Phospholipase C-ε (PLC-ε) is a highly elaborated PLC required for a diverse set of signaling pathways. Here we use a combination of cellular assays and studies with purified proteins to show that activated RhoA and Ras isoforms directly engage distinct regions of PLC-ε to stimulate its phospholipase activity. Purified PLC-ε was activated in a guanine nucleotide- and concentration-dependent fashion by purified lipidated K-Ras reconstituted in PtdIns(4,5)P2-containing phospholipid vesicles. Whereas mutation of two critical lysine residues within the second Ras-association domain of PLC-ε prevented K-Ras-dependent activation of the purified enzyme, guanine nucleotide-dependent activation by RhoA was retained. Deletion of a loop unique to PLC-ε eliminated its activation by RhoA but not H-Ras. In contrast, removal of the autoinhibitory X/Y-linker region of the catalytic core of PLC-ε markedly activates the enzyme (Hicks, S. N., Jezyk, M. R., Gershburg, S., Seifert, J. P., Harden, T. K., and Sondek, J. (2008) Mol. Cell, 31, 383–394), but PLC-ε lacking this regulatory region retained activation by both Rho and Ras GTPases. Additive activation of PLC-ε by RhoA and K- or H-Ras was observed in intact cell studies, and this additivity was recapitulated in experiments in which activation of purified PLC-ε was quantified with PtdIns(4,5)P2-containing phospholipid vesicles reconstituted with purified, isoprenylated GTPases. A maximally effective concentration of activated RhoA also increased the sensitivity of purified PLC-ε to activation by K-Ras. These results indicate that PLC-ε can be directly and concomitantly activated by both RhoA and individual Ras GTPases resulting in diverse upstream control of signaling cascades downstream of PLC-ε
