The regulation of phosphatidylcholine-hydrolyzing phospholipase D (PLD) by protein kinase C (PRC) in membranes of Chinese hamster lung fibroblasts (CCL39) was studied using conventional PKC isoforms cc, b and y isolated from rat brain and recombinant PKC isoforms. Cells were incubated with [Wlcholine to label endogenous phosphatidylcholine before membranes were prepared and assayed for release of [Wlcholine. PKCa was the most potent activator of PLD, producing a maximal effect at approximately 0.1 @ml. PKC/I also stimulated PLD but was less potent and less efficacious, whereas PKCy was ineffective. Stimulation required addition of a PKC activator, but the isoform specificity was the same whether phorbol 12-myristate 13-acetate (PMA) or Ca" was used. Recombinant Ca"-independent PKC isoforms 6, E, and [ failed to stimulate PLD, but recombinant PKQ?, stimulated PLD in a manner similar to the purified brain PKCB. Immunoblot analysis of the soluble fraction of CCL 39 fibroblasts detected only the a and [ isoforms of PKC. The results suggest that PKCa and /I are activators of PLD and that PKCa is responsible for the activation in these fibroblasts.

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