Cyclin-dependent kinases are inactivated by a combination of p21 and Thr-14/Tyr-15 phosphorylation after UV-induced DNA damage
Cyclin-dependent kinases are inactivated by a combination of p21 and Thr-14/Tyr-15 phosphorylation after UV-induced DNA damage
复制标题
DOI：
10.1074/jbc.271.22.13283
复制
发表时间：
1996-05-31
影响因子：
4.8
通讯作者：
Hunter, T
中科院分区：
文献类型：
--
作者：
Poon, RYC;Jiang, W;Hunter, T
细胞周期蛋白依赖性激酶（CDK）抑制剂p21是由肿瘤抑制基因产物p53诱导的，被认为对DNA损伤后细胞周期的阻滞很重要。在这里，我们研究了p21在抑制不同的细胞周期蛋白-CDK复合物，驱动不同的细胞周期转换后，紫外线照射诱导的DNA损伤在正常的人成纤维细胞和永生化的啮齿动物成纤维细胞的贡献。当细胞暴露于低剂量的紫外线照射时，p53和p21都被诱导;与Cdc 2，Cdk 2和Cdk 4相关的蛋白激酶活性被抑制;并且它们的抑制与p21的结合之间具有良好的相关性。单独的p21可能足以抑制Cdk 2，因为所有的Cdk 2的细胞周期蛋白复合物形式在照射后与p21相关。与此相反，只有一小部分的Cdk 4和Cdc 2与p21复合，尽管与p21或p27相关的Cdk 4水平在照射后增加。此外，重组p21添加到未照射的细胞裂解物中，在相同的水平，诱导辐射损伤抑制与Cdk 2相关的激酶活性。由于Cdc 2被酪氨酸磷酸化，因此Cdc 2可能被照射后Thr-14/Tyr-15磷酸化抑制，并且重组Cdc 25能够显著增加其激酶活性。综上所述，这些结果表明，不同的CDK被不同的机制抑制UV诱导的DNA损伤：Cdk 2被抑制的p21的水平升高; Cdk 4被抑制的p21与其他CDK抑制剂，如p27，并可能通过磷酸化的合作;和Cdc 2被抑制的Thr-14/Tyr-15磷酸化。很可能，这些潜在的机制，使CDKs的类似的其他类型的DNA损伤。
The cyclin-dependent kinase (CDK) inhibitor p21 is induced by the tumor suppressor gene product p53 and is thought to be important for the arrest of the cell cycle following DNA damage. Here we have investigated the contribution of p21 in inhibiting different cyclin-CDK complexes that drive different cell cycle transitions following UV irradiation-induced DNA damage in normal human fibroblasts and immortalized rodent fibroblasts. When cells were exposed to a low dose of UV irradiation, both p53 and p21 were induced; the protein kinase activities associated with Cdc2, Cdk2, and Cdk4 were inhibited; and there was a good correlation between their inhibition and binding to p21. p21 alone is likely to be sufficient for the inhibition of Cdk2 because all the cyclin-complexed forms of Cdk2 were associated with p21 after irradiation. In contrast, only a small proportion of Cdk4 and Cdc2 was complexed with p21, although the level of Cdk4 associated with either p21 or p27 was increased after irradiation. Furthermore, recombinant p21 added to an unirradiated cell lysate at the same level as that induced by irradiation damage inhibited only the kinase activity associated with Cdk2. Cdc2 is likely to be inhibited by Thr-14/Tyr-15 phosphorylation after irradiation because Cdc2 was tyrosine-phosphorylated, and recombinant Cdc25 was able to increase its kinase activity significantly. Taken together, these results suggest that different CDKs are inhibited by different mechanisms following UV-induced DNA damage: Cdk2 is inhibited by the elevated level of p21; Cdk4 is inhibited by cooperation of p21 with other CDK inhibitors, like p27, and possibly by phosphorylation; and Cdc2 is inhibited by Thr-14/Tyr-15 phosphorylation. It is likely that these underlying mechanisms that inactivate CDKs are similar for other kinds of DNA damage.