Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1993 Jan;13(1):155–162. doi: 10.1128/mcb.13.1.155

Ras activation by insulin and epidermal growth factor through enhanced exchange of guanine nucleotides on p21ras.

R H Medema 1, A M de Vries-Smits 1, G C van der Zon 1, J A Maassen 1, J L Bos 1
PMCID: PMC358895  PMID: 8417322

Abstract

A number of growth factors, including insulin and epidermal growth factor (EGF), induce accumulation of the GTP-bound form of p21ras. This accumulation could be caused either by an increase in guanine nucleotide exchange on p21ras or by a decrease in the GTPase activity of p21ras. To investigate whether insulin and EGF affect nucleotide exchange on p21ras, we measured binding of [alpha-32P]GTP to p21ras in cells permeabilized with streptolysin O. For this purpose, we used a cell line which expressed elevated levels of p21 H-ras and which was highly responsive to insulin and EGF. Stimulation with insulin or EGF resulted in an increase in the rate of nucleotide binding to p21ras. To determine whether this increased binding rate is due to the activation of a guanine nucleotide exchange factor, we made use of the inhibitory properties of a dominant negative mutant of p21ras, p21ras (Asn-17). Activation of p21ras by insulin and EGF in intact cells was abolished in cells infected with a recombinant vaccinia virus expressing p21ras (Asn-17). In addition, the enhanced nucleotide binding to p21ras in response to insulin and EGF in permeabilized cells was blocked upon expression of p21ras (Asn-17). From these data, we conclude that the activation of a guanine nucleotide exchange factor is involved in insulin- and EGF-induced activation of p21ras.

Full text

PDF
159

Images in this article

157Image
on p.157
158Image
on p.158
159Image
on p.159
160Image
on p.160
160Image
on p.160

Selected References

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

  1. Ballester R., Marchuk D., Boguski M., Saulino A., Letcher R., Wigler M., Collins F. The NF1 locus encodes a protein functionally related to mammalian GAP and yeast IRA proteins. Cell. 1990 Nov 16;63(4):851–859. doi: 10.1016/0092-8674(90)90151-4. [DOI] [PubMed] [Google Scholar]
  2. Barbacid M. ras genes. Annu Rev Biochem. 1987;56:779–827. doi: 10.1146/annurev.bi.56.070187.004023. [DOI] [PubMed] [Google Scholar]
  3. Basu T. N., Gutmann D. H., Fletcher J. A., Glover T. W., Collins F. S., Downward J. Aberrant regulation of ras proteins in malignant tumour cells from type 1 neurofibromatosis patients. Nature. 1992 Apr 23;356(6371):713–715. doi: 10.1038/356713a0. [DOI] [PubMed] [Google Scholar]
  4. Bollag G., McCormick F. Differential regulation of rasGAP and neurofibromatosis gene product activities. Nature. 1991 Jun 13;351(6327):576–579. doi: 10.1038/351576a0. [DOI] [PubMed] [Google Scholar]
  5. Bonfini L., Karlovich C. A., Dasgupta C., Banerjee U. The Son of sevenless gene product: a putative activator of Ras. Science. 1992 Jan 31;255(5044):603–606. doi: 10.1126/science.1736363. [DOI] [PubMed] [Google Scholar]
  6. Bourne H. R., Sanders D. A., McCormick F. The GTPase superfamily: a conserved switch for diverse cell functions. Nature. 1990 Nov 8;348(6297):125–132. doi: 10.1038/348125a0. [DOI] [PubMed] [Google Scholar]
  7. Bourne H. R., Sanders D. A., McCormick F. The GTPase superfamily: conserved structure and molecular mechanism. Nature. 1991 Jan 10;349(6305):117–127. doi: 10.1038/349117a0. [DOI] [PubMed] [Google Scholar]
  8. Broek D., Toda T., Michaeli T., Levin L., Birchmeier C., Zoller M., Powers S., Wigler M. The S. cerevisiae CDC25 gene product regulates the RAS/adenylate cyclase pathway. Cell. 1987 Mar 13;48(5):789–799. doi: 10.1016/0092-8674(87)90076-6. [DOI] [PubMed] [Google Scholar]
  9. Buller R. M., Chakrabarti S., Cooper J. A., Twardzik D. R., Moss B. Deletion of the vaccinia virus growth factor gene reduces virus virulence. J Virol. 1988 Mar;62(3):866–874. doi: 10.1128/jvi.62.3.866-874.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Burgering B. M., Medema R. H., Maassen J. A., van de Wetering M. L., van der Eb A. J., McCormick F., Bos J. L. Insulin stimulation of gene expression mediated by p21ras activation. EMBO J. 1991 May;10(5):1103–1109. doi: 10.1002/j.1460-2075.1991.tb08050.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Burgering B. M., Snijders A. J., Maassen J. A., van der Eb A. J., Bos J. L. Possible involvement of normal p21 H-ras in the insulin/insulinlike growth factor 1 signal transduction pathway. Mol Cell Biol. 1989 Oct;9(10):4312–4322. doi: 10.1128/mcb.9.10.4312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Créchet J. B., Poullet P., Mistou M. Y., Parmeggiani A., Camonis J., Boy-Marcotte E., Damak F., Jacquet M. Enhancement of the GDP-GTP exchange of RAS proteins by the carboxyl-terminal domain of SCD25. Science. 1990 May 18;248(4957):866–868. doi: 10.1126/science.2188363. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Downward J., Riehl R., Wu L., Weinberg R. A. Identification of a nucleotide exchange-promoting activity for p21ras. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5998–6002. doi: 10.1073/pnas.87.15.5998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Downward J., de Gunzburg J., Riehl R., Weinberg R. A. p21ras-induced responsiveness of phosphatidylinositol turnover to bradykinin is a receptor number effect. Proc Natl Acad Sci U S A. 1988 Aug;85(16):5774–5778. doi: 10.1073/pnas.85.16.5774. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Gaul U., Mardon G., Rubin G. M. A putative Ras GTPase activating protein acts as a negative regulator of signaling by the Sevenless receptor tyrosine kinase. Cell. 1992 Mar 20;68(6):1007–1019. doi: 10.1016/0092-8674(92)90073-l. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Gutierrez L., Magee A. I., Marshall C. J., Hancock J. F. Post-translational processing of p21ras is two-step and involves carboxyl-methylation and carboxy-terminal proteolysis. EMBO J. 1989 Apr;8(4):1093–1098. doi: 10.1002/j.1460-2075.1989.tb03478.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Janknecht R., de Martynoff G., Lou J., Hipskind R. A., Nordheim A., Stunnenberg H. G. Rapid and efficient purification of native histidine-tagged protein expressed by recombinant vaccinia virus. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):8972–8976. doi: 10.1073/pnas.88.20.8972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kamata T., Feramisco J. R. Epidermal growth factor stimulates guanine nucleotide binding activity and phosphorylation of ras oncogene proteins. Nature. 1984 Jul 12;310(5973):147–150. doi: 10.1038/310147a0. [DOI] [PubMed] [Google Scholar]
  24. Kazlauskas A., Ellis C., Pawson T., Cooper J. A. Binding of GAP to activated PDGF receptors. Science. 1990 Mar 30;247(4950):1578–1581. doi: 10.1126/science.2157284. [DOI] [PubMed] [Google Scholar]
  25. Leevers S. J., Marshall C. J. Activation of extracellular signal-regulated kinase, ERK2, by p21ras oncoprotein. EMBO J. 1992 Feb;11(2):569–574. doi: 10.1002/j.1460-2075.1992.tb05088.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Li B. Q., Kaplan D., Kung H. F., Kamata T. Nerve growth factor stimulation of the Ras-guanine nucleotide exchange factor and GAP activities. Science. 1992 Jun 5;256(5062):1456–1459. doi: 10.1126/science.1604323. [DOI] [PubMed] [Google Scholar]
  27. Margolis B., Li N., Koch A., Mohammadi M., Hurwitz D. R., Zilberstein A., Ullrich A., Pawson T., Schlessinger J. The tyrosine phosphorylated carboxyterminus of the EGF receptor is a binding site for GAP and PLC-gamma. EMBO J. 1990 Dec;9(13):4375–4380. doi: 10.1002/j.1460-2075.1990.tb07887.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Martegani E., Vanoni M., Zippel R., Coccetti P., Brambilla R., Ferrari C., Sturani E., Alberghina L. Cloning by functional complementation of a mouse cDNA encoding a homologue of CDC25, a Saccharomyces cerevisiae RAS activator. EMBO J. 1992 Jun;11(6):2151–2157. doi: 10.1002/j.1460-2075.1992.tb05274.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Martin G. A., Viskochil D., Bollag G., McCabe P. C., Crosier W. J., Haubruck H., Conroy L., Clark R., O'Connell P., Cawthon R. M. The GAP-related domain of the neurofibromatosis type 1 gene product interacts with ras p21. Cell. 1990 Nov 16;63(4):843–849. doi: 10.1016/0092-8674(90)90150-d. [DOI] [PubMed] [Google Scholar]
  30. Medema R. H., Wubbolts R., Bos J. L. Two dominant inhibitory mutants of p21ras interfere with insulin-induced gene expression. Mol Cell Biol. 1991 Dec;11(12):5963–5967. doi: 10.1128/mcb.11.12.5963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Moran M. F., Polakis P., McCormick F., Pawson T., Ellis C. Protein-tyrosine kinases regulate the phosphorylation, protein interactions, subcellular distribution, and activity of p21ras GTPase-activating protein. Mol Cell Biol. 1991 Apr;11(4):1804–1812. doi: 10.1128/mcb.11.4.1804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Satoh T., Endo M., Nakafuku M., Akiyama T., Yamamoto T., Kaziro Y. Accumulation of p21ras.GTP in response to stimulation with epidermal growth factor and oncogene products with tyrosine kinase activity. Proc Natl Acad Sci U S A. 1990 Oct;87(20):7926–7929. doi: 10.1073/pnas.87.20.7926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Satoh T., Endo M., Nakafuku M., Nakamura S., Kaziro Y. Platelet-derived growth factor stimulates formation of active p21ras.GTP complex in Swiss mouse 3T3 cells. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5993–5997. doi: 10.1073/pnas.87.15.5993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Shou C., Farnsworth C. L., Neel B. G., Feig L. A. Molecular cloning of cDNAs encoding a guanine-nucleotide-releasing factor for Ras p21. Nature. 1992 Jul 23;358(6384):351–354. doi: 10.1038/358351a0. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. 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]
  38. 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]
  39. Tanaka K., Nakafuku M., Satoh T., Marshall M. S., Gibbs J. B., Matsumoto K., Kaziro Y., Toh-e A. S. cerevisiae genes IRA1 and IRA2 encode proteins that may be functionally equivalent to mammalian ras GTPase activating protein. Cell. 1990 Mar 9;60(5):803–807. doi: 10.1016/0092-8674(90)90094-u. [DOI] [PubMed] [Google Scholar]
  40. Thomas S. M., DeMarco M., D'Arcangelo G., Halegoua S., Brugge J. S. Ras is essential for nerve growth factor- and phorbol ester-induced tyrosine phosphorylation of MAP kinases. Cell. 1992 Mar 20;68(6):1031–1040. doi: 10.1016/0092-8674(92)90075-n. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. West M., Kung H. F., Kamata T. A novel membrane factor stimulates guanine nucleotide exchange reaction of ras proteins. FEBS Lett. 1990 Jan 1;259(2):245–248. doi: 10.1016/0014-5793(90)80019-f. [DOI] [PubMed] [Google Scholar]
  43. Wolfman A., Macara I. G. A cytosolic protein catalyzes the release of GDP from p21ras. Science. 1990 Apr 6;248(4951):67–69. doi: 10.1126/science.2181667. [DOI] [PubMed] [Google Scholar]
  44. Wood K. W., Sarnecki C., Roberts T. M., Blenis J. ras mediates nerve growth factor receptor modulation of three signal-transducing protein kinases: MAP kinase, Raf-1, and RSK. Cell. 1992 Mar 20;68(6):1041–1050. doi: 10.1016/0092-8674(92)90076-o. [DOI] [PubMed] [Google Scholar]
  45. Xu G. F., O'Connell P., Viskochil D., Cawthon R., Robertson M., Culver M., Dunn D., Stevens J., Gesteland R., White R. The neurofibromatosis type 1 gene encodes a protein related to GAP. Cell. 1990 Aug 10;62(3):599–608. doi: 10.1016/0092-8674(90)90024-9. [DOI] [PubMed] [Google Scholar]
  46. Zhang K., Papageorge A. G., Lowy D. R. Mechanistic aspects of signaling through Ras in NIH 3T3 cells. Science. 1992 Jul 31;257(5070):671–674. doi: 10.1126/science.1496380. [DOI] [PubMed] [Google Scholar]
  47. de Vries-Smits A. M., Burgering B. M., Leevers S. J., Marshall C. J., Bos J. L. Involvement of p21ras in activation of extracellular signal-regulated kinase 2. Nature. 1992 Jun 18;357(6379):602–604. doi: 10.1038/357602a0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES