Protein kinase C (PKC) isoforms translocate to Triton-insoluble fractions in stimulated human neutrophils: correlation of conventional PKC with activation of NADPH oxidase.
The responses of human neutrophils (PMN) involve reorganization and phosphorylation of cytoskeletal components. We investigated the translocation of protein kinase C (PKC) isoforms to PMN cytoskeletal (Triton-insoluble) fractions, in conjunction with activation of the respiratory burst enzyme NADPH oxidase. In resting PMN, PKC-delta (29%) and small amounts of PKC-alpha (0.6%), but not PKC-betaII, were present in cytoskeletal fractions. Upon stimulation with the PKC agonist PMA, the levels of PKC-alpha, PKC-betaII, and PKC-delta increased in the cytoskeletal fraction, concomitant with a decrease in the noncytoskeletal (Triton-soluble) fractions. PKC-delta maximally associated with cytoskeletal fractions at 160 nM PMA and then declined, while PKC-alpha and PKC-betaII plateaued at 300 nM PMA. Translocation of PKC-delta was maximal by 2 min and sustained for at least 10 min. Translocation of PKC-alpha and PKC-betaII was biphasic, plateauing at 2-3 min and then increasing up to 10 min. Under maximal stimulation conditions, PKC isoforms were entirely cytoskeletal associated. Translocation of the NADPH oxidase component p47phox to the cytoskeletal fraction correlated with translocation of PKC-alpha and PKC-betaII, but not with translocation of PKC-delta. Oxidase activity in cytoskeletal fractions paralleled translocation of PKC-alpha, PKC-betaII, and p47phox. Stimulation with 1,2-dioctanoylglycerol resulted in little translocation of PKC isoforms or p47phox, and in minimal oxidase activity. We conclude that conventional PKC isoforms (PKC-alpha and/or PKC-betaII) may regulate PMA-stimulated cytoskeletal association and activation of NADPH oxidase. PKC-delta may modulate other PMN responses that involve cytoskeletal components.
Nixon
J B
JB
Department of Biochemistry, Division of Infectious Diseases, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA.
McPhail
L C
LC
D000328:Adult; D001692:Biological Transport / Q000187:drug effects / Q000276:immunology; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D004075:Diglycerides / Q000494:pharmacology; D004306:Dose-Response Relationship, Immunologic; D004789:Enzyme Activation / Q000187:drug effects / Q000276:immunology; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism*; D018375:Neutrophil Activation / Q000187:drug effects; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology* / Q000276:immunology; D017830:Octoxynol; D010743:Phospholipids / Q000032:analysis; D010750:Phosphoproteins / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D064546:Protein Kinase C beta; D051745:Protein Kinase C-delta; D011506:Proteins / Q000032:analysis; D012995:Solubility; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism; D013997:Time Factors
D004075:Diglycerides; D007527:Isoenzymes; D010743:Phospholipids; D010750:Phosphoproteins; D011506:Proteins; C044295:1,2-dioctanoylglycerol; D017830:Octoxynol; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; C497258:PRKCD protein, human; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D051745:Protein Kinase C-delta; D013755:Tetradecanoylphorbol Acetate