Abstract
There are two key methods which yield information about the cellular function of a protein. One can introduce into cells or whole organisms a form of the protein that is constitutively active, and which therefore functions independent of the normal regulatory mechanisms. This technique has been used very successfully with many small GTPases and is based largely on the early work on “natural” Ras mutants (associated with cancers), which were found to contain amino-acid substitutions predominantly at residues 12 and 61 of Ras (1). Although introducing GTPases with such mutations can reveal dramatic effects on cell signaling, membrane trafficking, or cytoskeletal architecture, this method must be complemented by a second technique-studying the loss of protein function. This method can be introduced by genetic manipulation, anti-sense DNA/RNA, specific antibodies, drugs, or expression of so-called “dominant inhibitory” proteins. These proteins have emerged as popular tools to accomplish GTPase inhibition in mammalian cells; mutated proteins can interfere with the function of their normal cellular counterparts or with the proteins that interact with them. This approach has been used extensively in the elucidation of signal-transduction cascades involving Ras-superfamily proteins. This chapter examines the mechanism underlying such dominant-inhibitory Ras proteins and some potential problems associated with their use.
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References
Lowy, D. R. and Willumsen, B. M. (1993) Function and regulation of ras. Annu.Rev. Biochem. 62, 851–891.
McCoy, M. S., Toole, J. J., Cunningham, J. M., Chang, E. H., Lowy, D. R.,and Weinberg, R. A. (1983) Characterization of a human colon/lung carcinoma oncogene. Nature 302, 79–81.
Katz, M. E. and McCormick, F. (1997) Signal transduction from multiple Ras effectors. Curr. Opin. Genet. Dev. 7, 75–79.
Nassar, N., Horn, G., Herrmann, C., Scherer, A., McCormick, F., and Wittinghofer, A. (1995) The 2.2 A crystal structure of the Ras-binding domain of the serine/threonine kinase c-Raf1 in complex with Rap1A and a GTP analogue.Nature 375, 554–560.
Pacold, M. E., Suire, S., Perisic, O., Lara-Gonzalez, S., Davis, C.T., Walker, E. H.,et al. (2000) Crystal structure and functional analysis of Ras binding to its effector phosphoinositide 3-kinase gamma. Cell 103, 931–943.
Feig, L. A. and Cooper, G. M. (1988) Inhibition of NIH 3T3 cell proliferation by a mutant Ras protein with preferential affinity for GDP. Mol. Cell. Biol. 8, 3235–3243.
Powers, S., O’Neill, K., and Wigler, M. (1989) Dominant yeast and mammalian Ras mutants that interfere with the CDC25-dependent activation of wild-type RAS in Saccharomyces cerevisiae. Mol. Cell. Biol. 9, 390–395.
Quilliam, L. A., Kato, K., Rabun, K. M., Hisaka, M. M., Huff, S. Y., Campbell-Burk, S., et al. (1994) Identification of residues critical for Ras(17N) growth-inhibitory phenotype and for Ras interaction with guanine nucleotide exchange factors. Mol. Cell. Biol. 14, 1113–1121.
Crechet J. B., Bernardi, A., and Parmeggiani, A. (1996) Distal switch II region of Ras2p is required for interaction with guanine nucleotide exchange factor. J.Biol. Chem. 271, 17,234–17,240.
Farnsworth, C. L. and Feig, L. A. (1991) Dominant inhibitory mutations in the Mg(2+)-binding site of RasH prevent its activation by GTP. Mol. Cell. Biol. 11, 4822–4829.
John, J., Rensland, H., Schlichting, I., Vetter, I., Borasio, G. D., Goody, R. S., et al. (1993) Kinetic and structural analysis of the Mg(2+)-binding site of the guanine nucleotide-binding protein p21H-ras. J. Biol. Chem. 268, 923–929.
Milburn, M. V., Tong, L., deVos, A. M., Brunger, A., Yamaizumi, Z., Nishimura, S., et al. (1990) Molecular switch for signal transduction: structural differences between active and inactive forms of protooncogenic ras proteins. Science 247, 939–945.
Stacey, D. W., Feig, L. A., and Gibbs, J. B. (1991) Dominant inhibitory Ras mutants selectively inhibit the activity of either cellular or oncogenic Ras. Mol.Cell. Biol. 11, 4053–4064.
Peterson, S. N., Trabalzini, L., Brtva, T. R., Fischer, T., Altschuler, D. L., Martelli, P.,et al. (1996) Identification of a novel RalGDS-related protein as a candidate effector for Ras and Rap1. J. Biol. Chem. 271, 29,903–29,908.
Lenzen, C., Cool, R. H., Prinz, H., Kuhlmann, J., and Wittinghofer, A. (1998) Kinetic analysis by fluorescence of the interaction between Ras and the catalytic domain of the guanine nucleotide exchange factor Cdc25Mm. Biochemistry 37, 7420–7430.
Lai, C. C., Boguski, M., Broek, D., and Powers, S. (1993) Influence of guanine nucleotides on complex formation between Ras and CDC25 proteins. Mol. Cell.Biol. 13, 1345–1352.
Farnsworth, C. L., Marshall, M. S., Gibbs, J. B., Stacey, D. W., and Feig, L. A.(1991) Preferential inhibition of the oncogenic form of RasH by mutations in the GAP binding/“effector” domain. Cell 64, 625–633.
Graham, S. M., Vojtek, A. B., Huff, S. Y., Cox, A. D., Clark, G. J., Cooper, J. A.,et al. (1996) TC21 causes transformation by Raf-independent signaling pathways.Mol. Cell. Biol. 16, 6132–6140.
Rosario, M., Paterson, H. F., and Marshall, C. J. (1999) Activation of the Raf/MAP kinase cascade by the Ras-related protein TC21 is required for the TC21-mediated transformation of NIH 3T3 cells. EMBO J. 18, 1270–1279.
Gotoh, T., Niino, Y., Tokuda, M., Hatase, O., Nakamura, S., Matsuda, M., et al.(1997) Activation of R-Ras by Ras-guanine nucleotide-releasing factor. J. Biol.Chem. 272, 18,602–18,607.
Mott, H. R., Carpenter, J. W., and Campbell, S. L. (1997) Structural and functional analysis of a mutant Ras protein that is insensitive to nitric oxide activation.Biochemistry 36, 3640–3644.
Downward, J., Graves, J. D., Warne, P. H., Rayter, S., and Cantrell, D. A. (1990) Stimulation of p21ras upon T-cell activation. Nature 346, 719–723.
Marais, R., Light, Y., Mason, C., Paterson, H., Olson, M. F., and Marshall, C. J.(1998) Requirement of Ras-GTP-Raf complexes for activation of Raf-1 by protein kinase C. Science 280, 109–112.
Szeberenyi, J., Cai, H., and Cooper, G. M. (1990) Effect of a dominant inhibitory Ha-ras mutation on neuronal differentiation of PC12 cells. Mol. Cell. Biol. 10, 5324–5332.
van den Berghe, N., Cool, R. H., Horn, G., and Wittinghofer, A. (1997) Biochemical characterization of C3G: an exchange factor that discriminates between Rap1 and Rap2 and is not inhibited by Rap1A(S17N). Oncogene 15, 845–850.
Cerione, R. A. and Zheng, Y. (1996) The Dbl family of oncogenes. Curr. Opin.Cell Biol. 8, 216–222.
Ridley, A. J. and Hall, A. (1992) The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors.Cell 70, 389–399.
Harden, N., Ricos, M., Ong, Y. M., Chia, W., and Lim, L. (1999) Participation of small GTPases in dorsal closure of the Drosophila embryo: distinct roles for Rho subfamily proteins in epithelial morphogenesis. J. Cell Sci. 112, 273–284.
Missy, K., Van Poucke, V., Raynal, P., Viala, C., Mauco, G., Plantavid, M., et al.(1998) Lipid products of phosphoinositide 3-kinase interact with Rac1 GTPase and stimulate GDP dissociation. J. Biol. Chem. 273, 30,279–30,286.
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Manser, E.J. (2002). The GTPase Cycle How Dominant Inhibitory Mutants Block the Biological Functions of Small GTPases. In: Manser, E., Leung, T. (eds) GTPase Protocols. Methods in Molecular Biology™, vol 189. Springer, Totowa, NJ. https://doi.org/10.1385/1-59259-281-3:003
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DOI: https://doi.org/10.1385/1-59259-281-3:003
Publisher Name: Springer, Totowa, NJ
Print ISBN: 978-0-89603-934-6
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