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• W1992258488 abstract "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. Biochem. J. 1995; 308: 697-711Crossref PubMed Scopus (498) Google Scholar, 2Sherr C.J. Roberts J.M. Genes Dev. 1995; 9: 1149-1163Crossref PubMed Scopus (3209) Google Scholar, 3Sherr C.J. Roberts J.M. Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5131) Google Scholar, 4King R.W. Deshaies R.J. Peters J.-M. Kirschner M.W. Science. 1996; 274: 1652-1659Crossref PubMed Scopus (1117) Google Scholar, 5Morgan D.O. Curr. Opin. Cell Biol. 1996; 8: 767-772Crossref PubMed Scopus (88) Google Scholar, 6Morgan D.O. Annu. Rev. Cell Dev. Biol. 1997; 13: 261-291Crossref PubMed Scopus (1796) Google Scholar, 7Solomon M.J. Kaldis P. Pagano M. Results and Problems in Cell Differentiation “Cell Cycle Control”. Springer, Heidelberg, Germany1998: 79-109Google Scholar). In the budding yeast Saccharomyces cerevisiae, Cdc28p is the main CDK involved in regulating the cell division cycle. On the other hand, Cdc2 (Cdk1), Cdk2, Cdk4, and Cdk6 control cell cycle progression in higher eukaryotes. 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 (498) Google Scholar, 4King R.W. Deshaies R.J. Peters J.-M. Kirschner M.W. Science. 1996; 274: 1652-1659Crossref PubMed Scopus (1117) Google Scholar, 8Evans T. Rosenthal E.T. Youngblom J. Distel D. Hunt T. Cell. 1983; 33: 389-396Abstract Full Text PDF PubMed Scopus (1003) Google Scholar, 9Sherr C.J. Cell. 1994; 79: 551-555Abstract Full Text PDF PubMed Scopus (2586) 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 (1796) 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 (1172) 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 (95) Google Scholar, 12Gould K.L. Moreno S. Owen D.J. Sazer S. Nurse P. EMBO J. 1991; 10: 3297-3309Crossref PubMed Scopus (326) Google Scholar, 13Lee T.H. Solomon M.J. Mumby M.C. Kirschner M.W. Cell. 1991; 64: 415-423Abstract Full Text PDF PubMed Scopus (163) Google Scholar, 14Krek W. Nigg E.A. The New Biologist. 1992; 4: 323-329PubMed Google Scholar, 15Solomon M.J. Lee T. Kirschner M.W. Mol. Biol. Cell. 1992; 3: 13-27Crossref PubMed Scopus (354) Google Scholar, 16Cismowski M.J. Laff G.M. Solomon M.J. Reed S.I. Mol. Cell. Biol. 1995; 15: 2983-2992Crossref PubMed Scopus (189) Google Scholar). This activating phosphorylation is carried out by Cdk-activating kinases (CAKs). Higher eukaryotic CAK, primarily localized to the nucleus, is composed of p40MO15/Cdk7, cyclin H, and an assembly factor, MAT1. These proteins also function as components of basal transcription factor IIH (17Feaver W.J. Svejstrup J.Q. Henry N.L. Kornberg R.D. Cell. 1994; 79: 1103-1109Abstract Full Text PDF PubMed Scopus (359) Google Scholar, 18Roy R. Adamczewski J.P. Seroz T. Vermeulen W. 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 (388) 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 (308) 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 (363) 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 (146) 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 (1322) Google Scholar, 27Hannon G.J. Casso D. Beach D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1731-1735Crossref PubMed Scopus (112) 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 (5230) 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 (127) 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 (127) 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 (2151) 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 (384) 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 (2151) 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 (127) Google Scholar, 33Stark M.J.R. Yeast. 1996; 12: 1647-1675Crossref PubMed Scopus (170) 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 (105) Google Scholar, 35Mann D.J. Campbell D.G. McGowan C.H. Cohen P.T. Biochim. Biophys. Acta. 1992; 1130: 100-104Crossref PubMed Scopus (60) Google Scholar, 36Wenk J. Trompeter H.I. Pettrich K.G. Cohen P.T. Campbell D.G. Mieskes G. FEBS Lett. 1992; 297: 135-138Crossref PubMed Scopus (70) Google Scholar, 37Guthridge M.A. Bellosta P. Tavoloni N. Basilico C. Mol. Cell. Biol. 1997; 17: 5485-5498Crossref PubMed Scopus (52) Google Scholar, 38Travis S.M. Welsh M.J. FEBS Lett. 1997; 412: 415-419Crossref PubMed Scopus (47) Google Scholar, 39Murray M.V. Kobayashi R. Krainer A.R. Genes Dev. 1999; 13: 87-97Crossref PubMed Scopus (73) Google Scholar, 40Tong Y. Quirion R. Shen S.H. J. Biol. Chem. 1998; 273: 35282-35290Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 41Fiscella M. Zhang H. Fan S. Sakaguchi K. Shen S. Mercer W.E. Vande Woude G.F. O'Connor P.M. Apella E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6048-6053Crossref PubMed Scopus (458) Google Scholar, 42Kitani T. Ishida A. Okuno S. Takeuchi M. Kameshita I. Fujisawa H. J. Biochem. ( Tokyo ). 1999; 125: 1022-1028Crossref PubMed Scopus (51) Google Scholar, 43Labes M. Roder J. Roach A. Mol. Cell Neurosci. 1998; 12: 29-47Crossref PubMed Scopus (17) Google Scholar). In addition, human PP2Cα has two isoforms resulting from alternative splicing (44Takekawa M. Maeda T. Saito H. EMBO J. 1998; 17: 4744-4752Crossref PubMed Scopus (239) Google Scholar), and murine PP2Cβ has five isoforms (for comparison, see Ref. 45Kusuda K. Kobayashi T. Ikeda S. Ohnishi M. Chida N. Yanagawa Y. Shineha R. Nishihira T. Satomi S. Hiraga A. Tamura S. Biochem. J. 1998; 332: 243-250Crossref PubMed Scopus (41) Google Scholar). In this study, we further characterized the PP2C activities capable of dephosphorylating human Cdk2 and Cdk6 in a HeLa cell extract. Fractionation of proteins by DEAE-Sepharose, Superdex-200, and Mono Q chromatographies demonstrated that Cdk2 and Cdk6 phosphatase activities co-purified with PP2Cα, a previously reported PP2C isoform, and PP2Cβ2, a novel human PP2Cβ isoform apparently resulting from alternative splicing. Recombinant PP2Cα and PP2Cβ2 effectively dephosphorylated monomeric- but not cyclin-bound Cdk2 and Cdk6 in vitro, confirming previous studies 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 (127) Google Scholar). Further enzymatic characterization showed that crude HeLa cell extract, partially purified PP2Cα and PP2Cβ2, and recombinant PP2Cα and PP2Cβ2 all exhibited a substrate preference for wild-type (Thr160) Cdk2 compared with a mutant Cdk2 protein containing an altered site of activating phosphorylation (Ser160). These results support the conclusion that PP2Cα and PP2Cβ2 are the PP2C isoforms that dephosphorylate human CDKsin vivo. Tissue culture medium, TRIzol, and SUPERSCRIPT II RNase H− reverse transcriptase were from Life Technologies (Grand Island, NY). DEAE-Sepharose Fast Flow, Superdex-200, and Mono Q HR5/5 were from Amersham Pharmacia Biotech. An antibody recognizing PP2Cα and PP2Cβ isoforms (catalog number 539548) was from Calbiochem (San Diego, CA). Sheep anti-PP2Cα antibodies (catalog number 06-523) were from Upstate Biotechnology (Lake Placid, NY). [γ-32P]ATP (3000 Ci/mmol) was from PerkinElmer Life Sciences. Horseradish peroxidase-conjugated secondary antibodies and SuperSignalTM ECL reagents were from Pierce (Rockford, IL).Pfu Turbo DNA polymerase and pBlueScript II KS(−) were from Stratagene (La Jolla, CA). All other chemicals were from Sigma unless indicated otherwise. 1 × protease inhibitors contained 1 mm phenylmethylsulfonyl fluoride and 10 μg/ml each of leupeptin, chymostatin, and pepstatin. HeLa S3 cells were lysed with a Dounce homogenizer in hypotonic buffer (10 mmHepes, pH 7.9 (at 4 °C), 1.5 mm MgCl2, 10 mm KCl, 1 mm dithiothreitol, 1 × protease inhibitors) and clarified by centrifugation at 100,000 ×g for 1 h in a 60-Ti rotor (46Kaldis P. Solomon M.J. Eur. J. Biochem. 2000; 267: 4213-4221Crossref PubMed Scopus (50) Google Scholar). The supernatant was frozen in liquid nitrogen and stored at −80 °C until use. Clarified extract (25 ml, protein concentration 6.3 mg/ml) was applied to a DEAE-Sepharose Fast Flow column (1.5 × 10 cm) pre-equilibrated with buffer A (20 mm Tris-HCl, pH 7.4, 0.1 mmEDTA, 0.1 mm EGTA, 0.01% Chaps, 0.1% β-mercaptoethanol, 1 × protease inhibitors) at a flow rate of 1 ml/min. The column was washed with buffer A until the absorbance of the elute at 280 nm was <0.05. Bound proteins were eluted with a 20-ml linear gradient from 0 to 1.0 m NaCl at a flow rate of 1 ml/min. One-ml fractions were collected, and Cdk2/Cdk6 phosphatase activities in the fractions were assayed (see below). The active fractions were pooled, concentrated using a Vivaspin concentrator (10,000 MWCO; Vivascience LTD., Binbrook Hill, United Kingdom), and loaded onto a Superdex-200 column pre-equilibrated with buffer B (20 mmtriethanolamine-HCl, pH 7.0 (at 25 °C), 5% glycerol, 0.01% Chaps, 0.1 mm EDTA, 0.1 mm EGTA, 0.1% β-mercaptoethanol, 1 × protease inhibitors) at a flow rate of 0.5 ml/min. One-ml fractions were collected, and Cdk2/Cdk6 phosphatase activities in the fractions were assayed (see below). The active fractions (∼45-kDa) were pooled, loaded onto a Mono Q HR5/5 column pre-equilibrated with buffer B, and developed with a linear salt gradient from 0 to 700 mm NaCl in buffer B at a flow rate of 0.5 ml/min as described (47McGowan C.H. Cohen P. Methods Enzymol. 1988; 159: 416-426Crossref PubMed Scopus (112) Google Scholar). 0.5-ml fractions were collected, assayed for activity, and immunoblotted (see below). To identify novel PP2Cβ isoforms, a BLASTN search of a human EST data base was performed using the last 420 nucleotides in the 3′-coding region of human PP2Cβ as the query. In addition to PP2Cβ, a second group of EST clones was found that only matched the first ∼120 nucleotides of the query, indicating that they encode a novel PP2Cβ isoform, which was named PP2Cβ2. Total RNA was isolated from HeLa cells with TRIzol reagent according to the manufacturer's protocol. The full-length coding regions of human PP2Cα and PP2Cβ2 isoforms were amplified from total RNA by RT-PCR with the following primers (start and stop codons are underlined): PP2Cα: 5′-CCCCATATGGGAGCATTTTTAGACAAG-3′ and 5′-CCCCAAGCTTTTACCACATATCATCTGTTG-3′; PP2Cβ2: 5′-GCCCCATGGGTGCATTTTTGGATAAACC-3′ and 5′-CGGGCTCGAGCTACCATGGGTCTTCTAGATC-3′. PCR fragments were inserted into pBlueScript II KS(−) and sequenced. For PP2Cβ2, a second COOH-terminal primer was used to eliminate an internalNcoI site before the stop codon without changing the amino acid residue. To express non-tagged proteins, the full-length coding regions of PP2Cα and PP2Cβ2 were inserted into the NdeI/HindIII sites of pET21a and into the NcoI/XhoI sites of pET21d, respectively. For the expression of hexahistidine-tagged (His6) enzymes, PP2Cα (with its stop codon) was amplified by PCR and inserted into the BamHI-HindIII sites of pET28a, which resulted in an NH2-terminal hexahistidine-tagged fusion protein. PP2Cβ2 (without its stop codon) was amplified by PCR and inserted into theNcoI-XhoI sites of pET21d to produce a COOH-terminal hexahistidine-tagged protein. For expression of NH2-terminal His6-fused PP2Cα1–308 (PP2CαΔC) and COOH-terminal His6-tagged PP2Cβ21–312 (PP2Cβ2ΔC), the following COOH-terminal primers were used: PP2Cα1–308, 5′-CCCCAAGCTTTTACAACTCTGCCTCCTTCTCAC-3′ and PP2Cβ1–312, 5′-CCCCTCGAGCAACTCTGAATCTTTTTTCAC-3′. PP2CαΔC and PP2Cβ2ΔC were amplified by PCR and inserted into pET28a and pET21d, respectively. All constructs were confirmed by DNA sequencing. GST-Cdk2, GST-Cdk2Ser-160, and His6-tagged PP2Cα, PP2Cβ2, PP2CαΔC, and PP2Cβ2ΔC were expressed in Escherichia coli and purified as described (30Cheng A. Ross K.E. Kaldis P. Solomon M.J. Genes Dev. 1999; 13: 2947-2957Crossref Scopus (127) Google Scholar). PP2Cγ was expressed in BL21(DE3) cells containing pET19b-PP2Cγ (provided by M. V. Murray and A. R. Krainer) and purified as described (39Murray M.V. Kobayashi R. Krainer A.R. Genes Dev. 1999; 13: 87-97Crossref PubMed Scopus (73) Google Scholar). The GST-Wip1 plasmid was provided by E. Appella and GST-Wip1 was expressed in E. coli and purified as described (41Fiscella M. Zhang H. Fan S. Sakaguchi K. Shen S. Mercer W.E. Vande Woude G.F. O'Connor P.M. Apella E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6048-6053Crossref PubMed Scopus (458) Google Scholar). GST-cyclin A173–432 (30Cheng A. Ross K.E. Kaldis P. Solomon M.J. Genes Dev. 1999; 13: 2947-2957Crossref Scopus (127) Google Scholar), GST-Cak1p (23Kaldis P. Sutton A. Solomon M.J. Cell. 1996; 86: 553-564Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar), and GST-Cdk6 (63Kaldis P. Russo A.A. Chou H.S. Pavletich N.P. Solomon M.J. Mol. Biol. Cell. 1998; 9: 2545-2560Crossref PubMed Scopus (92) Google Scholar) have been described previously. Purified GST-KSHV-cyclin was a gift from L. Tong and N. Pavletich. Cdk2, GST-Cdk2, GST-Cdk6, and GST-Cdk2Ser-160 were phosphorylated by GST-Cak1p in the presence of [γ-32P]ATP as described (30Cheng A. Ross K.E. Kaldis P. Solomon M.J. Genes Dev. 1999; 13: 2947-2957Crossref Scopus (127) Google Scholar). GST-Cdk2Thr-160 and GST-Cdk2Ser-160 were labeled by Cak1p to comparable and nearly saturated levels. The CDK phosphatase activity and casein phosphatase activity were determined as described (30Cheng A. Ross K.E. Kaldis P. Solomon M.J. Genes Dev. 1999; 13: 2947-2957Crossref Scopus (127) Google Scholar, 47McGowan C.H. Cohen P. Methods Enzymol. 1988; 159: 416-426Crossref PubMed Scopus (112) Google Scholar). The protein phosphatase activities of HeLa cell lysate and recombinant PP2Cs were assayed in the presence of 5 and 20 mm MgCl2, respectively. Briefly, 5 μl of each fraction was incubated with 50 ng of 32P-labeled CDKs in a 20-μl reaction for 15 min at room temperature. Reactions were terminated by addition of 10 μl of 3 × sample buffer, separated by 10% SDS-PAGE, and analyzed by autoradiography and PhosphorImager (Molecular Imager GS-250, Bio-Rad). Samples were resolved by SDS-PAGE (10% total acrylamide) and transferred to a polyvinylidene difluoride membrane (Immobilon-P, Millipore) with a semi-dry blotting apparatus (TransBlot-SD, Bio-Rad). After blocking at room temperature for 2 h in Blotto (1 × TBST containing 5% nonfat dry milk), the membranes were incubated with anti-PP2C polyclonal antibody (1 μg/ml in Blotto) overnight at 4 °C followed by horseradish peroxidase-conjugated goat anti-rabbit secondary antibody or rabbit anti-sheep secondary antibody (Pierce, 1:2000 dilution in Blotto) at room temperature for 2 h. Antibodies were detected with SuperSignal ECL reagents (Pierce). We previously found that PP2Cs are responsible for the vast majority (>99%) of phosphatase activity toward 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 (127) Google Scholar). The activity required Mg2+ and was insensitive to okadaic acid or sodium vanadate. To identify the responsible phosphatase(s), we fractionated a HeLa cell lysate on DEAE-Sepharose Fast Flow, Superdex-200, and Mono-Q columns and assayed fractions for their abilities to dephosphorylate Cdk2 phosphorylated on Thr-160 (Fig. 1) and Cdk6 phosphorylated on Thr177 (data not show). The Cdk2 and Cdk6 phosphatase activities were present in the same fractions and showed only a single sharp peak in the first two columns. Mono-Q chromatography, however, showed a broader distribution of Cdk2/Cdk6 phosphatase activity (Fig. 1D), suggesting the presence of more than one CDK phosphatase. PP2Cα and PP2Cβ isoforms are the mammalian PP2Cs most closely related to Ptc2p and Ptc3p, the budding yeast phosphatases responsible for dephosphorylating Thr169 of Cdc28p. We speculated that the CDK phosphatases in HeLa cells might be PP2Cα and/or PP2Cβ isoforms. This suggestion was supported by the observation that overexpression of human PP2Cα in yeast led to synthetic lethality incak1–22 ts cells at a semi-permissive temperature. 2A. Cheng and M. J. Solomon, unpublished data. We determined whether the active Mono Q fractions contained PP2Cα and/or PP2Cβ proteins by immunoblotting. 0.5-ml fractions were collected to provide higher resolution than in Fig. 1 D. Affinity purified antibodies that specifically recognize both PP2Cα and β detected two proteins in a HeLa cell lysate (Fig.2 B). Their sizes of about 45 and 42 kDa were similar to the calculated molecular mass of PP2Cα (∼42-kDa). Immunoblotting of Mono-Q fractions showed that the 45- and 42-kDa PP2Cα/βs co-purified with the Cdk2/Cdk6 phosphatase activities (Fig. 2, A and B). The total amounts of these isoforms correlated well with Cdk2/Cdk6 phosphatase activity, suggesting that they were likely candidates for the CDK phosphatases in HeLa cells. The elution profiles of the 45- and 42-kDa PP2Cα/βs on Mono Q chromatography were similar to those of the previously described rabbit PP2C1 and PP2C2 isoenzymes, respectively (48McGowan C.H. Cohen P. Eur. J. Biochem. 1987; 166: 713-722Crossref PubMed Scopus (55) Google Scholar). In addition, the 45- and 42-kDa PP2Cα/β isoforms were partially separable by Mono-Q chromatography (Fig. 2 B): fractions 30 and 31 contained only the 45-kDa PP2Cα/β and factions 34 and 35 contained almost exclusively the 42-kDa PP2Cα/β. Since the CDK phosphatase activity in fraction 34 was similar to that in fraction 30 (Fig. 2 A), it appears that both the 45- and 42-kDa PP2Cα/β proteins are capable of dephosphorylating Cdk2 and Cdk6. We identified the 45- and 42-kDa PP2C isoforms using a combination of immunoblotting and molecular cloning. An antibody specific for PP2Cα isoforms recognized the 45-kDa PP2C but not the 42-kDa PP2C (Fig. 2 C), indicating that the 45-kDa protein is a PP2Cα isoform and that the 42-kDa protein is a PP2Cβ isoform. Moreover, both the 45-kDa PP2Cα isoform and recombinant PP2Cα exhibited the same mobility in SDS-PAGE (Fig. 2 D, lanes 1and 2), indicating that the 45-kDa PP2C was likely the previously reported human PP2Cα. In contrast, the 42-kDa PP2Cβ isoform was much smaller than the previously reported human PP2Cβ, which has 479 amino acid residues and an apparent molecular mass of ∼55-kDa in SDS-PAGE (49Marley A.E. Kline A. Crabtree G. Sullivan J.E. Beri R.K. FEBS Lett. 1998; 431: 121-124Crossref PubMed Scopus (23) Google Scholar). Instead, the size of the 42-kDa PP2Cβ isoform was similar to those of reported PP2Cβs from rabbit, mouse, and rat (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 (105) Google Scholar, 35Mann D.J. Campbell D.G. McGowan C.H. Cohen P.T. Biochim. Biophys. Acta. 1992; 1130: 100-104Crossref PubMed Scopus (60) Google Scholar, 36Wenk J. Trompeter H.I. Pettrich K.G. Cohen P.T. Campbell D.G. Mieskes G. FEBS Lett. 1992; 297: 135-138Crossref PubMed Scopus (70) Google Scholar). We speculated that the 42-kDa PP2Cβ might be an unreported human ortholog of PP2Cβs in these other mammals. By searching an EST data base, we identified and cloned a novel human PP2Cβ isoform (“PP2Cβ2”) from HeLa cells by RT-PCR. The encoded protein is predicted to be 387 amino acids, compared with 479 amino acids for human PP2Cβ and 390 amino acids for mouse and rat PP2Cβ. Since the first 1134 nucleotides (378 amino acids) in the human PP2Cβ and PP2Cβ2 coding regions were identical, PP2Cβ and PP2Cβ2 appear to arise via alternative splicing. Human PP2Cβ2 showed ∼95% identity to mouse and rat PP2Cβs (Fig.3). Recombinant human PP2Cβ2 exhibited the same mobility as the 42-kDa PP2C on SDS-PAGE (Fig. 2 D, lanes 3 and 4), suggesting that the 42-kDa HeLa PP2C is PP2Cβ2. Interestingly, the mobility of PP2Cα was less than that of PP2Cβ2 on SDS-PAGE even though PP2Cα (382 aa) is slightly smaller than PP2Cβ2 (387 amino acids). We next determined whether recombinant human PP2Cs could dephosphorylate human Cdk2 and Cdk6 in vitro. PP2Cα and PP2Cβ2 were expressed in E. coli and purified as NH2-terminal and COOH-terminal hexahistidine-tagged (His6) proteins, respectively. His6-PP2Cγ was expressed and purified from BL21(DE3) cells bearing pET19b-PP2Cγ as described (39Murray M.V. Kobayashi R. Krainer A.R. Genes Dev. 1999; 13: 87-97Crossref PubMed Scopus (73) Google Scholar). Since the COOH-terminal portions of PP2Cs may regulate substrate specificity (45Kusuda K. Kobayashi T. Ikeda S. Ohnishi M. Chida N. Yanagawa Y. Shineha R. Nishihira T. Satomi S. Hiraga A. Tamura S. Biochem. J. 1998; 332: 243-250Crossref PubMed Scopus (41) Google Scholar), we also prepared His6-tagged COOH-terminal deletions of these proteins, PP2CαΔC (PP2Cα1–308) and PP2Cβ2ΔC (PP2Cβ21–312), which only contained the minimal regions necessary for activity (45Kusuda K. Kobayashi T. Ikeda S. Ohnishi M. Chida N. Yanagawa Y. Shineha R. Nishihira T. Satomi S. Hiraga A. Tamura S. Biochem. J. 1998; 332: 243-250Crossref PubMed Scopus (41) Google Scholar, 50Marley A.E. Sullivan J.E. Carling D. Abbott W.M. Smith G.J. Taylor I.W. Carey F. Beri R.K. Biochem. J. 1996; 320: 801-806Crossref PubMed Scopus (76) Google Scholar). The casein phosphatase activities of recombinant PP2Cα, PP2Cβ2, PP2CαΔC, and PP2Cβ2ΔC were comparable to each other and ranged from 91 to 109 milliunits/mg in the presence of 20 mm Mg2+ (Fig.4 A). Under the same conditions, the casein phosphatase activity of recombinant PP2Cγ was about 40 milliunits/mg. As shown in Fig. 4 B, PP2Cα and PP2Cβ2 effectively dephosphorylated Cdk2, as did the COOH-terminal truncated PP2Cα and PP2Cβ2 enzymes. The activities of recombinant human PP2Cβ against Cdk2 and casein were also similar to those of PP2Cα and PP2Cβ2. 3A. Cheng and M. J. Solomon, unpublished results. In contrast, PP2Cγ had very low activity toward Cdk2. We estimated from the linear phases of these reactions (when less than 30% of the substrate was used) that recombinant PP2Cα and PP2Cβ2 were at least 40-fold more active toward Cdk2 than recombinant PP2Cγ. Similar results were obtained with PP2Cα, PP2Cβ2, and PP2Cγ using GST-Cdk6 as the substrate (Fig. 4 C). Wip1, a p53-induced type 2C protein phosphatase, was incapable of dephosphorylating Cdk2 (data not shown). PP2Cα has been observed to show a 20-fold preference for a phosphothreonine peptide substrate compared with an equivalent phosphoserine substratein vitro (51Donella Deana A. McGowan C.H. Cohen P. Marciori F. Meyer H.E. Pinna L.A. Biochim. Biophys. Acta. 1990; 1051: 199-202Crossref PubMed Scopus (72) Google Scholar), leading to the suggestion that PP2C substrates are generally phosphorylated on threonine residues (32Das A.K. Helps N.R. Cohen P.T.W. Barford D. EMBO J. 1996; 15: 6798-6809Crossref PubMed Scopus (384) Google Scholar). We, therefore, compared the abilities of PP2Cs to dephosphorylate wild-type Cdk2Thr-160 and mutant Cdk2Ser-160. We previously showed that a HeLa cell extract dephosphorylated Cdk2Thr-160 about 4 times as fast as Cdk2Ser-160 (52Kaldis P. Cheng A. Solomon M.J. J. Biol. Chem. 2000; 275: 32578-32584Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Fig.5 A confirms this preference. Fig. 5 B shows that the partially purified HeLa PP2Cs (Mono-Q fractions 31 and 34) exhibited the same qualitative preference for Cdk2Thr-160. To quantitate this effect, we used recombinant PP2Cα and PP2Cβ2 and performed assays within the linear range (when less than 30% of the substrate was dephosphorylated). This analysis showed that PP2Cα and PP2Cβ2 have 3- and 2.7-fold preferences for Cdk2Thr-160 over Cdk2Ser-160 (Fig. 5 C), confirming the qualitative observations with the native enzymes (Fig.5 B). Since the binding of cyclins to CDKs blocked the dephosphorylation of yeast Cdc28p and human Cdk2 by Ptc2p, Ptc3p, and KAP (29Poon R.Y.C. Hunter T. Science. 1995; 270: 90-93Crossref PubMed Scopus (149) Google Scholar, 30Cheng A. Ross K.E. Kaldis P. Solomon M.J. Genes Dev. 1999; 13: 2947-2957Crossref Scopus (127) Google Scholar), we tested whether the binding of cyclin could also inhibit the dephosphorylation of Cdk2 and Cdk6 by human PP2C isoforms. Preincubation of Cdk2 with excess GST-cyclin A blocked the dephosphorylation of Cdk2 by PP2Cα, PP2Cβ2, PP2CαΔC, and PP2Cβ2ΔC (Fig. 6 A). Similarly, the dephosphorylation of GST-Cdk6 by PP2Cα and PP2Cβ2 was blocked by the binding of a viral D-type cyclin, KSHV-cyclin (Fig.6 B). In contrast, these cyclins had no effects on the casein phosphatase activities of PP2Cα, PP2Cβ2, PP2Cγ, PP2CαΔC, and PP2Cβ2ΔC (Fig. 6 C), indicating that cyclins did not inhibit PP2C activity directly. We previously reported that two yeast PP2Cs, Ptc2p and Ptc3p, are the major 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 (127) Google Scholar). We now provide evidence that the PP2C-like activities in HeLa cell extract are due to PP2Cβ2, a novel PP2Cβ isoform, and to PP2Cα. PP2Cα/β-specific antibodies detected two proteins with apparent molecular masses of 45- and 42-kDa in HeLa cell lysate. Mg2+-dependent Cdk2 and Cdk6 phosphatase activity co-purified with 45- and 42-kDa PP2Cα/β isoforms during chromatography on DEAE-Sepharose, Superdex-200, and Mono-Q columns. The 45-kDa PP2C was also recognized by a PP2Cα-specific antibody and had the same electrophoretic mobility as recombinant PP2Cα. The 42-kDa PP2Cα/β did not react with the PP2Cα-specific antibody and exhibited the same electrophoretic mobility as a novel PP2Cβ isoform (PP2Cβ2). PP2Cβ2 possesses 387 amino acid residues and shows ∼95% identity to PP2Cβ's from mouse, rat, and rabbit. We found that recombinant PP2Cα and PP2Cβ2 efficiently dephosphorylated monomeric Cdk2 and Cdk6 in vitro but that two other PP2C isoforms, PP2Cγ and Wip1, did not. Further biochemical analysis demonstrated that Cdk2Ser-160was a relatively poor substrate compared with wild-type Cdk2Thr-160 for HeLa cell extract, the partially purified 45- and 42-kDa PP2Cs, and recombinant PP2Cα and PP2Cβ2. Similar to budding yeast Ptc2p and Ptc3p, PP2Cα and PP2Cβ2 could not dephosphorylate cyclin-bound CDKs. These results indicate that human PP2Cα and PP2Cβ2 represent the PP2C activity responsible for removing the activating phosphorylation from Cdk2 in HeLa cells. These studies also provide evidence that Cdk6, like Cdk2, is a substrate for PP2Cs. Besides CDKs, a number of MAP kinases appear to be substrates for type 2C protein phosphatases. For instance, PP2Cs have been implicated in negatively regulating stress-responsive protein kinase cascades in eukaryotic cells. In both budding yeast and fission yeast, genetic studies have shown that PP2C-like enzymes oppose the activation of the MAP kinase pathway that is activated in response to osmotic and heat shocks (53Maeda T. Wurgler-Murphy S.M. Saito H. Nature. 1994; 369: 242-245Crossref PubMed Scopus (937) Google Scholar, 54Shiozaki K.H. Akhavan-Niaki H. McGowan C.H. Russell P. Mol. Cell. Biol. 1994; 14: 3742-3751Crossref PubMed Scopus (56) Google Scholar, 55Shiozaki K. Russell P. EMBO J. 1995; 14: 492-502Crossref PubMed Scopus (151) Google Scholar). In human cells, PP2Cα can reverse the activation of the p38 and JNK MAPKs induced by stress and cytokines (44Takekawa M. Maeda T. Saito H. EMBO J. 1998; 17: 4744-4752Crossref PubMed Scopus (239) Google Scholar). Biochemically, a human PP2Cα isoform dephosphorylated a similar threonine within the activation loop of the p38 MAPK (44Takekawa M. Maeda T. Saito H. EMBO J. 1998; 17: 4744-4752Crossref PubMed Scopus (239) Google Scholar). Recently, Ptc1 and Ptc3 in Schizosaccharomyces pombe were shown to dephosphorylate Thr171 of the p38 homolog (Spc1) in its activating loop (56Nguyen A.N. Shiozaki K. Genes Dev. 1999; 13: 1653-1663Crossref PubMed Scopus (106) Google Scholar). Given the similarity between the sites of activating phosphorylation in MAPKs and CDKs, we have proposed that PP2C-like enzymes could be general T-loop protein phosphatases (30Cheng A. Ross K.E. Kaldis P. Solomon M.J. Genes Dev. 1999; 13: 2947-2957Crossref Scopus (127) Google Scholar). The activities of PP2Cs toward a variety of substrates can be affected by whether the site to be dephosphorylated is a serine or a threonine and by regulatory factors that bind to the substrates. Biochemically, PP2Cα has been seen to dephosphorylate a phosphothreonine substrate 20-fold more efficiently than the corresponding phosphoserine substratein vitro (51Donella Deana A. McGowan C.H. Cohen P. Marciori F. Meyer H.E. Pinna L.A. Biochim. Biophys. Acta. 1990; 1051: 199-202Crossref PubMed Scopus (72) Google Scholar) and PP2C substrates have been proposed to be phosphorylated on threonine residues in vivo (32Das A.K. Helps N.R. Cohen P.T.W. Barford D. EMBO J. 1996; 15: 6798-6809Crossref PubMed Scopus (384) Google Scholar). Using human Cdk2, a likely physiological substrate for PP2Cs, we confirmed that PP2Cα and PP2Cβ2 removed the phosphate from a phosphoserine substrate slower (∼3-fold) than from the phosphothreonine substrate (Fig. 5). Indeed, many PP2C substrates, including all known CDKs undergoing activating phosphorylation, AMPK (57Clarke P.R. Moore F. Hardie D.G. Adv. Protein Phosphatases. 1991; 6: 187-209Google Scholar, 58Moore F. Weekes J. Hardie D.G. Eur. J. Biochem. 1991; 199: 691-697Crossref PubMed Scopus (192) Google Scholar, 59Davies S.P. Helps N.R. Cohen P.T. Hardie D.G. FEBS Lett. 1995; 377: 421-425Crossref PubMed Scopus (497) Google Scholar), moesin (62Hishiya A. Ohnishi M. Tamura S. Nakamura F. J. Biol. Chem. 1999; 274: 26705-26712Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), and p38 MAPK (44Takekawa M. Maeda T. Saito H. EMBO J. 1998; 17: 4744-4752Crossref PubMed Scopus (239) Google Scholar), are phosphorylated on threonine residues. However, some PP2C substrates such as axin (60Strovel E.T. Wu D. Sussman D.J. J. Biol. Chem. 2000; 275: 2399-2403Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) and CFTR (61Travis S.M. Berger H.A. Welsh M.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11055-11060Crossref PubMed Scopus (59) Google Scholar) may be phosphorylated on serines. Since exchanges of serines and threonines often have little effect on protein functions, the replacement of a threonine with a serine could be used to control the duration of phosphorylation. In addition, the binding of ligands or regulatory proteins to substrates also influences the rate of dephosphorylation by PP2Cs. For example, the dephosphorylation of AMPK is inhibited in the presence of 5′-AMP (59Davies S.P. Helps N.R. Cohen P.T. Hardie D.G. FEBS Lett. 1995; 377: 421-425Crossref PubMed Scopus (497) Google Scholar), and the dephosphorylation of CDKs is blocked by the binding of cyclins (Fig. 6,A-B, and Ref. 30Cheng A. Ross K.E. Kaldis P. Solomon M.J. Genes Dev. 1999; 13: 2947-2957Crossref Scopus (127) Google Scholar). The binding of ligands and regulatory proteins could induce conformational changes, block dephosphorylation, and preserve the phosphorylated state of the substrate for a period of time. Dephosphorylation could occur rapidly following removal of the ligand or of the regulatory proteins, such as happens with CDKs after cyclin degradation. Given that no regulatory subunits for PP2Cα/β have been found and that PP2Cα/β isoforms only differ significantly in their COOH-terminal segments, it is tempting to speculate that the diverse COOH-terminal regions are involved in regulating PP2C activity. Our studies showed that the COOH-terminal truncated forms of PP2Cα and PP2Cβ2 dephosphorylated Cdk2 and Cdk6 as well as the full-length enzymes (Fig. 4), indicating that the carboxyl regions may not directly regulate enzymatic activity or substrate specificity. Additional work will be required to determine whether these segments might facilitate interactions with substrates, localization within the cell, or other properties of these enzymes. In addition to the COOH-terminal regions, mammalian PP2Cα and -β isoforms possess potentialN-myristoylation sites similar to those in budding yeast Ptc2p and Ptc3p. In the crystal structure of human PP2Cα, the NH2-terminal glycine residue is close to its catalytic center (32Das A.K. Helps N.R. Cohen P.T.W. Barford D. EMBO J. 1996; 15: 6798-6809Crossref PubMed Scopus (384) Google Scholar), therefore, it is possible that mammalian PP2Cα and PP2Cβ are regulated by N-myristoylation in vivo. Further experiments will be necessary to test this possibility. We thank L. Tong and N. Pavletich for GST-KSHV cyclin, M. V. Murray and A. R. Krainer for PP2Cγ, E. Appella for GST-Wip1, and A. Natrillo for technical assistance. For helpful discussions and critical reading of the manuscript, we thank J. Burton, D. Ostapenko, K.E. Ross, and V. Tsakraklides." @default.
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• W1992258488 title "Dephosphorylation of Human Cyclin-dependent Kinases by Protein Phosphatase Type 2Cα and β2 Isoforms" @default.
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