Stimulation of phospholipase C-β2by the Rho GTPases Cdc42Hs and Rac1

Abstract

Neutrophils contain a soluble guanine‐nucleotidebinding protein, made up of two components with molecular masses of 23 and 26 kDa, that mediates stimulation of phospholipase C‐β₂ (PLCβ₂). We have identified the two components of the stimulatory heterodimer by amino acid sequencing as a Rho GTPase and the Rho guanine nucleotide dissociation inhibitor LyGDI. Using recombinant Rho GTPases and LyGDI, we demonstrate that PLCβ₂ is stimulated by guanosine 5′‐O‐(3‐thiotriphosphate) (GTP[S])‐activated Cdc42Hs×LyGDI, but not by RhoA×LyGDI. Stimulation of PLCβ₂, which was also observed for GTP[S]‐activated recombinant Rac1, was independent of LyGDI, but required C‐terminal processing of Cdc42Hs/Rac1. Cdc42Hs/Rac1 also stimulated PLCβ₂ in a system made up of purified recombinant proteins, suggesting that this function is mediated by direct protein–protein interaction. The Cdc42Hs mutants F37A and Y40C failed to stimulate PLCβ₂, indicating that the Cdc42Hs effector site is involved in this interaction. The results identify PLCβ₂ as a novel effector of the Rho GTPases Cdc42Hs and Rac1, and as the first mammalian effector directly regulated by both heterotrimeric and low‐molecular‐mass GTP‐binding proteins.