Abstract
Rho GTPases including RhoA, Cdc42, and Rac1 are master regulators of cell cytoskeleton dynamic, thus controlling essential cellular processes notably cell polarity, migration and cytokinesis. These GTPases undergo a spatiotemporal regulation primarily controlled by cellular factors inducing both the exchange of GDP for GTP and the hydrolysis of GTP into GDP. Recent findings have unveiled another layer of complexity in the regulation of Rho proteins consisting in their ubiquitylation followed by their proteasomal degradation. Here, we describe how to assess the level of ubiquitylation of Rho proteins in cells, taking Rac1 as an example.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Heasman SJ, Ridley AJ (2008) Mammalian Rho GTPases: new insights into their functions from in vivo studies. Nat Rev Mol Cell Biol 9, 690–701.
Jaffe AB, Hall A (2005) RHO GTPases: Biochemistry and Biology. Annu Rev Cell Dev Biol 21, 247–269.
Aktories, K., and Barbieri J.T. (2005) Bacterial cytotoxins: targeting eukaryotic switches. Nat Rev Microbiol 3, 397–410.
Visvikis, O., Maddugoda, M.P., and Lemichez, E. (2010) Direct modifications of Rho proteins: deconstructing GTPase regulation. Biol Cell 102, 377–389.
Kerscher, O., Felberbaum, R., and Hochstrasser, M. (2006) Modification of proteins by ubiquitin and ubiquitin-like proteins. Annu Rev Cell Dev Biol 22, 159–180.
Doye, A., Mettouchi, A., Bossis, G., Clément, R., Buisson-Touati, C., Flateau, G., Gagnoux, L., Piechaczyk, M., Boquet, P., and Lemichez, (2002) CNF1 exploits the ubiquitin-proteasome machinery to restrict Rho GTPase activation for bacterial host cell invasion. Cell 111, 553–564.
Lemonnier, M., Landraud, L., and Lemichez, E. (2007) Rho GTPase-activating bacterial toxins: from bacterial virulence regulation to eukaryotic cell biology. FEMS Microbiol Rev 31, 515–534.
Walther, D.J., Peter, J.U., Winter S, Höltje, M., Paulmann, N., Grohmann, M., Vowinckel, J., Alamo-Bethencourt, V., Wilhelm, C.S., Ahnert-Hilger, G., and Bader, M. (2003) Serotonylation of small GTPases is a signal transduction pathway that triggers platelet alpha-granule release. Cell 115, 851–862.
Lynch, E.A., Stall, J., Schmidt, G., Chavrier, P., and D’Souza-Schorey, C. (2006) Proteasome-mediated degradation of Rac1-GTP during epithelial cell scattering. Mol Biol Cell 17, 2236–2242.
Nethe, M., Anthony, E.C, Fernandez-Borja, M., Dee, R., Geerts, D., Hensbergen, P.J., Deelder, A.M., Schmidt, G., and Hordijk, P.L. (2010) Focal-adhesion targeting links caveolin-1 to a Rac1-degradation pathway. J Cell Sci 123, 1948–1958.
Ozdamar, B., Bose, R., Barrios-Rodiles, M., Wang, H.R., Zhang, Y., and Wrana, J.L. (2005) Regulation of the polarity protein Par6 by TGFbeta receptors controls epithelial cell plasticity. Science 307, 1603–1609.
Wang, H.R., Zhang, Y., Ozdamar, B., Ogunjimi, A.A., Alexandrova, E., Thomsen, G.H., and Wrana, J.L. (2003) Regulation of cell polarity and protrusion formation by targeting RhoA for degradation. Science 302, 1775–1779.
Boyer, L., Turchi, L., Desnues, B., Doye, A., Ponzio, G., Mege, J.L., Yamashita, M., Zhang, Y.E., Bertoglio, J., Flatau, G., Boquet, P., and Lemichez, E. (2006) CNF1-induced ubiquitylation and proteasome destruction of activated RhoA is impaired in Smurf1−/− cells. Mol Biol Cell 17, 2489–2497.
Chen, Y., Yang, Z., Meng, M., Zhao, Y., Dong, N., Yan, H., Liu, L., Ding, M., Peng, H.B., and Shao, F. (2009) Cullin mediates degradation of RhoA through evolutionarily conserved BTB adaptors to control actin cytoskeleton structure and cell movement. Mol Cell 35, 841–855.
Acknowledgments
This work was supported by an institutional funding from the INSERM, from the Agence Nationale de la Recherche (ANR-07-MIME-007 and ANR-07-BLAN-0046), and from the Association pour la Recherche sur le Cancer (ARC 3800).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Doye, A., Mettouchi, A., Lemichez, E. (2012). Assessing Ubiquitylation of Rho GTPases in Mammalian Cells. In: Rivero, F. (eds) Rho GTPases. Methods in Molecular Biology, vol 827. Springer, New York, NY. https://doi.org/10.1007/978-1-61779-442-1_5
Download citation
DOI: https://doi.org/10.1007/978-1-61779-442-1_5
Published:
Publisher Name: Springer, New York, NY
Print ISBN: 978-1-61779-441-4
Online ISBN: 978-1-61779-442-1
eBook Packages: Springer Protocols