STIMULATION OF PHOSPHOLIPASE C-BETA(2) BY THE RHO-GTPASES CDC42HS ANDRAC1

Neutrophils contain a soluble guanine-nucleotide-binding protein, made up of two components with molecular masses of 23 and 26 kDa, that med iates stimulation of phospholipase C-beta(2) (PLC beta(2)). We have id entified the two components of the stimulatory heterodimer by amino ac id sequencing as a Rho GTPase and the Rho guanine nucleotide dissociat ion inhibitor LyGDI, Using recombinant Rho GTPases and LyGDI, we demon strate that PLC beta(2) is stimulated by guanosine 5'-O-(3-thiotriphos phate) (GTP[S])-activated Cdc42Hs x LyGDI, but not by RhoA x LyGDI. St imulation of PLC beta(2), which was also observed for GTP[S]-activated recombinant Rad, was independent of LyGDI, but required C-terminal pr ocessing of Cdc42Hs/Rac1, Cdc42Hs/Rac1 also stimulated PLC beta(2) in a system made up of purified recombinant proteins, suggesting that thi s function is mediated by direct protein-protein interaction, The Cdc4 2Hs mutants F37A and Y40C failed to stimulate PLC beta(2), indicating that the Cdc42Hs effector site is involved in this interaction. The re sults identify PLC beta(2) as a novel effector of the Rho GTPases Cdc4 2Hs and Rad, and as the first mammalian effector directly regulated by both heterotrimeric and low-molecular-mass GTP-binding proteins.