REGULATION OF CAK KINASE-ACTIVITY BY P53

The growth suppressor p53 is an important key element which controls c ell cycle progression in response to cellular stress like DNA damage. Its ability to act as transcriptional activator or repressor links tra nscription and cell cycle control. Several target genes selectively tr ansactivated by p53 are implicated in growth control, apoptosis and DN A repair. Here we report the interaction of p53 with another important dual player of cell cycle control and transcription, the protein kina se complex CDK7/cyclin H/Mat1 (CDK activating kinase, CAK kinase). Thi s is implicated in the activating phosphorylation of CDK2/cyclin A kin ase required to allow cells to proceed through the G(1)/S transition, and on the other hand, as a component of the basal transcription facto r TFIIH found to be necessary for CTD phosphorylation of RNA polymeras e II in order to allow elongation of transcription. Based on previous binding studies of p53 with other C-terminal interaction partners of p 53 we demonstrate a direct physical interaction of p53 with cyclin H i ll vitro and in vivo. As a consequence of this interaction we tested t he influence of p53 on the kinase activity of CAK kinase for CTD and C DK2 phosphorylation. The addition of wild type p53 to the kinase react ions resulted in a significant downregulation of CDK2 phosphorylation and CTD phosphorylation by the CDK activating kinase. On the other han d addition of a mutant p53His175 failed to downregulate CDK2 and CTD p hosphorylation by the CDK activating kinase. In an attempt to support our findings in vivo we measured CAK kinase activity in p21(-/-) and p 53(-/-) mice embryonal fibroblasts under conditions when p53 gets acti vated by irradiation. In the case of p21(-/-) cells this led to a sign ificant reduction of CTD phosphorylation activity of the CDK activatin g kinase by irradiation of the cells. On the other hand in p53(-/-) ce lls no downregulation of CTD phosphorylation activity of CAK kinase wa s observed indicating that this kind of negative regulation of CAK kin ase activity is exclusively due to a functional p53. These findings im ply a direct involvement of p53 in triggering growth arrest by its int eraction with the CDK activating kinase complex without the need of cy clin-dependent kinase inhibitors (CKIs) and potentially suggest a new mechanism for p53-dependent apoptosis.