Activation of Raf kinase by phorbol ester does not correlate with enhanced phosphorylation or transactivation activity of tumor suppressor protein p53

The tumor suppressor protein p53 appears to be regulated by various means including phosphorylation. We investigated a possible influence of the raf kinase on phosphorylation and transactivation of p53. The phosphorylation pattern of p53 was analysed in normal or v-raf-transformed rat cells or in insect cells which were co-infected with recombinant baculoviruses encoding p53, protein kinase C and c-Raf. The presence of activated v-raf kinase or treatment of cells with PMA, which resulted in activation of both v-raf and c-Raf kinases, did not reveal any additional phosphorylation sites in p53 nor did any of the known sites show enhanced phosphorylation, neither in rat nor in baculovirus-infected insect cells. Instead, PMA treatment led to reduced phosphorylation of p53. This latter effect was similar in normal and v-raf-transformed cells suggesting that it is mediated by a PKC-dependent but raf-independent mechanism. Okadaic acid treatment compensated this reduced phosphorylation of p53 suggesting that it was due to activation of phosphatase(s). The transactivation activity of p53 was assayed under the same conditions, using an mdm2-promoter luciferase reporter gene construct. In this case, we obtained conflicting results: in normal rat cells both PMA and okadaic acid had a stimulatory effect which was additive. In the v-raf transformed cells, on the other hand, PMA alone reduced, whereas okadaic acid alone enhanced transcriptional activation and application of both drugs resulted in a slight net increase in transactivation. Thus, PMA seemed to have separable effects on phosphorylation and transactivation activity of p53. Our data suggest i) that neither Raf-l kinase nor PKC phosphorylate p53 directly in vivo, ii) that the reduced phosphorylation observed upon PMA treatment is independent of Raf, and perhaps mediated by indirect activation of phosphatases, iii) that the effects of PMA on p53-dependent transcription are not mediated by changes in the phosphorylation state of p53 itself.