Chk1 is a wee1 kinase in the G2 DNA damage checkpoint inhibiting cdc2 by Y15 phosphorylation.
The G2 DNA damage checkpoint ensures maintenance of cell viability by delaying progression into mitosis in cells which have suffered genomic damage. It is controlled by a number of proteins which are hypothesized to transduce signals through cell cycle regulators to delay activation of p34cdc2. Studies in mammalian cells have correlated induction of inhibitory tyrosine 15 (Y15) phosphorylation on p34cdc2 with the response to DNA damage. However, genetic studies in fission yeast have suggested that the major Y15 kinase, p107wee1, is not required for the cell cycle delay in response to DNA damage, although it is required for survival after irradiation. Thus, the target of the checkpoint, and hence the mechanism of cell cycle delay, remains unknown. We show here that Y15 phosphorylation is maintained in checkpoint-arrested fission yeast cells. Further, wee1 is required for cell cycle arrest induced by up-regulation of an essential component of this checkpoint, chk1. We observed that p107wee1 is hyperphosphorylated in cells delayed by chk1 overexpression or UV irradiation, and that p56chk1 can phosphorylate p107wee1 directly in vitro. These observations suggest that in response to DNA damage p107wee1 is phosphorylated by p56chk1 in vivo, and this results in maintenance of Y15 phosphorylation and hence G2 delay. In the absence of wee1, other Y15 kinases, such as p66mik1, may partially substitute for p107wee1 to induce cell cycle delay, but this wee1-independent delay is insufficient to maintain full viability. This study establishes a link between a G2 DNA damage checkpoint function and a core cell cycle regulator.
O'Connell
M J
MJ
Trescowthick Research Laboratories, Peter MacCallum Cancer Institute, Melbourne, Victoria, Australia.
Raleigh
J M
JM
Verkade
H M
HM
Nurse
P
P
D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000037:antagonists & inhibitors*; D002453:Cell Cycle / Q000502:physiology; D018797:Cell Cycle Proteins; D002470:Cell Survival; D000071877:Checkpoint Kinase 1; D004249:DNA Damage / Q000235:genetics*; D005434:Flow Cytometry; D005656:Fungal Proteins / Q000378:metabolism / Q000494:pharmacology; D016195:G2 Phase / Q000502:physiology*; D015966:Gene Expression Regulation, Fungal / Q000235:genetics; D008954:Models, Biological; D009687:Nuclear Proteins; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000032:analysis; D011233:Precipitin Tests; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins; D014466:Ultraviolet Rays
D018797:Cell Cycle Proteins; D005656:Fungal Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D029702:Schizosaccharomyces pombe Proteins; D019000:Phosphotyrosine; D011494:Protein Kinases; C068689:wee1 protein, S pombe; D011505:Protein-Tyrosine Kinases; D000071877:Checkpoint Kinase 1; C000605873:Chk1 protein, S pombe; D016203:CDC2 Protein Kinase