title: Effect of a dominant inhibitory Ha-ras mutation on mitogenic signal transduction in NIH 3T3 cells.
abstract: We used a dominant inhibitory mutation of c-Ha-ras which changes Ser-17 to Asn-17 in the gene product p21 [p21(Asn-17)Ha-ras] to investigate ras function in mitogenic signal transduction. An NIH 3T3 cell line [NIH(M17)] was isolated that displayed inducible expression of the mutant Ha-ras gene (Ha-ras Asn-17) via the mouse mammary tumor virus long terminal repeat and was growth inhibited by dexamethasone. The effect of dexamethasone induction on response of quiescent NIH(M17) cells to mitogens was then analyzed. Stimulation of DNA synthesis by epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) was completely blocked by p21(Asn-17) expression, and stimulation by serum, fibroblast growth factor, and platelet-derived growth factor was partially inhibited. However, the induction of fos, jun, and myc by EGF and TPA was not significantly inhibited in this cell line. An effect of p21(Asn-17) on fos induction was, however, demonstrated in transient expression assays in which quiescent NIH 3T3 cells were cotransfected with a fos-cat receptor plasmid plus a Ha-ras Asn-17 expression vector. In this assay, p21(Asn-17) inhibited chloramphenicol acetyltransferase expression induced by EGF and other growth factors. In contrast to its effect on DNA synthesis, however, Ha-ras Asn-17 expression did not inhibit fos-cat expression induced by TPA. Conversely, downregulation of protein kinase C did not inhibit fos-cat induction by activated ras or other oncogenes. These results suggest that ras proteins are involved in at least two parallel mitogenic signal transduction pathways, one of which is independent of protein kinase C. Although either pathway alone appears to be sufficient to induce fos, both appear to be necessary to induce the full mitogenic response.
authors.affiliation: Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: H
authors.initials: H
authors.lastname: Cai
authors.forename: J
authors.initials: J
authors.lastname: Szeberényi
authors.forename: G M
authors.initials: GM
authors.lastname: Cooper
mesh_terms: D000818:Animals; D002460:Cell Line; D004247:DNA / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000235:genetics; D005799:Genes, Dominant; D006133:Growth Substances / Q000494:pharmacology; D051379:Mice; D008934:Mitogens / Q000494:pharmacology*; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics; D015689:Oncogene Protein p21(ras) / Q000502:physiology*; D009857:Oncogenes; D011493:Protein Kinase C / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000235:genetics; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D016271:Proto-Oncogene Proteins c-myc; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics; D014162:Transfection
chemical_list: D004268:DNA-Binding Proteins; D006133:Growth Substances; D008934:Mitogens; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D016271:Proto-Oncogene Proteins c-myc; D014157:Transcription Factors; D004247:DNA; D011493:Protein Kinase C; D015689:Oncogene Protein p21(ras); D013755:Tetradecanoylphorbol Acetate