Dephosphorylation of human cyclin-dependent kinases by protein phosphatase type 2C alpha and beta 2 isoforms

J Biol Chem. 2000 Nov 3;275(44):34744-9. doi: 10.1074/jbc.M006210200.

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 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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Chromatography, Ion Exchange
  • Cyclin-Dependent Kinases / metabolism*
  • Cyclins / metabolism
  • HeLa Cells
  • Humans
  • Isoenzymes / chemistry
  • Isoenzymes / isolation & purification
  • Isoenzymes / metabolism*
  • Mice
  • Molecular Sequence Data
  • Phosphoprotein Phosphatases / chemistry
  • Phosphoprotein Phosphatases / isolation & purification
  • Phosphoprotein Phosphatases / metabolism*
  • Phosphorylation
  • Protein Phosphatase 2
  • Protein Phosphatase 2C
  • Rats
  • Saccharomyces cerevisiae Proteins*
  • Sequence Homology, Amino Acid
  • Substrate Specificity

Substances

  • Cyclins
  • Isoenzymes
  • Saccharomyces cerevisiae Proteins
  • Cyclin-Dependent Kinases
  • PPM1A protein, human
  • PTC1 protein, S cerevisiae
  • Phosphoprotein Phosphatases
  • Ppm1a protein, mouse
  • Protein Phosphatase 2
  • Protein Phosphatase 2C

Associated data

  • GENBANK/AF294792