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

Abstract

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 pp90RSKphosphorylation 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. 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 pp90RSKphosphorylation 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. cAMP response element-binding protein CREB-binding protein compound 5 or 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone epidermal growth factor extracellular signal-regulated kinase glutathione S-transferase luciferase mitogen-activated protein kinase MAPK/ERK kinase minimum essential medium nerve growth factor ribosomal S6 kinase 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 (1Shaywitz A.J. Greenberg M.E. Annu. Rev. Biochem. 1999; 68: 821-861Google Scholar, 2Mayr B. Montminy M. Nature Rev. 2001; 2: 599-609Google Scholar). 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 (3Bonni A. Ginty D.D. Dudek H. Greenberg M.E. Mol. Cell. Neurosci. 1995; 6: 168-183Google Scholar). 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 (4Xing J. Ginty D. Greenberg M.E. Science. 1996; 273: 959-963Google Scholar). For instance, both CREB phosphorylation and c-fostranscriptional 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 (5De Cesare D. Jacquot S. Hanauer A. Sassone-Corsi P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12202-12207Google Scholar, 6Trivier E.D. De Cesare D. Jacquot S. Pannetier S. Zackai E. Young I. Mandel J.L. Sassone-Corsi P. Hanauer A. Nature. 1996; 384: 567-570Google Scholar). 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 (7Chrivia J.C. Kwok R.P.S. Lamb N. Hagiwara M. Montminy M.R. Goodman R.H. Nature. 1993; 365: 855-859Google Scholar, 8Kwok R.P.S. Lundblad J.R. Chrivia J.C. Richards J.P. Bachinger H.P. Brennan R.G. Roberts S.G.E. Green M.R. Goodman R.H. Nature. 1994; 370: 223-226Google Scholar). Protein kinase A-mediated phosphorylation of a critical Ser-133 on CREB is required for the complex formation between CREB and CBP (9Parker D. Ferreri K. Nakajima T. LaMorte V.J. Evans R. Koerber S.C. Hoeger C. Montminy M.R. Mol. Cell. Biol. 1996; 16: 694-703Google Scholar). 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 (10Shikama N. Lyon J. La Thangue N.B. Trends Cell Biol. 1997; 7: 230-236Google Scholar). 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 (11Nakajima T. Fukamizu A. Takahashi J. Gage F.H. Fisher T. Blenis J. Montminy M.R. Cell. 1996; 86: 465-474Google Scholar). CBP and RSK-2 associate in a complex in quiescent cells, and they dissociate within a few min of mitogenic stimulation (12Merienne K. Pannetier S. Harel-Bellan A. Sassone-Corsi P. Mol. Cell. Biol. 2001; 21: 7089-7096Google Scholar). CBP interacts preferentially with unphosphorylated RSK-2 in a complex where both RSK-2 kinase activity and CBP acetylase activity are inhibited (12Merienne K. Pannetier S. Harel-Bellan A. Sassone-Corsi P. Mol. Cell. Biol. 2001; 21: 7089-7096Google Scholar). 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 (13Giebler H.A. Lemasson I. Nyborg J.K. Mol. Cell. Biol. 2000; 20: 4849-4858Google Scholar). These reports provide evidence that CBP has a pivotal effect on gene regulation and plays an important role in cellular differentiation and development (14Liu Y.-Z. Chrivia J.C. Latchman D.S. J. Biol. Chem. 1998; 273: 32400-32407Google Scholar). MAPKs are a family of second messenger kinases that are essential for transferring signals from the cell surface to the nucleus (15Davis R.J. J. Biol. Chem. 1993; 268: 14533-14556Google Scholar, 16Pawson A.J. Adv. Oncol. 1997; 13: 3-9Google Scholar). 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 (17Traverse S. Gomez N. Paterson H. Marshall C. Cohen P. Biochem. J. 1992; 288: 351-355Google Scholar). 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 (17Traverse S. Gomez N. Paterson H. Marshall C. Cohen P. Biochem. J. 1992; 288: 351-355Google Scholar, 18Pumiglia K.M. Decker S.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 448-452Google Scholar, 19Pang L. Sawada T. Decker S.J. Saltier A.R. J. Biol. Chem. 1995; 270: 13585-13588Google Scholar,21Adachi T. Kar S. Wang M. Carr B.I. J. Cell. Physiol. 2002; 192: 151-159Google Scholar). After activation, phospho-ERK is translocated to the nucleus (20Gonzalez F.A. Seth A. Raden D.L. Bowman D.S. Fay F.S. Davis R.J. J. Cell Biol. 1993; 122: 1089-1101Google Scholar,21Adachi T. Kar S. Wang M. Carr B.I. J. Cell. Physiol. 2002; 192: 151-159Google Scholar), where it can phosphorylate transcription factors, leading to altered gene expression (19Pang L. Sawada T. Decker S.J. Saltier A.R. J. Biol. Chem. 1995; 270: 13585-13588Google Scholar, 22Gille H. Kortenjann M. Thomae O. Moomaw C. Slaughter C. Cobb M.H. Shaw P.E. EMBO J. 1995; 14: 951-962Google Scholar, 23Treisman R. Curr. Opin. Genet. Dev. 1994; 4: 96-101Google Scholar). 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 (24Vogt A. Adachi T. Ducruet A.P. Chesebrough J. Nemoto K. Carr B.I. Lazo J.S. J. Biol. Chem. 2001; 276: 20544-20550Google Scholar, 25Wang Z. Wang M. Carr B.I. J. Cell. Physiol. 2000; 183: 338-346Google Scholar). 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. 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 (25Wang Z. Wang M. Carr B.I. J. Cell. Physiol. 2000; 183: 338-346Google Scholar). 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 cell were on a 10% and were saline with and bovine serum for then with the for After with the were with to of the Hep3B cell nuclear were as described with E. P. Scholar). or Cpd 5-treated Hep3B cells were washed with cold phosphate-buffered then in μl of buffer After a on cells were for min at at were by the and then washed with μl of buffer without and for min at at The nuclear were in 100 μl of buffer and on for and was to buffer and buffer After at for were and at Protein was protein assay For nuclear were with and protein at The protein were washed with radioimmune precipitation assay in μl of buffer for 5 and in The was then with or to the phosphorylation of CBP and the of the CBP·RSK were with by kinase For binding of were with 5 of nuclear and of in binding buffer at for were by in a in buffer and by For a of cold or were in the expression encoding C430S Cdc25A in a were by Roberts K. Roberts Mol. Cell. Biol. 1999; Scholar). were by the the Hep3B cells were plated in and transfected with DNA in After a the medium was with growth and the cells were to for Cells were treated without or with Cpd 5, and cell were by or Hep3B cells were plated in and with and expression with in for Cells were to by to growth medium and were then treated with Cpd 5 at the After twice with phosphate-buffered cells were lysed in 100 μl of buffer in the luciferase assay and to the cell The luciferase activity was in a The activity was as described previously R.H. Scholar). To the the luciferase activity was as a of to the activity from the cell We have shown previously that Cpd 5, a Cdc25A protein phosphatase inhibitor, persistent ERK phosphorylation in rat hepatocytes and Hep3B cells, which was in related to cell growth inhibition (24Vogt A. Adachi T. Ducruet A.P. Chesebrough J. Nemoto K. Carr B.I. Lazo J.S. J. Biol. Chem. 2001; 276: 20544-20550Google Scholar, 25Wang Z. Wang M. Carr B.I. J. Cell. Physiol. 2000; 183: 338-346Google Scholar, Z. Wang M. Carr B.I. J. 2002; Scholar). To explore the mechanism(s) by which persistent ERK activation causes cell growth inhibition, we examined the transcription factor CREB in Hep3B cells various on CREB and its by its phosphorylation (1Shaywitz A.J. Greenberg M.E. Annu. Rev. Biochem. 1999; 68: 821-861Google Scholar). Hep3B cells were grown in MEM with 10% fetal bovine serum about 75% confluence, then cells were for 24 EGF or Cpd 5 was then to the medium for from to After cells were lysed in radioimmune precipitation assay and were that EGF a ERK phosphorylation with a at which then to line at min and by However, Cpd 5 a persistent ERK phosphorylation for at least For CREB phosphorylation nuclear were from Hep3B cells treated with either EGF or Cpd 5 as described and was In to ERK phosphorylation, EGF CREB phosphorylation, Cpd 5 inhibited it To support on CREB phosphorylation we also examined the DNA binding activity. Hep3B cells were treated with EGF or Cpd 5 for and nuclear were used for DNA binding assay as described We found that EGF CREB DNA binding activity, Cpd 5 inhibited it in a 5 inhibited CREB DNA binding activity in a Hep3B cells were treated with EGF or Cpd 5 for and assay were as described It has been reported that CREB phosphorylation at Ser-133 is required for transcription in Montminy M.R. Cell. Scholar). We examined Cpd CREB phosphorylation might effect its transcriptional activity. We first activity in Hep3B cells the and found that Cpd 5 strongly inhibited the luciferase activity in a we examined well of gene cyclin D1 and that EGF effect on the expression of either cyclin D1 or both gene were strongly by Cpd 5, as as h This is with in rat hepatocytes that Cpd rat and DNA were with Bcl-2 and cyclin D1 inhibition Z. Wang M. Carr B.I. J. 2002; B.I. Wang Z. Wang M. Kar S. K. E. Lazo J.S. J. Mol. Biol. Scholar). Cpd 5 persistent ERK phosphorylation and CREB activity, we the inhibition of CREB activity was the result of ERK Hep3B cells were treated with inhibitors PD 98059 or for h the addition of Cpd 5. We found that both PD 98059 and antagonized the effects of Cpd 5 on ERK and CREB phosphorylation We the effects of inhibitors on transcriptional activity. Hep3B cells were transfected with After the cells were treated with PD 98059 or for h the addition of Cpd 5. that Cpd 5 inhibited luciferase activity and that both PD 98059 and antagonized this inhibition by about and These data that the inhibition of CREB activity by Cpd 5 is regulated at least in part by the phosphorylation of ERK pathway. The of kinases has been shown to be activated by the ERK kinase pathway in response to growth factors, including EGF R. C. Blenis J. Mol. Cell. Biol. 1992; Scholar, K. S. S. J. Proc. Natl. Acad. Sci. U. S. A. 1994; Scholar), and it to phosphorylate transcription factors such as CREB (1Shaywitz A.J. Greenberg M.E. Annu. Rev. Biochem. 1999; 68: 821-861Google Scholar). the that Cpd 5 a persistent ERK activation and CREB activity to examine pp90RSK was in Cpd 5 We first the effects of EGF and Cpd 5 on phosphorylation at and the phosphorylation at is thought to be related to ERK kinase activity N. J. Cohen P. J. Biol. Chem. 1998; 273: Scholar). We found that EGF phosphorylation at Cpd 5 an effect on Cpd 5 strongly inhibited phosphorylation at and and inhibited phosphorylation at To examine the between and ERK phosphorylation, we used PD 98059 and to inhibitors could the effects of Cpd 5 on 5 that both PD 98059 and antagonized Cpd 5-mediated hypophosphorylation at that persistent ERK activation by Cpd 5 inhibition of The pathway has been shown to a number of nuclear factors via a of kinases to phosphorylate factors in response to mitogenic experiments that the Cpd persistent ERK phosphorylation kinase and subsequently CREB activity. These to that CBP might play an important role in gene expression it has been reported that insulin or NGF CREB transcriptional activity via recruitment of to CBP (11Nakajima T. Fukamizu A. Takahashi J. Gage F.H. Fisher T. Blenis J. Montminy M.R. Cell. 1996; 86: 465-474Google Scholar). To Cpd persistent ERK phosphorylation increased recruitment of to CBP, Hep3B cells were serum starved for 24 h and then treated with EGF or Cpd 5 for the were and with and then with and that stimulation by CBP phosphorylation and its binding to were Cpd 5 treatment both CBP phosphorylation and complex Cdc25A protein phosphatase has protein and Cpd persistent ERK phosphorylation is related to its effects on Cdc25A activity, we examined Cdc25A was to phosphorylated CBP directly and inhibition of Cdc25A activity by Cpd 5 could result in CBP We used Cpd 5-treated Hep3B cell nuclear as the of phosphorylated CBP, which were with and with of protein in the or of Cpd 5 in phosphatase buffer at for We found that effect on phosphorylated CBP, and of with Cpd 5 CBP phosphorylation either These data provide evidence that ERK phosphorylation is with phosphorylation of CBP and an in the binding of CBP to This increased binding of CBP to is an important in the regulation of CREB activity. We have reported previously that Cdc25A has a direct effect on ERK kinase, and the inhibition of Cdc25A activity by Cpd 5 to persistent ERK phosphorylation (24Vogt A. Adachi T. Ducruet A.P. Chesebrough J. Nemoto K. Carr B.I. Lazo J.S. J. Biol. Chem. 2001; 276: 20544-20550Google Scholar). We that Cdc25A might cause a ERK phosphorylation, which also negatively activity. To Hep3B cells were transfected with C430S Cdc25A and then treated with or without Cpd 5 for that C430S Cdc25A in basal ERK phosphorylation with Hep3B cells. Cpd 5 treatment also ERK phosphorylation in of C430S transfected cells, which is caused by the of an of the cells. CREB Ser-133 and phosphorylation were in C430S Hep3B cells. Furthermore, CBP and experiments that Cdc25A caused a association in cells the Cdc25A with the Cpd 5-treated cells. These data support that persistent ERK phosphorylation caused by Cdc25A inhibition down-regulates CREB phosphorylation and activity, likely via the recruitment of CBP to We have reported previously that both EGF and Cpd 5, a protein phosphatase Cdc25A inhibitor, the pathway in normal rat human hepatoma cells, and cell T. Kar S. Wang M. Carr B.I. J. Cell. Physiol. 2002; 192: 151-159Google Z. Wang M. Carr B.I. J. Cell. Physiol. 2000; 183: 338-346Google Scholar, S. K. Carr B.I. J. Mol. Biol. 2001; Scholar). However, EGF DNA and Hep3B cell growth, Cpd 5 mediates an effect on the growth of both cell This is caused by the and of the ERK EGF a and ERK phosphorylation without nuclear translocation, and Cpd 5 a persistent and ERK phosphorylation with nuclear T. Kar S. Wang M. Carr B.I. J. Cell. Physiol. 2002; 192: 151-159Google Scholar, A. Adachi T. Ducruet A.P. Chesebrough J. Nemoto K. Carr B.I. Lazo J.S. J. Biol. Chem. 2001; 276: 20544-20550Google Scholar, 25Wang Z. Wang M. Carr B.I. J. Cell. Physiol. 2000; 183: 338-346Google Scholar). In the current study, we to explore the mechanism(s) by which persistent ERK activation cell growth. We a persistent ERK phosphorylation Cpd 5 as a tool to examine it regulates the of the CREB transcription We found that Cpd 5 inhibited CREB activity, including CREB phosphorylation and DNA binding and subsequently Bcl-2 and cyclin D1 The effect of Cpd 5 on CREB activity was likely by ERK phosphorylation inhibitors PD 98059 and Cpd 5-mediated CREB hypophosphorylation and inhibition of gene We role that may also be in Cpd suppression of CREB transcriptional activity. We previously that Cpd 5 protein Carr B.I. J. Biol. Chem. 1998; 273: Scholar), implying that are likely Furthermore, in Cdc25A Hep3B cells, CREB was in a hypophosphorylation to Hep3B cells treated with Cpd 5 Cdc25A is thought to be an ERK and C430S is to induce a ERK phosphorylation (24Vogt A. Adachi T. Ducruet A.P. Chesebrough J. Nemoto K. Carr B.I. Lazo J.S. J. Biol. Chem. 2001; 276: 20544-20550Google Scholar). Although the pathway has been shown to in the stimulation of cellular Cell. 1995; Scholar), we provide evidence that persistent ERK activation can also to suppression of CREB activity and inhibition of cell growth. role for pathway in growth arrest or cellular differentiation is For instance, it has been reported that is for differentiation of J. R. Nature. 1995; Scholar), and in and cells, cellular differentiation and cell cycle arrest are accompanied by a in activity and by inhibition of activity through induction of the inhibitor K.M. Decker S.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 448-452Google Scholar, J. Neurosci. 1997; Scholar, D.S. J. Biol. Chem. 1996; Scholar). In to which an increased transcriptional activity by persistent ERK activation, that Cpd persistent ERK phosphorylation in an inhibition of CREB transcriptional activity. CREB was originally identified as a target of the cAMP studies on activation of that CREB is a target of including ERK (1Shaywitz A.J. Greenberg M.E. Annu. Rev. Biochem. 1999; 68: 821-861Google Scholar). ERK phosphorylation and nuclear are thought to phosphorylate and CREB kinase which then phosphorylate CREB at Ser-133, inducing its transcriptional activity R. C. Blenis J. Mol. Cell. Biol. 1992; Scholar, K. S. S. J. Proc. Natl. Acad. Sci. U. S. A. 1994; Scholar, N. J. Cohen P. J. Biol. Chem. 1998; 273: Scholar). We examined different phosphorylation and which were to be regulated by ERK phosphorylation and related to activity N. J. Cohen P. J. Biol. Chem. 1998; 273: Scholar, M. S. Mol. Cell. 1999; Scholar), and we found that EGF the phosphorylation of of Cpd 5 strongly inhibited These that CREB hypophosphorylation was caused by inhibition of its kinase activity. Cpd ERK phosphorylation and nuclear in ERK kinase activity, and protein as T. Kar S. Wang M. Carr B.I. J. Cell. Physiol. 2002; 192: 151-159Google Scholar). is it that ERK activation can phosphorylate Cpd persistent ERK phosphorylation in an effect on phosphorylation and on cell This has to that CBP may with the interaction it has been reported that growth activation of pathway can and CREB activity by inducing recruitment of CBP to (11Nakajima T. Fukamizu A. Takahashi J. Gage F.H. Fisher T. Blenis J. Montminy M.R. Cell. 1996; 86: 465-474Google Scholar, K. Pannetier S. Harel-Bellan A. Sassone-Corsi P. Mol. Cell. Biol. 2001; 21: 7089-7096Google Scholar). CBP was originally as a protein, of the basal transcription complex with transcription factors R. T. Nature. 1996; Scholar, N. Goodman R.H. J. Biol. Chem. 2001; 276: Scholar). However, recent studies have found that CBP as a coactivator for CBP has been shown to interact with ERK and and to be phosphorylated by ERK For instance, NGF can induce formation of the which to be essential for induction of (11Nakajima T. Fukamizu A. Takahashi J. Gage F.H. Fisher T. Blenis J. Montminy M.R. Cell. 1996; 86: 465-474Google Scholar), and ERK has been found to interact with CBP and mediate of the transcriptional activity of CBP, through CBP phosphorylation (14Liu Y.-Z. Chrivia J.C. Latchman D.S. J. Biol. Chem. 1998; 273: 32400-32407Google Scholar, K. S. Mol. Cell. 2001; 7: Scholar). data that Cpd ERK activation in CBP phosphorylation and its binding to To the of direct we used Cdc25A to phosphorylated CBP, and direct CBP was We that increased association of CBP with for phosphorylation by ERK kinase, and in to phosphorylate leading to transcriptional suppression and cell growth inhibition This is with a current that CBP preferentially interacts with unphosphorylated RSK-2 in a complex where both RSK-2 kinase and CBP acetylase activity are inhibited (12Merienne K. Pannetier S. Harel-Bellan A. Sassone-Corsi P. Mol. Cell. Biol. 2001; 21: 7089-7096Google Scholar). we provide evidence that activated and nuclear translocated ERK is to phosphorylate coactivator CBP, which then to and with phosphorylation by leading to regulation of gene transcription and cell This can the of persistent activation of ERK It that the of persistent are caused by the and of the different effects on gene transcription and cell growth. Although we can the activity of persistent ERK phosphorylation with one of and phosphorylation with we have to in