The role of protein kinase C in the mechanism of phospholipase D activation by platelet-derived growth factor and 12-O-tetradecanoylphorbol- 13-acetate was studied in Swiss/3T3 fibroblasts that overexpress protein kinase C-α. Production of [3H]phosphatidylpropanol (specific product of the phospholipase D-catalyzed transphosphatidylation reaction) was determined in cells which were prelabeled with [3H]oleic acid. Accumulation of [3H]phosphatidylpropanol in response to platelet-derived growth factor and 12-O-tetradecanoylphorbol-13-acetate was 2-3-fold greater in protein kinase C-α-overexpressing SF1.4 cells compared with the vector control cells, SC1. Basal [3H] phosphatidylpropanol 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-tetradecanoylphorbol-13- acetate was comparable in the two cell lines and was not significantly altered by the overexpression of protein kinase C-α. Similarly, overexpression of protein kinase C-α did not affect either the kinetics of phospholipase D activation nor its dependence on platelet-derived growth factor or 12-O-tetradecanoylphorbol-13-acetate concentration. In vitro assay of phospholipase D activity in membranes isolated from the cells, utilizing exogenous [3H]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 indicated that the apparent V(max) and K(m) of phospholipase D derived from SF1.4 and SF3.2 (protein kinase C-α-overexpressing) cells are significantly higher than those of phospholipase D from control cells. These results indicate that in Swiss/3T3 cells overexpression of protein kinase C-α elevates basal and agonist- stimulated phospholipase D activity in intact cells as well as phospholipase D activity in vitro. These data are consistent with the hypothesis that overexpression of protein kinase C-α up-regulates phospholipase D, leading to a constitutive higher level of enzyme activity. Thus, protein kinase C-α may play a role in regulating phospholipase D expression.
|Number of pages||5|
|Journal||Journal of Biological Chemistry|
|State||Published - 1 Jan 1993|