B. Jaitner et al., ACTIVATION OF RAF KINASE BY PHORBOL ESTER DOES NOT CORRELATE WITH ENHANCED PHOSPHORYLATION OR TRANSACTIVATION ACTIVITY OF TUMOR-SUPPRESSOR PROTEIN P53, International journal of oncology, 10(3), 1997, pp. 649-657
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.