Hormonal regulation of phospholipase Cε through distinct and overlapping pathways involving G12 and Ras family G-proteins

PLCepsilon (phospholipase Cepsilon) is a novel PLC that has a CDC25 guanine nucleotide exchange factor domain and two RA (Ras-association) domains of which the second (RA2) is critical for Ras activation of the enzyme. In the present studies, we examined hormonal stimulation to elucidate receptor-mediated pathways that functionally regulate PLCepsilon. We demonstrate that EGF (epidermal growth factor), a receptor tyrosine kinase agonist, and LPA (lysophosphatildic acid), SIP (sphingosine I-phosphate) and thrombin, GPCR (G-protein-coupled receptor) agonists, stimulate PLCepsilon overexpressed in COS-7 cells. EGF stimulated PLCE in an RA2-dependent manner through Ras and Rap. In contrast, LPA, S IP and thrombin stimulated PLCepsilon by both RA2-independent and -dependent mechanisms. To determine the G-proteins that mediate the effects of these GPCR agonists, we co-expressed constitutively active G-proteins with PLCepsilon and found that G(alpha12), G(alpha13), Rho, Rac and Ra1 stimulate PLCepsilon in an RA2-independent manner; whereas TC21, Rap1A, Rap2A and Rap2B stimulate PLCepsilon in an RA2-dependent manner similar to H-Ras. Of these G-proteins, we show that G(alpha12)/G(alpha13) and Rap partly mediate the effects of LPA, S1P and thrombin to stimulate PLCepsilon. In addition, the stimulation by LPA and SIP is also partly sensitive to pertussis toxin. These studies demonstrate diverse hormonal regulation of PLCepsilon by distinct and overlapping pathways.