Determination of substrate specificity and putative substrates of Chk2 kinase.
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.
Seo
Gil-Ju
GJ
Department of Molecular Cellular Biology, Sungkyunkwan University School of Medicine, Samsung Biomedical Research Institute, 300 Chunchun-Dong, Changan-Ku, 440-746, Suwon, Kyunggi-Do, Republic of Korea.
Kim
Se-Eun
SE
Lee
Young-Man
YM
Lee
Jeong-Won
JW
Lee
Jae-Rin
JR
Hahn
Myong-Joon
MJ
Kim
Seong-Tae
ST
D000595:Amino Acid Sequence; D002460:Cell Line; D064447:Checkpoint Kinase 2; D016384:Consensus Sequence; D004252:DNA Mutational Analysis; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins; D016415:Sequence Alignment; D013379:Substrate Specificity; D020687:cdc25 Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D010455:Peptides; D011993:Recombinant Fusion Proteins; D029702:Schizosaccharomyces pombe Proteins; D005982:Glutathione Transferase; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C578634:CHEK2 protein, human; C578636:Cds1 protein, S pombe; D017346:Protein Serine-Threonine Kinases; C517004:CDC25A protein, human; D020687:cdc25 Phosphatases