Epidermal growth factor stimulates phospholipase C gamma 1 in cultured rabbit corneal epithelial cells

Phospholipase C gamma 1 (PLC gamma 1) catalyses hydrolysis of phosphatidyli nositol 4,5-bisphosphate to generate diacylglycerol and inositol 1,4,5-tris phosphate (IP3), two second messengers which play important roles in cell p roliferation and differentiation. The purpose of the current study was to i dentify PLC gamma 1 in corneal epithelial cells and investigate whether epi dermal growth factor (EGF) stimulates the activity of this enzyme. Addition of EGF to [H-3]myo-inositol-labeled, cultured corneal epithelial cells sti mulated production of IP3, indicating activation of PLC. Western immunoblot analysis and an in vitro assay of PLC activity revealed that EGF activates gamma 1 isoform of PLC, which is localized predominantly in the cytosolic fraction of the epithelial cells. EGF receptors were detected in the epithe lial cells by EGF receptor antibody. Addition of EGF to the cells caused ty rosine phosphorylation of the receptors, translocation of PLC gamma 1 from cytosol to plasma membrane, and phosphorylation of the enzyme at tyrosine r esidues. Addition of tyrphostin A-25, an inhibitor of receptor tyrosine kin ase, attenuated the tyrosine phosphorylation of PLC gamma 1 as well as its enzyme activity. These findings suggest that EGF stimulates PLC gamma 1 in rabbit corneal epithelial cells, and that this effect is probably mediated by tyrosine phosphorylation of the enzyme. (C) 2000 Academic Press.