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-beta2 (PLCbeta2). 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 PLCbeta2 is stimulated by guanosine 5'-O-(3-thiotriphosphate) (GTP[S])-activated Cdc42HsxLyGDI, but not by RhoAxLyGDI. Stimulation of PLCbeta2, 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 PLCbeta2 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 PLCbeta2, indicating that the Cdc42Hs effector site is involved in this interaction. The results identify PLCbeta2 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.

Rac2 Regulation of Phospholipase C-β2 Activity and Mode of Membrane Interactions in Intact Cells

“…Evidence for a tight connection between PLC␤ 2 and Rho GTPases in cells is provided by the chemoattractant receptor system, whose activation stimulates PLC␤ 2 and Rac1, Rac2 and Cdc42 (8 -11). Moreover, in accord with our in vitro studies on PLC␤ 2 activation by Rho GTPases (4,6), a recent study conducted on myeloid-differentiated HL-60 cells demonstrated that dominant-negative Cdc42 disrupted the stimulation of inositol 1,4,5-trisphosphate formation mediated via the chemoattractant receptors while inhibiting Rac2 activation (12). These findings strongly support the notion that Rac2 and possibly Rac1 and Cdc42 are critically involved in receptormediated stimulation of PLC␤ 2 activity.…”

“…as the mutant PLC␤ 2 ⌬ that lacks the Phe 819 -Glu 1166 segment, are resistant to stimulation by ␣ q but are susceptible to activation by Rho GTPases and G protein ␤␥ subunits (3,4,7). Recent studies (4 -6) show that ␤␥ dimers and Rho GTPases activate PLC␤ 2 by interacting with different regions of the effector enzyme.…”

Differential Regulation of Phospholipase C-β2 Activity and Membrane Interaction by Gαq, Gβ1γ2, and Rac2

“…After 20 h, the cells were serum-starved for another 24 h, homogenized, and fractionated into postnuclear particulate (P) and soluble (S) fractions as described under "Experimental Procedures." The two fractions were well separated, as controlled by immunoblotting for RhoGDI␣ (cytosolic) and G␤ [1][2][3][4][5] (membrane-bound, not shown). Equal proportions (v/v) of the P and S fractions of each sample were subjected to SDS-PAGE and quantified by immunoblotting and densitometry using anti-GFP antibodies.…”

Stimulation of phospholipase Cβ by membrane interactions, interdomain movement, and G protein binding — How many ways can you activate an enzyme?

“…Rac1, 2, 3, and to a lesser extent Cdc42Hs, were shown to activate PLC-β2 and β3 [60,61] by binding to their PH domains [11,61]. This binding was thought to activate the PLC-βs by recruiting them to the membrane surface [62].…”