Diversity and versatility of GTPase activating proteins for the p21rho subfamily of ras G proteins detected by a novel overlay assay

The p21ras superfamily, involved in diverse processes including cell growth and intracellular trafficking, possesses intrinsic GTPase activity and cycles between GTP-bound active and GDP-bound quiescent states. This intrinsic activity, which results in down-regulation, is accelerated by GTPase activating proteins (GAPs). Other proteins regulating the GDP/GTP cycle include exchange proteins and dissociation inhibitors. The p21s rho, rac, and cdc42Hs constitute a subfamily implicated in cytoskeletal organization. BCR and n-chimaerin are prototypes of a new GAP family for these p21s. To investigate proteins modulating GTP hydrolysis of the three p21s, we developed a novel overlay assay applicable to tissue extracts. Diverse GAPs with different specificities were identified in all rat tissues. Brain contained rac1 GAPs of 45, 50, 85, 100, and 150 kDa. The p50 and p150 GAPs also act on rhoA and cdc42Hs and are ubiquitous, while the p45-GAP, n-chimaerin, is brain- and testis-specific and acts preferentially on rac1; the p100 GAP acts on both rac1 and cdc42Hs and is brain-specific. A new class of p21-interacting proteins was also identified. This diversity, versatility, and tissue specificity of GAPs may be required for fine control of the down-regulation of GTP-bound p21s and the suggested specific downstream effects of individual GAPs, which could involve "cross-talk" between GAPs and p21s.