Isozyme-specific stimulation of phospholipase C-γ2 by Rac GTPases

Abstract

The regulation of the two isoforms of phospholipase C-γ, PLCγ1 and PLCγ2, by cell surface receptors involves protein tyrosine phosphorylation as well as interaction with adapter proteins and phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3) generated by inositol phospholipid 3-kinases (PI3Ks). All three processes may lead to recruitment of the PLCγ isozymes to the plasma membrane and/or stimulation of their catalytic activity. Recent evidence suggests that PLCγ may also be regulated by Rho GTPases. In this study, PLCγ1 and PLCγ2 were reconstituted in intact cells and in a cell-free system with Rho GTPases to examine their influence on PLCγ activity. PLCγ2, but not PLCγ1, was markedly activated in intact cells by constitutively active Rac1 G12V, Rac2G12V, and Rac3G12V but not by Cdc42G12V and RhoAG14V. The mechanism of PLCγ2 activation was apparently independent of phosphorylation of tyrosine residues known to be modified by PLCγ2-activating protein-tyrosine kinases. Activation of PLCγ2 by Rac2 G12V in intact cells coincided with a translocation of PLCγ2 from the soluble to the particulate fraction. PLCγ isozyme-specific activation of PLCγ2 by Rac GTPases (Rac1 ≈ Rac2 > Rac3), but not by Cdc42 or RhoA, was also observed in a cell-free system. Herein, activation of wild-type Rac GTPases with guanosine 5′-(3-O-thio)triphosphate caused a marked stimulation of PLCγ2 but had no effect on the activity of PLCγ 1. PLCγ1 and PLCγ2 have previously been shown to be indiscriminately activated by PtdInsP3 in vitro. Thus, the results suggest a novel mechanism of PLCγ2 activation by Rac GTPases involving neither protein tyrosine phosphorylation nor PI3K-mediated generation of PtdInsP3. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.