PROTEIN-KINASE-C INHIBITS EPIDERMAL GROWTH FACTOR-DEPENDENT TYROSINE PHOSPHORYLATION OF PHOSPHOLIPASE C-GAMMA AND ACTIVATION OF PHOSPHOINOSITIDE HYDROLYSIS

Recent studies have shown that the receptor for epidermal growth factor (EGF) can associate with and tyrosinephosphorylate the γ-isozyme of phosphoinositide (Ptdlns)-specific phospholipase C (PLC γ), suggesting a possible mechanism for activation of Ptdlns hydrolysis by EGF. In the present study, the coupling between Ptdlns hydrolysis and PLCγ tyrosine phosphorylation in WB liver epithelial cells was examined. Peak levels of [P-Tyr]PLCγ, measured by anti-P-Tyr immunoblotting, occurred at 0.5-2 min of EGF treatment and coincided with the onset of [3H]inositol phosphate production. The termination of Ptdlns hydrolysis after EGF stimulation was accompanied by return of [P-Tyr]PLCγ to near-basal levels. Activation of protein kinase C (PKC) with a phorbol ester inhibited (IC50 = 3-10 nM) both EGF-dependent Ptdlns hydrolysis and PLCγ phosphorylation by more than 90%. Both EGF-stimulated responses were potentiated in cells depleted of PKC by prolonged phorbol ester treatment. At physiological ionic strength, monoclonal antibodies to PLCγ specifically precipitated (in addition to PLCγ) the EGF receptor and at least six other [P-Tyr]proteins from extracts of EGF-treated cells. PKC activation had differential effects on the tyrosine phosphorylation of these coprecipitating proteins, i.e. the relative abundance of certain [P-Tyr] proteins decreased, whereas that of another protein increased. In conclusion, EGF-stimulated tyrosine phosphorylation of PLCγ is broadly correlated with stimulation of Ptdlns hydrolysis, consistent with a role for tyrosine phosphorylation in PLC activation. The attendant diacylglycerol release and activation of PKC may terminate PLCγ activation, in part by inhibiting PLCγ phosphorylation by the EGF receptor. Our results suggest further that PKC may exert regulatory effects by altering the relationship of PLCγ to its associated [P-Tyr]proteins. © 1990 by The Endocrine Society.