Phosphorylation activates Chk1 and is required for checkpoint-mediated cell cycle arrest.
In the fission yeast Schizosaccharomyces pombe, the protein kinase Chk1 has an essential role in transducing a delay signal to the cell cycle machinery in the presence of DNA damage. Fission yeast cells lacking the chk1 gene do not delay progression of the cell cycle in response to damage and are thus sensitive to DNA damaging agents. We have previously shown that Chk1 is phosphorylated following DNA damage induced by a variety of agents and that this is dependent on the integrity of the DNA damage checkpoint pathway, including Rad3, the ATR homolog. Through a combination of mutagenesis and phospho-specific antibodies, we have shown that serine at position 345 (S345) is phosphorylated in vivo in response to DNA damage, and that S345 phosphorylation is required for an intact checkpoint response. We have developed a kinase assay for Chk1, and have shown that basal Chk1 kinase activity is increased in response to DNA damage and that this increase, but not the basal activity, is dependent on S345. Furthermore, we show that S345 phosphorylation is required for Chk1 to associate with Rad24, a 14-3-3 protein, upon DNA damage. These results are consistent with a model whereby Chk1 phosphorylation results in increased Chk1 kinase activity that is necessary for both checkpoint delay and cellular survival following damage to the genome. These data are similar to observations made in mammalian cells and Xenopus oocyte extracts, suggesting that mechanisms leading to Chk1 activation have been conserved in evolution.
Capasso
Holly
H
Department of Pharmacology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.
Palermo
Carmela
C
Wan
Shanhong
S
Rao
Hui
H
John
Ulrik P
UP
O'Connell
Matthew J
MJ
Walworth
Nancy C
NC
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002166:Camptothecin / Q000494:pharmacology; D002453:Cell Cycle; D002460:Cell Line; D002470:Cell Survival / Q000528:radiation effects; D000071877:Checkpoint Kinase 1; D004249:DNA Damage / Q000187:drug effects / Q000528:radiation effects; D004307:Dose-Response Relationship, Radiation; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000201:enzymology; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014466:Ultraviolet Rays; D029867:Xenopus Proteins; D014982:Xenopus laevis
D029702:Schizosaccharomyces pombe Proteins; D029867:Xenopus Proteins; D010768:Phosphoserine; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; C000605875:Chek1 protein, Xenopus; C000605869:Chek1 protein, mouse; C000605873:Chk1 protein, S pombe; D002166:Camptothecin