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Effector Recruitment Method to Study Spatially Regulated Activation of Ras and Rho GTPases

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Ras Signaling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1120))

Abstract

Ras and Rho family GTPases control a wide variety of cellular processes, and the signaling downstream of these GTPases is influenced by their subcellular localization when activated. Since only a minority of total cellular GTPases is active, observation of the total subcellular distribution of GTPases does not reveal where active GTPases are localized. In this chapter, we describe the use of effector recruitment assays to monitor the subcellular localization of active Ras and Rho family GTPases. The recruitment assay relies on preferential binding of downstream effectors to active GTPases versus inactive GTPases. Tagging the GTPase-binding-domain (GBD) of a downstream effector with a fluorescent protein produces a probe that is recruited to compartments where GTPases are active. We describe an example of a recruitment assay using the GBD of PAK1 to monitor Rac1 activity and explain how the assay can be expanded to determine the subcellular localization of activation of other GTPases.

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Authors and Affiliations

  1. Department of Radiation Oncology, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Lauren P. Huff, Molly J. DeCristo & Adrienne D. Cox

  2. Departments of Pharmacology, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Lauren P. Huff

  3. Department of Pharmacology, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Molly J. DeCristo & Adrienne D. Cox

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  1. Lauren P. Huff
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  2. Molly J. DeCristo
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Editors and Affiliations

  1. University of Siena, Italy

    Lorenza Trabalzini

  2. University of Torino, Italy

    Saverio Francesco Retta

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Huff, L.P., DeCristo, M.J., Cox, A.D. (2014). Effector Recruitment Method to Study Spatially Regulated Activation of Ras and Rho GTPases. In: Trabalzini, L., Retta, S. (eds) Ras Signaling. Methods in Molecular Biology, vol 1120. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-791-4_18

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  • DOI: https://doi.org/10.1007/978-1-62703-791-4_18

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