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• W2000862518 abstract "Platelet-derived growth factor (PDGF)-induced Ras activation is required for G1 progression in Chinese hamster embryo fibroblasts (IIC9 cells). Ras stimulates both extracellular signal-related kinase (ERK) activation and RhoA activation in response to PDGF stimulation. Inhibition of either of these Ras-stimulated pathways results in growth arrest. We have shown previously that Ras-stimulated ERK activation is essential for the induction and continued G1 expression of cyclin D1. In this study we examine the role of Ras-induced RhoA activity in G1 progression. Unstimulated IIC9 cells expressed high levels of the G1 cyclin-dependent kinase inhibitor p27KIP1. Stimulation with PDGF resulted in a dramatic decrease in p27KIP1 protein expression. This decrease was attributed to increased p27KIP1 protein degradation. Overexpression of dominant-negative forms of Ras or RhoA completely blocked PDGF-induced p27KIP1 degradation, but only dominant-negative Ras inhibited cyclin D1 protein expression. C3 transferase also inhibited PDGF-induced p27KIP1 degradation, thus further implicating RhoA in p27KIP1 regulation. Overexpression of dominant-negative ERK resulted in inhibition of PDGF-induced cyclin D1 expression but had no effect on PDGF-induced p27KIP1 degradation. These data suggest that Ras coordinates the independent regulation of cyclin D1 and p27KIP1 expression by the respective activation of ERK and RhoA and that these pathways converge to determine the activation state of complexes of cyclin D1 and cyclin-dependent kinase in response to mitogen. Platelet-derived growth factor (PDGF)-induced Ras activation is required for G1 progression in Chinese hamster embryo fibroblasts (IIC9 cells). Ras stimulates both extracellular signal-related kinase (ERK) activation and RhoA activation in response to PDGF stimulation. Inhibition of either of these Ras-stimulated pathways results in growth arrest. We have shown previously that Ras-stimulated ERK activation is essential for the induction and continued G1 expression of cyclin D1. In this study we examine the role of Ras-induced RhoA activity in G1 progression. Unstimulated IIC9 cells expressed high levels of the G1 cyclin-dependent kinase inhibitor p27KIP1. Stimulation with PDGF resulted in a dramatic decrease in p27KIP1 protein expression. This decrease was attributed to increased p27KIP1 protein degradation. Overexpression of dominant-negative forms of Ras or RhoA completely blocked PDGF-induced p27KIP1 degradation, but only dominant-negative Ras inhibited cyclin D1 protein expression. C3 transferase also inhibited PDGF-induced p27KIP1 degradation, thus further implicating RhoA in p27KIP1 regulation. Overexpression of dominant-negative ERK resulted in inhibition of PDGF-induced cyclin D1 expression but had no effect on PDGF-induced p27KIP1 degradation. These data suggest that Ras coordinates the independent regulation of cyclin D1 and p27KIP1 expression by the respective activation of ERK and RhoA and that these pathways converge to determine the activation state of complexes of cyclin D1 and cyclin-dependent kinase in response to mitogen. Progression through the G1 phase of the mammalian cell cycle is mediated in part through the early induction of D-type cyclins by mitogenic stimulation (1Sherr C.J. Trends Biochem. Sci. 1995; 20: 187-190Abstract Full Text PDF PubMed Scopus (871) Google Scholar, 2Sherr C.J. Cell. 1994; 79: 551-555Abstract Full Text PDF PubMed Scopus (2576) Google Scholar, 3Matsushime H. Roussel M.F. Ashmun R.A. Sherr C.J. Cell. 1991; 65: 701-713Abstract Full Text PDF PubMed Scopus (988) Google Scholar). Cell cycle progression is orchestrated by distinct families of cyclin-dependent kinases (CDKs) 1The abbreviations used are: CDK(s), cyclin-dependent kinase(s); Rb, retinoblastoma; MAP kinase, mitogen-activated protein kinase; ERK, extracellular signal-related kinase; PDGF, platelet-derived growth factor; dn, dominant-negative; PBS, phosphate-buffered saline; GST, glutathione S-transferase; MEK, MAP kinase/ERK kinase. 1The abbreviations used are: CDK(s), cyclin-dependent kinase(s); Rb, retinoblastoma; MAP kinase, mitogen-activated protein kinase; ERK, extracellular signal-related kinase; PDGF, platelet-derived growth factor; dn, dominant-negative; PBS, phosphate-buffered saline; GST, glutathione S-transferase; MEK, MAP kinase/ERK kinase. whose activities depend upon cyclin binding, positive and negative phosphorylation, and association with inhibitory polypeptides (10Grana X. Reddy E.P. Oncogene. 1995; 11: 211-219PubMed Google Scholar). Progression through the G1 phase of the cell cycle is controlled by one of three D-type cyclins (D1, D2, or D3), which assemble with their catalytic partner CDK4 or CDK6, and cyclin E, which assembles with its catalytic partner CDK2 (1Sherr C.J. Trends Biochem. Sci. 1995; 20: 187-190Abstract Full Text PDF PubMed Scopus (871) Google Scholar, 2Sherr C.J. Cell. 1994; 79: 551-555Abstract Full Text PDF PubMed Scopus (2576) Google Scholar, 3Matsushime H. Roussel M.F. Ashmun R.A. Sherr C.J. Cell. 1991; 65: 701-713Abstract Full Text PDF PubMed Scopus (988) Google Scholar, 4Matsushime H. Quelle D.E. Shurtleff S.A. Shibuya M. Sherr C.J. Kato J.-Y. Mol. Cell. Biol. 1994; 14: 2066-2076Crossref PubMed Scopus (1023) Google Scholar, 5Bates S. Bonetta L. MacAllan D. Parry D. Holder A. Dickson C. Peters G. Oncogene. 1994; 9: 71-79PubMed Google Scholar, 6Matsushime H. Ewen M.E. Strom D.K. Kato J.-Y. Hanks S.K. Roussel M.F. Sherr C.J. Cell. 1992; 71: 323-334Abstract Full Text PDF PubMed Scopus (770) Google Scholar, 7Sherr C.J. Roberts J.M. Genes Dev. 1995; 9: 1149-1163Crossref PubMed Scopus (3200) Google Scholar, 8Xiong Y. Shang H. Beach D. Cell. 1992; 71: 505-514Abstract Full Text PDF PubMed Scopus (894) Google Scholar, 9Lukas J. Bartkova J. Welcker M. Peterson O.W. Peters G. Strauss M. Bartek J. Oncogene. 1995; 10: 2125-2134PubMed Google Scholar). D- and E-type CDKs are required for G1progression, and both contribute to the phosphorylation and inactivation of the retinoblastoma (Rb) protein thus canceling its growth-inhibitory properties (1Sherr C.J. Trends Biochem. Sci. 1995; 20: 187-190Abstract Full Text PDF PubMed Scopus (871) Google Scholar, 2Sherr C.J. Cell. 1994; 79: 551-555Abstract Full Text PDF PubMed Scopus (2576) Google Scholar, 5Bates S. Bonetta L. MacAllan D. Parry D. Holder A. Dickson C. Peters G. Oncogene. 1994; 9: 71-79PubMed Google Scholar, 7Sherr C.J. Roberts J.M. Genes Dev. 1995; 9: 1149-1163Crossref PubMed Scopus (3200) Google Scholar, 10Grana X. Reddy E.P. Oncogene. 1995; 11: 211-219PubMed Google Scholar, 11Dowdy S.F. Hinds P.W. Louie K. Reed S.I. Arnold A. Weinberg R.A. Cell. 1993; 73: 499-511Abstract Full Text PDF PubMed Scopus (688) Google Scholar, 12Resnitzky D. Gossen M. Bujard H. Reed S.I. Mol. Cell. Biol. 1994; 14: 1669-1679Crossref PubMed Scopus (987) Google Scholar, 13Kato J.-Y. Matsushime H. Hiebert S.W. Ewen M.E. Sherr C.J. Genes Dev. 1993; 7: 331-342Crossref PubMed Scopus (1080) Google Scholar, 14Ewen M.E. Sluss H.K. Sherr C.J. Matsushime H. Kato J.-Y. Livingston D.M. Cell. 1993; 73: 487-497Abstract Full Text PDF PubMed Scopus (914) Google Scholar, 15Hatakeyama M. Brill J.A. Fink G.R. Weinberg R.A. Genes Dev. 1994; 8: 1759-1771Crossref PubMed Scopus (221) Google Scholar, 16Resnitzky D. Reed S.I. Mol. Cell. Biol. 1995; 15: 3463-3469Crossref PubMed Scopus (432) Google Scholar, 17Horton L.E. Quian Y. Templeton D.J. Cell Growth Differ. 1995; 6: 397-407Google Scholar). The activation of CDK4/CDK6 following association with cyclin D is critical for G1 progression. Inhibition of cyclin D1 expression through antisense cDNA or microinjection of antibodies specific to cyclin D results in G1 growth arrest (18Quelle D.E. Ashmun R.A. Shurtleff S.A. Kato J.-Y. Bar-Sagi D. Roussel M.F. Sherr C.J. Genes Dev. 1993; 7: 1559-1571Crossref PubMed Scopus (978) Google Scholar, 19Baldin V. Lukas J. Marcote M.J. Pagano M. Draetta G. Genes Dev. 1993; 7: 812-821Crossref PubMed Scopus (1416) Google Scholar). D-type cyclins have been referred to as G1 mitogenic sensors because their induction requires mitogen, and removal of mitogen in G1results in their rapid degradation and subsequent growth arrest (1Sherr C.J. Trends Biochem. Sci. 1995; 20: 187-190Abstract Full Text PDF PubMed Scopus (871) Google Scholar, 2Sherr C.J. Cell. 1994; 79: 551-555Abstract Full Text PDF PubMed Scopus (2576) Google Scholar, 3Matsushime H. Roussel M.F. Ashmun R.A. Sherr C.J. Cell. 1991; 65: 701-713Abstract Full Text PDF PubMed Scopus (988) Google Scholar). The Ras/MAP kinase (ERK) pathway has been implicated in transducing mitogenic signals from growth factor receptors to the cell cycle machinery. Inhibition of the Ras/ERK pathway blocks mitogen-induced expression of cyclin D1 in Chinese hamster fibroblasts, demonstrating the importance of this pathway in mediating the mitogenic signals responsible for cyclin D1 induction (20Lavoie J.N. L'Allemain G. Brunet A. Muller R. Pouyssegur J. J. Biol. Chem. 1996; 271: 20608-20616Abstract Full Text Full Text PDF PubMed Scopus (1069) Google Scholar, 21Weber J.D. Raben D.M. Phillips P.J. Baldassare J.J. Biochem J. 1997; 326: 61-68Crossref PubMed Scopus (374) Google Scholar, 22Weber J.D. Cheng J. Raben D.M. Gardner A. Baldassare J.J. J. Biol. Chem. 1997; 272: 17320-17326Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). We have shown recently that PDGF induces the sustained activation of ERK and that this sustained activation is required for the continued accumulation of cyclin D1, implicating ERK activation in the regulation of cyclin D1 expression (21Weber J.D. Raben D.M. Phillips P.J. Baldassare J.J. Biochem J. 1997; 326: 61-68Crossref PubMed Scopus (374) Google Scholar). Concomitant with increased G1 cyclin D expression, cyclin D·CDK-associated activity increases in G1 (1Sherr C.J. Trends Biochem. Sci. 1995; 20: 187-190Abstract Full Text PDF PubMed Scopus (871) Google Scholar, 2Sherr C.J. Cell. 1994; 79: 551-555Abstract Full Text PDF PubMed Scopus (2576) Google Scholar, 3Matsushime H. Roussel M.F. Ashmun R.A. Sherr C.J. Cell. 1991; 65: 701-713Abstract Full Text PDF PubMed Scopus (988) Google Scholar, 4Matsushime H. Quelle D.E. Shurtleff S.A. Shibuya M. Sherr C.J. Kato J.-Y. Mol. Cell. Biol. 1994; 14: 2066-2076Crossref PubMed Scopus (1023) Google Scholar, 5Bates S. Bonetta L. MacAllan D. Parry D. Holder A. Dickson C. Peters G. Oncogene. 1994; 9: 71-79PubMed Google Scholar, 6Matsushime H. Ewen M.E. Strom D.K. Kato J.-Y. Hanks S.K. Roussel M.F. Sherr C.J. Cell. 1992; 71: 323-334Abstract Full Text PDF PubMed Scopus (770) Google Scholar, 7Sherr C.J. Roberts J.M. Genes Dev. 1995; 9: 1149-1163Crossref PubMed Scopus (3200) Google Scholar, 8Xiong Y. Shang H. Beach D. Cell. 1992; 71: 505-514Abstract Full Text PDF PubMed Scopus (894) Google Scholar, 9Lukas J. Bartkova J. Welcker M. Peterson O.W. Peters G. Strauss M. Bartek J. Oncogene. 1995; 10: 2125-2134PubMed Google Scholar, 20Lavoie J.N. L'Allemain G. Brunet A. Muller R. Pouyssegur J. J. Biol. Chem. 1996; 271: 20608-20616Abstract Full Text Full Text PDF PubMed Scopus (1069) Google Scholar, 21Weber J.D. Raben D.M. Phillips P.J. Baldassare J.J. Biochem J. 1997; 326: 61-68Crossref PubMed Scopus (374) Google Scholar, 22Weber J.D. Cheng J. Raben D.M. Gardner A. Baldassare J.J. J. Biol. Chem. 1997; 272: 17320-17326Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). The increase in cyclin D·CDK activity is a result of both an increase in cyclin D and a decrease in G1cyclin-dependent kinase inhibitor expression (1Sherr C.J. Trends Biochem. Sci. 1995; 20: 187-190Abstract Full Text PDF PubMed Scopus (871) Google Scholar, 2Sherr C.J. Cell. 1994; 79: 551-555Abstract Full Text PDF PubMed Scopus (2576) Google Scholar, 7Sherr C.J. Roberts J.M. Genes Dev. 1995; 9: 1149-1163Crossref PubMed Scopus (3200) Google Scholar). Although several cyclin-dependent kinase inhibitors have been identified as potent inhibitors of cyclin·CDK complexes, p27KIP1 is the only cyclin-dependent kinase inhibitor whose protein expression decreases as mitogen-induced cells enter the cell cycle (7Sherr C.J. Roberts J.M. Genes Dev. 1995; 9: 1149-1163Crossref PubMed Scopus (3200) Google Scholar, 23Winston J. Dong F. Pledger W.J. J. Biol. Chem. 1996; 271: 11253-11260Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 24Nourse J. Firpo E. Flanagan W.M. Coats S. Polyak K. Lee M.H. Massague J. Crabtree G.R. Roberts J.M. Nature. 1994; 372: 570-573Crossref PubMed Scopus (899) Google Scholar, 25Firpo E.J. Koff A. Solomon M.J. Roberts J.M. Mol. Cell. Biol. 1994; 14: 4889-4901Crossref PubMed Scopus (275) Google Scholar). The decrease in p27KIP1 expression occurs through protein degradation via the ubiquitin-proteasome pathway (26Pagano M. Tam S.W. Theodoras A.M. Beer-Romero P. Del Sal G. Chau V. Yew P.R. Draetta G.F. Rolfe M. Science. 1995; 269: 682-685Crossref PubMed Scopus (1731) Google Scholar). The retention of inhibitory levels of p27KIP1 appears to be involved in the growth-inhibitory properties of transforming growth factor-β, rapamycin, and cyclic AMP (27Polyak K. Kato J.Y. Solomon M.J. Sherr C.J. Massague J. Roberts J.M. Koff A. Genes Dev. 1994; 8: 9-22Crossref PubMed Scopus (1814) Google Scholar, 28Toyoshima H. Hunter T. Cell. 1994; 78: 67-74Abstract Full Text PDF PubMed Scopus (1925) Google Scholar, 29Kato J.Y. Matsuoka M. Polyak K. Massague J. Sherr C.J. Cell. 1994; 79: 487-496Abstract Full Text PDF PubMed Scopus (709) Google Scholar). In contrast, overexpression of p27KIP1 antisense cDNA results in mitogen-independent G1 progression, demonstrating the importance of p27KIP1 in maintaining cell quiescence (30Coats S. Flanagan W.M. Nourse J. Roberts J.M. Science. 1996; 272: 877-880Crossref PubMed Scopus (646) Google Scholar, 31Rivard N. L'Allemain G. Bartek J. Pouyssegur J. J. Biol. Chem. 1996; 271: 18337-18341Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). The mitogenic signals responsible for p27KIP1 degradation have not been defined clearly. PDGF stimulation causes the rapid activation of Ras and the subsequent downstream activation of ERK (21Weber J.D. Raben D.M. Phillips P.J. Baldassare J.J. Biochem J. 1997; 326: 61-68Crossref PubMed Scopus (374) Google Scholar, 22Weber J.D. Cheng J. Raben D.M. Gardner A. Baldassare J.J. J. Biol. Chem. 1997; 272: 17320-17326Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). In addition, Ras also stimulates the downstream activation of RhoA presumably to induce changes in cytoskeleton structure associated with growth (32Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3788) Google Scholar, 33Olson M.F. Ashworth A. Hall A. Science. 1995; 269: 1270-1272Crossref PubMed Scopus (1053) Google Scholar, 34Nobles C.D. Hall A. Cell. 1995; 81: 1-20Abstract Full Text PDF PubMed Scopus (260) Google Scholar, 35Denhardt D.T. Biochem J. 1996; 318: 729-747Crossref PubMed Scopus (449) Google Scholar, 36Tapon N. Hall A. Curr. Opin. Cell Biol. 1997; 9: 86-92Crossref PubMed Scopus (686) Google Scholar). However, RhoA activation has not been linked directly to the regulation of the cell cycle. In this study we demonstrate that Ras coordinates G1progression through two independent pathways: ERK regulation of cyclin D1 expression and RhoA regulation of p27KIP1 degradation to ensure the proper activation state of cyclin D1·CDK complexes following mitogenic stimulation. Cell Culture and Reagents—IIC9 cells, a subclone of Chinese hamster embryo fibroblasts (37Low D.A. Scott R.W. Baker J.B. Cunningham D.D. Nature. 1982; 298: 476-478Crossref PubMed Scopus (50) Google Scholar), were grown and maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 10% fetal calf serum and 2 mml-glutamine (Sigma). Subconfluent (60–70%) were growth arrested by washing once with fresh Dulbecco's modified Eagle's medium and reculturing in serum-free Dulbecco's modified Eagle's medium for 48 h. Human recombinant PDGF-BB (Calbiochem) was added to cultures at 10 ng/ml in all experiments. Growth-arrested IIC9 cells were preincubated with 10 μm PD98059 (New England Biolabs, Beverly, MA) before the addition of PDGF. Dominant-negative ERK2 (dnERK−) was a generous gift from Dr. Jacques Pouyssegur (University of Nice, France). Dominant-negative Ras (dnRas−) and RhoA (dnRhoA19) and constitutively active RhoA (RhoA63) were constructed as described previously through site-directed mutagenesis of Thr to Asn at codon 17 and 19 or Gln to Leu at codon 63, respectively, with the TransformerTM site-directed mutagenesis kit (CLONTECH) (38Qui R.G. Chen J. McCormick F. Symons M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11781-11785Crossref PubMed Scopus (486) Google Scholar). Transient transfection of IIC9 cells (50–60% confluence) using LipofectAMINE (Life Technologies, Inc.) as recommended by the manufacturer consistently resulted in >90% expression efficiency as visualized by β-galactosidase staining. PDGF was added to growth-arrested IIC9 cells in the presence (+) or absence (−) of PD98059 (10 μm), C3 transferase (40 μg/ml), or various dominant-negative plasmids. Cells were harvested 0–24 h after the addition of PDGF by scraping in cold 1 × PBS. Harvested cells were lysed and sonicated in solubilization buffer (25 mm Hepes; 300 mmNaCl; 0.2 mm EDTA; 1.5 mm MgCl2; 0.1% Triton X-100; 20 mm β-glycerophosphate; 0.1 mm sodium vanadate; 10 μg/ml each aprotinin, leupeptin, and pepstatin; and 0.5 mm phenylmethylsulfonyl fluoride). Protein concentrations were determined by Bio-Rad protein assay as recommended by the manufacturer. Western blots were performed on lysates/proteins (20 μg) as described previously (21Weber J.D. Raben D.M. Phillips P.J. Baldassare J.J. Biochem J. 1997; 326: 61-68Crossref PubMed Scopus (374) Google Scholar, 22Weber J.D. Cheng J. Raben D.M. Gardner A. Baldassare J.J. J. Biol. Chem. 1997; 272: 17320-17326Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Membranes were probed with cyclin D1, p27KIP1, or CDK4 polyclonal antibodies (Santa Cruz Biotechnology), conjugated with goat anti-rabbit IgG (H+L) horseradish peroxidase, and developed with ECL (enhanced chemiluminescence; Amersham Corp.) as recommended by the manufacturer. [3H]Thymidine incorporation into IIC9 cells was measured as described previously (21Weber J.D. Raben D.M. Phillips P.J. Baldassare J.J. Biochem J. 1997; 326: 61-68Crossref PubMed Scopus (374) Google Scholar,22Weber J.D. Cheng J. Raben D.M. Gardner A. Baldassare J.J. J. Biol. Chem. 1997; 272: 17320-17326Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Briefly, growth-arrested IIC9 cells were stimulated with PDGF (10 ng/ml) for 20 h. Approximately 17 h after the addition of PDGF, 1 μCi of [3H]thymidine (NEN Life Science Products) was added, and the cells were incubated for an additional 3 h. Cells were washed twice with cold 1 × PBS and incubated for an additional 30 min with 5% trichloroacetic acid. Trichloroacetic acid-precipitated DNA was washed with cold 5% trichloroacetic acid and solubilized with 2% sodium bicarbonate and 0.1 n NaOH. After neutralization with 5% trichloroacetic acid, precipitated [3H]DNA was quantitated by scintillation counting. ERK1 activity was measured as described previously (21Weber J.D. Raben D.M. Phillips P.J. Baldassare J.J. Biochem J. 1997; 326: 61-68Crossref PubMed Scopus (374) Google Scholar, 22Weber J.D. Cheng J. Raben D.M. Gardner A. Baldassare J.J. J. Biol. Chem. 1997; 272: 17320-17326Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Briefly, growth-arrested or PDGF-stimulated IIC9 cells were washed once with cold PBS and lysed in 300 μl of solubilization buffer (20 mm Tris-HCl, pH 8; 1 mm sodium vanadate; 10% glycerol; 1 mmphenylmethylsulfonyl fluoride; 2 mm EDTA; 1% Triton X-100; 50 mm β-glycerophosphate; and 10 μg/ml each aprotinin, leupeptin, and pepstatin). ERK1 was immunoprecipitated with a monoclonal ERK1 antibody (Santa Cruz) and protein A-Sepharose (Sigma). ERK1 immune complexes were assayed for their ability to phosphorylate myelin basic protein as described previously (21Weber J.D. Raben D.M. Phillips P.J. Baldassare J.J. Biochem J. 1997; 326: 61-68Crossref PubMed Scopus (374) Google Scholar, 22Weber J.D. Cheng J. Raben D.M. Gardner A. Baldassare J.J. J. Biol. Chem. 1997; 272: 17320-17326Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Cyclin D1·CDK activity was measured as described previously (4Matsushime H. Quelle D.E. Shurtleff S.A. Shibuya M. Sherr C.J. Kato J.-Y. Mol. Cell. Biol. 1994; 14: 2066-2076Crossref PubMed Scopus (1023) Google Scholar). Briefly, IIC9 cells were washed twice with 1 × PBS and lysed in IP buffer (50 mmHepes; 150 mm NaCl; 0.1 mm sodium vanadate; 1 mm EDTA; 2.5 mm EGTA; 1 mmdithiothreitol; 10 mm β-glycerophosphate; 0.1% Tween 20; 10% glycerol; 1 mm phenylmethylsulfonyl fluoride; and 10 μg/ml each aprotinin, leupeptin, and pepstatin) and sonicated briefly. Cyclin D1 complexes were immunoprecipitated with a monoclonal cyclin D1 antibody bound to protein G-Sepharose (Santa Cruz Biotechnology) and washed three times with IP buffer. Cyclin D1 immune complexes were resuspended in reaction buffer (50 mm Hepes, 10 mm MgCl2, 1 mm dithiothreitol, 2.5 mm EGTA, 10 mm β-glycerophosphate, 0.1 mm sodium vanadate, and 20 μm ATP). Resuspended complexes were incubated with 2 μg of soluble GST-Rb fusion protein (generous gift from Dr. Mark Ewen) and 5 μCi of [γ-32P]ATP. Samples were subjected to SDS-polyacrylamide gel electrophoresis and developed on a PhosphorImager. Protein levels of p27KIP1 are increased in contact-inhibited or serum-deprived cells and decrease when cells are stimulated by mitogen to enter the cell cycle (7Sherr C.J. Roberts J.M. Genes Dev. 1995; 9: 1149-1163Crossref PubMed Scopus (3200) Google Scholar, 23Winston J. Dong F. Pledger W.J. J. Biol. Chem. 1996; 271: 11253-11260Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 24Nourse J. Firpo E. Flanagan W.M. Coats S. Polyak K. Lee M.H. Massague J. Crabtree G.R. Roberts J.M. Nature. 1994; 372: 570-573Crossref PubMed Scopus (899) Google Scholar, 25Firpo E.J. Koff A. Solomon M.J. Roberts J.M. Mol. Cell. Biol. 1994; 14: 4889-4901Crossref PubMed Scopus (275) Google Scholar). Various mitogens including epidermal growth factor, PDGF, and serum are capable of stimulating cell cycle entry and p27KIP1 degradation (23Winston J. Dong F. Pledger W.J. J. Biol. Chem. 1996; 271: 11253-11260Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 24Nourse J. Firpo E. Flanagan W.M. Coats S. Polyak K. Lee M.H. Massague J. Crabtree G.R. Roberts J.M. Nature. 1994; 372: 570-573Crossref PubMed Scopus (899) Google Scholar, 25Firpo E.J. Koff A. Solomon M.J. Roberts J.M. Mol. Cell. Biol. 1994; 14: 4889-4901Crossref PubMed Scopus (275) Google Scholar). However, the mechanism by which these mitogens stimulate p27KIP1 degradation remains unclear. We have shown previously that PDGF is a potent mitogen for IIC9 cells, and addition of PDGF to quiescent IIC9 cells resulted in up-regulation of cyclin D1 protein expression and D-type cyclin-dependent kinase activity (21Weber J.D. Raben D.M. Phillips P.J. Baldassare J.J. Biochem J. 1997; 326: 61-68Crossref PubMed Scopus (374) Google Scholar, 22Weber J.D. Cheng J. Raben D.M. Gardner A. Baldassare J.J. J. Biol. Chem. 1997; 272: 17320-17326Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Stimulation with PDGF also resulted in the time-dependent degradation of p27KIP1 protein (Fig. 1). 2 h after PDGF stimulation, p27KIP1 protein levels decreased approximately 50%, and by 24 h they were nearly undetectable (Fig. 1). Levels of CDK4, which we have shown previously do not increase with PDGF stimulation (21Weber J.D. Raben D.M. Phillips P.J. Baldassare J.J. Biochem J. 1997; 326: 61-68Crossref PubMed Scopus (374) Google Scholar), were measured to ensure equal protein loading (Fig. 1). Previous studies have shown that loss of p27KIP1 protein occurs via a ubiquitin-mediated degradation pathway (26Pagano M. Tam S.W. Theodoras A.M. Beer-Romero P. Del Sal G. Chau V. Yew P.R. Draetta G.F. Rolfe M. Science. 1995; 269: 682-685Crossref PubMed Scopus (1731) Google Scholar). In agreement with these observations, incubation of IIC9 cells with a calpain I inhibitor resulted in the appearance of polyubiquitinated forms of p27KIP1 (data not shown). We have shown previously that PDGF-induced G1 progression requires the sustained activation of ERK in a MAP kinase/ERK kinase 1 (MEK1)-dependent manner (21Weber J.D. Raben D.M. Phillips P.J. Baldassare J.J. Biochem J. 1997; 326: 61-68Crossref PubMed Scopus (374) Google Scholar). The sustained activation of ERK following PDGF stimulation was responsible for the continued accumulation of cyclin D1, and inhibition of this activity resulted in the loss of cyclin D1 protein expression (21Weber J.D. Raben D.M. Phillips P.J. Baldassare J.J. Biochem J. 1997; 326: 61-68Crossref PubMed Scopus (374) Google Scholar). To determine whether PDGF-induced ERK activation also contributed to the degradation of p27KIP1, we overexpressed a dnERK− in IIC9 cells. Although dnERK− inhibits PDGF-induced G1 progression (21Weber J.D. Raben D.M. Phillips P.J. Baldassare J.J. Biochem J. 1997; 326: 61-68Crossref PubMed Scopus (374) Google Scholar), it did not inhibit the PDGF-induced loss of p27KIP1 (Fig.2 A). IIC9 cells preincubated with an inhibitor of MEK1 activation, PD98059, displayed normal PDGF-induced p27KIP1 protein degradation with p27KIP1 protein levels being reduced to 10% maximal levels by 16 h (Fig.2 B). These data suggest that downstream effectors of MEK1 and ERK are not responsible for the degradation of p27KIP1. We next looked at Ras, an upstream activator of the MAP kinase pathway, which we have shown previously is activated rapidly by PDGF (22Weber J.D. Cheng J. Raben D.M. Gardner A. Baldassare J.J. J. Biol. Chem. 1997; 272: 17320-17326Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). The addition of PDGF to IIC9 cells overexpressing dnRas− did not affect p27KIP1 protein levels, demonstrating the requirement of Ras activation for PDGF-induced p27KIP1 degradation. These data demonstrate clearly that mitogen-regulated destruction of p27KIP1 is downstream of Ras. It has become apparent that both MAP kinase and Rho pathways are important in the control of cell proliferation (20Lavoie J.N. L'Allemain G. Brunet A. Muller R. Pouyssegur J. J. Biol. Chem. 1996; 271: 20608-20616Abstract Full Text Full Text PDF PubMed Scopus (1069) Google Scholar, 21Weber J.D. Raben D.M. Phillips P.J. Baldassare J.J. Biochem J. 1997; 326: 61-68Crossref PubMed Scopus (374) Google Scholar, 22Weber J.D. Cheng J. Raben D.M. Gardner A. Baldassare J.J. J. Biol. Chem. 1997; 272: 17320-17326Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 32Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3788) Google Scholar, 33Olson M.F. Ashworth A. Hall A. Science. 1995; 269: 1270-1272Crossref PubMed Scopus (1053) Google Scholar, 34Nobles C.D. Hall A. Cell. 1995; 81: 1-20Abstract Full Text PDF PubMed Scopus (260) Google Scholar, 35Denhardt D.T. Biochem J. 1996; 318: 729-747Crossref PubMed Scopus (449) Google Scholar, 36Tapon N. Hall A. Curr. Opin. Cell Biol. 1997; 9: 86-92Crossref PubMed Scopus (686) Google Scholar, 39Vouret-Craviari V. Van Obberghen-Schilling E. Scimeca J.C. Van Obberghen E. Pouyssegur J. Biochem. J. 1993; 289: 209-214Crossref PubMed Scopus (143) Google Scholar, 40Albanese C. Johnson J. Watanabe G. Eklund N. Vu D. Arnold A. Pestell R.G. J. Biol. Chem. 1995; 270: 23589-23597Abstract Full Text Full Text PDF PubMed Scopus (759) Google Scholar). Whereas the role of the MAP kinase cascade has been shown clearly to regulate cyclin D1 expression (20Lavoie J.N. L'Allemain G. Brunet A. Muller R. Pouyssegur J. J. Biol. Chem. 1996; 271: 20608-20616Abstract Full Text Full Text PDF PubMed Scopus (1069) Google Scholar, 21Weber J.D. Raben D.M. Phillips P.J. Baldassare J.J. Biochem J. 1997; 326: 61-68Crossref PubMed Scopus (374) Google Scholar, 22Weber J.D. Cheng J. Raben D.M. Gardner A. Baldassare J.J. J. Biol. Chem. 1997; 272: 17320-17326Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 40Albanese C. Johnson J. Watanabe G. Eklund N. Vu D. Arnold A. Pestell R.G. J. Biol. Chem. 1995; 270: 23589-23597Abstract Full Text Full Text PDF PubMed Scopus (759) Google Scholar, 42Watanabe G. Lee R.J. Albanese C. Rainey W.E. Batle D. Pestell R.G. J. Biol. Chem. 1996; 271: 22570-22577Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), the role of the Rho cascade in cell cycle progression is unknown. To investigate the importance of PDGF-induced RhoA activity, we transfected IIC9 cells with dnRhoA19 and examined the effect of dnRhoA19 expression on several proteins that control progression through G1. Overexpression of dnRhoA19 inhibited PDGF-induced reduction of p27KIP1 protein levels in IIC9 cells (Fig. 3 A) similar to that seen in dnRas−-transfected cells (Fig. 2 A), suggesting that RhoA is a downstream Ras-dependent signaling molecule required for PDGF-induced p27KIP1 degradation. Incubation with C3 transferase, an inhibitor of RhoA activity, also resulted in the inhibition of PDGF-induced p27KIP1 degradation, further implicating RhoA activation in p27KIP1 destruction (Fig.4 A). Overexpression of a constitutively active RhoA mutant, RhoA63, resulted in the mitogen-independent decrease in p27KIP1 protein expression (Fig. 3 B) identical to that of PDGF-stimulated IIC9 cells. These data demonstrate that activated RhoA alone is sufficient for loss of p27KIP1. The requirement of RhoA for PDGF-induced p27KIP1 degradation and the ability of RhoA63 mutant to stimulate p27KIP1 degradation independently show clearly that RhoA activation has an important role in G1progres" @default.
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• W2000862518 title "Ras-stimulated Extracellular Signal-related Kinase 1 and RhoA Activities Coordinate Platelet-derived Growth Factor-induced G1 Progression through the Independent Regulation of Cyclin D1 and p27KIP1" @default.
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