Regulation of RhoGAP specificity by phospholipids and prenylation

Among the key protein regulators of the various and numerous small GTPases are the GTPase activating proteins (GAPs). Experimental studies of some of the approximately 170 GAPs predicted by the human genome indicate that their catalytic GAP activity is regulated by a variety of mechanisms, including phosphorylation, protein-protein interactions, proteolysis, and interactions with lipids. Most reported biochemical studies to address the specificity of GAPs for particular GTPases have been conducted in vitro with bacterially produced GTPases. Thus, the potential influence of these various regulatory mechanisms in the context of GAP-GTPase specificity may be overlooked in such assays. Here, we present experimental studies that highlight the role of lipids in modulating the GTPase specificity for some of the Rho GAPs. We find that particular phospholipids can substantially alter the substrate "preference" for the p190 GAPs. We find that C-terminal prenylation of GTPases can influence the specificity of GAP interactions as well. These observations emphasize the limitations of standard in vitro GAP assays in definitively establishing the physiologically relevant GTPase targets for particular GAPs.