INTERACTION OF RAC1 WITH GTPASE-ACTIVATING PROTEINS AND PUTATIVE EFFECTERS - A COMPARISON WITH CDC42 AND RHOA

The intrinsic GTPase activity of the Rho family GTP-binding protein Ra d is drastically stimulated upon interaction with its GTPase-activatin g proteins (GAPs) and is significantly inhibited when coupled to certa in effector targets such as the pal-activated kinases (PAKs) and IQGAP s, Here we have characterized the interaction of Rad with a panel of m ammalian GAPs and putative effecters by measuring the kinetic and bind ing parameters involved and made comparisons with similar interactions for Cdc42 and RhoA. In contrast with Cdc42 (for which the GAP domain of p50RhoGAP is 50-fold more efficient than those of p190, Bcr, and 3B P-1) and with RhoA (toward which only p50RhoGAP and p190 displayed hig h efficiencies), the catalytic efficiencies (K-cat/K-m) of the GAP dom ains of p50RhoGAP, p190, Bcr, and 3BP-1 on Rad are found to be compara ble in a range between 0.9 and 2.6 min(-1) mu M-1. However, similar to the cases of Cdc42 and RhoA, the K-m values of the GAP domains on Rad compare web to the binding affinity to the guanylyl imidodiphosphate- bound Rad, which ranges from 10.5 to 40.5 mu M suggesting a rapid equi librium reaction mechanism. The dissociation constants of the pal-bind ing domains of PAK1, PAK2, and the RasGAP-related domain of IQGAP1, wh ich all cause significant reduction of the intrinsic rate of GTP hydro lysis upon binding to Rac1-GTP, are found to be 0.71, 0.26, and 2.13 m u M for Rac1-GTP, compared with that determined for Cdc42-GTP at 2.9, 20.5, and 0.39 mu M, respectively, under similar conditions, These res ults suggest that p50RhoGAP, p190, Bcr, and 3BP-1 are all capable of a cting as a negative regulator for Rad-mediated signaling, and that, al though PAK1 and IQGAP1 can couple tightly with both Rad and Cdc42, PAK 2 is likely to be a specific effector for Rad instead of Cdc42.