Epidermal growth factor stimulates phospholipase cgamma1 in cultured rabbit corneal epithelial cells.
Phospholipase Cgamma1 (PLCgamma1) catalyses hydrolysis of phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol and inositol 1,4,5-trisphosphate (IP(3)), two second messengers which play important roles in cell proliferation and differentiation. The purpose of the current study was to identify PLCgamma1 in corneal epithelial cells and investigate whether epidermal growth factor (EGF) stimulates the activity of this enzyme. Addition of EGF to [(3)H]myo-inositol-labeled, cultured corneal epithelial cells stimulated production of IP(3), indicating activation of PLC. Western immunoblot analysis and an in vitro assay of PLC activity revealed that EGF activates gamma1 isoform of PLC, which is localized predominantly in the cytosolic fraction of the epithelial cells. EGF receptors were detected in the epithelial cells by EGF receptor antibody. Addition of EGF to the cells caused tyrosine phosphorylation of the receptors, translocation of PLCgamma1 from cytosol to plasma membrane, and phosphorylation of the enzyme at tyrosine residues. Addition of tyrphostin A-25, an inhibitor of receptor tyrosine kinase, attenuated the tyrosine phosphorylation of PLCgamma1 as well as its enzyme activity. These findings suggest that EGF stimulates PLCgamma1 in rabbit corneal epithelial cells, and that this effect is probably mediated by tyrosine phosphorylation of the enzyme.
Islam
M
M
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia 30912, USA.
Akhtar
R A
RA
D000818:Animals; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D004847:Epithelial Cells / Q000201:enzymology*; D019573:Epithelium, Corneal / Q000201:enzymology*; D066246:ErbB Receptors / Q000378:metabolism; D006868:Hydrolysis / Q000187:drug effects; D007527:Isoenzymes / Q000378:metabolism*; D051966:Phospholipase C gamma; D010766:Phosphorylation; D011817:Rabbits; D020794:Receptor Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D011994:Recombinant Proteins / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism; D020032:Tyrphostins / Q000494:pharmacology
D007527:Isoenzymes; D011994:Recombinant Proteins; D020032:Tyrphostins; C079836:tyrphostin 25; D014443:Tyrosine; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D020794:Receptor Protein-Tyrosine Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma