Degradation of Cyclin A Does Not Require Its Phosphorylation by CDC2 and Cyclin-dependent Kinase 2

Many cyclins are degraded by the ubiquitination/proteasome pathways involving the anaphase-promoting complex and SCF complexes. These degradations are frequently dependent on phosphorylation by cyclin-dependent kinases (CDKs), providing a self-limiting mechanism for CDK activity. Here we present evidence from in vitro and in vivo assay systems that the degradation of human cyclin A can be inhibited by kinase-inactive mutants of CDK2 and CDC2. One obvious interpretation of these results is that like other cyclins, CDK-dependent phosphorylation of the cyclin A may be involved in cyclin A degradation. Our data indicated that CDK2 can phosphorylate cyclin A on Ser-154. Site-directed mutagenesis of Ser-154 abolished the phosphorylation by recombinant CDK2 in vitro and the majority of cyclin A phosphorylation in the cell. Activation of CDK2 and binding to SKP2 or p27KIP1 were not affected by the phosphorylation of Ser-154. Surprising, in marked contrast to cyclin E, where phosphorylation of Thr-380 by CDK2 is required for proteolysis, degradation of cyclin A was not affected by Ser-154 phosphorylation. It is likely that the stabilization of cyclin A by the kinase-inactive CDKs was mainly due to a cell cycle effect. These data suggest an important difference between the regulation of cyclin A and cyclin E. Many cyclins are degraded by the ubiquitination/proteasome pathways involving the anaphase-promoting complex and SCF complexes. These degradations are frequently dependent on phosphorylation by cyclin-dependent kinases (CDKs), providing a self-limiting mechanism for CDK activity. Here we present evidence from in vitro and in vivo assay systems that the degradation of human cyclin A can be inhibited by kinase-inactive mutants of CDK2 and CDC2. One obvious interpretation of these results is that like other cyclins, CDK-dependent phosphorylation of the cyclin A may be involved in cyclin A degradation. Our data indicated that CDK2 can phosphorylate cyclin A on Ser-154. Site-directed mutagenesis of Ser-154 abolished the phosphorylation by recombinant CDK2 in vitro and the majority of cyclin A phosphorylation in the cell. Activation of CDK2 and binding to SKP2 or p27KIP1 were not affected by the phosphorylation of Ser-154. Surprising, in marked contrast to cyclin E, where phosphorylation of Thr-380 by CDK2 is required for proteolysis, degradation of cyclin A was not affected by Ser-154 phosphorylation. It is likely that the stabilization of cyclin A by the kinase-inactive CDKs was mainly due to a cell cycle effect. These data suggest an important difference between the regulation of cyclin A and cyclin E. cyclin-dependent kinase anaphase-promoting complex polymerase chain reaction glutathioneS-transferase phosphate-buffered saline hemagglutinin polyacrylamide gel electrophoresis CDK-activating kinase Cyclins and cyclin-dependent kinases (CDKs)1 are key regulators of the eukaryotic cell cycle. Cyclin B is associated with CDC2 and the cyclin B-CDC2 complexes regulate entry into M phase (1.King R.W. Jackson P.K. Kirschner M.W. Cell. 1994; 79: 563-571Abstract Full Text PDF PubMed Scopus (685) Google Scholar). Cyclin A-CDK2 and cyclin E-CDK2 complexes are important for progression through S phase and the G1/S transition, respectively (2.Sherr C.J. Cell. 1994; 79: 551-555Abstract Full Text PDF PubMed Scopus (2594) Google Scholar, 3.Heichman K.A. Roberts J.M. Cell. 1994; 79: 557-562Abstract Full Text PDF PubMed Scopus (240) Google Scholar). D-type cyclins are associated with CDK4 and CDK6, and the complexes are required for G1 progression (2.Sherr C.J. Cell. 1994; 79: 551-555Abstract Full Text PDF PubMed Scopus (2594) Google Scholar). The kinase activity of CDK is tightly regulated by an intricate system of phosphorylation and protein-protein interactions (4.Poon R.Y.C. Bertino J.R. Encyclopedia of Cancer. Academic Press, San Diego1996: 246-255Google Scholar, 5.Morgan D.O. Annu. Rev. Cell Dev. Biol. 1997; 13: 261-291Crossref PubMed Scopus (1810) Google Scholar). By definition, the activation of CDKs is dependent on the association with a cyclin subunit. The post-translational regulation of cyclins occurs mainly through degradation (6.Koepp D.M. Harper J.W. Elledge S.J. Cell. 1999; 97: 431-434Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar). The mitotic cyclins are degraded near the end of anaphase by the ubiquitin-proteasome system, consisting of a nonspecific ubiquitin-activating enzyme, a ubiquitin-carrier protein, a cyclin-specific ubiquitin ligase known as the cyclosome or anaphase-promoting complex (APC), and a constitutively active proteasome complex. The N-terminal destruction box sequences of the mitotic cyclins are required for their degradation (7.Yamano H. Tsurumi C. Gannon J. Hunt T. EMBO J. 1998; 17: 5670-5678Crossref PubMed Scopus (94) Google Scholar), possibly because they are recognized by the cyclin-specific ubiquitin ligase enzyme. Recent biochemical and genetic studies have demonstrated that vertebrate APC contains at least eight subunits (APC1–APC8) (8.Morgan D.O. Nat. Cell Biol. 1999; 1: E47-E53Crossref PubMed Scopus (308) Google Scholar). The activity of APC is highly regulated, turning on in anaphase and persist until late G1 (9.Brandeis M. Hunt T. EMBO J. 1996; 15: 5280-5289Crossref PubMed Scopus (248) Google Scholar, 10.Amon A. Irniger S. Nasmyth K. Cell. 1994; 77: 1037-1050Abstract Full Text PDF PubMed Scopus (399) Google Scholar). Association between APC and the WD40 repeat-containing protein Hct1p (also known as Cdh1p) is required for the degradation of mitotic cyclins in yeast (11.Schwab M. Lutum A.S. Seufert W. Cell. 1997; 90: 683-693Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar). From S phase onward, phosphorylation of Hct1p by CDKs blocked the Hct1p-APC interaction (12.Zachariae W. Schwab M. Nasmyth K. Seufert W. Science. 1998; 282: 1721-1724Crossref PubMed Scopus (442) Google Scholar), allowing the mitotic cyclins to accumulate until anaphase. After anaphase, cyclin destruction is initiated by the dephosphorylation of Hct1p by the phosphatase Cdc14p (13.Visintin R. Craig K. Hwang E.S. Prinz S. Tyers M. Amon A. Mol. Cell. 1998; 2: 709-718Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar, 14.Jaspersen S.L. Charles J.F. Morgan D.O. Curr. Biol. 1999; 9: 227-236Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). Cdc14p is sequestered in the nucleolus by anchoring to Cfi1p for most of the cell cycle and is only released from the nucleolus to act on its targets during anaphase (15.Visintin R. Hwang E.S. Amon A. Nature. 1999; 398: 818-823Crossref PubMed Scopus (489) Google Scholar). Polo-like kinase (Cdc5p in Saccharomyces cerevisiae) is activated by cyclin B-CDC2, and activated Polo-like kinase phosphorylates components of APC and promotes the ubiquitination of cyclin B (16.Shirayama M. Zachariae W. Ciosk R. Nasmyth K. EMBO J. 1998; 17: 1336-1349Crossref PubMed Scopus (440) Google Scholar, 17.Charles J.F. Jaspersen S.L. Tinker-Kulberg R.L. Hwang L. Szidon A. Morgan D.O. Curr. 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Unlike the mitotic cyclins, the G1 cyclins do not have a destruction box but do contain PEST sequences at the C-terminal portion of the protein that are partly responsible for the relatively short half-life of these cyclins (19.Salama S.R. Hendricks K.B. Thorner J. Mol. Cell. Biol. 1994; 14: 7953-7966Crossref PubMed Scopus (90) Google Scholar). In yeast, CDK-dependent phosphorylation of the Cln is involved in the ubiquitination-dependent turnover of these cyclins (20.Lanker S. Valdivieso M.H. Wittenberg C. Science. 1996; 271: 1597-1600Crossref PubMed Scopus (195) Google Scholar, 21.Yaglom J. Linskens M.H. Sadis S. Rubin D.M. Futcher B. Finley D. Mol. Cell. Biol. 1995; 15: 731-741Crossref PubMed Google Scholar). Similarly, phosphorylation of human cyclin E (on Thr-380) and cyclin D1 (on Thr-286) is important for their ubiquitin-dependent degradation (22.Clurman B.E. Sheaff R.J. Thress K. Groudine M. Roberts J.M. Genes Dev. 1996; 10: 1979-1990Crossref PubMed Scopus (417) Google Scholar, 23.Won K.A. Reed S.I. EMBO J. 1996; 15: 4182-4193Crossref PubMed Scopus (306) Google Scholar, 24.Diehl J.A. Zindy F. C.J. Genes Dev. 1997; PubMed Scopus Google Scholar). the CDK2 of cyclin E can phosphorylate cyclin E at but the CDK4 of cyclin D1 is not responsible for cyclin D1 phosphorylation. can be by cyclin D1 turnover to pathways J.A. M. C.J. Genes Dev. 1998; PubMed Scopus Google Scholar). The ubiquitination of Cln cyclins their association with SCF complexes. The is that in with and an protein, into a cyclin-specific ubiquitin ligase complex protein and SCF complexes the and but may for the Cln cyclins, to and and targets their destruction Zachariae W. A. S. M. J. S. Nasmyth K. R.J. Genes Dev. 1999; 13: PubMed Scopus Google Scholar, D. D.M. T. J.W. Elledge S.J. Harper J.W. Science. 1999; PubMed Scopus Google Scholar, D. L. D. Craig Tyers M. Genes Dev. 1998; PubMed Scopus Google Scholar, S. C. 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Surprising, we that of the cyclin A phosphorylation to not the degradation of cyclin A. of cyclin E was inhibited by kinase-inactive in contrast to cyclin A, of the CDK phosphorylation in cyclin E to inhibited the degradation of cyclin E. cyclin A in were from human cyclin Cyclin A in from was by with the A and A and into R.Y.C. Mol. Cell. Biol. 1999; PubMed Scopus Google A in or cyclin A in of cyclin A in was into to A in Site-directed mutagenesis of cyclin A was by a as at Press, Scholar), cyclin A and cyclin A and the and its to the the was and into or Cyclin A in was with and into to in Cyclin in was a from Hunt A in was as K. Hunt T. J. EMBO J. PubMed Scopus Google Scholar). cyclin in from The was with and and into to cyclin in Cyclin E in was by with the and the the was into the from The Site-directed mutagenesis of cyclin E was as cyclin A the and its to the the was to cyclin in cyclin in was a from Hunt Cyclin in was with and into to in in and in were by the of in and in K. Hunt T. J. EMBO J. PubMed Scopus Google Scholar), into CDC2 and in were from and on the for in were from of in and in were as R.Y.C. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar). in and in R.Y.C. Mol. Cell. Biol. 1999; PubMed Scopus Google were by with the and and into from to in and in SKP2 in was as R.Y.C. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar). in was by the and into the of in was by of in R.Y.C. T. 1998; PubMed Scopus Google into in was by of the in R.Y.C. H. T. Mol. Biol. Cell. 1995; PubMed Scopus Google by the and by and into in was as R.Y.C. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar). ubiquitin for was a from Hunt from are with the M. H. S. A. PubMed Scopus Google and can into the or in the of to were in with in a at in were as R.Y.C. H. T. Mol. Biol. Cell. 1995; PubMed Scopus Google Scholar). (also was at for were blocked at by in for G1 were released from the were blocked in S phase by in for were with by the F. R. R. D. J. J. K. in Scholar). 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Cyclin A not cyclin is are at with like the degradation of cyclin A by cyclin A in in the of and we the cyclin A with of that were in of the cell cycle. A that cyclin A was with a In cyclin A was not with cell from in G1, or phase of the cell cycle. The destruction of the cyclin A by was inhibited by the of the of the of cyclin A can be in the of and SKP2 were of cyclin A as a and that the of SKP2 was not with the we of the SKP2 that was due to phosphorylation of by CDKs R.Y.C. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar). The destruction of cyclin A in in vitro system was dependent on the of the N-terminal destruction B that of the to inhibited the destruction of cyclin A by the cyclin was of cyclin A, we that cyclin was in G1 phase cell but not by S phase or these data that we have an in vitro assay for cyclin A destruction by that was for cyclin A but not for cyclin next the destruction of cyclin A by was affected by cyclin cyclin A and SKP2 were with A cell from with or a of CDK2 to were to the that as cyclin A was SKP2 was relatively in the of inhibited the destruction of cyclin A a of components of complexes not the of cyclin A likely of these results are as the of cyclin A phosphorylation by inhibited cyclin A the of blocked the cell cycle at G1 and inhibited the degradation of cyclin A. The two are to but we the in the present The that of blocked the in G1 phase R.Y.C. Mol. Cell. Biol. 1999; PubMed Scopus Google to the the destruction of cyclin A is affected by kinase-inactive CDK2 in we a assay to the of cyclin A in were with that cyclin A the of The were into and the of cyclin A was by of to the A that the of cyclin A its was the of the cyclin A in assay was not by the cyclin A was with the for its In contrast to cyclin A, the of relatively that a of was in in the in vitro A was was affected the of cyclin A in A was with CDK2 or were and the of A was as B that the of cyclin A was with in to with that at the of the the of the of A was in the of in the of CDK2 The of these of to the protein have the that the of the was not into to protein results not with the in vitro degradation these data suggest that the of cyclin A can be in the of a kinase-inactive of due to CDK phosphorylation or to an of cell cycle In contrast to cyclin B and cyclin E, cyclin A with two of CDKs in the CDK2 and CDC2. next like a kinase-inactive of CDC2 can cyclin A. A was with CDC2 or in a as that the of cyclin A was in that were with in to that with CDC2. that the of cyclin A was by with the CDK not a to cyclin A, we next cyclin E and cyclin to the Cyclin E was with or not in to that with The half-life of cyclin E in the of was to that with CDK2 not the other hand, we that the of was the was with CDK2 or these that cyclin A and cyclin E, but not cyclin was by is to cyclin A, the cyclin was in H. W. A. and R. C. but in contrast to cyclin A and cyclin E, cyclin not to next cyclin A can be by cyclin A was with in the of we that the cyclin A was at the cyclin A was or cyclin A was of the protein of human cyclin A, is only phosphorylation by a at Ser-154. is in the sequences of cyclin and cyclin in next Ser-154 is by CDK2 in of cyclin A and Ser-154 to a were and the was with The cyclin A and was with in the of B that only cyclin A, but not cyclin was by Protein of the gel a of cyclin A and cyclin was present in the be that the of phosphorylation of cyclin was not due to a of CDK2 kinase activity cyclin associated with the kinase were that cyclin A-CDK2 and cyclin can phosphorylate that recombinant cyclin was as active as cyclin A-CDK2 complex. next cyclin A is the cell. were with the were with cyclin A, or cyclin Cell were and were and with a A that A was a protein in the cell of Ser-154 to abolished most of the phosphorylation of cyclin A that Ser-154 was the phosphorylation in cyclin A. of the indicated that cyclin A and cyclin were at In with Ser-154 as the phosphorylation of the cyclin A indicated that the majority of the phosphorylation was on that the phosphorylation of the was mainly on not The cyclin A was a protein in the cell and and we that the phosphorylation was mainly on not cyclin can and cyclin A or cyclin was in and with an A that CDK2 and can be in the with an and kinase can be to with A and the CDK p27KIP1 was with the cyclin A, complexes between p27KIP1 and of cyclin A can be the kinase activity associated with of cyclin A was abolished by with a of the CDKs that associated with the These data that cyclin can to CDKs and that cyclin A could be a for we next the can with B that SKP2 can be with cyclin A and cyclin and In SKP2 was not cyclin A was not The of cyclin A and cyclin was by with that SKP2 can with cyclin A of can be on Ser-154. were not to binding to cyclin in is likely due to the high of in the cell. cyclin A can the cell cycle in or M with cyclin A or cyclin and a for the of the After the cell cycle of the and was by that of cyclin A or cyclin in a in the of in or M phase of the cell cycle. These data suggest that at least the of and of cyclin A on CDK SKP2 and on the cell cycle were the next the destruction of cyclin A was affected by the Ser-154 phosphorylation. the destruction of cyclin the in vitro system as in cyclin A or cyclin was with and were at for cyclin and cyclin were in the A that cyclin A was cyclin and cyclin were relatively difference was cyclin was of cyclin A inhibited the destruction of of cyclin A and were for of cyclin A in the of and These data suggest that the ubiquitination and destruction of cyclin A were of Ser-154 can be or next the destruction of cyclin with the assay a we cyclin E and the cyclin Thr-380 to an the A that cyclin was cyclin E. These results that at least assay was of in the of cyclins due to phosphorylation. In were between cyclin A and cyclin we that cyclin can be with These data that cyclin E, where phosphorylation by CDK2 on Thr-380 is required for its phosphorylation of cyclin A on Ser-154 by CDK2 is to be involved in the ubiquitination and degradation of cyclin A. Here we that cyclin A can be by a kinase-inactive in a assay in of the like and in the in vitro degradation assay the that a kinase-inactive of CDC2 and the CDK could the degradation of cyclin that the of cyclin A phosphorylation by inhibited cyclin A degradation or the of blocked the cell cycle and inhibited the degradation of cyclin A. that cyclin E to the CDK as cyclin is regulated by degradation K.A. Reed S.I. EMBO J. 1996; 15: 4182-4193Crossref PubMed Scopus (306) Google Scholar), we the obvious that of cyclin A by may be required for its degradation. that Ser-154 is the CDK2 phosphorylation in cyclin A because of the Ser-154 is the only in cyclin abolished the phosphorylation of by CDK in abolished most of the phosphorylation of cyclin A in the cell. Ser-154 is likely to be the only CDK phosphorylation in cyclin A, Ser-154 is not the only phosphorylation in cyclin A in the cell Ser-154 phosphorylation is not involved in the degradation of cyclin A, is that the phosphorylation of other in cyclin A by other protein kinases may cyclin A for degradation or the activity of cyclin A. other are phosphorylation of cyclin A by CDK2 not the stabilization of cyclin A with One of is that affected the phosphorylation of cyclin other cyclin A It is that phosphorylation of other by cyclin is required for the degradation of cyclin A. interpretation of the is that other cyclins were inhibited by which in affected the degradation of cyclin A. can to cyclin A and cyclin E and act as mutants for It is that cyclin E-CDK2 activity may be required for cyclin A degradation. inhibited the activity of cyclin E as as cyclin E degradation through of Thr-380 the results are with the that cyclin E degradation is required for cyclin A degradation. that these involving cyclin E are because the kinase-inactive CDC2 can cyclin A but not cyclin E but abolished the degradation of cyclin A with the CDC2 kinase-inactive is by the that cyclin E was not the of cyclin A and cyclin B were of the activity of cyclin B is involved in the degradation of cyclin A is phosphorylation of cyclin A by CDK2 is not involved in the destruction of cyclin A, is likely that the stabilization of cyclin A by was mainly due to a cell cycle effect. The that the of cyclin A and cyclin are they are in the of and cyclin the is responsible for its during The of cyclin is by binding of to the A. C. P. M. J. EMBO J. 1998; 17: PubMed Scopus Google Scholar, J. E.S. J. S. Genes Dev. 1998; PubMed Scopus (283) Google Scholar, F. T. A. M. E. EMBO J. 1998; 17: PubMed Scopus Google Scholar). of by CDK in the of cyclin is important for its possibly through the of interaction J. E.S. J. S. Genes Dev. 1998; PubMed Scopus (283) Google Scholar). mechanism the of cyclin to the the complexes are It is that phosphorylation of Ser-154 by CDKs may the of cyclin A. we difference in the between cyclin A and cyclin It is that the CDK phosphorylation in cyclin A, is the binding of the protein M. S. Hunt T. T. H. EMBO J. 1999; PubMed Scopus Google Scholar). is to the yeast protein and contains a at the of to cyclin A the degradation of cyclin A in It be to phosphorylation of Ser-154 the binding to to cyclin A. we that not the of cyclin A, we do not that phosphorylation of Ser-154 a on are to and for and of the for their of