THE RAS-RELATED GTPASE RAC1 BINDS TUBULIN

The Ras-related Rho family are involved in controlling actin-based cha nges in cell morphology. Microinjection of Rac1, RhoA, and Cdc42Hs int o Swiss 3T3 cells induces pinocytosis and membrane ruffling, stress fi ber formation, and filopodia formation, respectively. To identify targ et proteins involved in these signaling pathways cell extracts immobil ized on nitrocellulose have been probed with [gamma-P-32]GTP-labeled R ac1, RhoA, and Gdc42Hs. We have identified two 55-kDa brain proteins w hich bind Rac1 but not RhoA or Cdc42Hs. These 55-kDa proteins were abu ndant, had pI values of around 5.5, and could be purified by Q-Sepharo se chromatography. The characteristics on two dimensional gel analysis suggested the proteins comprised alpha- and P-tubulin. Indeed, beta-t ubulin specific antibodies detected one of the purified 55-kDa protein s. Rad bound pure tubulin (purified by cycles of polymerization and de polymerization) only in the GTP-bound state. The GTPase negative Rac1 point mutants, G12V and Q61L, did not significantly affect the ability of Rac1 to interact with tubulin while the ''effector-site'' mutant D 38A prevented interaction. These results suggest that the Rac1-tubulin interaction may play a role in Rac1 function.