Role of diacylglycerol-regulated protein kinase C isotypes in growth factor activation of the Raf-1 protein kinase.
The Raf protein kinases function downstream of Ras guanine nucleotide-binding proteins to transduce intracellular signals from growth factor receptors. Interaction with Ras recruits Raf to the plasma membrane, but the subsequent mechanism of Raf activation has not been established. Previous studies implicated hydrolysis of phosphatidylcholine (PC) in Raf activation; therefore, we investigated the role of the epsilon isotype of protein kinase C (PKC), which is stimulated by PC-derived diacylglycerol, as a Raf activator. A dominant negative mutant of PKC epsilon inhibited both proliferation of NIH 3T3 cells and activation of Raf in COS cells. Conversely, overexpression of active PKC epsilon stimulated Raf kinase activity in COS cells and overcame the inhibitory effects of dominant negative Ras in NIH 3T3 cells. PKC epsilon also stimulated Raf kinase in baculovirus-infected Spodoptera frugiperda Sf9 cells and was able to directly activate Raf in vitro. Consistent with its previously reported activity as a Raf activator in vitro, PKC alpha functioned similarly to PKC epsilon in both NIH 3T3 and COS cell assays. In addition, constitutively active mutants of both PKC alpha and PKC epsilon overcame the inhibitory effects of dominant negative mutants of the other PKC isotype, indicating that these diacylglycerol-regulated PKCs function as redundant activators of Raf-1 in vivo.
Cai
H
H
Division of Molecular Genetics, Dana-Farber Cancer Institute, and Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Smola
U
U
Wixler
V
V
Eisenmann-Tappe
I
I
Diaz-Meco
M T
MT
Moscat
J
J
Rapp
U
U
Cooper
G M
GM
D016475:3T3 Cells; D000818:Animals; D019556:COS Cells; D002455:Cell Division; D002460:Cell Line; D004075:Diglycerides / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology*; D051379:Mice; D008934:Mitogens / Q000494:pharmacology; D009154:Mutation; D011493:Protein Kinase C / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology*; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D011993:Recombinant Fusion Proteins; D015398:Signal Transduction / Q000502:physiology*; D018411:Spodoptera; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D004075:Diglycerides; D007527:Isoenzymes; D008934:Mitogens; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D004815:Epidermal Growth Factor; C497256:Prkce protein, mouse; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; C496630:Prkca protein, mouse; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D013755:Tetradecanoylphorbol Acetate