Utilizing protein phosphatase inhibitors to define PP2A as a regulator of ataxia-telangiectasia mutated

Methods Mol Biol. 2007:365:47-59. doi: 10.1385/1-59745-267-X:47.

Abstract

Ataxia-telangiectasia mutated (ATM) is a serine/threonine protein kinase that plays a central role in controlling the cellular response to DNA double-strand breaks caused by ionizing radiation. Ionizing radiation induces the autophosphorylation of ATM on serine 1981; however, the precise mechanisms that regulate ATM autophosphorylation are not fully understood. By treating cells with okadaic acid, a cell-permeable protein phosphatase inhibitor, together with assays to quantify the activity of particular protein phosphatases, we have demonstrated that the autophosphorylation of ATM on serine 1981 is regulated by a protein phosphatase 2A-like activity. Here, we describe the series of experiments that employed protein phosphatase inhibitors to establish that ATM was regulated by a type-2A protein phosphatase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Ataxia Telangiectasia Mutated Proteins
  • Cell Cycle Proteins / metabolism*
  • Cell Line, Tumor
  • Cells, Cultured
  • DNA-Binding Proteins / metabolism*
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Inhibitors / pharmacology*
  • Humans
  • Immunoblotting
  • Immunoprecipitation
  • Okadaic Acid / pharmacology
  • Phosphoprotein Phosphatases / antagonists & inhibitors
  • Phosphoprotein Phosphatases / metabolism*
  • Phosphoprotein Phosphatases / physiology
  • Phosphorylation / drug effects
  • Protein Phosphatase 2
  • Protein Serine-Threonine Kinases / metabolism*
  • Tumor Suppressor Proteins / metabolism*

Substances

  • Cell Cycle Proteins
  • DNA-Binding Proteins
  • Enzyme Inhibitors
  • Tumor Suppressor Proteins
  • Okadaic Acid
  • ATM protein, human
  • Ataxia Telangiectasia Mutated Proteins
  • Protein Serine-Threonine Kinases
  • Phosphoprotein Phosphatases
  • Protein Phosphatase 2