Regulation of Chk2 phosphorylation by interaction with protein phosphatase 2A via its B' regulatory subunit

Biol Cell. 2004 Sep;96(7):509-17. doi: 10.1016/j.biolcel.2004.04.010.

Abstract

Chk2 is a key player of the DNA damage signalling pathway. To identify new regulators of this kinase, we performed a yeast two-hybrid screen and found that Chk2 associated with the B' regulatory subunit of protein phosphatase PP2A. In vitro GST-Chk2 pulldowns demonstrated that B'gamma isoforms bound to Chk2 with the strongest apparent affinity. This was confirmed in cellulo by co-immunoprecipitation after overexpression of the respective partners in HEK293 cells. The A and C subunits of PP2A were present in the complexes, suggesting that Chk2 was associated with a functionnal PP2A. In vitro kinase assays showed that B'gamma3 was a potent Chk2 substrate. This phosphorylation increased the catalytic phosphatase activity of PP2A measured on MAP kinase-phosphorylated myelin basic protein as well as on autophosphorylated Chk2. Finally, we demonstrated that overexpressing B'gamma3 in HEK293 suppressed the phosphorylation of Chk2 induced by a genotoxic treatment, suggesting that PP2A may counteract the action of the checkpoint kinase in living cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Checkpoint Kinase 2
  • Humans
  • Phosphoprotein Phosphatases / genetics
  • Phosphoprotein Phosphatases / metabolism*
  • Phosphorylation
  • Protein Binding / genetics
  • Protein Binding / physiology
  • Protein Phosphatase 2
  • Protein Structure, Tertiary / genetics
  • Protein Structure, Tertiary / physiology
  • Protein Subunits / genetics
  • Protein Subunits / metabolism*
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism*
  • Signal Transduction / genetics
  • Signal Transduction / physiology
  • Two-Hybrid System Techniques

Substances

  • Protein Subunits
  • Checkpoint Kinase 2
  • CHEK2 protein, human
  • Protein-Serine-Threonine Kinases
  • Phosphoprotein Phosphatases
  • Protein Phosphatase 2