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INTRODUCTION |
Extracellular stimuli elicit changes in gene expression in target
cells by activating intracellular protein kinase cascades that
phosphorylate transcription factors within the nucleus. The first and
one of the best characterized stimulus-induced transcription factors
with activity shown to be regulated by phosphorylation, is the cyclic
AMP (cAMP) response element (CRE)-binding protein, or
CREB1 (1, 2). In addition to
its role as a cAMP-responsive activator, CREB can also be
phosphorylated in response to several growth factor and stress signals,
which have been shown to promote phosphorylation of CREB at Ser-133,
with comparable stoichiometry and kinetics (3). This growth factor
activation of CREB has been shown to be Ras-dependent and
to involve the mitogen-activated protein kinases (MAPKs). Although a
number of kinases downstream from the MAPKs may also be implicated,
members of the pp90RSK (ribosomal S6 kinase, RSK) family
have been identified as mitogen-responsive CREB kinases (4). For
instance, both CREB phosphorylation and c-fos
transcriptional induction are drastically impaired in response to
epidermal growth factor (EGF) in human fibroblasts derived from
Coffin-Lowry syndrome patients, which carry mutations in the gene
encoding the RSK-2 kinase (5, 6).
CREB-binding protein (CBP) is a large pleiotropic cellular coactivator
protein which is critical to the execution of a large variety of
cellular programs, including cell growth, differentiation, and
apoptosis. CBP was originally discovered through its interaction with
the cellular transcription factor CREB (7, 8). Protein kinase
A-mediated phosphorylation of a critical Ser-133 on CREB is required
for the complex formation between CREB and CBP (9). However, recent
studies have shown that CBP can also interact with a multitude of
structurally unrelated cellular transcription factors and components of
the basal transcription apparatus (10). It has been reported that
insulin or nerve growth factor (NGF)-stimulated activation of the Ras
pathway represses CREB activity by inducing recruitment of
pp90RSK to CBP (11). CBP and RSK-2 associate in a complex
in quiescent cells, and they dissociate within a few min of mitogenic
stimulation (12). CBP interacts preferentially with unphosphorylated
RSK-2 in a complex where both RSK-2 kinase activity and CBP acetylase activity are inhibited (12). Moreover, both CREB and p53 can interact
directly with CBP, and phosphorylated CREB mediates recruitment of CBP
to p53-responsive promoters through direct interaction with p53 (13).
These reports provide evidence that CBP has a pivotal effect on gene
regulation and plays an important role in cellular differentiation and
development (14).
MAPKs are a family of second messenger kinases that are essential for
transferring signals from the cell surface to the nucleus (15, 16).
Among them, extracellular signal-regulated kinase (ERK) is activated in
response to proliferative factors such as epidermal growth factor (EGF)
as well as in response to differentiative factors such as NGF (17).
Although ERK is thought to play a key role in the proliferative
process, recent studies have also suggested that persistent activation
of ERK might mediate cell cycle arrest and differentiation (17-19,
21). After activation, phospho-ERK is translocated to the nucleus (20,
21), where it can phosphorylate transcription factors, leading to
altered gene expression (19, 22, 23). We have shown previously that a
Cdc25A protein phosphatase inhibitor,
2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone, or compound 5 (Cpd
5), can induce persistent ERK phosphorylation and nuclear
translocation, which is related to cell growth inhibition in both
normal rat hepatocytes and Hep3B human hepatoma cells (24, 25). In the
current study, we used Cpd 5 as a tool to explore the mechanism(s) by
which persistent ERK phosphorylation regulates transcription factor
activity. We found that in Cpd 5-treated Hep3B cells, CREB activity was
strongly inhibited, and this inhibition was antagonized by MAPK/ERK
kinase (MEK) inhibitors PD 98059 and U-0126. Furthermore, Cpd 5 treatment increased CBP binding to CREB kinase pp90RSK,
leading to pp90RSK hypophosphorylation followed by
suppression of CREB transcriptional activity. We propose that
persistent ERK activation negatively regulates CREB activity via
increased association of CBP to pp90RSK.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Cells of the Hep3B human hepatoma cell line
were maintained in Eagle's MEM supplemented with 10% fetal bovine
serum. For experiments comparing the effects of EGF and Cpd 5 on CREB
phosphorylation, Hep3B cells were grown in MEM with 10% fetal bovine
serum. After about 75% confluence, Hep3B cells were serum starved for
24 h and then treated with either EGF or Cpd 5 at different time
intervals. Cpd 5, a synthetic vitamin K analog, was synthesized as
described previously (25).
Western Blot Analysis--
Hep3B cells were plated in 100-mm
tissue culture dishes and treated with or without Cpd 5 for various
times. After treatment, the cells were washed twice with cold
phosphate-buffered saline and then lysed in 100 µl of radioimmune
precipitation assay buffer (150 mM NaCl, 50 mM
Tris-HCl, pH 8.0, 0.1% SDS, 1% Triton X-100, 1 mM
orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml leupeptin, 10 mg/ml aprotinin). Whole cell extracts (20 µg) were resolved on a 10% SDS-polyacrylamide gel and transferred onto Hybond
polyvinylidene difluoride membranes (Amersham Biosciences). Membranes
were blocked using Tris-buffered saline with Tween 20 (TBST, 150 mM NaCl, 10 mM Tris-HCl, pH 8.0, and 0.05%
Tween 20) containing 1% bovine serum albumin for 1 h, then probed
with the indicated primary antibody for 1 h. After washing four
times with TBST, the membranes were probed with horseradish
peroxidase-conjugated secondary antibody to allow detection of the
appropriate bands using enhanced chemiluminescence (Amersham
Biosciences).
Nuclear Extract Preparation and Immunoprecipitation
Assay--
Hep3B cell nuclear extracts were prepared as described
previously, with some modifications (26). Briefly, EGF- or Cpd
5-treated Hep3B cells were washed with cold phosphate-buffered saline,
then resuspended in 200 µl of buffer A (10 mM HEPES, pH
7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5%
Nonidet P-40). After a 10-min incubation on ice, cells were centrifuged
for 15 min at 5,000 rpm at 4 °C. Nuclei were collected by removing
the supernatant and then washed with 500 µl of buffer A without
Nonidet P-40 and centrifuged for 15 min at 5,000 rpm at 4 °C. The
nuclear pellets were resuspended in 100 µl of buffer B (20 mM HEPES, pH 7.9, 25% glycerol (v/v), 1.5 mM
MgCl2, 0.5 mM EDTA, 0.5 M KCl) and
incubated on ice for 60 min. A stock solution giving 0.5 mM
dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 1 mg/ml leupeptin, 1 mg/ml pepstatin A, and 1 mg/ml chymostatin was added
to buffer A and buffer B before use. After centrifugation at 14,000 rpm
for 30 min, supernatants were collected, aliquoted, and stored at
80 °C. Protein concentration was measured using bicinchoninic acid
(BCA) protein assay reagent (Pierce).
For immunoprecipitation assay, 100-µg nuclear extracts were
immunoprecipitated with anti-CBP antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and protein A-agarose (Sigma) at 4 °C overnight. The
protein A-agarose pellets were washed three times with radioimmune precipitation assay buffer, boiled in 40 µl of 2 × sample
buffer for 5 min, and resolved in 8% SDS-PAGE. The transferred
polyvinylidene difluoride membrane was then probed with anti-RSK-1 or
anti-phosphoserine antibody to detect the phosphorylation status of CBP
and the existence of the CBP·RSK immunocomplex.
Electrophoretic Mobility Shift Assay--
Consensus CRE
oligonucleotides (5'-AGA GAT TGC CTG ACG TCA GAG AGC TAG-3') (Santa
Cruz Biotechnology) were end labeled with [
-32P]ATP by
T4 polynucleotide kinase (Roche Molecular Biochemicals). For binding
reactions, 1 × 105 cpm of labeled oligonucleotide
probes were incubated with 5 µg of nuclear extracts and 1 µg of
poly(dI-dC) in binding buffer (4% (v/v) glycerol, 1 mM
MgCl2, 0.5 mM EDTA, 0.5 mM
dithiothreitol, 50 mM NaCl, 10 mM Tris-Cl, pH
7.5) at room temperature for 20 min. Protein·DNA complexes were
separated by electrophoresis in a 5% nondenaturing polyacrylamide gel
in 0.5 × TBE buffer and visualized by autoradiography. For
competition experiments, a 100-fold molar excess of cold or mutant
oligonucleotides (5'-AGA GAT TGC CTG ATA TCA GAG AGC TAG-3', Santa Cruz
Biotechnology) were included in the mixture.
Cell Transfection--
A mammalian expression plasmid encoding
catalytically inactive C430S mutant Cdc25A in a pcDNA3 vector were
generously provided by Dr. Thomas Roberts (Dana Farber Cancer
Institute, Boston, MA) (27). Transfections were carried out by the
LipofectAMINE method following the manufacturer's instructions
(Invitrogen). Briefly, Hep3B cells (100,000/well) were plated in
six-well plates and transfected with 1.0 µg/well plasmid DNA in
Opti-MEM using LipofectAMINE Plus reagent (Invitrogen) After a 5-h
transfection, the medium was replaced with complete growth medium, and
the cells were allowed to recover for 48 h. Cells were treated
without or with 20 µM Cpd 5, and cell lysates were
analyzed by Western blot or immunoprecipitation assay.
Luciferase Activity Assay--
Hep3B cells were plated in
six-well plates and cotransfected with 1.0 µg/well pCRE-Luc plasmid
(Invitrogen) and 0.2 µg/well pIETLacZ (
-galactosidase expression
plasmid with cytomegalovirus promoter) (Invitrogen) in Opti-MEM using
LipofectAMINE Plus reagent for 6 h. Cells were allowed to recover
by changing to complete growth medium overnight and were then treated
with Cpd 5 at the indicated concentrations. After washing twice with
phosphate-buffered saline, cells were lysed in 100 µl of lysis buffer
provided in the Promega luciferase assay kit and centrifuged to remove
the cell debris. The luciferase activity was measured in relative light
units using a Monolight Luminometer. The
-galactosidase activity was
measured as described previously (28). To normalize the transfection
efficiency, the luciferase activity was expressed as a ratio of
relative light units to the
-galactosidase activity obtained from
the same cell lysates.
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RESULTS |
Cpd 5 Induces Persistent ERK Phosphorylation and Represses CREB
Activity--
We have shown previously that Cpd 5, a Cdc25A protein
phosphatase inhibitor, induced persistent ERK phosphorylation in rat hepatocytes and Hep3B cells, which was in turn related to cell growth
inhibition (24, 25, 29). To explore the mechanism(s) by which
persistent ERK activation causes cell growth inhibition, we examined
the transcription factor CREB in Hep3B cells because various signaling
routes converge on CREB and control its function by modulating its
phosphorylation status (1). Hep3B cells were grown in MEM with 10%
fetal bovine serum until about 75% confluence, then cells were
serum-starved for 24 h. 10 ng/ml EGF or 20 µM Cpd 5 was then added to the medium for periods from 15 to 180 min. After
harvest, cells were lysed in radioimmune precipitation assay buffer,
and Western blots were performed using anti-phospho-ERK antibody. Fig.
1A shows that EGF induced a
transient ERK phosphorylation with a peak at 15 min, which then
returned to base line levels at 60 min and disappeared by 180 min.
However, Cpd 5 induced a persistent ERK phosphorylation for at least
180 min. For CREB phosphorylation experiments, nuclear extracts were
prepared from Hep3B cells treated with either EGF or Cpd 5 as described
above, and Western blot analysis was performed using anti-phospho-CREB (Ser-133) antibody. In contrast to ERK phosphorylation, EGF gradually induced CREB phosphorylation, whereas Cpd 5 inhibited it (Fig. 1B). To support our findings on CREB phosphorylation
further, we also examined the CRE DNA binding activity. Hep3B cells
were treated with 10 ng/ml EGF or 5-20 µM Cpd 5 for 30 min, and nuclear extracts were used for DNA binding assay as described
under "Experimental Procedures." We found that EGF stimulated CREB
DNA binding activity, whereas Cpd 5 inhibited it in a
dose-dependent manner (Fig.
2).

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Fig. 1.
Effects of EGF and Cpd 5 on ERK and CREB
phosphorylation. A, Hep3B cells were treated with 10 ng/ml EGF or 20 µM Cpd 5 from 15 to 180 min. Cell lysates
were immunoblotted with anti-phospho-ERK and anti-ERK-2 antibodies.
B, EGF- or Cpd 5-treated Hep3B cell nuclear extracts were
immunoblotted with anti-phospho-CREB and anti-CREB antibodies.
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Fig. 2.
Cpd 5 inhibited CREB DNA binding activity in
a dose-dependent manner. Hep3B cells were treated with
10 ng/ml EGF or 5-20 µM Cpd 5 for 30 min. Nuclear
extract preparation and electrophoretic mobility shift assay were
performed as described under "Experimental Procedures."
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Cpd 5 Inhibits CRE-dependent Gene
Transcription--
It has been reported that CREB phosphorylation at
Ser-133 is required for signal-induced transcription in vivo
(30). We therefore examined whether Cpd 5-inhibited CREB
phosphorylation might effect its transcriptional activity. We first
measured CRE-dependent promoter activity in Hep3B cells
using the pCRE-Luc reporter plasmid transfection and found that Cpd 5 strongly inhibited the luciferase activity in a
dose-dependent manner (Fig.
3A). Then, we examined two
well known products of CREB-regulated gene expression, namely cyclin D1
and Bcl-2. Fig. 3B shows that EGF had little effect on the
expression of either cyclin D1 or Bcl-2, whereas both gene products
were strongly suppressed by Cpd 5, as early as 1 h after treatment. This is consistent with our previous findings in rat hepatocytes that Cpd 5-induced rat hepatocyte apoptosis and DNA synthesis were associated with Bcl-2 and cyclin D1 inhibition (29,
31).

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Fig. 3.
A, Cpd 5 inhibits CREB transcriptional
activity in a dose-dependent manner. Hep3B cells
were treated with 5, 10, and 20 µM Cpd 5 for 60 min.
Luciferase activity was measured as described under "Experimental
Procedures" and expressed as a percentage of control levels.
B, Cpd 5, but not EGF, inhibits CREB-regulated Bcl-2 and
cyclin D1 expression. Hep3B cells were treated with 10 ng/ml EGF or 20 µM Cpd 5 from 1 to 24 h. Whole cell lysates were
immunoblotted with anti-Bcl-2 and anti-cyclin D1 antibodies,
respectively.
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The Inhibition of CREB Transcriptional Activity by Cpd 5 Involves
Persistent ERK Phosphorylation--
Because Cpd 5 induced persistent
ERK phosphorylation and suppressed CREB activity, we investigated
whether the inhibition of CREB activity was the result of ERK
phosphorylation. Hep3B cells were treated with MEK inhibitors PD 98059 (20 µM) or U-0126 (5 µM) for 1 h
before the addition of 20 µM Cpd 5. We found that both PD
98059 and U-0126 effectively antagonized the effects of Cpd 5 on ERK
and CREB phosphorylation (Fig.
4A). We further evaluated the
effects of these MEK inhibitors on CREB-dependent
transcriptional activity. Hep3B cells were transiently transfected with
pCRE-Luc plasmids. After recovery, the cells were treated with PD 98059 or U-0126 for 1 h before the addition of 20 µM Cpd
5. Fig. 4B shows that Cpd 5 almost completely inhibited
luciferase activity and that both PD 98059 and U-0126 antagonized this
inhibition by about 60 and 50%, respectively. These data suggest that
the inhibition of CREB activity by Cpd 5 is regulated at least in part
by the phosphorylation of ERK pathway.

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Fig. 4.
A, the effects of Cpd 5 on ERK and CREB
phosphorylation are antagonized by MEK inhibitors PD 98059 and U-126.
Hep3B cells were treated with 20 µM PD 98059 or 5 µM U-126 for 1 h before the addition of 20 µM Cpd 5 for 1 additional h. Cell lysates or nuclear
extracts were immunoblotted with anti-phospho-ERK or anti-phospho-CREB
antibodies. B, the Cpd 5-induced inhibition of CREB
transcriptional activity is blocked by MEK inhibitors. Hep3B cells were
transiently transfected with pCRE-Luc plasmids as described under
"Experimental Procedures." After recovery, the transfected Hep3B
cells were treated with 20 µM PD 98059 or 5 µM U-126 for 1 h before the addition of 20 µM Cpd 5. After a 1-h culture, the cells were harvested
for luciferase activity assay as described under "Experimental
Procedures."
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Persistent ERK Activation Is Related to Inhibition of
pp90RSK Phosphorylation--
The pp90RSK
family of serine/threonine kinases has been shown to be activated by
the ERK kinase pathway in response to many growth factors, including
EGF (32, 33), and it functions to phosphorylate transcription factors
such as CREB (1). Thus, the fact that Cpd 5 induced a persistent ERK
activation and suppressed CREB activity prompted us to examine whether
pp90RSK was involved in Cpd 5 actions. We first compared
the effects of EGF and Cpd 5 on RSK-1 phosphorylation at residues
Ser-380, Thr-359/Ser-363, and Thr-574 because the phosphorylation at
these residues is thought to be related to ERK kinase activity (34). We
found that EGF moderately stimulated RSK-1 phosphorylation at these
residues, but Cpd 5 had an inhibitory effect on them. Cpd 5 strongly
inhibited RSK-1 phosphorylation at Ser-380 and Thr-574 and moderately
inhibited phosphorylation at Thr-359/Ser-363 (Fig.
5A). To examine the
relationship between RSK and ERK phosphorylation, we used PD 98059 and
U-0126 to determine whether these MEK inhibitors could reverse the
inhibitory effects of Cpd 5 on RSK phosphorylation. Fig. 5B
shows that both PD 98059 and U-0126 effectively antagonized Cpd
5-mediated RSK hypophosphorylation at Ser-380, suggesting that
persistent ERK activation by Cpd 5 mediated inhibition of RSK
phosphorylation.

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Fig. 5.
Cpd 5 inhibits pp90RSK
phosphorylation, and this inhibitory effect is antagonized by MEK
inhibitors PD 98059 and U-126. A, Hep3B cells were
treated with 10 ng/ml EGF or 20 µM Cpd 5 for 5-60 min.
Cell lysates were immunoblotted with three different anti-phospho-RSK-1
antibodies. B, Hep3B cells were pretreated with MEK
inhibitor PD 98059 or U-126 for 1 h, then 20 µM Cpd
5 was added for 1 more h. Cell lysates were immunoblotted with
anti-phospho-RSK-1 (Ser-380) antibody.
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Cpd 5 Increases the Association of pp90RSK with
CBP--
The Ras-ERK pathway has been shown to up-regulate a number of
nuclear factors via a cascade of kinases to phosphorylate these factors
in response to mitogenic signals. Our experiments showed that the
growth-inhibiting Cpd 5-induced persistent ERK phosphorylation suppressed RSK kinase and subsequently CREB activity. These results led
us to speculate that CBP might play an important role in controlling CREB-regulated gene expression because it has been reported that insulin or NGF suppressed CREB transcriptional activity via recruitment of RSK to CBP (11). To investigate whether Cpd 5-induced persistent ERK
phosphorylation increased recruitment of RSK to CBP, Hep3B cells were
serum starved for 24 h and then treated with EGF or Cpd 5 for the
indicated times (15-60 min). Nuclear extracts were prepared and
immunoprecipitated with anti-CBP antibody and then Western blotted with
anti-RSK1 and anti-phosphoserine antibodies. Fig.
6 shows that after stimulation by EGF,
CBP serine phosphorylation and its binding to RSK-1 were decreased,
whereas Cpd 5 treatment enhanced both CBP phosphorylation and
CBP·RSK-1 complex formation. Because Cdc25A protein phosphatase has
multiple protein substrates and Cpd 5-induced persistent ERK
phosphorylation is partially related to its inhibitory effects on
Cdc25A activity, we thus examined whether Cdc25A was able to
dephosphorylate phosphorylated CBP directly and inhibition of Cdc25A
activity by Cpd 5 could result in CBP phosphorylation. We used Cpd
5-treated Hep3B cell nuclear extracts as the source of phosphorylated
CBP, which were immunoprecipitated with anti-CBP antibody and incubated
with 25 units of recombinant active GST-Cdc25A protein (Upstate
Biotechnology) in the presence or absence of 10 µM Cpd 5 pretreatment in 1 × phosphatase buffer at 30 °C for 10 min. We
found that GST-Cdc25A had no dephosphorylation effect on phosphorylated
CBP, and pretreatment of GST-Cdc25A with Cpd 5 did not alter CBP
phosphorylation status either (data not shown). These data provide
evidence that persistent, but not transient, ERK phosphorylation is
associated with enhanced phosphorylation of CBP and thus an increase in
the binding of CBP to RSK-1. This increased binding of CBP to RSK is an
important mechanism in the negative regulation of CREB activity.

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Fig. 6.
Cpd 5 enhances CBP phosphorylation and its
recruitment to RSK-1. Hep3B cells were treated with 10 ng/ml EGF
or 20 µM Cpd 5 for 15-60 min. Cell lysates were
immunoprecipitated with anti-CBP antibody, and the immunoprecipitates
were blotted with anti-phosphoserine, anti-RSK-1, and anti-CBP
antibodies, respectively.
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The Effects of C430S Cdc25A Mutant Transfection on ERK
Phosphorylation Mimic Cpd 5 Actions--
We have reported previously
that Cdc25A has a direct dephosphorylation effect on ERK kinase, and
the inhibition of Cdc25A activity by Cpd 5 contributes to persistent
ERK phosphorylation (24). We therefore expected that Cdc25A mutation
might cause a constitutive-like ERK phosphorylation, which would also
negatively regulate CREB·RSK activity. To confirm our hypothesis,
Hep3B cells were transiently transfected with C430S Cdc25A mutant and
then treated with or without Cpd 5 for 60 min. Fig.
7, top lane, shows that C430S
Cdc25A transfection resulted in strong basal ERK phosphorylation compared with wild-type Hep3B cells. Cpd 5 treatment also induced ERK
phosphorylation in cultures of C430S transfected cells, which is caused
by the coexistence of an admixture of the wild-type cells. CREB Ser-133
and RSK-1 Ser-380 phosphorylation were almost completely suppressed in
C430S Hep3B cells. Furthermore, CBP and RSK-1 coimmunoprecipitation
experiments showed that Cdc25A mutation caused a much stronger
CBP·RSK-1 association in untreated cells containing the Cdc25A mutant
compared with the Cpd 5-treated wild-type cells. These data further
support our above observations that persistent ERK phosphorylation
caused by Cdc25A inhibition down-regulates CREB phosphorylation and
activity, likely via the recruitment of CBP to hypophosphorylated
RSK-1.

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Fig. 7.
Cdc25A mutant C430S-induced constitutive-like
ERK activation mimics the effects of Cpd 5 on the ERK-CREB
pathway. Hep3B cells were transiently transfected with C430S
plasmid as described under "Experimental Procedures." The wild-type
and C430S-transfected Hep3B cells were treated without or with 20 µM Cpd 5 for 1 h. Whole cell lysates or nuclear
extracts were immunoblotted with anti-phospho-ERK, anti-phospho-CREB,
and anti-phospho-RSK-1 (Ser-380) antibodies. For the
immunoprecipitation assay, cell nuclear extracts were
immunoprecipitated with anti-CBP antibody and blotted with anti-RSK-1
antibody.
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DISCUSSION |
We have reported previously that both EGF and Cpd 5, a protein
phosphatase Cdc25A inhibitor, activate the MAPK pathway in normal rat
hepatocytes, human hepatoma cells, and multiple other cell types (21,
25, 35). However, EGF induces hepatocyte DNA synthesis and Hep3B cell
growth, whereas Cpd 5 mediates an inhibitory effect on the growth of
both cell types. This paradox is probably caused by the duration and
strength of the ERK phosphorylation: EGF induces a transient and weak
ERK phosphorylation without significant nuclear translocation, and Cpd
5 induces a persistent and strong ERK phosphorylation with dramatic
nuclear translocation (21, 24, 25). In the current study, we extended
our previous investigations to explore the mechanism(s) by which
persistent ERK activation inhibits cell growth. We established a
persistent endogenous ERK phosphorylation model using Cpd 5 as a tool
to examine how it regulates the function of the CREB transcription
factor. We found that Cpd 5 inhibited CREB activity, including CREB
phosphorylation and DNA binding ability, and subsequently
CREB-regulated Bcl-2 and cyclin D1 expression. The inhibitory effect of
Cpd 5 on CREB activity was most likely mediated by ERK phosphorylation
because MEK inhibitors PD 98059 and U-0126 partially blocked Cpd
5-mediated CREB hypophosphorylation and inhibition of CREB-regulated
gene transcription. We cannot role out, however, that other signal pathways may also be involved in regulating Cpd 5-induced suppression of CREB transcriptional activity. We showed previously that Cpd 5 induced multiple tyrosine-phosphorylated protein bands (36), implying
that multiple phosphatases are likely involved. Furthermore, in Cdc25A
mutant C430S-transfected Hep3B cells, CREB was in a hypophosphorylation
state similar to wild-type Hep3B cells treated with Cpd 5 because
Cdc25A is thought to be an ERK phosphatase, and C430S
transfection is able to induce a constitutive-like ERK phosphorylation (24).
Although the MAPK pathway has been shown to function in the stimulation
of cellular proliferation (37), we provide evidence here that
persistent ERK activation can also lead to suppression of CREB activity
and inhibition of cell growth. A role for MAPK pathway signaling in
growth arrest or cellular differentiation is not unprecedented. For
instance, it has been reported that MAPK is necessary for
differentiation of thymocytes (38), and in PC12 and NIH 3T3 cells,
NGF-induced cellular differentiation and cell cycle arrest are
accompanied by a prolonged increase in MAPK activity and by inhibition
of CDK activity through induction of the CDK inhibitor
p21Cip1/WAF1 (18, 39, 40). In contrast to these
reports, which showed an increased transcriptional activity by
persistent ERK activation, our results indicated that Cpd 5-induced
persistent ERK phosphorylation resulted in an inhibition of CREB
transcriptional activity. CREB was originally identified as a target of
the cAMP signaling pathway, but studies on activation of
immediate-early genes revealed that CREB is a target of other signaling
pathways, including ERK (1). ERK phosphorylation and nuclear
translocation are thought to phosphorylate and activate CREB kinase
RSKs, which then phosphorylate CREB at Ser-133, thus inducing its
transcriptional activity (32-34). We therefore examined three
different RSK-1 phosphorylation sites, Ser-380, Thr-359/Ser-363, and
Thr-574, which were believed to be regulated by ERK phosphorylation and
related to RSK activity (34, 41), and we found that EGF slightly
induced the phosphorylation of each of these three sites, but Cpd 5 strongly inhibited their phosphorylation. These results imply that CREB
hypophosphorylation was caused by inhibition of its kinase RSK-1 activity.
Cpd 5-induced ERK phosphorylation and nuclear translocation resulted in
enhanced ERK kinase activity, using Elk-1 and myelin-based protein as
substrates (21). So why is it that EGF-induced transient ERK activation
can phosphorylate RSK-1, whereas Cpd 5-induced persistent ERK
phosphorylation results in an opposite effect on RSK-1 phosphorylation
and on cell growth? This puzzle has prompted us to consider that CBP
may interfere with the ERK-RSK interaction because it has been reported
that growth factor-dependent activation of MAPK pathway can
repress RSK and CREB activity by inducing recruitment of CBP to RSK
(11, 12). CBP was originally regarded as a bridging protein, bringing
together proteins of the RNA polymerase II-dependent basal
transcription complex with specific transcription factors (42, 43).
However, recent studies have found that CBP functions as a coactivator
for many signal-dependent factors. Indeed, CBP has been
shown to interact with ERK and RSK and even to be phosphorylated by ERK
kinase. For instance, NGF can induce formation of the
pp90RSK·CBP complex, which appears to be essential for
induction of NGF-responsive genes (11), and ERK has been found to
interact functionally with CBP and mediate NGF-induced up-regulation of
the transcriptional activity of CBP, possibly through stimulating CBP
phosphorylation (14, 44). Our data show that Cpd 5-induced ERK
activation resulted in CBP phosphorylation and enhanced its binding to
RSK-1, thus inhibiting RSK-1 phosphorylation. To rule out the
possibility of Cdc25A-CBP direct interaction, we used purified active
Cdc25A to dephosphorylate phosphorylated CBP, and no direct CBP
dephosphorylation was found. We hypothesize that increased association
of CBP with RSK-1 makes RSK-1 unavailable for phosphorylation by ERK
kinase, and inactivated RSK-1 in turn fails to phosphorylate CREB,
eventually leading to transcriptional suppression and cell growth
inhibition (Fig. 8). This finding is
consistent with a current report showing that CBP preferentially
interacts with unphosphorylated RSK-2 in a complex where both RSK-2
kinase and CBP acetylase activity are inhibited (12). Taken together,
we provide evidence here that persistently activated and nuclear
translocated ERK is able to phosphorylate coactivator CBP, which then
binds to RSK and interferes with RSK phosphorylation by ERK, leading to
negative regulation of CREB-mediated gene transcription and cell
proliferation. This observation can partially explain our puzzle over
the functional significance of transient versus persistent
activation of ERK signaling. It seems that the differential end results
of transient versus persistent signaling are caused entirely
by the intensity and duration of the same molecules having different
effects on gene transcription and cell growth. Although we can clearly
correlate the activity of persistent ERK phosphorylation with one set
of molecular consequences and transient phosphorylation correlates with
another set, we have still to explain in molecular terms how these
differential consequences occur.