Abstract
Rho-like GTPases, including RhoA, Rac1 and Cdc42, have been implicated in the control of a wide range of biological processes such as the organization of cytoskeletal structures, adhesion, motility, transcriptional activation, and cell-cycle progression (1–3). The Rho-like GTPases comprise a subfamily of the Ras superfamily of small guanosine triphosphate (GTP)-binding proteins. Like all members of the Ras superfamily, the Rho-GTPases function as molecular switches, cycling between an inactive guanosine diphosphate (GDP)-bound state and an active GTP-bound state. Among all Ras superfamily members, specific guanine-nucleotide-exchange factors (GEFs) activate Rho-like GTPases by promoting the transition from the GDP-bound to the GTP-bound state, while GTPase-activating proteins (GAPs), which increase the intrinsic rate of hydrolysis of bound GTP, inactivate Rho-like proteins. Rho-like GTPases are further regulated by guanine-nucleotide dissociation inhibitors (GDIs), which can inhibit both the exchange of GTP and the hydrolysis of bound GTP. Rho GDIs, as well as members of the ERM family (ezrin, radixin, and moesin), also appear to play a crucial role in the translocation of Rho-like GTPases between membranes and the cytoplasm-a process also associated with their activation. During recent years, numerous downstream effector proteins that bind Rho, Rac, and Cdc42 in their GTP-bound form have only been identified in an attempt to reveal the underlying molecular mechanisms by which the Rho-like GTPases mediate their various effects.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
van Aelst, L. and D’Souza-Schorey, C. (1997) Rho GTPases and signaling networks. Genes Dev. 11, 2295–2322.
Hall, A. (1998) Rho GTPases and the actin cytoskeleton. Science 279, 509–514.
Zohn, I. M., Campbell, S. L., Khosravi-Far, R., Rossman, K. L., and Der, C. J. (1998) Rho family proteins and Ras transformation: the RHOad less traveled gets congested. Oncogene 17, 1415–1438.
Manser, E., Leung, T., Salihuddin, H., Zhao, Z. S., and Lim, L. (1994) A brain serine threonine protein kinase activated by Cdc42 and Rac1. Nature 367, 40–46.
Manser, E. and Lim, L. (1999) Roles of PAK family kinases. Prog. Mol. Subcell. Biol. 22, 115–133.
Knaus, U. G. and Bokoch, G. M. (1998) The p21Rac/Cdc42-activated kinases (PAKs). Int. J. Biochem. Cell Biol. 30, 857–862.
Sander, E. E., van Delft, S., ten Klooster, J. P., Reid, T., van der Kammen, R. A., Michiels, F., et al. (1998) Matrix-dependent Tiam1/Rac signaling in epithelial cells promotes either cell-cell adhesion or cell migration and is regulated by phosphatidylinositol 3-kinase.J. Cell Biol. 143, 1385–1398.
Franke, B., Akkerman, J. W. N., and Bos, J. L. (1997) Rapid Ca2+-mediated activation of Rap1 in human platelets. EMBO J. 16, 252–259.
de Rooij, J. and Bos, J. L. (1997) Minimal Ras-binding domain of Raf1 can be used as an activation-specific probe for Ras. Oncogene 14, 623–625.
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.
Sander, E. E., ten Klooster, J. P., van Delft, S., van der Kammen, R. A., and Collard, J. G. (1999) Rac downregulates Rho activity: reciprocal balance between both GTPases determines cellular morphology and migratory behavior. J. Cell Biol. 147, 1009–1022.
Zondag, G. C., Evers, E. E., ten Klooster, J. P., Janssen, L., van der Kammen, R. A., and Collard, J. G. (2000) Oncogenic Ras downregulates Rac activity, which leads to increased Rho activity and epithelial-mesenchymal transition. J. Cell Biol. 149, 775–782.
Reid, T., Bathoorn, A., Ahmadian, M. R., and Collard, J. G. (1999) Identification and characterization of hPEM-2, a guanine nucleotide exchange factor specific for Cdc42. J. Biol. Chem. 274, 33,587–33,593.
del Pozo, M. A., Price, L. S., Alderson, N. B., Ren, X. D., and Schwartz, M. A. (2000) Adhesion to the extracellular matrix regulates the coupling of the small GTPase Rac to its effector PAK. EMBO J. 19, 2008–2014.
Michiels, F., Habets, G. G. M., Stam, J. C., van der Kammen, R. A., and Collard, J. G. (1995) A role for Rac in Tiam1-induced membrane ruffling and invasion. Nature 375, 338–340.
Qiu, R. G., Chen, J., Kirn, D., McCormick, F., and Symons, M. (1995) An essential role for Rac in Ras transformation. Nature 374, 457–59.
Mira, J. P., Benard, V., Groffen, J., Sanders, L. C., and Knaus, U. G. (2000) Endogenous, hyperactive Rac3 controls proliferation of breast cancer cells by a p21-activated kinase-dependent pathway. Proc. Natl. Acad. Sci. USA 97, 185–189.
Reid, T., Furayashiki, T., Ishizaki, T., Watanabe, G., Watanabe, N., Fujisawa, K., et al. (1996) Rhotekin, a new putative target for Rho bearing homology to a serine/threonine kinase, PKN, and rhophilin in the Rho-binding domain. J. Biol. Chem. 271, 13,556–13,560.
Michiels, F., Stam, J. C., Hordijk, P. L., van der Kammen, R. A., Ruulsvanstalle, L., Feltkamp, C. A., et al. (1997) Regulated membrane localization of Tiam1, mediated by the NH2-terminal pleckstrin homology domain, is required for Rac-dependent membrane ruffling and c-jun NH2-terminal kinase activation. J. Cell Biol. 137, 387–398.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2002 Humana Press Inc.
About this protocol
Cite this protocol
Malliri, A., Ten Klooster, J.P., Olivo, C., Collard, J.G. (2002). Determination of the Activity of Rho-Like GTPases in Cells. In: Manser, E., Leung, T. (eds) GTPase Protocols. Methods in Molecular Biology™, vol 189. Springer, Totowa, NJ. https://doi.org/10.1385/1-59259-281-3:099
Download citation
DOI: https://doi.org/10.1385/1-59259-281-3:099
Publisher Name: Springer, Totowa, NJ
Print ISBN: 978-0-89603-934-6
Online ISBN: 978-1-59259-281-4
eBook Packages: Springer Protocols