Eberhard Schneider

Regulation of CAK kinase activity by p53

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.

Wild‐type p53 inhibits protein kinase CK2 activity

The growth suppressor protein p53 and the protein kinase CK2 are both implicated in cellular growth regulation. We previously found that p53 binds to protein kinase CK2 via its regulatory β‐subunit. In the present study, we analyzed the consequences of the binding of p53 to CK2 for the enzymatic activity of CK2 in vitro and in vivo. We found that the carboxy‐terminus of p53 which is a potent transforming agent stimulated CK2 activity whereas full length wild‐type p53 which is a growth suppressor inhibited the activity of protein kinase CK2. Inhibition of protein kinase CK2 by p53 was dose‐dependent and was seen for various CK2 substrates. Experiments with heat‐denatured p53 and the conformational mutant p53R175H revealed that an intact conformation of p53 seemed to be necessary. Transfection of wild‐type and of mutant p53 into p53−/− cells showed that the inhibition of p53 on CK2 activity was also detectable in intact cells and specific for wild‐type p53 indicating that the growth suppressing function of p53 might at least be partially achieved by down‐regulation of protein kinase CK2. J. Cell. Biochem. 81:172–183, 2001.

The cyclin H/cdk7/Mat1 kinase activity is regulated by CK2 phosphorylation of cyclin H

Cyclin dependent kinases are regulated by phosphorylation and dephosphorylation of the catalytic cdk subunits, by assembly with specific cyclins and by specific inhibitor molecules. Recently, it turned out that cyclins are also phosphoproteins, which means that they are also potential targets for a regulation by phosphorylation and dephosphorylation. Here, we show that cyclin H was phosphorylated by protein kinase CK2. Like most other CK2 substrates cyclin H was much better phosphorylated by the CK2 holoenzyme than by the α-subunit alone. By using point mutants derived from the cyclin H sequence we mapped the CK2 phosphorylation site at threonine 315 at the C-terminal end of cyclin H. Phosphorylation at this position had no influence on the assembly of the cyclin H/cdk7/Mat1 complex. However, phosphorylation at amino acid 315 of cyclin H turned out to be critical for a full cyclin H/cdk7/Mat1 kinase activity when the CTD peptide of RNA polymerase II or cdk2 was used as a substrate.

Regulation of p53 mediated transactivation by the β-subunit of protein kinase CK2

The growth suppressor protein p53 plays a main part in cellular growth control. Two of its key functions are sequence specific DNA binding and transactivation. Functions of p53 in growth control are regulated at least in part by its interaction with protein kinases. p53 binds to protein kinase CK2, formerly known as casein kinase 2, and it is phosphorylated by this enzyme. CK2 is composed of two regulating β‐subunits and two catalytic α‐ or α′‐subunits and the interaction with p53 is mediated by the regulatory β‐subunit of CK2. Recently we showed that the β‐subunit could inhibit the sequence specific DNA binding activity of p53 in vitro. Based on this finding, we asked if a coexpression of the β‐subunit of CK2 with p53 in mammalian cells could inhibit the DNA binding activity of p53 in a physiological context. We found that the coexpression of the β‐subunit showed the same inhibitory effect as in the previous assays with purified proteins. Then, we investigated the effects of the coexpression of the β‐subunit of CK2 on the transactivation and transrepression activity of p53. We found that transactivation of the mdm2, p21WAF1/CIP1 and cyclin G promoter was inhibited in three different cell lines whereas transactivation of the bax promoter was not affected in COS1 cells but down‐regulated in MCO1 and SaosS138V21 cells. p53 mediated transrepression of the fos promoter was not influenced by coexpression of the CK2 β‐subunit. Taken together we propose a cell type dependent fine regulation of the p53 transactivation function by the CK2 β‐subunit in vivo, which does not affect p53 mediated transrepression.