Abstract
Rap1 is a small Ras-related GTPase which when over-expressed is able to revert transformation by Ki-Ras. We have investigated the role of Rap1 in regulating 'normal' Ras function by studying the activation of the mitogen-activated protein (MAP) kinases ERK1 and ERK2 by two fundamentally different growth factors, epidermal growth factor (EGF) and 1-oleoyl-lyso-phosphatidic acid (LPA). Conditional expression of RasN17 (a dominant-negative mutant) in Rat-1 cells inhibited activation of MAP kinases by EGF and also LPA, the first time a defined G-protein-coupled receptor mitogen has been shown to require Ras to exert its effects. Conditional or constitutive expression of even low levels of RapV12 (a mutant insensitive to Rap-GAP) attenuated activation of MAP kinases by EGF and LPA, but did not interfere with growth factor-stimulated increases in Ras-GTP, indicating that signalling from receptors to Ras was not impaired. Inhibition of Ras-mediated signalling with either RasN17 or RapV12 attenuated DNA synthesis by EGF and LPA. We conclude that receptor tyrosine kinases and G-protein-coupled receptors use Ras as a common step in signalling to MAP kinases and that Rap-GTP (RapV12) at physiological levels interferes with downstream signalling from Ras to MAP kinases in vivo.
Full text
PDF










Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Adari H., Lowy D. R., Willumsen B. M., Der C. J., McCormick F. Guanosine triphosphatase activating protein (GAP) interacts with the p21 ras effector binding domain. Science. 1988 Apr 22;240(4851):518–521. doi: 10.1126/science.2833817. [DOI] [PubMed] [Google Scholar]
- Barbacid M. ras genes. Annu Rev Biochem. 1987;56:779–827. doi: 10.1146/annurev.bi.56.070187.004023. [DOI] [PubMed] [Google Scholar]
- Bokoch G. M. Biology of the Rap proteins, members of the ras superfamily of GTP-binding proteins. Biochem J. 1993 Jan 1;289(Pt 1):17–24. doi: 10.1042/bj2890017. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Bollag G., McCormick F. Regulators and effectors of ras proteins. Annu Rev Cell Biol. 1991;7:601–632. doi: 10.1146/annurev.cb.07.110191.003125. [DOI] [PubMed] [Google Scholar]
- 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]
- Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Downward J. Ras regulation: putting back the GTP. Curr Biol. 1992 Jun;2(6):329–331. doi: 10.1016/0960-9822(92)90897-j. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Frech M., John J., Pizon V., Chardin P., Tavitian A., Clark R., McCormick F., Wittinghofer A. Inhibition of GTPase activating protein stimulation of Ras-p21 GTPase by the Krev-1 gene product. Science. 1990 Jul 13;249(4965):169–171. doi: 10.1126/science.2164710. [DOI] [PubMed] [Google Scholar]
- Hagag N., Halegoua S., Viola M. Inhibition of growth factor-induced differentiation of PC12 cells by microinjection of antibody to ras p21. Nature. 1986 Feb 20;319(6055):680–682. doi: 10.1038/319680a0. [DOI] [PubMed] [Google Scholar]
- Hariharan I. K., Carthew R. W., Rubin G. M. The Drosophila roughened mutation: activation of a rap homolog disrupts eye development and interferes with cell determination. Cell. 1991 Nov 15;67(4):717–722. doi: 10.1016/0092-8674(91)90066-8. [DOI] [PubMed] [Google Scholar]
- Hata Y., Kikuchi A., Sasaki T., Schaber M. D., Gibbs J. B., Takai Y. Inhibition of the ras p21 GTPase-activating protein-stimulated GTPase activity of c-Ha-ras p21 by smg p21 having the same putative effector domain as ras p21s. J Biol Chem. 1990 May 5;265(13):7104–7107. [PubMed] [Google Scholar]
- Hudziak R. M., Schlessinger J., Ullrich A. Increased expression of the putative growth factor receptor p185HER2 causes transformation and tumorigenesis of NIH 3T3 cells. Proc Natl Acad Sci U S A. 1987 Oct;84(20):7159–7163. doi: 10.1073/pnas.84.20.7159. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jelinek M. A., Hassell J. A. Reversion of middle T antigen-transformed Rat-2 cells by Krev-1: implications for the role of p21c-ras in polyomavirus-mediated transformation. Oncogene. 1992 Sep;7(9):1687–1698. [PubMed] [Google Scholar]
- Kahan C., Seuwen K., Meloche S., Pouysségur J. Coordinate, biphasic activation of p44 mitogen-activated protein kinase and S6 kinase by growth factors in hamster fibroblasts. Evidence for thrombin-induced signals different from phosphoinositide turnover and adenylylcyclase inhibition. J Biol Chem. 1992 Jul 5;267(19):13369–13375. [PubMed] [Google Scholar]
- Kitayama H., Sugimoto Y., Matsuzaki T., Ikawa Y., Noda M. A ras-related gene with transformation suppressor activity. Cell. 1989 Jan 13;56(1):77–84. doi: 10.1016/0092-8674(89)90985-9. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- McCormick F. ras GTPase activating protein: signal transmitter and signal terminator. Cell. 1989 Jan 13;56(1):5–8. doi: 10.1016/0092-8674(89)90976-8. [DOI] [PubMed] [Google Scholar]
- McNeall J., Sánchez A., Gray P. P., Chesterman C. N., Sleigh M. J. Hyperinducible gene expression from a metallothionein promoter containing additional metal-responsive elements. Gene. 1989 Mar 15;76(1):81–88. doi: 10.1016/0378-1119(89)90010-3. [DOI] [PubMed] [Google Scholar]
- Moolenaar W. H. G-protein-coupled receptors, phosphoinositide hydrolysis, and cell proliferation. Cell Growth Differ. 1991 Jul;2(7):359–364. [PubMed] [Google Scholar]
- 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]
- Plevin R., MacNulty E. E., Palmer S., Wakelam M. J. Differences in the regulation of endothelin-1- and lysophosphatidic-acid-stimulated Ins(1,4,5)P3 formation in rat-1 fibroblasts. Biochem J. 1991 Dec 15;280(Pt 3):609–615. doi: 10.1042/bj2800609. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pouysségur J., Seuwen K. Transmembrane receptors and intracellular pathways that control cell proliferation. Annu Rev Physiol. 1992;54:195–210. doi: 10.1146/annurev.ph.54.030192.001211. [DOI] [PubMed] [Google Scholar]
- Qui M. S., Green S. H. PC12 cell neuronal differentiation is associated with prolonged p21ras activity and consequent prolonged ERK activity. Neuron. 1992 Oct;9(4):705–717. doi: 10.1016/0896-6273(92)90033-a. [DOI] [PubMed] [Google Scholar]
- Quilliam L. A., Mueller H., Bohl B. P., Prossnitz V., Sklar L. A., Der C. J., Bokoch G. M. Rap1A is a substrate for cyclic AMP-dependent protein kinase in human neutrophils. J Immunol. 1991 Sep 1;147(5):1628–1635. [PubMed] [Google Scholar]
- Rubinfeld B., Crosier W. J., Albert I., Conroy L., Clark R., McCormick F., Polakis P. Localization of the rap1GAP catalytic domain and sites of phosphorylation by mutational analysis. Mol Cell Biol. 1992 Oct;12(10):4634–4642. doi: 10.1128/mcb.12.10.4634. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rubinfeld B., Munemitsu S., Clark R., Conroy L., Watt K., Crosier W. J., McCormick F., Polakis P. Molecular cloning of a GTPase activating protein specific for the Krev-1 protein p21rap1. Cell. 1991 Jun 14;65(6):1033–1042. doi: 10.1016/0092-8674(91)90555-d. [DOI] [PubMed] [Google Scholar]
- Sakoda T., Kaibuchi K., Kishi K., Kishida S., Doi K., Hoshino M., Hattori S., Takai Y. smg/rap1/Krev-1 p21s inhibit the signal pathway to the c-fos promoter/enhancer from c-Ki-ras p21 but not from c-raf-1 kinase in NIH3T3 cells. Oncogene. 1992 Sep;7(9):1705–1711. [PubMed] [Google Scholar]
- 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]
- Seuwen K., Kahan C., Hartmann T., Pouyssegur J. Strong and persistent activation of inositol lipid breakdown induces early mitogenic events but not Go to S phase progression in hamster fibroblasts. Comparison of thrombin and carbachol action in cells expressing M1 muscarinic acetylcholine receptors. J Biol Chem. 1990 Dec 25;265(36):22292–22299. [PubMed] [Google Scholar]
- Seuwen K., Magnaldo I., Kobilka B. K., Caron M. G., Regan J. W., Lefkowitz R. J., Pouysségur J. Alpha 2-adrenergic agonists stimulate DNA synthesis in Chinese hamster lung fibroblasts transfected with a human alpha 2-adrenergic receptor gene. Cell Regul. 1990 May;1(6):445–451. doi: 10.1091/mbc.1.6.445. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Siess W., Winegar D. A., Lapetina E. G. Rap1-B is phosphorylated by protein kinase A in intact human platelets. Biochem Biophys Res Commun. 1990 Jul 31;170(2):944–950. doi: 10.1016/0006-291x(90)92182-y. [DOI] [PubMed] [Google Scholar]
- Simon M. A., Bowtell D. D., Dodson G. S., Laverty T. R., Rubin G. M. Ras1 and a putative guanine nucleotide exchange factor perform crucial steps in signaling by the sevenless protein tyrosine kinase. Cell. 1991 Nov 15;67(4):701–716. doi: 10.1016/0092-8674(91)90065-7. [DOI] [PubMed] [Google Scholar]
- Stacey D. W., Roudebush M., Day R., Mosser S. D., Gibbs J. B., Feig L. A. Dominant inhibitory Ras mutants demonstrate the requirement for Ras activity in the action of tyrosine kinase oncogenes. Oncogene. 1991 Dec;6(12):2297–2304. [PubMed] [Google Scholar]
- 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]
- 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]
- Vouret-Craviari V., Van Obberghen-Schilling E., Rasmussen U. B., Pavirani A., Lecocq J. P., Pouysségur J. Synthetic alpha-thrombin receptor peptides activate G protein-coupled signaling pathways but are unable to induce mitogenesis. Mol Biol Cell. 1992 Jan;3(1):95–102. doi: 10.1091/mbc.3.1.95. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wong G., Arnheim N., Clark R., McCabe P., Innis M., Aldwin L., Nitecki D., McCormick F. Detection of activated Mr 21,000 protein, the product of ras oncogenes, using antibodies with specificity for amino acid 12. Cancer Res. 1986 Dec;46(12 Pt 1):6029–6033. [PubMed] [Google Scholar]
- 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]
- Yatani A., Quilliam L. A., Brown A. M., Bokoch G. M. Rap1A antagonizes the ability of Ras and Ras-Gap to inhibit muscarinic K+ channels. J Biol Chem. 1991 Nov 25;266(33):22222–22226. [PubMed] [Google Scholar]
- 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]
- Zachary I., Sinnett-Smith J., Rozengurt E. Bombesin, vasopressin, and endothelin stimulation of tyrosine phosphorylation in Swiss 3T3 cells. Identification of a novel tyrosine kinase as a major substrate. J Biol Chem. 1992 Sep 25;267(27):19031–19034. [PubMed] [Google Scholar]
- Zhang K., Noda M., Vass W. C., Papageorge A. G., Lowy D. R. Identification of small clusters of divergent amino acids that mediate the opposing effects of ras and Krev-1. Science. 1990 Jul 13;249(4965):162–165. doi: 10.1126/science.2115210. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- van Corven E. J., Groenink A., Jalink K., Eichholtz T., Moolenaar W. H. Lysophosphatidate-induced cell proliferation: identification and dissection of signaling pathways mediated by G proteins. Cell. 1989 Oct 6;59(1):45–54. doi: 10.1016/0092-8674(89)90868-4. [DOI] [PubMed] [Google Scholar]
- van Corven E. J., Hordijk P. L., Medema R. H., Bos J. L., Moolenaar W. H. Pertussis toxin-sensitive activation of p21ras by G protein-coupled receptor agonists in fibroblasts. Proc Natl Acad Sci U S A. 1993 Feb 15;90(4):1257–1261. doi: 10.1073/pnas.90.4.1257. [DOI] [PMC free article] [PubMed] [Google Scholar]
- van der Bend R. L., Brunner J., Jalink K., van Corven E. J., Moolenaar W. H., van Blitterswijk W. J. Identification of a putative membrane receptor for the bioactive phospholipid, lysophosphatidic acid. EMBO J. 1992 Jul;11(7):2495–2501. doi: 10.1002/j.1460-2075.1992.tb05314.x. [DOI] [PMC free article] [PubMed] [Google Scholar]