Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1994 Dec 20;91(26):12609–12613. doi: 10.1073/pnas.91.26.12609

Identification of the guanine nucleotide dissociation stimulator for Ral as a putative effector molecule of R-ras, H-ras, K-ras, and Rap.

M Spaargaren 1, J R Bischoff 1
PMCID: PMC45488  PMID: 7809086

Abstract

To identify proteins that bind to the Ras-related protein R-ras we performed a yeast two-hybrid cDNA library screen. Several clones were obtained encoding the C-terminal region of the guanine nucleotide dissociation stimulator for Ral (RalGDS). The R-ras-binding domain of RalGDS (RalGDS-RBD) is distinct from the conserved catalytic exchange factor regions. Using the two-hybrid system, we show that RalGDS-RBD interacts with H-ras, K-ras, and Rap, and with active but not with inactive point mutants of these Ras-like GTPases. Moreover, using purified proteins, we demonstrate the direct GTP-dependent interaction of the Ras-like GTPases with RalGDS-RBD and full-length RalGDS in vitro. Furthermore, we show that RalGDS-RBD and the Ras-binding domain of Raf-1 compete for binding to the Ras-like GTPases. These data indicate that RalGDS is a putative effector molecule for R-ras, H-ras, K-ras, and Rap.

Full text

PDF
12609

Images in this article

12610Fig. 1
on p.12610
12611Fig. 2
on p.12611
12611Fig. 3
on p.12611
12612Fig. 5
on p.12612

Selected References

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

  1. Albright C. F., Giddings B. W., Liu J., Vito M., Weinberg R. A. Characterization of a guanine nucleotide dissociation stimulator for a ras-related GTPase. EMBO J. 1993 Jan;12(1):339–347. doi: 10.1002/j.1460-2075.1993.tb05662.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boguski M. S., McCormick F. Proteins regulating Ras and its relatives. Nature. 1993 Dec 16;366(6456):643–654. doi: 10.1038/366643a0. [DOI] [PubMed] [Google Scholar]
  3. Bos J. L. ras oncogenes in human cancer: a review. Cancer Res. 1989 Sep 1;49(17):4682–4689. [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. Buday L., Downward J. Epidermal growth factor regulates p21ras through the formation of a complex of receptor, Grb2 adapter protein, and Sos nucleotide exchange factor. Cell. 1993 May 7;73(3):611–620. doi: 10.1016/0092-8674(93)90146-h. [DOI] [PubMed] [Google Scholar]
  7. Chardin P., Tavitian A. The ral gene: a new ras related gene isolated by the use of a synthetic probe. EMBO J. 1986 Sep;5(9):2203–2208. doi: 10.1002/j.1460-2075.1986.tb04485.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chen S. Y., Huff S. Y., Lai C. C., Der C. J., Powers S. Ras-15A protein shares highly similar dominant-negative biological properties with Ras-17N and forms a stable, guanine-nucleotide resistant complex with CDC25 exchange factor. Oncogene. 1994 Sep;9(9):2691–2698. [PubMed] [Google Scholar]
  9. Chien C. T., Bartel P. L., Sternglanz R., Fields S. The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9578–9582. doi: 10.1073/pnas.88.21.9578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cook S. J., Rubinfeld B., Albert I., McCormick F. RapV12 antagonizes Ras-dependent activation of ERK1 and ERK2 by LPA and EGF in Rat-1 fibroblasts. EMBO J. 1993 Sep;12(9):3475–3485. doi: 10.1002/j.1460-2075.1993.tb06022.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Crews C. M., Alessandrini A., Erikson R. L. The primary structure of MEK, a protein kinase that phosphorylates the ERK gene product. Science. 1992 Oct 16;258(5081):478–480. doi: 10.1126/science.1411546. [DOI] [PubMed] [Google Scholar]
  12. Dent P., Haser W., Haystead T. A., Vincent L. A., Roberts T. M., Sturgill T. W. Activation of mitogen-activated protein kinase kinase by v-Raf in NIH 3T3 cells and in vitro. Science. 1992 Sep 4;257(5075):1404–1407. doi: 10.1126/science.1326789. [DOI] [PubMed] [Google Scholar]
  13. Fernandez-Sarabia M. J., Bischoff J. R. Bcl-2 associates with the ras-related protein R-ras p23. Nature. 1993 Nov 18;366(6452):274–275. doi: 10.1038/366274a0. [DOI] [PubMed] [Google Scholar]
  14. Fields S., Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989 Jul 20;340(6230):245–246. doi: 10.1038/340245a0. [DOI] [PubMed] [Google Scholar]
  15. Finney R. E., Robbins S. M., Bishop J. M. Association of pRas and pRaf-1 in a complex correlates with activation of a signal transduction pathway. Curr Biol. 1993 Dec 1;3(12):805–812. doi: 10.1016/0960-9822(93)90214-9. [DOI] [PubMed] [Google Scholar]
  16. Garrett M. D., Self A. J., van Oers C., Hall A. Identification of distinct cytoplasmic targets for ras/R-ras and rho regulatory proteins. J Biol Chem. 1989 Jan 5;264(1):10–13. [PubMed] [Google Scholar]
  17. Guarente L. Strategies for the identification of interacting proteins. Proc Natl Acad Sci U S A. 1993 Mar 1;90(5):1639–1641. doi: 10.1073/pnas.90.5.1639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hancock J. F., Hall A. A novel role for RhoGDI as an inhibitor of GAP proteins. EMBO J. 1993 May;12(5):1915–1921. doi: 10.1002/j.1460-2075.1993.tb05840.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hancock J. F., Paterson H., Marshall C. J. 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. doi: 10.1016/0092-8674(90)90294-o. [DOI] [PubMed] [Google Scholar]
  20. Hannon G. J., Demetrick D., Beach D. Isolation of the Rb-related p130 through its interaction with CDK2 and cyclins. Genes Dev. 1993 Dec;7(12A):2378–2391. doi: 10.1101/gad.7.12a.2378. [DOI] [PubMed] [Google Scholar]
  21. Howe L. R., Leevers S. J., Gómez N., Nakielny S., Cohen P., Marshall C. J. Activation of the MAP kinase pathway by the protein kinase raf. Cell. 1992 Oct 16;71(2):335–342. doi: 10.1016/0092-8674(92)90361-f. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Koide H., Satoh T., Nakafuku M., Kaziro Y. GTP-dependent association of Raf-1 with Ha-Ras: identification of Raf as a target downstream of Ras in mammalian cells. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8683–8686. doi: 10.1073/pnas.90.18.8683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kyriakis J. M., App H., Zhang X. F., Banerjee P., Brautigan D. L., Rapp U. R., Avruch J. Raf-1 activates MAP kinase-kinase. Nature. 1992 Jul 30;358(6385):417–421. doi: 10.1038/358417a0. [DOI] [PubMed] [Google Scholar]
  25. Leevers S. J., Paterson H. F., Marshall C. J. Requirement for Ras in Raf activation is overcome by targeting Raf to the plasma membrane. Nature. 1994 Jun 2;369(6479):411–414. doi: 10.1038/369411a0. [DOI] [PubMed] [Google Scholar]
  26. Lowe D. G., Capon D. J., Delwart E., Sakaguchi A. Y., Naylor S. L., Goeddel D. V. Structure of the human and murine R-ras genes, novel genes closely related to ras proto-oncogenes. Cell. 1987 Jan 16;48(1):137–146. doi: 10.1016/0092-8674(87)90364-3. [DOI] [PubMed] [Google Scholar]
  27. Lowe D. G., Goeddel D. V. Heterologous expression and characterization of the human R-ras gene product. Mol Cell Biol. 1987 Aug;7(8):2845–2856. doi: 10.1128/mcb.7.8.2845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lowe D. G., Ricketts M., Levinson A. D., Goeddel D. V. Chimeric proteins define variable and essential regions of Ha-ras-encoded protein. Proc Natl Acad Sci U S A. 1988 Feb;85(4):1015–1019. doi: 10.1073/pnas.85.4.1015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Macdonald S. G., Crews C. M., Wu L., Driller J., Clark R., Erikson R. L., McCormick F. Reconstitution of the Raf-1-MEK-ERK signal transduction pathway in vitro. Mol Cell Biol. 1993 Nov;13(11):6615–6620. doi: 10.1128/mcb.13.11.6615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. McCabe P. C., Haubruck H., Polakis P., McCormick F., Innis M. A. Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae. Mol Cell Biol. 1992 Sep;12(9):4084–4092. doi: 10.1128/mcb.12.9.4084. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Moodie S. A., Willumsen B. M., Weber M. J., Wolfman A. Complexes of Ras.GTP with Raf-1 and mitogen-activated protein kinase kinase. Science. 1993 Jun 11;260(5114):1658–1661. doi: 10.1126/science.8503013. [DOI] [PubMed] [Google Scholar]
  32. Olofsson B., Chardin P., Touchot N., Zahraoui A., Tavitian A. Expression of the ras-related ralA, rho12 and rab genes in adult mouse tissues. Oncogene. 1988 Aug;3(2):231–234. [PubMed] [Google Scholar]
  33. Porfiri E., Evans T., Chardin P., Hancock J. F. Prenylation of Ras proteins is required for efficient hSOS1-promoted guanine nucleotide exchange. J Biol Chem. 1994 Sep 9;269(36):22672–22677. [PubMed] [Google Scholar]
  34. Rey I., Taylor-Harris P., van Erp H., Hall A. R-ras interacts with rasGAP, neurofibromin and c-raf but does not regulate cell growth or differentiation. Oncogene. 1994 Mar;9(3):685–692. [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. Ruggieri R., Macdonald S. G., Callow M., McCormick F. Raf-1 interferes with Ras and Rap1A effector functions in yeast. Mol Biol Cell. 1994 Feb;5(2):173–181. doi: 10.1091/mbc.5.2.173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Spaargaren M., Martin G. A., McCormick F., Fernandez-Sarabia M. J., Bischoff J. R. The Ras-related protein R-ras interacts directly with Raf-1 in a GTP-dependent manner. Biochem J. 1994 Jun 1;300(Pt 2):303–307. doi: 10.1042/bj3000303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Stokoe D., Macdonald S. G., Cadwallader K., Symons M., Hancock J. F. Activation of Raf as a result of recruitment to the plasma membrane. Science. 1994 Jun 3;264(5164):1463–1467. doi: 10.1126/science.7811320. [DOI] [PubMed] [Google Scholar]
  40. Tsai M. H., Hall A., Stacey D. W. Inhibition by phospholipids of the interaction between R-ras, rho, and their GTPase-activating proteins. Mol Cell Biol. 1989 Nov;9(11):5260–5264. doi: 10.1128/mcb.9.11.5260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Van Aelst L., Barr M., Marcus S., Polverino A., Wigler M. Complex formation between RAS and RAF and other protein kinases. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6213–6217. doi: 10.1073/pnas.90.13.6213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Vojtek A. B., Hollenberg S. M., Cooper J. A. Mammalian Ras interacts directly with the serine/threonine kinase Raf. Cell. 1993 Jul 16;74(1):205–214. doi: 10.1016/0092-8674(93)90307-c. [DOI] [PubMed] [Google Scholar]
  43. Warne P. H., Viciana P. R., Downward J. Direct interaction of Ras and the amino-terminal region of Raf-1 in vitro. Nature. 1993 Jul 22;364(6435):352–355. doi: 10.1038/364352a0. [DOI] [PubMed] [Google Scholar]
  44. Wildey G. M., Viggeswarapu M., Rim S., Denker J. K. Isolation of cDNA clones and tissue expression of rat ral A and ral B GTP-binding proteins. Biochem Biophys Res Commun. 1993 Jul 15;194(1):552–559. doi: 10.1006/bbrc.1993.1855. [DOI] [PubMed] [Google Scholar]
  45. Zhang X. F., Settleman J., Kyriakis J. M., Takeuchi-Suzuki E., Elledge S. J., Marshall M. S., Bruder J. T., Rapp U. R., Avruch J. Normal and oncogenic p21ras proteins bind to the amino-terminal regulatory domain of c-Raf-1. Nature. 1993 Jul 22;364(6435):308–313. doi: 10.1038/364308a0. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES