UP-REGULATION OF PHOSPHOLIPASE-D ACTIVITY-INDUCED BY OVEREXPRESSION OF PROTEIN-KINASE C-ALPHA - STUDIES IN INTACT SWISS 3T3 CELLS AND IN DETERGENT-SOLUBILIZED MEMBRANES INVITRO/

The role of protein kinase C in the mechanism of phospholipase D activ ation by platelet-derived growth factor and 12-O-tetradecanoylphorbol- 13-acetate was studied in Swiss/3T3 fibroblasts that overexpress prote in kinase C-alpha. Production of [H-3]phosphatidylpropanol (specific p roduct of the phospholipase D-catalyzed transphosphatidylation reactio n) was determined in cells which were prelabeled with [H-3]oleic acid. Accumulation of [H-3]phosphatidylpropanol in response to platelet-der ived growth factor and 12-O-tetradecanoylphorbol-13-acetate was 2-3-fo ld greater in protein kinase C-alpha-overexpressing SF1.4 cells compar ed with the vector control cells, SC1. Basal [H-3] phosphatidylpropano l production also was 2-fold higher in SF1.4 cells than in SC1 cells. Hence, -fold stimulation of basal phospholipase D activity by platelet -derived growth factor and 12-O-tetradecanoyl-phorbol-13-acetate was c omparable in the two cell lines and was not significantly altered by t he overexpression of protein kinase C-alpha. Similarly, overexpression of protein kinase C-alpha did not affect either the kinetics of phosp holipase D activation nor its dependence on platelet-derived growth fa ctor or 12-O-tetradecanoylphorbol-13-acetate concentration. In vitro a ssay of phospholipase D activity in membranes isolated from the cells, utilizing exogenous [H-3]phosphatidylcholine as a substrate, revealed nearly 2-fold higher phospholipase D activity in SF1.4 cell membranes . Kinetic analysis of detergent-solubilized phospholipase D activity i ndicated that the apparent V(max) and K(m) of phospholipase D derived from SF1.4 and SF3.2 (protein kinase C-alpha-overexpressing) cells are significantly higher than those of phospholipase D from control cells . These results indicate that in Swiss/3T3 cells overexpression of pro tein kinase C-alpha elevates basal and agonist-stimulated phospholipas e D activity in intact cells as well as phospholipase D activity in vi tro. These data are consistent with the hypothesis that overexpression of protein kinase C-alpha up-regulates phospholipase D, leading to a constitutive higher level of enzyme activity. Thus, protein kinase C-a lpha may play a role in regulating phospholipase D expression.