Constitutive association of c-N-Ras with c-Raf-1 and protein kinase C epsilon in latent signaling modules.
Phorbol ester stimulation of the MAPK cascade is believed to be mediated through the protein kinase C (PKC)-dependent activation of Raf-1. Although several studies suggest that phorbol ester stimulation of MAPK is insensitive to dominant-negative Ras, a requirement for Ras in Raf-1 activation by PKC has been suggested recently. We now demonstrate that in normal, quiescent mouse fibroblasts, endogenous c-N-Ras is constitutively associated with both c-Raf-1 and PKC epsilon in a biochemically silent, but latent, signaling module. Chemical inhibition of novel PKCs blocks phorbol 12-myristate 13-acetate (PMA)-mediated activation of MAPKs. Down-regulation of PKC epsilon protein levels by antisense oligodeoxyribonucleotides blocks MAPK activation in response to PMA stimulation, demonstrating that PKC epsilon activity is required for MAPK activation by PMA. c-Raf-1 activity in immunoprecipitated c-N-Ras.c-Raf-1.PKC epsilon complexes is stimulated by PMA and is inhibited by GF109203X, thereby linking c-Raf-1 activation in this complex to PKC activation. These observations suggest that in quiescent cells Ras is organized into ordered, inactive signaling modules. Furthermore, the regulation of the MAPK cascade by both Ras and PKC is intimately linked, converging at the plasma membrane through their association with c-Raf-1.
Hamilton
M
M
Department of Cell Biology, The Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA. hamilton_mark@lilly.com
Liao
J
J
Cathcart
M K
MK
Wolfman
A
A
D000818:Animals; D002460:Cell Line; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000378:metabolism; D016895:Culture Media, Serum-Free; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005347:Fibroblasts; D011905:Genes, ras; D006160:Guanosine Triphosphate / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology; D011493:Protein Kinase C / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D051744:Protein Kinase C-epsilon; D019908:Proto-Oncogene Proteins c-raf / Q000302:isolation & purification / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014162:Transfection
D016895:Culture Media, Serum-Free; D007527:Isoenzymes; D020319:Oligodeoxyribonucleotides, Antisense; D011993:Recombinant Fusion Proteins; D004815:Epidermal Growth Factor; D006160:Guanosine Triphosphate; C497256:Prkce protein, mouse; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon; D016283:Proto-Oncogene Proteins p21(ras); D013755:Tetradecanoylphorbol Acetate