Regulation of phospholipase C-epsilon by Rho and Ras family proteins

Inositol phospholipids are a unique collection of signaling molecules involved in an extensive variety of signaling events including actin remodeling, membrane trafficking, and calcium mobilization. Phosphoinositides regulate signaling responses by binding specific effector proteins, or through the generation of second messenger molecules. The most well characterized phosphoinositide signaling pathway is the hydrolysis of phosphatidylinositol (4,5)-bisphosphate by the phospholipase C (PLC) family of enzymes. PLCs cleaves the inositol headgroup from the glycerol backbone of PtdIns(4,5)P2, generating the second messenger molecules inositol (1,4,5)-trisphosphate and diacylglycerol. An extensive array of extracellular signaling molecules elicit their physiological effects through receptor-mediated stimulation of PLC isozymes. All PLC enzymes contain a conserved catalytic region; however, elaboration of the catalytic core with various protein binding and/or regulatory domains gives rise to multiple modes of regulation and different PLC subfamilies. Recent work has implicated Ras superfamily GTPases in the regulation of several PLC isoforms adding a new level of complexity to phosphoinositide metabolism.; Phospholipase C-epsilon (PLC-epsilon) is an elaborate PLC isoform that is regulated by several signaling pathways, including downstream of both heterotrimeric and Ras superfamily GTPases. The work described herein examines the regulation of PLC-epsilon enzyme activity through direct interactions with Ras superfamily GTPases. We describe the purification and functional characterization of a catalytically active fragment of PLC-epsilon with the goal of providing incontrovertible evidence regarding direct Rho and Ras family regulation of enzyme activity. Reconstitution of purified proteins in model phospholipid vesicles demonstrates RhoA and K-Ras individually are sufficient to increase the activity of PLC-epsilon in both a concentration- and GTPgammaS-dependent manner. Moreover, RhoA and K-Ras required mutually exclusive sites of interaction to confer increased PLC-epsilon activity. Finally, when maximally activated by RhoA or K-Ras, PLC-epsilon activity is increased further by the addition of the complementary GTPase, and the enzyme displayed increased sensitivity to K-Ras in the presence of a maximally activating concentration of RhoA. These results identify PLC-epsilon as a novel enzyme that is regulated by Rho and Ras family GTPases through distinct domains, leading to individual and dual regulation of enzyme activity.