Nonradioactive Methods for Detecting Activation of Ras-Related Small G Proteins

  • Douglas A. Andres
Part of the Methods in Molecular Biology book series (MIMB, volume 284)

Abstract

Ras-related small GTPases serve as critical regulators for a wide range of cellular signaling pathways and are activated by the conversion of the GDP-bound state to the GTP-bound conformation. Until recently, measurement of the GTP-bound active form of Ras-related G proteins involved immunoprecipitation of 32P-labeled protein followed by separation of the labeled GTP/GDP bound to GTPase. A new method based on the large affinity difference of the GTP- and GDP-bound form of Ras proteins for specific binding domains of effector proteins in vitro has been developed. By using glutathione S-transferase (GST) fusion proteins containing these binding domains, the GTP-bound form of the GTPase can be precipitated from cell lysates. In principle, this method can be used for all members of the Ras superfamily. Here we describe a general procedure to monitor the GTP-bound form of Ras-related GTPases.

Key Words

GTP-bound Ras GTPase activation probe 

References

  1. 1.
    Macara, I. G., Lounsbury, K. M., Richards, S. A., et al. (1996) The Ras superfamily of GTPases. FASEB J. 10, 625–630.PubMedGoogle Scholar
  2. 2.
    Campbell, S. L., Khosravi-Far, R., Rossman, K. L., et al. (1998) Increasing complexity of Ras signaling. Oncogene 17, 1395–1413.PubMedCrossRefGoogle Scholar
  3. 3.
    Hall, A. (1998) Rho GTPases and the actin cytoskeleton. Science 279, 509–514.PubMedCrossRefGoogle Scholar
  4. 4.
    Malumbres, M. and Pellicer, A. (1998) RAS pathways to cell cycle control and cell transformation. Front Biosci. 3, d887–d912.PubMedGoogle Scholar
  5. 5.
    Satoh, T. and Kaziro, Y. (1995) Measurement of Ras-bound guanine nucleotide in stimulated hematopoietic cells. Methods Enzymol. 255, 149–155.PubMedCrossRefGoogle Scholar
  6. 6.
    Gibbs, J. B. (1995) Determination of guanine nucleotides bound to Ras in mammalian cells. Methods Enzymol. 255, 118–125.PubMedCrossRefGoogle Scholar
  7. 7.
    Carey, K. D. and Stork, P. J. (2002) Nonisotopic methods for detecting activation of small G proteins. Methods Enzymol. 345, 383–397.PubMedCrossRefGoogle Scholar
  8. 8.
    Benard, V. and Bokoch, G. M. (2002) Assay of Cdc42, Rac, and Rho GTPase activation by affinity methods. Methods Enzymol. 345, 349–359.PubMedCrossRefGoogle Scholar
  9. 9.
    Taylor, S. J., Resnick, R. J., and Shalloway, D. (2001) Nonradioactive determination of Ras-GTP levels using activated ras interaction assay. Methods Enzymol. 333, 333–342.PubMedCrossRefGoogle Scholar
  10. 10.
    van Triest, M., de Rooij, J., and Bos, J. L. (2001) Measurement of GTP-bound Ras-like GTPases by activation-specific probes. Methods Enzymol. 333, 343–348.PubMedCrossRefGoogle Scholar
  11. 11.
    Taylor, S. J. and Shalloway, D. (1996) Cell cycle-dependent activation of Ras Curr. Biol. 6, 1621–1627.PubMedCrossRefGoogle Scholar
  12. 12.
    Spaargaren, M. and Bischoff, J. R. (1994) Identification of the guanine nucleotide dissociation stimulator for Ral as a putative effector molecule of R-ras, H-ras, K-ras, and Rap. Proc. Natl. Acad. Sci. USA 91, 12,609–12,613.PubMedCrossRefGoogle Scholar
  13. 13.
    Franke, B., Akkerman, J. W., and Bos, J. L. (1997) Rapid Ca2+-mediated activation of Rap1 in human platelets. EMBO J. 16, 252–259.PubMedCrossRefGoogle Scholar
  14. 14.
    Ohba, Y., Mochizuki, N., Matsuo, K., et al. (2000) Rap2 as a slowly responding molecular switch in the Rap1 signaling cascade. Mol. Cell Biol. 20, 6074–6083.PubMedCrossRefGoogle Scholar
  15. 15.
    Reedquist, K. A. and Bos, J. L. (1998) Costimulation through CD28 suppresses T cell receptor-dependent activation of the Ras-like small GTPase Rap1 in human T lymphocytes. J. Biol. Chem. 273, 4944–4949.PubMedCrossRefGoogle Scholar
  16. 16.
    Dell’Angelica, E. C., Puertollano, R., Mullins, C., et al. (2000) GGAs: A family of ADP ribosylation factor-binding proteins related to adaptors and associated with the Golgi complex. J. Cell Biol. 149, 81–94.CrossRefGoogle Scholar
  17. 17.
    Santy, L. C. and Casanova, J. E. (2001) Activation of ARF6 by ARNO stimulates epithelial cell migration through downstream activation of both Rac1 and phospholipase D. J. Cell Biol. 154, 599–610.PubMedCrossRefGoogle Scholar
  18. 18.
    Goi, T., Rusanescu, G., Urano, T., and Feig, L. A. (1999) Ral-specific guanine nucleotide exchange factor activity opposes other Ras effectors in PC12 cells by inhibiting neurite outgrowth. Mol. Cell Biol. 19, 1731–1741.PubMedGoogle Scholar
  19. 19.
    Shao, H. and Andres, D. A. (2000) Anovel RalGEF-like protein, RGL3, as a candidate effector for rit and Ras [In Process Citation]. J. Biol. Chem. 275, 26,914–26,924.PubMedGoogle Scholar
  20. 20.
    Spencer, M. L., Shao, H., Tucker, H. M., and Andres, D. A. (2002) Nerve Growth Factor-dependent Activation of the Small GTPase Rin. J. Biol. Chem. 277, 17,605–17,615.PubMedCrossRefGoogle Scholar
  21. 21.
    Self, A. J. and Hall, A. (1995) Measurement of intrinsic nucleotide exchange and GTP hydrolysis rates. Methods Enzymol. 256, 67–76.PubMedCrossRefGoogle Scholar
  22. 22.
    Posern, G., Weber, C. K., Rapp, U. R., and Feller, S. M. (1998) Activity of Rap1 is regulated by bombesin, cell adhesion, and cell density in NIH3T3 fibroblasts. J. Biol. Chem. 273, 24,297–24,300.PubMedCrossRefGoogle Scholar
  23. 23.
    Benard, V., Bohl, B. P., and Bokoch, G. M. (1999) Characterization of rac and cdc42 activation in chemoattractant-stimulated human neutrophils using a novel assay for active GTPases. J. Biol. Chem. 274, 13,198–13,204.PubMedCrossRefGoogle Scholar
  24. 24.
    Bagrodia, S., Taylor, S. J., Jordon, K. A., et al. (1998) A novel regulator of p21-activated kinases. J. Biol. Chem. 273, 23,633–23,636.PubMedCrossRefGoogle Scholar
  25. 25.
    Ren, X. D., Kiosses, W. B., and Schwartz, M. A. (1999) Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton. EMBO J. 18, 578–585.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2004

Authors and Affiliations

  • Douglas A. Andres
    • 1
  1. 1.Department of Molecular and Cellular BiochemistryUniversity of Kentucky College of MedicineLexington

Personalised recommendations