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Regulation of CAK kinase activity by p53
E Schneider1, M Montenarh, P Wagner
1Medical Biochemistry and Molecular Biology, University of the Saarland, Homburg/Saar, Germany.
Abstract:
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.
Insights
The tumor suppressor p53 directly interacts with CDK7/cyclin H/Mat1 (CAK kinase), downregulating its activity. This interaction, crucial for cell cycle control and transcription, suggests a new p53-mediated pathway for growth arrest and apoptosis.
Area of Science:
- Molecular Biology
- Cell Cycle Regulation
- Cancer Biology
Background:
- The tumor suppressor p53 is a critical regulator of cell cycle progression in response to DNA damage.
- p53 acts as a transcriptional activator or repressor, linking transcription and cell cycle control.
- The CDK7/cyclin H/Mat1 (CAK kinase) complex plays dual roles in cell cycle progression and transcription.
Purpose of the Study:
- To investigate the physical interaction between p53 and the CAK kinase complex.
- To determine the effect of p53 on the kinase activity of CAK kinase.
- To elucidate the in vivo relevance of p53's interaction with CAK kinase in regulating cell cycle control.
Main Methods:
- In vitro and in vivo binding assays to demonstrate p53-cyclin H interaction.
- Kinase assays measuring CDK2 and CTD phosphorylation by CAK kinase in the presence of wild-type and mutant p53.
- Measurement of CAK kinase activity in p21-/- and p53-/- mouse embryonic fibroblasts after irradiation.
Main Results:
- Direct physical interaction between p53 and cyclin H was demonstrated.
- Wild-type p53 significantly downregulated CDK2 and CTD phosphorylation by CAK kinase in vitro.
- Mutant p53 (His175) failed to downregulate CAK kinase activity.
- In vivo, irradiation led to reduced CAK kinase activity in p21-/- cells but not in p53-/- cells, indicating p53-dependent regulation.
Conclusions:
- p53 directly interacts with the CAK kinase complex, inhibiting its activity.
- This interaction provides a novel mechanism for p53-mediated growth arrest independent of cyclin-dependent kinase inhibitors (CKIs).
- The findings suggest a new role for p53 in apoptosis through its regulation of CAK kinase.