Dephosphorylation of human cyclin-dependent kinases by protein phosphatase type 2C alpha and beta 2 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 PP2C beta 2, a novel PP2C beta isoform, and to PP2C alpha. PP2C alpha and PP2C beta 2 co-purified with Mg(2+)-dependent Cdk2/Cdk6 phosphatase activity in DEAE-Sepharose, Superdex-200, and Mono Q chromatographies. Moreover, purified recombinant PP2C alpha and PP2C beta 2 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 PP2C alpha and PP2C beta 2 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.
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520-8114, USA.
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Cheng
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Kaldis
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Solomon
D000595:Amino Acid Sequence; D000818:Animals; D002852:Chromatography, Ion Exchange; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D054648:Protein Phosphatase 2; D000071636:Protein Phosphatase 2C; D051381:Rats; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D016213:Cyclins; D007527:Isoenzymes; D029701:Saccharomyces cerevisiae Proteins; D018844:Cyclin-Dependent Kinases; C000607456:PPM1A protein, human; C087358:PTC1 protein, S cerevisiae; D010749:Phosphoprotein Phosphatases; C000607466:Ppm1a protein, mouse; D054648:Protein Phosphatase 2; D000071636:Protein Phosphatase 2C