Role of prenylation in the interaction of Rho-family small GTPases with GTPase activating proteins.
The role of prenylation in the interaction of Rho-family small GTPases with their GTPase activating proteins (GAPs) was investigated. Prenylated and nonprenylated small GTPases were expressed in Sf9 insect cells and Escherichia coli, respectively. Nucleotide binding to and hydrolysis by prenylated and nonprenylated proteins were identical, but three major differences were observed in their reactions with GAPs. (1) Membrane-associated GAPs accelerate GTP hydrolysis only on prenylated Rac1 and RhoA, but they are inactive on the nonprenylated form of these proteins. The difference is independent of the presence of detergents. In contrast to Rac1 and RhoA, nonprenylated Cdc42 is able to interact with membrane-localized GAPs. (2) Full-length p50RhoGAP and p190RhoGAP react less intensely with nonprenylated Rac1 than with the prenylated protein, whereas no difference was observed in the reaction of isolated GAP domains of either p50RhoGAP or Bcr with the different types of Rac1. (3) Fluoride exerts a significant inhibitory effect only on the interaction of prenylated Rac1 with the isolated GAP domains of p50RhoGAP or Bcr. The effect of fluoride is not influenced by addition or chelation of Al(3+). This is the first detailed study demonstrating that prenylation of the small GTPase is an important factor in determining its reaction with GAPs. It is suggested that both intramolecular interactions and membrane targeting of GAP proteins represent potential mechanisms regulating Rac signaling.
Molnár
G
G
Department of Physiology, Semmelweis University, P.O. Box 259, H-1444 Budapest, Hungary.
Dagher
M C
MC
Geiszt
M
M
Settleman
J
J
Ligeti
E
E
D004789:Enzyme Activation; D005459:Fluorides / Q000494:pharmacology; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000378:metabolism; D009504:Neutrophils / Q000378:metabolism; D017368:Protein Prenylation; D011994:Recombinant Proteins / Q000378:metabolism; D013347:Subcellular Fractions; D020744:rac GTP-Binding Proteins / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000378:metabolism
D020690:GTPase-Activating Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D020558:GTP Phosphohydrolases; D020744:rac GTP-Binding Proteins; D020741:rho GTP-Binding Proteins; D005459:Fluorides