	O
apoptotic	O
signals	O
can	O
inactivate	O
Rb	B-Gene_or_gene_product
via	O
the	O
p38	B-Gene_or_gene_product
pathway	O
.	O

Here	O
we	O
show	O
that	O
Rb	B-Gene_or_gene_product
associates	O
with	O
the	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
ASK1	B-Gene_or_gene_product
in	O
response	O
to	O
specific	O
apoptotic	O
signals	O
.	O

An	O
LXCXE	O
motif	O
on	O
ASK1	B-Gene_or_gene_product
was	O
required	O
for	O
Rb	B-Gene_or_gene_product
binding	O
;	O
this	O
correlated	O
with	O
increased	O
E2F1	B-Gene_or_gene_product
transcriptional	O
activity	O
and	O
up	O
-	O
regulation	O
of	O
the	O
proapoptotic	O
protein	O
p73	B-Gene_or_gene_product
.	O

Overexpression	O
of	O
Rb	B-Gene_or_gene_product
inhibited	O
ASK1	B-Gene_or_gene_product
-	O
induced	O
apoptosis	O
;	O
in	O
addition	O
,	O
an	O
ASK1	B-Gene_or_gene_product
mutant	O
incapable	O
of	O
binding	O
Rb	B-Gene_or_gene_product
could	O
not	O
induce	O
apoptosis	O
,	O
indicating	O
that	O
ASK1	B-Gene_or_gene_product
has	O
to	O
overcome	O
the	O
antiapoptotic	O
properties	O
of	O
Rb	B-Gene_or_gene_product
to	O
kill	O
cells	O
.	O

Chromatin	B-Cellular_component
immunoprecipitation	O
assays	O
show	O
that	O
in	O
asynchronous	O
cells	O
the	O
p73P1	O
promoter	O
is	O
occupied	O
predominantly	O
by	O
E2F3	B-Gene_or_gene_product
;	O
upon	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
stimulation	O
,	O
E2F3	B-Gene_or_gene_product
is	O
dissociated	O
from	O
the	O
promoter	O
and	O
replaced	O
by	O
E2F1	B-Gene_or_gene_product
.	O

At	O
the	O
same	O
time	O
,	O
TNF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
stimulation	O
causes	O
Rb	B-Gene_or_gene_product
to	O
dissociate	O
from	O
the	O
p73P1	O
promoter	O
.	O

These	O
are	O
promoter	O
-	O
specific	O
events	O
because	O
Rb	B-Gene_or_gene_product
binds	O
to	O
the	O
mitogenic	O
cdc25A	O
promoter	O
upon	O
TNF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
stimulation	O
.	O

These	O
studies	O
suggest	O
that	O
Rb	B-Gene_or_gene_product
acts	O
as	O
a	O
link	O
between	O
apoptotic	O
and	O
proliferative	O
pathways	O
by	O
interacting	O
with	O
distinct	O
kinases	O
and	O
occupying	O
different	O
promoters	O
.	O

p105	B-Gene_or_gene_product
,	O
the	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
p50	B-Gene_or_gene_product
precursor	O
protein	O
,	O
is	O
one	O
of	O
the	O
cellular	O
proteins	O
complexed	O
with	O
the	O
v	B-Gene_or_gene_product
-	I-Gene_or_gene_product
Rel	I-Gene_or_gene_product
oncoprotein	O
in	O
transformed	O
chicken	O
spleen	O
cells	O
.	O

Active	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
-	O
like	O
transcription	O
complexes	O
are	O
multimers	O
consisting	O
of	O
one	O
or	O
two	O
members	O
of	O
a	O
family	O
of	O
proteins	O
related	O
to	O
the	O
c	B-Gene_or_gene_product
-	I-Gene_or_gene_product
Rel	I-Gene_or_gene_product
proto	O
-	O
oncoprotein	O
.	O

We	O
have	O
isolated	O
a	O
chicken	O
cDNA	O
encoding	O
p105	B-Gene_or_gene_product
,	O
the	O
precursor	O
protein	O
for	O
the	O
p50	B-Gene_or_gene_product
subunit	O
of	O
NF	B-Complex
-	I-Complex
kappa	I-Complex
B	I-Complex
.	O

Sequence	O
analysis	O
shows	O
that	O
chicken	O
p105	B-Gene_or_gene_product
is	O
approximately	O
70	O
%	O
identical	O
to	O
the	O
mouse	O
and	O
human	O
p105	B-Gene_or_gene_product
proteins	O
,	O
containing	O
the	O
Rel	B-Gene_or_gene_product
homology	O
domain	O
in	O
its	O
N	O
-	O
terminal	O
370	O
amino	O
acids	O
and	O
several	O
ankyrinlike	O
repeats	O
in	O
the	O
C	O
-	O
terminal	O
portion	O
of	O
the	O
protein	O
.	O

The	O
Rel	B-Gene_or_gene_product
homology	O
domain	O
is	O
particularly	O
highly	O
conserved	O
between	O
chicken	O
and	O
mammalian	O
p50	B-Gene_or_gene_product
,	O
and	O
an	O
in	O
vitro	O
-	O
synthesized	O
,	O
truncated	O
chicken	O
p105	B-Gene_or_gene_product
protein	O
,	O
containing	O
sequences	O
that	O
correspond	O
to	O
the	O
predicted	O
p50	B-Gene_or_gene_product
protein	O
,	O
bound	O
to	O
a	O
consensus	O
kappa	B-Complex
B	I-Complex
site	O
in	O
an	O
electrophoretic	O
mobility	O
shift	O
assay	O
.	O

In	O
v	B-Gene_or_gene_product
-	I-Gene_or_gene_product
Rel	I-Gene_or_gene_product
-	O
transformed	O
chicken	O
spleen	O
cells	O
,	O
v	B-Gene_or_gene_product
-	I-Gene_or_gene_product
Rel	I-Gene_or_gene_product
is	O
found	O
in	O
high	O
-	O
molecular	O
-	O
weight	O
complexes	O
which	O
include	O
cellular	O
proteins	O
of	O
approximately	O
124	O
kDa	O
(	O
p124	B-Gene_or_gene_product
)	O
and	O
115	O
kDa	O
(	O
p115	B-Gene_or_gene_product
)	O
.	O

Here	O
we	O
report	O
that	O
in	O
vitro	O
-	O
produced	O
p105	B-Gene_or_gene_product
comigrates	O
with	O
p124	B-Gene_or_gene_product
from	O
v	B-Gene_or_gene_product
-	I-Gene_or_gene_product
Rel	I-Gene_or_gene_product
-	O
transformed	O
spleen	O
cells	O
and	O
that	O
p105	B-Gene_or_gene_product
and	O
p124	B-Gene_or_gene_product
appear	O
to	O
be	O
identical	O
by	O
partial	O
proteolytic	O
mapping	O
with	O
V8	B-Gene_or_gene_product
protease	O
.	O

Furthermore	O
,	O
both	O
p105	B-Gene_or_gene_product
and	O
p50	B-Gene_or_gene_product
can	O
complex	O
directly	O
with	O
v	B-Gene_or_gene_product
-	I-Gene_or_gene_product
Rel	I-Gene_or_gene_product
and	O
chicken	O
c	B-Gene_or_gene_product
-	I-Gene_or_gene_product
Rel	I-Gene_or_gene_product
in	O
vitro	O
.	O

However	O
,	O
in	O
vitro	O
association	O
with	O
p105	B-Gene_or_gene_product
by	O
v	B-Gene_or_gene_product
-	I-Gene_or_gene_product
Rel	I-Gene_or_gene_product
does	O
not	O
necessarily	O
correlate	O
with	O
transformation	O
,	O
since	O
one	O
nontransforming	O
v	B-Gene_or_gene_product
-	I-Gene_or_gene_product
Rel	I-Gene_or_gene_product
mutant	O
can	O
associate	O
with	O
p105	B-Gene_or_gene_product
in	O
vitro	O
.	O

Repression	O
of	O
fibroblast	B-Gene_or_gene_product
growth	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
receptor	I-Gene_or_gene_product
1	I-Gene_or_gene_product
gene	O
expression	O
by	O
E2F4	B-Gene_or_gene_product
in	O
skeletal	O
muscle	O
cells	O
.	O

Fibroblast	B-Gene_or_gene_product
growth	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
receptor	I-Gene_or_gene_product
1	I-Gene_or_gene_product
(	O
FGFR1	B-Gene_or_gene_product
)	O
gene	O
expression	O
is	O
positively	O
and	O
negatively	O
regulated	O
during	O
muscle	O
differentiation	O
.	O

We	O
recently	O
reported	O
that	O
FGFR1	B-Gene_or_gene_product
gene	O
expression	O
was	O
up	O
-	O
regulated	O
by	O
Sp	B-Gene_or_gene_product
transcription	O
factors	O
in	O
proliferating	O
myoblasts	O
.	O

However	O
,	O
the	O
mechanism	O
of	O
down	O
-	O
regulation	O
of	O
this	O
gene	O
during	O
differentiation	O
is	O
unknown	O
.	O

We	O
have	O
identified	O
the	O
transcription	O
factor	O
E2F4	B-Gene_or_gene_product
as	O
a	O
negative	O
regulator	O
of	O
FGFR1	B-Gene_or_gene_product
gene	O
expression	O
.	O

Immunodetection	O
studies	O
revealed	O
that	O
endogenous	O
E2F1	B-Gene_or_gene_product
and	O
E2F2	B-Gene_or_gene_product
proteins	O
were	O
cytoplasmic	B-Cellular_component
in	O
myoblasts	O
and	O
myotubes	O
,	O
whereas	O
E2F4	B-Gene_or_gene_product
was	O
abundant	O
in	O
the	O
nuclei	B-Cellular_component
of	O
both	O
.	O

Upon	O
overexpression	O
,	O
E2F4	B-Gene_or_gene_product
repressed	O
FGFR1	B-Gene_or_gene_product
promoter	O
activity	O
in	O
a	O
dose	O
-	O
dependent	O
manner	O
in	O
myoblasts	O
and	O
Drosophila	O
SL2	O
cells	O
,	O
and	O
mutation	O
of	O
the	O
E2F4	B-Gene_or_gene_product
binding	O
site	O
increased	O
FGFR1	B-Gene_or_gene_product
promoter	O
activity	O
and	O
reduced	O
E2F4	B-Gene_or_gene_product
-	O
mediated	O
repression	O
.	O

Gel	O
shift	O
assays	O
detected	O
E2F4	B-Gene_or_gene_product
binding	O
to	O
a	O
synthetic	O
FGFR1	B-Gene_or_gene_product
E2F4	B-Gene_or_gene_product
binding	O
site	O
and	O
chromatin	B-Cellular_component
immunoprecipitation	O
assays	O
detected	O
E2F4	B-Gene_or_gene_product
binding	O
to	O
the	O
endogenous	O
FGFR1	B-Gene_or_gene_product
promoter	O
in	O
proliferating	O
myoblasts	O
and	O
myotubes	O
.	O

The	O
results	O
indicate	O
that	O
FGFR1	B-Gene_or_gene_product
promoter	O
activity	O
in	O
skeletal	O
muscle	O
cells	O
is	O
repressed	O
by	O
E2F4	B-Gene_or_gene_product
.	O

TRIP6	B-Gene_or_gene_product
is	O
a	O
RIP2	B-Gene_or_gene_product
-	O
associated	O
common	O
signaling	O
component	O
of	O
multiple	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
activation	O
pathways	O
.	O

Receptor	B-Gene_or_gene_product
-	I-Gene_or_gene_product
interacting	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
2	I-Gene_or_gene_product
(	O
RIP2	B-Gene_or_gene_product
)	O
is	O
a	O
member	O
of	O
the	O
RIP	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
family	O
that	O
has	O
been	O
shown	O
to	O
be	O
crucially	O
involved	O
in	O
inflammation	O
,	O
innate	O
and	O
adaptive	O
immune	O
responses	O
.	O

The	O
physiological	O
and	O
pathological	O
roles	O
of	O
RIP2	B-Gene_or_gene_product
are	O
mediated	O
through	O
its	O
involvement	O
in	O
multiple	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
activation	O
pathways	O
,	O
including	O
those	O
triggered	O
by	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
interleukin	B-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
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
TLR2	B-Gene_or_gene_product
)	O
,	O
TLR3	B-Gene_or_gene_product
,	O
TLR4	B-Gene_or_gene_product
and	O
Nod1	B-Gene_or_gene_product
.	O

In	O
this	O
report	O
,	O
we	O
identified	O
the	O
LIM	O
-	O
domain	O
-	O
containing	O
protein	O
TRIP6	B-Gene_or_gene_product
as	O
a	O
RIP2	B-Gene_or_gene_product
-	O
interacting	O
protein	O
in	O
yeast	O
two	O
-	O
hybrid	O
screens	O
.	O

In	O
mammalian	O
cells	O
,	O
TRIP6	B-Gene_or_gene_product
interacts	O
with	O
RIP2	B-Gene_or_gene_product
in	O
a	O
TNF	B-Gene_or_gene_product
-	O
or	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
-	O
dependent	O
manner	O
.	O

Overexpression	O
of	O
TRIP6	B-Gene_or_gene_product
potentiates	O
RIP2	B-Gene_or_gene_product
-	O
mediated	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
activation	O
in	O
a	O
dose	O
-	O
dependent	O
manner	O
.	O

The	O
LIM	O
domains	O
of	O
TRIP6	B-Gene_or_gene_product
are	O
responsible	O
for	O
its	O
interaction	O
with	O
RIP2	B-Gene_or_gene_product
.	O

TRIP6	B-Gene_or_gene_product
also	O
interacts	O
with	O
TRAF2	B-Gene_or_gene_product
,	O
a	O
protein	O
that	O
is	O
crucially	O
involved	O
in	O
TNF	B-Gene_or_gene_product
signaling	O
,	O
as	O
well	O
as	O
the	O
IL	B-Complex
-	I-Complex
1	I-Complex
receptor	I-Complex
,	O
TLR2	B-Gene_or_gene_product
and	O
Nod1	B-Gene_or_gene_product
.	O

Overexpression	O
of	O
TRIP6	B-Gene_or_gene_product
potentiates	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
activation	O
by	O
TNF	B-Gene_or_gene_product
,	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
,	O
TLR2	B-Gene_or_gene_product
or	O
Nod1	B-Gene_or_gene_product
,	O
whereas	O
a	O
dominant	O
negative	O
mutant	O
or	O
RNA	O
-	O
interference	O
construct	O
of	O
TRIP6	B-Gene_or_gene_product
inhibits	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
activation	O
by	O
TNF	B-Gene_or_gene_product
,	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
,	O
TLR2	B-Gene_or_gene_product
or	O
Nod1	B-Gene_or_gene_product
.	O

Moreover	O
,	O
TRIP6	B-Gene_or_gene_product
also	O
potentiates	O
RIP2	B-Gene_or_gene_product
-	O
and	O
Nod1	B-Gene_or_gene_product
-	O
mediated	O
ERK	B-Gene_or_gene_product
activation	O
.	O

These	O
data	O
have	O
established	O
a	O
physical	O
and	O
functional	O
association	O
between	O
TRIP6	B-Gene_or_gene_product
and	O
RIP2	B-Gene_or_gene_product
,	O
and	O
suggest	O
that	O
RIP2	B-Gene_or_gene_product
'	O
s	O
involvement	O
in	O
multiple	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
and	O
ERK	B-Gene_or_gene_product
activation	O
pathways	O
is	O
mediated	O
through	O
TRIP6	B-Gene_or_gene_product
.	O

Induction	O
of	O
p21	B-Gene_or_gene_product
by	O
p53	B-Gene_or_gene_product
following	O
DNA	O
damage	O
inhibits	O
both	O
Cdk4	B-Gene_or_gene_product
and	O
Cdk2	B-Gene_or_gene_product
activities	O
.	O

DNA	O
damage	O
often	O
activates	O
the	O
p53	B-Gene_or_gene_product
-	O
p21	B-Gene_or_gene_product
pathway	O
and	O
causes	O
G	O
(	O
1	O
)	O
-	O
phase	O
arrest	O
in	O
mammalian	O
cells	O
.	O

Although	O
there	O
is	O
ample	O
evidence	O
that	O
p21	B-Gene_or_gene_product
induction	O
by	O
p53	B-Gene_or_gene_product
leads	O
to	O
Cdk2	B-Gene_or_gene_product
inhibition	O
,	O
it	O
is	O
unclear	O
whether	O
this	O
checkpoint	O
event	O
also	O
leads	O
to	O
Cdk4	B-Gene_or_gene_product
inhibition	O
.	O

Diaminocyclohexane	B-Simple_chemical
(	I-Simple_chemical
trans	I-Simple_chemical
-	I-Simple_chemical
diacetato	I-Simple_chemical
)	I-Simple_chemical
(	I-Simple_chemical
dichloro	I-Simple_chemical
)	I-Simple_chemical
platinum	I-Simple_chemical
(	I-Simple_chemical
IV	I-Simple_chemical
)	I-Simple_chemical
(	O
DAP	B-Simple_chemical
)	O
,	O
a	O
platinum	B-Simple_chemical
-	O
based	O
coordination	O
complex	O
,	O
is	O
a	O
DNA	O
-	O
damaging	O
agent	O
that	O
is	O
effective	O
against	O
a	O
variety	O
of	O
tumor	O
cells	O
resistant	O
to	O
the	O
parental	O
drug	O
cisplatin	B-Simple_chemical
.	O

Our	O
previous	O
studies	O
established	O
that	O
treatment	O
of	O
human	O
cancer	O
cells	O
with	O
low	O
effective	O
concentrations	O
of	O
DAP	B-Simple_chemical
specifically	O
activates	O
the	O
G	O
(	O
1	O
)	O
-	O
phase	O
checkpoint	O
and	O
simultaneously	O
inhibit	O
Cdk4	B-Gene_or_gene_product
and	O
Cdk2	B-Gene_or_gene_product
activities	O
.	O

Here	O
we	O
demonstrate	O
that	O
DAP	B-Simple_chemical
treatment	O
of	O
human	O
cancer	O
cells	O
activates	O
the	O
p53	B-Gene_or_gene_product
-	O
p21	B-Gene_or_gene_product
pathway	O
without	O
activating	O
other	O
known	O
mechanisms	O
that	O
inhibit	O
Cdk4	B-Gene_or_gene_product
and	O
Cdk2	B-Gene_or_gene_product
activities	O
.	O

The	O
induced	O
p21	B-Gene_or_gene_product
binds	O
to	O
both	O
the	O
Cdk4	B-Complex
/	I-Complex
cyclin	I-Complex
D	I-Complex
and	O
Cdk2	B-Complex
/	I-Complex
cyclin	I-Complex
E	I-Complex
complexes	O
and	O
inhibits	O
both	O
of	O
their	O
kinase	O
activities	O
.	O

Conversely	O
,	O
inhibition	O
of	O
p21	B-Gene_or_gene_product
induction	O
by	O
cycloheximide	B-Simple_chemical
or	O
by	O
p21	B-Gene_or_gene_product
gene	O
deletion	O
prevents	O
DAP	B-Simple_chemical
-	O
induced	O
inhibition	O
of	O
Cdk4	B-Gene_or_gene_product
and	O
Cdk2	B-Gene_or_gene_product
activities	O
.	O

Attenuated	O
p53	B-Gene_or_gene_product
expression	O
and	O
p21	B-Gene_or_gene_product
induction	O
also	O
eliminates	O
DAP	B-Simple_chemical
-	O
induced	O
G	O
(	O
1	O
)	O
-	O
phase	O
arrest	O
and	O
inhibition	O
of	O
Cdk4	B-Gene_or_gene_product
and	O
Cdk2	B-Gene_or_gene_product
activities	O
.	O

Together	O
,	O
these	O
findings	O
establish	O
that	O
activation	O
of	O
the	O
p53	B-Gene_or_gene_product
-	O
p21	B-Gene_or_gene_product
pathway	O
is	O
responsible	O
for	O
the	O
DAP	B-Simple_chemical
-	O
induced	O
G	O
(	O
1	O
)	O
-	O
phase	O
checkpoint	O
response	O
and	O
provide	O
the	O
first	O
solid	O
evidence	O
that	O
p21	B-Gene_or_gene_product
induction	O
by	O
p53	B-Gene_or_gene_product
during	O
a	O
DNA	O
damage	O
-	O
induced	O
G	O
(	O
1	O
)	O
-	O
phase	O
checkpoint	O
response	O
inhibits	O
both	O
Cdk4	B-Gene_or_gene_product
and	O
Cdk2	B-Gene_or_gene_product
activities	O
.	O

The	O
sphingoid	B-Simple_chemical
long	O
chain	O
base	O
phytosphingosine	B-Simple_chemical
activates	O
AGC	O
-	O
type	O
protein	O
kinases	O
in	O
Saccharomyces	O
cerevisiae	O
including	O
Ypk1	B-Gene_or_gene_product
,	O
Ypk2	B-Gene_or_gene_product
,	O
and	O
Sch9	B-Gene_or_gene_product
.	O

The	O
Pkh1	B-Gene_or_gene_product
protein	O
kinase	O
of	O
Saccharomyces	O
cerevisiae	O
,	O
a	O
homolog	O
of	O
the	O
mammalian	O
3	B-Gene_or_gene_product
-	I-Gene_or_gene_product
phosphoinositide	I-Gene_or_gene_product
-	I-Gene_or_gene_product
dependent	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
(	O
PDK1	B-Gene_or_gene_product
)	O
,	O
regulates	O
downstream	O
AGC	O
-	O
type	O
protein	O
kinases	O
including	O
Ypk1	B-Gene_or_gene_product
/	O
2	B-Gene_or_gene_product
and	O
Pkc1	B-Gene_or_gene_product
,	O
which	O
control	O
cell	B-Cellular_component
wall	I-Cellular_component
integrity	O
,	O
growth	O
,	O
and	O
other	O
processes	O
.	O

Phytosphingosine	B-Simple_chemical
(	O
PHS	B-Simple_chemical
)	O
,	O
a	O
sphingoid	B-Simple_chemical
long	O
chain	O
base	O
,	O
is	O
hypothesized	O
to	O
be	O
a	O
lipid	B-Simple_chemical
activator	O
of	O
Pkh1	B-Gene_or_gene_product
and	O
thereby	O
controls	O
the	O
activity	O
of	O
Ypk1	B-Gene_or_gene_product
/	O
2	B-Gene_or_gene_product
.	O

Here	O
we	O
present	O
biochemical	O
evidence	O
supporting	O
this	O
hypothesis	O
,	O
and	O
in	O
addition	O
we	O
demonstrate	O
that	O
PHS	B-Simple_chemical
also	O
stimulates	O
autophosphorylation	O
and	O
activation	O
of	O
Ypk1	B-Gene_or_gene_product
/	O
2	B-Gene_or_gene_product
.	O

Greatest	O
stimulation	O
of	O
Ypk1	B-Gene_or_gene_product
/	O
2	B-Gene_or_gene_product
phosphorylation	O
and	O
activity	O
are	O
achieved	O
by	O
inclusion	O
of	O
both	O
PHS	B-Simple_chemical
and	O
Pkh1	B-Gene_or_gene_product
in	O
an	O
in	O
vitro	O
kinase	O
reaction	O
.	O

We	O
also	O
demonstrate	O
for	O
the	O
first	O
time	O
that	O
Pkh1	B-Gene_or_gene_product
phosphorylates	O
the	O
Sch9	B-Gene_or_gene_product
protein	O
kinase	O
in	O
vitro	O
and	O
that	O
such	O
phosphorylation	O
is	O
stimulated	O
by	O
PHS	B-Simple_chemical
.	O

This	O
is	O
the	O
first	O
biochemical	O
demonstration	O
of	O
Sch9	B-Gene_or_gene_product
activators	O
,	O
and	O
the	O
results	O
further	O
support	O
roles	O
for	O
long	O
chain	O
bases	O
in	O
heat	O
stress	O
resistance	O
in	O
addition	O
to	O
implying	O
roles	O
in	O
chronological	O
aging	O
and	O
cell	O
size	O
determination	O
,	O
since	O
Sch9	B-Gene_or_gene_product
functions	O
in	O
these	O
processes	O
.	O

Thus	O
,	O
our	O
data	O
support	O
a	O
model	O
in	O
which	O
PHS	B-Simple_chemical
,	O
rather	O
than	O
simply	O
being	O
an	O
upstream	O
activator	O
of	O
Pkh1	B-Gene_or_gene_product
,	O
also	O
activates	O
kinases	O
that	O
are	O
downstream	O
targets	O
of	O
Pkh1	B-Gene_or_gene_product
including	O
Ypk1	B-Gene_or_gene_product
/	O
2	B-Gene_or_gene_product
and	O
Sch9	B-Gene_or_gene_product
.	O

A	O
role	O
for	O
E2F6	B-Gene_or_gene_product
in	O
the	O
restriction	O
of	O
male	O
-	O
germ	O
-	O
cell	O
-	O
specific	O
gene	O
expression	O
.	O

E2F	B-Gene_or_gene_product
transcription	O
factors	O
play	O
a	O
pivotal	O
role	O
in	O
the	O
regulation	O
of	O
cellular	O
proliferation	O
and	O
can	O
be	O
subdivided	O
into	O
activating	O
and	O
repressing	O
family	O
members	O
[	O
1	O
]	O
.	O

Like	O
other	O
E2Fs	B-Gene_or_gene_product
,	O
E2F6	B-Gene_or_gene_product
binds	O
to	O
E2F	B-Gene_or_gene_product
consensus	O
sites	O
,	O
but	O
in	O
contrast	O
to	O
E2F1	B-Gene_or_gene_product
-	O
5	B-Gene_or_gene_product
,	O
it	O
lacks	O
an	O
Rb	B-Gene_or_gene_product
binding	O
domain	O
and	O
functions	O
as	O
an	O
Rb	B-Gene_or_gene_product
-	O
independent	O
transcriptional	O
repressor	O
[	O
2	O
,	O
3	O
,	O
4	O
and	O
5	O
]	O
.	O

Instead	O
,	O
E2F6	B-Gene_or_gene_product
has	O
been	O
shown	O
to	O
complex	O
with	O
Polycomb	B-Gene_or_gene_product
(	O
PcG	B-Gene_or_gene_product
)	O
group	O
proteins	O
[	O
6	O
and	O
7	O
]	O
,	O
which	O
have	O
a	O
well	O
-	O
established	O
role	O
in	O
gene	O
silencing	O
.	O

Here	O
,	O
we	O
show	O
that	O
E2F6	B-Gene_or_gene_product
plays	O
an	O
unexpected	O
and	O
essential	O
role	O
in	O
the	O
tissue	O
specificity	O
of	O
gene	O
expression	O
.	O

E2F6	B-Gene_or_gene_product
-	O
deficient	O
mice	O
ubiquitously	O
express	O
the	O
alpha	B-Gene_or_gene_product
-	I-Gene_or_gene_product
tubulin	I-Gene_or_gene_product
3	I-Gene_or_gene_product
and	O
7	B-Gene_or_gene_product
genes	O
,	O
which	O
are	O
expressed	O
strictly	O
testis	O
-	O
specifically	O
in	O
control	O
mice	O
.	O

Like	O
an	O
additional	O
E2F6	B-Gene_or_gene_product
target	O
gene	O
,	O
Tex12	B-Gene_or_gene_product
,	O
that	O
we	O
identified	O
,	O
tubulin	B-Gene_or_gene_product
3	I-Gene_or_gene_product
and	O
7	B-Gene_or_gene_product
are	O
normally	O
expressed	O
in	O
male	O
germ	O
cells	O
only	O
.	O

The	O
promoters	O
of	O
the	O
alpha	B-Gene_or_gene_product
-	I-Gene_or_gene_product
tubulin	I-Gene_or_gene_product
and	O
Tex12	B-Gene_or_gene_product
genes	O
share	O
a	O
perfectly	O
conserved	O
E2F	B-Gene_or_gene_product
site	O
,	O
which	O
E2F6	B-Gene_or_gene_product
binds	O
to	O
.	O

Mechanistically	O
,	O
E2F6	B-Gene_or_gene_product
-	O
mediated	O
repression	O
involves	O
CpG	B-Gene_or_gene_product
hypermethylation	O
locking	O
target	O
promoters	O
in	O
an	O
inactive	O
state	O
.	O

Thus	O
,	O
E2F6	B-Gene_or_gene_product
is	O
essential	O
for	O
the	O
long	O
-	O
term	O
somatic	O
silencing	O
of	O
certain	O
male	O
-	O
germ	O
-	O
cell	O
-	O
specific	O
genes	O
,	O
but	O
it	O
is	O
dispensable	O
for	O
cell	O
-	O
cycle	O
regulation	O
.	O

mTOR	B-Gene_or_gene_product
function	O
in	O
skeletal	O
muscle	O
hypertrophy	O
:	O
increased	O
ribosomal	B-Cellular_component
RNA	O
via	O
cell	O
cycle	O
regulators	O
.	O

The	O
purpose	O
of	O
this	O
study	O
was	O
to	O
identify	O
the	O
potential	O
downstream	O
functions	O
associated	O
with	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
signaling	O
during	O
myotube	O
hypertrophy	O
.	O

Terminally	O
differentiated	O
myotubes	O
were	O
serum	O
stimulated	O
for	O
3	O
,	O
6	O
,	O
12	O
,	O
24	O
,	O
and	O
48	O
h	O
.	O

This	O
treatment	O
resulted	O
in	O
significant	O
myotube	O
hypertrophy	O
(	O
protein	O
/	O
DNA	O
)	O
and	O
increased	O
RNA	O
content	O
(	O
RNA	O
/	O
DNA	O
)	O
with	O
no	O
changes	O
in	O
DNA	O
content	O
or	O
indices	O
of	O
cell	O
proliferation	O
.	O

During	O
myotube	O
hypertrophy	O
,	O
the	O
increase	O
in	O
RNA	O
content	O
was	O
accompanied	O
by	O
an	O
increase	O
in	O
tumor	O
suppressor	O
protein	O
retinoblastoma	B-Gene_or_gene_product
(	O
Rb	B-Gene_or_gene_product
)	O
phosphorylation	O
and	O
a	O
corresponding	O
increase	O
in	O
the	O
availability	O
of	O
the	O
ribosomal	B-Cellular_component
DNA	O
transcription	O
factor	O
upstream	B-Gene_or_gene_product
binding	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
(	O
UBF	B-Gene_or_gene_product
)	O
.	O

Serum	O
stimulation	O
also	O
induced	O
an	O
increase	O
in	O
cyclin	B-Gene_or_gene_product
D1	I-Gene_or_gene_product
protein	O
expression	O
in	O
the	O
differentiated	O
myotubes	O
with	O
a	O
concomitant	O
increase	O
in	O
cyclin	B-Gene_or_gene_product
D1	I-Gene_or_gene_product
-	O
dependent	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
CDK	I-Gene_or_gene_product
)	I-Gene_or_gene_product
-	I-Gene_or_gene_product
4	I-Gene_or_gene_product
activity	O
toward	O
Rb	B-Gene_or_gene_product
.	O

The	O
increases	O
in	O
myotube	O
hypertrophy	O
and	O
RNA	O
content	O
were	O
blocked	O
by	O
rapamycin	B-Simple_chemical
treatment	O
,	O
which	O
also	O
prevented	O
the	O
increase	O
in	O
cyclin	B-Gene_or_gene_product
D1	I-Gene_or_gene_product
protein	O
expression	O
,	O
CDK	B-Gene_or_gene_product
-	I-Gene_or_gene_product
4	I-Gene_or_gene_product
activity	O
,	O
Rb	B-Gene_or_gene_product
phosphorylation	O
,	O
and	O
the	O
increase	O
in	O
UBF	B-Gene_or_gene_product
availability	O
.	O

Our	O
findings	O
demonstrate	O
that	O
activation	O
of	O
mTOR	B-Gene_or_gene_product
is	O
necessary	O
for	O
myotube	O
hypertrophy	O
and	O
suggest	O
that	O
the	O
role	O
of	O
mTOR	B-Gene_or_gene_product
is	O
in	O
part	O
to	O
modulate	O
cyclin	B-Gene_or_gene_product
D1	I-Gene_or_gene_product
-	O
dependent	O
CDK	B-Gene_or_gene_product
-	I-Gene_or_gene_product
4	I-Gene_or_gene_product
activity	O
in	O
the	O
regulation	O
of	O
Rb	B-Gene_or_gene_product
and	O
ribosomal	B-Cellular_component
RNA	O
synthesis	O
.	O

On	O
the	O
basis	O
of	O
these	O
results	O
,	O
we	O
propose	O
that	O
common	O
molecular	O
mechanisms	O
contribute	O
to	O
the	O
regulation	O
of	O
myotube	O
hypertrophy	O
and	O
growth	O
during	O
the	O
G1	O
phase	O
of	O
the	O
cell	O
cycle	O
.	O

Molecular	O
genetic	O
analysis	O
of	O
the	O
yeast	O
repressor	O
Rfx1	B-Gene_or_gene_product
/	O
Crt1	B-Gene_or_gene_product
reveals	O
a	O
novel	O
two	O
-	O
step	O
regulatory	O
mechanism	O
.	O

In	O
Saccharomyces	O
cerevisiae	O
,	O
the	O
repressor	O
Crt1	B-Gene_or_gene_product
and	O
the	O
global	O
corepressor	O
Ssn6	B-Complex
-	I-Complex
Tup1	I-Complex
repress	O
the	O
DNA	O
damage	O
-	O
inducible	O
ribonucleotide	B-Gene_or_gene_product
reductase	I-Gene_or_gene_product
(	O
RNR	B-Gene_or_gene_product
)	O
genes	O
.	O

Initiation	O
of	O
DNA	O
damage	O
signals	O
causes	O
the	O
release	O
of	O
Crt1	B-Gene_or_gene_product
and	O
Ssn6	B-Complex
-	I-Complex
Tup1	I-Complex
from	O
the	O
promoter	O
,	O
coactivator	O
recruitment	O
,	O
and	O
derepression	O
of	O
transcription	O
,	O
indicating	O
that	O
Crt1	B-Gene_or_gene_product
plays	O
a	O
crucial	O
role	O
in	O
the	O
switch	O
between	O
gene	O
repression	O
and	O
activation	O
.	O

Here	O
we	O
have	O
mapped	O
the	O
functional	O
domains	O
of	O
Crt1	B-Gene_or_gene_product
and	O
identified	O
two	O
independent	O
repression	O
domains	O
and	O
a	O
region	O
required	O
for	O
gene	O
activation	O
.	O

The	O
N	O
terminus	O
of	O
Crt1	B-Gene_or_gene_product
is	O
the	O
major	O
repression	O
domain	O
,	O
it	O
directly	O
binds	O
to	O
the	O
Ssn6	B-Complex
-	I-Complex
Tup1	I-Complex
complex	O
,	O
and	O
its	O
repression	O
activities	O
are	O
dependent	O
upon	O
Ssn6	B-Complex
-	I-Complex
Tup1	I-Complex
and	O
histone	B-Gene_or_gene_product
deacetylases	I-Gene_or_gene_product
(	O
HDACs	B-Gene_or_gene_product
)	O
.	O

In	O
addition	O
,	O
we	O
identified	O
a	O
C	O
-	O
terminal	O
repression	O
domain	O
,	O
which	O
is	O
independent	O
of	O
Ssn6	B-Complex
-	I-Complex
Tup1	I-Complex
and	O
HDACs	B-Gene_or_gene_product
and	O
functions	O
at	O
native	O
genes	O
in	O
vivo	O
.	O

Furthermore	O
,	O
we	O
show	O
that	O
TFIID	B-Gene_or_gene_product
and	O
SWI	B-Complex
/	I-Complex
SNF	I-Complex
bind	O
to	O
a	O
region	O
within	O
the	O
N	O
terminus	O
of	O
Crt1	B-Gene_or_gene_product
,	O
overlapping	O
with	O
but	O
distinct	O
from	O
the	O
Ssn6	B-Complex
-	I-Complex
Tup1	I-Complex
binding	O
and	O
repression	O
domain	O
,	O
suggesting	O
that	O
Crt1	B-Gene_or_gene_product
may	O
have	O
activator	O
functions	O
.	O

Crt1	B-Gene_or_gene_product
mutants	O
were	O
constructed	O
to	O
dissect	O
its	O
activator	O
and	O
repressor	O
functions	O
.	O

All	O
of	O
the	O
mutants	O
were	O
competent	O
for	O
repression	O
of	O
the	O
DNA	O
damage	O
-	O
inducible	O
genes	O
,	O
but	O
a	O
majority	O
were	O
"	O
derepression	O
-	O
defective	O
"	O
mutants	O
.	O

Further	O
characterization	O
of	O
these	O
mutants	O
indicated	O
that	O
they	O
are	O
capable	O
of	O
receiving	O
DNA	O
damage	O
signals	O
and	O
releasing	O
the	O
Ssn6	B-Complex
-	I-Complex
Tup1	I-Complex
complex	O
from	O
the	O
promoter	O
but	O
are	O
selectively	O
impaired	O
for	O
TFIID	B-Gene_or_gene_product
and	O
SWI	B-Complex
/	I-Complex
SNF	I-Complex
recruitment	O
.	O

These	O
results	O
imply	O
a	O
two	O
-	O
step	O
activation	O
model	O
of	O
the	O
DNA	O
damage	O
-	O
inducible	O
genes	O
and	O
that	O
Crt1	B-Gene_or_gene_product
functions	O
as	O
a	O
signal	O
-	O
dependent	O
dual	O
-	O
transcription	O
activator	O
and	O
repressor	O
that	O
acts	O
in	O
a	O
transient	O
manner	O
.	O

Mechanisms	O
of	O
tumor	O
suppression	O
by	O
the	O
SCF	B-Complex
(	I-Complex
Fbw7	I-Complex
)	I-Complex
.	O

SCF	B-Complex
ubiquitin	B-Gene_or_gene_product
ligases	O
regulate	O
the	O
degradation	O
of	O
many	O
proteins	O
involved	O
in	O
the	O
control	O
of	O
cell	O
division	O
and	O
growth	O
.	O

F	O
-	O
box	O
proteins	O
are	O
the	O
SCF	B-Complex
components	O
that	O
bind	O
to	O
substrates	O
,	O
and	O
this	O
binding	O
is	O
usually	O
signaled	O
by	O
substrate	O
phosphorylation	O
.	O

The	O
Fbw7	B-Gene_or_gene_product
/	O
hCdc4	B-Gene_or_gene_product
F	O
-	O
box	O
protein	O
was	O
first	O
recognized	O
by	O
its	O
ability	O
to	O
bind	O
cyclin	B-Gene_or_gene_product
E	I-Gene_or_gene_product
,	O
and	O
the	O
SCF	B-Complex
(	I-Complex
Fbw7	I-Complex
)	I-Complex
is	O
now	O
known	O
to	O
target	O
c	B-Gene_or_gene_product
-	I-Gene_or_gene_product
Myc	I-Gene_or_gene_product
,	O
c	B-Gene_or_gene_product
-	I-Gene_or_gene_product
Jun	I-Gene_or_gene_product
and	O
Notch	B-Gene_or_gene_product
for	O
degradation	O
in	O
addition	O
to	O
its	O
role	O
in	O
cyclin	B-Gene_or_gene_product
E	I-Gene_or_gene_product
proteolysis	O
.	O

Fbw7	B-Gene_or_gene_product
thus	O
negatively	O
regulates	O
several	O
key	O
oncoproteins	O
.	O

Accordingly	O
,	O
Fbw7	B-Gene_or_gene_product
is	O
a	O
tumor	O
suppressor	O
that	O
is	O
mutated	O
in	O
a	O
wide	O
spectrum	O
of	O
human	O
cancers	O
,	O
and	O
Fbw7	B-Gene_or_gene_product
functions	O
as	O
a	O
haploin	O
sufficient	O
tumor	O
suppressor	O
in	O
mice	O
.	O

Because	O
there	O
are	O
three	O
Fbw7	B-Gene_or_gene_product
isoforms	O
that	O
reside	O
in	O
different	O
subcellular	B-Cellular_component
compartments	I-Cellular_component
,	O
as	O
well	O
as	O
multiple	O
Fbw7	B-Gene_or_gene_product
substrates	O
that	O
are	O
the	O
products	O
of	O
proto	O
-	O
oncogenes	O
,	O
the	O
mechanisms	O
of	O
tumor	O
suppression	O
by	O
Fbw7	B-Gene_or_gene_product
are	O
complex	O
and	O
incompletely	O
understood	O
.	O

In	O
this	O
review	O
we	O
discuss	O
the	O
activities	O
of	O
the	O
SCF	B-Complex
(	I-Complex
Fbw7	I-Complex
)	I-Complex
in	O
the	O
context	O
of	O
its	O
role	O
as	O
a	O
tumor	O
suppressor	O
and	O
highlight	O
recent	O
findings	O
demonstrating	O
that	O
dominant	O
oncogenes	O
disable	O
Fbw7	B-Gene_or_gene_product
function	O
.	O

Identification	O
of	O
an	O
intestinal	O
heme	B-Simple_chemical
transporter	O
.	O

Dietary	O
heme	B-Simple_chemical
iron	B-Simple_chemical
is	O
an	O
important	O
nutritional	O
source	O
of	O
iron	B-Simple_chemical
in	O
carnivores	O
and	O
omnivores	O
that	O
is	O
more	O
readily	O
absorbed	O
than	O
non	O
-	O
heme	B-Simple_chemical
iron	B-Simple_chemical
derived	O
from	O
vegetables	O
and	O
grain	O
.	O

Most	O
heme	B-Simple_chemical
is	O
absorbed	O
in	O
the	O
proximal	O
intestine	O
,	O
with	O
absorptive	O
capacity	O
decreasing	O
distally	O
.	O

We	O
utilized	O
a	O
subtractive	O
hybridization	O
approach	O
to	O
isolate	O
a	O
heme	B-Simple_chemical
transporter	O
from	O
duodenum	O
by	O
taking	O
advantage	O
of	O
the	O
intestinal	O
gradient	O
for	O
heme	B-Simple_chemical
absorption	O
.	O

Here	O
we	O
show	O
a	O
membrane	B-Cellular_component
protein	O
named	O
HCP	B-Gene_or_gene_product
1	I-Gene_or_gene_product
(	O
heme	B-Gene_or_gene_product
carrier	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
1	I-Gene_or_gene_product
)	O
,	O
with	O
homology	O
to	O
bacterial	O
metal	B-Simple_chemical
-	O
tetracycline	B-Simple_chemical
transporters	O
,	O
mediates	O
heme	B-Simple_chemical
uptake	O
by	O
cells	O
in	O
a	O
temperature	O
-	O
dependent	O
and	O
saturable	O
manner	O
.	O

HCP	B-Gene_or_gene_product
1	I-Gene_or_gene_product
mRNA	O
was	O
highly	O
expressed	O
in	O
duodenum	O
and	O
regulated	O
by	O
hypoxia	O
.	O

HCP	B-Gene_or_gene_product
1	I-Gene_or_gene_product
protein	O
was	O
iron	B-Simple_chemical
regulated	O
and	O
localized	O
to	O
the	O
brush	O
-	O
border	O
membrane	O
of	O
duodenal	O
enterocytes	O
in	O
iron	B-Simple_chemical
deficiency	O
.	O

Our	O
data	O
indicate	O
that	O
HCP	B-Gene_or_gene_product
1	I-Gene_or_gene_product
is	O
the	O
long	O
-	O
sought	O
intestinal	O
heme	B-Simple_chemical
transporter	O
.	O

Rapid	O
,	O
Wnt	B-Gene_or_gene_product
-	O
induced	O
changes	O
in	O
GSK3beta	B-Gene_or_gene_product
associations	O
that	O
regulate	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
stabilization	O
are	O
mediated	O
by	O
Galpha	B-Gene_or_gene_product
proteins	O
.	O

BACKGROUND	O
:	O
In	O
the	O
absence	O
of	O
Wnt	B-Gene_or_gene_product
stimulation	O
,	O
the	O
transcriptional	O
cofactor	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
is	O
destabilized	O
via	O
phosphorylation	O
by	O
protein	O
kinase	O
GSK3beta	B-Gene_or_gene_product
in	O
complex	O
with	O
Axin	B-Gene_or_gene_product
family	O
members	O
.	O

In	O
the	O
"	O
canonical	O
"	O
Wnt	B-Gene_or_gene_product
signaling	O
pathway	O
,	O
Disheveled	B-Gene_or_gene_product
(	O
Dvl	B-Gene_or_gene_product
)	O
is	O
required	O
to	O
functionally	O
inhibit	O
the	O
activity	O
of	O
the	O
GSK3beta	B-Complex
/	I-Complex
Axin	I-Complex
complex	O
and	O
thereby	O
stabilize	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
.	O

Yet	O
,	O
the	O
mechanisms	O
that	O
underlie	O
Wnt	B-Gene_or_gene_product
regulation	O
of	O
GSK3	B-Gene_or_gene_product
and	O
stabilization	O
of	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
are	O
still	O
not	O
fully	O
appreciated	O
.	O

RESULTS	O
:	O
Here	O
,	O
we	O
examine	O
time	O
-	O
dependent	O
changes	O
in	O
protein	O
-	O
protein	O
interactions	O
that	O
occur	O
in	O
response	O
to	O
Wnt	B-Gene_or_gene_product
treatment	O
.	O

We	O
show	O
that	O
GSK3beta	B-Complex
/	I-Complex
Axin	I-Complex
complexes	O
are	O
rapidly	O
(	O
t1	O
/	O
2	O
<	O
3	O
min	O
)	O
disrupted	O
upon	O
Wnt	B-Gene_or_gene_product
stimulation	O
and	O
that	O
changes	O
in	O
GSK3beta	B-Complex
/	I-Complex
Axin	I-Complex
association	O
substantially	O
precede	O
both	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
stabilization	O
and	O
Axin	B-Gene_or_gene_product
degradation	O
.	O

We	O
further	O
demonstrate	O
that	O
depletion	O
of	O
Galpha	B-Gene_or_gene_product
(	I-Gene_or_gene_product
o	I-Gene_or_gene_product
)	I-Gene_or_gene_product
or	O
Galpha	B-Gene_or_gene_product
(	I-Gene_or_gene_product
q	I-Gene_or_gene_product
)	I-Gene_or_gene_product
will	O
inhibit	O
,	O
respectively	O
,	O
the	O
Wnt	B-Gene_or_gene_product
-	O
induced	O
disruption	O
of	O
GSK3beta	B-Complex
/	I-Complex
Axin2	I-Complex
and	O
GSK3beta	B-Complex
/	I-Complex
Axin	I-Complex
complexes	O
and	O
diminish	O
Wnt	B-Gene_or_gene_product
stabilization	O
of	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
.	O

We	O
also	O
show	O
that	O
direct	O
activation	O
of	O
G	B-Gene_or_gene_product
proteins	I-Gene_or_gene_product
in	O
vivo	O
with	O
GTPgammaS	B-Gene_or_gene_product
in	O
the	O
absence	O
of	O
exogenous	O
Wnt	B-Gene_or_gene_product
will	O
disrupt	O
GSK3beta	B-Complex
/	I-Complex
Axin2	I-Complex
complexes	O
and	O
stabilize	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
.	O

Finally	O
,	O
we	O
demonstrate	O
an	O
association	O
of	O
Galpha	B-Gene_or_gene_product
(	I-Gene_or_gene_product
o	I-Gene_or_gene_product
)	I-Gene_or_gene_product
with	O
Fz	B-Gene_or_gene_product
that	O
is	O
also	O
very	O
rapidly	O
(	O
t1	O
/	O
2	O
<	O
1	O
min	O
)	O
perturbed	O
upon	O
Wnt	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3a	I-Gene_or_gene_product
stimulation	O
and	O
that	O
the	O
Wnt	B-Gene_or_gene_product
-	O
dependent	O
effects	O
on	O
both	O
GSK3beta	B-Complex
/	I-Complex
Axin2	I-Complex
and	O
Galpha	B-Complex
(	I-Complex
o	I-Complex
)	I-Complex
/	I-Complex
Fz	I-Complex
are	O
pertussis	O
-	O
toxin	O
sensitive	O
.	O

Collectively	O
,	O
these	O
data	O
implicate	O
a	O
role	O
for	O
G	B-Gene_or_gene_product
proteins	I-Gene_or_gene_product
in	O
the	O
regulation	O
of	O
Wnt	B-Gene_or_gene_product
-	O
mediated	O
protein	O
-	O
protein	O
interactions	O
and	O
signaling	O
to	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
.	O

CONCLUSIONS	O
:	O
We	O
conclude	O
that	O
rapid	O
disruption	O
of	O
GSK3beta	B-Complex
/	I-Complex
Axin	I-Complex
interactions	O
in	O
response	O
to	O
Wnt	B-Gene_or_gene_product
leads	O
to	O
the	O
initial	O
stabilization	O
of	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
and	O
that	O
Galpha	B-Gene_or_gene_product
(	I-Gene_or_gene_product
o	I-Gene_or_gene_product
)	I-Gene_or_gene_product
and	O
Galpha	B-Gene_or_gene_product
(	I-Gene_or_gene_product
q	I-Gene_or_gene_product
)	I-Gene_or_gene_product
signaling	O
contributes	O
to	O
Wnt	B-Gene_or_gene_product
-	O
mediated	O
GSK3beta	B-Complex
/	I-Complex
Axin	I-Complex
disruption	O
and	O
the	O
ultimate	O
stabilization	O
of	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
.	O

Activation	O
of	O
the	O
checkpoint	O
kinase	O
Rad53	B-Gene_or_gene_product
by	O
the	O
phosphatidyl	B-Gene_or_gene_product
inositol	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
-	I-Gene_or_gene_product
like	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
Mec1	B-Gene_or_gene_product
.	O

Saccharomyces	O
cerevisiae	O
Rad53	B-Gene_or_gene_product
,	O
the	O
ortholog	O
of	O
mammalian	O
Chk2	B-Gene_or_gene_product
,	O
is	O
an	O
essential	O
protein	O
kinase	O
in	O
DNA	O
damage	O
and	O
DNA	O
replication	O
checkpoint	O
pathways	O
.	O

Consecutive	O
phosphatidyl	B-Gene_or_gene_product
inositol	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
-	I-Gene_or_gene_product
like	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
(	O
PIKK	B-Gene_or_gene_product
)	O
-	O
dependent	O
and	O
PIKK	B-Gene_or_gene_product
-	O
independent	O
steps	O
in	O
activation	O
of	O
Rad53	B-Gene_or_gene_product
are	O
key	O
steps	O
for	O
controlling	O
and	O
transmitting	O
diverse	O
downstream	O
responses	O
to	O
DNA	O
damage	O
.	O

However	O
,	O
these	O
activities	O
have	O
not	O
been	O
demonstrated	O
in	O
vitro	O
in	O
defined	O
systems	O
.	O

Here	O
,	O
we	O
have	O
shown	O
that	O
enzymatically	O
dephosphorylated	O
purified	O
Rad53	B-Gene_or_gene_product
autoactivates	O
in	O
vitro	O
through	O
a	O
phosphorylation	O
-	O
dependent	O
mechanism	O
.	O

Kinetic	O
analysis	O
demonstrated	O
that	O
autophosphorylation	O
results	O
in	O
a	O
more	O
than	O
9	O
-	O
fold	O
increase	O
in	O
protein	O
kinase	O
activity	O
.	O

Autophosphorylation	O
was	O
Rad53	B-Gene_or_gene_product
concentration	O
-	O
dependent	O
,	O
indicating	O
that	O
the	O
reaction	O
follows	O
an	O
intermolecular	O
mechanism	O
.	O

DNA	O
damage	O
induced	O
oligomerization	O
of	O
a	O
subset	O
of	O
Rad53	B-Gene_or_gene_product
molecules	O
in	O
vivo	O
.	O

At	O
low	O
concentrations	O
of	O
Rad53	B-Gene_or_gene_product
,	O
preincubation	O
of	O
Rad53	B-Gene_or_gene_product
with	O
immune	O
complexes	O
containing	O
the	O
Mec1	B-Complex
/	I-Complex
Ddc2	I-Complex
complex	O
can	O
activate	O
Rad53	B-Gene_or_gene_product
kinase	O
activity	O
.	O

Our	O
findings	O
showed	O
that	O
Mec1	B-Complex
/	I-Complex
Ddc2	I-Complex
complexes	O
can	O
directly	O
activate	O
Rad53	B-Gene_or_gene_product
through	O
a	O
phosphorylation	O
-	O
dependent	O
mechanism	O
,	O
and	O
more	O
generally	O
,	O
supported	O
the	O
hypothesis	O
that	O
PIKKs	B-Gene_or_gene_product
regulate	O
Chk2	B-Gene_or_gene_product
orthologs	O
through	O
phosphorylation	O
.	O

Moreover	O
,	O
this	O
work	O
has	O
substantiated	O
a	O
model	O
for	O
PIKK	B-Gene_or_gene_product
-	O
independent	O
amplification	O
of	O
Rad53	B-Gene_or_gene_product
activation	O
(	O
and	O
by	O
extension	O
,	O
activation	O
of	O
other	O
Chk2	B-Gene_or_gene_product
orthologs	O
)	O
mediated	O
by	O
inter	O
-	O
Rad53	B-Gene_or_gene_product
phosphorylation	O
.	O

The	O
mechanisms	O
of	O
regulation	O
of	O
Hdm2	B-Gene_or_gene_product
protein	O
level	O
by	O
serum	O
growth	O
factors	O
.	O

Cell	O
cycle	O
progression	O
in	O
response	O
to	O
serum	O
growth	O
factors	O
is	O
dependent	O
on	O
the	O
expression	O
of	O
functional	O
Hdm2	B-Gene_or_gene_product
(	O
Mdm2	B-Gene_or_gene_product
)	O
,	O
which	O
inhibits	O
p53	B-Gene_or_gene_product
-	O
dependent	O
transcription	O
of	O
anti	O
-	O
proliferative	O
genes	O
.	O

In	O
a	O
well	O
characterised	O
non	O
-	O
transformed	O
human	O
fibroblast	O
model	O
,	O
growth	O
factors	O
induce	O
the	O
expression	O
of	O
Hdm2	B-Gene_or_gene_product
with	O
rapid	O
kinetics	O
.	O

Here	O
we	O
dissect	O
the	O
mechanistic	O
basis	O
for	O
this	O
critical	O
response	O
.	O

In	O
contrast	O
to	O
previous	O
studies	O
in	O
which	O
components	O
of	O
the	O
growth	O
factor	O
signalling	O
pathways	O
were	O
overexpressed	O
,	O
hdm2	B-Gene_or_gene_product
mRNA	O
expression	O
is	O
not	O
induced	O
with	O
immediate	O
-	O
early	O
kinetics	O
in	O
these	O
cells	O
.	O

Rather	O
,	O
the	O
elevated	O
Hdm2	B-Gene_or_gene_product
protein	O
levels	O
which	O
follow	O
growth	O
factor	O
stimulation	O
are	O
primarily	O
a	O
consequence	O
of	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
dependent	O
stabilisation	O
of	O
the	O
Hdm2	B-Gene_or_gene_product
protein	O
combined	O
with	O
a	O
global	O
increase	O
in	O
protein	O
synthesis	O
.	O

Phosphorylation	O
of	O
the	O
chromosomal	B-Cellular_component
passenger	O
protein	O
Bir1	B-Gene_or_gene_product
is	O
required	O
for	O
localization	O
of	O
Ndc10	B-Gene_or_gene_product
to	O
the	O
spindle	B-Cellular_component
during	O
anaphase	O
and	O
full	O
spindle	B-Cellular_component
elongation	O
.	O

The	O
Saccharomyces	O
cerevisiae	O
inhibitor	B-Gene_or_gene_product
of	I-Gene_or_gene_product
apoptosis	I-Gene_or_gene_product
(	O
IAP	B-Gene_or_gene_product
)	O
repeat	O
protein	O
Bir1	B-Gene_or_gene_product
localizes	O
as	O
a	O
chromosomal	B-Cellular_component
passenger	O
.	O

A	O
deletion	O
analysis	O
of	O
Bir1	B-Gene_or_gene_product
identified	O
two	O
regions	O
important	O
for	O
function	O
.	O

The	O
C	O
-	O
terminal	O
region	O
is	O
essential	O
for	O
growth	O
,	O
binds	O
Sli15	B-Gene_or_gene_product
,	O
and	O
is	O
necessary	O
and	O
sufficient	O
for	O
the	O
localization	O
of	O
Bir1	B-Gene_or_gene_product
as	O
a	O
chromosomal	B-Cellular_component
passenger	O
.	O

The	O
middle	O
region	O
is	O
not	O
essential	O
but	O
is	O
required	O
to	O
localize	O
the	O
inner	O
kinetochore	B-Cellular_component
protein	O
Ndc10	B-Gene_or_gene_product
to	O
the	O
spindle	B-Cellular_component
during	O
anaphase	O
and	O
to	O
the	O
midzone	B-Cellular_component
at	O
telophase	O
.	O

In	O
contrast	O
,	O
precise	O
deletion	O
of	O
the	O
highly	O
conserved	O
IAP	B-Gene_or_gene_product
repeats	O
conferred	O
no	O
phenotype	O
and	O
did	O
not	O
alter	O
the	O
cell	O
cycle	O
delay	O
caused	O
by	O
loss	O
of	O
cohesin	B-Gene_or_gene_product
.	O

Bir1	B-Gene_or_gene_product
is	O
phosphorylated	O
in	O
a	O
cell	O
cycle	O
-	O
dependent	O
manner	O
.	O

Mutation	O
of	O
all	O
nine	O
CDK	B-Gene_or_gene_product
consensus	O
sites	O
in	O
the	O
middle	O
region	O
of	O
Bir1	B-Gene_or_gene_product
significantly	O
decreased	O
the	O
level	O
of	O
phosphorylation	O
and	O
blocked	O
localization	O
of	O
Ndc10	B-Gene_or_gene_product
to	O
the	O
spindle	B-Cellular_component
at	O
anaphase	O
.	O

Moreover	O
,	O
immunoprecipitation	O
of	O
Ndc10	B-Gene_or_gene_product
with	O
Bir1	B-Gene_or_gene_product
was	O
dependent	O
on	O
phosphorylation	O
.	O

The	O
loss	O
of	O
Ndc10	B-Gene_or_gene_product
from	O
the	O
anaphase	O
spindle	B-Cellular_component
prevented	O
elongation	O
of	O
the	O
spindle	B-Cellular_component
beyond	O
7	O
microm	O
.	O

We	O
conclude	O
that	O
phosphorylation	O
of	O
the	O
middle	O
region	O
of	O
Bir1	B-Gene_or_gene_product
is	O
required	O
to	O
bring	O
Ndc10	B-Gene_or_gene_product
to	O
the	O
spindle	B-Cellular_component
at	O
anaphase	O
,	O
which	O
is	O
required	O
for	O
full	O
spindle	B-Cellular_component
elongation	O
.	O

Evidence	O
that	O
the	O
human	O
cytomegalovirus	O
IE2	B-Gene_or_gene_product
-	I-Gene_or_gene_product
86	I-Gene_or_gene_product
protein	O
binds	O
mdm2	B-Gene_or_gene_product
and	O
facilitates	O
mdm2	B-Gene_or_gene_product
degradation	O
.	O

Levels	O
of	O
the	O
p53	B-Gene_or_gene_product
tumor	O
suppressor	O
protein	O
are	O
increased	O
in	O
human	O
cytomegalovirus	O
(	O
HCMV	O
)	O
-	O
infected	O
cells	O
and	O
may	O
be	O
important	O
for	O
HCMV	O
pathogenesis	O
.	O

In	O
normal	O
cells	O
p53	B-Gene_or_gene_product
levels	O
are	O
kept	O
low	O
due	O
to	O
an	O
autoregulatory	O
feedback	O
loop	O
where	O
p53	B-Gene_or_gene_product
activates	O
the	O
transcription	O
of	O
mdm2	B-Gene_or_gene_product
and	O
mdm2	B-Gene_or_gene_product
binds	O
and	O
ubiquitinates	O
p53	B-Gene_or_gene_product
,	O
targeting	O
p53	B-Gene_or_gene_product
for	O
proteasomal	O
degradation	O
.	O

Here	O
we	O
report	O
that	O
,	O
in	O
contrast	O
to	O
uninfected	O
cells	O
,	O
mdm2	B-Gene_or_gene_product
was	O
undetectable	O
upon	O
treatment	O
of	O
infected	O
fibroblasts	O
with	O
the	O
proteasome	O
inhibitor	O
MG132	B-Simple_chemical
.	O

Cellular	O
depletion	O
of	O
mdm2	B-Gene_or_gene_product
was	O
reproducible	O
in	O
p53	B-Gene_or_gene_product
-	O
null	O
cells	O
transfected	O
with	O
the	O
HCMV	O
IE2	B-Gene_or_gene_product
-	I-Gene_or_gene_product
86	I-Gene_or_gene_product
protein	O
,	O
but	O
not	O
with	O
IE172	B-Gene_or_gene_product
,	O
independently	O
of	O
the	O
endogenous	O
mdm2	B-Gene_or_gene_product
promoter	O
.	O

IE2	B-Gene_or_gene_product
-	I-Gene_or_gene_product
86	I-Gene_or_gene_product
also	O
prevented	O
the	O
emergence	O
of	O
presumably	O
ubiquitinated	O
species	O
of	O
p53	B-Gene_or_gene_product
.	O

The	O
regions	O
of	O
IE2	B-Gene_or_gene_product
-	I-Gene_or_gene_product
86	I-Gene_or_gene_product
important	O
for	O
mdm2	B-Gene_or_gene_product
depletion	O
were	O
those	O
containing	O
the	O
sequences	O
corresponding	O
to	O
the	O
putative	O
zinc	B-Simple_chemical
finger	O
and	O
C	O
-	O
terminal	O
acidic	O
motifs	O
.	O

mdm2	B-Gene_or_gene_product
and	O
IE2	B-Gene_or_gene_product
-	I-Gene_or_gene_product
86	I-Gene_or_gene_product
coimmunoprecipitated	O
in	O
transfected	O
and	O
infected	O
cell	O
lysates	O
and	O
in	O
a	O
cell	O
-	O
free	O
system	O
.	O

IE2	B-Gene_or_gene_product
-	I-Gene_or_gene_product
86	I-Gene_or_gene_product
blocked	O
mdm2	B-Gene_or_gene_product
'	O
s	O
p53	B-Gene_or_gene_product
-	O
independent	O
transactivation	O
of	O
the	O
cyclin	B-Gene_or_gene_product
A	I-Gene_or_gene_product
promoter	O
in	O
transient	O
-	O
transfection	O
experiments	O
.	O

Pulse	O
-	O
chase	O
experiments	O
revealed	O
that	O
IE2	B-Gene_or_gene_product
-	I-Gene_or_gene_product
86	I-Gene_or_gene_product
but	O
not	O
IE1	B-Gene_or_gene_product
-	I-Gene_or_gene_product
72	I-Gene_or_gene_product
or	O
several	O
loss	O
-	O
of	O
-	O
function	O
IE2	B-Gene_or_gene_product
-	I-Gene_or_gene_product
86	I-Gene_or_gene_product
mutants	O
increased	O
the	O
half	O
-	O
life	O
of	O
p53	B-Gene_or_gene_product
and	O
reduced	O
the	O
half	O
-	O
life	O
of	O
mdm2	B-Gene_or_gene_product
.	O

Short	O
interfering	O
RNA	O
-	O
mediated	O
depletion	O
of	O
IE2	B-Gene_or_gene_product
-	I-Gene_or_gene_product
86	I-Gene_or_gene_product
restored	O
the	O
ability	O
of	O
HCMV	O
-	O
infected	O
cells	O
to	O
accumulate	O
mdm2	B-Gene_or_gene_product
in	O
response	O
to	O
proteasome	O
inhibition	O
.	O

Taken	O
together	O
,	O
the	O
data	O
suggest	O
that	O
specific	O
interactions	O
between	O
IE2	B-Gene_or_gene_product
-	I-Gene_or_gene_product
86	I-Gene_or_gene_product
and	O
mdm2	B-Gene_or_gene_product
cause	O
proteasome	O
-	O
independent	O
degradation	O
of	O
mdm2	B-Gene_or_gene_product
and	O
that	O
this	O
may	O
be	O
important	O
for	O
the	O
accumulation	O
of	O
p53	B-Gene_or_gene_product
in	O
HCMV	O
-	O
infected	O
cells	O
.	O

Activity	O
of	O
TSC2	B-Gene_or_gene_product
is	O
inhibited	O
by	O
AKT	B-Gene_or_gene_product
-	O
mediated	O
phosphorylation	O
and	O
membrane	B-Cellular_component
partitioning	O
.	O

Loss	O
of	O
tuberin	B-Gene_or_gene_product
,	O
the	O
product	O
of	O
TSC2	B-Gene_or_gene_product
gene	O
,	O
increases	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
signaling	O
,	O
promoting	O
cell	O
growth	O
and	O
tumor	O
development	O
.	O

However	O
,	O
in	O
cells	O
expressing	O
tuberin	B-Gene_or_gene_product
,	O
it	O
is	O
not	O
known	O
how	O
repression	O
of	O
mTOR	B-Gene_or_gene_product
signaling	O
is	O
relieved	O
to	O
activate	O
this	O
pathway	O
in	O
response	O
to	O
growth	O
factors	O
and	O
how	O
hamartin	B-Gene_or_gene_product
participates	O
in	O
this	O
process	O
.	O

We	O
show	O
that	O
hamartin	B-Gene_or_gene_product
colocalizes	O
with	O
hypophosphorylated	O
tuberin	B-Gene_or_gene_product
at	O
the	O
membrane	B-Cellular_component
,	O
where	O
tuberin	B-Gene_or_gene_product
exerts	O
its	O
GTPase	O
-	O
activating	O
protein	O
(	O
GAP	O
)	O
activity	O
to	O
repress	O
Rheb	B-Gene_or_gene_product
signaling	O
.	O

In	O
response	O
to	O
growth	O
signals	O
,	O
tuberin	B-Gene_or_gene_product
is	O
phosphorylated	O
by	O
AKT	B-Gene_or_gene_product
and	O
translocates	O
to	O
the	O
cytosol	B-Cellular_component
,	O
relieving	O
Rheb	B-Gene_or_gene_product
repression	O
.	O

Phosphorylation	O
of	O
tuberin	B-Gene_or_gene_product
at	O
serines	B-Simple_chemical
939	I-Simple_chemical
and	O
981	B-Simple_chemical
does	O
not	O
alter	O
its	O
intrinsic	O
GAP	O
activity	O
toward	O
Rheb	B-Gene_or_gene_product
but	O
partitions	O
tuberin	B-Gene_or_gene_product
to	O
the	O
cytosol	B-Cellular_component
,	O
where	O
it	O
is	O
bound	O
by	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
proteins	O
.	O

Thus	O
,	O
tuberin	B-Gene_or_gene_product
bound	O
by	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
in	O
response	O
to	O
AKT	B-Gene_or_gene_product
phosphorylation	O
is	O
sequestered	O
away	O
from	O
its	O
membrane	B-Cellular_component
-	O
bound	O
activation	O
partner	O
(	O
hamartin	B-Gene_or_gene_product
)	O
and	O
its	O
target	O
GTPase	O
(	O
Rheb	B-Gene_or_gene_product
)	O
to	O
relieve	O
the	O
growth	O
inhibitory	O
effects	O
of	O
this	O
tumor	O
suppressor	O
.	O

Adeno	O
-	O
associated	O
virus	O
type	O
2	O
increases	O
proteosome	O
-	O
dependent	O
degradation	O
of	O
p21WAF1	B-Gene_or_gene_product
in	O
a	O
human	O
papillomavirus	O
type	O
31b	O
-	O
positive	O
cervical	O
carcinoma	O
line	O
.	O

Adeno	O
-	O
associated	O
virus	O
type	O
2	O
(	O
AAV2	O
)	O
seropositivity	O
is	O
negatively	O
correlated	O
with	O
the	O
development	O
of	O
human	O
papillomavirus	O
(	O
HPV	O
)	O
-	O
associated	O
cervical	O
cancer	O
.	O

We	O
have	O
begun	O
analysis	O
of	O
the	O
molecular	O
mechanisms	O
underlying	O
AAV2	O
-	O
mediated	O
onco	O
-	O
suppression	O
through	O
cell	O
cycle	O
regulation	O
in	O
HPV	O
-	O
infected	O
keratinocytes	O
isolated	O
from	O
a	O
low	O
-	O
grade	O
cervical	O
lesion	O
.	O

AAV2	O
superinfection	O
of	O
HPV	O
type	O
31b	O
(	O
HPV31b	O
)	O
-	O
positive	O
cells	O
at	O
early	O
times	O
postinfection	O
resulted	O
in	O
degradation	O
of	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
p21	B-Gene_or_gene_product
(	O
WAF1	B-Gene_or_gene_product
)	O
protein	O
in	O
a	O
proteosome	O
-	O
dependent	O
manner	O
.	O

Downstream	O
consequences	O
of	O
lowering	O
p21	B-Gene_or_gene_product
(	O
WAF1	B-Gene_or_gene_product
)	O
levels	O
included	O
a	O
proportional	O
loss	O
of	O
cyclin	B-Complex
E	I-Complex
/	I-Complex
CDK2	I-Complex
complexes	O
bound	O
to	O
p21	B-Gene_or_gene_product
(	O
WAF1	B-Gene_or_gene_product
)	O
.	O

The	O
loss	O
of	O
stable	O
p21	B-Complex
(	I-Complex
WAF1	I-Complex
)	I-Complex
/	I-Complex
cyclin	I-Complex
E	I-Complex
/	I-Complex
CDK2	I-Complex
complexes	O
coincided	O
with	O
an	O
increase	O
in	O
CDK2	B-Gene_or_gene_product
-	I-Gene_or_gene_product
associated	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
activity	O
and	O
cyclin	B-Gene_or_gene_product
E	I-Gene_or_gene_product
levels	O
.	O

Both	O
events	O
have	O
the	O
potential	O
to	O
enhance	O
the	O
G	O
(	O
1	O
)	O
/	O
S	O
transition	O
point	O
mediated	O
by	O
active	O
cyclin	B-Complex
E	I-Complex
/	I-Complex
CDK2	I-Complex
complexes	O
.	O

Concurrently	O
,	O
cyclin	B-Gene_or_gene_product
A	I-Gene_or_gene_product
and	O
E2F	B-Gene_or_gene_product
levels	O
were	O
decreased	O
,	O
conditions	O
reminiscent	O
of	O
delayed	O
entrance	O
into	O
the	O
S	O
phase	O
of	O
the	O
cell	O
cycle	O
.	O

On	O
the	O
other	O
hand	O
,	O
infection	O
of	O
primary	O
human	O
foreskin	O
keratinocytes	O
with	O
AAV2	O
resulted	O
in	O
upregulation	O
of	O
p21	B-Gene_or_gene_product
(	O
WAF1	B-Gene_or_gene_product
)	O
protein	O
levels	O
,	O
reminiscent	O
of	O
a	O
block	O
in	O
G	O
(	O
1	O
)	O
phase	O
progression	O
.	O

We	O
propose	O
that	O
by	O
down	O
regulating	O
p21	B-Gene_or_gene_product
(	O
WAF1	B-Gene_or_gene_product
)	O
,	O
AAV2	O
initiates	O
cell	O
cycle	O
activities	O
leading	O
to	O
enhanced	O
G	O
(	O
1	O
)	O
/	O
S	O
phase	O
-	O
like	O
conditions	O
which	O
may	O
be	O
favorable	O
for	O
AAV2	O
-	O
specific	O
functions	O
and	O
may	O
lead	O
to	O
downstream	O
interference	O
with	O
HPV	O
-	O
associated	O
cervical	O
cancer	O
progression	O
.	O

Increased	O
p21	B-Gene_or_gene_product
expression	O
and	O
complex	O
formation	O
with	O
cyclin	B-Complex
E	I-Complex
/	I-Complex
CDK2	I-Complex
in	O
retinoid	O
-	O
induced	O
pre	O
-	O
B	O
lymphoma	O
cell	O
apoptosis	O
.	O

Cip	B-Gene_or_gene_product
/	O
Kip	B-Gene_or_gene_product
family	O
protein	O
p21	B-Gene_or_gene_product
,	O
a	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
,	O
is	O
directly	O
transactivated	O
by	O
retinoic	B-Gene_or_gene_product
acid	I-Gene_or_gene_product
receptor	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
(	O
RARalpha	B-Gene_or_gene_product
)	O
upon	O
retinoic	B-Simple_chemical
acid	I-Simple_chemical
(	O
RA	B-Simple_chemical
)	O
:	O
RARalpha	B-Gene_or_gene_product
binding	O
.	O

Yet	O
the	O
role	O
of	O
p21	B-Gene_or_gene_product
upregulation	O
by	O
RA	B-Simple_chemical
in	O
lymphoma	O
cells	O
remains	O
unknown	O
.	O

Here	O
,	O
we	O
show	O
that	O
,	O
in	O
human	O
pre	O
-	O
B	O
lymphoma	O
Nalm6	O
cells	O
,	O
RA	B-Simple_chemical
-	O
induced	O
proliferation	O
inhibition	O
results	O
from	O
massive	O
cell	O
death	O
characterized	O
by	O
apoptosis	O
.	O

Upregulated	O
p21	B-Gene_or_gene_product
by	O
RA	B-Simple_chemical
accompanies	O
caspase	B-Gene_or_gene_product
-	I-Gene_or_gene_product
3	I-Gene_or_gene_product
activation	O
and	O
precedes	O
the	O
occurrence	O
of	O
apoptosis	O
.	O

p21	B-Gene_or_gene_product
induction	O
leads	O
to	O
increased	O
p21	B-Gene_or_gene_product
complex	O
formation	O
with	O
cyclin	B-Complex
E	I-Complex
/	I-Complex
CDK2	I-Complex
,	O
which	O
occurs	O
when	O
cyclin	B-Gene_or_gene_product
E	I-Gene_or_gene_product
and	O
CDK2	B-Gene_or_gene_product
levels	O
remain	O
constant	O
.	O

CDK2	B-Gene_or_gene_product
can	O
alternatively	O
promote	O
apoptosis	O
,	O
but	O
the	O
mechanisms	O
remain	O
unknown	O
.	O

Data	O
presented	O
here	O
suggest	O
a	O
novel	O
RA	B-Simple_chemical
-	O
signaling	O
,	O
by	O
which	O
RA	B-Simple_chemical
-	O
induced	O
p21	B-Gene_or_gene_product
induction	O
and	O
complex	O
formation	O
with	O
cyclin	B-Complex
E	I-Complex
/	I-Complex
CDK2	I-Complex
diverts	O
CDK2	B-Gene_or_gene_product
function	O
from	O
normally	O
driving	O
proliferation	O
to	O
alternatively	O
promoting	O
apoptosis	O
.	O

p53	B-Gene_or_gene_product
transcription	O
-	O
dependent	O
and	O
-	O
independent	O
regulation	O
of	O
cerebellar	O
neural	O
precursor	O
cell	O
apoptosis	O
.	O

Regulation	O
of	O
cerebellar	O
neural	O
precursor	O
cell	O
(	O
NPC	O
)	O
death	O
is	O
important	O
for	O
both	O
normal	O
brain	O
development	O
and	O
prevention	O
of	O
brain	O
tumor	O
formation	O
.	O

The	O
tumor	O
suppressor	O
p53	B-Gene_or_gene_product
is	O
an	O
important	O
regulator	O
of	O
NPC	O
apoptosis	O
,	O
but	O
the	O
precise	O
mechanism	O
of	O
p53	B-Gene_or_gene_product
-	O
regulated	O
cerebellar	O
NPC	O
death	O
remains	O
largely	O
unknown	O
.	O

Here	O
,	O
by	O
using	O
primary	O
cerebellar	O
NPCs	O
and	O
a	O
mouse	O
cerebellar	O
NPC	O
line	O
,	O
we	O
compared	O
the	O
molecular	O
regulation	O
of	O
cerebellar	O
NPC	O
death	O
produced	O
by	O
staurosporine	B-Simple_chemical
(	O
STS	B-Simple_chemical
)	O
,	O
a	O
broad	O
-	O
spectrum	O
kinase	O
inhibitor	O
,	O
with	O
that	O
caused	O
by	O
genotoxic	O
agents	O
.	O

We	O
found	O
that	O
both	O
STS	B-Simple_chemical
-	O
and	O
genotoxin	O
-	O
induced	O
cerebellar	O
NPC	O
death	O
were	O
markedly	O
inhibited	O
by	O
p53	B-Gene_or_gene_product
or	O
Bax	B-Gene_or_gene_product
deficiency	O
.	O

Genotoxin	O
-	O
induced	O
cerebellar	O
NPC	O
death	O
required	O
new	O
protein	O
synthesis	O
and	O
PUMA	B-Gene_or_gene_product
,	O
a	O
p53	B-Gene_or_gene_product
transcriptionally	O
regulated	O
BH3	O
-	O
only	O
molecule	O
.	O

In	O
contrast	O
,	O
STS	B-Simple_chemical
caused	O
cerebellar	O
NPC	O
death	O
without	O
requiring	O
new	O
protein	O
synthesis	O
or	O
PUMA	B-Gene_or_gene_product
expression	O
.	O

In	O
addition	O
,	O
genotoxic	O
agents	O
increased	O
nuclear	B-Cellular_component
p53	B-Gene_or_gene_product
immunoreactivity	O
,	O
whereas	O
STS	B-Simple_chemical
produced	O
rapid	O
cytoplasmic	B-Cellular_component
p53	B-Gene_or_gene_product
accumulation	O
.	O

Interestingly	O
,	O
STS	B-Simple_chemical
-	O
induced	O
death	O
of	O
cerebellar	O
granule	O
neurons	O
was	O
p53	B-Gene_or_gene_product
-	O
independent	O
,	O
indicating	O
a	O
differentiation	O
-	O
dependent	O
feature	O
of	O
neuronal	O
apoptotic	O
regulation	O
.	O

These	O
results	O
suggest	O
that	O
STS	B-Simple_chemical
-	O
induced	O
cerebellar	O
NPC	O
death	O
requires	O
a	O
direct	O
effect	O
of	O
p53	B-Gene_or_gene_product
on	O
cytoplasmic	B-Cellular_component
apoptotic	O
mediators	O
,	O
whereas	O
genotoxin	O
-	O
induced	O
death	O
requires	O
p53	B-Gene_or_gene_product
-	O
dependent	O
gene	O
transcription	O
of	O
PUMA	B-Gene_or_gene_product
.	O

Thus	O
,	O
p53	B-Gene_or_gene_product
has	O
multiple	O
death	O
promoting	O
mechanisms	O
in	O
cerebellar	O
NPCs	O
.	O

Cdk	B-Gene_or_gene_product
-	O
inhibitory	O
activity	O
and	O
stability	O
of	O
p27Kip1	B-Gene_or_gene_product
are	O
directly	O
regulated	O
by	O
oncogenic	O
tyrosine	B-Simple_chemical
kinases	O
.	O

p27Kip1	B-Gene_or_gene_product
controls	O
cell	O
proliferation	O
by	O
binding	O
to	O
and	O
regulating	O
the	O
activity	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
.	O

Here	O
we	O
show	O
that	O
Cdk	B-Gene_or_gene_product
inhibition	O
and	O
p27	B-Gene_or_gene_product
stability	O
are	O
regulated	O
through	O
direct	O
phosphorylation	O
by	O
tyrosine	B-Simple_chemical
kinases	O
.	O

A	O
conserved	O
tyrosine	B-Simple_chemical
residue	O
(	O
Y88	B-Simple_chemical
)	O
in	O
the	O
Cdk	B-Gene_or_gene_product
-	O
binding	O
domain	O
of	O
p27	B-Gene_or_gene_product
can	O
be	O
phosphorylated	O
by	O
the	O
Src	B-Gene_or_gene_product
-	O
family	O
kinase	O
Lyn	B-Gene_or_gene_product
and	O
the	O
oncogene	O
product	O
BCR	B-Gene_or_gene_product
-	I-Gene_or_gene_product
ABL	I-Gene_or_gene_product
.	O

Y88	B-Simple_chemical
phosphorylation	O
does	O
not	O
prevent	O
p27	B-Gene_or_gene_product
binding	O
to	O
cyclin	B-Complex
A	I-Complex
/	I-Complex
Cdk2	I-Complex
.	O

Instead	O
,	O
it	O
causes	O
phosphorylated	O
Y88	B-Simple_chemical
and	O
the	O
entire	O
inhibitory	O
3	O
(	O
10	O
)	O
-	O
helix	O
of	O
p27	B-Gene_or_gene_product
to	O
be	O
ejected	O
from	O
the	O
Cdk2	B-Gene_or_gene_product
active	O
site	O
,	O
thus	O
restoring	O
partial	O
Cdk	B-Gene_or_gene_product
activity	O
.	O

Importantly	O
,	O
this	O
allows	O
Y88	B-Simple_chemical
-	O
phosphorylated	O
p27	B-Gene_or_gene_product
to	O
be	O
efficiently	O
phosphorylated	O
on	O
threonine	B-Simple_chemical
187	I-Simple_chemical
by	O
Cdk2	B-Gene_or_gene_product
which	O
in	O
turn	O
promotes	O
its	O
SCF	O
-	O
Skp2	B-Gene_or_gene_product
-	O
dependent	O
degradation	O
.	O

This	O
direct	O
link	O
between	O
transforming	O
tyrosine	B-Simple_chemical
kinases	O
and	O
p27	B-Gene_or_gene_product
may	O
provide	O
an	O
explanation	O
for	O
Cdk	B-Gene_or_gene_product
kinase	O
activities	O
observed	O
in	O
p27	B-Gene_or_gene_product
complexes	O
and	O
for	O
premature	O
p27	B-Gene_or_gene_product
elimination	O
in	O
cells	O
that	O
have	O
been	O
transformed	O
by	O
activated	O
tyrosine	B-Simple_chemical
kinases	O
.	O

A	O
systems	O
-	O
biology	O
analysis	O
of	O
feedback	O
inhibition	O
in	O
the	O
Sho1	B-Gene_or_gene_product
osmotic	O
-	O
stress	O
-	O
response	O
pathway	O
.	O

BACKGROUND	O
:	O
A	O
common	O
property	O
of	O
signal	O
transduction	O
systems	O
is	O
that	O
they	O
rapidly	O
lose	O
their	O
ability	O
to	O
respond	O
to	O
a	O
given	O
stimulus	O
.	O

For	O
instance	O
in	O
yeast	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
(	I-Gene_or_gene_product
MAP	I-Gene_or_gene_product
)	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
Hog1	B-Gene_or_gene_product
is	O
activated	O
and	O
inactivated	O
within	O
minutes	O
,	O
even	O
when	O
the	O
osmotic	O
-	O
stress	O
stimulus	O
is	O
sustained	O
.	O

RESULTS	O
:	O
Here	O
,	O
we	O
used	O
a	O
combination	O
of	O
experimental	O
and	O
computational	O
analyses	O
to	O
investigate	O
the	O
dynamic	O
behavior	O
of	O
Hog1	B-Gene_or_gene_product
activation	O
in	O
vivo	O
.	O

Computational	O
modeling	O
suggested	O
that	O
a	O
negative	O
-	O
feedback	O
loop	O
operates	O
early	O
in	O
the	O
pathway	O
and	O
leads	O
to	O
rapid	O
attenuation	O
of	O
Hog1	B-Gene_or_gene_product
signaling	O
.	O

Experimental	O
analysis	O
revealed	O
that	O
the	O
membrane	B-Cellular_component
-	O
bound	O
osmosensor	O
Sho1	B-Gene_or_gene_product
is	O
phosphorylated	O
by	O
Hog1	B-Gene_or_gene_product
and	O
that	O
phosphorylation	O
occurs	O
on	O
Ser	B-Simple_chemical
-	I-Simple_chemical
166	I-Simple_chemical
.	O

Moreover	O
,	O
Sho1	B-Gene_or_gene_product
exists	O
in	O
a	O
homo	O
-	O
oligomeric	O
complex	O
,	O
and	O
phosphorylation	O
by	O
Hog1	B-Gene_or_gene_product
promotes	O
a	O
transition	O
from	O
the	O
oligomeric	O
to	O
monomeric	O
state	O
.	O

A	O
phosphorylation	O
-	O
site	O
mutation	O
(	O
Sho1	B-Gene_or_gene_product
(	I-Gene_or_gene_product
S166E	I-Gene_or_gene_product
)	I-Gene_or_gene_product
)	O
diminishes	O
the	O
formation	O
of	O
Sho1	B-Gene_or_gene_product
-	O
oligomers	O
,	O
dampens	O
activation	O
of	O
the	O
Hog1	B-Gene_or_gene_product
kinase	O
,	O
and	O
impairs	O
growth	O
in	O
high	O
-	O
salt	B-Simple_chemical
or	O
sorbitol	B-Simple_chemical
conditions	O
.	O

CONCLUSIONS	O
:	O
These	O
findings	O
reveal	O
a	O
novel	O
phosphorylation	O
-	O
dependent	O
feedback	O
loop	O
leading	O
to	O
diminished	O
cellular	O
responses	O
to	O
an	O
osmotic	O
-	O
stress	O
stimulus	O
.	O

The	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
is	O
a	O
physiological	O
substrate	O
of	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
complex	I-Gene_or_gene_product
1	I-Gene_or_gene_product
.	O

The	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
kilodaltons	I-Gene_or_gene_product
(	O
PRAS40	B-Gene_or_gene_product
)	O
was	O
identified	O
as	O
a	O
raptor	B-Gene_or_gene_product
-	O
binding	O
protein	O
that	O
is	O
phosphorylated	O
directly	O
by	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
but	O
not	O
mTORC2	B-Complex
in	O
vitro	O
,	O
predominantly	O
at	O
PRAS40	B-Gene_or_gene_product
(	O
Ser	B-Simple_chemical
(	I-Simple_chemical
183	I-Simple_chemical
)	I-Simple_chemical
)	O
.	O

The	O
binding	O
of	O
S6K1	B-Gene_or_gene_product
and	O
4E	B-Gene_or_gene_product
-	I-Gene_or_gene_product
BP1	I-Gene_or_gene_product
to	O
raptor	B-Gene_or_gene_product
requires	O
a	O
TOR	B-Gene_or_gene_product
signaling	O
(	O
TOS	O
)	O
motif	O
,	O
which	O
contains	O
an	O
essential	O
Phe	B-Simple_chemical
followed	O
by	O
four	O
alternating	O
acidic	O
and	O
small	O
hydrophobic	O
amino	O
acids	O
.	O

PRAS40	B-Gene_or_gene_product
binding	O
to	O
raptor	B-Gene_or_gene_product
was	O
severely	O
inhibited	O
by	O
mutation	O
of	O
PRAS40	B-Gene_or_gene_product
(	O
Phe	B-Simple_chemical
(	I-Simple_chemical
129	I-Simple_chemical
)	I-Simple_chemical
to	O
Ala	B-Simple_chemical
)	O
.	O

Immediately	O
carboxyl	O
-	O
terminal	O
to	O
Phe	B-Simple_chemical
(	I-Simple_chemical
129	I-Simple_chemical
)	I-Simple_chemical
are	O
two	O
small	O
hydrophobic	O
amino	O
acid	O
followed	O
by	O
two	O
acidic	O
residues	O
.	O

PRAS40	B-Gene_or_gene_product
binding	O
to	O
raptor	B-Gene_or_gene_product
was	O
also	O
abolished	O
by	O
mutation	O
of	O
the	O
major	O
mTORC1	B-Complex
phosphorylation	O
site	O
,	O
Ser	B-Simple_chemical
(	I-Simple_chemical
183	I-Simple_chemical
)	I-Simple_chemical
,	O
to	O
Asp	B-Simple_chemical
.	O

PRAS40	B-Gene_or_gene_product
(	O
Ser	B-Simple_chemical
(	I-Simple_chemical
183	I-Simple_chemical
)	I-Simple_chemical
)	O
was	O
phosphorylated	O
in	O
intact	O
cells	O
;	O
this	O
phosphorylation	O
was	O
inhibited	O
by	O
rapamycin	B-Simple_chemical
,	O
by	O
2	B-Simple_chemical
-	I-Simple_chemical
deoxyglucose	I-Simple_chemical
,	O
and	O
by	O
overexpression	O
of	O
the	O
tuberous	B-Complex
sclerosis	I-Complex
complex	I-Complex
heterodimer	O
.	O

PRAS40	B-Gene_or_gene_product
(	O
Ser	B-Simple_chemical
(	I-Simple_chemical
183	I-Simple_chemical
)	I-Simple_chemical
)	O
phosphorylation	O
was	O
also	O
inhibited	O
reversibly	O
by	O
withdrawal	O
of	O
all	O
or	O
of	O
only	O
the	O
branched	O
chain	O
amino	O
acids	O
;	O
this	O
inhibition	O
was	O
reversed	O
by	O
overexpression	O
of	O
the	O
Rheb	B-Gene_or_gene_product
GTPase	O
.	O

Overexpressed	O
PRAS40	B-Gene_or_gene_product
suppressed	O
the	O
phosphorylation	O
of	O
S6K1	B-Gene_or_gene_product
and	O
4E	B-Gene_or_gene_product
-	I-Gene_or_gene_product
BP1	I-Gene_or_gene_product
at	O
their	O
rapamycin	B-Simple_chemical
-	O
sensitive	O
phosphorylation	O
sites	O
,	O
and	O
reciprocally	O
,	O
overexpression	O
of	O
S6K1	B-Gene_or_gene_product
or	O
4E	B-Gene_or_gene_product
-	I-Gene_or_gene_product
BP1	I-Gene_or_gene_product
suppressed	O
phosphorylation	O
of	O
PRAS40	B-Gene_or_gene_product
(	O
Ser	B-Simple_chemical
(	I-Simple_chemical
183	I-Simple_chemical
)	I-Simple_chemical
)	O
and	O
its	O
binding	O
to	O
raptor	B-Gene_or_gene_product
.	O

RNA	O
interference	O
-	O
induced	O
depletion	O
of	O
PRAS40	B-Gene_or_gene_product
enhanced	O
the	O
amino	O
acid	O
-	O
stimulated	O
phosphorylation	O
of	O
both	O
S6K1	B-Gene_or_gene_product
and	O
4E	B-Gene_or_gene_product
-	I-Gene_or_gene_product
BP1	I-Gene_or_gene_product
.	O

These	O
results	O
establish	O
PRAS40	B-Gene_or_gene_product
as	O
a	O
physiological	O
mTORC1	B-Complex
substrate	O
that	O
contains	O
a	O
variant	O
TOS	O
motif	O
.	O

Moreover	O
,	O
they	O
indicate	O
that	O
the	O
ability	O
of	O
raptor	B-Gene_or_gene_product
to	O
bind	O
endogenous	O
substrates	O
is	O
limiting	O
for	O
the	O
activity	O
of	O
mTORC1	B-Complex
in	O
vivo	O
and	O
is	O
therefore	O
a	O
potential	O
locus	O
of	O
regulation	O
.	O

MDM2	B-Gene_or_gene_product
mRNA	O
expression	O
in	O
the	O
p53	B-Gene_or_gene_product
pathway	O
may	O
predict	O
the	O
potential	O
of	O
invasion	O
and	O
liver	O
metastasis	O
in	O
colorectal	O
cancer	O
.	O

PURPOSE	O
:	O
The	O
p53	B-Gene_or_gene_product
/	O
MDM2	B-Gene_or_gene_product
/	O
p14ARF	B-Gene_or_gene_product
pathway	O
is	O
one	O
of	O
the	O
major	O
signaling	O
cascades	O
involved	O
in	O
the	O
regulation	O
of	O
apoptosis	O
.	O

Although	O
many	O
tumors	O
have	O
been	O
reported	O
to	O
show	O
disruption	O
of	O
the	O
p53	B-Gene_or_gene_product
/	O
MDM2	B-Gene_or_gene_product
/	O
p14ARF	B-Gene_or_gene_product
pathway	O
,	O
few	O
studies	O
have	O
examined	O
p53	B-Gene_or_gene_product
,	O
MDM2	B-Gene_or_gene_product
,	O
and	O
p14ARF	B-Gene_or_gene_product
simultaneously	O
in	O
colorectal	O
carcinoma	O
.	O

The	O
present	O
study	O
was	O
undertaken	O
to	O
clarify	O
whether	O
correlations	O
exist	O
among	O
MDM2	B-Gene_or_gene_product
,	O
p53	B-Gene_or_gene_product
,	O
and	O
p14ARF	B-Gene_or_gene_product
in	O
colorectal	O
cancer	O
.	O

METHODS	O
:	O
We	O
determined	O
the	O
presence	O
of	O
mutations	O
in	O
the	O
p53	B-Gene_or_gene_product
gene	O
,	O
MDM2	B-Gene_or_gene_product
expression	O
,	O
and	O
methylation	O
status	O
of	O
the	O
p14ARF	B-Gene_or_gene_product
in	O
97	O
primary	O
colorectal	O
carcinoma	O
specimens	O
.	O

Associations	O
with	O
survival	O
and	O
clinicopathologic	O
factors	O
were	O
investigated	O
.	O

RESULTS	O
:	O
At	O
least	O
one	O
abnormality	O
of	O
these	O
three	O
molecules	O
was	O
found	O
in	O
82	O
(	O
84	O
percent	O
)	O
tumors	O
.	O

We	O
observed	O
a	O
significant	O
inverse	O
association	O
between	O
MDM2	B-Gene_or_gene_product
expression	O
and	O
tumor	O
invasion	O
(	O
P	O
=	O
0	O
.	O
01	O
)	O
.	O

Furthermore	O
,	O
the	O
presence	O
of	O
liver	O
metastasis	O
was	O
also	O
significantly	O
associated	O
with	O
low	O
MDM2	B-Gene_or_gene_product
expression	O
(	O
P	O
=	O
0	O
.	O
02	O
)	O
.	O

CONCLUSIONS	O
:	O
The	O
results	O
suggest	O
that	O
disruption	O
of	O
the	O
p53	B-Gene_or_gene_product
/	O
MDM2	B-Gene_or_gene_product
/	O
p14ARF	B-Gene_or_gene_product
pathway	O
may	O
frequently	O
participate	O
in	O
colonic	O
carcinogenesis	O
and	O
that	O
MDM2	B-Gene_or_gene_product
expression	O
status	O
may	O
be	O
a	O
factor	O
in	O
the	O
prediction	O
of	O
potential	O
invasion	O
and	O
liver	O
metastasis	O
of	O
colorectal	O
carcinomas	O
.	O

Eco1	B-Gene_or_gene_product
-	O
dependent	O
cohesin	B-Complex
acetylation	O
during	O
establishment	O
of	O
sister	B-Cellular_component
chromatid	I-Cellular_component
cohesion	O
.	O

Replicated	O
chromosomes	B-Cellular_component
are	O
held	O
together	O
by	O
the	O
chromosomal	B-Cellular_component
cohesin	B-Complex
complex	O
from	O
the	O
time	O
of	O
their	O
synthesis	O
in	O
S	O
phase	O
onward	O
.	O

This	O
requires	O
the	O
replication	O
fork	O
-	O
associated	O
acetyl	B-Simple_chemical
transferase	O
Eco1	B-Gene_or_gene_product
,	O
but	O
Eco1	B-Gene_or_gene_product
'	O
s	O
mechanism	O
of	O
action	O
is	O
not	O
known	O
.	O

We	O
identified	O
spontaneous	O
suppressors	O
of	O
the	O
thermosensitive	O
eco1	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
allele	O
in	O
budding	O
yeast	O
.	O

An	O
acetylation	O
-	O
mimicking	O
mutation	O
of	O
a	O
conserved	O
lysine	B-Simple_chemical
in	O
cohesin	B-Complex
'	O
s	O
Smc3	B-Gene_or_gene_product
subunit	O
makes	O
Eco1	B-Gene_or_gene_product
dispensable	O
for	O
cell	O
growth	O
,	O
and	O
we	O
show	O
that	O
Smc3	B-Gene_or_gene_product
is	O
acetylated	O
in	O
an	O
Eco1	B-Gene_or_gene_product
-	O
dependent	O
manner	O
during	O
DNA	O
replication	O
to	O
promote	O
sister	B-Cellular_component
chromatid	I-Cellular_component
cohesion	O
.	O

A	O
second	O
set	O
of	O
eco1	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
suppressors	O
inactivate	O
the	O
budding	O
yeast	O
ortholog	O
of	O
the	O
cohesin	B-Complex
destabilizer	O
Wapl	B-Gene_or_gene_product
.	O

Our	O
results	O
indicate	O
that	O
Eco1	B-Gene_or_gene_product
modifies	O
cohesin	B-Complex
to	O
stabilize	O
sister	B-Cellular_component
chromatid	I-Cellular_component
cohesion	O
in	O
parallel	O
with	O
a	O
cohesion	O
establishment	O
reaction	O
that	O
is	O
in	O
principle	O
Eco1	B-Gene_or_gene_product
-	O
independent	O
.	O

Influence	O
of	O
tetramerisation	O
on	O
site	O
-	O
specific	O
post	O
-	O
translational	O
modifications	O
of	O
p53	B-Gene_or_gene_product
:	O
comparison	O
of	O
human	O
and	O
murine	O
p53	B-Gene_or_gene_product
tumor	O
suppressor	O
protein	O
.	O

The	O
tumor	O
suppressor	O
protein	O
p53	B-Gene_or_gene_product
is	O
considered	O
the	O
"	O
Guardian	O
of	O
the	O
Genome	O
"	O
,	O
crucial	O
for	O
cell	O
cycle	O
control	O
and	O
mutated	O
in	O
over	O
50	O
%	O
of	O
human	O
cancers	O
.	O

Following	O
cellular	O
stress	O
,	O
post	O
-	O
translational	O
modifications	O
such	O
as	O
phosphorylation	O
and	O
acetylation	O
stabilise	O
and	O
activate	O
p53	B-Gene_or_gene_product
for	O
cell	O
cycle	O
arrest	O
,	O
DNA	O
repair	O
,	O
apoptosis	O
or	O
senescence	O
.	O

p53	B-Gene_or_gene_product
protein	O
functions	O
as	O
a	O
tetramer	O
and	O
we	O
have	O
shown	O
that	O
loss	O
of	O
tetramerisation	O
and	O
changes	O
at	O
the	O
N	O
-	O
terminus	O
influence	O
the	O
recovery	O
of	O
wild	O
type	O
p53	B-Gene_or_gene_product
'	O
status	O
'	O
.	O

To	O
investigate	O
the	O
relationship	O
between	O
tetramerisation	O
and	O
post	O
-	O
translational	O
modifications	O
we	O
examined	O
a	O
range	O
of	O
site	O
-	O
specific	O
modifications	O
in	O
wild	O
type	O
and	O
dimeric	O
mutant	O
(	O
M340Q	O
/	O
L344R	O
)	O
murine	O
p53	B-Gene_or_gene_product
expressed	O
in	O
MEFs	O
p53	B-Gene_or_gene_product
(	O
-	O
/	O
-	O
)	O
and	O
in	O
wild	O
type	O
,	O
monomeric	O
(	O
L344P	O
)	O
and	O
dimeric	O
(	O
M340Q	O
/	O
L344R	O
)	O
human	O
p53	B-Gene_or_gene_product
expressed	O
in	O
HCT116	O
p53	B-Gene_or_gene_product
(	O
-	O
/	O
-	O
)	O
cells	O
.	O

Using	O
site	O
-	O
specific	O
antibodies	O
we	O
demonstrate	O
that	O
in	O
murine	O
p53	B-Gene_or_gene_product
,	O
S15	B-Simple_chemical
is	O
phosphorylated	O
in	O
a	O
tetramerisation	O
-	O
dependent	O
manner	O
.	O

In	O
contrast	O
,	O
human	O
p53	B-Gene_or_gene_product
S15	B-Simple_chemical
phosphorylation	O
is	O
not	O
tetramerisation	O
-	O
dependent	O
.	O

Inability	O
to	O
form	O
tetramers	O
in	O
human	O
p53	B-Gene_or_gene_product
proteins	O
reduced	O
site	O
-	O
specific	O
N	O
-	O
terminal	O
phosphorylation	O
at	O
S6	B-Simple_chemical
,	O
S9	B-Simple_chemical
and	O
S46	B-Simple_chemical
and	O
reduced	O
C	O
-	O
terminal	O
phosphorylation	O
and	O
acetylation	O
at	O
S315	B-Simple_chemical
and	O
K382	B-Simple_chemical
respectively	O
.	O

In	O
addition	O
,	O
p53	B-Gene_or_gene_product
tetramerisation	O
is	O
required	O
for	O
efficient	O
p21	B-Gene_or_gene_product
and	O
hdm2	B-Gene_or_gene_product
transcription	O
and	O
protein	O
expression	O
and	O
recruitment	O
of	O
p53	B-Gene_or_gene_product
to	O
specific	O
promoter	O
regions	O
of	O
p21	B-Gene_or_gene_product
and	O
hdm2	B-Gene_or_gene_product
.	O

Analysis	O
of	O
chemical	O
shift	O
changes	O
reveals	O
the	O
binding	O
modes	O
of	O
isoindolinone	B-Simple_chemical
inhibitors	O
of	O
the	O
MDM2	B-Gene_or_gene_product
-	O
p53	B-Gene_or_gene_product
interaction	O
.	O

In	O
this	O
study	O
we	O
present	O
a	O
method	O
for	O
defining	O
the	O
binding	O
modes	O
of	O
a	O
set	O
of	O
structurally	O
related	O
isoindolinone	B-Simple_chemical
inhibitors	O
of	O
the	O
MDM2	B-Gene_or_gene_product
-	O
p53	B-Gene_or_gene_product
interaction	O
.	O

This	O
approach	O
derives	O
the	O
location	O
and	O
orientation	O
of	O
isoindolinone	B-Simple_chemical
binding	O
,	O
based	O
on	O
an	O
analysis	O
of	O
the	O
patterns	O
of	O
magnitude	O
and	O
direction	O
of	O
chemical	O
shift	O
perturbations	O
for	O
a	O
series	O
of	O
inhibitors	O
of	O
the	O
MDM2	B-Gene_or_gene_product
-	O
p53	B-Gene_or_gene_product
interaction	O
.	O

The	O
MDM2	B-Complex
-	I-Complex
p53	I-Complex
complex	O
is	O
an	O
attractive	O
target	O
for	O
therapeutic	O
intervention	O
in	O
cancer	O
cells	O
with	O
intact	O
tumor	O
suppressor	O
p53	B-Gene_or_gene_product
,	O
as	O
it	O
offers	O
the	O
possibility	O
of	O
releasing	O
p53	B-Gene_or_gene_product
by	O
blocking	O
the	O
MDM2	B-Gene_or_gene_product
-	O
p53	B-Gene_or_gene_product
binding	O
site	O
with	O
a	O
small	O
molecule	O
antagonist	O
to	O
promote	O
apoptosis	O
.	O

Isoindolinones	B-Simple_chemical
are	O
a	O
novel	O
class	O
of	O
MDM2	B-Gene_or_gene_product
-	O
antagonists	O
of	O
moderate	O
affinity	O
,	O
which	O
still	O
require	O
the	O
development	O
of	O
more	O
potent	O
candidates	O
for	O
clinical	O
applications	O
.	O

As	O
the	O
applicability	O
of	O
conventional	O
structural	O
methods	O
to	O
this	O
system	O
is	O
limited	O
by	O
a	O
number	O
of	O
fundamental	O
factors	O
,	O
the	O
exploitation	O
of	O
the	O
information	O
contained	O
in	O
chemical	O
shift	O
perturbations	O
has	O
offered	O
a	O
useful	O
route	O
to	O
obtaining	O
structural	O
information	O
to	O
guide	O
the	O
development	O
of	O
more	O
potent	O
compounds	O
.	O

For	O
a	O
set	O
of	O
12	O
structurally	O
related	O
isoindolinones	B-Simple_chemical
,	O
the	O
data	O
suggests	O
4	O
different	O
orientations	O
of	O
binding	O
,	O
caused	O
by	O
subtle	O
changes	O
in	O
the	O
chemical	O
structure	O
of	O
the	O
inhibitors	O
.	O

SMAR1	B-Gene_or_gene_product
forms	O
a	O
ternary	O
complex	O
with	O
p53	B-Gene_or_gene_product
-	O
MDM2	B-Gene_or_gene_product
and	O
negatively	O
regulates	O
p53	B-Gene_or_gene_product
-	O
mediated	O
transcription	O
.	O

The	O
intra	O
-	O
cellular	O
level	O
of	O
tumor	O
suppressor	O
protein	O
p53	B-Gene_or_gene_product
is	O
tightly	O
controlled	O
by	O
an	O
autoregulatory	O
feedback	O
loop	O
between	O
the	O
protein	O
and	O
its	O
negative	O
regulator	O
MDM2	B-Gene_or_gene_product
.	O

The	O
role	O
of	O
MDM2	B-Gene_or_gene_product
in	O
down	O
-	O
regulating	O
the	O
p53	B-Gene_or_gene_product
response	O
in	O
unstressed	O
conditions	O
and	O
in	O
the	O
post	O
-	O
stress	O
recovery	O
phase	O
is	O
well	O
documented	O
.	O

However	O
,	O
interplay	O
between	O
the	O
N	O
-	O
terminal	O
phosphorylations	O
and	O
C	O
-	O
terminal	O
acetylations	O
of	O
p53	B-Gene_or_gene_product
in	O
this	O
context	O
remains	O
unclear	O
.	O

Here	O
,	O
we	O
show	O
that	O
an	O
MAR	B-Gene_or_gene_product
binding	O
protein	O
SMAR1	B-Gene_or_gene_product
interacts	O
with	O
MDM2	B-Gene_or_gene_product
and	O
the	O
Ser15	B-Simple_chemical
phosphorylated	O
form	O
of	O
p53	B-Gene_or_gene_product
,	O
forming	O
a	O
ternary	O
complex	O
in	O
the	O
post	O
stress	O
-	O
recovery	O
phase	O
.	O

This	O
triple	O
complex	O
formation	O
between	O
p53	B-Gene_or_gene_product
,	O
MDM2	B-Gene_or_gene_product
and	O
SMAR1	B-Gene_or_gene_product
results	O
in	O
recruitment	O
of	O
HDAC1	B-Gene_or_gene_product
to	O
deacetylate	O
p53	B-Gene_or_gene_product
.	O

The	O
deacetylated	O
p53	B-Gene_or_gene_product
binds	O
poorly	O
to	O
the	O
target	O
promoter	O
(	O
p21	B-Gene_or_gene_product
)	O
,	O
which	O
results	O
in	O
switching	O
off	O
the	O
p53	B-Gene_or_gene_product
response	O
,	O
essential	O
for	O
re	O
-	O
entry	O
into	O
the	O
cell	O
cycle	O
.	O

Interestingly	O
,	O
the	O
knock	O
-	O
down	O
of	O
SMAR1	B-Gene_or_gene_product
using	O
siRNA	O
leads	O
to	O
a	O
prolonged	O
cell	O
-	O
cycle	O
arrest	O
in	O
the	O
post	O
stress	O
recovery	O
phase	O
due	O
to	O
ablation	O
of	O
p53	B-Gene_or_gene_product
-	O
MDM2	B-Gene_or_gene_product
-	O
HDAC1	B-Gene_or_gene_product
interaction	O
.	O

Thus	O
,	O
the	O
results	O
presented	O
here	O
for	O
the	O
first	O
time	O
highlight	O
the	O
role	O
of	O
SMAR1	B-Gene_or_gene_product
in	O
masking	O
the	O
active	O
phosphorylation	O
site	O
of	O
p53	B-Gene_or_gene_product
,	O
enabling	O
the	O
deacetylation	O
of	O
p53	B-Gene_or_gene_product
by	O
HDAC1	B-Complex
-	I-Complex
MDM2	I-Complex
complex	O
,	O
thereby	O
regulating	O
the	O
p53	B-Gene_or_gene_product
transcriptional	O
response	O
during	O
stress	O
rescue	O
.	O

Modification	O
of	O
cysteine	B-Simple_chemical
179	I-Simple_chemical
of	O
IkappaBalpha	B-Gene_or_gene_product
kinase	O
by	O
nimbolide	B-Simple_chemical
leads	O
to	O
down	O
-	O
regulation	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
-	O
regulated	O
cell	O
survival	O
and	O
proliferative	O
proteins	O
and	O
sensitization	O
of	O
tumor	O
cells	O
to	O
chemotherapeutic	O
agents	O
.	O

Reverse	O
pharmacology	O
,	O
also	O
called	O
the	O
"	O
bedside	O
to	O
bench	O
"	O
approach	O
,	O
that	O
deals	O
with	O
new	O
uses	O
for	O
a	O
well	O
known	O
molecular	O
entity	O
has	O
been	O
used	O
extensively	O
in	O
cancer	O
drug	O
development	O
to	O
identify	O
novel	O
compounds	O
and	O
delineate	O
their	O
mechanisms	O
of	O
action	O
.	O

Here	O
,	O
we	O
show	O
that	O
nimbolide	B-Simple_chemical
,	O
a	O
triterpenoid	B-Simple_chemical
isolated	O
from	O
Azadirachta	O
indica	O
,	O
enhanced	O
the	O
apoptosis	O
induced	O
by	O
inflammatory	O
cytokines	O
and	O
chemotherapeutic	O
agents	O
in	O
tumor	O
cells	O
.	O

This	O
limonoid	B-Simple_chemical
abrogated	O
the	O
expression	O
of	O
proteins	O
associated	O
with	O
cell	O
survival	O
(	O
Bcl	B-Gene_or_gene_product
-	I-Gene_or_gene_product
2	I-Gene_or_gene_product
,	O
Bcl	B-Gene_or_gene_product
-	I-Gene_or_gene_product
xL	I-Gene_or_gene_product
,	O
IAP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
,	O
and	O
IAP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
2	I-Gene_or_gene_product
)	O
,	O
proliferation	O
(	O
cyclin	B-Gene_or_gene_product
D1	I-Gene_or_gene_product
)	O
,	O
invasion	O
(	O
MMP	B-Gene_or_gene_product
-	I-Gene_or_gene_product
9	I-Gene_or_gene_product
)	O
,	O
and	O
angiogenesis	O
(	O
VEGF	B-Gene_or_gene_product
)	O
,	O
all	O
regulated	O
by	O
nuclear	B-Complex
factor	I-Complex
(	I-Complex
NF	I-Complex
)	I-Complex
-	I-Complex
kappaB	I-Complex
.	O

Nimbolide	O
inhibited	O
the	O
activation	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
induced	O
by	O
carcinogens	O
and	O
inflammatory	O
stimuli	O
.	O

Constitutively	O
active	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
found	O
in	O
most	O
tumor	O
cells	O
was	O
also	O
inhibited	O
.	O

We	O
found	O
that	O
suppression	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
activation	O
by	O
nimbolide	B-Simple_chemical
was	O
caused	O
by	O
inhibition	O
of	O
IkappaB	B-Complex
kinase	I-Complex
(	O
IKK	B-Complex
)	O
,	O
which	O
led	O
to	O
suppression	O
of	O
IkappaBalpha	B-Gene_or_gene_product
phosphorylation	O
and	O
degradation	O
,	O
nuclear	B-Cellular_component
translocation	O
,	O
DNA	O
binding	O
,	O
and	O
gene	O
transcription	O
.	O

Reducing	O
agent	O
reversed	O
the	O
action	O
of	O
the	O
limonoid	B-Simple_chemical
,	O
suggesting	O
the	O
involvement	O
of	O
a	O
cysteine	B-Simple_chemical
residue	O
.	O

Replacement	O
of	O
Cys	B-Simple_chemical
(	I-Simple_chemical
179	I-Simple_chemical
)	I-Simple_chemical
of	O
IKK	B-Gene_or_gene_product
-	I-Gene_or_gene_product
beta	I-Gene_or_gene_product
with	O
alanine	B-Simple_chemical
abolished	O
the	O
effect	O
of	O
nimbolide	B-Simple_chemical
,	O
suggesting	O
that	O
Cys	B-Simple_chemical
(	I-Simple_chemical
179	I-Simple_chemical
)	I-Simple_chemical
plays	O
a	O
critical	O
role	O
in	O
inhibiting	O
the	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
activation	O
.	O

Overall	O
,	O
our	O
results	O
indicate	O
that	O
nimbolide	B-Simple_chemical
can	O
sensitize	O
tumor	O
cells	O
to	O
chemotherapeutic	O
agents	O
through	O
interaction	O
with	O
IKK	B-Complex
,	O
leading	O
to	O
inhibition	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
-	O
regulated	O
proteins	O
.	O

Phosphorylation	O
of	O
p50	B-Gene_or_gene_product
NF	B-Complex
-	I-Complex
kappaB	I-Complex
at	O
a	O
single	O
serine	B-Simple_chemical
residue	O
by	O
DNA	B-Complex
-	I-Complex
dependent	I-Complex
protein	I-Complex
kinase	I-Complex
is	O
critical	O
for	O
VCAM	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
expression	O
upon	O
TNF	B-Gene_or_gene_product
treatment	O
.	O

The	O
DNA	O
binding	O
activity	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
is	O
critical	O
for	O
VCAM	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
expression	O
during	O
inflammation	O
.	O

DNA	B-Complex
-	I-Complex
dependent	I-Complex
protein	I-Complex
kinase	I-Complex
(	O
DNA	B-Complex
-	I-Complex
PK	I-Complex
)	O
is	O
thought	O
to	O
be	O
involved	O
in	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
activation	O
.	O

Here	O
we	O
show	O
that	O
DNA	B-Complex
-	I-Complex
PK	I-Complex
is	O
required	O
for	O
VCAM	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
expression	O
in	O
response	O
to	O
TNF	B-Gene_or_gene_product
.	O

The	O
phosphorylation	O
and	O
subsequent	O
degradation	O
of	O
I	B-Gene_or_gene_product
-	I-Gene_or_gene_product
kappaBalpha	I-Gene_or_gene_product
as	O
well	O
as	O
the	O
serine	B-Simple_chemical
536	I-Simple_chemical
phosphorylation	O
and	O
nuclear	B-Cellular_component
translocation	O
of	O
p65	B-Gene_or_gene_product
NF	B-Complex
-	I-Complex
kappaB	I-Complex
were	O
insufficient	O
for	O
VCAM	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
expression	O
in	O
response	O
to	O
TNF	B-Gene_or_gene_product
.	O

The	O
requirement	O
for	O
p50	B-Gene_or_gene_product
NF	B-Complex
-	I-Complex
kappaB	I-Complex
in	O
TNF	B-Gene_or_gene_product
-	O
induced	O
VCAM	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
expression	O
may	O
be	O
associated	O
with	O
its	O
interaction	O
with	O
and	O
phosphorylation	O
by	O
DNA	B-Complex
-	I-Complex
PK	I-Complex
,	O
which	O
appears	O
to	O
be	O
dominant	O
over	O
the	O
requirement	O
for	O
p65	B-Gene_or_gene_product
NF	B-Complex
-	I-Complex
kappaB	I-Complex
activation	O
.	O

p50	B-Gene_or_gene_product
NF	B-Complex
-	I-Complex
kappaB	I-Complex
binding	O
to	O
its	O
consensus	O
sequence	O
increased	O
its	O
susceptibility	O
to	O
phosphorylation	O
by	O
DNA	B-Complex
-	I-Complex
PK	I-Complex
.	O

Additionally	O
,	O
DNA	B-Complex
-	I-Complex
PK	I-Complex
activity	O
appeared	O
to	O
increase	O
the	O
association	O
between	O
p50	B-Gene_or_gene_product
/	O
p50	B-Gene_or_gene_product
and	O
p50	B-Gene_or_gene_product
/	O
p65	B-Gene_or_gene_product
NF	B-Complex
-	I-Complex
kappaB	I-Complex
dimers	O
upon	O
binding	O
to	O
DNA	O
and	O
after	O
binding	O
of	O
p50	B-Gene_or_gene_product
NF	B-Complex
-	I-Complex
kappaB	I-Complex
to	O
the	O
VCAM	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
promoter	O
.	O

Analyses	O
of	O
the	O
p50	B-Gene_or_gene_product
NF	B-Complex
-	I-Complex
kappaB	I-Complex
protein	O
sequence	O
revealed	O
that	O
both	O
serine	B-Simple_chemical
20	I-Simple_chemical
and	O
serine	B-Simple_chemical
227	I-Simple_chemical
at	O
the	O
amino	O
terminus	O
of	O
the	O
protein	O
are	O
putative	O
sites	O
for	O
phosphorylation	O
by	O
DNA	B-Complex
-	I-Complex
PK	I-Complex
.	O

Mutation	O
of	O
serine	B-Simple_chemical
20	I-Simple_chemical
completely	O
eliminated	O
phosphorylation	O
of	O
p50	B-Gene_or_gene_product
NF	B-Complex
-	I-Complex
kappaB	I-Complex
by	O
DNA	B-Complex
-	I-Complex
PK	I-Complex
,	O
suggesting	O
that	O
serine	B-Simple_chemical
20	I-Simple_chemical
is	O
the	O
only	O
site	O
in	O
p50	B-Gene_or_gene_product
NF	B-Complex
-	I-Complex
kappaB	I-Complex
for	O
phosphorylation	O
by	O
DNA	B-Complex
-	I-Complex
PK	I-Complex
.	O

Re	O
-	O
establishing	O
wild	O
-	O
type	O
p50	B-Gene_or_gene_product
NF	B-Complex
-	I-Complex
kappaB	I-Complex
,	O
but	O
not	O
its	O
serine	B-Simple_chemical
20	I-Simple_chemical
/	O
alanine	B-Simple_chemical
mutant	O
,	O
in	O
p50	B-Gene_or_gene_product
NF	B-Complex
-	I-Complex
kappaB	I-Complex
(	O
-	O
/	O
-	O
)	O
fibroblasts	O
reversed	O
VCAM	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
expression	O
after	O
TNF	B-Gene_or_gene_product
treatment	O
,	O
demonstrating	O
the	O
importance	O
of	O
the	O
serine	B-Simple_chemical
20	I-Simple_chemical
phosphorylation	O
site	O
in	O
the	O
induction	O
of	O
VCAM	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
expression	O
.	O

Together	O
,	O
these	O
results	O
elucidate	O
a	O
novel	O
mechanism	O
for	O
the	O
involvement	O
of	O
DNA	B-Complex
-	I-Complex
PK	I-Complex
in	O
the	O
positive	O
regulation	O
of	O
p50	B-Gene_or_gene_product
NF	B-Complex
-	I-Complex
kappaB	I-Complex
to	O
drive	O
VCAM	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
expression	O
.	O

NF	B-Complex
-	I-Complex
kappaB	I-Complex
p65	B-Gene_or_gene_product
represses	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
-	O
activated	O
transcription	O
of	O
cyclin	B-Gene_or_gene_product
D1	I-Gene_or_gene_product
.	O

Signaling	O
crosstalk	O
between	O
the	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
and	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
pathways	O
represents	O
a	O
functional	O
network	O
.	O

To	O
test	O
whether	O
the	O
crosstalk	O
also	O
occurs	O
on	O
their	O
common	O
target	O
genes	O
,	O
the	O
cyclin	B-Gene_or_gene_product
D1	I-Gene_or_gene_product
promoter	O
was	O
used	O
as	O
a	O
model	O
because	O
it	O
contains	O
binding	O
sites	O
for	O
both	O
proteins	O
.	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
activated	O
transcription	O
from	O
the	O
cyclin	B-Gene_or_gene_product
D1	I-Gene_or_gene_product
promoter	O
,	O
while	O
co	O
-	O
expression	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
p65	B-Gene_or_gene_product
reduced	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
-	O
induced	O
transcription	O
.	O

Chromatin	B-Gene_or_gene_product
immunoprecipitation	O
revealed	O
lithium	B-Simple_chemical
chloride	I-Simple_chemical
-	O
induced	O
binding	O
of	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
on	O
one	O
of	O
the	O
T	B-Gene_or_gene_product
-	I-Gene_or_gene_product
cell	I-Gene_or_gene_product
activating	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
binding	O
sites	O
.	O

More	O
interestingly	O
,	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
binding	O
was	O
greatly	O
reduced	O
by	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
p65	B-Gene_or_gene_product
,	O
possibly	O
by	O
the	O
protein	O
-	O
protein	O
interaction	O
between	O
the	O
two	O
proteins	O
.	O

Such	O
a	O
dynamic	O
and	O
complex	O
binding	O
of	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
and	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
on	O
promoters	O
might	O
contribute	O
to	O
the	O
regulated	O
expression	O
of	O
their	O
target	O
genes	O
.	O

Zac1	B-Gene_or_gene_product
is	O
a	O
histone	B-Gene_or_gene_product
acetylation	O
-	O
regulated	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
suppressor	O
that	O
mediates	O
histone	B-Gene_or_gene_product
deacetylase	I-Gene_or_gene_product
inhibitor	I-Gene_or_gene_product
-	O
induced	O
apoptosis	O
.	O

Histone	B-Gene_or_gene_product
deacetylase	I-Gene_or_gene_product
(	I-Gene_or_gene_product
HDAC	I-Gene_or_gene_product
)	I-Gene_or_gene_product
inhibitors	I-Gene_or_gene_product
are	O
a	O
class	O
of	O
promising	O
anticancer	O
reagents	O
.	O

They	O
are	O
able	O
to	O
induce	O
apoptosis	O
in	O
embryonic	O
carcinoma	O
(	O
EC	O
)	O
cells	O
.	O

However	O
,	O
the	O
underlying	O
mechanism	O
remains	O
poorly	O
understood	O
.	O

Here	O
we	O
show	O
that	O
increased	O
expression	O
of	O
zinc	B-Gene_or_gene_product
-	I-Gene_or_gene_product
finger	I-Gene_or_gene_product
protein	I-Gene_or_gene_product
regulator	I-Gene_or_gene_product
of	I-Gene_or_gene_product
apoptosis	I-Gene_or_gene_product
and	I-Gene_or_gene_product
cell	I-Gene_or_gene_product
-	I-Gene_or_gene_product
cycle	I-Gene_or_gene_product
arrest	I-Gene_or_gene_product
(	O
Zac1	B-Gene_or_gene_product
)	O
is	O
implicated	O
in	O
HDAC	B-Gene_or_gene_product
inhibitor	I-Gene_or_gene_product
-	O
induced	O
apoptosis	O
in	O
F9	O
and	O
P19	O
EC	O
cells	O
.	O

By	O
chromatin	B-Cellular_component
immunoprecipitation	O
analysis	O
we	O
identified	O
that	O
increased	O
Zac1	B-Gene_or_gene_product
expression	O
is	O
mediated	O
by	O
histone	B-Gene_or_gene_product
acetylation	O
of	O
the	O
Zac1	B-Gene_or_gene_product
promoter	O
region	O
.	O

Knockdown	O
of	O
Zac1	B-Gene_or_gene_product
inhibited	O
HDAC	B-Gene_or_gene_product
inhibitor	I-Gene_or_gene_product
-	O
induced	O
cell	O
apoptosis	O
.	O

Moreover	O
,	O
HDAC	B-Gene_or_gene_product
inhibitors	I-Gene_or_gene_product
repressed	O
nuclear	B-Complex
factor	I-Complex
-	I-Complex
kappaB	I-Complex
(	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
)	O
activity	O
,	O
and	O
this	O
effect	O
is	O
abrogated	O
by	O
Zac1	B-Gene_or_gene_product
knockdown	O
.	O

Consistently	O
,	O
Zac1	B-Gene_or_gene_product
overexpression	O
suppressed	O
cellular	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
activity	O
.	O

Further	O
investigation	O
showed	O
that	O
Zac1	B-Gene_or_gene_product
inhibits	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
activity	O
by	O
interacting	O
with	O
the	O
C	O
-	O
terminus	O
of	O
the	O
p65	B-Gene_or_gene_product
subunit	O
,	O
which	O
suppresses	O
the	O
phosphorylation	O
of	O
p65	B-Gene_or_gene_product
at	O
Ser468	B-Simple_chemical
and	O
Ser536	B-Simple_chemical
residues	O
.	O

These	O
results	O
indicate	O
that	O
Zac1	B-Gene_or_gene_product
is	O
a	O
histone	B-Gene_or_gene_product
acetylation	O
-	O
regulated	O
suppressor	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
,	O
which	O
is	O
induced	O
and	O
implicated	O
in	O
HDAC	B-Gene_or_gene_product
inhibitor	I-Gene_or_gene_product
-	O
mediated	O
EC	O
cell	O
apoptosis	O
.	O

Crosstalk	O
between	O
the	O
canonical	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
and	O
Notch	B-Gene_or_gene_product
signaling	O
pathways	O
inhibits	O
Ppargamma	B-Gene_or_gene_product
expression	O
and	O
promotes	O
pancreatic	O
cancer	O
progression	O
in	O
mice	O
.	O

The	O
majority	O
of	O
human	O
pancreatic	O
cancers	O
have	O
activating	O
mutations	O
in	O
the	O
KRAS	B-Gene_or_gene_product
proto	O
-	O
oncogene	O
.	O

These	O
mutations	O
result	O
in	O
increased	O
activity	O
of	O
the	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
pathway	O
and	O
the	O
subsequent	O
constitutive	O
production	O
of	O
proinflammatory	O
cytokines	O
.	O

Here	O
,	O
we	O
show	O
that	O
inhibitor	B-Gene_or_gene_product
of	I-Gene_or_gene_product
kappaB	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
2	I-Gene_or_gene_product
(	O
Ikk2	B-Gene_or_gene_product
)	O
,	O
a	O
component	O
of	O
the	O
canonical	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
signaling	O
pathway	O
,	O
synergizes	O
with	O
basal	O
Notch	B-Gene_or_gene_product
signaling	O
to	O
upregulate	O
transcription	O
of	O
primary	O
Notch	B-Gene_or_gene_product
target	O
genes	O
,	O
resulting	O
in	O
suppression	O
of	O
antiinflammatory	O
protein	O
expression	O
and	O
promotion	O
of	O
pancreatic	O
carcinogenesis	O
in	O
mice	O
.	O

We	O
found	O
that	O
in	O
the	O
Kras	B-Gene_or_gene_product
(	O
G12D	O
)	O
Pdx1	B-Gene_or_gene_product
-	O
cre	B-Gene_or_gene_product
mouse	O
model	O
of	O
pancreatic	O
cancer	O
,	O
genetic	O
deletion	O
of	O
Ikk2	B-Gene_or_gene_product
in	O
initiated	O
pre	O
-	O
malignant	O
epithelial	O
cells	O
substantially	O
delayed	O
pancreatic	O
oncogenesis	O
and	O
resulted	O
in	O
downregulation	O
of	O
the	O
classical	O
Notch	B-Gene_or_gene_product
target	O
genes	O
Hes1	B-Gene_or_gene_product
and	O
Hey1	B-Gene_or_gene_product
.	O

Tnf	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
stimulated	O
canonical	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
signaling	O
and	O
,	O
in	O
collaboration	O
with	O
basal	O
Notch	B-Gene_or_gene_product
signals	O
,	O
induced	O
optimal	O
expression	O
of	O
Notch	B-Gene_or_gene_product
targets	O
.	O

Mechanistically	O
,	O
Tnf	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
stimulation	O
resulted	O
in	O
phosphorylation	O
of	O
histone	B-Gene_or_gene_product
H3	I-Gene_or_gene_product
at	O
the	O
Hes1	B-Gene_or_gene_product
promoter	O
,	O
and	O
this	O
signal	O
was	O
lost	O
with	O
Ikk2	B-Gene_or_gene_product
deletion	O
.	O

Hes1	B-Gene_or_gene_product
suppresses	O
expression	O
of	O
Pparg	B-Gene_or_gene_product
,	O
which	O
encodes	O
the	O
antiinflammatory	O
nuclear	B-Cellular_component
receptor	O
Ppargamma	B-Gene_or_gene_product
.	O

Thus	O
,	O
crosstalk	O
between	O
Tnf	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
/	O
Ikk2	B-Gene_or_gene_product
and	O
Notch	B-Gene_or_gene_product
sustains	O
the	O
intrinsic	O
inflammatory	O
profile	O
of	O
transformed	O
cells	O
.	O

These	O
findings	O
reveal	O
what	O
we	O
believe	O
to	O
be	O
a	O
novel	O
interaction	O
between	O
oncogenic	O
inflammation	O
and	O
a	O
major	O
cell	O
fate	O
pathway	O
and	O
show	O
how	O
these	O
pathways	O
can	O
cooperate	O
to	O
promote	O
cancer	O
progression	O
.	O

Notch1	B-Gene_or_gene_product
promotes	O
glioma	O
cell	O
migration	O
and	O
invasion	O
by	O
stimulating	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
and	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
signaling	O
via	O
AKT	B-Gene_or_gene_product
activation	O
.	O

The	O
Notch	B-Gene_or_gene_product
signaling	O
pathway	O
has	O
been	O
implicated	O
in	O
both	O
developmental	O
processes	O
and	O
tumorigenesis	O
.	O

Aberrant	O
Notch	B-Gene_or_gene_product
signaling	O
has	O
been	O
repeatedly	O
demonstrated	O
to	O
facilitate	O
the	O
proliferation	O
and	O
survival	O
of	O
glioma	O
cells	O
by	O
regulating	O
downstream	O
effectors	O
or	O
other	O
signaling	O
pathways	O
.	O

In	O
glioblastoma	O
multiforme	O
specimens	O
from	O
59	O
patients	O
,	O
Notch1	B-Gene_or_gene_product
was	O
highly	O
expressed	O
in	O
tumor	O
tissues	O
compared	O
with	O
normal	O
brain	O
tissues	O
,	O
and	O
this	O
expression	O
was	O
correlated	O
with	O
elevated	O
AKT	B-Gene_or_gene_product
phosphorylation	O
and	O
Snail	B-Gene_or_gene_product
expression	O
.	O

Increased	O
nuclear	B-Cellular_component
localization	O
of	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
and	O
p50	B-Gene_or_gene_product
as	O
well	O
as	O
enhanced	O
IKKalpha	B-Gene_or_gene_product
/	O
AKT	B-Gene_or_gene_product
interaction	O
were	O
also	O
observed	O
in	O
glioma	O
tissues	O
.	O

In	O
U87MG	O
cells	O
,	O
the	O
activation	O
of	O
Notch1	B-Gene_or_gene_product
by	O
DLL4	B-Gene_or_gene_product
stimulation	O
or	O
by	O
the	O
overexpression	O
of	O
Notch	B-Gene_or_gene_product
intracellular	B-Cellular_component
domain	O
(	O
NICD	O
)	O
resulted	O
in	O
AKT	B-Gene_or_gene_product
activation	O
and	O
thereby	O
promoted	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
activity	O
and	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
signaling	O
.	O

Inhibition	O
of	O
EGFR	B-Gene_or_gene_product
partially	O
blocked	O
the	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
and	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
signaling	O
stimulated	O
by	O
Notch1	B-Gene_or_gene_product
activation	O
.	O

Furthermore	O
,	O
NICD	O
overexpression	O
in	O
U87MG	O
cells	O
led	O
to	O
the	O
upregulated	O
expression	O
of	O
several	O
metastasis	O
-	O
associated	O
molecules	O
,	O
which	O
could	O
be	O
abrogated	O
by	O
the	O
knockdown	O
of	O
either	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
or	O
p50	B-Gene_or_gene_product
.	O

In	O
U87MG	O
and	O
U251	O
cells	O
,	O
DLL4	B-Gene_or_gene_product
-	O
induced	O
cellular	O
migration	O
and	O
invasion	O
could	O
be	O
inhibited	O
by	O
either	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
or	O
a	O
p50	B-Gene_or_gene_product
inhibitor	O
.	O

Collectively	O
,	O
these	O
results	O
indicate	O
that	O
Notch	B-Gene_or_gene_product
activation	O
could	O
stimulate	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
and	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
signaling	O
through	O
AKT	B-Gene_or_gene_product
activation	O
in	O
glioma	O
cells	O
.	O

Thus	O
,	O
Notch	B-Gene_or_gene_product
activation	O
-	O
stimulated	O
beta	B-Gene_or_gene_product
-	I-Gene_or_gene_product
catenin	I-Gene_or_gene_product
and	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
signaling	O
synergistically	O
promote	O
the	O
migratory	O
and	O
invasive	O
properties	O
of	O
glioma	O
cells	O
.	O

The	O
p65	B-Gene_or_gene_product
subunit	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
and	O
PARP1	B-Gene_or_gene_product
assist	O
Snail1	B-Gene_or_gene_product
in	O
activating	O
fibronectin	B-Gene_or_gene_product
transcription	O
.	O

Snail1	B-Gene_or_gene_product
is	O
a	O
transcriptional	O
repressor	O
of	O
E	B-Gene_or_gene_product
-	I-Gene_or_gene_product
cadherin	I-Gene_or_gene_product
that	O
triggers	O
epithelial	O
-	O
mesenchymal	O
transition	O
(	O
EMT	O
)	O
.	O

Here	O
,	O
we	O
report	O
assisted	O
Snail1	B-Gene_or_gene_product
interaction	O
with	O
the	O
promoter	O
of	O
a	O
typical	O
mesenchymal	O
gene	O
,	O
fibronectin	B-Gene_or_gene_product
(	O
FN1	B-Gene_or_gene_product
)	O
,	O
both	O
in	O
epithelial	O
cells	O
undergoing	O
EMT	O
and	O
in	O
fibroblasts	O
.	O

Together	O
with	O
Snail1	B-Gene_or_gene_product
,	O
the	O
p65	B-Gene_or_gene_product
subunit	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
and	O
PARP1	B-Gene_or_gene_product
bound	O
to	O
the	O
FN1	B-Gene_or_gene_product
promoter	O
.	O

We	O
detected	O
nuclear	B-Cellular_component
interaction	O
of	O
these	O
proteins	O
and	O
demonstrated	O
the	O
requirement	O
of	O
all	O
three	O
for	O
FN1	B-Gene_or_gene_product
transcription	O
.	O

Moreover	O
,	O
other	O
genes	O
involved	O
in	O
cell	O
movement	O
mimic	O
FN1	B-Gene_or_gene_product
expression	O
induced	O
by	O
Snail1	B-Gene_or_gene_product
or	O
TGF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
beta1	I-Gene_or_gene_product
treatment	O
and	O
recruit	O
p65NF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
kappaB	I-Gene_or_gene_product
and	O
Snail1	B-Gene_or_gene_product
to	O
their	O
promoters	O
.	O

The	O
molecular	O
cooperation	O
between	O
Snail1	B-Gene_or_gene_product
and	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
in	O
transcription	O
activation	O
provides	O
a	O
new	O
insight	O
into	O
how	O
Snail1	B-Gene_or_gene_product
can	O
modulate	O
a	O
variety	O
of	O
cell	O
programs	O
.	O

Double	O
plant	O
homeodomain	O
(	O
PHD	O
)	O
finger	O
proteins	O
DPF3a	B-Gene_or_gene_product
and	O
-	B-Gene_or_gene_product
3b	I-Gene_or_gene_product
are	O
required	O
as	O
transcriptional	O
co	O
-	O
activators	O
in	O
SWI	B-Complex
/	I-Complex
SNF	I-Complex
complex	O
-	O
dependent	O
activation	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
RelA	I-Complex
/	I-Complex
p50	I-Complex
heterodimer	O
.	O

We	O
have	O
previously	O
shown	O
that	O
DPF2	B-Gene_or_gene_product
(	O
requiem	B-Gene_or_gene_product
/	O
REQ	B-Gene_or_gene_product
)	O
functions	O
as	O
a	O
linker	O
protein	O
between	O
the	O
SWI	B-Complex
/	I-Complex
SNF	I-Complex
complex	O
and	O
RelB	B-Complex
/	I-Complex
p52	I-Complex
NF	I-Complex
-	I-Complex
kappaB	I-Complex
heterodimer	O
and	O
plays	O
important	O
roles	O
in	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
transactivation	O
via	O
its	O
noncanonical	O
pathway	O
.	O

Using	O
sensitive	O
293FT	O
reporter	O
cell	O
clones	O
that	O
had	O
integrated	O
a	O
SWI	B-Complex
/	I-Complex
SNF	I-Complex
-	O
dependent	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
reporter	O
gene	O
,	O
we	O
find	O
in	O
this	O
study	O
that	O
the	O
overexpression	O
of	O
DPF1	B-Gene_or_gene_product
,	O
DPF2	B-Gene_or_gene_product
,	O
DPF3a	B-Gene_or_gene_product
,	O
DPF3b	B-Gene_or_gene_product
,	O
and	O
PHF10	B-Gene_or_gene_product
significantly	O
potentiates	O
the	O
transactivating	O
activity	O
of	O
typical	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
dimers	O
.	O

Knockdown	O
analysis	O
using	O
293FT	O
reporter	O
cells	O
that	O
endogenously	O
express	O
these	O
five	O
proteins	O
at	O
low	O
levels	O
clearly	O
showed	O
that	O
DPF3a	B-Gene_or_gene_product
and	O
DPF3b	B-Gene_or_gene_product
,	O
which	O
are	O
produced	O
from	O
the	O
DPF3	B-Gene_or_gene_product
gene	O
by	O
alternative	O
splicing	O
,	O
are	O
the	O
most	O
critical	O
for	O
the	O
RelA	B-Complex
/	I-Complex
p50	I-Complex
NF	I-Complex
-	I-Complex
kappaB	I-Complex
heterodimer	O
transactivation	O
induced	O
by	O
TNF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
stimulation	O
.	O

Our	O
data	O
further	O
show	O
that	O
this	O
transactivation	O
requires	O
the	O
SWI	B-Complex
/	I-Complex
SNF	I-Complex
complex	O
.	O

DPF3a	B-Gene_or_gene_product
and	O
DPF3b	B-Gene_or_gene_product
are	O
additionally	O
shown	O
to	O
interact	O
directly	O
with	O
RelA	B-Gene_or_gene_product
,	O
p50	B-Gene_or_gene_product
,	O
and	O
several	O
subunits	O
of	O
the	O
SWI	B-Complex
/	I-Complex
SNF	I-Complex
complex	O
in	O
vitro	O
and	O
to	O
be	O
co	O
-	O
immunoprecipitated	O
with	O
RelA	B-Complex
/	I-Complex
p50	I-Complex
and	O
the	O
SWI	B-Complex
/	I-Complex
SNF	I-Complex
complex	O
from	O
the	O
nuclear	B-Cellular_component
fractions	I-Cellular_component
of	O
cells	O
treated	O
with	O
TNF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
.	O

In	O
ChIP	O
experiments	O
,	O
we	O
further	O
found	O
that	O
endogenous	O
DPF3a	B-Gene_or_gene_product
/	O
b	B-Gene_or_gene_product
and	O
the	O
SWI	B-Complex
/	I-Complex
SNF	I-Complex
complex	O
are	O
continuously	O
present	O
on	O
HIV	O
-	O
1	O
LTR	B-Gene_or_gene_product
,	O
whereas	O
the	O
kinetics	O
of	O
RelA	B-Complex
/	I-Complex
p50	I-Complex
recruitment	O
after	O
TNF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
treatment	O
correlate	O
well	O
with	O
the	O
viral	O
transcriptional	O
activation	O
levels	O
.	O

Additionally	O
,	O
re	O
-	O
ChIP	O
experiments	O
showed	O
DPF3a	B-Gene_or_gene_product
/	O
b	B-Gene_or_gene_product
and	O
the	O
SWI	B-Complex
/	I-Complex
SNF	I-Complex
complex	O
associate	O
with	O
RelA	B-Gene_or_gene_product
on	O
the	O
endogenous	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
6	I-Gene_or_gene_product
promoter	O
after	O
TNF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
alpha	I-Gene_or_gene_product
treatment	O
.	O

In	O
conclusion	O
,	O
our	O
present	O
data	O
indicate	O
that	O
by	O
linking	O
RelA	B-Complex
/	I-Complex
p50	I-Complex
to	O
the	O
SWI	B-Complex
/	I-Complex
SNF	I-Complex
complex	O
,	O
DPF3a	B-Gene_or_gene_product
/	O
b	B-Gene_or_gene_product
induces	O
the	O
transactivation	O
of	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
target	O
gene	O
promoters	O
in	O
relatively	O
inactive	O
chromatin	B-Cellular_component
contexts	O
.	O

Structure	O
-	O
based	O
analysis	O
of	O
the	O
molecular	O
recognitions	O
between	O
HIV	O
-	O
1	O
TAR	B-Gene_or_gene_product
-	O
RNA	O
and	O
transcription	O
factor	O
nuclear	B-Complex
factor	I-Complex
-	I-Complex
kappaB	I-Complex
(	O
NFkB	B-Complex
)	O
.	O

In	O
this	O
paper	O
we	O
applied	O
the	O
"	O
macromolecular	O
docking	O
"	O
procedure	O
to	O
perform	O
molecular	O
modeling	O
with	O
the	O
aim	O
of	O
screening	O
transcription	O
factor	O
sequences	O
for	O
possible	O
interaction	O
to	O
the	O
HIV	O
-	O
1	O
TAR	B-Gene_or_gene_product
-	O
RNA	O
,	O
employing	O
the	O
software	O
Hex	O
version	O
4	O
.	O
2	O
.	O

The	O
molecular	O
modeling	O
data	O
were	O
compared	O
with	O
electrophoretic	O
mobility	O
shift	O
assays	O
(	O
EMSA	O
)	O
and	O
surface	O
plasmon	O
resonance	O
(	O
SPR	O
)	O
based	O
biospecific	O
interaction	O
analysis	O
(	O
BIA	O
)	O
using	O
an	O
optical	O
biosensor	O
.	O

Finally	O
the	O
specific	O
interactions	O
between	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
and	O
RNA	O
have	O
been	O
calculated	O
utilizing	O
the	O
AMBER	O
-	O
MM	O
and	O
FMO	O
calculations	O
.	O

The	O
results	O
obtained	O
clearly	O
indicate	O
that	O
(	O
a	O
)	O
NF	B-Complex
-	I-Complex
kB	I-Complex
p50	B-Gene_or_gene_product
transcription	O
factor	O
can	O
bind	O
TAR	B-Gene_or_gene_product
-	O
RNA	O
;	O
(	O
b	O
)	O
this	O
binding	O
efficiency	O
is	O
lower	O
than	O
that	O
displayed	O
by	O
NF	B-Complex
-	I-Complex
kB	I-Complex
factor	O
in	O
respect	O
to	O
DNA	O
sequences	O
;	O
(	O
c	O
)	O
other	O
structured	O
RNAs	O
used	O
as	O
controls	O
do	O
not	O
bind	O
to	O
NF	B-Complex
-	I-Complex
kB	I-Complex
;	O
(	O
d	O
)	O
TAR	B-Gene_or_gene_product
-	O
RNA	O
is	O
capable	O
to	O
bind	O
pre	O
-	O
formed	O
NF	B-Complex
-	I-Complex
kB	I-Complex
/	I-Complex
DNA	I-Complex
complexes	O
.	O

Despite	O
the	O
fact	O
that	O
our	O
data	O
do	O
not	O
indicate	O
whether	O
NF	B-Complex
-	I-Complex
kB	I-Complex
/	I-Complex
TAR	I-Complex
-	I-Complex
RNA	I-Complex
complexes	O
play	O
a	O
role	O
in	O
the	O
early	O
steps	O
of	O
HIV	O
-	O
1	O
transcriptional	O
activation	O
,	O
the	O
results	O
presented	O
strongly	O
indicate	O
that	O
interactions	O
between	O
transcription	O
factors	O
recruited	O
at	O
the	O
level	O
of	O
HIV	O
-	O
1	O
LTR	O
might	O
interact	O
with	O
the	O
TAR	B-Gene_or_gene_product
-	O
RNA	O
and	O
deserve	O
further	O
studies	O
aimed	O
to	O
determine	O
its	O
role	O
in	O
the	O
HIV	O
-	O
1	O
life	O
cycle	O
.	O

Oroxylin	B-Simple_chemical
A	I-Simple_chemical
prevents	O
inflammation	O
-	O
related	O
tumor	O
through	O
down	O
-	O
regulation	O
of	O
inflammatory	O
gene	O
expression	O
by	O
inhibiting	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
signaling	O
.	O

Increasing	O
evidence	O
suggests	O
that	O
inflammatory	O
microenvironment	O
plays	O
a	O
critical	O
role	O
at	O
different	O
stages	O
of	O
tumor	O
development	O
.	O

However	O
,	O
the	O
molecular	O
mechanisms	O
of	O
the	O
interaction	O
between	O
inflammation	O
and	O
proliferation	O
of	O
cancer	O
cells	O
remain	O
poorly	O
defined	O
.	O

Here	O
we	O
reported	O
the	O
inhibitory	O
effects	O
of	O
oroxylin	B-Simple_chemical
A	I-Simple_chemical
on	O
the	O
inflammation	O
-	O
stimulated	O
proliferation	O
of	O
tumor	O
cells	O
and	O
delineated	O
the	O
mechanism	O
of	O
its	O
action	O
.	O

The	O
results	O
indicated	O
that	O
treatment	O
with	O
oroxylin	B-Simple_chemical
A	I-Simple_chemical
inhibited	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
p65	B-Gene_or_gene_product
nuclear	B-Cellular_component
translocation	O
and	O
phosphorylation	O
of	O
IkappaBalpha	B-Gene_or_gene_product
and	O
IKKalpha	B-Gene_or_gene_product
/	O
beta	B-Gene_or_gene_product
in	O
both	O
human	O
colon	O
tumor	O
HCT116	O
cells	O
and	O
human	O
monocytes	O
THP	O
-	O
1	O
cells	O
.	O

In	O
addition	O
,	O
in	O
THP	O
-	O
1	O
cells	O
,	O
oroxylin	B-Simple_chemical
A	I-Simple_chemical
significantly	O
suppressed	O
lipopolysaccharide	B-Simple_chemical
(	O
LPS	B-Simple_chemical
)	O
-	O
induced	O
secretion	O
of	O
prototypical	O
proinflammatory	O
cytokine	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
6	I-Gene_or_gene_product
but	O
not	O
IL	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1beta	I-Gene_or_gene_product
,	O
and	O
it	O
was	O
confirmed	O
at	O
the	O
transcription	O
level	O
.	O

Moreover	O
,	O
oroxylin	B-Simple_chemical
A	I-Simple_chemical
inhibited	O
the	O
proliferation	O
of	O
HCT116	O
cells	O
stimulated	O
by	O
LPS	B-Simple_chemical
-	O
induced	O
THP	O
-	O
1	O
cells	O
in	O
co	O
-	O
culture	O
microenvironment	O
.	O

In	O
summary	O
,	O
oroxylin	B-Simple_chemical
A	I-Simple_chemical
modulated	O
NF	B-Complex
-	I-Complex
kappaB	I-Complex
signaling	O
pathway	O
involved	O
in	O
inflammation	O
-	O
induced	O
cancer	O
initiation	O
and	O
progression	O
and	O
therefore	O
could	O
be	O
a	O
potential	O
cancer	O
chemoprevention	O
agent	O
for	O
inflammation	O
-	O
related	O
cancer	O
.	O

(	O
c	O
)	O
2012	O
Wiley	O
Periodicals	O
,	O
Inc	O
.	O

Steric	O
and	O
conformational	O
features	O
of	O
the	O
aconitase	B-Gene_or_gene_product
mechanism	O
.	O

Crystal	O
structures	O
of	O
mitochondrial	B-Cellular_component
aconitase	B-Gene_or_gene_product
with	O
alpha	B-Simple_chemical
-	I-Simple_chemical
methylisocitrate	I-Simple_chemical
and	O
with	O
sulfate	B-Simple_chemical
bound	O
have	O
been	O
solved	O
and	O
refined	O
at	O
2	O
.	O
0	O
A	O
resolution	O
with	O
R	O
factors	O
of	O
18	O
.	O
2	O
and	O
16	O
.	O
8	O
%	O
,	O
respectively	O
.	O

The	O
steric	O
factors	O
and	O
conformational	O
effects	O
observed	O
in	O
both	O
new	O
structures	O
support	O
the	O
proposed	O
mechanism	O
for	O
the	O
overall	O
reaction	O
catalyzed	O
by	O
aconitase	B-Gene_or_gene_product
.	O

The	O
alternate	O
substrate	O
alpha	B-Simple_chemical
-	I-Simple_chemical
methylisocitrate	I-Simple_chemical
is	O
derived	O
from	O
alpha	B-Simple_chemical
-	I-Simple_chemical
methyl	I-Simple_chemical
-	I-Simple_chemical
cis	I-Simple_chemical
-	I-Simple_chemical
aconitate	I-Simple_chemical
during	O
crystallization	O
and	O
is	O
observed	O
to	O
bind	O
in	O
the	O
active	O
site	O
in	O
a	O
manner	O
very	O
similar	O
to	O
that	O
observed	O
for	O
isocitrate	B-Simple_chemical
.	O

The	O
methyl	B-Simple_chemical
group	O
is	O
accommodated	O
by	O
favorable	O
contact	O
with	O
Ile	B-Simple_chemical
-	I-Simple_chemical
425	I-Simple_chemical
.	O

However	O
,	O
the	O
other	O
potential	O
hydration	O
product	O
of	O
alpha	B-Simple_chemical
-	I-Simple_chemical
methyl	I-Simple_chemical
-	I-Simple_chemical
cis	I-Simple_chemical
-	I-Simple_chemical
aconitate	I-Simple_chemical
,	O
alpha	B-Simple_chemical
-	I-Simple_chemical
methylcitrate	I-Simple_chemical
,	O
cannot	O
be	O
accommodated	O
in	O
the	O
active	O
site	O
due	O
to	O
steric	O
conflict	O
of	O
the	O
methyl	B-Simple_chemical
group	O
with	O
Asp	B-Simple_chemical
-	I-Simple_chemical
165	I-Simple_chemical
.	O

The	O
results	O
are	O
consistent	O
with	O
the	O
requirement	O
that	O
cis	B-Simple_chemical
-	I-Simple_chemical
aconitate	I-Simple_chemical
must	O
bind	O
in	O
two	O
ways	O
,	O
in	O
the	O
citrate	B-Simple_chemical
mode	O
and	O
in	O
the	O
isocitrate	B-Simple_chemical
mode	O
.	O

Crystals	O
of	O
aconitase	B-Gene_or_gene_product
with	O
sulfate	B-Simple_chemical
bound	O
are	O
isomorphous	O
to	O
those	O
with	O
isocitrate	B-Simple_chemical
bound	O
.	O

However	O
,	O
the	O
structure	O
displays	O
significant	O
conformational	O
changes	O
,	O
providing	O
a	O
model	O
for	O
the	O
substrate	O
-	O
free	O
state	O
of	O
enzyme	O
.	O

Three	O
water	B-Simple_chemical
molecules	O
bind	O
in	O
place	O
of	O
the	O
C	O
alpha	O
-	O
and	O
C	O
beta	O
-	O
hydroxyl	B-Simple_chemical
and	O
carboxyl	B-Simple_chemical
groups	O
of	O
isocitrate	B-Simple_chemical
,	O
while	O
sulfate	B-Simple_chemical
binds	O
in	O
place	O
of	O
the	O
C	O
gamma	O
-	O
carboxyl	B-Simple_chemical
group	O
.	O

Side	O
chains	O
of	O
Ser	B-Simple_chemical
-	I-Simple_chemical
642	I-Simple_chemical
and	O
Arg	B-Simple_chemical
-	I-Simple_chemical
447	I-Simple_chemical
in	O
the	O
active	O
site	O
rotate	O
to	O
pair	O
with	O
other	O
side	O
chains	O
in	O
the	O
absence	O
of	O
substrate	O
.	O

The	O
new	O
conformation	O
of	O
Arg	B-Simple_chemical
-	I-Simple_chemical
447	I-Simple_chemical
triggers	O
a	O
concerted	O
set	O
of	O
shifts	O
which	O
transmits	O
conformational	O
change	O
to	O
the	O
surface	O
of	O
the	O
protein	O
,	O
30	O
A	O
from	O
the	O
active	O
site	O
.	O

In	O
the	O
absence	O
of	O
substrate	O
,	O
a	O
chain	O
segment	O
containing	O
the	O
[	B-Simple_chemical
4Fe	I-Simple_chemical
-	I-Simple_chemical
4S	I-Simple_chemical
]	I-Simple_chemical
ligand	O
Cys	B-Simple_chemical
-	I-Simple_chemical
358	I-Simple_chemical
also	O
shifts	O
,	O
resulting	O
in	O
the	O
net	O
translation	O
and	O
reorientation	O
of	O
the	O
Fe	B-Simple_chemical
-	I-Simple_chemical
S	I-Simple_chemical
cluster	O
.	O

Phosphatidylcholine	B-Gene_or_gene_product
translocase	I-Gene_or_gene_product
:	O
a	O
physiological	O
role	O
for	O
the	O
mdr2	B-Gene_or_gene_product
gene	O
.	O

P	B-Gene_or_gene_product
-	I-Gene_or_gene_product
glycoproteins	I-Gene_or_gene_product
(	O
P	B-Gene_or_gene_product
-	I-Gene_or_gene_product
gps	I-Gene_or_gene_product
)	O
encoded	O
by	O
the	O
mouse	O
mdr2	B-Gene_or_gene_product
and	O
mdr3	B-Gene_or_gene_product
genes	O
were	O
expressed	O
in	O
secretory	B-Cellular_component
vesicles	I-Cellular_component
(	O
SVs	B-Cellular_component
)	O
from	O
the	O
yeast	O
mutant	O
sec6	O
-	O
4	O
,	O
and	O
their	O
capacity	O
to	O
function	O
as	O
a	O
lipid	B-Gene_or_gene_product
translocase	I-Gene_or_gene_product
/	O
flippase	B-Gene_or_gene_product
was	O
tested	O
.	O

An	O
assay	O
that	O
uses	O
a	O
fluorescent	O
phosphatidylcholine	B-Simple_chemical
(	O
PC	B-Simple_chemical
)	O
analog	O
was	O
developed	O
to	O
quantitate	O
asymmetric	O
lipid	B-Simple_chemical
distribution	O
in	O
the	O
outer	O
and	O
inner	O
leaflets	O
of	O
the	O
lipid	B-Cellular_component
bilayer	I-Cellular_component
of	O
these	O
vesicles	B-Cellular_component
.	O

Mdr2	B-Gene_or_gene_product
expression	O
in	O
SVs	B-Cellular_component
caused	O
a	O
time	O
-	O
and	O
temperature	O
-	O
dependent	O
enhancement	O
of	O
PC	B-Simple_chemical
translocation	O
to	O
the	O
inner	B-Cellular_component
leaflet	I-Cellular_component
of	O
the	O
membrane	B-Cellular_component
.	O

The	O
Mdr2	B-Gene_or_gene_product
-	O
mediated	O
effect	O
was	O
specific	O
since	O
expression	O
of	O
Mdr3	B-Gene_or_gene_product
in	O
these	O
vesicles	B-Cellular_component
was	O
without	O
effect	O
on	O
the	O
membrane	B-Cellular_component
distribution	O
of	O
PC	B-Simple_chemical
.	O

Increased	O
Mdr2	B-Gene_or_gene_product
-	O
mediated	O
PC	B-Simple_chemical
translocation	O
was	O
strictly	O
ATP	B-Simple_chemical
and	O
Mg2	B-Simple_chemical
+	I-Simple_chemical
dependent	O
,	O
was	O
abrogated	O
by	O
the	O
ATPase	B-Gene_or_gene_product
inhibitor	O
vanadate	B-Simple_chemical
and	O
the	O
P	B-Gene_or_gene_product
-	I-Gene_or_gene_product
gp	I-Gene_or_gene_product
modulator	O
verapamil	B-Simple_chemical
,	O
but	O
was	O
insensitive	O
to	O
the	O
presence	O
of	O
excess	O
of	O
the	O
multidrug	O
resistance	O
drugs	O
colchicine	B-Simple_chemical
and	O
vinblastine	B-Simple_chemical
.	O

The	O
SRF	B-Gene_or_gene_product
accessory	O
protein	O
Elk	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
contains	O
a	O
growth	O
factor	O
-	O
regulated	O
transcriptional	O
activation	O
domain	O
.	O

The	O
Elk	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
and	O
SRF	B-Gene_or_gene_product
transcription	O
factors	O
form	O
a	O
ternary	O
complex	O
at	O
the	O
c	B-Gene_or_gene_product
-	I-Gene_or_gene_product
fos	I-Gene_or_gene_product
serum	O
response	O
element	O
(	O
SRE	O
)	O
.	O

Growth	O
factor	O
stimulation	O
rapidly	O
induces	O
a	O
reversible	O
change	O
in	O
the	O
electrophoretic	O
mobility	O
of	O
the	O
ternary	O
complex	O
,	O
accompanied	O
by	O
increased	O
phosphorylation	O
of	O
the	O
Elk	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
C	O
-	O
terminal	O
region	O
and	O
by	O
the	O
activation	O
of	O
a	O
42	O
kd	O
cellular	O
Elk	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
kinase	O
.	O

Phosphorylation	O
of	O
Elk	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
in	O
vitro	O
by	O
partially	O
purified	O
p42	B-Gene_or_gene_product
/	I-Gene_or_gene_product
p44	I-Gene_or_gene_product
MAP	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
induces	O
a	O
similar	O
reduction	O
in	O
ternary	O
complex	O
mobility	O
but	O
has	O
little	O
effect	O
on	O
the	O
efficiency	O
of	O
its	O
formation	O
.	O

In	O
vitro	O
,	O
MAP	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
phosphorylates	O
the	O
Elk	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
C	O
-	O
terminal	O
region	O
at	O
multiple	O
sites	O
,	O
which	O
are	O
also	O
phosphorylated	O
following	O
growth	O
factor	O
stimulation	O
in	O
vivo	O
.	O

The	O
Elk	B-Gene_or_gene_product
-	I-Gene_or_gene_product
1	I-Gene_or_gene_product
C	O
-	O
terminal	O
region	O
functions	O
as	O
a	O
regulated	O
transcriptional	O
activation	O
domain	O
whose	O
activity	O
in	O
vivo	O
is	O
dependent	O
on	O
the	O
integrity	O
of	O
the	O
MAP	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
sites	O
.	O

These	O
findings	O
directly	O
link	O
transcriptional	O
activation	O
by	O
the	O
SRE	O
to	O
the	O
growth	O
factor	O
-	O
regulated	O
phosphorylation	O
of	O
an	O
SRE	O
-	O
binding	O
protein	O
.	O

Physical	O
association	O
of	O
the	O
small	O
GTPase	O
Rho	B-Gene_or_gene_product
with	O
a	O
68	O
-	O
kDa	O
phosphatidylinositol	B-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
in	O
Swiss	O
3T3	O
cells	O
.	O

Our	O
previous	O
work	O
showed	O
that	O
post	O
-	O
translationally	O
modified	O
Rho	B-Gene_or_gene_product
in	O
its	O
GTP	B-Simple_chemical
-	O
bound	O
state	O
stimulated	O
phosphatidylinositol	B-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
(	O
PIP5K	B-Gene_or_gene_product
)	O
activity	O
in	O
mouse	O
fibroblast	O
lysates	O
.	O

To	O
investigate	O
whether	O
Rho	B-Gene_or_gene_product
physically	O
interacts	O
with	O
PIP5K	B-Gene_or_gene_product
,	O
we	O
incubated	O
immobilized	O
Rho	B-Gene_or_gene_product
-	I-Gene_or_gene_product
GST	I-Gene_or_gene_product
with	O
Swiss	O
3T3	O
cell	O
lysates	O
and	O
tested	O
for	O
retained	O
PIP5K	B-Gene_or_gene_product
activity	O
.	O

Rho	B-Gene_or_gene_product
-	I-Gene_or_gene_product
GST	I-Gene_or_gene_product
,	O
but	O
not	O
Ras	B-Gene_or_gene_product
-	I-Gene_or_gene_product
GST	I-Gene_or_gene_product
or	O
GST	B-Gene_or_gene_product
alone	O
,	O
bound	O
significant	O
PIP5K	B-Gene_or_gene_product
activity	O
.	O

The	O
binding	O
of	O
PIP5K	B-Gene_or_gene_product
was	O
independent	O
of	O
whether	O
Rho	B-Gene_or_gene_product
was	O
in	O
a	O
GTP	B-Simple_chemical
-	O
or	O
GDP	B-Simple_chemical
-	O
bound	O
state	O
.	O

An	O
antibody	O
against	O
a	O
68	O
-	O
kDa	O
human	O
erythrocyte	O
type	O
I	O
PIP5K	B-Gene_or_gene_product
recognized	O
a	O
single	O
68	O
-	O
kDa	O
protein	O
eluted	O
from	O
Rho	B-Gene_or_gene_product
-	I-Gene_or_gene_product
GST	I-Gene_or_gene_product
column	O
.	O

The	O
Rho	B-Gene_or_gene_product
-	O
associated	O
PIP5K	B-Gene_or_gene_product
responded	O
to	O
phosphatidic	B-Simple_chemical
acid	I-Simple_chemical
differentially	O
from	O
the	O
erythrocyte	O
type	O
I	O
PIP5K	B-Gene_or_gene_product
,	O
suggesting	O
that	O
it	O
could	O
be	O
a	O
distinct	O
isoform	O
not	O
reported	O
previously	O
.	O

Rho	B-Gene_or_gene_product
co	O
-	O
immunoprecipitated	O
with	O
the	O
68	O
-	O
kDa	O
PIP5K	B-Gene_or_gene_product
from	O
Swiss	O
3T3	O
lysates	O
,	O
demonstrating	O
that	O
endogenous	O
Rho	B-Gene_or_gene_product
also	O
interacts	O
with	O
PIP5K	B-Gene_or_gene_product
.	O

ADP	B-Simple_chemical
-	O
ribosylation	O
of	O
Rho	B-Gene_or_gene_product
with	O
C3	B-Gene_or_gene_product
exoenzyme	O
enhanced	O
PIP5K	B-Gene_or_gene_product
binding	O
by	O
approximately	O
eightfold	O
,	O
consistent	O
with	O
the	O
ADP	B-Simple_chemical
-	O
ribosylated	O
Rho	B-Gene_or_gene_product
functioning	O
as	O
a	O
dominant	O
negative	O
inhibitor	O
.	O

These	O
results	O
demonstrate	O
that	O
Rho	B-Gene_or_gene_product
physically	O
interacts	O
with	O
a	O
68	O
-	O
kDa	O
PIP5K	B-Gene_or_gene_product
,	O
although	O
whether	O
the	O
association	O
is	O
direct	O
or	O
indirect	O
is	O
unknown	O
.	O

Cloning	O
,	O
expression	O
,	O
purification	O
,	O
and	O
characterization	O
of	O
the	O
human	O
broad	O
specificity	O
lysosomal	B-Cellular_component
acid	O
alpha	B-Gene_or_gene_product
-	I-Gene_or_gene_product
mannosidase	I-Gene_or_gene_product
.	O

We	O
have	O
cloned	O
and	O
expressed	O
two	O
cDNAs	O
encoding	O
the	O
human	O
lysosomal	B-Cellular_component
alpha	B-Gene_or_gene_product
-	I-Gene_or_gene_product
mannosidase	I-Gene_or_gene_product
(	O
EC	B-Gene_or_gene_product
3	I-Gene_or_gene_product
.	I-Gene_or_gene_product
2	I-Gene_or_gene_product
.	I-Gene_or_gene_product
1	I-Gene_or_gene_product
.	I-Gene_or_gene_product
24	I-Gene_or_gene_product
)	O
by	O
RT	O
-	O
PCR	O
of	O
human	O
spleen	O
mRNA	O
.	O

This	O
enzyme	O
is	O
required	O
for	O
the	O
degradation	O
of	O
N	O
-	O
linked	O
carbohydrates	B-Simple_chemical
during	O
glycoprotein	O
catabolism	O
in	O
eucaryotic	O
cells	O
.	O

The	O
shorter	O
of	O
the	O
two	O
cDNAs	O
(	O
3	O
kilobases	O
(	O
kb	O
)	O
)	O
was	O
found	O
to	O
encode	O
an	O
open	O
reading	O
frame	O
of	O
2964	O
base	O
pairs	O
and	O
,	O
when	O
expressed	O
in	O
Pichia	O
pastoris	O
,	O
was	O
found	O
to	O
encode	O
an	O
enzyme	O
that	O
could	O
cleave	O
high	O
mannose	B-Simple_chemical
oligosaccharides	B-Simple_chemical
,	O
oligosaccharides	B-Simple_chemical
isolated	O
from	O
alpha	O
-	O
mannosidosis	O
fibroblasts	O
,	O
and	O
p	B-Simple_chemical
-	I-Simple_chemical
nitrophenyl	I-Simple_chemical
-	I-Simple_chemical
alpha	I-Simple_chemical
-	I-Simple_chemical
D	I-Simple_chemical
-	I-Simple_chemical
mannopyranoside	I-Simple_chemical
substrates	O
.	O

In	O
addition	O
,	O
the	O
Pichia	O
-	O
expressed	O
enzyme	O
was	O
inhibited	O
by	O
swainsonine	B-Simple_chemical
,	O
and	O
had	O
a	O
pH	O
optimum	O
,	O
Km	O
,	O
and	O
Vmax	O
characteristic	O
of	O
the	O
enzyme	O
purified	O
previously	O
from	O
human	O
liver	O
.	O

The	O
second	O
,	O
larger	O
RT	O
-	O
PCR	O
product	O
(	O
3	O
.	O
6	O
kb	O
)	O
was	O
found	O
to	O
contain	O
an	O
insertion	O
and	O
a	O
deletion	O
relative	O
to	O
the	O
3	O
-	O
kb	O
spleen	O
amplimer	O
and	O
encoded	O
a	O
truncated	O
coding	O
region	O
,	O
indicating	O
that	O
it	O
resulted	O
from	O
alternate	O
transcript	O
splicing	O
.	O

No	O
alpha	B-Gene_or_gene_product
-	I-Gene_or_gene_product
mannosidase	I-Gene_or_gene_product
activity	O
could	O
be	O
detected	O
in	O
Pichia	O
transformants	O
containing	O
this	O
coding	O
region	O
,	O
indicating	O
that	O
it	O
did	O
not	O
encode	O
a	O
functional	O
enzyme	O
.	O

Antiserum	O
raised	O
to	O
the	O
recombinant	O
product	O
of	O
the	O
3	O
-	O
kb	O
alpha	B-Gene_or_gene_product
-	I-Gene_or_gene_product
mannosidase	I-Gene_or_gene_product
cDNA	O
immunoprecipitated	O
lysosomal	B-Cellular_component
alpha	B-Gene_or_gene_product
-	I-Gene_or_gene_product
mannosidase	I-Gene_or_gene_product
activity	O
from	O
human	O
fibroblast	O
extracts	O
.	O

Northern	O
blots	O
identified	O
a	O
3	O
-	O
kb	O
RNA	O
transcript	O
in	O
all	O
human	O
tissues	O
tested	O
,	O
including	O
alpha	O
-	O
mannosidosis	O
fibroblasts	O
,	O
while	O
minor	O
transcripts	O
of	O
3	O
.	O
6	O
kb	O
were	O
also	O
present	O
in	O
several	O
adult	O
tissues	O
.	O

Human	O
chromosome	B-Cellular_component
mapping	O
of	O
the	O
mannosidase	B-Gene_or_gene_product
gene	O
confirmed	O
that	O
the	O
functional	O
gene	O
maps	O
to	O
the	O
MANB	O
locus	O
on	O
chromosome	B-Cellular_component
19	I-Cellular_component
.	O

Sequence	O
comparisons	O
were	O
made	O
to	O
previously	O
published	O
human	O
cDNA	O
sequences	O
encoding	O
a	O
putative	O
lysosomal	B-Cellular_component
alpha	B-Gene_or_gene_product
-	I-Gene_or_gene_product
mannosidase	I-Gene_or_gene_product
(	O
Nebes	O
,	O
V	O
.	O
L	O
.	O
,	O
and	O
Schmidt	O
,	O
M	O
.	O
C	O
.	O
(	O
1994	O
)	O
Biochem	O
.	O
Biophys	O
.	O
Res	O
.	O
Commun	O
.	O
200	O
,	O
239	O
-	O
245	O
)	O
and	O
several	O
differences	O
were	O
found	O
relative	O
to	O
the	O
functional	O
lysosomal	B-Cellular_component
alpha	B-Gene_or_gene_product
-	I-Gene_or_gene_product
mannosidase	I-Gene_or_gene_product
encoded	O
by	O
the	O
3	O
-	O
kb	O
spleen	O
cDNA	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
kinases	I-Gene_or_gene_product
activate	O
the	O
serine	B-Simple_chemical
/	O
threonine	B-Simple_chemical
kinases	O
Mnk1	B-Gene_or_gene_product
and	O
Mnk2	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
(	I-Gene_or_gene_product
MAP	I-Gene_or_gene_product
)	I-Gene_or_gene_product
kinases	I-Gene_or_gene_product
bind	O
tightly	O
to	O
many	O
of	O
their	O
physiologically	O
relevant	O
substrates	O
.	O

We	O
have	O
identified	O
a	O
new	O
subfamily	O
of	O
murine	O
serine	B-Simple_chemical
/	O
threonine	B-Simple_chemical
kinases	O
,	O
whose	O
members	O
,	O
MAP	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
-	I-Gene_or_gene_product
interacting	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
1	I-Gene_or_gene_product
(	O
Mnk1	B-Gene_or_gene_product
)	O
and	O
Mnk2	B-Gene_or_gene_product
,	O
bind	O
tightly	O
to	O
the	O
growth	O
factor	O
-	O
regulated	O
MAP	B-Gene_or_gene_product
kinases	I-Gene_or_gene_product
,	O
Erk1	B-Gene_or_gene_product
and	O
Erk2	B-Gene_or_gene_product
.	O

MNK1	B-Gene_or_gene_product
,	O
but	O
not	O
Mnk2	B-Gene_or_gene_product
,	O
also	O
binds	O
strongly	O
to	O
the	O
stress	O
-	O
activated	O
kinase	O
,	O
p38	B-Gene_or_gene_product
.	O

MNK1	B-Gene_or_gene_product
complexes	O
more	O
strongly	O
with	O
inactive	O
than	O
active	O
Erk	B-Gene_or_gene_product
,	O
implying	O
that	O
Mnk	B-Gene_or_gene_product
and	O
Erk	B-Gene_or_gene_product
may	O
dissociate	O
after	O
mitogen	O
stimulation	O
.	O

Erk	B-Gene_or_gene_product
and	O
p38	B-Gene_or_gene_product
phosphorylate	O
MNK1	B-Gene_or_gene_product
and	O
Mnk2	B-Gene_or_gene_product
,	O
which	O
stimulates	O
their	O
in	O
vitro	O
kinase	O
activity	O
toward	O
a	O
substrate	O
,	O
eukaryotic	B-Gene_or_gene_product
initiation	I-Gene_or_gene_product
factor	I-Gene_or_gene_product
-	I-Gene_or_gene_product
4E	I-Gene_or_gene_product
(	O
eIF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
4E	I-Gene_or_gene_product
)	O
.	O

Initiation	O
factor	O
eIF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
4E	I-Gene_or_gene_product
is	O
a	O
regulatory	O
phosphoprotein	O
whose	O
phosphorylation	O
is	O
increased	O
by	O
insulin	B-Gene_or_gene_product
in	O
an	O
Erk	B-Gene_or_gene_product
-	O
dependent	O
manner	O
.	O

In	O
vitro	O
,	O
MNK1	B-Gene_or_gene_product
rapidly	O
phosphorylates	O
eIF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
4E	I-Gene_or_gene_product
at	O
the	O
physiologically	O
relevant	O
site	O
,	O
Ser209	B-Simple_chemical
.	O

In	O
cells	O
,	O
Mnk1	B-Gene_or_gene_product
is	O
post	O
-	O
translationally	O
modified	O
and	O
enzymatically	O
activated	O
in	O
response	O
to	O
treatment	O
with	O
either	O
peptide	O
growth	O
factors	O
,	O
phorbol	B-Simple_chemical
esters	I-Simple_chemical
,	O
anisomycin	B-Simple_chemical
or	O
UV	O
.	O

Mitogen	O
-	O
and	O
stress	O
-	O
mediated	O
MNK1	B-Gene_or_gene_product
activation	O
is	O
blocked	O
by	O
inhibitors	O
of	O
MAP	B-Gene_or_gene_product
kinase	I-Gene_or_gene_product
kinase	I-Gene_or_gene_product
1	I-Gene_or_gene_product
(	O
Mkk1	B-Gene_or_gene_product
)	O
and	O
p38	B-Gene_or_gene_product
,	O
demonstrating	O
that	O
Mnk1	B-Gene_or_gene_product
is	O
downstream	O
of	O
multiple	O
MAP	B-Gene_or_gene_product
kinases	I-Gene_or_gene_product
.	O

MNK1	B-Gene_or_gene_product
may	O
define	O
a	O
convergence	O
point	O
between	O
the	O
growth	O
factor	O
-	O
activated	O
and	O
one	O
of	O
the	O
stress	O
-	O
activated	O
protein	O
kinase	O
cascades	O
and	O
is	O
a	O
candidate	O
to	O
phosphorylate	O
eIF	B-Gene_or_gene_product
-	I-Gene_or_gene_product
4E	I-Gene_or_gene_product
in	O
cells	O
.	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
isozymes	O
catalyze	O
the	O
synthesis	O
of	O
3	B-Simple_chemical
-	I-Simple_chemical
phosphate	I-Simple_chemical
-	O
containing	O
phosphatidylinositol	B-Simple_chemical
signaling	O
molecules	O
.	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
kinases	I-Gene_or_gene_product
(	O
PIP5Ks	B-Gene_or_gene_product
)	O
utilize	O
phosphatidylinositols	B-Simple_chemical
containing	O
D	O
-	O
3	O
-	O
position	O
phosphates	B-Simple_chemical
as	O
substrates	O
to	O
form	O
phosphatidylinositol	B-Simple_chemical
3	I-Simple_chemical
,	I-Simple_chemical
4	I-Simple_chemical
-	I-Simple_chemical
bisphosphate	I-Simple_chemical
.	O

In	O
addition	O
,	O
type	B-Gene_or_gene_product
I	I-Gene_or_gene_product
PIP5Ks	I-Gene_or_gene_product
phosphorylate	O
phosphatidylinositol	B-Simple_chemical
3	I-Simple_chemical
,	I-Simple_chemical
4	I-Simple_chemical
-	I-Simple_chemical
bisphosphate	I-Simple_chemical
to	O
phosphatidylinositol	B-Simple_chemical
3	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
while	O
type	B-Gene_or_gene_product
II	I-Gene_or_gene_product
kinases	I-Gene_or_gene_product
have	O
less	O
activity	O
toward	O
this	O
substrate	O
.	O

Remarkably	O
,	O
these	O
kinases	O
can	O
convert	O
phosphatidylinositol	B-Simple_chemical
3	I-Simple_chemical
-	I-Simple_chemical
phosphate	I-Simple_chemical
to	O
phosphatidylinositol	B-Simple_chemical
3	I-Simple_chemical
,	I-Simple_chemical
4	I-Simple_chemical
,	I-Simple_chemical
5	I-Simple_chemical
-	I-Simple_chemical
trisphosphate	I-Simple_chemical
in	O
a	O
concerted	O
reaction	O
.	O

Kinase	O
activities	O
toward	O
the	O
3	O
-	O
position	O
phosphoinositides	B-Simple_chemical
are	O
comparable	O
with	O
those	O
seen	O
with	O
phosphatidylinositol	B-Simple_chemical
4	I-Simple_chemical
-	I-Simple_chemical
phosphate	I-Simple_chemical
as	O
the	O
substrate	O
.	O

Therefore	O
,	O
the	O
PIP5Ks	B-Gene_or_gene_product
can	O
synthesize	O
phosphatidylinositol	B-Simple_chemical
4	I-Simple_chemical
,	I-Simple_chemical
5	I-Simple_chemical
-	I-Simple_chemical
bisphosphate	I-Simple_chemical
and	O
two	O
3	B-Simple_chemical
-	I-Simple_chemical
phosphate	I-Simple_chemical
-	O
containing	O
polyphosphoinositides	B-Simple_chemical
.	O

These	O
unexpected	O
activities	O
position	O
the	O
PIP5Ks	B-Gene_or_gene_product
as	O
potential	O
participants	O
in	O
the	O
generation	O
of	O
all	O
polyphosphoinositide	B-Simple_chemical
signaling	O
molecules	O
.	O

A	O
novel	O
p53	B-Gene_or_gene_product
-	O
inducible	O
gene	O
,	O
PAG608	B-Gene_or_gene_product
,	O
encodes	O
a	O
nuclear	B-Cellular_component
zinc	O
finger	O
protein	O
whose	O
overexpression	O
promotes	O
apoptosis	O
.	O

The	O
biological	O
effects	O
of	O
the	O
p53	B-Gene_or_gene_product
tumor	O
suppressor	O
protein	O
are	O
elicited	O
,	O
at	O
least	O
in	O
part	O
,	O
through	O
sequence	O
-	O
specific	O
transactivation	O
of	O
a	O
battery	O
of	O
target	O
genes	O
.	O

The	O
differential	O
display	O
method	O
was	O
employed	O
towards	O
identifying	O
additional	O
p53	B-Gene_or_gene_product
target	O
genes	O
,	O
with	O
emphasis	O
on	O
genes	O
whose	O
induction	O
may	O
contribute	O
to	O
p53	B-Gene_or_gene_product
-	O
mediated	O
apoptosis	O
.	O

We	O
report	O
here	O
the	O
cloning	O
of	O
a	O
novel	O
p53	B-Gene_or_gene_product
-	O
inducible	O
gene	O
,	O
designated	O
PAG608	B-Gene_or_gene_product
.	O

PAG608	B-Gene_or_gene_product
transcripts	O
are	O
induced	O
by	O
DNA	O
damage	O
in	O
a	O
p53	B-Gene_or_gene_product
-	O
dependent	O
manner	O
.	O

PAG608	B-Gene_or_gene_product
encodes	O
a	O
nuclear	B-Cellular_component
zinc	O
finger	O
protein	O
,	O
which	O
appears	O
to	O
localize	O
preferentially	O
to	O
nucleoli	B-Cellular_component
when	O
expressed	O
at	O
moderate	O
levels	O
in	O
transfected	O
cells	O
.	O

Transient	O
overexpression	O
of	O
PAG608	B-Gene_or_gene_product
in	O
human	O
tumor	O
-	O
derived	O
cells	O
leads	O
to	O
distinctive	O
changes	O
in	O
nuclear	B-Cellular_component
morphology	O
,	O
and	O
can	O
promote	O
apoptosis	O
.	O

Together	O
with	O
additional	O
p53	B-Gene_or_gene_product
target	O
genes	O
,	O
PAG608	B-Gene_or_gene_product
may	O
therefore	O
play	O
a	O
role	O
in	O
mediating	O
the	O
biological	O
activities	O
of	O
p53	B-Gene_or_gene_product
.	O

Organic	B-Gene_or_gene_product
anion	I-Gene_or_gene_product
transporting	I-Gene_or_gene_product
polypeptide	I-Gene_or_gene_product
mediates	O
organic	B-Simple_chemical
anion	I-Simple_chemical
/	O
HCO3	B-Simple_chemical
-	I-Simple_chemical
exchange	O
.	O

Organic	B-Gene_or_gene_product
anion	I-Gene_or_gene_product
transporting	I-Gene_or_gene_product
polypeptide	I-Gene_or_gene_product
(	O
oatp	B-Gene_or_gene_product
)	O
is	O
an	O
integral	O
membrane	B-Cellular_component
protein	O
cloned	O
from	O
rat	O
liver	O
that	O
mediates	O
Na	B-Simple_chemical
+	I-Simple_chemical
-	O
independent	O
transport	O
of	O
organic	B-Simple_chemical
anions	I-Simple_chemical
such	O
as	O
sulfobromophthalein	B-Simple_chemical
and	O
taurocholic	B-Simple_chemical
acid	I-Simple_chemical
.	O

Previous	O
studies	O
in	O
rat	O
hepatocytes	O
suggested	O
that	O
organic	B-Simple_chemical
anion	I-Simple_chemical
uptake	O
is	O
associated	O
with	O
base	O
exchange	O
.	O

To	O
better	O
characterize	O
the	O
mechanism	O
of	O
oatp	B-Gene_or_gene_product
-	O
mediated	O
organic	B-Simple_chemical
anion	I-Simple_chemical
uptake	O
,	O
we	O
examined	O
transport	O
of	O
taurocholate	B-Simple_chemical
in	O
a	O
HeLa	O
cell	O
line	O
stably	O
transfected	O
with	O
oatp	B-Gene_or_gene_product
under	O
the	O
regulation	O
of	O
a	O
zinc	B-Simple_chemical
-	O
inducible	O
promoter	O
(	O
Shi	O
,	O
X	O
.	O
,	O
Bai	O
,	O
S	O
.	O
,	O
Ford	O
,	O
A	O
.	O
C	O
.	O
,	O
Burk	O
,	O
R	O
.	O
D	O
.	O
,	O
Jacquemin	O
,	O
E	O
.	O
,	O
Hagenbuch	O
,	O
B	O
.	O
,	O
Meier	O
,	O
P	O
.	O
J	O
.	O
,	O
and	O
Wolkoff	O
,	O
A	O
.	O
W	O
.	O
(	O
1995	O
)	O
J	O
.	O
Biol	O
.	O
Chem	O
.	O
270	O
,	O
25591	O
-	O
25595	O
)	O
.	O

Whereas	O
noninduced	O
transfected	O
cells	O
showed	O
virtually	O
no	O
uptake	O
of	O
[	B-Simple_chemical
3H	I-Simple_chemical
]	I-Simple_chemical
taurocholate	I-Simple_chemical
,	O
taurocholate	B-Simple_chemical
uptake	O
by	O
induced	O
cells	O
was	O
Na	B-Simple_chemical
+	I-Simple_chemical
-	O
independent	O
and	O
saturable	O
(	O
Km	O
=	O
19	O
.	O
4	O
+	O
/	O
-	O
3	O
.	O
3	O
microM	O
;	O
Vmax	O
=	O
62	O
.	O
2	O
+	O
/	O
-	O
1	O
.	O
4	O
pmol	O
/	O
min	O
/	O
mg	O
protein	O
;	O
n	O
=	O
3	O
)	O
.	O

To	O
test	O
whether	O
organic	B-Simple_chemical
anion	I-Simple_chemical
transport	O
is	O
coupled	O
to	O
HCO3	B-Simple_chemical
-	I-Simple_chemical
extrusion	O
,	O
we	O
compared	O
the	O
rates	O
of	O
taurocholate	B-Simple_chemical
-	O
dependent	O
HCO3	B-Simple_chemical
-	I-Simple_chemical
efflux	O
from	O
alkali	B-Simple_chemical
-	O
loaded	O
noninduced	O
and	O
induced	O
cells	O
.	O

Monolayers	O
grown	O
on	O
glass	O
coverslips	O
were	O
loaded	O
with	O
the	O
pH	O
-	O
sensitive	O
dye	O
2	B-Simple_chemical
'	I-Simple_chemical
,	I-Simple_chemical
7	I-Simple_chemical
'	I-Simple_chemical
-	I-Simple_chemical
bis	I-Simple_chemical
(	I-Simple_chemical
carboxyethyl	I-Simple_chemical
)	I-Simple_chemical
-	I-Simple_chemical
5	I-Simple_chemical
(	I-Simple_chemical
6	I-Simple_chemical
)	I-Simple_chemical
-	I-Simple_chemical
carboxyfluorescein	I-Simple_chemical
;	O
intracellular	B-Cellular_component
pH	O
(	O
pHi	O
)	O
was	O
measured	O
by	O
excitation	O
ratio	O
fluorometry	O
.	O

Noninduced	O
and	O
induced	O
cells	O
were	O
alkalinized	O
to	O
an	O
equivalent	O
pHi	O
(	O
approximately	O
7	O
.	O
7	O
)	O
by	O
transient	O
exposure	O
to	O
a	O
50	O
mM	O
HCO3	B-Simple_chemical
-	I-Simple_chemical
,	O
Cl	B-Simple_chemical
-	I-Simple_chemical
-	O
free	O
solution	O
.	O

In	O
the	O
absence	O
of	O
extracellular	B-Cellular_component
Cl	B-Simple_chemical
-	I-Simple_chemical
and	O
taurocholate	B-Simple_chemical
,	O
isohydric	O
reduction	O
of	O
superfusate	O
HCO3	B-Simple_chemical
-	I-Simple_chemical
concentration	O
from	O
50	O
to	O
25	O
mM	O
resulted	O
in	O
an	O
insignificant	O
change	O
in	O
pHi	O
over	O
time	O
(	O
dpHi	O
/	O
dt	O
)	O
in	O
both	O
groups	O
.	O

Addition	O
of	O
25	O
microM	O
taurocholate	B-Simple_chemical
to	O
the	O
superfusate	O
led	O
to	O
a	O
rapid	O
fall	O
in	O
pHi	O
in	O
induced	O
(	O
-	O
0	O
.	O
037	O
+	O
/	O
-	O
0	O
.	O
011	O
pH	O
units	O
/	O
min	O
to	O
pHi	O
of	O
7	O
.	O
41	O
+	O
/	O
-	O
0	O
.	O
14	O
)	O
but	O
not	O
in	O
noninduced	O
(	O
0	O
.	O
003	O
+	O
/	O
-	O
0	O
.	O
006	O
pH	O
units	O
/	O
min	O
to	O
pHi	O
of	O
7	O
.	O
61	O
+	O
/	O
-	O
0	O
.	O
08	O
)	O
cells	O
(	O
p	O
<	O
0	O
.	O
03	O
)	O
.	O

These	O
data	O
indicate	O
that	O
oatp	B-Gene_or_gene_product
-	O
mediated	O
taurocholate	B-Simple_chemical
transport	O
is	O
Na	B-Simple_chemical
+	I-Simple_chemical
-	O
independent	O
,	O
saturable	O
,	O
and	O
accompanied	O
by	O
HCO3	B-Simple_chemical
-	I-Simple_chemical
exchange	O
.	O

We	O
conclude	O
that	O
organic	B-Simple_chemical
anion	I-Simple_chemical
/	O
base	O
exchange	O
is	O
an	O
important	O
,	O
potentially	O
regulatable	O
component	O
of	O
oatp	B-Gene_or_gene_product
function	O
.	O

Cdc53	B-Gene_or_gene_product
is	O
a	O
scaffold	O
protein	O
for	O
multiple	O
Cdc34	B-Complex
/	I-Complex
Skp1	I-Complex
/	I-Complex
F	I-Complex
-	I-Complex
box	I-Complex
proteincomplexes	O
that	O
regulate	O
cell	O
division	O
and	O
methionine	B-Simple_chemical
biosynthesis	O
in	O
yeast	O
.	O

In	O
budding	O
yeast	O
,	O
ubiquitination	O
of	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
Sic1	B-Gene_or_gene_product
is	O
catalyzed	O
by	O
the	O
E2	O
ubiquitin	B-Gene_or_gene_product
conjugating	O
enzyme	O
Cdc34	B-Gene_or_gene_product
in	O
conjunction	O
with	O
an	O
E3	O
ubiquitin	B-Gene_or_gene_product
ligase	O
complex	O
composed	O
of	O
Skp1	B-Gene_or_gene_product
,	O
Cdc53	B-Gene_or_gene_product
and	O
the	O
F	O
-	O
box	O
protein	O
,	O
Cdc4	B-Gene_or_gene_product
(	O
the	O
SCFCdc4	B-Complex
complex	O
)	O
.	O

Skp1	B-Gene_or_gene_product
binds	O
a	O
motif	O
called	O
the	O
F	O
-	O
box	O
and	O
in	O
turn	O
F	O
-	O
box	O
proteins	O
appear	O
to	O
recruit	O
specific	O
substrates	O
for	O
ubiquitination	O
.	O

We	O
find	O
that	O
Skp1	B-Gene_or_gene_product
interacts	O
with	O
Cdc53	B-Gene_or_gene_product
in	O
vivo	O
,	O
and	O
that	O
Skp1	B-Gene_or_gene_product
bridges	O
Cdc53	B-Gene_or_gene_product
to	O
three	O
different	O
F	O
-	O
box	O
proteins	O
,	O
Cdc4	B-Gene_or_gene_product
,	O
Met30	B-Gene_or_gene_product
,	O
and	O
Grr1	B-Gene_or_gene_product
.	O

Cdc53	B-Gene_or_gene_product
contains	O
independent	O
binding	O
sites	O
for	O
Cdc34	B-Gene_or_gene_product
and	O
Skp1	B-Gene_or_gene_product
suggesting	O
it	O
functions	O
as	O
a	O
scaffold	O
protein	O
within	O
an	O
E2	O
/	O
E3	O
core	O
complex	O
.	O

F	O
-	O
box	O
proteins	O
show	O
remarkable	O
functional	O
specificity	O
in	O
vivo	O
:	O
Cdc4	B-Gene_or_gene_product
is	O
specific	O
for	O
degradation	O
of	O
Sic1	B-Gene_or_gene_product
,	O
Grr1	B-Gene_or_gene_product
is	O
specific	O
for	O
degradation	O
of	O
the	O
G1	B-Gene_or_gene_product
cyclin	I-Gene_or_gene_product
Cln2	B-Gene_or_gene_product
,	O
and	O
Met30	B-Gene_or_gene_product
is	O
specific	O
for	O
repression	O
of	O
methionine	B-Simple_chemical
biosynthesis	O
genes	O
.	O

In	O
contrast	O
,	O
the	O
Cdc34	B-Complex
-	I-Complex
Cdc53	I-Complex
-	I-Complex
Skp1	I-Complex
E2	O
/	O
E3	O
core	O
complex	O
is	O
required	O
for	O
all	O
three	O
functions	O
.	O

Combinatorial	O
control	O
of	O
SCF	B-Gene_or_gene_product
complexes	O
may	O
provide	O
a	O
basis	O
for	O
the	O
regulation	O
of	O
diverse	O
cellular	O
processes	O
.	O

Rat	O
liver	O
mitochondria	B-Cellular_component
can	O
hydrolyse	O
thiamine	B-Simple_chemical
pyrophosphate	I-Simple_chemical
to	O
thiamine	B-Simple_chemical
monophosphate	I-Simple_chemical
which	O
can	O
cross	O
the	O
mitochondrial	B-Cellular_component
membrane	I-Cellular_component
in	O
a	O
carrier	O
-	O
mediated	O
process	O
.	O

We	O
show	O
here	O
that	O
TPP	B-Simple_chemical
-	O
-	O
>	O
TMP	B-Simple_chemical
conversion	O
can	O
take	O
place	O
in	O
rat	O
liver	O
mitochondria	B-Cellular_component
.	O

This	O
occurs	O
via	O
the	O
novel	O
,	O
putative	O
TPP	B-Gene_or_gene_product
pyrophosphatase	I-Gene_or_gene_product
localised	O
in	O
the	O
mitochondrial	B-Cellular_component
matrix	I-Cellular_component
,	O
as	O
shown	O
both	O
by	O
digitonin	B-Simple_chemical
titration	O
and	O
by	O
an	O
HPLC	O
enzyme	O
assay	O
carried	O
out	O
on	O
the	O
mitochondrial	B-Cellular_component
matrix	I-Cellular_component
fraction	O
.	O

Certain	O
features	O
of	O
the	O
reaction	O
,	O
including	O
the	O
substrate	O
and	O
pH	O
dependence	O
,	O
are	O
reported	O
.	O

Additional	O
evidence	O
is	O
given	O
that	O
externally	O
added	O
TMP	B-Simple_chemical
can	O
cross	O
the	O
mitochondrial	B-Cellular_component
membrane	I-Cellular_component
in	O
a	O
manner	O
consistent	O
with	O
the	O
occurrence	O
of	O
a	O
carrier	O
-	O
mediated	O
process	O
.	O

This	O
can	O
occur	O
both	O
via	O
the	O
TPP	B-Simple_chemical
translocator	O
and	O
via	O
a	O
novel	O
translocator	O
,	O
inhibited	O
by	O
CAT	B-Simple_chemical
but	O
different	O
from	O
the	O
ADP	B-Simple_chemical
/	O
ATP	B-Simple_chemical
carrier	O
.	O

Maturation	O
of	O
human	O
cyclin	B-Gene_or_gene_product
E	I-Gene_or_gene_product
requires	O
the	O
function	O
of	O
eukaryotic	O
chaperonin	O
CCT	B-Complex
.	O

Cyclin	B-Gene_or_gene_product
E	I-Gene_or_gene_product
,	O
a	O
partner	O
of	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
Cdk2	I-Gene_or_gene_product
,	O
has	O
been	O
implicated	O
in	O
positive	O
control	O
of	O
the	O
G1	O
/	O
S	O
phase	O
transition	O
.	O

Whereas	O
degradation	O
of	O
cyclin	B-Gene_or_gene_product
E	I-Gene_or_gene_product
has	O
been	O
shown	O
to	O
be	O
exquisitely	O
regulated	O
by	O
ubiquitination	O
and	O
proteasomal	O
action	O
,	O
little	O
is	O
known	O
about	O
posttranscriptional	O
aspects	O
of	O
its	O
biogenesis	O
.	O

In	O
a	O
yeast	O
-	O
based	O
screen	O
designed	O
to	O
identify	O
human	O
proteins	O
that	O
interact	O
with	O
human	O
cyclin	B-Gene_or_gene_product
E	I-Gene_or_gene_product
,	O
we	O
identified	O
components	O
of	O
the	O
eukaryotic	O
cytosolic	B-Cellular_component
chaperonin	O
CCT	B-Complex
.	O

We	O
found	O
that	O
the	O
endogenous	O
CCT	B-Complex
complex	O
in	O
yeast	O
was	O
essential	O
for	O
the	O
maturation	O
of	O
cyclin	B-Gene_or_gene_product
E	I-Gene_or_gene_product
in	O
vivo	O
.	O

Under	O
conditions	O
of	O
impaired	O
CCT	B-Complex
function	O
,	O
cyclin	B-Gene_or_gene_product
E	I-Gene_or_gene_product
failed	O
to	O
accumulate	O
.	O

Furthermore	O
,	O
newly	O
translated	O
cyclin	B-Gene_or_gene_product
E	I-Gene_or_gene_product
,	O
both	O
in	O
vitro	O
in	O
reticulocyte	O
lysate	O
and	O
in	O
vivo	O
in	O
human	O
cells	O
in	O
culture	O
,	O
is	O
efficiently	O
bound	O
and	O
processed	O
by	O
the	O
CCT	B-Complex
.	O

In	O
vitro	O
,	O
in	O
the	O
presence	O
of	O
ATP	B-Simple_chemical
,	O
the	O
bound	O
protein	O
is	O
folded	O
and	O
released	O
in	O
order	O
to	O
become	O
associated	O
with	O
Cdk2	B-Gene_or_gene_product
.	O

Thus	O
,	O
both	O
the	O
acquisition	O
of	O
the	O
native	O
state	O
and	O
turnover	O
of	O
cyclin	B-Gene_or_gene_product
E	I-Gene_or_gene_product
involve	O
ATP	B-Simple_chemical
-	O
dependent	O
processes	O
mediated	O
by	O
large	O
oligomeric	O
assemblies	O
.	O

A	O
human	O
homologue	O
of	O
the	O
checkpoint	O
kinase	O
Cds1	B-Gene_or_gene_product
directly	O
inhibits	O
Cdc25	B-Gene_or_gene_product
phosphatase	O
.	O

BACKGROUND	O
:	O
In	O
human	O
cells	O
,	O
the	O
mitosis	O
-	O
inducing	O
kinase	O
Cdc2	B-Gene_or_gene_product
is	O
inhibited	O
by	O
phosphorylation	O
on	O
Thr14	B-Simple_chemical
and	O
Tyr15	B-Simple_chemical
.	O

Disruption	O
of	O
these	O
phosphorylation	O
sites	O
abrogates	O
checkpoint	O
-	O
mediated	O
regulation	O
of	O
Cdc2	B-Gene_or_gene_product
and	O
renders	O
cells	O
highly	O
sensitive	O
to	O
agents	O
that	O
damage	O
DNA	O
.	O

Phosphorylation	O
of	O
these	O
sites	O
is	O
controlled	O
by	O
the	O
opposing	O
activities	O
of	O
the	O
Wee1	B-Gene_or_gene_product
/	O
Myt1	B-Gene_or_gene_product
kinases	O
and	O
the	O
Cdc25	B-Gene_or_gene_product
phosphatase	O
.	O

The	O
regulation	O
of	O
these	O
enzymes	O
is	O
therefore	O
likely	O
to	O
be	O
crucial	O
for	O
the	O
operation	O
of	O
the	O
G2	O
-	O
M	O
DNA	O
-	O
damage	O
checkpoint	O
.	O

RESULTS	O
:	O
Here	O
,	O
we	O
show	O
that	O
the	O
activity	O
of	O
Cdc25	B-Gene_or_gene_product
decreased	O
following	O
exposure	O
to	O
ionizing	O
radiation	O
.	O

The	O
irradiation	O
-	O
induced	O
decrease	O
in	O
Cdc25	B-Gene_or_gene_product
activity	O
was	O
suppressed	O
by	O
wortmannin	B-Simple_chemical
,	O
an	O
inhibitor	O
of	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
kinases	I-Gene_or_gene_product
,	O
and	O
was	O
dependent	O
on	O
the	O
function	O
of	O
the	O
gene	O
that	O
is	O
mutated	B-Gene_or_gene_product
in	I-Gene_or_gene_product
ataxia	I-Gene_or_gene_product
telangiectasia	I-Gene_or_gene_product
.	O

We	O
also	O
identified	O
two	O
human	O
kinases	O
that	O
phosphorylate	O
and	O
inactivate	O
Cdc25	B-Gene_or_gene_product
in	O
vitro	O
.	O

One	O
is	O
the	O
previously	O
characterized	O
Chk1	B-Gene_or_gene_product
kinase	O
.	O

The	O
second	O
is	O
novel	O
and	O
is	O
homologous	O
to	O
the	O
Cds1	B-Gene_or_gene_product
/	I-Gene_or_gene_product
Rad53	I-Gene_or_gene_product
family	O
of	O
checkpoint	O
kinases	O
in	O
yeast	O
.	O

Human	O
Cds1	B-Gene_or_gene_product
was	O
found	O
to	O
be	O
activated	O
in	O
response	O
to	O
DNA	O
damage	O
.	O

CONCLUSIONS	O
:	O
These	O
results	O
suggest	O
that	O
,	O
in	O
human	O
cells	O
,	O
the	O
DNA	O
-	O
damage	O
checkpoint	O
involves	O
direct	O
inactivation	O
of	O
Cdc25	B-Gene_or_gene_product
catalyzed	O
by	O
Cds1	B-Gene_or_gene_product
and	O
/	O
or	O
Chk1	B-Gene_or_gene_product
.	O

