Effect of a dominant inhibitory Ha-ras mutation on mitogenic signal transduction in NIH 3T3 cells (original) (raw)

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.

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Selected References

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  1. Beckner S. K., Hattori S., Shih T. Y. The ras oncogene product p21 is not a regulatory component of adenylate cyclase. Nature. 1985 Sep 5;317(6032):71–72. doi: 10.1038/317071a0. [DOI] [PubMed] [Google Scholar]
  2. Blair D. G., McClements W. L., Oskarsson M. K., Fischinger P. J., Vande Woude G. F. Biological activity of cloned Moloney sarcoma virus DNA: Terminally redundant sequences may enhance transformation efficiency. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3504–3508. doi: 10.1073/pnas.77.6.3504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Copeland N. G., Cooper G. M. Transfection by exogenous and endogenous murine retrovirus DNAs. Cell. 1979 Feb;16(2):347–356. doi: 10.1016/0092-8674(79)90011-4. [DOI] [PubMed] [Google Scholar]
  4. Feig L. A., Cooper G. M. Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP. Mol Cell Biol. 1988 Aug;8(8):3235–3243. doi: 10.1128/mcb.8.8.3235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Feramisco J. R., Gross M., Kamata T., Rosenberg M., Sweet R. W. Microinjection of the oncogene form of the human H-ras (T-24) protein results in rapid proliferation of quiescent cells. Cell. 1984 Aug;38(1):109–117. doi: 10.1016/0092-8674(84)90531-2. [DOI] [PubMed] [Google Scholar]
  6. Fleischman L. F., Chahwala S. B., Cantley L. ras-transformed cells: altered levels of phosphatidylinositol-4,5-bisphosphate and catabolites. Science. 1986 Jan 24;231(4736):407–410. doi: 10.1126/science.3001936. [DOI] [PubMed] [Google Scholar]
  7. Fujii M., Shalloway D., Verma I. M. Gene regulation by tyrosine kinases: src protein activates various promoters, including c-fos. Mol Cell Biol. 1989 Jun;9(6):2493–2499. doi: 10.1128/mcb.9.6.2493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fukumoto Y., Kaibuchi K., Oku N., Hori Y., Takai Y. Activation of the c-fos serum-response element by the activated c-Ha-ras protein in a manner independent of protein kinase C and cAMP-dependent protein kinase. J Biol Chem. 1990 Jan 15;265(2):774–780. [PubMed] [Google Scholar]
  9. Furth M. E., Davis L. J., Fleurdelys B., Scolnick E. M. Monoclonal antibodies to the p21 products of the transforming gene of Harvey murine sarcoma virus and of the cellular ras gene family. J Virol. 1982 Jul;43(1):294–304. doi: 10.1128/jvi.43.1.294-304.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gauthier-Rouvière C., Fernandez A., Lamb N. J. ras-induced c-fos expression and proliferation in living rat fibroblasts involves C-kinase activation and the serum response element pathway. EMBO J. 1990 Jan;9(1):171–180. doi: 10.1002/j.1460-2075.1990.tb08093.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jamal S., Ziff E. Transactivation of c-fos and beta-actin genes by raf as a step in early response to transmembrane signals. Nature. 1990 Mar 29;344(6265):463–466. doi: 10.1038/344463a0. [DOI] [PubMed] [Google Scholar]
  13. Lacal J. C., Fleming T. P., Warren B. S., Blumberg P. M., Aaronson S. A. Involvement of functional protein kinase C in the mitogenic response to the H-ras oncogene product. Mol Cell Biol. 1987 Nov;7(11):4146–4149. doi: 10.1128/mcb.7.11.4146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lacal J. C., Moscat J., Aaronson S. A. Novel source of 1,2-diacylglycerol elevated in cells transformed by Ha-ras oncogene. Nature. 1987 Nov 19;330(6145):269–272. doi: 10.1038/330269a0. [DOI] [PubMed] [Google Scholar]
  15. Lacal J. C., de la Peña P., Moscat J., Garcia-Barreno P., Anderson P. S., Aaronson S. A. Rapid stimulation of diacylglycerol production in Xenopus oocytes by microinjection of H-ras p21. Science. 1987 Oct 23;238(4826):533–536. doi: 10.1126/science.2821623. [DOI] [PubMed] [Google Scholar]
  16. Lloyd A. C., Paterson H. F., Morris J. D., Hall A., Marshall C. J. p21H-ras-induced morphological transformation and increases in c-myc expression are independent of functional protein kinase C. EMBO J. 1989 Apr;8(4):1099–1104. doi: 10.1002/j.1460-2075.1989.tb03479.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Morris J. D., Price B., Lloyd A. C., Self A. J., Marshall C. J., Hall A. Scrape-loading of Swiss 3T3 cells with ras protein rapidly activates protein kinase C in the absence of phosphoinositide hydrolysis. Oncogene. 1989 Jan;4(1):27–31. [PubMed] [Google Scholar]
  18. Mulcahy L. S., Smith M. R., Stacey D. W. Requirement for ras proto-oncogene function during serum-stimulated growth of NIH 3T3 cells. Nature. 1985 Jan 17;313(5999):241–243. doi: 10.1038/313241a0. [DOI] [PubMed] [Google Scholar]
  19. Pan B. T., Cooper G. M. Role of phosphatidylinositide metabolism in ras-induced Xenopus oocyte maturation. Mol Cell Biol. 1990 Mar;10(3):923–929. doi: 10.1128/mcb.10.3.923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Powers S., O'Neill K., Wigler M. Dominant yeast and mammalian RAS mutants that interfere with the CDC25-dependent activation of wild-type RAS in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Feb;9(2):390–395. doi: 10.1128/mcb.9.2.390. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Preiss J., Loomis C. R., Bishop W. R., Stein R., Niedel J. E., Bell R. M. Quantitative measurement of sn-1,2-diacylglycerols present in platelets, hepatocytes, and ras- and sis-transformed normal rat kidney cells. J Biol Chem. 1986 Jul 5;261(19):8597–8600. [PubMed] [Google Scholar]
  22. Sassone-Corsi P., Sisson J. C., Verma I. M. Transcriptional autoregulation of the proto-oncogene fos. Nature. 1988 Jul 28;334(6180):314–319. doi: 10.1038/334314a0. [DOI] [PubMed] [Google Scholar]
  23. Shalloway D., Zelenetz A. D., Cooper G. M. Molecular cloning and characterization of the chicken gene homologous to the transforming gene of Rous sarcoma virus. Cell. 1981 May;24(2):531–541. doi: 10.1016/0092-8674(81)90344-5. [DOI] [PubMed] [Google Scholar]
  24. Smith M. R., DeGudicibus S. J., Stacey D. W. Requirement for c-ras proteins during viral oncogene transformation. Nature. 1986 Apr 10;320(6062):540–543. doi: 10.1038/320540a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
  26. Stacey D. W., Kung H. F. Transformation of NIH 3T3 cells by microinjection of Ha-ras p21 protein. Nature. 1984 Aug 9;310(5977):508–511. doi: 10.1038/310508a0. [DOI] [PubMed] [Google Scholar]
  27. Stacey D. W., Watson T., Kung H. F., Curran T. Microinjection of transforming ras protein induces c-fos expression. Mol Cell Biol. 1987 Jan;7(1):523–527. doi: 10.1128/mcb.7.1.523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Stanton V. P., Jr, Nichols D. W., Laudano A. P., Cooper G. M. Definition of the human raf amino-terminal regulatory region by deletion mutagenesis. Mol Cell Biol. 1989 Feb;9(2):639–647. doi: 10.1128/mcb.9.2.639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Szeberényi J., Cai H., Cooper G. M. Effect of a dominant inhibitory Ha-ras mutation on neuronal differentiation of PC12 cells. Mol Cell Biol. 1990 Oct;10(10):5324–5332. doi: 10.1128/mcb.10.10.5324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Toda T., Uno I., Ishikawa T., Powers S., Kataoka T., Broek D., Cameron S., Broach J., Matsumoto K., Wigler M. In yeast, RAS proteins are controlling elements of adenylate cyclase. Cell. 1985 Jan;40(1):27–36. doi: 10.1016/0092-8674(85)90305-8. [DOI] [PubMed] [Google Scholar]
  31. Wakelam M. J., Davies S. A., Houslay M. D., McKay I., Marshall C. J., Hall A. Normal p21N-ras couples bombesin and other growth factor receptors to inositol phosphate production. Nature. 1986 Sep 11;323(6084):173–176. doi: 10.1038/323173a0. [DOI] [PubMed] [Google Scholar]
  32. Wasylyk C., Imler J. L., Wasylyk B. Transforming but not immortalizing oncogenes activate the transcription factor PEA1. EMBO J. 1988 Aug;7(8):2475–2483. doi: 10.1002/j.1460-2075.1988.tb03094.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wasylyk C., Wasylyk B., Heidecker G., Huleihel M., Rapp U. R. Expression of raf oncogenes activates the PEA1 transcription factor motif. Mol Cell Biol. 1989 May;9(5):2247–2250. doi: 10.1128/mcb.9.5.2247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wolfes H., Kogawa K., Millette C. F., Cooper G. M. Specific expression of nuclear proto-oncogenes before entry into meiotic prophase of spermatogenesis. Science. 1989 Aug 18;245(4919):740–743. doi: 10.1126/science.2475907. [DOI] [PubMed] [Google Scholar]
  35. Wolfman A., Macara I. G. Elevated levels of diacylglycerol and decreased phorbol ester sensitivity in ras-transformed fibroblasts. Nature. 1987 Jan 22;325(6102):359–361. doi: 10.1038/325359a0. [DOI] [PubMed] [Google Scholar]
  36. Yu C. L., Tsai M. H., Stacey D. W. Cellular ras activity and phospholipid metabolism. Cell. 1988 Jan 15;52(1):63–71. doi: 10.1016/0092-8674(88)90531-4. [DOI] [PubMed] [Google Scholar]
  37. Zaret K. S., Yamamoto K. R. Reversible and persistent changes in chromatin structure accompany activation of a glucocorticoid-dependent enhancer element. Cell. 1984 Aug;38(1):29–38. doi: 10.1016/0092-8674(84)90523-3. [DOI] [PubMed] [Google Scholar]