REGULATION OF PHOSPHOLIPASE-D BY PROTEIN-KINASE-C IN HUMAN NEUTROPHILS - CONVENTIONAL ISOFORMS OF PROTEIN-KINASE-C PHOSPHORYLATE A PHOSPHOLIPASE D-RELATED COMPONENT IN THE PLASMA-MEMBRANE

In a variety of intact cells, phorbol esters are known to activate pho spholipase D. In a cell-free system consisting of plasma membrane and cytosol from human neutrophils, phorbol esters activated phospholipase D in an adenosine nucleotide triphosphate-dependent manner. ATP gamma S (adenosine 5'-O-(thiotriphosphate)) was 2-3-fold more effective tha n ATP, while ADP and AppNHp (adenyl-5'-yl imidodiphosphate) were ineff ective, and activation was blocked by the kinase inhibitor staurospori ne, In cytosol depleted of protein kinase C by chromatography on threo nine Sepharose, phorbol ester-dependent activation was lost, but was r estored upon addition of purified rat brain protein kinase C, The targ et for phosphorylation was shown to be the plasma membrane: plasma mem brane was phosphorylated using ATP gamma S/phorbol 12,13-dibutyrate an d protein kinase C and was reisolated to remove activators, Upon addin g nucleotide depleted cytosol, activator-independent phospho lipase D activity was seen, Using this prephosphorylation protocol, PKC-depende nt activation of plasma membranes was found to require micromolar calc ium, implicating a conventional protein kinase C, Using recombinant is oforms of protein kinase C, only the conventional isoforms showed sign ificant activation, with the following rank order of potency: beta(1) > alpha > gamma; the beta(2), delta, epsilon, eta, and zeta isoforms s howed little or no activity. Thus, conventional isoform(s) of protein kinase C activate neutrophil phospholipase D by phosphorylating a targ et protein located in the plasma membrane.