PROTEIN KINASE-C-MEDIATED SERINE PHOSPHORYLATION DIRECTLY ACTIVATES RAF-1 IN MURINE HEMATOPOIETIC-CELLS

We have previously found that Raf-1, which is activated by hematopoiet ic growth factors in association with phosphorylation, is required for hematopoietic cell proliferation. Recently, 12-O-tetradecanoylphorbol 13-acetate has been found to mediate Raf-l phosphorylation, suggestin g that protein kinase C (PKC) may be involved in the Raf-l activation mechanism(s). Since PKC can be activated by hematopoietic growth facto rs, it was investigated as a potential ''Raf-l kinase-kinase.'' Result s demonstrate that bryostatin 1, a pharmacologic activator of PKC, ind uces activation of Raf-l in FDC-P1 cells. PKC inhibitors H7 and stauro sporine block both bryostatin 1- and interleukin-3-mediated Raf-l phos phorylation and FDC-P1 cell proliferation. Additionally, an antisense c-raf oligodeoxyribonucleotide specifically inhibits bryostatin 1-medi ated proliferation, indicating a necessary role for Raf-l in PKC signa ling. Purified PKC can phosphorylate Raf-l serine residues to high sto ichiometry in vitro. Comparative phosphopeptide maps localize two PKC phosphorylation sites to Raf-l phosphopeptides isolated from hematopoi etic growth factor- or bryostatin 1-stimulated cells. The sites of PKC -mediated Raf-l phosphorylation are deduced to be Ser497 and Ser619. F urthermore, PKC-mediated serine phosphorylation is sufficient to activ ate the enzymatic function of Raf-l in vitro. These findings demonstra te that activated PKC can promote hematopoietic cell growth by regulat ing the enzymatic activity of Raf-1 through direct serine phosphorylat ion.