0013-7227/90/1274-1697$02.00/0 Endocrinology Copyright© 1990 by The Endocrine Society

Vol. 127, No. 4 Printed in U.S.A.

Protein Kinase C Inhibits Epidermal Growth FactorDependent Tyrosine Phosphorylation of Phospholipase and Activation of Phosphoinositide Hydrolysis*1^ WILLIAM R. HUCKLE, JOHN R. HEPLER, SUE GOO RHEE, T. KENDALL HARDEN, AND H. SHELTON EARP Lineberger Cancer Research Center (W.R.H., H.S.E.) and Departments of Pharmacology (J.R.H., T.K.H., H.S.E.) and Medicine (H.S.E.), School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 and Laboratory of Biochemistry (S.G.R.), National Heart, Lung and Blood Institute, Bethesda, Maryland 20892

ABSTRACT. Recent studies have shown that the receptor for epidermal growth factor (EGF) can associate with and tyrosinephosphorylate the 7-isozyme of phosphoinositide (Ptdlns)-specific phospholipase C (PLC7), suggesting a possible mechanism for activation of Ptdlns hydrolysis by EGF. In the present study, the coupling between Ptdlns hydrolysis and PLC7 tyrosine phosphorylation in WB liver epithelial cells was examined. Peak levels of [P-Tyr]PLC7, 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]PLC7 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 PLC7 phosphorylation by more than 90%. Both EGF-stimulated responses were potentiated in cells depleted of PKC by prolonged phorbol

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PIDERMAL growth factor (EGF) is a mitogenic peptide that stimulates the growth of variety of cell types (1). The cell-surface EGF receptor, a 170-kDa transmembrane glycoprotein, contains a ligand-activated, tyrosine-specific protein kinase essential for cellular responses such as Ca2+ mobilization and mitogenesis (2-6). This finding suggests that the effects of EGF are mediated by specific protein substrates phosphorylated on tyrosine residues by the EGF receptor. However, the Received May 15,1990. Address all correspondence and requests for reprints to: Dr. Shelton Earp, Lineberger Cancer Research Center, University of North Carolina, CB #7295, Chapel Hill, North Carolina 27599. * This work was supported by NIH grants DK31386 (to H.S.F.), GM29536 and GM38213 (to T.K.H.), and Training Grant T32CA09156 to the Lineberger Cancer Research Center (W.R.H.). Portions of this work were presented at the 29th annual meeting of the American Society for Cell Biology, Houston, Texas, November 1989. t Abbreviations used are: EGF, epidermal growth factor; PDGF, platelet-derived growth factor; PKC, protein kinase C; PLC, phosphoinositide-specific phospholipase C; PMA, /3-phorbol 12-myristate, 13acetate; Ptdlns, phosphoinositide; InsP, total inositol phosphates; p93, protein of 93 kDa; CSF-1, colony-stimulating factor-1.

ester treatment. At physiological ionic strength, monoclonal antibodies to PLC7 specifically precipitated (in addition to PLC7) 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 PLC7 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 PLC7 activation, in part by inhibiting PLC7 phosphorylation by the EGF receptor. Our results suggest further that PKC may exert regulatory effects by altering the relationship of PLC7 to its associated [P-Tyr]proteins. (Endocrinology 127: 1697-1705, 1990)

identities and functions of the proteins phosphorylated in response to EGF remain largely unknown. One of the earlier measurable responses of certain cells to EGF is the hydrolysis of phosphatidylinositol 4,5bisphosphate catalyzed by phosphoinositide (Ptdlns) specific phospholipase C (PLC) (7-9), producing the second messengers inositol 1,4,5-triphosphate and 1,2diacylglycerol. We previously have studied the regulation of this process by EGF and other agents using WB cells, a line of nontransformed rat liver epithelial cells expressing levels of EGF receptor (200,000-300,000 per cell) similar to those found in normal hepatocytes (10, 11). Ptdlns hydrolysis stimulated by EGF, angiotensin II, or [Arg8]vasopressin in these cells (12) is inhibited by exogenous activators of protein kinase C (PKC) and is potentiated in cells depleted of PKC (13), suggesting that each of these agents activates a PKC-dependent negative feedback pathway. Based on its requirement for EGF receptor tyrosine kinase activity (6), the pathway of PLC activation by EGF has been anticipated to involve tyro-

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PLC 7 PHOSPHORYLATION AND Ptdlns HYDROLYSIS

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sine phosphorylation. Indeed, several laboratories recently have reported that the 7-isozyme of PLC (formerly designated type II; 14) is rapidly phosphorylated on tyrosine residues after EGF treatment of A431 epidermoid carcinoma cells (15-19). The suggestion that the EGF receptor itself phosphorylates PLC7 in intact cells has been strengthened by the demonstration that the purified EGF receptor is able to phosphorylate purified PLC7 (18, 19). Thus, it has been postulated that EGF-dependent tyrosine phosphorylation of PLC7 may regulate the activity of this enzyme, although phosphorylation-dependent alterations in the intrinsic properties of PLC have not been demonstrated. The present studies were undertaken to test the hypothesis that tyrosine phosphorylation of PLC7 is involved in the regulation of Ptdlns hydrolysis by both the EGF receptor and PKC. Using anti-P-Tyr immunoblotting of anti-PLC7 immunoprecipitates, we have examined the relationship between levels of [P-Tyr]PLC7 and accumulation of [3H]InsP in intact WB cells treated with EGF and phorbol ester. Materials and Methods Materials EGF was purified from mouse salivary glands as described (20); purity was confirmed by amino acid analysis. Characterization of pooled monoclonal antibodies against PLC7 (from hybridoma clones F-7-2, B-6-4, E-8-4, B-20-3, D-7-3, and E-94) and PLC/3 (hybridomas K-32-3, K-82-3, and K-92-3) has been described (21, 22). Monoclonal anti-c-src antibody 327 was provided by Dr. Joan Brugge, University of Pennsylvania. Phorbol 12-myristate 13-acetate (PMA; Sigma Chemical Co., St. Louis, MO) was prepared as a 10 mM stock solution in dimethylsulfoxide; final vehicle concentration was

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 tyrosine-phosphorylate the gamma-isozyme of phosp...
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