Stimulation of the Raf/MEK/ERK Cascade Is Necessary and Sufficient for Activation and Thr-160 Phosphorylation of a Nuclear-targeted CDK2

The activity of cyclin-dependent kinase 2 is required for G1-S-phase progression of the eukaryotic cell cycle. In this study, we examine the activation of CDK2-cyclin E by constructing a CDK2 that is constitutively targeted to the nucleus. Activation of CDK2 requires the removal of two inhibitory phosphates (Thr-14 and Tyr-15) and the addition of one activating phosphate (Thr-160) by a nuclear localized CDK-activating kinase, which is thought to be constitutively active. Surprisingly, nuclear localized CDK2-NLS and CDK2-NLS(A14,F15), which lacks the inhibitory phosphorylation sites, require serum to become active, despite complexing with expressed cyclin E. We show that inhibition of mitogen-mediated ERK activation by treatment with U0126, a selective MEK inhibitor, or expression of dominant-negative ERK markedly reduces the phosphorylation of Thr-160 and enzymatic activity of both CDK2-NLS constructs. Consistent with a role for ERK in Thr-160 phosphorylation, expression of constitutively active Raf-1 induces Thr-160 phosphorylation of CDK2-NLS in serum-arrested cells, an effect that is blocked by treatment with U0126. Taken together, these data show a new role for ERK in G1 cell cycle progression: In addition to its role in stimulating cyclin D1 expression and nuclear translocation of CDK2, ERK regulates Thr-160 phosphorylation of CDK2-cyclin E. The activity of cyclin-dependent kinase 2 is required for G1-S-phase progression of the eukaryotic cell cycle. In this study, we examine the activation of CDK2-cyclin E by constructing a CDK2 that is constitutively targeted to the nucleus. Activation of CDK2 requires the removal of two inhibitory phosphates (Thr-14 and Tyr-15) and the addition of one activating phosphate (Thr-160) by a nuclear localized CDK-activating kinase, which is thought to be constitutively active. Surprisingly, nuclear localized CDK2-NLS and CDK2-NLS(A14,F15), which lacks the inhibitory phosphorylation sites, require serum to become active, despite complexing with expressed cyclin E. We show that inhibition of mitogen-mediated ERK activation by treatment with U0126, a selective MEK inhibitor, or expression of dominant-negative ERK markedly reduces the phosphorylation of Thr-160 and enzymatic activity of both CDK2-NLS constructs. Consistent with a role for ERK in Thr-160 phosphorylation, expression of constitutively active Raf-1 induces Thr-160 phosphorylation of CDK2-NLS in serum-arrested cells, an effect that is blocked by treatment with U0126. Taken together, these data show a new role for ERK in G1 cell cycle progression: In addition to its role in stimulating cyclin D1 expression and nuclear translocation of CDK2, ERK regulates Thr-160 phosphorylation of CDK2-cyclin E. In eukaryotic cells, growth factors activate signaling pathways that stimulate cells to divide. The sequential activation of the cyclin-dependent kinases (CDKs) 1The abbreviations used are: CDK, cyclin-dependent kinase; FCS, fetal calf serum; ERK, extracellular signal-regulated kinase; CAK, CDK-activating kinase; NLS, nuclear localization signal; pRb, retinoblastoma protein; PBS, phosphate-buffered saline; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase controls the orderly progression of cell cycle events (1Sherr C.J. Science. 1996; 274: 1672-1677Google Scholar, 2Sherr C.J. Cell. 1994; 79: 551-555Google Scholar). As the name implies, CDKs require the binding of a cyclin subunit for full activation (3Ohtsubo M. Roberts J.M. Science. 1993; 259: 1908-1912Google Scholar, 4Sherr C.J. Cell. 1993; 73: 1059-1065Google Scholar). The first CDK to become active in G1 is CDK4/6, which requires the accumulation of cyclin D (5Matsushime H. Ewen M.E. Strom D.K. Kato J.Y. Hanks S.K. Roussel M.F. Sherr C.J. Cell. 1992; 71: 323-334Google Scholar, 6Meyerson M. Harlow E. Mol. Cell. Biol. 1994; 14: 2077-2086Google Scholar, 7Bates S. Bonetta L. MacAllan D. Parry D. Holder A. Dickson C. Peters G. Oncogene. 1994; 9: 71-79Google Scholar, 8Tam S.W. Theodoras A.M. Shay J.W. Draetta G.F. Pagano M. Oncogene. 1994; 9: 2663-2674Google Scholar). Two signaling pathways, the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase cascade and the phosphatidylinositol 3-kinase pathway are required for cyclin D1 accumulation, and therefore, cell cycle progression in IIC9 cells (9Phillips-Mason P.J. Raben D.M. Baldassare J.J. J. Biol. Chem. 2000; 275: 18046-18053Google Scholar, 10Weber J.D. Raben D.M. Phillips P.J. Baldassare J.J. Biochem. J. 1997; 326: 61-68Google Scholar). To promote progression through the G1 phase of the cell cycle, mitogen-induced signals are required through the G1-S phase transition, for reasons that are not fully understood (11Donjerkovic D. Scott D.W. Cell Res. 2000; 10: 1-16Google Scholar, 12Pardee A.B. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 1286-1290Google Scholar, 13Dou Q.P. Levin A.H. Zhao S. Pardee A.B. Cancer Res. 1993; 53: 1493-1497Google Scholar). In vitro and in vivo studies indicate that the major and perhaps only function of cyclin d-CDK4/6 is to regulate the accumulation of the next activating cyclin, cyclin E (14Kato J. Matsushime H. Hiebert S.W. Ewen M.E. Sherr C.J. Genes Dev. 1993; 7: 331-342Google Scholar,15Geng Y. Whoriskey W. Park M.Y. Bronson R.T. Medema R.H. Li T. Weinberg R.A. Sicinski P. Cell. 1999; 97: 767-777Google Scholar). Cyclin E is required for the late G1 activation of CDK2 (16Koff A. Cross F. Fisher A. Schumacher J. Leguellec K. Philippe M. Roberts J.M. Cell. 1991; 66: 1217-1228Google Scholar, 17Koff A. Giordano A. Desai D. Yamashita K. Harper J.W. Elledge S. Nishimoto T. Morgan D.O. Franza B.R. Roberts J.M. Science. 1992; 257: 1689-1694Google Scholar). CDK2-cyclin E has many reported substrates and is critical for regulating several cell-cycle components necessary for progression into S phase (18Winey M. Curr. Biol. 1999; 9: R449-R452Google Scholar, 19Zhao J. Dynlacht B. Imai T. Hori T. Harlow E. Genes Dev. 1998; 12: 456-461Google Scholar, 20Dynlacht B.D. Flores O. Lees J.A. Harlow E. Genes Dev. 1994; 8: 1772-1786Google Scholar, 21Knoblich J.A. Sauer K. Jones L. Richardson H. Saint R. Lehner C.F. Cell. 1994; 77: 107-120Google Scholar, 22Tsai L.H. Lees E. Faha B. Harlow E. Riabowol K. Oncogene. 1993; 8: 1593-1602Google Scholar). In addition to cyclin E accumulation, activation of CDK2 requires additional modifications to become active. Recently, CDK2-cyclin E translocation to the nucleus has been shown to be necessary for enzymatic activation (23Keenan S.M. Bellone C. Baldassare J.J. J. Biol. Chem. 2001; 276: 22404-22409Google Scholar, 24Blanchard D.A. Mouhamad S. Auffredou M.T. Pesty A. Bertoglio J. Leca G. Vazquez A. Oncogene. 2000; 19: 4184-4189Google Scholar, 25Dietrich C. Wallenfang K. Oesch F. Wieser R. Exp. Cell Res. 1997; 232: 72-78Google Scholar). This is, presumably, because of post-translational modifications that occur only in the nucleus and are required for CDK2 activity. One such nuclear modification is phosphorylation on Thr-160. The phosphorylation site, located on the “T-loop” is highly conserved among all CDKs and is essential for proper alignment of the kinase domain (26Russo A.A. Jeffrey P.D. Pavletich N.P. Nat. Struct. Biol. 1996; 3: 696-700Google Scholar, 27Jeffrey P.D. Russo A.A. Polyak K. Gibbs E. Hurwitz J. Massague J. Pavletich N.P. Nature. 1995; 376: 313-320Google Scholar). Thr-160 phosphorylation is thought to be carried out by a CDK-activating kinase (CAK) activity. The identity of CAK is reported to be the heterotrimeric complex consisting of p40MO15 (CDK7), cyclin H, and menage-a-trois (MAT1), which is believed to be constitutively active and nuclear (28Larochelle S. Pandur J. Fisher R.P. Salz H.K. Suter B. Genes Dev. 1998; 12: 370-381Google Scholar, 29Harper J.W. Elledge S.J. Genes Dev. 1998; 12: 285-289Google Scholar, 30Yankulov K.Y. Bentley D.L. EMBO J. 1997; 16: 1638-1646Google Scholar, 31Fisher R.P. Morgan D.O. Cell. 1994; 78: 713-724Google Scholar). Another post-translational modification that is required for CDK2 activity is the removal of inhibitory phosphorylations on Thr-14 and Tyr-15 (32Sebastian B. Kakizuka A. Hunter T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3521-3524Google Scholar). These phosphates are added to CDK2 by the cytosolic mixed-lineage kinase Wee-1. They are removed by cdc25A phosphatase (32Sebastian B. Kakizuka A. Hunter T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3521-3524Google Scholar, 34Sexl V. Diehl J.A. Sherr C.J. Ashmun R. Beach D. Roussel M.F. Oncogene. 1999; 18: 573-582Google Scholar). The regulation and localization of cdc25A is not well understood (35Blomberg I. Hoffmann I. Mol. Cell. Biol. 1999; 19: 6183-6194Google Scholar). Our laboratory recently found a second role for ERK in G1progression. In that study (23Keenan S.M. Bellone C. Baldassare J.J. J. Biol. Chem. 2001; 276: 22404-22409Google Scholar), we show that U0126, a MEK inhibitor, blocks nuclear translocation and activation of CDK2-cyclin E, indicating a role for ERK kinase in CDK2-cyclin E nuclear translocation. However, these studies did not address whether other mitogen-mediated modifications are critical for nuclear CDK2-cyclin E activation. To address this question, we targeted CDK2-cyclin E to the nucleus, examined the activation of CDK2-cyclin E complexes, and found a novel role for ERK in the activation of nuclear targeted CDK2-cyclin E (CDK2-NLS). Human HU4 cyclin E fragment expression plasmid was generously provided by James Roberts (16Koff A. Cross F. Fisher A. Schumacher J. Leguellec K. Philippe M. Roberts J.M. Cell. 1991; 66: 1217-1228Google Scholar). CDK2-NLS was generated by subcloning human CDK2 cDNA (plus HA tag) into the Strategene pShooter vector pCMV/myc/nuc according to manufacturer's protocol. CDK2-NLS(A14,F15) was generated by Strategene One-ShotTM site-directed mutagenesis; mutations were confirmed by sequencing (Beckman-Coulter). ERK1(K71R) was generated by site-directed mutagenesis from ERK1 cDNA generously provided by Melanie Cobb (36Boulton T.G. Yancopoulos G.D. Gregory J.S. Slaughter C. Moomaw C. Hsu J. Cobb M.H. Science. 1990; 249: 64-67Scopus (474) Google Scholar). Constitutively active Raf-1 was generously provided by Thomas Sturgill (37Dent P. Reardon D.B. Morrison D.K. Sturgill T.W. Mol. Cell. Biol. 1995; 15: 4125-4135Google Scholar). E2F1 expression plasmid was generously provided by Jason Weber. RbΔCDK expression plasmid was generously provided by J. Wade Harper (38Leng X. Connell-Crowley L. Goodrich D. Harper J.W. Curr. Biol. 1997; 7: 709-712Google Scholar). IIC9 cells (Chinese Hamster Embryonic Fibroblasts) were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 4. 5 g/liter glucose and 2 mm l-glutamine (BioWhittaker, Walkersville, MD) supplemented with 5% (v/v) fetal calf serum, 100 units/ml penicillin, and 100 mg/ml streptomycin (all from Sigma). Growth-arrested (Go) cells were established by washing subconfluent (80%) or transfected cells once with phosphate-buffered saline (PBS) followed by a 48-h incubation with α-MEM containing 2 mm l-glutamine (BioWhittaker) supplemented with 100 units/ml penicillin and 100 mg/ml streptomycin (basal medium). Growth-arrested IIC9 cells were stimulated with 10% (v/v) fetal calf serum. U0126 at 10 μm(PerkinElmer Life Sciences) and LY294002 at 15 μm(Calbiochem, San Diego, CA) were added to cells 30 min before stimulation. IIC9 cells were grown to subconfluency (80%) in DMEM containing 4. 5 g/liter glucose and 2 mm l-glutamine (BioWhittaker) supplemented with 5% (v/v) fetal calf serum (FCS), 100 units/ml penicillin, and 100 mg/ml of streptomycin (Sigma). Cells were transfected with a solution containing 5 μl/ml PlusTM, 5 μl/ml LipofectAMINETM (Invitrogen) and 2 μg of total DNA per 1 ml in Opti-MEM medium (Invitrogen) following the manufacturer's protocol. Five hours post-transfection, DMEM supplemented with FCS (final serum concentration of 0.1% v/v), 100 units/ml penicillin, and 100 mg/ml streptomycin was added. After 12 h, the cells were growth-arrested for 24 h in basal medium before stimulation. Growth-arrested IIC9 cells were incubated in the absence or presence of 10% FCS after pre-incubation in the absence or presence of 15 μm LY294002 or 10 μm U0126 for 30 min. After the indicated time, cells were washed twice with cold PBS and scraped into cold lysis buffer (50 mm HEPES, pH 7. 5, 150 mm NaCl, 1 mm EDTA, 2.5 mm EGTA, 0.1% (v/v) Tween 20, 10% (v/v) glycerol, 1 mm phenylmethylsulfonyl fluoride, 2 μm sodium vanadate, 20 mmsodium fluoride, μm 10 10 and 10 were and was by at at for 2 min. were by the Cell (50 μg of were incubated with 1 μg of or 1 μg of CDK2 at with The were by incubation with protein at with were by at and washed two with lysis two with cold PBS, and once with cold buffer (50 pH 1 mm and 10 mm were in 30 of buffer (50 mm HEPES, pH 1 mm 10 mm 2.5 mm EGTA, 10 μm and 20 μm and incubated with μg of and of at 30 for min. The was by the addition of of were to The were and CDK2-cyclin E activity was a and growth-arrested IIC9 cells grown on were incubated in the absence or presence of 10% After h of cells were in a solution for 10 min at followed by a incubation in at and supplemented with 1 Sigma). The cells were washed in PBS twice and blocked in 1 ml of buffer of serum in 100 ml of for 2 h at was added at a and incubated at for 2 The were washed with The was added in for min at the were washed with PBS and CA) and were a and growth-arrested IIC9 cells were incubated in the presence or absence of 10% FCS after in the presence or absence of 15 μm LY294002 or 10 μm U0126 for 30 min. the indicated cells were washed twice with cold PBS and scraped into cold lysis buffer (50 mm HEPES, pH 150 mm NaCl, 1 mm EDTA, 2.5 mm EGTA, 0.1% (v/v) Tween 20, 10% (v/v) glycerol, 1 fluoride, 2 mm sodium vanadate, 20 mm sodium fluoride, 10 10 and 10 were and the was by at at for 2 min. were by the were incubated with 5 μg of or 5 μg of at with were by incubation with protein at with The were by at and washed with cold lysis buffer and two with cold PBS supplemented with 1 fluoride, 2 mm sodium vanadate, 20 mm sodium fluoride, 10 10 and 10 were in PBS in by and to a by the were with CDK2 or cyclin E were by by the we found that mitogen-induced ERK activity is required for CDK2 nuclear translocation (23Keenan S.M. Bellone C. Baldassare J.J. J. Biol. Chem. 2001; 276: 22404-22409Google Scholar). In several studies shown that CAK CDK2 in the nucleus in late G1 P. J. Biochem. 2000; Scholar). CAK is reported to be constitutively active and localized to the nucleus, we that CDK2-cyclin E to the nucleus in activation of CDK2 in serum-arrested we a that human CDK2 cDNA with a This a which to CDK2-NLS from with the nuclear in both serum-arrested and was not in cells not We not of the nucleus, indicating that CDK2-NLS to the nucleus, of serum stimulation. full activation of CDK2 requires with a cyclin we next examined whether CDK2-NLS a complex with expressed cyclin E. These expression of and expression of cyclin E in complex is of To were from serum-arrested cells cyclin E, or and with the from basal cell not cyclin E only CDK2-NLS is expressed Cyclin E with CDK2 CDK2-NLS and cyclin E are in basal cells 2 As a we and examined whether the CDK2 or cyclin E 2 Consistent with the of the of the CDKs with cyclin, cyclin E CDK2 with 2 These data indicate that CDK2-NLS not require serum to a complex with expressed cyclin E. We next whether CDK2 requires to become active. To examine we transfected CDK2-NLS into IIC9 cells, with cyclin E, and in vitro kinase activity from serum-arrested and Surprisingly, in serum-arrested cells, CDK2-NLS kinase activity 2 However, addition of serum induces an activation of CDK2-NLS indicating that CDK2-NLS requires signaling to become active. activity we 1 which an for Thr-160 and be by 2 1 and This is found in the nucleus, of serum 1 These data show that E to the nucleus is not for activation. of the serum of E activation is the presence of CDK2 of the in the E complex in not stimulated cells J.W. K. Elledge S.J. Cell. 1993; Scholar, K. M.H. H. A. Roberts J.M. P. Massague J. Cell. 1994; 78: Scholar, J.D. W. Raben D.M. Baldassare J.J. J. Biol. Chem. 1997; Scholar). The CDK2 in serum-arrested cells, of serum induces a in protein 1 H. Hunter T. Cell. 1994; 78: Scholar, W. Bellone C.J. Baldassare J.J. J. Biol. Chem. 1999; 274: Scholar). However, we were to in the E in basal or stimulated cells we were to in complex with E not is that or blocks CDK2-NLS activation in serum-arrested cyclin E accumulation, CDK2 nuclear and the post-translational which are required for occur late in G1 (23Keenan S.M. Bellone C. Baldassare J.J. J. Biol. Chem. 2001; 276: 22404-22409Google Scholar, W. Bellone C.J. Baldassare J.J. J. Biol. Chem. 1999; 274: Scholar), CDK2-cyclin E active late in We E to become active CDK2, because to the nucleus in serum-arrested Consistent with this serum induces a in E activity in h, with full activation in only 5 h of serum In with CDK2 not become fully active 12 h of serum These data are with the that CDK2 is in the nucleus. because nuclear translocation of CDK2-cyclin E 12 h after serum addition (23Keenan S.M. Bellone C. Baldassare J.J. J. Biol. Chem. 2001; 276: 22404-22409Google and E is after 5 h the that the enzymatic required for the post-translational modifications of CDK2 are hours before nuclear translocation. In addition to with cyclin E, CDK2 requires post-translational modifications in to become phosphorylations on Thr-14 and which are added by the cytosolic mixed-lineage kinase and removed by the phosphatase be removed for CDK2 to become active V. Diehl J.A. Sherr C.J. Ashmun R. Beach D. Roussel M.F. Oncogene. 1999; 18: 573-582Google Scholar). is the regulation of CDK2-NLS requires serum to become active is that cdc25A requires to Thr-14 and To examine we CDK2-NLS(A14,F15), which an and for Thr-14 and This to the nucleus not this be at these sites, cdc25A phosphatase activity is not required for activation. with cyclin E, CDK2-NLS(A14,F15) not activity in serum-arrested cells, with the addition of serum 5 the of activation of CDK2-NLS(A14,F15) that of fully active in 5 h not This that the of Thr-14 and Tyr-15 is not the required for CDK2-NLS activation. CDK2-NLS(A14,F15) requires serum to become active, we that the for E activation is the phosphorylation on Thr-160. We this by Thr-160 phosphorylation of at after of serum, an for of CDK2-NLS we that serum induces the phosphorylation of Thr-160 in h, with CDK2-NLS in 5 h The Thr-160 phosphorylation of CDK2-NLS(A14,F15) is by serum These indicate CDK2-NLS to the nucleus, serum is required for the phosphorylation of and therefore, activity. This that CAK activity is growth and required for CDK2-NLS of U0126 on CDK2-NLS activity and Thr-160 IIC9 cells were with CDK2-NLS and cyclin E and growth-arrested for 24 Cells were incubated with the indicated of U0126 or 30 min before stimulation. Cells were stimulated by FCS to a 10% (v/v) concentration for were by into cold lysis and protein was with 1 μg of were for the to in IIC9 cells were with CDK2-NLS(A14,F15) and cyclin E and growth-arrested for 24 Cells were incubated in the presence or absence of 10 μm U0126 or 15 30 min before The IIC9 cells were stimulated by addition of FCS to 10% for indicated of were by into cold lysis and protein was with μg of were examined by CDK2 or are of at In an to the signaling that are required for CDK2-NLS we of selective of phosphatidylinositol 3-kinase and the ERK two pathways that are critical for cell-cycle laboratory that inhibition of CDK2-cyclin E activity by a phosphatidylinositol 3-kinase inhibitor, not an ERK cascade inhibitor, be by of cyclin E in stimulated cells (23Keenan S.M. Bellone C. Baldassare J.J. J. Biol. Chem. 2001; 276: 22404-22409Google Scholar). of cells with the MEK U0126, which the MEK activation of ERK, the activation of CDK2-NLS in a fully activation at 10 μm In addition of 20 μm a concentration which phosphatidylinositol 3-kinase and cell-cycle progression in IIC9 cells (9Phillips-Mason P.J. Raben D.M. Baldassare J.J. J. Biol. Chem. 2000; 275: 18046-18053Google Scholar, R. P.J. Raben D.M. Baldassare J.J. J. Biol. Chem. Scholar), not effect CDK2-NLS activity we found the phosphorylation of CDK2-NLS on Thr-160 to be the in and because MEK activity is critical for we that ERK regulates the phosphorylation of CDK2 on Thr-160. we found that U0126 Thr-160 phosphorylation of CDK2-NLS(A14,F15) in cells, is The was found with CDK2-NLS These data indicate a critical role for ERK in the phosphorylation of CDK2 on Thr-160. the of Thr-160 phosphorylation of CDK2-NLS and CDK2-NLS(A14,F15) were with the of enzymatic activation ERK activity is required for the phosphorylation of CDK2-NLS on of a dominant-negative ERK Thr-160 As in cells, expression of Thr-160 phosphorylation of CDK2-NLS to that of basal that ERK activity is necessary for this To the role of the ERK cascade in Thr-160 phosphorylation, we expressed a constitutively active Raf-1 in basal Raf-1 is the protein kinase kinase of the ERK and therefore, constitutively active Raf-1 the ERK cascade of growth factors J.M. H. P. D.L. J. Nature. 1992; Scholar, L. J. R. F. Mol. Cell. Biol. 1993; Scholar, W. Biochem. J. 2000; Scholar). expression of constitutively active Raf-1 Thr-160 phosphorylation of CDK2-NLS in basal cells indicating that of the pathway is for Thr-160 this effect was blocked by treatment with the MEK Taken together, these data and indicate a role for the ERK pathway in regulating CDK2 phosphorylation on Thr-160. The phosphorylation of the retinoblastoma protein by and CDK2 the of the J.W. A.A. Cell. 1999; Scholar, P. Mol. Cell. Biol. 1996; 16: Scholar, M.E. H.K. Sherr C.J. Matsushime H. Kato J. D.M. Cell. 1993; 73: Scholar). and cyclin E is of we that the activation of E is of is a that be by CDKs and not from on serum (38Leng X. Connell-Crowley L. Goodrich D. Harper J.W. Curr. Biol. 1997; 7: 709-712Google Scholar). of been shown to cyclin and cell-cycle M. H. and J. J. expression of not Thr-160 phosphorylation of CDK2-NLS a role for in the phosphorylation of CDK2-NLS on Thr-160. Consistent with of E2F1 in basal cells not Thr-160 phosphorylation of CDK2-NLS These with the that the role for ERK in Thr-160 phosphorylation of CDK2-NLS is through a other The enzymatic activation of the first CDK to become active in has been in However, is the regulation of CDK2-cyclin E, an activity for progression into S The role for ERK in the accumulation of cyclin D1 in G1 is well In the of ERK in regulating CDK2 nuclear a second role for ERK in G1 progression was found (23Keenan S.M. Bellone C. Baldassare J.J. J. Biol. Chem. 2001; 276: 22404-22409Google Scholar). In the study, a CDK2 that localized to the nucleus of and cyclin E, we found role for ERK in G1 phosphorylation and activation of CDK2-cyclin E. ERK activity is regulating cyclin D1 CDK2-cyclin E nuclear and the phosphorylation and activation of CDK2-cyclin E In an study, M. E. J.S. Biochem. J. 2000; used ERK cascade and that CDK2 to become on Thr-160 serum stimulation. However, because the role for ERK in CDK2 nuclear translocation was not the were a of CDK2 to to the nucleus, Thr-160 phosphorylation is reported to The of CDK2 the to examine Thr-160 phosphorylation of nuclear translocation. The activity thought to be for this phosphorylation is CAK, the identity of CAK is reported to be the complex (28Larochelle S. Pandur J. Fisher R.P. Salz H.K. Suter B. Genes Dev. 1998; 12: 370-381Google Scholar, 31Fisher R.P. Morgan D.O. Cell. 1994; 78: 713-724Google Scholar, A. A.M. D. M. EMBO J. 1995; 14: Scholar). The by which ERK regulates the activity of CAK, at In IIC9 cells, to full activity in and cells, H. and J. J. that ERK is not regulating activation. studies A.M. M. F. M. EMBO J. 1997; 16: M. I. J.M. EMBO J. 1997; 16: that growth factors that ERK regulate the of to CDK2-cyclin E. Another is the localization of for be with the complex in serum-arrested cells and become in late is that ERK regulates a CAK activity a CAK activity that is not has been reported in and human cells V. J. Biol. Chem. Google Scholar, P. J. Biochem. 2000; Scholar, H. A.M. P. Proc. Natl. Acad. Sci. U. S. A. 1999; Scholar). As CAK activity is in the ERK that CAK regulation become