Effector recruitment method to study spatially regulated activation of Ras and Rho GTPases.
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.
Department of Radiation Oncology, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
Lauren P
LP
Huff
Molly J
MJ
DeCristo
Adrienne D
AD
Cox
D002460:Cell Line; D004789:Enzyme Activation; D042541:Intracellular Space / Q000378:metabolism; D057054:Molecular Imaging; D021381:Protein Transport; D013194:Staining and Labeling; D014162:Transfection; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D018631:ras Proteins; D020741:rho GTP-Binding Proteins