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 fac tor (PDGF), prostaglandin F2alpha and 12-O-tetradecanoylphorbol 13-ace tate (TPA) was studied in NIH-3T3 fibroblasts. PLD activation was dete rmined by measuring the production of both [H-3]phosphatidic acid and [H-3]phosphatidylpropanol (products of the PLD-catalyzed hydrolysis an d 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 prostagla ndin F2alpha and PDGF was transient and declined to near basal levels by 15 min and 55 min, respectively. In contrast, TPA-induced activatio n of PLD was sustained for at least 60 min of incubation. A combinatio n 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 kina se inhibitor staurosporine inhibited PLD activation by PDGF or TPA wit h almost identical dose/response curves. In contrast, staurosporine po tentiated prostaglandin-F2alpha-induced PLD activation. The specific p rotein kinase C inhibitor GF109203X (a bisindolylmaleimide) inhibited PLD activation by prostaglandin F2alpha and PDGF at concentrations hig her than those required for inhibition of PLD activation induced by TP A. Depletion of cellular protein kinase C abolished PLD activation by all three mitogens without affecting in vitro activity of membrane-bou nd PLD. The distinct kinetics of PLD activation and its differential s usceptibility to protein kinase inhibitors suggest the existence of ag onist-specific activation and/or inactivation mechanisms. The results indicate also that protein kinase C participates in the mechanism of P LD activation via PDGF, while the effect of prostaglandin F2alpha invo lves a pathway independent of protein kinase C.