Raf-1 protein kinase is required for growth of induced NIH/3T3 cells.
Many growth factors regulate the cytoplasmic Raf-1 protein kinase, consistent with its having a central role in transduction of growth signals. The kinase is ubiquitously expressed and can promote proliferation, presumably in a manner dependent on growth-factor receptors and membrane-associated oncogenes. We have now examined the dependence of serum- and TPA (12-O-tetradecanoylphorbol-13-acetate)-regulated NIH/3T3 cell growth on RAF-1 kinase to determine whether Raf-1 is essential for receptor signalling. We inhibited Raf-1 function by expressing c-raf-1 antisense RNA or kinase-defective c-raf-1 mutants. Antisense RNA for c-raf-1 interferes with proliferation of normal NIH/3T3 cells and reverts raf-transformed cells. In revertant cells, DNA replication induced by serum or TPA was eliminated or reduced proportionately to the reduction in Raf protein levels. Expression of a kinase-defective Raf-1 mutant (craf301) or a regulatory domain fragment (HCR) inhibited serum-induced NIH/3T3-cell proliferation and raf transformation even more efficiently. Inhibition by antisense RNA or craf301 blocked proliferation and transformation by Ki- and Ha-ras oncogenes. We conclude that raf functions as an essential signal transducer downstream of serum growth factor receptors, protein kinase C and ras.
Kolch
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W
Laboratory of Viral Carcinogenesis, NIH/NCI, Frederick Cancer Research and Development Center, Maryland 21702-1201.
Heidecker
G
G
Lloyd
P
P
Rapp
U R
UR
D000818:Animals; D002455:Cell Division; D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D051379:Mice; D010957:Plasmids; D011494:Protein Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D016372:RNA, Antisense; D015398:Signal Transduction; D014162:Transfection
D011518:Proto-Oncogene Proteins; D016372:RNA, Antisense; D011494:Protein Kinases; D019908:Proto-Oncogene Proteins c-raf