REQUIREMENT FOR PHOSPHOLIPASE C-GAMMA-1 ENZYMATIC-ACTIVITY IN GROWTH FACTOR-INDUCED MITOGENESIS

The cytoplasmic regions of the receptors for epidermal growth factor ( EGF) acid platelet-derived growth factor (PDGF) bind and activate phos pholipase C-gamma 1 (PLC-gamma 1) and other signaling proteins in resp onse to ligand binding outside the cell, Receptor binding by PLC-gamma 1 is a function of its SH2 domains and is required for growth factor- induced cell cycle progression into the S phase. Microinjection into M DCK epithelial cells and NIH 3T3 fibroblasts of a polypeptide correspo nding to the noncatalytic SH2-SH2-SH3 domains of PLC-gamma 1 (PLC-gamm a 1 SH2-SH2-SH3) blocked growth factor-induced S-phase entry, Treatmen t of cells with diacylglycerol (DAG) or DAG and microinjected inositol -1,4,5-triphosphate (IP3), the products of activated PLC-gamma 1, did not stimulate cellular DNA synthesis by themselves but did suppress th e inhibitory effects of the PLC-gamma 1 SH2-SH2-SH3 polypeptide but no t the cell cycle block imposed by inhibition of the adapter protein Gr b2 or p21 Ras, Two c-fos serum response element (SRE)-chloramphenicol acetyltransferase (CAT) reporter plasmids, a wild-type version, wtSRE- CAT, and a mutant, pm18, were used to investigate the function of PLC- gamma 1 in EGF- and PDGF-induced mitogenesis. wtSRE-CAT responds to bo th protein kinase C (PKC)-dependent and -independent signals, while th e mutant, pm18, responds only to PKC-independent signals, Microinjecti on of the dominant-negative PLC-gamma 1 SH2-SH2-SH3 polypeptide greatl y reduced the responses of wtSRE-CAT to EGF stimulation in MDCK cells and to PDGF stimulation in NIH 3T3 cells but had no effect on the resp onses of mutant pm18, These results indicate that in addition to Grb2- mediated activation of Ras, PLC-gamma 1-mediated DAG production is req uired for EGF-and PDGF-induced S-phase entry and gene expression, poss ibly through activation of PKC.