Blasina A, de Weyer I V, Laus M C, Luyten W H, Parker A E, McGowan C H
Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Curr Biol. 1999 Jan 14;9(1):1-10. doi: 10.1016/s0960-9822(99)80041-4.
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.
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.
These results suggest that, in human cells, the DNA-damage checkpoint involves direct inactivation of Cdc25 catalyzed by Cds1 and/or Chk1.
在人类细胞中，有丝分裂诱导激酶Cdc2在苏氨酸14和酪氨酸15位点被磷酸化而受到抑制。这些磷酸化位点的破坏消除了检查点介导的对Cdc2的调控，并使细胞对损伤DNA的试剂高度敏感。这些位点的磷酸化由Wee1/Myt1激酶和Cdc25磷酸酶的相反活性控制。因此，这些酶的调控对于G2-M期DNA损伤检查点的运作可能至关重要。
在这里，我们表明暴露于电离辐射后Cdc25的活性降低。渥曼青霉素（一种磷脂酰肌醇（PI）3激酶抑制剂）可抑制辐射诱导的Cdc25活性降低，且该降低依赖于共济失调毛细血管扩张症中发生突变的基因的功能。我们还鉴定出两种在体外使Cdc25磷酸化并使其失活的人类激酶。一种是先前已表征的Chk1激酶。另一种是新发现的，与酵母中的检查点激酶Cds1/Rad53家族同源。发现人类Cds1在DNA损伤时被激活。
这些结果表明，在人类细胞中，DNA损伤检查点涉及由Cds1和/或Chk1催化的Cdc25的直接失活。
