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 phospholipase 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 than ATP, while ADP and AppNHp (adenyl-5'-yl imidodiphosphate) were ineffective, and activation was blocked by the kinase inhibitor staurosporine. In cytosol deplete of protein kinase C by chromatography on threnoine-Sepharose, phorbol ester-dependent activation was lost, but was restored upon addition of purified rat brain protein kinase C. The target for phosphorylation was shown to be the plasma membrane plasma membrane was phosphorylated using ATP gamma S/phorbol 12,13-dibutyrate and protein kinase C and was reisolated to remove activators. Upon adding nucleotide-depleted cytosol, activator-independent phospholipase D activity was seen. Using this prephosphorylation protocol, PKC-dependent activation of plasma membranes was found to require micromolar calcium, implicating a conventional protein kinase C. Using recombinant isoforms of protein kinase C, only the conventional isoforms showed significant activation, with the following rank order of potency: beta 1 > alpha > gamma; the beta 2, delta, epsilon, eta, and sigma isoforms showed little or no activity. Thus, conventional isoform(s) of protein kinase C activate neutrophil phospholipase D by phosphorylating a target protein located in the plasma membrane.
Lopez
I
I
Department of Biochemistry, Emory University Medical School, Atlanta, Georgia 30322, USA.
Burns
D J
DJ
Lambeth
J D
JD
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D004789:Enzyme Activation; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology*; D010739:Phospholipase D / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D007527:Isoenzymes; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D010739:Phospholipase D; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium