A protein phosphatase feedback mechanism regulates the basal phosphorylation of Chk2 kinase in the absence of DNA damage

Biochim Biophys Acta. 2010 Oct;1803(10):1213-23. doi: 10.1016/j.bbamcr.2010.06.002. Epub 2010 Jun 22.

Abstract

The checkpoint kinase Chk2 is an effector component of the ATM-dependent DNA damage response (DDR) pathway. The activation of Chk2 by genotoxic stress involves its phosphorylation on T68 by ATM and additional auto/transphosphorylations. Here we demonstrate that in unperturbed cells, chemical inhibition of Chk2 by VRX0466617 (VRX) enhances the phosphorylation of Chk2-T68 throughout the cell cycle phases. This event, dependent on the presence of ATM and catalytically functional Chk2, is not consequential to DNA damage, as neither gamma-H2AX nuclear foci nor increased ATM activation is detected in VRX-treated cells, suggesting the involvement of other regulatory proteins. As serine/threonine protein phosphatases (PPs) regulate the phosphorylation and deactivation of proteins of the DDR pathway, we analyzed their role in phospho-T68-Chk2 regulation. We found that intracellular inhibition of PP1 and PP2A-like activities by okadaic acid markedly raised the accumulation of Chk2-pT68 without DNA damage induction, and this phenomenon was also seen when PP1-C, PP2A-C, and Wip1/PPM1D were simultaneously knockdown by siRNA. Altogether, these data indicate a novel mechanism in undamaged cells where PPs function to maintain the balance between ATM and its direct substrate Chk2 through a regulatory circuit.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Ataxia Telangiectasia Mutated Proteins
  • Blotting, Western
  • Cell Cycle Proteins / metabolism
  • Cell Line
  • Cell Line, Tumor
  • Checkpoint Kinase 2
  • DNA Damage*
  • DNA-Binding Proteins / metabolism
  • Enzyme Inhibitors / pharmacology
  • Feedback, Physiological*
  • Humans
  • Models, Biological
  • Okadaic Acid / pharmacology
  • Phosphoprotein Phosphatases / genetics
  • Phosphoprotein Phosphatases / metabolism*
  • Phosphorylation / drug effects
  • Protein Phosphatase 1 / genetics
  • Protein Phosphatase 1 / metabolism
  • Protein Phosphatase 2 / genetics
  • Protein Phosphatase 2 / metabolism
  • Protein Phosphatase 2C
  • Protein-Serine-Threonine Kinases / antagonists & inhibitors
  • Protein-Serine-Threonine Kinases / metabolism*
  • RNA Interference
  • Thiazoles / pharmacology
  • Threonine / metabolism
  • Tumor Suppressor Proteins / metabolism

Substances

  • Cell Cycle Proteins
  • DNA-Binding Proteins
  • Enzyme Inhibitors
  • Thiazoles
  • Tumor Suppressor Proteins
  • VRX0466617
  • Okadaic Acid
  • Threonine
  • Checkpoint Kinase 2
  • ATM protein, human
  • Ataxia Telangiectasia Mutated Proteins
  • CHEK2 protein, human
  • Protein-Serine-Threonine Kinases
  • PPM1D protein, human
  • Phosphoprotein Phosphatases
  • Protein Phosphatase 1
  • Protein Phosphatase 2
  • Protein Phosphatase 2C