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Mol Cell Biol. 1993 April; 13(4): 2420–2431.
PMCID: PMC359563

Plasma membrane-targeted ras GTPase-activating protein is a potent suppressor of p21ras function.


Although p21ras is localized to the plasma membrane, proteins it interacts with, such as the GTPase-activating proteins (GAPs) ras GAP and neurofibromin (NF1), are not, suggesting that one function of p21ras GTP may be to target such proteins to the plasma membrane. To investigate the effects of targeting ras GAP to the plasma membrane, ras C-terminal motifs sufficient for plasma membrane localization of p21ras were cloned onto the C terminus of ras GAP. Plasma membrane-targeted ras GAP is growth inhibitory to NIH 3T3 fibroblasts and COS cells. This growth inhibition correlates with GAP catalytic activity, since the plasma membrane-targeted C-terminal catalytic domain or the GAP-related domain of neurofibromin is inhibitory, whereas the similarly targeted N-terminal domain is not. Moreover, the inhibition is abrogated by the inactivating mutation L902I, which abolishes ras GAP catalytic activity. Coexpression of oncogenic mutant ras rescues cell viability, but the majority of rescued colonies are phenotypically untransformed. Furthermore, in focus assays, targeted ras GAP suppresses transformation by oncogenic mutant ras, and in reversion assays, targeted ras GAP can revert cells transformed by oncogenic mutant ras. Neither the targeted or nontargeted N-terminal domain nor the L902I mutant of ras GAP has any transforming activity. These data demonstrate that ras GAP can function as a negative regulator of ras and that plasma membrane localization potentiates this activity. However, if ras GAP is involved in the effector functions of p21ras, it can only be part of the effector complex for cell transformation.

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  • Adari H, Lowy DR, Willumsen BM, Der CJ, McCormick F. Guanosine triphosphatase activating protein (GAP) interacts with the p21 ras effector binding domain. Science. 1988 Apr 22;240(4851):518–521. [PubMed]
  • 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. [PubMed]
  • Ballester R, Michaeli T, Ferguson K, Xu HP, McCormick F, Wigler M. Genetic analysis of mammalian GAP expressed in yeast. Cell. 1989 Nov 17;59(4):681–686. [PubMed]
  • Barbacid M. ras genes. Annu Rev Biochem. 1987;56:779–827. [PubMed]
  • Basu TN, Gutmann DH, Fletcher JA, Glover TW, Collins FS, Downward J. Aberrant regulation of ras proteins in malignant tumour cells from type 1 neurofibromatosis patients. Nature. 1992 Apr 23;356(6371):713–715. [PubMed]
  • Bollag G, McCormick F. Differential regulation of rasGAP and neurofibromatosis gene product activities. Nature. 1991 Jun 13;351(6327):576–579. [PubMed]
  • Buchberg AM, Cleveland LS, Jenkins NA, Copeland NG. Sequence homology shared by neurofibromatosis type-1 gene and IRA-1 and IRA-2 negative regulators of the RAS cyclic AMP pathway. Nature. 1990 Sep 20;347(6290):291–294. [PubMed]
  • Calés C, Hancock JF, Marshall CJ, Hall A. The cytoplasmic protein GAP is implicated as the target for regulation by the ras gene product. Nature. 1988 Apr 7;332(6164):548–551. [PubMed]
  • Cawthon RM, Weiss R, Xu GF, Viskochil D, Culver M, Stevens J, Robertson M, Dunn D, Gesteland R, O'Connell P, et al. A major segment of the neurofibromatosis type 1 gene: cDNA sequence, genomic structure, and point mutations. Cell. 1990 Jul 13;62(1):193–201. [PubMed]
  • Chu G, Hayakawa H, Berg P. Electroporation for the efficient transfection of mammalian cells with DNA. Nucleic Acids Res. 1987 Feb 11;15(3):1311–1326. [PMC free article] [PubMed]
  • DeClue JE, Cohen BD, Lowy DR. Identification and characterization of the neurofibromatosis type 1 protein product. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):9914–9918. [PubMed]
  • DeClue JE, Papageorge AG, Fletcher JA, Diehl SR, Ratner N, Vass WC, Lowy DR. Abnormal regulation of mammalian p21ras contributes to malignant tumor growth in von Recklinghausen (type 1) neurofibromatosis. Cell. 1992 Apr 17;69(2):265–273. [PubMed]
  • DeClue JE, Stone JC, Blanchard RA, Papageorge AG, Martin P, Zhang K, Lowy DR. A ras effector domain mutant which is temperature sensitive for cellular transformation: interactions with GTPase-activating protein and NF-1. Mol Cell Biol. 1991 Jun;11(6):3132–3138. [PMC free article] [PubMed]
  • DeClue JE, Zhang K, Redford P, Vass WC, Lowy DR. 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. [PMC free article] [PubMed]
  • Dominguez I, Marshall MS, Gibbs JB, García de Herreros A, Cornet ME, Graziani G, Diaz-Meco MT, Johansen T, McCormick F, Moscat J. Role of GTPase activating protein in mitogenic signalling through phosphatidylcholine-hydrolysing phospholipase C. EMBO J. 1991 Nov;10(11):3215–3220. [PubMed]
  • 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. [PubMed]
  • Feig LA, Cooper GM. 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. [PMC free article] [PubMed]
  • Gaul U, Mardon G, Rubin GM. 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. [PubMed]
  • Gibbs JB, Marshall MS, Scolnick EM, Dixon RA, Vogel US. 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]
  • Gibbs JB, Schaber MD, Schofield TL, Scolnick EM, Sigal IS. Xenopus oocyte germinal-vesicle breakdown induced by [Val12]Ras is inhibited by a cytosol-localized Ras mutant. Proc Natl Acad Sci U S A. 1989 Sep;86(17):6630–6634. [PubMed]
  • Gutierrez L, Magee AI, Marshall CJ, Hancock JF. Post-translational processing of p21ras is two-step and involves carboxyl-methylation and carboxy-terminal proteolysis. EMBO J. 1989 Apr;8(4):1093–1098. [PubMed]
  • Hall A. ras and GAP--who's controlling whom? Cell. 1990 Jun 15;61(6):921–923. [PubMed]
  • Hancock JF, Cadwallader K, Marshall CJ. Methylation and proteolysis are essential for efficient membrane binding of prenylated p21K-ras(B). EMBO J. 1991 Mar;10(3):641–646. [PubMed]
  • Hancock JF, Cadwallader K, Paterson H, Marshall CJ. A CAAX or a CAAL motif and a second signal are sufficient for plasma membrane targeting of ras proteins. EMBO J. 1991 Dec;10(13):4033–4039. [PubMed]
  • Hancock JF, Magee AI, Childs JE, Marshall CJ. All ras proteins are polyisoprenylated but only some are palmitoylated. Cell. 1989 Jun 30;57(7):1167–1177. [PubMed]
  • Hancock JF, Paterson H, Marshall CJ. A polybasic domain or palmitoylation is required in addition to the CAAX motif to localize p21ras to the plasma membrane. Cell. 1990 Oct 5;63(1):133–139. [PubMed]
  • Hattori S, Maekawa M, Nakamura S. Identification of neurofibromatosis type I gene product as an insoluble GTPase-activating protein toward ras p21. Oncogene. 1992 Mar;7(3):481–485. [PubMed]
  • Imai Y, Miyake S, Hughes DA, Yamamoto M. Identification of a GTPase-activating protein homolog in Schizosaccharomyces pombe. Mol Cell Biol. 1991 Jun;11(6):3088–3094. [PMC free article] [PubMed]
  • Kaplan DR, Morrison DK, Wong G, McCormick F, Williams LT. PDGF beta-receptor stimulates tyrosine phosphorylation of GAP and association of GAP with a signaling complex. Cell. 1990 Apr 6;61(1):125–133. [PubMed]
  • Kazlauskas A, Ellis C, Pawson T, Cooper JA. Binding of GAP to activated PDGF receptors. Science. 1990 Mar 30;247(4950):1578–1581. [PubMed]
  • Li Y, Bollag G, Clark R, Stevens J, Conroy L, Fults D, Ward K, Friedman E, Samowitz W, Robertson M, et al. Somatic mutations in the neurofibromatosis 1 gene in human tumors. Cell. 1992 Apr 17;69(2):275–281. [PubMed]
  • Marshall CJ, Hall A, Weiss RA. A transforming gene present in human sarcoma cell lines. Nature. 1982 Sep 9;299(5879):171–173. [PubMed]
  • Marshall MS, Hill WS, Ng AS, Vogel US, Schaber MD, Scolnick EM, Dixon RA, Sigal IS, Gibbs JB. A C-terminal domain of GAP is sufficient to stimulate ras p21 GTPase activity. EMBO J. 1989 Apr;8(4):1105–1110. [PubMed]
  • Martin GA, Viskochil D, Bollag G, McCabe PC, Crosier WJ, Haubruck H, Conroy L, Clark R, O'Connell P, Cawthon RM, et al. The GAP-related domain of the neurofibromatosis type 1 gene product interacts with ras p21. Cell. 1990 Nov 16;63(4):843–849. [PubMed]
  • Martin GA, Yatani A, Clark R, Conroy L, Polakis P, Brown AM, McCormick F. GAP domains responsible for ras p21-dependent inhibition of muscarinic atrial K+ channel currents. Science. 1992 Jan 10;255(5041):192–194. [PubMed]
  • Miller J, Germain RN. Efficient cell surface expression of class II MHC molecules in the absence of associated invariant chain. J Exp Med. 1986 Nov 1;164(5):1478–1489. [PMC free article] [PubMed]
  • Molloy CJ, Bottaro DP, Fleming TP, Marshall MS, Gibbs JB, Aaronson SA. PDGF induction of tyrosine phosphorylation of GTPase activating protein. Nature. 1989 Dec 7;342(6250):711–714. [PubMed]
  • Moran MF, 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. [PMC free article] [PubMed]
  • Morgenstern JP, Land H. Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res. 1990 Jun 25;18(12):3587–3596. [PMC free article] [PubMed]
  • Nori M, Vogel US, Gibbs JB, Weber MJ. Inhibition of v-src-induced transformation by a GTPase-activating protein. Mol Cell Biol. 1991 May;11(5):2812–2818. [PMC free article] [PubMed]
  • Schweighoffer F, Barlat I, Chevallier-Multon MC, Tocque B. Implication of GAP in Ras-dependent transactivation of a polyoma enhancer sequence. Science. 1992 May 8;256(5058):825–827. [PubMed]
  • Settleman J, Narasimhan V, Foster LC, Weinberg RA. Molecular cloning of cDNAs encoding the GAP-associated protein p190: implications for a signaling pathway from ras to the nucleus. Cell. 1992 May 1;69(3):539–549. [PubMed]
  • Skinner RH, Bradley S, Brown AL, Johnson NJ, Rhodes S, Stammers DK, Lowe PN. Use of the Glu-Glu-Phe C-terminal epitope for rapid purification of the catalytic domain of normal and mutant ras GTPase-activating proteins. J Biol Chem. 1991 Aug 5;266(22):14163–14166. [PubMed]
  • Stacey DW, Feig LA, Gibbs JB. Dominant inhibitory Ras mutants selectively inhibit the activity of either cellular or oncogenic Ras. Mol Cell Biol. 1991 Aug;11(8):4053–4064. [PMC free article] [PubMed]
  • Tanaka K, Nakafuku M, Satoh T, Marshall MS, Gibbs JB, 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. [PubMed]
  • Tanaka K, Nakafuku M, Tamanoi F, Kaziro Y, Matsumoto K, Toh-e A. IRA2, a second gene of Saccharomyces cerevisiae that encodes a protein with a domain homologous to mammalian ras GTPase-activating protein. Mol Cell Biol. 1990 Aug;10(8):4303–4313. [PMC free article] [PubMed]
  • 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. [PubMed]
  • Trahey M, Wong G, Halenbeck R, Rubinfeld B, Martin GA, Ladner M, Long CM, Crosier WJ, Watt K, Koths K, et al. Molecular cloning of two types of GAP complementary DNA from human placenta. Science. 1988 Dec 23;242(4886):1697–1700. [PubMed]
  • Viskochil D, Buchberg AM, Xu G, Cawthon RM, Stevens J, Wolff RK, Culver M, Carey JC, Copeland NG, Jenkins NA, et al. Deletions and a translocation interrupt a cloned gene at the neurofibromatosis type 1 locus. Cell. 1990 Jul 13;62(1):187–192. [PubMed]
  • Vogel US, Dixon RA, Schaber MD, Diehl RE, Marshall MS, Scolnick EM, Sigal IS, Gibbs JB. Cloning of bovine GAP and its interaction with oncogenic ras p21. Nature. 1988 Sep 1;335(6185):90–93. [PubMed]
  • Wallace MR, Marchuk DA, Andersen LB, Letcher R, Odeh HM, Saulino AM, Fountain JW, Brereton A, Nicholson J, Mitchell AL, et al. Type 1 neurofibromatosis gene: identification of a large transcript disrupted in three NF1 patients. Science. 1990 Jul 13;249(4965):181–186. [PubMed]
  • Wang Y, Boguski M, Riggs M, Rodgers L, Wigler M. sar1, a gene from Schizosaccharomyces pombe encoding a protein that regulates ras1. Cell Regul. 1991 Jun;2(6):453–465. [PMC free article] [PubMed]
  • Wong G, Müller O, Clark R, Conroy L, Moran MF, Polakis P, McCormick F. Molecular cloning and nucleic acid binding properties of the GAP-associated tyrosine phosphoprotein p62. Cell. 1992 May 1;69(3):551–558. [PubMed]
  • Xu GF, Lin B, Tanaka K, Dunn D, Wood D, Gesteland R, White R, Weiss R, Tamanoi F. The catalytic domain of the neurofibromatosis type 1 gene product stimulates ras GTPase and complements ira mutants of S. cerevisiae. Cell. 1990 Nov 16;63(4):835–841. [PubMed]
  • Xu GF, O'Connell P, Viskochil D, Cawthon R, Robertson M, Culver M, Dunn D, Stevens J, Gesteland R, White R, et al. The neurofibromatosis type 1 gene encodes a protein related to GAP. Cell. 1990 Aug 10;62(3):599–608. [PubMed]
  • Yatani A, Okabe K, Polakis P, Halenbeck R, McCormick F, Brown AM. ras p21 and GAP inhibit coupling of muscarinic receptors to atrial K+ channels. Cell. 1990 Jun 1;61(5):769–776. [PubMed]
  • Zhang K, DeClue JE, Vass WC, Papageorge AG, McCormick F, Lowy DR. Suppression of c-ras transformation by GTPase-activating protein. Nature. 1990 Aug 23;346(6286):754–756. [PubMed]

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