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 hematopoietic growth factors in association with phosphorylation, is required for hematopoietic cell proliferation. Recently, 12-O-tetradecanoylphorbol 13-acetate has been found to mediate Raf-1 phosphorylation, suggesting that protein kinase C (PKC) may be involved in the Raf-1 activation mechanism(s). Since PKC can be activated by hematopoietic growth factors, it was investigated as a potential "Raf-1 kinase-kinase." Results demonstrate that bryostatin 1, a pharmacologic activator of PKC, induces activation of Raf-1 in FDC-P1 cells. PKC inhibitors H7 and staurosporine block both bryostatin 1- and interleukin-3-mediated Raf-1 phosphorylation and FDC-P1 cell proliferation. Additionally, an antisense c-raf oligodeoxyribonucleotide specifically inhibits bryostatin 1-mediated proliferation, indicating a necessary role for Raf-1 in PKC signaling. Purified PKC can phosphorylate Raf-1 serine residues to high stoichiometry in vitro. Comparative phosphopeptide maps localize two PKC phosphorylation sites to Raf-1 phosphopeptides isolated from hematopoietic growth factor- or bryostatin 1-stimulated cells. The sites of PKC-mediated Raf-1 phosphorylation are deduced to be Ser497 and Ser619. Furthermore, PKC-mediated serine phosphorylation is sufficient to activate the enzymatic function of Raf-1 in vitro. These findings demonstrate that activated PKC can promote hematopoietic cell growth by regulating the enzymatic activity of Raf-1 through direct serine phosphorylation.
Carroll
M P
MP
Johns Hopkins Oncology Center, Baltimore, Maryland 21287.
May
W S
WS
D000595:Amino Acid Sequence; D054713:Bryostatins; D002455:Cell Division; D004789:Enzyme Activation; D006801:Humans; D066298:In Vitro Techniques; D007377:Interleukin-3 / Q000502:physiology; D007783:Lactones / Q000494:pharmacology; D018942:Macrolides; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011493:Protein Kinase C / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D015398:Signal Transduction
D054713:Bryostatins; D007377:Interleukin-3; D007783:Lactones; D018942:Macrolides; D016376:Oligonucleotides, Antisense; D010455:Peptides; D011518:Proto-Oncogene Proteins; D010768:Phosphoserine; C046785:bryostatin 1; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C