Differential regulation of the p72-74 RAF-1 kinase in 3T3 fibroblasts expressing ras or src oncogenes.
The c-raf-1 protooncogene encodes a p72-74 serine/threonine-specific kinase that has been implicated in growth factor-mediated signal transduction and malignant transformation. Here, we compared the effects of Ha-c-ras and v-src oncogenes on the regulation of p72-74 RAF-1 kinase in NIH3T3 cells. In both serum-starved and platelet-derived growth factor-treated v-src-transformed cells, the RAF-1 kinase was constitutively activated, displaying characteristic retarded mobility in electrophoretic gels and elevated activity in in vitro kinase assays. In contrast, the RAF-1 protein from quiescent ras-transformed cells did not exhibit constitutively shifted gel mobility or elevated kinase activity but did respond normally with regard to platelet-derived growth factor- and phorbol myristate acetate-induced changes in p72-74 RAF-1 phosphorylation and kinase activity. 3T3 cells transformed by ras, however, contained elevated levels of p72-74 RAF-1 protein (as determined by immunoblotting), suggesting an indirect influence on this kinase. Quantitative differences in the levels and subcellular distribution of immunodetectable protein kinase C enzymes did not account for the differences between src- and ras-transformed 3T3 cells with regard to regulation of the RAF kinase. These findings in serum-deprived 3T3 cells demonstrate that expression of a ras oncogene can be insufficient for full activation of the p72-74 RAF-1 kinase, implying necessity for an additional growth factor-mediated stimulus.
Reed
J C
JC
Department of Pathology, University of Pennsylvania School of Medicine, Philadelphia 19104-6082.
Yum
S
S
Cuddy
M P
MP
Turner
B C
BC
Rapp
U R
UR
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D002471:Cell Transformation, Neoplastic / Q000378:metabolism; D016391:Genes, src; D066298:In Vitro Techniques; D051379:Mice; D008970:Molecular Weight; D015689:Oncogene Protein p21(ras) / Q000502:physiology; D015688:Oncogene Protein pp60(v-src) / Q000502:physiology; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D010750:Phosphoproteins; D010982:Platelet-Derived Growth Factor; D011518:Proto-Oncogene Proteins; D015688:Oncogene Protein pp60(v-src); D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D015689:Oncogene Protein p21(ras); D013755:Tetradecanoylphorbol Acetate