Phospholipase D activation in fibroblast membranes by the α and β isoforms of protein kinase C

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 α, β and γ isolated from rat brain and recombinant PKC isoforms. Cells were incubated with [¹⁴C]choline to label endogenous phosphatidylcholine before membranes were prepared and assayed for release of ¹⁴C]choline. PKCα was the most potent activator of PLD, producing a maximal effect at approximately 0.1 . PKCβ also stimulated PLD but was less potent and less efficacious, whereas PKCγ was ineffective. Stimulation required addition of a PKC activator, but the isoform specificity was the same whether phorbol 12-myristate 13-acetate (PMA) or Ca²⁺ was used. Recombinant Ca²⁺-independent PKC isoforms δ, ϵ, and ζ, failed to stimulate PLD, but recombinant PKCβ₁ stimulated PLD in a manner similar to the purified brain PKCβ. Immunoblot analysis of the soluble fraction of CCL 39 fibroblasts detected only the α and ζ isoforms of PKC. The results suggest that PKCα and β are activators of PLD and that PKCα is responsible for the activation in these fibroblasts.