Ataxia telangiectasia-mutated phosphorylates Chk2 in vivo and in vitro

Proc Natl Acad Sci U S A. 2000 Sep 12;97(19):10389-94. doi: 10.1073/pnas.190030497.

Abstract

The protein kinase Chk2, the mammalian homolog of the budding yeast Rad53 and fission yeast Cds1 checkpoint kinases, is phosphorylated and activated in response to DNA damage by ionizing radiation (IR), UV irradiation, and replication blocks by hydroxyurea (HU). Phosphorylation and activation of Chk2 are ataxia telangiectasia-mutated (ATM) dependent in response to IR, whereas Chk2 phosphorylation is ATM-independent when cells are exposed to UV or HU. Here we show that in vitro, ATM phosphorylates the Ser-Gln/Thr-Gln (SQ/TQ) cluster domain (SCD) on Chk2, which contains seven SQ/TQ motifs, and Thr68 is the major in vitro phosphorylation site by ATM. ATM- and Rad3-related also phosphorylates Thr68 in addition to Thr26 and Ser50, which are not phosphorylated to a significant extent by ATM in vitro. In vivo, Thr68 is phosphorylated in an ATM-dependent manner in response to IR, but not in response to UV or HU. Substitution of Thr68 with Ala reduced the extent of phosphorylation and activation of Chk2 in response to IR, and mutation of all seven SQ/TQ motifs blocked all phosphorylation and activation of Chk2 after IR. These results suggest that in vivo, Chk2 is directly phosphorylated by ATM in response to IR and that Chk2 is regulated by phosphorylation of the SCD.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Ataxia Telangiectasia Mutated Proteins
  • Cell Cycle Proteins
  • Cell Line
  • Checkpoint Kinase 2
  • DNA-Binding Proteins
  • Humans
  • Phosphorylation
  • Protein Kinases / chemistry
  • Protein Kinases / metabolism*
  • Protein-Serine-Threonine Kinases / metabolism*
  • Recombinant Proteins / metabolism
  • Tumor Suppressor Proteins

Substances

  • Cell Cycle Proteins
  • DNA-Binding Proteins
  • Recombinant Proteins
  • Tumor Suppressor Proteins
  • Protein Kinases
  • Checkpoint Kinase 2
  • ATM protein, human
  • Ataxia Telangiectasia Mutated Proteins
  • CHEK2 protein, human
  • Protein-Serine-Threonine Kinases