Role of Prenylation in the Interaction of Rho-Family Small GTPases with GTPase Activating Proteins

摘要

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 Racl 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 Racl and RhoA, nonprenylated Cdc42 is able to interact with membrane-localized GAPs. (2) Full-length p50RhoGAP and p190RhoGAP react less intensely with nonprenylated Racl than with the prenylated protein, whereas no difference was observed in the reaction of isolated GAP domains of either p50RhoGAP or Vcr with the different types of Racl. (3) Fluoride exerts a significant inhibitory effect only on the interaction of prenylated Racl with the isolated GAP domains of p50RhoGAP or Vcr. 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 represent potential mechanisms regulating Rac Signaling.