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 me ans including phosphorylation. We investigated a possible influence of the raf kinase on phosphorylation and transactivation of p53. The pho sphorylation pattern of p53 was analysed in normal or v-raf-transforme d 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 result ed in activation of both v-raf and c-Raf kinases, did not reveal any a dditional phosphorylation sites in p53 nor did any of the known sites show enhanced phosphorylation, neither in rat nor in baculovirus-infec ted insect cells. Instead, PMA treatment led to reduced phosphorylatio n of p53. This latter effect was similar in normal and v-raf-transform ed cells suggesting that it is mediated by a PKC-dependent but raf-ind ependent mechanism. Okadaic acid treatment compensated this reduced ph osphorylation of p53 suggesting that it was due to activation of phosp hatase(s). The transactivation activity of p53 was assayed under the s ame conditions, using an mdm2-promoter luciferase reporter gene constr uct. In this case, we obtained conflicting results: in normal rat cell s both PMA and okadaic acid had a stimulatory effect which was additiv e. In the v-raf transformed cells, on the other hand, PMA alone reduce d, whereas okadaic acid alone enhanced transcriptional activation and application of both drugs resulted in a slight net increase in transac tivation. Thus, PMA seemed to have separable effects on phosphorylatio n and transactivation activity of p53. Our data suggest i) that neithe r Raf-l kinase nor PKC phosphorylate p53 directly ill vivo, ii) that t he reduced phosphorylation observed upon PMA treatment is independent of Raf, and perhaps mediated by indirect activation of phosphatases, i ii) that the effects of PMA on p53-dependent transcription are not med iated by changes in the phosphorylation state of p53 itself.