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• W2055296088 abstract "Distinct protein kinase C (PKC) isoforms differentially regulate cellular proliferation in rat microvascular endothelial cells (EC). Overexpression of PKCα has little effect on proliferation, whereas PKCδ slows endothelial cell proliferation and induces S-phase arrest. Analyses were performed on EC overexpressing PKCα (PKCαEC) or PKCδ (PKCδEC) to determine the role of specific cell cycle regulatory proteins in the PKCδ-induced cell cycle arrest. Serum-induced stimulation of cyclins D1, E, and A-associated kinase activity was delayed by 12 h in the PKCδEC line in association with S-phase arrest. However, the protein levels for cyclins D1, E, and A were similar. Nuclear accumulation of cyclin D1 protein in response to serum was also delayed in PKCδEC. In the PKCδEC line, serum induced p27Kip1 but not p16Ink4a or p21Cip1. Serum did not affect p27Kip1 levels in the control vascular endothelial cell line. Immunoprecipitation-Western blotting analysis of p27Kip1 showed serum stimulation of the vascular endothelial cell line resulted in increased amounts of cyclin D1 bound to p27Kip1. In the PKCδEC line, serum did not increase the amount of cyclin D1 bound to p27Kip1. Transfection of full-length p27Kip1 antisense into the PCKδEC line reversed the S-phase arrest and resulted in normal cell cycle progression, suggesting a critical role for p27Kip1 in the PKCδ-mediated S-phase arrest. Distinct protein kinase C (PKC) isoforms differentially regulate cellular proliferation in rat microvascular endothelial cells (EC). Overexpression of PKCα has little effect on proliferation, whereas PKCδ slows endothelial cell proliferation and induces S-phase arrest. Analyses were performed on EC overexpressing PKCα (PKCαEC) or PKCδ (PKCδEC) to determine the role of specific cell cycle regulatory proteins in the PKCδ-induced cell cycle arrest. Serum-induced stimulation of cyclins D1, E, and A-associated kinase activity was delayed by 12 h in the PKCδEC line in association with S-phase arrest. However, the protein levels for cyclins D1, E, and A were similar. Nuclear accumulation of cyclin D1 protein in response to serum was also delayed in PKCδEC. In the PKCδEC line, serum induced p27Kip1 but not p16Ink4a or p21Cip1. Serum did not affect p27Kip1 levels in the control vascular endothelial cell line. Immunoprecipitation-Western blotting analysis of p27Kip1 showed serum stimulation of the vascular endothelial cell line resulted in increased amounts of cyclin D1 bound to p27Kip1. In the PKCδEC line, serum did not increase the amount of cyclin D1 bound to p27Kip1. Transfection of full-length p27Kip1 antisense into the PCKδEC line reversed the S-phase arrest and resulted in normal cell cycle progression, suggesting a critical role for p27Kip1 in the PKCδ-mediated S-phase arrest. The vascular endothelium is a dynamic organ controlling hemostasis, vasodilation, and wound healing. The endothelium is influenced by shear stress, hypoxia, and chemotactic/mitogenic gradients that promote migration and division of its cells. Endothelial cellular division is an important component of the angiogenic response to many stimuli (2Yang E.Y. Moses H.L. J. Cell Biol. 1990; 111: 731-741Crossref PubMed Scopus (420) Google Scholar, 3Ausprunk D.H. Folkman J. Microvasc. Res. 1977; 14: 53-65Crossref PubMed Scopus (1047) Google Scholar, 4Yancopoulos G.D. Klagsbrun M. Folkman J. Cell. 1998; 93: 661-664Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). As several different external agents promote or inhibit endothelial cell proliferation, the secondary messengers mediating these responses are being actively investigated. The protein kinase C (PKC) 1The abbreviations used are: PKC, protein kinase C; CDK, cyclin-dependent kinase; CKI, CDK inhibitor; EC, endothelial cell(s); V-EC, vector EC (cell line); PBS, phosphate-buffered saline; HEPES, 4(2-hydroxyethyl)-1-piperazineethanesulfonic acid; GST, glutathioneS-transferase; MACS, magnetic activated cell separation system; pRB, retinoblastoma protein; JAB, JAK-binding protein1The abbreviations used are: PKC, protein kinase C; CDK, cyclin-dependent kinase; CKI, CDK inhibitor; EC, endothelial cell(s); V-EC, vector EC (cell line); PBS, phosphate-buffered saline; HEPES, 4(2-hydroxyethyl)-1-piperazineethanesulfonic acid; GST, glutathioneS-transferase; MACS, magnetic activated cell separation system; pRB, retinoblastoma protein; JAB, JAK-binding protein family of Ser-Thr kinases is a common intracellular signaling pathway that coordinates a diverse array of signals that arise in the extracellular environment. Activation of the PKC pathway by phorbol esters, for example, induces endothelial cell proliferation and angiogenesisin vivo (5Montesano R. Orci L. Cell. 1985; 42: 469-477Abstract Full Text PDF PubMed Scopus (370) Google Scholar, 6Hu D.E. Fan T.P. Inflammation. 1995; 19: 39-54Crossref PubMed Scopus (26) Google Scholar, 7Wright P.S. Cross-Doersen D. Miller J.A. Jones W.D. Bitonti A.J. J. Cell. Physiol. 1992; 152: 448-457Crossref PubMed Scopus (22) Google Scholar). In contrast, inhibition of the PKC pathway by prolonged treatment with phorbol esters inhibits mitogenesis of endothelial cells (8Presta M. Tiberio L. Rusnati M. Dell'Era P. Ragnotti G. Cell Regul. 1991; 2: 719-726Crossref PubMed Scopus (57) Google Scholar, 9Daviet I. Herbert J.M. Maffrand J.P. FEBS Lett. 1990; 259: 315-317Crossref PubMed Scopus (35) Google Scholar). The molecular mechanisms regulating endothelial cell proliferation in response to mitogens and PKC activation are poorly understood. However, it is likely that specific components of the cell cycle regulatory apparatus may govern these responses. Recent studies have suggested that individual isozymes modulate specific cell cycle transitions in specific cell types. The G1-S transition is regulated by the PKCη isozyme in NIH3T3 cells (10Livneh E. Shimon T. Bechor E. Doki Y. Schieren I. Weinstein I.B. Oncogene. 1996; 12: 311-320Google Scholar), whereas in vascular smooth muscle cells PKCα/ε regulate this transition (11Sasaguri T. Kosaka C. Hirata M. Masuda J. Shimokado K. Fujishima M. Ogata J. Exp. Cell Res. 1993; 208: 311-320Crossref PubMed Scopus (65) Google Scholar). Overexpression of PKCα and PKCδ affect cellular proliferation and cell cycle progression in several different cell types. PKCα promotes cellular proliferation in human breast cancer and other cells (12Hirai S. Izumi Y. Higa K. Kaibucji K. Mizuno K. Osada S. Suzuki K. Ohno S. EMBO J. 1994; 13: 2331-2340Crossref PubMed Scopus (110) Google Scholar, 13Ways D.K. Kukoly C.A. deVente J. Hooker J.L. Bryant W.O. Posekany K.J. Fletcher D.J. Cook P.P. Parker P.J. J. Clin. Invest. 1995; 95: 1906-1915Crossref PubMed Scopus (266) Google Scholar). In contrast, overexpression of the PKCδ isoform in Chinese hamster ovary fibroblasts in the presence of phorbol ester induces G2/M-phase arrest (14Watanabe T. Ono Y. Taniyama Y. Hazama K. Igarashi K. Ogita K. Kikkawa U. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10159-10163Crossref PubMed Scopus (297) Google Scholar).The components of the cell cycle regulatory apparatus governing progression through the G1 phase are increasingly well understood (15Pestell R.G. Albanese C. Reutens A.T. Lee R.J. Arnold A. Endocr. Rev. 1999; (in press)PubMed Google Scholar, 16Weinberg R.A. Cell. 1995; 81: 323-330Abstract Full Text PDF PubMed Scopus (4295) Google Scholar, 17Sherr C.J. Cell. 1994; 79: 551-555Abstract Full Text PDF PubMed Scopus (2579) Google Scholar). The cyclin-dependent kinases (CDKs) are serine-threonine holoenzymes, consisting of a regulatory and catalytic subunit that phosphorylate target substrates to promote progression through the G1 phase of the cell cycle. The phosphorylation of the pRB protein is mediated in part by cyclin D1/Cdk4 and cyclin E/Cdk2 (18Resnitzky D. Reed S.I. Mol. Cell. Biol. 1995; 15: 3463-3469Crossref PubMed Scopus (435) Google Scholar, 19Xiong W. Pestell R.G. Watanabe G. Rosner M.R. Hershenson M.B. Am. J. Physiol. 1997; 272: L1205-L1210PubMed Google Scholar). The phosphorylation of pRB inactivates its ability to block G1 phase progression. Phosphorylation of pRB is associated with release of E2F/DP proteins from their binding site on the pRB protein and progression through G1 into a phase of DNA synthesis. The activity of the CDKs is inhibited by members of the p21Waf1/Cip1 family (p21Cip1, p27Kip1, p57Kip2) and the INK family (p16Ink4a, p15Ink4b, p18Ink4c, and p19Ink4d). These proteins inhibit CDK enzymatic activity in part through binding to the CDK regulatory subunit, thereby inhibiting holoenzyme association. The p21 family proteins are referred to as “universal inhibitors” because of the ability to block the activity of the cyclin D, cyclin E, and cyclin protein kinase A. As the CDK holoenzymatic activity is directed at nuclear substrates, the activity of the CDK inhibitor (CKI) is in part determined by its subcellular distribution. Thus, the CKI is inhibitory in the nuclear but not the cytoplasmic location.In recent studies we showed that overexpression of PKCδ, but not PKCα, in EC inhibited cellular proliferation through an arrest in S-phase (1Harrington E.O. Loffler J. Nelson P.R. Kent K.C. Simons M. Ware J.A. J. Biol. Chem. 1997; 272: 7390-7397Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). These findings were consistent with several other studies in which the loss of PKCδ expression was associated with increased cellular proliferation or transformation (20Lu Z. Hornia A. Jiang Y.-W. Zang Q. Foster D.A. Mol. Cell. Biol. 1997; 17: 3418-3428Crossref PubMed Google Scholar, 21Zang Q. Lu Z. Curto M. Barile N. Shalloway D. Foster D.A. J. Biol. Chem. 1997; 272: 13275-13280Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 22Denning M.F. Dlugosz A.A. Howett M.K. Yuspa S.H. J. Biol. Chem. 1993; 268: 26079-26081Abstract Full Text PDF PubMed Google Scholar). Together these studies suggested a role for PKCδ as an inhibitor of cellular proliferation, which may play an important role in slowing cell cycle progression in normal cells. The current studies were performed to understand the molecular mechanisms governing PKCδ-mediated cell cycle arrest. We report here that PKCδ overexpression (PKCδEC) delays serum-induced expression of kinase activity associated with cyclins D1, E, and A. However, the cyclin protein levels induced by serum in the PKCδEC were unchanged compared with the induction seen in control cells. Expression of the INK family members (p16Ink4a and p18Ink4c) and the CKI p21Cip1 was unchanged. PKCδEC contained higher nuclear levels of the cyclin-dependent kinase inhibitor p27Kip1 than vector controls. p27Kip1-antisense reduced p27Kip1 levels and relieved the cell cycle defect induced by PKCδ, strongly suggesting that increased expression of p27Kip1 was responsible for the prolongation of S-phase in PKCδEC.DISCUSSIONThe molecular mechanisms by which specific isozymes of the PKC family regulate cellular proliferation are poorly understood. The current studies extend our previous findings that PKCδ delays S-phase progression in rat microvascular endothelial cells (1Harrington E.O. Loffler J. Nelson P.R. Kent K.C. Simons M. Ware J.A. J. Biol. Chem. 1997; 272: 7390-7397Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The PKCδ-mediated inhibition of S-phase progression was associated with a delay in the induction of the kinase activities associated with cyclins D1, E, and A. An investigation of the abundance of the cyclin-dependent kinase inhibitors associated with the inhibition of the cyclin kinase activity revealed an increase in the abundance of the “universal inhibitor,” p27Kip1. The relative abundance of p21Cip1 and the INK4 protein family was unchanged, suggesting that the induction of p27Kip1 was a relatively specific change. In addition, overexpression of an antisense expression plasmid for p27Kip1, which was shown to reduce p27Kip1 protein levels, was also shown to reverse the S-phase arrest observed in the PKCδEC lines. These studies are consistent with a model in which the induction of p27Kip1may play an important and specific role in PKCδ-mediated S-phase arrest in microvascular endothelial cells.The pRB protein is a critical regulator of cell cycle progression, and the phosphorylation of pRB during G1 phase coincides with passage of the cell through the restriction point in G1(15Pestell R.G. Albanese C. Reutens A.T. Lee R.J. Arnold A. Endocr. Rev. 1999; (in press)PubMed Google Scholar, 16Weinberg R.A. Cell. 1995; 81: 323-330Abstract Full Text PDF PubMed Scopus (4295) Google Scholar). pRB undergoes continued phosphorylation throughout the S-phase (40Mittnacht S. Lees J.A. Desai D. Morgan D.O. Weinberg R.A. EMBO J. 1994; 13: 118-127Crossref PubMed Scopus (126) Google Scholar), and the cyclin D1-dependent phosphorylation, which is required for cyclin D1 to promote cell cycle progression, occurs on specific phosphorylation sites that can be assessed in immunoprecipitation assays using a pRB fragment containing this site (25Watanabe G. Pena P. Shambaugh III, G.E. Haines III, G.K. Pestell R.G. Dev. Brain Res. 1998; 108: 77-87Crossref PubMed Scopus (36) Google Scholar, 26Watanabe G. Albanese C. Lee R.J. Reutens A. Vairo G. Henglein B. Pestell R.G. Mol. Cell. Biol. 1998; 18: 3212-3222Crossref PubMed Scopus (145) Google Scholar, 27Watanabe G. Lee R.J. Albanese C. Rainey W.E. Batlle D. Pestell R.G. J. Biol. Chem. 1996; 271: 22570-22577Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 28Lee R.J. Watanabe G. Pestell R.G. Methods in Molecular Medicine: Fibroblast Growth Factor Family of Polypeptides. Humana Press Inc., 1999Google Scholar). Upon phosphorylation by cyclin D1, cyclin E kinase further phosphorylates pRB at distinct sites (41Lundberg A.S. Weinberg R.A. Mol. Cell. Biol. 1998; 18: 753-761Crossref PubMed Scopus (852) Google Scholar). Cyclin E kinase activity phosphorylates and inactivates additional substrates that contribute to cell cycle progression in a pRB-independent manner (42Zhao J. Dynlacht B. Imai T. Hori T. Harlow E. Genes & Dev. 1998; 12: 456-461Crossref PubMed Scopus (183) Google Scholar). In the current studies the induction of cyclin D1-dependent pRB phosphorylation was maximally induced at 24 h in the V-EC and PKCαEC but occurred in a delayed manner in the PKCδEC. The induction of cyclin E kinase activity by serum was also delayed in the PKCδEC. These data are consistent with the role of p27Kip1 as a “universal inhibitor” of both cyclin E and cyclin D1 kinase activity. The phosphorylation of pRB coincides with the loss of the ability of pRB to bind and inhibit E2F/DP complexes. The corresponding induction of “free E2F activity” activates genes involved in DNA synthesis. In the current studies, the delayed induction of cyclin A kinase activity, a marker of S-phase entry, in the PKCδEC line, is consistent with the delayed entry into S-phase. pRB is a poor substrate for cyclin E kinase, and cyclin E overexpression can promote S-phase entry independently of pRB, suggesting that cyclins D1 and E function in parallel pathways to promote S-phase entry (21Zang Q. Lu Z. Curto M. Barile N. Shalloway D. Foster D.A. J. Biol. Chem. 1997; 272: 13275-13280Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 42Zhao J. Dynlacht B. Imai T. Hori T. Harlow E. Genes & Dev. 1998; 12: 456-461Crossref PubMed Scopus (183) Google Scholar). The current studies suggest that PKCδ inhibits these parallel pathways in EC lines.In the current studies, PKCδ induced p27Kip1 in rat microvascular endothelial cells. The induction of p27Kip1by serum was enhanced in the PKCδEC in association with S-phase arrest. Antisense p27Kip1 expression blocked the PKCδEC-induced S-phase arrest. Overexpression of p27Kip1, initially characterized as a protein homologous to p21Cip1(32Toyoshima H. Hunter T. Cell. 1994; 78: 67-74Abstract Full Text PDF PubMed Scopus (1927) Google Scholar), can delay cell cycle progression in fibroblasts (43Coats S. Flanagan W.M. Nourse J. Roberts J.M. Science. 1996; 272: 877-880Crossref PubMed Scopus (648) Google Scholar, 44Rivard 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). In recent studies p27Kip1 reduced the proliferation of smooth muscle induced by angioplasty and mediated the inhibition of smooth muscle cell proliferation by fibrillar collagen (45Chen D. Krasinski K. Chen D. Sylvester A. Chen J. Nisen P.D. Andres V. J. Clin. Invest. 1997; 99: 2334-2341Crossref PubMed Scopus (174) Google Scholar, 46Koyama H. Raines E.W. Bornfeldt K.E. Roberts J.M. Ross R. Cell. 1996; 87: 1069-1078Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar). Together these studies suggest that p27Kip1 may be an important inhibitor of vascular remodeling (45Chen D. Krasinski K. Chen D. Sylvester A. Chen J. Nisen P.D. Andres V. J. Clin. Invest. 1997; 99: 2334-2341Crossref PubMed Scopus (174) Google Scholar). Our finding that p27Kip1 is involved in the cell cycle delay by PKCδ extends the known cytostatic signaling pathways in which p27Kip1 is involved. p27Kip1 also mediates the cytostatic effects of rapamycin and cAMP (32Toyoshima H. Hunter T. Cell. 1994; 78: 67-74Abstract Full Text PDF PubMed Scopus (1927) Google Scholar, 47Polyak 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 (1824) Google Scholar, 48Kato J.-y. Matsuoka M. Stromm D.K. Sherr C.J. Mol. Cell. Biol. 1994; 14: 2713-2721Crossref PubMed Scopus (262) Google Scholar, 49Luo Y. Marx S.O. Kiyokawa H. Koff A. Massague J. Marks A.R. Mol. Cell. Biol. 1996; 16: 6744-6751Crossref PubMed Scopus (204) Google Scholar).In the current studies, p27Kip1 immunoprecipitation assays were performed to assess the effect of the PKC isoforms on the multimeric complexes bound to p27Kip1. The cyclin/CDK complex to which p27Kip1 is bound determines its functional activity. p27Kip1 associates with cyclin E in a variety of cell types during quiescence (47Polyak 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 (1824) Google Scholar, 50Agrawal D. Dong K.F. Wang Y.Z. Kayda D. Pledger W.J. Cell Growth & Differ. 1995; 6: 1199-1205PubMed Google Scholar). When bound to cyclin D1/Cdk4, p27Kip1 may not be inhibitory (47Polyak 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 (1824) Google Scholar, 51Polyak K. Lee M.H. Erdjument-Bromage H. Koff A. Roberts J.M. Tempst P. Massague J. Cell. 1994; 78: 59-66Abstract Full Text PDF PubMed Scopus (2048) Google Scholar, 52Reynisdottir I. Polyak K. Iavarone A. Massague J. Genes & Dev. 1995; 9: 1831-1845Crossref PubMed Scopus (888) Google Scholar, 53Soos T. Kiyokawa J.H. Yan J.S. Rubin M.S. Giordano A. DeBlasio A. Bottega S. Wong B. Mendelsohn J. Koff A. Cell Growth & Differ. 1996; 7: 135-146PubMed Google Scholar), whereas cyclin E/Cdk2 activity is inhibited by p27Kip1. In the current studies, we compared the relative proportion of p27Kip1bound to either cyclin D1 or cyclin E after 36 h of serum stimulation. In the PKCδEC line, p27Kip1 was bound to both cyclins D1 and E after 36 h stimulation; however, there was relatively more cyclin D1 bound to p27Kip1 in the V-EC line (Fig. 4). Thus, in the PKCδEC line the serum-induced increase in cyclin D1 binding to p27Kip1 is reduced. It is thought that the removal of p27Kip1 from the cyclin E/CDK complex is an essential step for S-phase entry. Through binding cyclin D1/Cdk4, p27Kip1 is sequestered from cyclin E/Cdk2, reducing its inhibition by p27Kip1 (47Polyak 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 (1824) Google Scholar, 51Polyak K. Lee M.H. Erdjument-Bromage H. Koff A. Roberts J.M. Tempst P. Massague J. Cell. 1994; 78: 59-66Abstract Full Text PDF PubMed Scopus (2048) Google Scholar, 52Reynisdottir I. Polyak K. Iavarone A. Massague J. Genes & Dev. 1995; 9: 1831-1845Crossref PubMed Scopus (888) Google Scholar, 53Soos T. Kiyokawa J.H. Yan J.S. Rubin M.S. Giordano A. DeBlasio A. Bottega S. Wong B. Mendelsohn J. Koff A. Cell Growth & Differ. 1996; 7: 135-146PubMed Google Scholar). Thus, in the PKCδEC line it may be expected that p27Kip1 is incorporated proportionately more in an inhibitory complex with cyclin E than is the case in the V-EC line. The failure of p27Kip1 to bind increasing amounts of cyclin D1 may be the result of the delayed nuclear entry of cyclin D1 in response to serum (Fig. 3). Thus, these studies suggest that PKCδ overexpression both increases the amount of p27Kip1 induced in the cell in response to serum stimulation and also alters the multiprotein complex with which p27Kip1 is associated in the cell.The mechanisms responsible for the increased p27Kip1 levels in the PKCδEC line remain to be fully evaluated. The abundance of p27Kip1 is regulated primarily at a post-translational level, and p27Kip1 protein levels decrease after mitogenic stimulation in quiescent NIH3T3 cells (50Agrawal D. Dong K.F. Wang Y.Z. Kayda D. Pledger W.J. Cell Growth & Differ. 1995; 6: 1199-1205PubMed Google Scholar, 54Winston J. Dong F. Pledger W.J. J. Biol. Chem. 1996; 271: 11253-11260Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 55Firpo E.J. Koff A. Solomon E. Flanagan W.M. Coats S. Polyak K. Lee M.H. Massague J. Crabtree G.R. Roberts J.M. Mol. Cell. Biol. 1994; 14: 4889-4901Crossref PubMed Scopus (275) Google Scholar). The degradation of p27Kip1 upon mitogen stimulation is dependent upon prior phosphorylation. Cyclin E/Cdk2-induced phosphorylation of p27Kip1 on T187 in murine fibroblasts (56Sheaff R.J. Groudine M. Gordon M. Roberts J.M. Clurman B.E. Genes & Dev. 1997; 11: 1464-1478Crossref PubMed Scopus (790) Google Scholar) and phosphorylation of p27Kip1 by cyclin E/Cdk2 enhanced degradation of p27Kip1. The delayed induction of cyclin E/Cdk2 activity in the PKCδEC line (Fig. 1C) may have delayed phosphorylation of p27Kip1, in turn delaying its phosphorylation-dependent degradation. The binding of the JAB-1 gene product to p27Kip1 causes p27Kip1degradation (57Tomoda K. Kubota Y. Kato J-Y. Nature. 1999; 398: 160-164Crossref PubMed Scopus (550) Google Scholar). An alternate mechanism may be that JAB-1 is a downstream target of PKCδ, with phosphorylation resulting in functional inactivation of JAB-1.The mechanism by which p27Kip1 inhibits cell cycle progression may vary with the cell type, although our studies are consistent with the model in which p27Kip1 inhibits cell cycle progression in part by binding to Cdk2 and thereby reducing cyclin E/Cdk2 kinase activity (32Toyoshima H. Hunter T. Cell. 1994; 78: 67-74Abstract Full Text PDF PubMed Scopus (1927) Google Scholar). Increased p27Kip1levels induced by overexpression in VSMCs was associated with reduced Cdk2 activity (45Chen D. Krasinski K. Chen D. Sylvester A. Chen J. Nisen P.D. Andres V. J. Clin. Invest. 1997; 99: 2334-2341Crossref PubMed Scopus (174) Google Scholar). Consistent with our findings overexpression of p27Kip1 in the nuclear, rather than the cytoplasmic, compartment was required for the cell cycle arrest (58Reynisdottir I. Massague J. Genes & Dev. 1997; 11: 492-503Crossref PubMed Scopus (307) Google Scholar), indicating that the subcellular distribution of p27Kip1 is important in the inhibition of cellular proliferation. In recent studies an alternate mechanism of p27Kip1 action was proposed. In LAP-3 cells, derived from NIH3T3 cells, p27Kip1overexpression induced a cell cycle arrest associated with a specific E2F pocket protein complex. One consequence of the p27Kip1-Cdk2 association was disruption of the interaction between Cdk2 and both the E2F-p130 and the E2F-p107 repressor complexes (59Shiyanov P. Hayes S. Chen N. Pestov D.G. Lau L.F. Raychaudhuri P. Mol. Biol. Cell. 1997; 8: 1815-1827Crossref PubMed Scopus (17) Google Scholar). The p130/p107 complexes that were induced by p27Kip1were similar to the complexes induced by serum starvation (59Shiyanov P. Hayes S. Chen N. Pestov D.G. Lau L.F. Raychaudhuri P. Mol. Biol. Cell. 1997; 8: 1815-1827Crossref PubMed Scopus (17) Google Scholar). Further studies will be directed at analyzing the effect of p27Kip1in the presence of the PKCδEC on components of the E2F-130 and E2F-p107 complexes. The present studies indicate, however, that alterations in cyclin E protein abundance do not appear to be important in the cell cycle effects mediated by p27Kip1, arguing against an indirect effect of p27Kip1 on E2F-p130/p107 complex activity.The results presented here are consistent with recent studies in which PKC isozymes were implicated in the inhibition of cellular proliferation and cell cycle progression. PKC has been shown to inhibit cell cycle progression in intestinal epithelial cells (60Frey M.R. Saxon M.L. Zhao X. Rollins A. Evans S.S. Black J.D. J. Biol. Chem. 1997; 272: 9424-9435Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), IMR-90 fibroblasts (61Hamada K. Takuwa N. Zhou W. Kumada M. Takuwa Y. Biochim. Biophys. Acta. 1996; 1310: 149-156Crossref PubMed Scopus (29) Google Scholar), melanoma cells (62Coppock D.L. Tansey J.B. Nathanson L. Cell. Growth & Differ. 1993; 3: 485-494Google Scholar), and vascular endothelial cells (63Zhou W. Takuwa N. Kumada M. Takuwa Y. J. Biol. Chem. 1993; 268: 23041-23048Abstract Full Text PDF PubMed Google Scholar, 64Kosaka C. Sasaguri T. Ishida A. Ogata J. Am. J. Physiol. 1996; 39: C170-C178Crossref Google Scholar). Both the PKCα and PKCδ isozymes have been implicated in the inhibition of cellular proliferation in different cell types. Overexpression of PKCα inhibited cell cycle progression in Chinese hamster ovary cells (65Yamaguchi K. Ogita K. Nakamura S. Nishizuka Y. Biochem. Biophys. Res. Commun. 1995; 210: 639-647Crossref PubMed Scopus (56) Google Scholar), B16 melanoma cells (66Gruber J.T. Ohno S. Niles R.M. J. Biol. Chem. 1992; 267: 13356-13360Abstract Full Text PDF PubMed Google Scholar), and F9 teratocarcinoma cells (67Kindregan H.C. Rosenbaum S.E. Ohno S. Niles R.M. J. Biol. Chem. 1994; 269: 27756-27761Abstract Full Text PDF PubMed Google Scholar). In rat microvascular capillary endothelial cells (EC), PKCα does not inhibit cell cycle progression but rather promotes migration of the endothelial cells in response to growth factors (1Harrington E.O. Loffler J. Nelson P.R. Kent K.C. Simons M. Ware J.A. J. Biol. Chem. 1997; 272: 7390-7397Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The studies suggest the cell cycle regulatory effect of the PKC isozymes may be cell type specific. PKCδ is the only isoform to undergo tyrosine phosphorylation (68Gschwendt M. Keilbassa K. Kittstein W. Marks F. FEBS Lett. 1994; 347: 85-89Crossref PubMed Scopus (98) Google Scholar), and PKCδ was inactivated by tyrosine phosphorylation in v-Src (21Zang Q. Lu Z. Curto M. Barile N. Shalloway D. Foster D.A. J. Biol. Chem. 1997; 272: 13275-13280Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) and v-Ras (22Denning M.F. Dlugosz A.A. Howett M.K. Yuspa S.H. J. Biol. Chem. 1993; 268: 26079-26081Abstract Full Text PDF PubMed Google Scholar) transformed cells, raising the possibility that inactivation of PKCδ may promote unregulated cellular proliferation and transformation. In view of the current findings that p27Kip1 is required for the cell cycle inhibitory function of PKCδ and the prior observations that PKCδ inactivation may play an important role in oncogene induced transformation, future studies are warranted to examine the role of p27Kip1 in oncogene/PKC-induced transformation. The vascular endothelium is a dynamic organ controlling hemostasis, vasodilation, and wound healing. The endothelium is influenced by shear stress, hypoxia, and chemotactic/mitogenic gradients that promote migration and division of its cells. Endothelial cellular division is an important component of the angiogenic response to many stimuli (2Yang E.Y. Moses H.L. J. Cell Biol. 1990; 111: 731-741Crossref PubMed Scopus (420) Google Scholar, 3Ausprunk D.H. Folkman J. Microvasc. Res. 1977; 14: 53-65Crossref PubMed Scopus (1047) Google Scholar, 4Yancopoulos G.D. Klagsbrun M. Folkman J. Cell. 1998; 93: 661-664Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). As several different external agents promote or inhibit endothelial cell proliferation, the secondary messengers mediating these responses are being actively investigated. The protein kinase C (PKC) 1The abbreviations used are: PKC, protein kinase C; CDK, cyclin-dependent kinase; CKI, CDK inhibitor; EC, endothelial cell(s); V-EC, vector EC (cell line); PBS, phosphate-buffered saline; HEPES, 4(2-hydroxyethyl)-1-piperazineethanesulfonic acid; GST, glutathioneS-transferase; MACS, magnetic activated cell separation system; pRB, retinoblastoma protein; JAB, JAK-binding protein1The abbreviations used are: PKC, protein kinase C; CDK, cyclin-dependent kinase; CKI, CDK inhibitor; EC, endothelial cell(s); V-EC, vector EC (cell line); PBS, phosphate-buffered saline; HEPES, 4(2-hydroxyethyl)-1-piperazineethanesulfonic acid; GST, glutathioneS-transferase; MACS, magnetic activated cell separation system; pRB, retinoblastoma protein; JAB, JAK-binding protein family of Ser-Thr kinases is a common intracellular signaling pathway that coordinates a diverse array of signals that arise in the extracellular environment. Activation of the PKC pathway by phorbol esters, for example, induces endothelial cell proliferation and angiogenesisin vivo (5Montesano R. Orci L. Cell. 1985; 42: 469-477Abstract Full Text PDF PubMed Scopus (370) Google Scholar, 6Hu D.E. Fan T.P. Inflammation. 1995; 19: 39-54Crossref PubMed Scopus (26) Google Scholar, 7Wright P.S. Cross-Doersen" @default.
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