Persistent ERK Phosphorylation Negatively Regulates cAMP Response Element-binding Protein (CREB) Activity via Recruitment of CREB-binding Protein to pp90RSK*

Ziqiu Wang, Baochun Zhang, Meifang Wang, and Brian I. CarrDagger

From the Liver Cancer Center, Thomas E. Starzl Transplantation Institute, Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania 15213

Received for publication, September 5, 2002, and in revised form, January 2, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Compound 5 (Cpd 5) or 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone, is an inhibitor of protein phosphatase Cdc25A and causes persistent activation of extracellular signal-regulated kinase (ERK) and cell growth inhibition. To study the mechanism(s) by which persistent ERK phosphorylation might induce cell growth inhibition, we used Cpd 5 as a tool to examine its effects on the activity of CREB (cAMP response element-binding protein) transcription factor in Hep3B human hepatoma cells. We found that CREB activity, including its DNA binding ability and phosphorylation on residue Ser-133, was strongly inhibited by Cpd 5, followed by suppression of CRE-mediated transcription of cyclin D1 and Bcl-2 genes. Cpd 5-mediated suppression of CREB phosphorylation and transcriptional activity was antagonized by mitogen-activated protein kinase kinase inhibitors PD 98059 and U-0126, implying that this inhibition of CREB activity was regulated at least in part by the ERK pathway. The phosphorylation of ribosomal S6 kinase (pp90RSK), a CREB kinase in response to mitogen stimulation, was also found to be inhibited by Cpd 5 action. This inhibition of pp90RSK phosphorylation is likely the result of its increased association with CREB-binding protein (CBP), which subsequently caused inhibition of CREB phosphorylation and activity. To support the hypothesis that Cpd 5 effects on Cdc25A inhibition with subsequent ERK activation could cause CREB inhibition, we examined the effects of Cdc25A inhibition without the use of Cpd 5. Hep3B cells were transfected with C430S Cdc25A mutant, and ERK was found to be phosphorylated in a constitutively activated manner, which was accompanied by decreased CREB phosphorylation and increased recruitment of CBP to pp90RSK. These data provide evidence that CBP·RSK complex formation in response to persistent ERK phosphorylation by Cpd 5 down-regulates CREB activity, leading to inhibition of both cAMP response element-mediated gene expression and cell growth.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 [gamma -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 (beta -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 beta -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 beta -galactosidase activity obtained from the same cell lysates.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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."

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.

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."

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.

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.

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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


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Fig. 8.   Hypothesis of mechanisms of persistent ERK phosphorylation-mediated growth inhibition.


    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant CA 82723 (to B. I. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom the reprints should be addressed: Transplantation Institute, Dept. of Surgery, University of Pittsburgh School of Medicine, E 1552 Biomedical Science Tower, 200 Lothrop St., Pittsburgh, PA 15213. Tel.: 412-624-6684; Fax: 412-624-6666; E-mail: _carrbi@msx.upmc.edu.

Published, JBC Papers in Press, January 22, 2003, DOI 10.1074/jbc.M209108200

    ABBREVIATIONS

The abbreviations used are: CREB, cAMP response element-binding protein; CBP, CREB-binding protein; Cpd 5, compound 5 or 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; GST, glutathione S-transferase; Luc, luciferase; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; MEM, minimum essential medium; NGF, nerve growth factor; RSK, ribosomal S6 kinase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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