Determination of substrate specificity and putative substrates of Chk2 kinase

Biochem Biophys Res Commun. 2003 May 2;304(2):339-43. doi: 10.1016/s0006-291x(03)00589-8.

Abstract

Chk2/hCds1, the human homolog of Saccharomyces cerevisiae Rad53p and Schizosaccharomyces pombe Cds1p, plays a critical role in the DNA damage checkpoint pathway. While several in vivo targets of Chk2 have been identified, the other target proteins of Chk2 responsible for multiple functions, such as cell cycle arrest, DNA repair, and apoptosis, remain to be elucidated. We utilized the GST-peptide approach to identify physiological substrates for Chk2. Mutational analyses using GST-linked Cdc25A containing serine 123 revealed that residues at positions -5 and -3 are critical determinants for the recognition of the Chk2 substrate. We determined the general phosphorylation consensus sequence and identified in vitro targets of Chk2 using GST peptides as substrates. The newly identified in vitro target proteins include Abl1, Bub1R, Bub1, Bub3, Psk-H1, Smc3, Plk1, Cdc25B, Dcamkl1, Mre11, Pms1, and Xrcc9.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Cell Line
  • Checkpoint Kinase 2
  • Consensus Sequence
  • DNA Mutational Analysis
  • Glutathione Transferase / genetics
  • Humans
  • Peptides / chemistry
  • Peptides / metabolism
  • Phosphorylation
  • Protein Kinases / metabolism*
  • Protein-Serine-Threonine Kinases*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Schizosaccharomyces pombe Proteins
  • Sequence Alignment
  • Substrate Specificity
  • cdc25 Phosphatases / chemistry
  • cdc25 Phosphatases / genetics
  • cdc25 Phosphatases / metabolism

Substances

  • Peptides
  • Recombinant Fusion Proteins
  • Schizosaccharomyces pombe Proteins
  • Glutathione Transferase
  • Protein Kinases
  • Checkpoint Kinase 2
  • CHEK2 protein, human
  • Cds1 protein, S pombe
  • Protein-Serine-Threonine Kinases
  • CDC25A protein, human
  • cdc25 Phosphatases