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.
Cheng
A
A
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520-8114, USA.
Kaldis
P
P
Solomon
M J
MJ
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