Schneider E, Montenarh M, Wagner P
Medical Biochemistry and Molecular Biology, University of the Saarland, Homburg/Saar, Germany.
Oncogene. 1998 Nov 26;17(21):2733-41. doi: 10.1038/sj.onc.1202504.
The growth suppressor p53 is an important key element which controls cell cycle progression in response to cellular stress like DNA damage. Its ability to act as transcriptional activator or repressor links transcription and cell cycle control. Several target genes selectively transactivated by p53 are implicated in growth control, apoptosis and DNA repair. Here we report the interaction of p53 with another important dual player of cell cycle control and transcription, the protein kinase complex CDK7/cyclin H/Mat1 (CDK activating kinase, CAK kinase). This is implicated in the activating phosphorylation of CDK2/cyclin A kinase required to allow cells to proceed through the G1/S transition, and on the other hand, as a component of the basal transcription factor TFIIH found to be necessary for CTD phosphorylation of RNA polymerase II in order to allow elongation of transcription. Based on previous binding studies of p53 with other C-terminal interaction partners of p53 we demonstrate a direct physical interaction of p53 with cyclin H in vitro and in vivo. As a consequence of this interaction we tested the influence of p53 on the kinase activity of CAK kinase for CTD and CDK2 phosphorylation. The addition of wild type p53 to the kinase reactions resulted in a significant downregulation of CDK2 phosphorylation and CTD phosphorylation by the CDK activating kinase. On the other hand addition of a mutant p53His175 failed to downregulate CDK2 and CTD phosphorylation by the CDK activating kinase. In an attempt to support our findings in vivo we measured CAK kinase activity in p21-/- and p53-/- mice embryonal fibroblasts under conditions when p53 gets activated by irradiation. In the case of p21-/- cells this led to a significant reduction of CTD phosphorylation activity of the CDK activating kinase by irradiation of the cells. On the other hand in p53 cells no downregulation of CTD phosphorylation activity of CAK kinase was observed indicating that this kind of negative regulation of CAK kinase activity is exclusively due to a functional p53. These findings imply a direct involvement of p53 in triggering growth arrest by its interaction with the CDK activating kinase complex without the need of cyclin-dependent kinase inhibitors (CKIs) and potentially suggest a new mechanism for p53-dependent apoptosis.
生长抑制因子p53是一个重要的关键元件，它能响应诸如DNA损伤等细胞应激来控制细胞周期进程。其作为转录激活因子或抑制因子的能力将转录与细胞周期调控联系起来。p53选择性反式激活的几个靶基因与生长控制、细胞凋亡及DNA修复有关。在此我们报道了p53与细胞周期调控和转录的另一个重要双重作用因子——蛋白激酶复合物CDK7/细胞周期蛋白H/Mat1（CDK激活激酶，CAK激酶）之间的相互作用。这与允许细胞通过G1/S期转换所需的CDK2/细胞周期蛋白A激酶的激活磷酸化有关，另一方面，作为基础转录因子TFIIH的一个组分，它被发现是RNA聚合酶II的CTD磷酸化从而允许转录延伸所必需的。基于之前p53与p53其他C末端相互作用伙伴的结合研究，我们在体外和体内证实了p53与细胞周期蛋白H之间存在直接的物理相互作用。作为这种相互作用的结果，我们测试了p53对CAK激酶CTD磷酸化和CDK2磷酸化激酶活性的影响。向激酶反应中加入野生型p53导致CDK激活激酶对CDK2磷酸化和CTD磷酸化显著下调。另一方面，加入突变型p53His175未能下调CDK激活激酶对CDK2和CTD的磷酸化。为了在体内支持我们的发现，我们在p53被辐射激活的条件下，测量了p21基因敲除小鼠和p53基因敲除小鼠胚胎成纤维细胞中的CAK激酶活性。在p21基因敲除细胞的情况下，对细胞进行辐射导致CDK激活激酶的CTD磷酸化活性显著降低。另一方面，在p53基因敲除细胞中未观察到CAK激酶CTD磷酸化活性的下调，这表明CAK激酶活性的这种负调控完全是由于功能性p53所致。这些发现意味着p53通过与CDK激活激酶复合物相互作用直接参与触发生长停滞，而无需细胞周期蛋白依赖性激酶抑制剂（CKIs），并可能提示了一种p53依赖性细胞凋亡的新机制。
