Suppression of epidermal growth factor-induced phospholipase C activation associated with actin rearrangement in rat hepatocytes in primary culture

Hepatocytes maintained in primary culture for periods of 1 to 24 hours exhi bited a rapid decline in epidermal growth factor (EGF)-induced activation o f phospholipase C (PLC), as was evident in a loss of EGF-induced inositol 1 ,4,5-trisphosphate (IP3) formation and mobilization of Ca2+ from intracellu lar Ca2+ stores. The loss of PLC activation was not the result of a decreas e in EGF receptor or phospholipase C-gamma1 (PLC gamma1) protein levels, no r the result of a loss of tyrosine phosphorylation of these proteins, but w as associated with a decrease in EGF-induced translocation of PLC gamma1 to the Triton-insoluble fraction, presumably reflecting binding to the actin cytoskeleton. Disruption of F-actin by treatment of cultured hepatocytes wi th cytochalasin D recovered the EGF-induced IP3 formation and Ca2+ mobiliza tion to the same level and with the same dose-response relationship as was obtained in freshly isolated cells. Analysis of PLC gamma1 colocalization w ith F-actin by confocal microscopy showed that PLC gamma1 was mostly distri buted diffusely in the cytosol, both in freshly plated cells and in cells i n culture for 24 hours, despite marked differences in actin structures, EGF stimulation caused a modest redistribution of PLC gamma1 and a detectable increase in colocalization with cortical actin structures in freshly plated cells or in cytochalasin D-treated cells, but in cells that had been maint ained and spread in culture only a limited PLC gamma1 relocation was detect ed to specific actin-structure associated with lamellipodia and membrane ru ffles. We conclude that actin cytoskeletal structures can exert negative co ntrol over PLC gamma1 activity in hepatocytes and the interaction of the en zyme with specific actin structures dissociates PLC gamma1 tyrosine phospho rylation from activation of its enzymatic activity.