CDK Inhibitors: Cell Cycle Regulators and Beyond
2008; Elsevier BV; Volume: 14; Issue: 2 Linguagem: Inglês
10.1016/j.devcel.2008.01.013
ISSN1878-1551
AutoresArnaud Besson, Steven F. Dowdy, James M. Roberts,
Tópico(s)Cancer, Hypoxia, and Metabolism
ResumoFirst identified as cell cycle inhibitors mediating the growth inhibitory cues of upstream signaling pathways, the cyclin-CDK inhibitors of the Cip/Kip family p21Cip1, p27Kip1, and p57Kip2 have emerged as multifaceted proteins with functions beyond cell cycle regulation. In addition to regulating the cell cycle, Cip/Kip proteins play important roles in apoptosis, transcriptional regulation, cell fate determination, cell migration and cytoskeletal dynamics. A complex phosphorylation network modulates Cip/Kip protein functions by altering their subcellular localization, protein-protein interactions, and stability. These functions are essential for the maintenance of normal cell and tissue homeostasis, in processes ranging from embryonic development to tumor suppression. First identified as cell cycle inhibitors mediating the growth inhibitory cues of upstream signaling pathways, the cyclin-CDK inhibitors of the Cip/Kip family p21Cip1, p27Kip1, and p57Kip2 have emerged as multifaceted proteins with functions beyond cell cycle regulation. In addition to regulating the cell cycle, Cip/Kip proteins play important roles in apoptosis, transcriptional regulation, cell fate determination, cell migration and cytoskeletal dynamics. A complex phosphorylation network modulates Cip/Kip protein functions by altering their subcellular localization, protein-protein interactions, and stability. These functions are essential for the maintenance of normal cell and tissue homeostasis, in processes ranging from embryonic development to tumor suppression. Progression through the cell-division cycle is regulated by the coordinated activities of cyclin/cyclin-dependent kinases (CDK) complexes. One level of regulation of these cyclin-CDK complexes is provided by their binding to CDK inhibitors (CKIs). In metazoans, two CKI gene families have been defined based on their evolutionary origins, structure, and CDK specificities. The INK4 gene family encodes p16INK4a, p15INK4b, p18INK4c, and p19INK4d, all of which bind to CDK4 and CDK6 and inhibit their kinase activities by interfering with their association with D-type cyclins (Sherr and Roberts, 1999Sherr C.J. Roberts J.M. CDK inhibitors: positive and negative regulators of G1-phase progression.Genes Dev. 1999; 13: 1501-1512Crossref PubMed Google Scholar). In contrast, CKIs of the Cip/Kip family bind to both cyclin and CDK subunits and can modulate the activities of cyclin D-, E-, A-, and B-CDK complexes (Sherr and Roberts, 1999Sherr C.J. Roberts J.M. CDK inhibitors: positive and negative regulators of G1-phase progression.Genes Dev. 1999; 13: 1501-1512Crossref PubMed Google Scholar). The Cip/Kip family members p21Cip1/Waf1/Sdi1 (p21, encoded by cdkn1a) (el-Deiry et al., 1993el-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. WAF1, a potential mediator of p53 tumor suppression.Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (6115) Google Scholar, Gu et al., 1993Gu Y. Turck C.W. Morgan D.O. Inhibition of CDK2 activity in vivo by an associated 20K regulatory subunit.Nature. 1993; 366: 707-710Crossref PubMed Scopus (527) Google Scholar, Harper et al., 1993Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases.Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (3942) Google Scholar, Xiong et al., 1993Xiong Y. Hannon G.J. Zhang H. Casso D. Kobayashi R. Beach D. p21 is a universal inhibitor of cyclin kinases.Nature. 1993; 366: 701-704Crossref PubMed Google Scholar), p27Kip1 (p27, cdkn1b) (Polyak et al., 1994aPolyak K. Kato J. Solomon M.J. Sherr C.J. Massague J. Roberts J.M. Koff A. p27Kip1, a cyclin-CDK inhibitor, links transforming growth factor β and contact inhibition to cell cycle arrest.Genes Dev. 1994; 8: 9-22Crossref PubMed Google Scholar, Polyak et al., 1994bPolyak K. Lee M.H. Erdjument-Bromage H. Koff A. Roberts J.M. Tempst P. Massague J. Cloning of p27Kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals.Cell. 1994; 78: 59-66Abstract Full Text PDF PubMed Google Scholar, Toyoshima and Hunter, 1994Toyoshima H. Hunter T. p27, a novel inhibitor of G1 cyclin-CDK protein kinase activity, is related to p21.Cell. 1994; 78: 67-74Abstract Full Text PDF PubMed Scopus (1539) Google Scholar), and p57Kip2 (p57, cdkn1c) (Lee et al., 1995Lee M.H. Reynisdottir I. Massague J. Cloning of p57KIP2, a cyclin-dependent kinase inhibitor with unique domain structure and tissue distribution.Genes Dev. 1995; 9: 639-649Crossref PubMed Google Scholar, Matsuoka et al., 1995Matsuoka S. Edwards M.C. Bai C. Parker S. Zhang P. Baldini A. Harper J.W. Elledge S.J. p57KIP2, a structurally distinct member of the p21CIP1 Cdk inhibitor family, is a candidate tumor suppressor gene.Genes Dev. 1995; 9: 650-662Crossref PubMed Google Scholar) share a conserved N-terminal domain that mediates binding to cyclins and CDKs but diverge in the remainder of their sequence, suggesting that each of these proteins could have distinct functions and regulation. A vast body of literature has described the importance of p21, p27, and p57 in restraining proliferation during development, differentiation, and response to cellular stresses (Sherr and Roberts, 1999Sherr C.J. Roberts J.M. CDK inhibitors: positive and negative regulators of G1-phase progression.Genes Dev. 1999; 13: 1501-1512Crossref PubMed Google Scholar), although each has specific biological functions that distinguish it from the other family members. Thus, different antiproliferative signals tend to cause elevated expression of only a subset of the Cip/Kip proteins. For example, p21 is an important transcriptional target of p53 and mediates DNA-damage-induced cell-cycle arrest in G1 and G2 (el-Deiry et al., 1993el-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. WAF1, a potential mediator of p53 tumor suppression.Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (6115) Google Scholar, Gartel and Tyner, 1999Gartel A.L. Tyner A.L. Transcriptional regulation of the p21((WAF1/CIP1)) gene.Exp. Cell Res. 1999; 246: 280-289Crossref PubMed Scopus (453) Google Scholar). In contrast to p21, p27 expression is usually elevated in mitogen-starved cells and other quiescent states, and the protein is rapidly downregulated as cells enter the cell cycle (Besson et al., 2006Besson A. Gurian-West M. Chen X. Kelly-Spratt K.S. Kemp C.J. Roberts J.M. A pathway in quiescent cells that controls p27Kip1 stability, subcellular localization, and tumor suppression.Genes Dev. 2006; 20: 47-64Crossref PubMed Scopus (107) Google Scholar, Coats et al., 1996Coats S. Flanagan W.M. Nourse J. Roberts J.M. Requirements of p27Kip1 for restriction point control of the fibroblast cell cycle.Science. 1996; 272: 877-880Crossref PubMed Google Scholar). Several lines of evidence point toward an important role for p57 in the regulation of the cell cycle during embryonic development. Unlike its ubiquitous siblings, p57 has a tissue-restricted expression pattern during embryogenesis and in the adult (Lee et al., 1995Lee M.H. Reynisdottir I. Massague J. Cloning of p57KIP2, a cyclin-dependent kinase inhibitor with unique domain structure and tissue distribution.Genes Dev. 1995; 9: 639-649Crossref PubMed Google Scholar, Matsuoka et al., 1995Matsuoka S. Edwards M.C. Bai C. Parker S. Zhang P. Baldini A. Harper J.W. Elledge S.J. p57KIP2, a structurally distinct member of the p21CIP1 Cdk inhibitor family, is a candidate tumor suppressor gene.Genes Dev. 1995; 9: 650-662Crossref PubMed Google Scholar). The transcriptional regulation of p57 is mediated by factors that play critical roles during embryogenesis such as Notch/Hes1, MyoD, BMP-2 and -6, and p73 (Blint et al., 2002Blint E. Phillips A.C. Kozlov S. Stewart C.L. Vousden K.H. Induction of p57(KIP2) expression by p73β.Proc. Natl. Acad. Sci. USA. 2002; 99: 3529-3534Crossref PubMed Scopus (76) Google Scholar, Georgia et al., 2006Georgia S. Soliz R. Li M. Zhang P. Bhushan A. p57 and Hes1 coordinate cell cycle exit with self-renewal of pancreatic progenitors.Dev. Biol. 2006; 298: 22-31Crossref PubMed Scopus (89) Google Scholar, Gosselet et al., 2007Gosselet F.P. Magnaldo T. Culerrier R.M. Sarasin A. Ehrhart J.C. BMP2 and BMP6 control p57(Kip2) expression and cell growth arrest/terminal differentiation in normal primary human epidermal keratinocytes.Cell. Signal. 2007; 19: 731-739Crossref PubMed Scopus (28) Google Scholar, Vaccarello et al., 2006Vaccarello G. Figliola R. Cramerotti S. Novelli F. Maione R. p57Kip2 is induced by MyoD through a p73-dependent pathway.J. Mol. Biol. 2006; 356: 578-588Crossref PubMed Scopus (23) Google Scholar). The cdkn1c gene is also an imprinted gene with preferred expression of the maternal allele (Matsuoka et al., 1996Matsuoka S. Thompson J.S. Edwards M.C. Bartletta J.M. Grundy P. Kalikin L.M. Harper J.W. Elledge S.J. Feinberg A.P. Imprinting of the gene encoding a human cyclin-dependent kinase inhibitor, p57KIP2, on chromosome 11p15.Proc. Natl. Acad. Sci. USA. 1996; 93: 3026-3030Crossref PubMed Google Scholar), which is recognized as a general mechanism to regulate embryonic growth (Andrews et al., 2007Andrews S.C. Wood M.D. Tunster S.J. Barton S.C. Surani M.A. John R.M. Cdkn1c (p57Kip2) is the major regulator of embryonic growth within its imprinted domain on mouse distal chromosome 7.BMC Dev. Biol. 2007; 7: 53Crossref PubMed Scopus (37) Google Scholar). Importantly, p57 is the only CKI to be required for embryonic development, as most mice lacking the cdkn1c gene have multiple developmental abnormalities and die at birth (Yan et al., 1997Yan Y. Frisen J. Lee M.H. Massague J. Barbacid M. Ablation of the CDK inhibitor p57Kip2 results in increased apoptosis and delayed differentiation during mouse development.Genes Dev. 1997; 11: 973-983Crossref PubMed Google Scholar, Zhang et al., 1997Zhang P. Liegeois N.J. Wong C. Finegold M. Hou H. Thompson J.C. Silverman A. Harper J.W. DePinho R.A. Elledge S.J. Altered cell differentiation and proliferation in mice lacking p57KIP2 indicates a role in Beckwith-Wiedemann syndrome.Nature. 1997; 387: 151-158Crossref PubMed Scopus (532) Google Scholar). The importance of the Cip/Kip proteins in cell-cycle regulation is underscored by the phenotypes of the knockout mice for each of these proteins. p27 null mice display an overall increased body size and multiple organ hyperplasia, revealing the importance of p27 in limiting growth (Fero et al., 1996Fero M.L. Rivkin M. Tasch M. Porter P. Carow C.E. Firpo E. Polyak K. Tsai L.H. Broudy V. Perlmutter R.M. et al.A syndrome of multiorgan hyperplasia with features of gigantism, tumorigenesis, and female sterility in p27(Kip1)-deficient mice.Cell. 1996; 85: 733-744Abstract Full Text Full Text PDF PubMed Scopus (1115) Google Scholar). Although mice lacking p21 do not display an overt hyperproliferative disorder, p21−/− cells fail to undergo DNA-damage-induced cell-cycle arrest and can reach higher saturation density (Deng et al., 1995Deng C. Zhang P. Harper J.W. Elledge S.J. Leder P. Mice lacking p21CIP1/WAF1 undergo normal development, but are defective in G1 checkpoint control.Cell. 1995; 82: 675-684Abstract Full Text PDF PubMed Google Scholar). Embryos lacking p57 exhibit hyperplasia in several organs and delayed differentiation, probably due to failure to exit the cell cycle in a timely fashion (Zhang et al., 1997Zhang P. Liegeois N.J. Wong C. Finegold M. Hou H. Thompson J.C. Silverman A. Harper J.W. DePinho R.A. Elledge S.J. Altered cell differentiation and proliferation in mice lacking p57KIP2 indicates a role in Beckwith-Wiedemann syndrome.Nature. 1997; 387: 151-158Crossref PubMed Scopus (532) Google Scholar). Although p21, p27, and p57 were initially considered as tumor suppressors based on their ability to block cell proliferation, it rapidly became clear that the situation was not so simple. p21, p27, and p57 are also involved in the regulation of cellular processes beyond cell-cycle regulation, including transcription, apoptosis, and migration, which may be oncogenic under certain circumstances. Moreover, it appears that the loss or subversion of the regulatory mechanisms governing Cip/Kip proteins may lead to the specific loss of the tumor suppressor function of the CKI while maintaining the oncogenic ones. Herein, we will examine these various functions, how they are regulated, and their significance in vivo, especially in the context of tumorigenesis. Cip/Kip proteins were initially characterized as strict inhibitors of all cyclin-CDK complexes, albeit displaying lower affinity toward cyclin B-CDK1 (Sherr and Roberts, 1999Sherr C.J. Roberts J.M. CDK inhibitors: positive and negative regulators of G1-phase progression.Genes Dev. 1999; 13: 1501-1512Crossref PubMed Google Scholar). The crystal structure of the N-terminal cyclin and CDK-binding domains of p27 (aa 22–106) bound to cyclin A-CDK2 revealed that the CKI occludes a substrate interaction domain on the cyclin subunit and inserts itself in the catalytic cleft of the CDK, thereby preventing ATP binding and catalytic activity (Russo et al., 1996Russo A.A. Jeffrey P.D. Patten A.K. Massague J. Pavletich N.P. Crystal structure of the p27Kip1 cyclin dependent kinase inhibitor bound to the cyclin A-Cdk2 complex.Nature. 1996; 382: 325-331Crossref PubMed Scopus (620) Google Scholar). However, subsequent studies reported that p21, p27, and p57 participated in the assembly of catalytically active cyclin D-CDK4/6 complexes (LaBaer et al., 1997LaBaer J. Garrett M.D. Stevenson L.F. Slingerland J.M. Sandhu C. Chou H.S. Fattaey A. Harlow E. New functional activities for the p21 family of CDK inhibitors.Genes Dev. 1997; 11: 847-862Crossref PubMed Google Scholar). Therefore, not only could Cip/Kip proteins promote cyclin D-dependent events, but the sequestration of CKIs into cyclin D-CDK4/6 complexes could allow the downstream activation of cyclin E-CDK2 (Cheng et al., 1998Cheng M. Sexl V. Sherr C.J. Roussel M.F. Assembly of cyclin D-dependent kinase and titration of p27Kip1 regulated by mitogen-activated protein kinase kinase (MEK1).Proc. Natl. Acad. Sci. USA. 1998; 95: 1091-1096Crossref PubMed Scopus (407) Google Scholar, Perez-Roger et al., 1999Perez-Roger I. Kim S.H. Griffiths B. Sewing A. Land H. Cyclins D1 and D2 mediate myc-induced proliferation via sequestration of p27(Kip1) and p21(Cip1).EMBO J. 1999; 18: 5310-5320Crossref PubMed Scopus (229) Google Scholar, Polyak et al., 1994aPolyak K. Kato J. Solomon M.J. Sherr C.J. Massague J. Roberts J.M. Koff A. p27Kip1, a cyclin-CDK inhibitor, links transforming growth factor β and contact inhibition to cell cycle arrest.Genes Dev. 1994; 8: 9-22Crossref PubMed Google Scholar, Reynisdottir et al., 1995Reynisdottir I. Polyak K. Iavarone A. Massague J. Kip/Cip and Ink4 Cdk inhibitors cooperate to induce cell cycle arrest in response to TGF-beta.Genes Dev. 1995; 9: 1831-1845Crossref PubMed Google Scholar). The latter is confirmed in cyclin D1 and D2 knockout mice, which display reduced CDK2-associated kinase activity, likely due to the increased availability of Cip/Kip proteins to bind to CDK2 (Geng et al., 2001Geng Y. Yu Q. Sicinska E. Das M. Bronson R.T. Sicinski P. Deletion of the p27Kip1 gene restores normal development in cyclin D1-deficient mice.Proc. Natl. Acad. Sci. USA. 2001; 98: 194-199Crossref PubMed Google Scholar, Perez-Roger et al., 1999Perez-Roger I. Kim S.H. Griffiths B. Sewing A. Land H. Cyclins D1 and D2 mediate myc-induced proliferation via sequestration of p27(Kip1) and p21(Cip1).EMBO J. 1999; 18: 5310-5320Crossref PubMed Scopus (229) Google Scholar). Nevertheless, there are other reports demonstrating inhibition of cyclin D-CDK4/6 complexes by Cip/Kip proteins, and therefore it was anticipated that the effect of these CKIs on CDK activity would be modulated by other factors. The Cip/Kip proteins are intrinsically unstructured, adopting specific tertiary conformations only after binding to other proteins (Adkins and Lumb, 2002Adkins J.N. Lumb K.J. Intrinsic structural disorder and sequence features of the cell cycle inhibitor p57Kip2.Proteins. 2002; 46: 1-7Crossref PubMed Scopus (44) Google Scholar, Esteve et al., 2003Esteve V. Canela N. Rodriguez-Vilarrupla A. Aligue R. Agell N. Mingarro I. Bachs O. Perez-Paya E. The structural plasticity of the C terminus of p21Cip1 is a determinant for target protein recognition.ChemBioChem. 2003; 4: 863-869Crossref PubMed Scopus (15) Google Scholar, Lacy et al., 2004Lacy E.R. Filippov I. Lewis W.S. Otieno S. Xiao L. Weiss S. Hengst L. Kriwacki R.W. p27 binds cyclin-CDK complexes through a sequential mechanism involving binding-induced protein folding.Nat. Struct. Mol. Biol. 2004; 11: 358-364Crossref PubMed Scopus (164) Google Scholar). This conformational flexibility suggests that phosphorylation events and protein-protein interactions may modify the folding of the CKIs, thereby modulating their ability to inhibit cyclin-CDK complexes. Likewise, it may explain why CKIs are capable of interacting with a wide diversity of proteins to regulate various cellular functions. Indeed, it appears that the binding specificity of Cip/Kip proteins is modulated by their phosphorylation on distinct residues, and their potency to inhibit cyclin-CDK complexes can be modified by binding to other proteins. For example, phosphorylation of p21 on Thr-57 (by CDK2 or glycogen synthase kinase 3β [GSK3β]) increases the ability of p21 to bind to cyclin B1-CDK1 complexes at the G2/M transition, without inhibiting the complexes, thus promoting cell-cycle progression (Dash and El-Deiry, 2005Dash B.C. El-Deiry W.S. Phosphorylation of p21 in G2/M promotes cyclin B-Cdc2 kinase activity.Mol. Cell. Biol. 2005; 25: 3364-3387Crossref PubMed Scopus (60) Google Scholar). Likewise, the phosphorylation of p27 on Tyr-74, -88 and/or -89 by Src, Lyn, or Abl, greatly decreased the ability of p27 to inhibit CDK2 containing complexes, as Tyr-88 is part of the 310-helix that normally inserts into the ATP-binding site of the CDK (Chu et al., 2007Chu I. Sun J. Arnaout A. Kahn H. Hanna W. Narod S. Sun P. Tan C.K. Hengst L. Slingerland J. p27 phosphorylation by Src regulates inhibition of cyclin E-Cdk2.Cell. 2007; 128: 281-294Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, Grimmler et al., 2007Grimmler M. Wang Y. Mund T. Cilensek Z. Keidel E.M. Waddell M.B. Jakel H. Kullmann M. Kriwacki R.W. Hengst L. Cdk-inhibitory activity and stability of p27Kip1 are directly regulated by oncogenic tyrosine kinases.Cell. 2007; 128: 269-280Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). In addition, p27 phosphorylation on Tyr-88 and -89 was reported to decrease its affinity for CDK2 while increasing that for CDK4 complexes (Kardinal et al., 2006Kardinal C. Dangers M. Kardinal A. Koch A. Brandt D.T. Tamura T. Welte K. Tyrosine phosphorylation modulates binding preference to cyclin-dependent kinases and subcellular localization of p27Kip1 in the acute promyelocytic leukemia cell line NB4.Blood. 2006; 107: 1133-1140Crossref PubMed Scopus (25) Google Scholar). A recent report suggested that Tyr-88 phosphorylation was cell-cycle regulated and modulated the ability of p27 to inhibit cyclin D-CDK4 complexes (James et al., 2008James M.K. Ray A. Leznova D. Blain S.W. Differential modification of p27Kip1 controls its cyclin D-cdk4 inhibitory activity.Mol. Cell. Biol. 2008; 28 (Published online October 1, 2007): 498-510https://doi.org/10.1128/MCB.02171-06Crossref PubMed Scopus (51) Google Scholar). p27 was a potent cyclin D-CDK4 inhibitor in quiescent cells, but not in cycling cells, in which it was tyrosine phosphorylated. Moreover, the weak inhibitory form of p27, phosphorylated on Tyr-88, could be converted to a potent inhibitor by treatment with protein tyrosine phosphatase (PTP) (James et al., 2008James M.K. Ray A. Leznova D. Blain S.W. Differential modification of p27Kip1 controls its cyclin D-cdk4 inhibitory activity.Mol. Cell. Biol. 2008; 28 (Published online October 1, 2007): 498-510https://doi.org/10.1128/MCB.02171-06Crossref PubMed Scopus (51) Google Scholar). However, the physiological significance of this regulatory pathway of p27 in vivo remains to be investigated. Several other phospho-sites on p21 and p27 also indirectly affect the ability of these proteins to bind to and inhibit cyclin-CDK complexes by controlling their subcellular localization (see below) (Borriello et al., 2007Borriello A. Cucciolla V. Oliva A. Zappia V. Della Ragione F. p27Kip1 metabolism: a fascinating labyrinth.Cell Cycle. 2007; 6: 1053-1061Crossref PubMed Google Scholar, Child and Mann, 2006Child E.S. Mann D.J. The intricacies of p21 phosphorylation: protein/protein interactions, subcellular localization and stability.Cell Cycle. 2006; 5: 1313-1319Crossref PubMed Google Scholar). A number of proteins can either enhance or diminish the inhibitory effect of CKIs on cyclin-CDK complexes by forming quaternary complexes and potentially altering the conformation of the CKI bound to these complexes. Human papillomavirus-16 E7 protein can bind to p21 and p27 and abrogate their inhibitory activity toward CDK2-containing complexes (Funk et al., 1997Funk J.O. Waga S. Harry J.B. Espling E. Stillman B. Galloway D.A. Inhibition of CDK activity and PCNA-dependent DNA replication by p21 is blocked by interaction with the HPV-16 E7 oncoprotein.Genes Dev. 1997; 11: 2090-2100Crossref PubMed Google Scholar, Jones et al., 1997Jones D.L. Alani R.M. Munger K. The human papillomavirus E7 oncoprotein can uncouple cellular differentiation and proliferation in human keratinocytes by abrogating p21Cip1-mediated inhibition of cdk2.Genes Dev. 1997; 11: 2101-2111Crossref PubMed Google Scholar). The nuclear protein Set/TAF1 (Template-activating factor-1) was found to associate with the C-terminal part of p21, which reversed the inhibition of cyclin E-CDK2 and enhanced the inhibition of cyclin B-CDK1 complexes (Canela et al., 2003Canela N. Rodriguez-Vilarrupla A. Estanyol J.M. Diaz C. Pujol M.J. Agell N. Bachs O. The SET protein regulates G2/M transition by modulating cyclin B-cyclin-dependent kinase 1 activity.J. Biol. Chem. 2003; 278: 1158-1164Crossref PubMed Scopus (77) Google Scholar, Estanyol et al., 1999Estanyol J.M. Jaumot M. Casanovas O. Rodriguez-Vilarrupla A. Agell N. Bachs O. The protein SET regulates the inhibitory effect of p21(Cip1) on cyclin E-cyclin-dependent kinase 2 activity.J. Biol. Chem. 1999; 274: 33161-33165Crossref PubMed Scopus (63) Google Scholar). Two other proteins, TOK1α (p21 and CDK-associated protein-1) (Ono et al., 2000Ono T. Kitaura H. Ugai H. Murata T. Yokoyama K.K. Iguchi-Ariga S.M. Ariga H. TOK-1, a novel p21Cip1-binding protein that cooperatively enhances p21-dependent inhibitory activity toward CDK2 kinase.J. Biol. Chem. 2000; 275: 31145-31154Crossref PubMed Scopus (69) Google Scholar) and the multifunctional domain protein TSG101 (Tumor susceptibility gene-101) (Oh et al., 2002Oh H. Mammucari C. Nenci A. Cabodi S. Cohen S.N. Dotto G.P. Negative regulation of cell growth and differentiation by TSG101 through association with p21(Cip1/WAF1).Proc. Natl. Acad. Sci. USA. 2002; 99: 5430-5435Crossref PubMed Scopus (40) Google Scholar), can also associate with p21 to enhance cyclin-CDK inhibition. Further studies are warranted to elucidate how the binding of these proteins may alter the conformation of the CKI bound to cyclin-CDK complexes and to determine how significant these interactions are in the control of p21's function. The general conclusion is that Cip/Kip proteins are potent inhibitors of cyclin-CDK complexes, although most of the findings in recent years have shown that their inhibitory potential is dependent on cellular context and regulated via phosphorylations and protein-protein interactions. Cip/Kip proteins also modulate cell-cycle progression independently of cyclins and CDKs via the inhibition of components of the replication machinery. p21 was first reported to bind to proliferating cell nuclear antigen (PCNA), a DNA polymerase δ processivity factor, via its C terminus (aa 143–160), thereby blocking processive DNA synthesis (Luo et al., 1995Luo Y. Hurwitz J. Massague J. Cell-cycle inhibition by independent CDK and PCNA binding domains in p21Cip1.Nature. 1995; 375: 159-161Crossref PubMed Google Scholar). This function of p21 is modulated by phosphorylations on Ser-145 (by PKB/Akt [protein kinase B] and possibly PKA), Ser-146 (by PKC ζ), or Ser-160 (by PKC), which prevent p21 from binding to PCNA (Child and Mann, 2006Child E.S. Mann D.J. The intricacies of p21 phosphorylation: protein/protein interactions, subcellular localization and stability.Cell Cycle. 2006; 5: 1313-1319Crossref PubMed Google Scholar). p57 was subsequently found to interact with PCNA (via aa 271–275), preventing its activity and blocking DNA replication (Watanabe et al., 1998Watanabe H. Pan Z.Q. Schreiber-Agus N. DePinho R.A. Hurwitz J. Xiong Y. Suppression of cell transformation by the cyclin-dependent kinase inhibitor p57KIP2 requires binding to proliferating cell nuclear antigen.Proc. Natl. Acad. Sci. USA. 1998; 95: 1392-1397Crossref PubMed Scopus (120) Google Scholar). Although no interaction between p27 and PCNA has been reported, p27 may also inhibit DNA synthesis via the interaction and inhibition of minichromosome maintenance-7 (MCM7), a subunit of the MCM2-7 replication fork helicase, an activity that lies within the C-terminal part of p27 (aa 144–198) (Nallamshetty et al., 2005Nallamshetty S. Crook M. Boehm M. Yoshimoto T. Olive M. Nabel E.G. The cell cycle regulator p27Kip1 interacts with MCM7, a DNA replication licensing factor, to inhibit initiation of DNA replication.FEBS Lett. 2005; 579: 6529-6536Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Phosphorylation on various amino acids controls many aspects of Cip/Kip protein biology, not only by altering the Cip/Kip proteins' affinity for specific cyclin-CDK complexes and other proteins, but also their stability (reviewed in Borriello et al., 2007Borriello A. Cucciolla V. Oliva A. Zappia V. Della Ragione F. p27Kip1 metabolism: a fascinating labyrinth.Cell Cycle. 2007; 6: 1053-1061Crossref PubMed Google Scholar, Child and Mann, 2006Child E.S. Mann D.J. The intricacies of p21 phosphorylation: protein/protein interactions, subcellular localization and stability.Cell Cycle. 2006; 5: 1313-1319Crossref PubMed Google Scholar), and their subcellular localization. Phosphorylation of p21 on two sites, Thr-145 and Ser-153, by PKB/Akt and PKC, respectively, promote the cytoplasmic retention of p21 (Child and Mann, 2006Child E.S. Mann D.J. The intricacies of p21 phosphorylation: protein/protein interactions, subcellular localization and stability.Cell Cycle. 2006; 5: 1313-1319Crossref PubMed Google Scholar, Rodriguez-Vilarrupla et al., 2005Rodriguez-Vilarrupla A. Jaumot M. Abella N. Canela N. Brun S. Diaz C. Estanyol J.M. Bachs O. Agell N. Binding of calmodulin to the carboxy-terminal region of p21 induces nuclear accumulation via inhibition of protein kinase C-mediated phosphorylation of Ser153.Mol. Cell. Biol. 2005; 25: 7364-7374Crossref PubMed Scopus (26) Google Scholar, Zhou et al., 2001Zhou B.P. Liao Y. Xia W. Spohn B. Lee M.H. Hung M.C. Cytoplasmic localization of p21Cip1/Waf1 by Akt-induced phosphorylation in HER-2/neu-overexpressing cells.Nat. Cell Biol. 2001; 3: 245-252Crossref PubMed Scopus (651) Google Scholar). The first site is proximal to p21's nuclear localization sequence (NLS) and prevents the interaction with importin when phosphorylated, and the second one occludes a binding site for calmodulin, thus blocking calmodulin-mediated nuclear import (Child and Mann, 2006Child E.S. Mann D.J. The intricacies of p21 phosphorylation: protein/protein interactions, subcellular localization and stability.Cell Cycle. 2006; 5: 1313-1319Crossref PubMed Google Scholar, Rodriguez-Vilarrupla et al., 2005Rodriguez-Vilarrupla A. Jaumot M. Abella N. Canela N. Brun S. Diaz C. Estanyol J.M. Bachs O. Agell N. Binding of calmodulin to the carboxy-terminal region of p21 induces nuclear accumulation via inhibition of protein kinase C-mediated phosphorylation of Ser153.Mol. Cell. Biol. 2005; 25: 7364-7374Crossref PubMed Scopus (26) Google Scholar, Zhou et al., 2001Zhou B.P. Liao Y. Xia W. Spohn B. Lee M.H. Hung M.C. Cytoplasmic localization of p21Cip1/Waf1 by Akt-induced phosphorylation in HER-2/neu-overexpressing cells.Nat. Cell Biol. 2001; 3: 245-252Crossref PubMed Scopus (651) Google Scholar). Three phosphorylation sites on p27 were found to cause cytoplasmic localization. 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Meloche S. p27 cytoplasmic localization is regulated by phosphorylation on Ser 10 and is not a prerequisite for its proteolysis.EMBO J. 2001; 20: 6672-6682Crossref PubMed Scopus (204) Google Scholar). In quiescent cells, Mirk/Dirk kinase has been proposed to phosphorylate this site, whereas in proliferating cells, there
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