DISTINCT MECHANISMS OF PHOSPHOLIPASE-D ACTIVATION AND ATTENUATION UTILIZED BY DIFFERENT MITOGENS IN NIH-3T3 FIBROBLASTS

The activation of phospholipase D (PLD) by platelet-derived growth factor (PDGF), prostaglandin F2alpha and 12-O-tetradecanoylphorbol 13-acetate (TPA) was studied in NIH-3T3 fibroblasts. PLD activation was determined by measuring the production of both [H-3]phosphatidic acid and [H-3]phosphatidylpropanol (products of the PLD-catalyzed hydrolysis and transphosphatidylation reactions, respectively), in cells that were metabolically pre-labeled with [H-3]oleic acid. All mitogens caused a rapid (within 2 min) activation of PLD. Activation of PLD by prostaglandin F2alpha and PDGF was transient and declined to near basal levels by 15 min and 55 min, respectively. In contrast, TPA-induced activation of PLD was sustained for at least 60 min of incubation. A combination of maximally effective concentrations of PDGF and TPA stimulated PLD activity in a non-additive manner, while the effect of prostaglandin F2alpha was additional to that of either PDGF or TPA. The protein kinase inhibitor staurosporine inhibited PLD activation by PDGF or TPA with almost identical dose/response curves. In contrast, staurosporine potentiated prostaglandin-F2alpha-induced PLD activation. The specific protein kinase C inhibitor GF109203X (a bisindolylmaleimide) inhibited PLD activation by prostaglandin F2alpha and PDGF at concentrations higher than those required for inhibition of PLD activation induced by TPA. Depletion of cellular protein kinase C abolished PLD activation by all three mitogens without affecting in vitro activity of membrane-bound PLD. The distinct kinetics of PLD activation and its differential susceptibility to protein kinase inhibitors suggest the existence of agonist-specific activation and/or inactivation mechanisms. The results indicate also that protein kinase C participates in the mechanism of PLD activation via PDGF, while the effect of prostaglandin F2alpha involves a pathway independent of protein kinase C.