Dephosphorylation of Human Cyclin-dependent Kinases by Protein Phosphatase Type 2Cα and β2 Isoforms

We previously reported that the activating phosphorylation on cyclin-dependent kinases in yeast (Cdc28p) and in humans (Cdk2) is removed by type 2C protein phosphatases. In this study, we characterize this PP2C-like activity in HeLa cell extract and determine that it is due to PP2Cβ2, a novel PP2Cβ isoform, and to PP2Cα. PP2Cα and PP2Cβ2 co-purified with Mg2+-dependent Cdk2/Cdk6 phosphatase activity in DEAE-Sepharose, Superdex-200, and Mono Q chromatographies. Moreover, purified recombinant PP2Cα and PP2Cβ2 proteins efficiently dephosphorylated monomeric Cdk2/Cdk6 in vitro. The dephosphorylation of Cdk2 and Cdk6 by PP2C isoforms was inhibited by the binding of cyclins. We found that the PP2C-like activity in HeLa cell extract, partially purified HeLa PP2Cα and PP2Cβ2 isoforms, and the recombinant PP2Cs exhibited a comparable substrate preference for a phosphothreonine containing substrate, consistent with the conservation of threonine residues at the site of activating phosphorylation in CDKs. We previously reported that the activating phosphorylation on cyclin-dependent kinases in yeast (Cdc28p) and in humans (Cdk2) is removed by type 2C protein phosphatases. In this study, we characterize this PP2C-like activity in HeLa cell extract and determine that it is due to PP2Cβ2, a novel PP2Cβ isoform, and to PP2Cα. PP2Cα and PP2Cβ2 co-purified with Mg2+-dependent Cdk2/Cdk6 phosphatase activity in DEAE-Sepharose, Superdex-200, and Mono Q chromatographies. Moreover, purified recombinant PP2Cα and PP2Cβ2 proteins efficiently dephosphorylated monomeric Cdk2/Cdk6 in vitro. The dephosphorylation of Cdk2 and Cdk6 by PP2C isoforms was inhibited by the binding of cyclins. We found that the PP2C-like activity in HeLa cell extract, partially purified HeLa PP2Cα and PP2Cβ2 isoforms, and the recombinant PP2Cs exhibited a comparable substrate preference for a phosphothreonine containing substrate, consistent with the conservation of threonine residues at the site of activating phosphorylation in CDKs. cyclin-dependent kinase Cdk-activating kinase Ser/Thr protein phosphatase Ser/Thr protein phosphatase type 2C reverse transcriptase-polymerase chain reaction glutathione S-transferase polyacrylamide gel electrophoresis mitogen-activated protein kinase 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid Eukaryotic cell cycle progression is driven by the ordered activation and inactivation of cyclin-dependent protein kinases (CDKs).1 To precisely control the cell cycle engine, extracellular and intracellular signals control CDK activities through a variety of mechanisms, including association with regulatory proteins (cyclins, inhibitors, and assembly factors), subcellular localization, transcriptional regulation, selective proteolysis, and reversible protein phosphorylation (1Pines J. 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Full activation of CDKs, which is necessary for normal cell cycle progression, requires binding of a cyclin, removal of inhibitory phosphorylations, and the presence of an activating phosphorylation. The cyclins are transcribed, synthesized, and degraded periodically during the cell cycle (1Pines J. Biochem. J. 1995; 308: 697-711Crossref PubMed Scopus (502) Google Scholar, 4King R.W. Deshaies R.J. Peters J.-M. Kirschner M.W. Science. 1996; 274: 1652-1659Crossref PubMed Scopus (1123) Google Scholar, 8Evans T. Rosenthal E.T. Youngblom J. Distel D. Hunt T. Cell. 1983; 33: 389-396Abstract Full Text PDF PubMed Scopus (1105) Google Scholar, 9Sherr C.J. Cell. 1994; 79: 551-555Abstract Full Text PDF PubMed Scopus (2623) Google Scholar). The inhibitory phosphorylations are carried out by the Wee1-like protein kinases, and removed by members of the Cdc25 family of dual specificity protein phosphatases (for reviews, see Refs. 6Morgan D.O. Annu. Rev. Cell Dev. Biol. 1997; 13: 261-291Crossref PubMed Scopus (1833) Google Scholar and 7Solomon M.J. Kaldis P. Pagano M. Results and Problems in Cell Differentiation “Cell Cycle Control”. Springer, Heidelberg, Germany1998: 79-109Google Scholar). Activating phosphorylation occurs within the “T-loop” (7Solomon M.J. Kaldis P. Pagano M. Results and Problems in Cell Differentiation “Cell Cycle Control”. Springer, Heidelberg, Germany1998: 79-109Google Scholar, 10Johnson L.N. Noble M.E.M. Owen D.J. Cell. 1996; 85: 149-158Abstract Full Text Full Text PDF PubMed Scopus (1193) Google Scholar) on a conserved threonine residue corresponding to Thr161 in human Cdc2 and Thr160 in human Cdk2. Mutation of the equivalent site to alanine in Cdc2 from a variety of species abolishes kinase activity and biological function (11Booher R. Beach D. Mol. Cell. Biol. 1986; 6: 3523-3530Crossref PubMed Scopus (115) Google Scholar, 12Gould K.L. Moreno S. Owen D.J. Sazer S. Nurse P. 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Tassan J.-P. Schaeffer L. Nigg E.A. Hoeijmakers J.H.J. Egly J.-M. Cell. 1994; 79: 1093-1101Abstract Full Text PDF PubMed Scopus (397) Google Scholar, 19Serizawa H. Mäkelä T.P. Conaway J.W. Conaway R.C. Weinberg R.A. Young R.A. Nature. 1995; 374: 280-282Crossref PubMed Scopus (310) Google Scholar, 20Shiekhattar R. Mermelstein F. Fisher R.P. Drapkin R. Dynlacht B. Wessling H.C. Morgan D.O. Reinberg D. Nature. 1995; 374: 283-287Crossref PubMed Scopus (371) Google Scholar, 21Adamczewski J.P. Rossignol M. Tassan J.-P. Nigg E.A. Moncollin V. Egly J.-M. EMBO J. 1996; 15: 1877-1884Crossref PubMed Scopus (114) Google Scholar). In contrast to CAK in higher eukaryotes, CAK from budding yeast (Cak1p or Civ1p) is only distantly related to p40MO15 (22Espinoza F.H. Farrell A. Erdjument-Bromage H. Tempst P. Morgan D.O. Science. 1996; 273: 1714-1717Crossref PubMed Scopus (147) Google Scholar, 23Kaldis P. Sutton A. Solomon M.J. Cell. 1996; 86: 553-564Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 24Thuret J.-Y. Valay J.-G. Faye G. Mann C. Cell. 1996; 86: 565-576Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar), functions as a monomer, and is predominantly cytoplasmic (25Kaldis P. Pitluk Z.W. Bany I.A. Enke D.A. Wagner M. Winter E. Solomon M.J. J. Cell Sci. 1998; 111: 3585-3596PubMed Google Scholar). Despite the large body of knowledge on CAK, there is relatively little information about the protein phosphatases that reverse the activating phosphorylation of the CDKs. A dual-specificity human protein phosphatase termed KAP (also Cdi1 and Cip2), which was identified by its interaction with Cdc2, Cdk2, and Cdk3 (26Gyuris J. Golemis E. Chertkov H. Brent R. Cell. 1993; 75: 791-803Abstract Full Text PDF PubMed Scopus (1333) Google Scholar, 27Hannon G.J. Casso D. Beach D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1731-1735Crossref PubMed Scopus (116) Google Scholar, 28Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5334) Google Scholar), was shown to dephosphorylate Thr160 on Cdk2 (29Poon R.Y.C. Hunter T. Science. 1995; 270: 90-93Crossref PubMed Scopus (149) Google Scholar). However, there is no obvious KAP homologue in budding yeast. We found recently that two budding yeast type 2C protein phosphatases (PP2Cs), Ptc2p and Ptc3p, are the predominant and physiological enzymes that dephosphorylate Thr169 on the Cdc28p cyclin-dependent kinase (30Cheng A. Ross K.E. Kaldis P. Solomon M.J. Genes Dev. 1999; 13: 2947-2957Crossref Scopus (128) Google Scholar). We also observed that PP2C-like activities were responsible for >99% of the phosphatase activity in HeLa cell extracts acting on Thr160 of Cdk2, indicating that the ability of type 2C protein phosphatases to remove the activating phosphorylation of CDKs is evolutionarily conserved (30Cheng A. Ross K.E. Kaldis P. Solomon M.J. Genes Dev. 1999; 13: 2947-2957Crossref Scopus (128) Google Scholar). Ser/Thr protein phosphatases (PPases) are classified into the PP1, PP2A, PP2B, and PP2C families based on their biochemical properties: PP1 and PP2A have no ion requirements and are sensitive to okadaic acid and microcystins; PP2B requires Ca2+ for full activity; and PP2C requires Mg2+ or Mn2+ (31Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2318) Google Scholar). Molecular cloning revealed that PP1, PP2A, and PP2B families share homology to each other whereas the PP2C family is structurally distinct (32Das A.K. Helps N.R. Cohen P.T.W. Barford D. EMBO J. 1996; 15: 6798-6809Crossref PubMed Scopus (393) Google Scholar). In addition to their catalytic subunits, the PP1, PP2A, and PP2B holoenzymes also contain one or two regulatory subunits that appear to determine the substrate specificities of these phosphatases (31Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2318) Google Scholar). Unlike PP1, PP2A, and PP2B, PP2C acts as a monomer. Thus, for PP2C, substrate specificity may result from the many PP2C family members, rather than from multiple regulatory subunits. In budding yeast, there are at least five PP2Cs (Ptc1p-Ptc5p) (30Cheng A. Ross K.E. Kaldis P. Solomon M.J. Genes Dev. 1999; 13: 2947-2957Crossref Scopus (128) Google Scholar, 33Stark M.J.R. Yeast. 1996; 12: 1647-1675Crossref PubMed Scopus (171) Google Scholar). The mammalian PP2C family includes PP2Cα, PP2Cβ, PP2Cγ (also called FIN13), PP2Cδ, Wip1, Ca2+/calmodulin-dependent kinase II phosphatase, and NERPP-2C (34Tamura S. Lynch K.R. Larner J. Fox J. Yasui A. Kikuchi K. Suzuki Y. Tsuiki S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1796-1800Crossref PubMed Scopus (113) Google Scholar, 35Mann D.J. Campbell D.G. McGowan C.H. Cohen 1992; PubMed Scopus Google Scholar, J. Cohen Campbell D.G. G. 1992; PubMed Scopus Google Scholar, P. N. C. Mol. Cell. Biol. 1997; PubMed Scopus Google Scholar, M.J. 1997; PubMed Scopus Google Scholar, R. Genes Dev. 1999; 13: PubMed Scopus Google Scholar, Y. R. J. Biol. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, M. H. S. K. S. E. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar, T. A. S. M. H. J. Biochem. 1999; PubMed Scopus Google Scholar, M. J. A. Mol. Cell 1998; 12: PubMed Scopus Google Scholar). In human PP2Cα two isoforms from M. T. H. EMBO J. 1998; PubMed Scopus Google Scholar), and PP2Cβ five isoforms (for see K. T. S. M. N. Y. R. T. S. A. S. Biochem. J. 1998; PubMed Scopus Google Scholar). In this study, we the PP2C activities of human Cdk2 and Cdk6 in a HeLa cell of proteins by DEAE-Sepharose, Superdex-200, and Mono Q that Cdk2 and Cdk6 phosphatase activities co-purified with PP2Cα, a previously reported PP2C isoform, and PP2Cβ2, a novel human PP2Cβ from PP2Cα and PP2Cβ2 dephosphorylated Cdk2 and Cdk6 in with yeast PP2Cs and human Cdk2 in a HeLa cell extract (30Cheng A. Ross K.E. Kaldis P. Solomon M.J. Genes Dev. 1999; 13: 2947-2957Crossref Scopus (128) Google Scholar). that HeLa cell extract, partially purified PP2Cα and PP2Cβ2, and recombinant PP2Cα and PP2Cβ2 exhibited a substrate preference for Cdk2 with a Cdk2 protein containing an site of activating phosphorylation These the that PP2Cα and PP2Cβ2 are the PP2C isoforms that dephosphorylate human and II reverse were from Superdex-200, and Mono Q were from PP2Cα and PP2Cβ isoforms was from were from was from and were from and II were from other were from and each of and HeLa were with a in and by at for in a P. Solomon M.J. J. Biochem. PubMed Scopus Google Scholar). The was in and at extract protein was to a with A at a of The was with A the of the at was proteins were with a from to at a of were and Cdk2/Cdk6 phosphatase activities in the were The were a and a with at a of were and Cdk2/Cdk6 phosphatase activities in the were The were a Mono Q with and with a from to in at a of as C.H. Cohen P. PubMed Scopus (116) Google Scholar). were for and To novel PP2Cβ isoforms, a of a human was the in the of human PP2Cβ as the In addition to PP2Cβ, a of was found that only the of the indicating that a novel PP2Cβ isoform, which was was from HeLa with to the The of human PP2Cα and PP2Cβ2 isoforms were from by with the and are and and were into II and PP2Cβ2, a was to an site the the acid To the of PP2Cα and PP2Cβ2 were into the of and into the of the of PP2Cα its was by and into the of which in an PP2Cβ2 its was by and into of to a of and the were and and were by and into and were by and PP2Cα, PP2Cβ2, and were in and purified as (30Cheng A. Ross K.E. Kaldis P. Solomon M.J. Genes Dev. 1999; 13: 2947-2957Crossref Scopus (128) Google Scholar). PP2Cγ was in containing by M. V. and A. R. and purified as R. Genes Dev. 1999; 13: PubMed Scopus Google Scholar). The was by E. and was in E. and purified as M. H. S. K. S. E. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). (30Cheng A. Ross K.E. Kaldis P. Solomon M.J. Genes Dev. 1999; 13: 2947-2957Crossref Scopus (128) Google Scholar), P. Sutton A. Solomon M.J. Cell. 1996; 86: 553-564Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar), and P. Solomon M.J. Mol. Biol. Cell. 1998; 9: PubMed Scopus Google Scholar) have was a from L. and N. Cdk2, and were by in the presence of as (30Cheng A. Ross K.E. Kaldis P. Solomon M.J. Genes Dev. 1999; 13: 2947-2957Crossref Scopus (128) Google Scholar). and were by to comparable and The CDK phosphatase activity and phosphatase activity were as (30Cheng A. Ross K.E. Kaldis P. Solomon M.J. Genes Dev. 1999; 13: 2947-2957Crossref Scopus (128) Google Scholar, C.H. Cohen P. PubMed Scopus (116) Google Scholar). The protein phosphatase activities of HeLa cell and recombinant PP2Cs were in the presence of and of each was with of CDKs in a reaction for at were by addition of of by and by and were by and to a with a at for in containing the were with in at by or in at for were with We previously found that PP2Cs are responsible for the of phosphatase activity Thr160 of Cdk2 in a HeLa cell extract (30Cheng A. Ross K.E. Kaldis P. Solomon M.J. Genes Dev. 1999; 13: 2947-2957Crossref Scopus (128) Google Scholar). The activity Mg2+ and was to okadaic acid or To the responsible we a HeLa cell on Superdex-200, and and for their to dephosphorylate Cdk2 on and Cdk6 on The Cdk2 and Cdk6 phosphatase activities were in the and only a in the two a of Cdk2/Cdk6 phosphatase activity the presence of than one CDK PP2Cα and PP2Cβ isoforms are the mammalian PP2Cs related to Ptc2p and Ptc3p, the budding yeast phosphatases responsible for Thr169 of We that the CDK phosphatases in HeLa PP2Cα PP2Cβ This was by the that of human PP2Cα in yeast to at a and M. J. We the Mono Q PP2Cα PP2Cβ proteins by were to higher than in D. purified that PP2Cα and two proteins in a HeLa cell of about and were to the of PP2Cα of that the and co-purified with the Cdk2/Cdk6 phosphatase activities A and The of these isoforms with Cdk2/Cdk6 phosphatase that were for the CDK phosphatases in HeLa The of the and on Mono Q were to of the previously and C.H. Cohen P. J. Biochem. PubMed Scopus Google Scholar). In the and isoforms were partially by and only the and and the the CDK phosphatase activity in was to that in it that the and proteins are of Cdk2 and We identified the and PP2C isoforms a of and for PP2Cα isoforms the PP2C the PP2C indicating that the protein is a PP2Cα and that the protein is a PP2Cβ Moreover, the PP2Cα and recombinant PP2Cα exhibited the in indicating that the PP2C was the previously reported human PP2Cα. In the PP2Cβ was than the previously reported human PP2Cβ, which acid residues and an of in A. G. 1998; PubMed Scopus Google Scholar). the of the PP2Cβ was to of reported from and (34Tamura S. Lynch K.R. Larner J. Fox J. Yasui A. Kikuchi K. Suzuki Y. Tsuiki S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1796-1800Crossref PubMed Scopus (113) Google Scholar, 35Mann D.J. Campbell D.G. McGowan C.H. Cohen 1992; PubMed Scopus Google Scholar, J. Cohen Campbell D.G. G. 1992; PubMed Scopus Google Scholar). We that the PP2Cβ an human of in these other an we identified and a novel human PP2Cβ from HeLa by The protein is to with for human PP2Cβ and for and the in the human PP2Cβ and PP2Cβ2 were PP2Cβ and PP2Cβ2 appear to PP2Cβ2 to and human PP2Cβ2 exhibited the as the PP2C on and that the HeLa PP2C is the of PP2Cα was than that of PP2Cβ2 on PP2Cα is than PP2Cβ2 We recombinant human PP2Cs dephosphorylate human Cdk2 and Cdk6 in vitro. PP2Cα and PP2Cβ2 were in E. and purified as and was and purified from as R. Genes Dev. 1999; 13: PubMed Scopus Google Scholar). the of PP2Cs may substrate specificity K. T. S. M. N. Y. R. T. S. A. S. Biochem. J. 1998; PubMed Scopus Google Scholar), we also of these and which only the necessary for activity K. T. S. M. N. Y. R. T. S. A. S. Biochem. J. 1998; PubMed Scopus Google Scholar, D. G.J. F. Biochem. J. 1996; PubMed Scopus Google Scholar). The phosphatase activities of recombinant PP2Cα, PP2Cβ2, and were comparable to each other and from to in the presence of Mg2+ the the phosphatase activity of recombinant PP2Cγ was about shown in PP2Cα and PP2Cβ2 dephosphorylated Cdk2, as the PP2Cα and PP2Cβ2 The activities of recombinant human PP2Cβ Cdk2 and were also to of PP2Cα and and M. J. In PP2Cγ activity Cdk2. We from the of these than of the substrate was that recombinant PP2Cα and PP2Cβ2 were at least Cdk2 than recombinant were with PP2Cα, PP2Cβ2, and PP2Cγ as the substrate Wip1, a type 2C protein phosphatase, was of Cdk2 PP2Cα observed to a preference for a phosphothreonine substrate with an equivalent A. McGowan C.H. Cohen P. F. PubMed Scopus Google Scholar), to the that PP2C are on threonine residues (32Das A.K. Helps N.R. Cohen P.T.W. Barford D. EMBO J. 1996; 15: 6798-6809Crossref PubMed Scopus (393) Google Scholar). the of PP2Cs to dephosphorylate and We previously that a HeLa cell extract dephosphorylated about as as P. A. Solomon M.J. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). A this that the partially purified HeLa PP2Cs and exhibited the preference for To this we recombinant PP2Cα and PP2Cβ2 and within the than of the substrate was This that PP2Cα and PP2Cβ2 have and for the with the enzymes the binding of cyclins to CDKs the dephosphorylation of yeast Cdc28p and human Cdk2 by Ptc3p, and KAP (29Poon R.Y.C. Hunter T. Science. 1995; 270: 90-93Crossref PubMed Scopus (149) Google Scholar, A. Ross K.E. Kaldis P. Solomon M.J. Genes Dev. 1999; 13: 2947-2957Crossref Scopus (128) Google Scholar), we the binding of cyclin also the dephosphorylation of Cdk2 and Cdk6 by human PP2C of Cdk2 with A the dephosphorylation of Cdk2 by PP2Cα, PP2Cβ2, and the dephosphorylation of by PP2Cα and PP2Cβ2 was by the binding of a cyclin, In these cyclins no on the phosphatase activities of PP2Cα, PP2Cβ2, and indicating that cyclins PP2C activity We previously reported that two yeast Ptc2p and Ptc3p, are the physiological protein phosphatases for the Cdc28p cyclin-dependent kinase in budding yeast and that PP2C-like activity was also responsible for >99% of the Cdk2 phosphatase activity in a HeLa cell extract (30Cheng A. Ross K.E. Kaldis P. Solomon M.J. Genes Dev. 1999; 13: 2947-2957Crossref Scopus (128) Google Scholar). We that the PP2C-like activities in HeLa cell extract are due to PP2Cβ2, a novel PP2Cβ isoform, and to PP2Cα. two proteins with of and in HeLa cell Mg2+-dependent Cdk2 and Cdk6 phosphatase activity co-purified with and isoforms during on DEAE-Sepharose, Superdex-200, and The PP2C was also by a and the as recombinant PP2Cα. The with the and exhibited the as a novel PP2Cβ PP2Cβ2 acid residues and to from and We found that recombinant PP2Cα and PP2Cβ2 efficiently dephosphorylated monomeric Cdk2 and Cdk6 in that two other PP2C isoforms, PP2Cγ and Wip1, biochemical that a relatively substrate with for HeLa cell extract, the partially purified and and recombinant PP2Cα and to budding yeast Ptc2p and Ptc3p, PP2Cα and PP2Cβ2 dephosphorylate CDKs. These that human PP2Cα and PP2Cβ2 the PP2C activity responsible for the activating phosphorylation from Cdk2 in HeLa These also that Cdk2, is a substrate for CDKs, a of kinases appear to for type 2C protein phosphatases. 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The binding of and regulatory proteins and the of the substrate for a of removal of the or of the regulatory as with CDKs cyclin that no regulatory subunits for have found and that isoforms only in their it is to that the are involved in regulating PP2C that the of PP2Cα and PP2Cβ2 dephosphorylated Cdk2 and Cdk6 as as the enzymes indicating that the may activity or substrate to determine these with within the or other of these In addition to the mammalian PP2Cα and isoforms to in budding yeast Ptc2p and In the of human PP2Cα, the residue is to its catalytic (32Das A.K. Helps N.R. Cohen P.T.W. Barford D. EMBO J. 1996; 15: 6798-6809Crossref PubMed Scopus (393) Google Scholar), it is that mammalian PP2Cα and PP2Cβ are by in necessary to this We L. and N. for cyclin, M. V. and A. R. for E. for and A. for and of the we J. D. K.E. and V.