A dominant-negative mutant of raf blocks mitogen-activated protein kinase activation by growth factors and oncogenic p21ras.
p74raf-1, a serine/threonine kinase, is structurally related to the protein kinase C (PKC) family and contains a cysteine motif in its N-terminal domain, which is essential for its regulation. It has been shown that p74raf-1 functions upstream of mitogen-activated protein (MAP) kinase kinase. We have constructed a p74raf-1 mutant (N delta raf) that only contains the N-terminal regulatory domain. When transiently expressed in COS-M6 cells, N delta raf efficiently blocked the activation of the MAP extracellular signal regulated kinase (ERK2), induced by either epidermal growth factor, phorbol ester, serum, or oncogenic p21ras. Similar constructs with the cysteine motifs from either PKC-alpha or diacylglycerol kinase did not inhibit activation of ERK2. Overexpression of full-length p74raf-1 rescued the inhibition of ERK2 by N delta raf in a stimulus dependent manner, indicating that N delta raf acts as a competitive inhibitor of wild-type p74raf-1. In contrast, overexpression of either PKC-alpha, -epsilon, or -zeta in N delta raf-containing cells could not rescue the inhibition of ERK2. We conclude that p74raf-1 is an essential mediator of epidermal growth factor- and phorbol ester-induced ERK2 activation and that the MAP kinase kinase activity of p74raf-1 cannot be substituted with either PKC-alpha, -epsilon or -zeta.
Schaap
D
D
Division of Cellular Biochemistry, The Netherlands Cancer Institute, Amsterdam.
van der Wal
J
J
Howe
L R
LR
Marshall
C J
CJ
van Blitterswijk
W J
WJ
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002460:Cell Line; D019852:Diacylglycerol Kinase; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D016259:Genes, myc; D006801:Humans; D007527:Isoenzymes / Q000032:analysis / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009842:Oligopeptides / Q000138:chemical synthesis / Q000276:immunology; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D010770:Phosphotransferases / Q000032:analysis / Q000378:metabolism; D011493:Protein Kinase C / Q000032:analysis / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D011519:Proto-Oncogenes; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection
D000911:Antibodies, Monoclonal; D007527:Isoenzymes; D009842:Oligopeptides; D011506:Proteins; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D015240:Phorbol 12,13-Dibutyrate; D004815:Epidermal Growth Factor; D010770:Phosphotransferases; D011494:Protein Kinases; D019852:Diacylglycerol Kinase; D011505:Protein-Tyrosine Kinases; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)