Jin P, Gu Y, Morgan D O
Department of Physiology, University of California, San Francisco 94143-0444, USA.
J Cell Biol. 1996 Aug;134(4):963-70. doi: 10.1083/jcb.134.4.963.
The activity of the mitosis-promoting kinase CDC2-cyclin B is normally suppressed in S phase and G2 by inhibitory phosphorylation at Thr14 and Tyr15. This work explores the possibility that these phosphorylations are responsible for the G2 arrest that occurs in human cells after DNA damage. HeLa cell lines were established in which CDC2AF, a mutant that cannot be phosphorylated at Thr14 and Tyr15, was expressed from a tetracycline-repressible promoter. Expression of CDC2AF did not induce mitotic events in cells arrested at the beginning of S phase with DNA synthesis inhibitors, but induced low levels of premature chromatin condensation in cells progressing through S phase and G2. Expression of CDC2AF greatly reduced the G2 delay that resulted when cells were X-irradiated in S phase. However, a significant G2 delay was still observed and was accompanied by high CDC2-associated kinase activity. Expression of wild-type CDC2, or the related kinase CDK2AF, had no effect on the radiation-induced delay. Thus, inhibitory phosphorylation of CDC2, as well as additional undefined mechanisms, delay mitosis after DNA damage.
有丝分裂促进激酶CDC2 - 细胞周期蛋白B的活性在S期和G2期通常通过苏氨酸14和酪氨酸15位点的抑制性磷酸化而受到抑制。这项研究探讨了这些磷酸化是否是DNA损伤后人类细胞中出现G2期阻滞的原因。构建了HeLa细胞系，其中从四环素可抑制启动子表达一种不能在苏氨酸14和酪氨酸15位点磷酸化的突变体CDC2AF。在被DNA合成抑制剂阻滞于S期起始阶段的细胞中，CDC2AF的表达并未诱导有丝分裂事件，但在经历S期和G2期的细胞中诱导了低水平的过早染色质凝聚。当细胞在S期受到X射线照射时，CDC2AF的表达极大地缩短了由此导致的G2期延迟。然而，仍观察到显著的G2期延迟，并且伴随着高CDC2相关激酶活性。野生型CDC2或相关激酶CDK2AF的表达对辐射诱导的延迟没有影响。因此，CDC2的抑制性磷酸化以及其他未明确的机制在DNA损伤后延迟了有丝分裂。
