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Protein kinase C-mediated serine phosphorylation directly activates Raf-1 in murine hematopoietic cells
Ist Teil von
The Journal of biological chemistry, 1994-01, Vol.269 (2), p.1249-1256
Ort / Verlag
Bethesda, MD: American Society for Biochemistry and Molecular Biology
Erscheinungsjahr
1994
Link zum Volltext
Quelle
MEDLINE
Beschreibungen/Notizen
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.