Modulation of β-Catenin Phosphorylation/Degradation by Cyclin-dependent Kinase 2

β-Catenin functions as a downstream component of the Wnt/Wingless signal transduction pathway, and inappropriate control of cytosolic β-catenin is a crucial step in the genesis of several human cancers. Here we demonstrate that cyclin-dependent kinase 2 (CDK2) in association with cyclin A or cyclin E directly binds to β-catenin. In vivo and in vitro kinase assays with cyclin-CDK2 demonstrate β-catenin phosphorylation on residues Ser33, Ser37, Thr41, and Ser45. This phosphorylation promotes rapid degradation of cytosolic β-catenin via the β-TrCP-mediated proteasome pathway. Moreover, cyclin E-CDK2 contributes to rapid degradation of cytosolic β-catenin levels during G1 phase by regulating β-catenin phosphorylation and subsequent degradation. In this way, CDK2 may “fine tune” β-catenin levels over the course of the cell cycle. β-Catenin functions as a downstream component of the Wnt/Wingless signal transduction pathway, and inappropriate control of cytosolic β-catenin is a crucial step in the genesis of several human cancers. Here we demonstrate that cyclin-dependent kinase 2 (CDK2) in association with cyclin A or cyclin E directly binds to β-catenin. In vivo and in vitro kinase assays with cyclin-CDK2 demonstrate β-catenin phosphorylation on residues Ser33, Ser37, Thr41, and Ser45. This phosphorylation promotes rapid degradation of cytosolic β-catenin via the β-TrCP-mediated proteasome pathway. Moreover, cyclin E-CDK2 contributes to rapid degradation of cytosolic β-catenin levels during G1 phase by regulating β-catenin phosphorylation and subsequent degradation. In this way, CDK2 may “fine tune” β-catenin levels over the course of the cell cycle. β-Catenin is a multifunctional protein that plays an essential role in the transduction of Wnt signals and the function of the intercellular adhesion molecule E-cadherin (1Barth A.I. Nathke I.S. Nelson W.J. Curr. Opin. Cell Biol. 1997; 9: 683-690Crossref PubMed Scopus (490) Google Scholar, 2Morin P.J. BioEssays. 1999; 21: 1021-1030Crossref PubMed Scopus (821) Google Scholar, 3Peifer M. Polakis P. Science. 2000; 287: 1606-1609Crossref PubMed Scopus (1154) Google Scholar). In the absence of Wnt signaling, cytosolic β-catenin is phosphorylated by a protein complex composed of adnomatous polyposis coli (APC), 1The abbreviations used are: APC, adnomatous polyposis coli; CDK, cyclin-dependent kinase; GSK3β, glycogen synthase kinase 3β; GST, glutathione S-transferase; Ab, antibody; WT, wild type; GFP, green fluorescent protein. Axin, and glycogen synthase kinase 3β (GSK3β) (4Zeng L. Fagotto F. Zhang T. Hsu W. Vasicek T.J. Perry W.L. Lee J.J. Tilghman S.M. Gumbiner B.M. Costantini F. Cell. 1997; 90: 181-192Abstract Full Text Full Text PDF PubMed Scopus (802) Google Scholar, 5Behrens J. Jerchow B.A. Wurtele M. Grimm J. Asbrand C. Wirtz R. Kuhl M. Wedlich D. Birchmeier W. Science. 1998; 280: 596-599Crossref PubMed Scopus (1126) Google Scholar, 6Hart M.J. del los Santos R. Albert I.N. Rubinfeld B. Polakis P. Curr. Biol. 1998; 8: 573-581Abstract Full Text Full Text PDF PubMed Google Scholar, 7Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1114) Google Scholar, 8Sakanaka C. Weiss J.B. Williams L.T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3020-3023Crossref PubMed Scopus (283) Google Scholar, 9Itoh K. Krupnik V.E. Sokol S.Y. Curr. Biol. 1998; 8: 591-594Abstract Full Text Full Text PDF PubMed Google Scholar, 10Salic A. Lee E. Mayer L. Kirschner M.W. Mol. Cell. 2000; 5: 523-532Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 11Kikuchi A. Cell. Signal. 1999; 11: 777-788Crossref PubMed Scopus (166) Google Scholar, 12Yost C. Torres M. Miller J.R. Huang E. Kimelman D. Moon R.T. Genes Dev. 1996; 10: 1443-1454Crossref PubMed Scopus (1033) Google Scholar), which leads to its rapid ubiquitination and proteasomal degradation (13Orford K. Crockett C. Jensen J.P. Weissman A.M. Byers S.W. J. Biol. Chem. 1997; 272: 24735-24738Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar, 14Aberle H. Bauer A. Stappert J. Kispert A. Kemler R. EMBO J. 1997; 16: 3797-3804Crossref PubMed Scopus (2201) Google Scholar), as the phospho-serine/threonine residues are targets for β-TrCP and the specificity component of the ubiquitination apparatus (15Winston J.T. Strack P. Beer-Romero P. Chu C.Y. Elledge S.J. Harper J.W. Genes Dev. 1999; 13: 270-283Crossref PubMed Scopus (827) Google Scholar, 16Kitagawa M. Hatakeyama S. Shirane M. Matsumoto M. Ishida N. Hattori K. Nakamichi I. Kikuchi A. Nakayama K. Nakayama K. EMBO J. 1999; 18: 2401-2410Crossref PubMed Scopus (487) Google Scholar, 17Hart M. Concordet J.P. Lassot I. Albert I. del los Santos R. Durand H. Perret C. Rubinfeld B. Margottin F. Bernarous R. Polakis P. Curr. Biol. 1999; 9: 207-210Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar, 18Latres E. Chiaur D.S. Pagano M. Oncogene. 1999; 18: 849-854Crossref PubMed Scopus (385) Google Scholar, 19Liu C. Kato Y. Zhang Z. Do V.M. Yankner B.A. He X. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6273-6278Crossref PubMed Scopus (343) Google Scholar). Wnt signaling appears to reduce the activity of GSK3β (2Morin P.J. BioEssays. 1999; 21: 1021-1030Crossref PubMed Scopus (821) Google Scholar, 3Peifer M. Polakis P. Science. 2000; 287: 1606-1609Crossref PubMed Scopus (1154) Google Scholar, 20van Leeuwen F. Samos C.H. Nusse R. Nature. 1994; 368: 342-344Crossref PubMed Scopus (176) Google Scholar), leading to accumulation of cytosolic β-catenin, which is believed to enter the nucleus and complex with Tcf and Lef family transcription factors and thereby stimulate activation of genes containing Tcf and Lef binding sites (21Molenaar M. van de Wetering M. Oosterwegel M. Peterson-Maduro J. Godsave S. Korinek V. Roose J. Destree O. Clevers H. Cell. 1996; 86: 391-399Abstract Full Text Full Text PDF PubMed Scopus (1636) Google Scholar, 22Behrens J. von Kries J.P. Kuhl M. Bruhn L. Wedlich D. Grosschedl R. Birchmeier W. Nature. 1996; 382: 638-642Crossref PubMed Scopus (2630) Google Scholar, 23Huber O. Korn R. McLaughlin J. Ohsugi M. Herrmann B.G. Kemler R. Mech. Dev. 1996; 59: 3-10Crossref PubMed Scopus (792) Google Scholar) (e.g. MYC and CCND1) (24He T.C. Sparks A.B. Rago C. Hermeking H. Zawel L. da Costa L.T. Morin P.J. Vogelstein B. Kinzler K.W. Science. 1998; 281: 1509-1512Crossref PubMed Scopus (4121) Google Scholar, 25Tetsu O. McCormick F. Nature. 1999; 398: 422-426Crossref PubMed Scopus (3291) Google Scholar). The fact that abnormal expression of β-catenin or mutation of its regulatory region is a crucial step in the genesis of several human cancers (2Morin P.J. BioEssays. 1999; 21: 1021-1030Crossref PubMed Scopus (821) Google Scholar, 26Voeller H.J. Truica C.I. Gelmann E.P. Cancer Res. 1998; 58: 2520-2523PubMed Google Scholar, 27Whitehead I. Kirk H. Kay R. Mol. Cell. Biol. 1995; 15: 704-710Crossref PubMed Google Scholar) suggests that β-catenin may act as an oncogene in mammalian cells. Consistent with that idea, disruption of β-catenin/TCF activity in colorectal cancer cells (28van de Wetering M. Sancho E. Verweij C. de Lau W. Oving I. Hurlstone A. van der Horn K. Batlle E. Coudreuse D. Haramis A.P. Tjon-Pon-Fong M. Moerer P. van den Born M. Soete G. Pals S. Eilers M. Medema R. Clevers H. Cell. 2002; 111: 241-250Abstract Full Text Full Text PDF PubMed Scopus (1762) Google Scholar) or overexpression of APC in normal cells induces rapid G1 arrest (29Baeg G.H. Matsumine A. Kuroda T. Bhattacharjee R.N. Miyashiro I. Toyoshima K. Akiyama T. EMBO J. 1995; 14: 5618-5625Crossref PubMed Scopus (167) Google Scholar). It thus appears that fine control of β-catenin levels probably contributes to the maintenance of a normal cell cycle. It is noteworthy in that regard that levels of cytosolic β-catenin fluctuate over the course of the cell cycle, increasing during S phase, peaking in late G2/M phase, and then abruptly declining in G1 phase (30Olmeda D. Castel S. Vilaro S. Cano A. Mol. Biol. Cell. 2003; 14: 2844-2860Crossref PubMed Scopus (169) Google Scholar, 31Orford K. Orford C.C. Byers S.W. J. Cell Biol. 1999; 146: 855-868Crossref PubMed Scopus (240) Google Scholar). The mechanism responsible for this oscillation in β-catenin levels remains unknown, however. Cyclin-dependent kinases (CDKs) are key regulators of cell cycle progression in eukaryotic cells (e.g. cyclin E-CDK2 and cyclin A-CDK2 promote progression from G1 phase into and through S phase) (32Morgan D.O. Annu. Rev. Cell Dev. Biol. 1997; 13: 261-291Crossref PubMed Scopus (1833) Google Scholar, 33Sherr C.J. Cell. 1993; 73: 1059-1065Abstract Full Text PDF PubMed Scopus (2046) Google Scholar), and periodic activation of cyclin-CDK complexes is largely responsible for the characteristic sequence of cell cycle events, including mitosis, DNA synthesis, chromatin assembly, and other biosynthetic processes. It is known that a short sequence motif (RXL) present in cyclin-CDK2 substrates is necessary for the binding of the enzyme and phosphorylation of the substrates (34Adams P.D. Sellers W.R. Sharma S.K. Wu A.D. Nalin C.M. Kaelin Jr., W.G. Mol. Cell. Biol. 1996; 16: 6623-6633Crossref PubMed Scopus Google Scholar, K. E. L. Mol. Cell. Biol. 1997; 17: PubMed Scopus Google Scholar, L. E. 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EMBO J. 2002; 21: PubMed Scopus Google Scholar) of and used for and to and cyclin cyclin and cyclin from from CDK2 and from Cell and and cells in with to the of DNA by the of In β-catenin or β-catenin a and protein on with in vitro in binding for with binding by and Cell by the cells in binding for by binding then as that of cell used of the in vitro The by by Cell and as M. A.M. N. Y. Acad. Sci. 1998; PubMed Scopus Google Scholar) cells. The as the cytosolic cells for in containing and a of and which the for in of cell with of or A or 2 of for The complexes then on protein or and with The to and by in an or which with the an β-catenin phosphorylation in cells in containing and for The several through a and for The into and to by In assays as T. C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) CDK2 complexes from cyclin A-CDK2 and cyclin E-CDK2 from B. A. R.T. EMBO J. 2002; 21: PubMed Scopus Google Scholar). of or used as with of or used as Cell and Cell cells the G1 or S phase, cells in the of for or 2 for The DNA of cells by Cell cycle as K. Orford C.C. Byers S.W. J. Cell Biol. 1999; 146: 855-868Crossref PubMed Scopus (240) Google Scholar). with to cell cycle regulators with β-catenin, we with cell and then the by in cyclin A and E to to β-catenin. are known to with the with Ab, which the of CDK2 that the CDK2 directly with β-catenin, we the of to in vitro that that β-catenin with cyclin A-CDK2 cyclin E-CDK2 complexes in assays and cells that complexes containing β-catenin, and CDK2 in The motif is for binding of cyclin-CDK2 complexes to substrates (34Adams P.D. Sellers W.R. Sharma S.K. Wu A.D. Nalin C.M. Kaelin Jr., W.G. Mol. Cell. Biol. 1996; 16: 6623-6633Crossref PubMed Scopus Google Scholar, K. E. L. Mol. Cell. Biol. 1997; 17: PubMed Scopus Google Scholar, L. E. 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PubMed Scopus Google Scholar). in we that phosphorylation Ser33, Ser37, Thr41, and residues on β-catenin. the of the cyclin-CDK2 phosphorylation sites in β-catenin, β-catenin in which or phosphorylation sites then in vitro kinase assays to phosphorylation of the and that the of the for and in that the that Ser33, Ser37, and are phosphorylation sites on β-catenin. In wild β-catenin as as and demonstrate that cyclin-CDK2 β-catenin on residues Ser33, Ser37, Thr41, and in CDK2 activity is responsible for β-catenin phosphorylation in cells with which to CDK2 or its P. B. T. I. J. R. Nature. 1996; PubMed Scopus Google Scholar, B.A. P. R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar, Jr., Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar). CDK2 the association CDK2 and β-catenin and β-catenin phosphorylation on residues Ser33, Ser37, Thr41, and in vivo and cyclin-CDK2 the residues in we cells with cyclin or cyclin which phosphorylation of β-catenin or of cyclin E-CDK2 and cyclin A-CDK2 phosphorylation of β-catenin on residues that cyclin E-CDK2 and cyclin A-CDK2 in phosphorylation on residues Ser33, Ser37, Thr41, and in the to which phosphorylation by cyclin-CDK2 degradation of cytosolic β-catenin, we cells with β-catenin with cyclin A-CDK2 or cyclin which cytosolic from the by of β-catenin to levels of cytosolic cytosolic β-catenin then by cyclin A-CDK2 and cyclin E-CDK2 β-catenin largely to cyclin E-CDK2 and cyclin A-CDK2 that degradation of cytosolic β-catenin is on its phosphorylation by β-Catenin via the β-TrCP-mediated β-catenin is known to to which is in the accumulation of S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1114) Google Scholar, 14Aberle H. Bauer A. Stappert J. Kispert A. Kemler R. EMBO J. 1997; 16: 3797-3804Crossref PubMed Scopus (2201) Google Scholar). of β-catenin via the cells with a S proteasome which the cell with or with to the accumulation of β-catenin by ubiquitination of the phosphorylated protein The for phosphorylation by the that accumulation of β-catenin by with β-TrCP-mediated ubiquitination the via which β-catenin is by the that expression of a β-TrCP to to phosphorylated β-catenin, to a complex M. Concordet J.P. Lassot I. Albert I. del los Santos R. Durand H. Perret C. Rubinfeld B. Margottin F. Bernarous R. Polakis P. Curr. 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Chem. 1999; Full Text Full Text PDF PubMed Scopus Google mechanism is in β-catenin during cell cycle. of in this that of β-catenin, and cyclin in β-catenin during G1 β-catenin with in vivo and in that a short sequence motif (RXL) is present in a of cyclin-CDK2 which is necessary for the binding of cyclin-CDK2 (34Adams P.D. Sellers W.R. Sharma S.K. Wu A.D. Nalin C.M. Kaelin Jr., W.G. Mol. Cell. Biol. 1996; 16: 6623-6633Crossref PubMed Scopus Google Scholar, K. E. L. Mol. Cell. Biol. 1997; 17: PubMed Scopus Google Scholar, L. E. Genes Dev. 1995; 9: PubMed Scopus Google Scholar). present in β-catenin to its binding to cyclin-CDK2 cyclin-CDK2 β-catenin on the residues by GSK3β Ser37, Thr41, and It is that cyclin-CDK2 β-catenin on residues Ser33, Ser37, Thr41, and residues that are in the CDK2 phosphorylation and The phosphorylation sequence for is a or by a Curr. Opin. Cell Biol. 1993; 5: PubMed Scopus Google Scholar). 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In the present the association of β-catenin with cyclin E-CDK2 and its kinase activity during G1 phase, which with levels of β-catenin GSK3β activity the cell cycle. cyclin-CDK2 of cytosolic β-catenin through the mechanism the β-TrCP-mediated S proteasome pathway. GSK3β β-catenin, which is by a specificity component of an ubiquitination apparatus (15Winston J.T. Strack P. Beer-Romero P. Chu C.Y. Elledge S.J. Harper J.W. Genes Dev. 1999; 13: 270-283Crossref PubMed Scopus (827) Google Scholar, 16Kitagawa M. Hatakeyama S. Shirane M. Matsumoto M. Ishida N. Hattori K. Nakamichi I. Kikuchi A. Nakayama K. Nakayama K. 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Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1114) Google Scholar, 8Sakanaka C. Weiss J.B. Williams L.T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3020-3023Crossref PubMed Scopus (283) Google Scholar, 9Itoh K. Krupnik V.E. Sokol S.Y. Curr. Biol. 1998; 8: 591-594Abstract Full Text Full Text PDF PubMed Google Scholar, 10Salic A. Lee E. Mayer L. Kirschner M.W. Mol. Cell. 2000; 5: 523-532Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 11Kikuchi A. Cell. Signal. 1999; 11: 777-788Crossref PubMed Scopus (166) Google Scholar, 12Yost C. Torres M. Miller J.R. Huang E. Kimelman D. Moon R.T. Genes Dev. 1996; 10: 1443-1454Crossref PubMed Scopus (1033) Google Scholar, M. van de Wetering M. Oosterwegel M. Peterson-Maduro J. Godsave S. Korinek V. Roose J. Destree O. Clevers H. Cell. 1996; 86: 391-399Abstract Full Text Full Text PDF PubMed Scopus (1636) Google Scholar, 22Behrens J. von Kries J.P. Kuhl M. Bruhn L. Wedlich D. Grosschedl R. Birchmeier W. Nature. 1996; 382: 638-642Crossref PubMed Scopus (2630) Google Scholar, 23Huber O. Korn R. McLaughlin J. Ohsugi M. Herrmann B.G. Kemler R. Mech. Dev. 1996; 59: 3-10Crossref PubMed Scopus (792) Google Scholar). cytosolic β-catenin over the course of the cell cycle (30Olmeda D. Castel S. Vilaro S. Cano A. Mol. Biol. Cell. 2003; 14: 2844-2860Crossref PubMed Scopus (169) Google Scholar, 31Orford K. Orford C.C. Byers S.W. J. Cell Biol. 1999; 146: 855-868Crossref PubMed Scopus (240) Google Scholar). GSK3β is probably responsible for of β-catenin of cell cycle, which is by the that GSK3β activity the cell cycle. In to GSK3β, cyclin-CDK2 is probably responsible for rapid of the β-catenin G1 phase, cyclin-CDK2 GSK3β the kinase cyclin-CDK2 act as a of β-catenin during the cell cycle. the functions of cyclin-CDK2 in β-catenin phosphorylation to that cyclin-CDK2 probably cytosolic β-catenin phosphorylation and its subsequent proteasomal and β-catenin by cyclin E-CDK2 may the fine of β-catenin in G1 are to for cyclin A-CDK2 and cyclin and for