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. 1993 Dec;13(12):7718–7724. doi: 10.1128/mcb.13.12.7718

Regulated and constitutive activity by CDC25Mm (GRF), a Ras-specific exchange factor.

H Cen 1, A G Papageorge 1, W C Vass 1, K E Zhang 1, D R Lowy 1
PMCID: PMC364843  PMID: 8246988

Abstract

Serum stimulates cells to increase their proportion of Ras protein in the active GTP-bound state. We have recently identified four types (I to IV) of apparently full-length cDNAs from a single mammalian gene, called CDC25Mm or GRF, which is homologous to the Ras-specific exchange factor CDC25 of S. cerevisiae. The largest cDNA (type IV) is brain specific, with the other three classes, although they have distinct 5' ends, essentially representing progressive N-terminal deletions of this cDNA. When placed in a retroviral expression vector, all four types of cDNAs induced morphologic transformation of NIH 3T3 cells and an increase in the basal level of GTP.Ras. Serum stimulation of these transformants lead to a further increase in GTP.Ras only in cells expressing the type IV cDNA. Each type of GRF protein was found in cytosolic and membrane fractions, and the protein in each fraction could stimulate guanine nucleotide release from GDP.Ras in vitro. When NIH 3T3 cells and cells expressing the type IV protein were transfected with two versions of a mutant ras gene, one encoding membrane-associated Ras protein and the other encoding a cytosolic Ras protein, the basal levels of GTP bound to both forms of the mutant Ras protein were significantly higher in the cells expressing the type IV protein. However, serum increased the level of GTP bound to the membrane-associated mutant Ras protein in NIH 3T3 cells and in cells expressing the type IV protein but not in cells expressing the cytosolic version of the Ras protein. We conclude that each type of CDC25Mm induces cell transformation via the ability of its C terminus to stimulate guanine nucleotide exchange on Ras, the presence of N-terminal sequences is associated with a serum-dependent change in GTP.Ras, and the serum-dependent increase in GTP.Ras by exogenous CDC25Mm or by endogenous exchange factors probably requires membrane association of both Ras and the exchange factor.

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Selected References

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  1. Barbacid M. ras genes. Annu Rev Biochem. 1987;56:779–827. doi: 10.1146/annurev.bi.56.070187.004023. [DOI] [PubMed] [Google Scholar]
  2. Barlat I., Schweighoffer F., Chevallier-Multon M. C., Duchesne M., Fath I., Landais D., Jacquet M., Tocque B. The Saccharomyces cerevisiae gene product SDC25 C-domain functions as an oncoprotein in NIH3T3 cells. Oncogene. 1993 Jan;8(1):215–218. [PubMed] [Google Scholar]
  3. Bowtell D., Fu P., Simon M., Senior P. Identification of murine homologues of the Drosophila son of sevenless gene: potential activators of ras. Proc Natl Acad Sci U S A. 1992 Jul 15;89(14):6511–6515. doi: 10.1073/pnas.89.14.6511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Buday L., Downward J. Epidermal growth factor regulates the exchange rate of guanine nucleotides on p21ras in fibroblasts. Mol Cell Biol. 1993 Mar;13(3):1903–1910. doi: 10.1128/mcb.13.3.1903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cen H., Papageorge A. G., Zippel R., Lowy D. R., Zhang K. Isolation of multiple mouse cDNAs with coding homology to Saccharomyces cerevisiae CDC25: identification of a region related to Bcr, Vav, Dbl and CDC24. EMBO J. 1992 Nov;11(11):4007–4015. doi: 10.1002/j.1460-2075.1992.tb05494.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cross F. R., Garber E. A., Pellman D., Hanafusa H. A short sequence in the p60src N terminus is required for p60src myristylation and membrane association and for cell transformation. Mol Cell Biol. 1984 Sep;4(9):1834–1842. doi: 10.1128/mcb.4.9.1834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. DeClue J. E., Vass W. C., Johnson M. R., Stacey D. W., Lowy D. R. Functional role of GTPase-activating protein in cell transformation by pp60v-src. Mol Cell Biol. 1993 Nov;13(11):6799–6809. doi: 10.1128/mcb.13.11.6799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Downward J. Regulatory mechanisms for ras proteins. Bioessays. 1992 Mar;14(3):177–184. doi: 10.1002/bies.950140308. [DOI] [PubMed] [Google Scholar]
  10. Egan S. E., Giddings B. W., Brooks M. W., Buday L., Sizeland A. M., Weinberg R. A. Association of Sos Ras exchange protein with Grb2 is implicated in tyrosine kinase signal transduction and transformation. Nature. 1993 May 6;363(6424):45–51. doi: 10.1038/363045a0. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. Gross E., Goldberg D., Levitzki A. Phosphorylation of the S. cerevisiae Cdc25 in response to glucose results in its dissociation from Ras. Nature. 1992 Dec 24;360(6406):762–765. doi: 10.1038/360762a0. [DOI] [PubMed] [Google Scholar]
  14. Hoshino M., Kawakita M., Hattori S. Characterization of a factor that stimulates hydrolysis of GTP bound to ras gene product p21 (GTPase-activating protein) and correlation of its activity to cell density. Mol Cell Biol. 1988 Oct;8(10):4169–4173. doi: 10.1128/mcb.8.10.4169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jacquet E., Vanoni M., Ferrari C., Alberghina L., Martegani E., Parmeggiani A. A mouse CDC25-like product enhances the formation of the active GTP complex of human ras p21 and Saccharomyces cerevisiae RAS2 proteins. J Biol Chem. 1992 Dec 5;267(34):24181–24183. [PubMed] [Google Scholar]
  16. Jones S., Vignais M. L., Broach J. R. The CDC25 protein of Saccharomyces cerevisiae promotes exchange of guanine nucleotides bound to ras. Mol Cell Biol. 1991 May;11(5):2641–2646. doi: 10.1128/mcb.11.5.2641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lai C. C., Boguski M., Broek D., Powers S. Influence of guanine nucleotides on complex formation between Ras and CDC25 proteins. Mol Cell Biol. 1993 Mar;13(3):1345–1352. doi: 10.1128/mcb.13.3.1345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Li N., Batzer A., Daly R., Yajnik V., Skolnik E., Chardin P., Bar-Sagi D., Margolis B., Schlessinger J. Guanine-nucleotide-releasing factor hSos1 binds to Grb2 and links receptor tyrosine kinases to Ras signalling. Nature. 1993 May 6;363(6424):85–88. doi: 10.1038/363085a0. [DOI] [PubMed] [Google Scholar]
  19. Lowy D. R., Willumsen B. M. Function and regulation of ras. Annu Rev Biochem. 1993;62:851–891. doi: 10.1146/annurev.bi.62.070193.004223. [DOI] [PubMed] [Google Scholar]
  20. Lugo T. G., Pendergast A. M., Muller A. J., Witte O. N. Tyrosine kinase activity and transformation potency of bcr-abl oncogene products. Science. 1990 Mar 2;247(4946):1079–1082. doi: 10.1126/science.2408149. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Mayer B. J., Ren R., Clark K. L., Baltimore D. A putative modular domain present in diverse signaling proteins. Cell. 1993 May 21;73(4):629–630. doi: 10.1016/0092-8674(93)90244-k. [DOI] [PubMed] [Google Scholar]
  23. Medema R. H., de Vries-Smits A. M., van der Zon G. C., Maassen J. A., Bos J. L. Ras activation by insulin and epidermal growth factor through enhanced exchange of guanine nucleotides on p21ras. Mol Cell Biol. 1993 Jan;13(1):155–162. doi: 10.1128/mcb.13.1.155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Olivier J. P., Raabe T., Henkemeyer M., Dickson B., Mbamalu G., Margolis B., Schlessinger J., Hafen E., Pawson T. A Drosophila SH2-SH3 adaptor protein implicated in coupling the sevenless tyrosine kinase to an activator of Ras guanine nucleotide exchange, Sos. Cell. 1993 Apr 9;73(1):179–191. doi: 10.1016/0092-8674(93)90170-u. [DOI] [PubMed] [Google Scholar]
  26. Patel G., MacDonald M. J., Khosravi-Far R., Hisaka M. M., Der C. J. Alternate mechanisms of ras activation are complementary and favor and formation of ras-GTP. Oncogene. 1992 Feb;7(2):283–288. [PubMed] [Google Scholar]
  27. Robinson L. C., Gibbs J. B., Marshall M. S., Sigal I. S., Tatchell K. CDC25: a component of the RAS-adenylate cyclase pathway in Saccharomyces cerevisiae. Science. 1987 Mar 6;235(4793):1218–1221. doi: 10.1126/science.3547648. [DOI] [PubMed] [Google Scholar]
  28. Rozakis-Adcock M., Fernley R., Wade J., Pawson T., Bowtell D. The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1. Nature. 1993 May 6;363(6424):83–85. doi: 10.1038/363083a0. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. 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]
  32. Simon M. A., Dodson G. S., Rubin G. M. An SH3-SH2-SH3 protein is required for p21Ras1 activation and binds to sevenless and Sos proteins in vitro. Cell. 1993 Apr 9;73(1):169–177. doi: 10.1016/0092-8674(93)90169-q. [DOI] [PubMed] [Google Scholar]
  33. Wei W., Mosteller R. D., Sanyal P., Gonzales E., McKinney D., Dasgupta C., Li P., Liu B. X., Broek D. Identification of a mammalian gene structurally and functionally related to the CDC25 gene of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):7100–7104. doi: 10.1073/pnas.89.15.7100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Willumsen B. M., Christensen A., Hubbert N. L., Papageorge A. G., Lowy D. R. The p21 ras C-terminus is required for transformation and membrane association. Nature. 1984 Aug 16;310(5978):583–586. doi: 10.1038/310583a0. [DOI] [PubMed] [Google Scholar]
  35. Willumsen B. M., Norris K., Papageorge A. G., Hubbert N. L., Lowy D. R. Harvey murine sarcoma virus p21 ras protein: biological and biochemical significance of the cysteine nearest the carboxy terminus. EMBO J. 1984 Nov;3(11):2581–2585. doi: 10.1002/j.1460-2075.1984.tb02177.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Willumsen B. M., Papageorge A. G., Kung H. F., Bekesi E., Robins T., Johnsen M., Vass W. C., Lowy D. R. Mutational analysis of a ras catalytic domain. Mol Cell Biol. 1986 Jul;6(7):2646–2654. doi: 10.1128/mcb.6.7.2646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Willumsen B. M., Vass W. C., Velu T. J., Papageorge A. G., Schiller J. T., Lowy D. R. The bovine papillomavirus E5 oncogene can cooperate with ras: identification of p21 amino acids critical for transformation by c-rasH but not v-rasH. Mol Cell Biol. 1991 Dec;11(12):6026–6033. doi: 10.1128/mcb.11.12.6026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. 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]

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