A human homologue of the checkpoint kinase Cds1 directly inhibits Cdc25 phosphatase.
BACKGROUND
In human cells, the mitosis-inducing kinase Cdc2 is inhibited by phosphorylation on Thr14 and Tyr15. Disruption of these phosphorylation sites abrogates checkpoint-mediated regulation of Cdc2 and renders cells highly sensitive to agents that damage DNA. Phosphorylation of these sites is controlled by the opposing activities of the Wee1/Myt1 kinases and the Cdc25 phosphatase. The regulation of these enzymes is therefore likely to be crucial for the operation of the G2-M DNA-damage checkpoint.


RESULTS
Here, we show that the activity of Cdc25 decreased following exposure to ionizing radiation. The irradiation-induced decrease in Cdc25 activity was suppressed by wortmannin, an inhibitor of phosphatidylinositol (PI) 3-kinases, and was dependent on the function of the gene that is mutated in ataxia telangiectasia. We also identified two human kinases that phosphorylate and inactivate Cdc25 in vitro. One is the previously characterized Chk1 kinase. The second is novel and is homologous to the Cds1/Rad53 family of checkpoint kinases in yeast. Human Cds1 was found to be activated in response to DNA damage.


CONCLUSIONS
These results suggest that, in human cells, the DNA-damage checkpoint involves direct inactivation of Cdc25 catalyzed by Cds1 and/or Chk1.
Blasina
A
A
Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
de Weyer
I V
IV
Laus
M C
MC
Luyten
W H
WH
Parker
A E
AE
McGowan
C H
CH
D000595:Amino Acid Sequence; D000730:Androstadienes / Q000494:pharmacology; D001345:Autoradiography; D015152:Blotting, Northern; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors* / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D002460:Cell Line; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D004249:DNA Damage; D004261:DNA Replication / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases; D016415:Sequence Alignment; D000077191:Wortmannin; D020687:cdc25 Phosphatases
D000730:Androstadienes; D018797:Cell Cycle Proteins; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C000605867:CHEK1 protein, human; C578634:CHEK2 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases; D010749:Phosphoprotein Phosphatases; D020687:cdc25 Phosphatases; D000077191:Wortmannin