Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1992 Mar;12(3):936–945. doi: 10.1128/mcb.12.3.936

Regulation of tetradecanoyl phorbol acetate-induced responses in NIH 3T3 cells by GAP, the GTPase-activating protein associated with p21c-ras.

M Nori 1, G L'Allemain 1, M J Weber 1
PMCID: PMC369525  PMID: 1545825

Abstract

Proteins of the ras family of oncogenes have been implicated in signal transduction pathways initiated by protein kinase C (PKC) and by tyrosine kinase oncogenes and receptors, but the role that ras plays in these diverse signalling systems is poorly defined. The activity of ras proteins has been shown to be controlled in part by a cellular protein, GAP (GTPase-activating protein), that negatively regulates p21c-ras by enhancing its intrinsic GTPase activity. Thus, overexpression of GAP provides a tool for determining the step(s) in signal transduction dependent on p21c-ras activity. In this paper, we report that overexpression of GAP blocks the phorbol ester (tetradecanoyl phorbol acetate [TPA])-induced activation of p42 mitogen-activated protein kinase (p42mapk), c-fos expression, and DNA synthesis. GAP overexpression did not block responses to serum or fluoroaluminate. Moreover, not all biochemical events elicited by TPA were affected by GAP overexpression, as increased glucose uptake and phosphorylation of MARCKS, a major PKC substrate, occurred normally. Reduction of GAP expression to near normal levels restored the ability of the cells to activate p42mapk in response to TPA. These findings suggest that ras and GAP together play a key role in a PKC-dependent signal transduction pathway which leads to p42mapk activation and cell proliferation.

Full text

PDF
945

Images in this article

938Image
on p.938
939Image
on p.939
939Image
on p.939
940Image
on p.940
941Image
on p.941
941Image
on p.941
942Image
on p.942

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Almoguera C., Shibata D., Forrester K., Martin J., Arnheim N., Perucho M. Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes. Cell. 1988 May 20;53(4):549–554. doi: 10.1016/0092-8674(88)90571-5. [DOI] [PubMed] [Google Scholar]
  2. Anderson D., Koch C. A., Grey L., Ellis C., Moran M. F., Pawson T. Binding of SH2 domains of phospholipase C gamma 1, GAP, and Src to activated growth factor receptors. Science. 1990 Nov 16;250(4983):979–982. doi: 10.1126/science.2173144. [DOI] [PubMed] [Google Scholar]
  3. Anderson N. G., Maller J. L., Tonks N. K., Sturgill T. W. Requirement for integration of signals from two distinct phosphorylation pathways for activation of MAP kinase. Nature. 1990 Feb 15;343(6259):651–653. doi: 10.1038/343651a0. [DOI] [PubMed] [Google Scholar]
  4. Balch W. E. Small GTP-binding proteins in vesicular transport. Trends Biochem Sci. 1990 Dec;15(12):473–477. doi: 10.1016/0968-0004(90)90301-q. [DOI] [PubMed] [Google Scholar]
  5. Barbacid M. ras genes. Annu Rev Biochem. 1987;56:779–827. doi: 10.1146/annurev.bi.56.070187.004023. [DOI] [PubMed] [Google Scholar]
  6. Benito M., Porras A., Nebreda A. R., Santos E. Differentiation of 3T3-L1 fibroblasts to adipocytes induced by transfection of ras oncogenes. Science. 1991 Aug 2;253(5019):565–568. doi: 10.1126/science.1857988. [DOI] [PubMed] [Google Scholar]
  7. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  8. Butler-Gralla E., Herschman H. R. Glucose uptake and ornithine decarboxylase activity in tetradecanoyl phorbol acetate non-proliferative variants. J Cell Physiol. 1983 Mar;114(3):317–320. doi: 10.1002/jcp.1041140310. [DOI] [PubMed] [Google Scholar]
  9. Butler-Gralla E., Herschman H. R. Variants of 3T3 cells lacking mitogenic response to the tumor promoter tetradecanoyl-phorbol-acetate. J Cell Physiol. 1981 Apr;107(1):59–67. doi: 10.1002/jcp.1041070108. [DOI] [PubMed] [Google Scholar]
  10. Cai H., Szeberényi J., Cooper G. M. Effect of a dominant inhibitory Ha-ras mutation on mitogenic signal transduction in NIH 3T3 cells. Mol Cell Biol. 1990 Oct;10(10):5314–5323. doi: 10.1128/mcb.10.10.5314. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chardin P. Small GTP-binding proteins of the ras family: a conserved functional mechanism? Cancer Cells. 1991 Apr;3(4):117–126. [PubMed] [Google Scholar]
  12. Cobb M. H., Boulton T. G., Robbins D. J. Extracellular signal-regulated kinases: ERKs in progress. Cell Regul. 1991 Dec;2(12):965–978. doi: 10.1091/mbc.2.12.965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cooper J. A., Sefton B. M., Hunter T. Diverse mitogenic agents induce the phosphorylation of two related 42,000-dalton proteins on tyrosine in quiescent chick cells. Mol Cell Biol. 1984 Jan;4(1):30–37. doi: 10.1128/mcb.4.1.30. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. DeClue J. E., Zhang K., Redford P., Vass W. C., Lowy D. R. Suppression of src transformation by overexpression of full-length GTPase-activating protein (GAP) or of the GAP C terminus. Mol Cell Biol. 1991 May;11(5):2819–2825. doi: 10.1128/mcb.11.5.2819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Downward J., Graves J. D., Warne P. H., Rayter S., Cantrell D. A. Stimulation of p21ras upon T-cell activation. Nature. 1990 Aug 23;346(6286):719–723. doi: 10.1038/346719a0. [DOI] [PubMed] [Google Scholar]
  16. Dugaiczyk A., Haron J. A., Stone E. M., Dennison O. E., Rothblum K. N., Schwartz R. J. Cloning and sequencing of a deoxyribonucleic acid copy of glyceraldehyde-3-phosphate dehydrogenase messenger ribonucleic acid isolated from chicken muscle. Biochemistry. 1983 Mar 29;22(7):1605–1613. doi: 10.1021/bi00276a013. [DOI] [PubMed] [Google Scholar]
  17. Ellis C., Moran M., McCormick F., Pawson T. Phosphorylation of GAP and GAP-associated proteins by transforming and mitogenic tyrosine kinases. Nature. 1990 Jan 25;343(6256):377–381. doi: 10.1038/343377a0. [DOI] [PubMed] [Google Scholar]
  18. Ely C. M., Oddie K. M., Litz J. S., Rossomando A. J., Kanner S. B., Sturgill T. W., Parsons S. J. A 42-kD tyrosine kinase substrate linked to chromaffin cell secretion exhibits an associated MAP kinase activity and is highly related to a 42-kD mitogen-stimulated protein in fibroblasts. J Cell Biol. 1990 Mar;110(3):731–742. doi: 10.1083/jcb.110.3.731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Erickson A. K., Payne D. M., Martino P. A., Rossomando A. J., Shabanowitz J., Weber M. J., Hunt D. F., Sturgill T. W. Identification by mass spectrometry of threonine 97 in bovine myelin basic protein as a specific phosphorylation site for mitogen-activated protein kinase. J Biol Chem. 1990 Nov 15;265(32):19728–19735. [PubMed] [Google Scholar]
  20. Forrester K., Almoguera C., Han K., Grizzle W. E., Perucho M. Detection of high incidence of K-ras oncogenes during human colon tumorigenesis. 1987 May 28-Jun 3Nature. 327(6120):298–303. doi: 10.1038/327298a0. [DOI] [PubMed] [Google Scholar]
  21. Gibbs J. B., Marshall M. S., Scolnick E. M., Dixon R. A., Vogel U. S. Modulation of guanine nucleotides bound to Ras in NIH3T3 cells by oncogenes, growth factors, and the GTPase activating protein (GAP). J Biol Chem. 1990 Nov 25;265(33):20437–20442. [PubMed] [Google Scholar]
  22. Gilbert P. X., Harris H. The role of the ras oncogene in the formation of tumours. J Cell Sci. 1988 Jul;90(Pt 3):433–446. doi: 10.1242/jcs.90.3.433. [DOI] [PubMed] [Google Scholar]
  23. Hoshi M., Nishida E., Sakai H. Activation of a Ca2+-inhibitable protein kinase that phosphorylates microtubule-associated protein 2 in vitro by growth factors, phorbol esters, and serum in quiescent cultured human fibroblasts. J Biol Chem. 1988 Apr 15;263(11):5396–5401. [PubMed] [Google Scholar]
  24. Kahn R. A. Fluoride is not an activator of the smaller (20-25 kDa) GTP-binding proteins. J Biol Chem. 1991 Aug 25;266(24):15595–15597. [PubMed] [Google Scholar]
  25. Kamata T., Sullivan N. F., Wooten M. W. Reduced protein kinase C activity in a ras-resistant cell line derived from Ki-MSV transformed cells. Oncogene. 1987 Mar;1(1):37–46. [PubMed] [Google Scholar]
  26. Kamps M. P., Sefton B. M. Identification of multiple novel polypeptide substrates of the v-src, v-yes, v-fps, v-ros, and v-erb-B oncogenic tyrosine protein kinases utilizing antisera against phosphotyrosine. Oncogene. 1988 Apr;2(4):305–315. [PubMed] [Google Scholar]
  27. Kanner S. B., Reynolds A. B., Wang H. C., Vines R. R., Parsons J. T. The SH2 and SH3 domains of pp60src direct stable association with tyrosine phosphorylated proteins p130 and p110. EMBO J. 1991 Jul;10(7):1689–1698. doi: 10.1002/j.1460-2075.1991.tb07693.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Koch C. A., Anderson D., Moran M. F., Ellis C., Pawson T. SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins. Science. 1991 May 3;252(5006):668–674. doi: 10.1126/science.1708916. [DOI] [PubMed] [Google Scholar]
  29. Kohno M. Diverse mitogenic agents induce rapid phosphorylation of a common set of cellular proteins at tyrosine in quiescent mammalian cells. J Biol Chem. 1985 Feb 10;260(3):1771–1779. [PubMed] [Google Scholar]
  30. Kremer N. E., D'Arcangelo G., Thomas S. M., DeMarco M., Brugge J. S., Halegoua S. Signal transduction by nerve growth factor and fibroblast growth factor in PC12 cells requires a sequence of src and ras actions. J Cell Biol. 1991 Nov;115(3):809–819. doi: 10.1083/jcb.115.3.809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. L'Allemain G., Pouyssegur J., Weber M. J. p42/mitogen-activated protein kinase as a converging target for different growth factor signaling pathways: use of pertussis toxin as a discrimination factor. Cell Regul. 1991 Aug;2(8):675–684. doi: 10.1091/mbc.2.8.675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. L'Allemain G., Sturgill T. W., Weber M. J. Defective regulation of mitogen-activated protein kinase activity in a 3T3 cell variant mitogenically nonresponsive to tetradecanoyl phorbol acetate. Mol Cell Biol. 1991 Feb;11(2):1002–1008. doi: 10.1128/mcb.11.2.1002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lacal J. C., Cuadrado A., Jones J. E., Trotta R., Burstein D. E., Thomson T., Pellicer A. Regulation of protein kinase C activity in neuronal differentiation induced by the N-ras oncogene in PC-12 cells. Mol Cell Biol. 1990 Jun;10(6):2983–2990. doi: 10.1128/mcb.10.6.2983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  35. Lim R. W., Varnum B. C., Herschman H. R. Cloning of tetradecanoyl phorbol ester-induced 'primary response' sequences and their expression in density-arrested Swiss 3T3 cells and a TPA non-proliferative variant. Oncogene. 1987;1(3):263–270. [PubMed] [Google Scholar]
  36. Liu X. Q., Pawson T. The epidermal growth factor receptor phosphorylates GTPase-activating protein (GAP) at Tyr-460, adjacent to the GAP SH2 domains. Mol Cell Biol. 1991 May;11(5):2511–2516. doi: 10.1128/mcb.11.5.2511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Lobaugh L. A., Blackshear P. J. Neuropeptide Y stimulation of myosin light chain phosphorylation in cultured aortic smooth muscle cells. J Biol Chem. 1990 Oct 25;265(30):18393–18399. [PubMed] [Google Scholar]
  38. Manne V., Yamazaki S., Kung H. F. Guanosine nucleotide binding by highly purified Ha-ras-encoded p21 protein produced in Escherichia coli. Proc Natl Acad Sci U S A. 1984 Nov;81(22):6953–6957. doi: 10.1073/pnas.81.22.6953. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Markwell M. A., Haas S. M., Bieber L. L., Tolbert N. E. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem. 1978 Jun 15;87(1):206–210. doi: 10.1016/0003-2697(78)90586-9. [DOI] [PubMed] [Google Scholar]
  40. McCormick F. The world according to GAP. Oncogene. 1990 Sep;5(9):1281–1283. [PubMed] [Google Scholar]
  41. McGrath J. P., Capon D. J., Goeddel D. V., Levinson A. D. Comparative biochemical properties of normal and activated human ras p21 protein. Nature. 1984 Aug 23;310(5979):644–649. doi: 10.1038/310644a0. [DOI] [PubMed] [Google Scholar]
  42. Molloy C. J., Bottaro D. P., Fleming T. P., Marshall M. S., Gibbs J. B., Aaronson S. A. PDGF induction of tyrosine phosphorylation of GTPase activating protein. Nature. 1989 Dec 7;342(6250):711–714. doi: 10.1038/342711a0. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. 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]
  45. Nishizuka Y. The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature. 1984 Apr 19;308(5961):693–698. doi: 10.1038/308693a0. [DOI] [PubMed] [Google Scholar]
  46. Nori M., Shawver L. K., Weber M. J. A Swiss 3T3 variant cell line resistant to the effects of tumor promoters cannot be transformed by src. Mol Cell Biol. 1990 Aug;10(8):4155–4162. doi: 10.1128/mcb.10.8.4155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Nori M., Vogel U. S., Gibbs J. B., Weber M. J. Inhibition of v-src-induced transformation by a GTPase-activating protein. Mol Cell Biol. 1991 May;11(5):2812–2818. doi: 10.1128/mcb.11.5.2812. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Payne D. M., Rossomando A. J., Martino P., Erickson A. K., Her J. H., Shabanowitz J., Hunt D. F., Weber M. J., Sturgill T. W. Identification of the regulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAP kinase). EMBO J. 1991 Apr;10(4):885–892. doi: 10.1002/j.1460-2075.1991.tb08021.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Ray L. B., Sturgill T. W. Rapid stimulation by insulin of a serine/threonine kinase in 3T3-L1 adipocytes that phosphorylates microtubule-associated protein 2 in vitro. Proc Natl Acad Sci U S A. 1987 Mar;84(6):1502–1506. doi: 10.1073/pnas.84.6.1502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Rossomando A. J., Payne D. M., Weber M. J., Sturgill T. W. Evidence that pp42, a major tyrosine kinase target protein, is a mitogen-activated serine/threonine protein kinase. Proc Natl Acad Sci U S A. 1989 Sep;86(18):6940–6943. doi: 10.1073/pnas.86.18.6940. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Rossomando A. J., Sanghera J. S., Marsden L. A., Weber M. J., Pelech S. L., Sturgill T. W. Biochemical characterization of a family of serine/threonine protein kinases regulated by tyrosine and serine/threonine phosphorylations. J Biol Chem. 1991 Oct 25;266(30):20270–20275. [PubMed] [Google Scholar]
  52. Satoh T., Nakamura S., Kaziro Y. Induction of neurite formation in PC12 cells by microinjection of proto-oncogenic Ha-ras protein preincubated with guanosine-5'-O-(3-thiotriphosphate). Mol Cell Biol. 1987 Dec;7(12):4553–4556. doi: 10.1128/mcb.7.12.4553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Seger R., Ahn N. G., Boulton T. G., Yancopoulos G. D., Panayotatos N., Radziejewska E., Ericsson L., Bratlien R. L., Cobb M. H., Krebs E. G. Microtubule-associated protein 2 kinases, ERK1 and ERK2, undergo autophosphorylation on both tyrosine and threonine residues: implications for their mechanism of activation. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6142–6146. doi: 10.1073/pnas.88.14.6142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. 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]
  55. Smrcka A. V., Hepler J. R., Brown K. O., Sternweis P. C. Regulation of polyphosphoinositide-specific phospholipase C activity by purified Gq. Science. 1991 Feb 15;251(4995):804–807. doi: 10.1126/science.1846707. [DOI] [PubMed] [Google Scholar]
  56. Stumpo D. J., Graff J. M., Albert K. A., Greengard P., Blackshear P. J. Molecular cloning, characterization, and expression of a cDNA encoding the "80- to 87-kDa" myristoylated alanine-rich C kinase substrate: a major cellular substrate for protein kinase C. Proc Natl Acad Sci U S A. 1989 Jun;86(11):4012–4016. doi: 10.1073/pnas.86.11.4012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Sturgill T. W., Wu J. Recent progress in characterization of protein kinase cascades for phosphorylation of ribosomal protein S6. Biochim Biophys Acta. 1991 May 17;1092(3):350–357. doi: 10.1016/s0167-4889(97)90012-4. [DOI] [PubMed] [Google Scholar]
  58. Sukumar S. ras oncogenes in chemical carcinogenesis. Curr Top Microbiol Immunol. 1989;148:93–114. doi: 10.1007/978-3-642-74700-7_3. [DOI] [PubMed] [Google Scholar]
  59. Taylor S. J., Chae H. Z., Rhee S. G., Exton J. H. Activation of the beta 1 isozyme of phospholipase C by alpha subunits of the Gq class of G proteins. Nature. 1991 Apr 11;350(6318):516–518. doi: 10.1038/350516a0. [DOI] [PubMed] [Google Scholar]
  60. Trahey M., McCormick F. A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants. Science. 1987 Oct 23;238(4826):542–545. doi: 10.1126/science.2821624. [DOI] [PubMed] [Google Scholar]
  61. Ullrich A., Schlessinger J. Signal transduction by receptors with tyrosine kinase activity. Cell. 1990 Apr 20;61(2):203–212. doi: 10.1016/0092-8674(90)90801-k. [DOI] [PubMed] [Google Scholar]
  62. Vaidya T. B., Weyman C. M., Teegarden D., Ashendel C. L., Taparowsky E. J. Inhibition of myogenesis by the H-ras oncogene: implication of a role for protein kinase C. J Cell Biol. 1991 Aug;114(4):809–820. doi: 10.1083/jcb.114.4.809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Vogel U. S., Dixon R. A., Schaber M. D., Diehl R. E., Marshall M. S., Scolnick E. M., Sigal I. S., Gibbs J. B. Cloning of bovine GAP and its interaction with oncogenic ras p21. Nature. 1988 Sep 1;335(6185):90–93. doi: 10.1038/335090a0. [DOI] [PubMed] [Google Scholar]
  64. Weber M. J. Hexose transport in normal and in Rous sarcoma virus-transformed cells. J Biol Chem. 1973 May 10;248(9):2978–2983. [PubMed] [Google Scholar]
  65. Wu J., Rossomando A. J., Her J. H., Del Vecchio R., Weber M. J., Sturgill T. W. Autophosphorylation in vitro of recombinant 42-kilodalton mitogen-activated protein kinase on tyrosine. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9508–9512. doi: 10.1073/pnas.88.21.9508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Zhang K., DeClue J. E., Vass W. C., Papageorge A. G., McCormick F., Lowy D. R. Suppression of c-ras transformation by GTPase-activating protein. Nature. 1990 Aug 23;346(6286):754–756. doi: 10.1038/346754a0. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES