title: Phosphorylation of p47phox sites by PKC alpha, beta II, delta, and zeta: effect on binding to p22phox and on NADPH oxidase activation.
abstract: Production of superoxide anions by the multicomponent enzyme of human neutrophil NADPH oxidase is accompanied by extensive phosphorylation of p47(phox), one of its cytosolic components. p47(phox) is an excellent substrate for protein kinase C (PKC), but the respective contribution of each PKC isoform to this process is not clearly defined. In this study, we found that PKC isoforms known to be present in human neutrophils (PKC alpha, beta, delta, and zeta) phosphorylate p47(phox) in a time- and concentration-dependent manner, with apparent K(m) values of 10.33, 3.37, 2.37, and 2.13 microM for PKC alpha, beta II, delta, and zeta, respectively. Phosphopeptide mapping of p47(phox) showed that, as opposed to PKC zeta, PKC alpha, beta II, and delta are able to phosphorylate all the major PKC sites. The use of p47(phox) mutants identified serines 303, 304, 315, 320, 328, 359, 370, and 379 as targets of PKC alpha, beta II, and delta. Comparison of the intensity of phosphopeptides suggests that Ser 328 is the most phosphorylated serine. The ability of each PKC isoform to induce p47(phox) to associate with p22(phox) was tested by using an overlay technique; the results showed that all the PKC isoforms that were studied induce p47(phox) binding to the cytosolic fragment of p22(phox). In addition, PKC alpha, beta II, delta, and zeta were able to induce production of superoxide anions in a cell-free system using recombinant cytosolic proteins. Surprisingly, PKC zeta, which phosphorylates a subset of selective p47(phox) sites, induced stronger activation of the NADPH oxidase. Taken together, these results suggest that PKC alpha, beta II, delta, and zeta expressed in human neutrophils can individually phosphorylate p47(phox) and induce both its translocation and NADPH oxidase activation. In addition, phosphorylation of some serines could have an inhibitory effect on oxidase activation.
authors.affiliation: Institut National de la Santé et de la Recherche Médicale U-479, Centre Hospitalier Universitaire Xavier Bichat, Paris, France.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Fontayne
authors.forename: Pham My-Chan
authors.initials: PM
authors.lastname: Dang
authors.forename: Marie-Anne
authors.initials: MA
authors.lastname: Gougerot-Pocidalo
authors.forename: Jamel
authors.initials: J
authors.lastname: El-Benna
mesh_terms: D000409:Alanine / Q000235:genetics; D002474:Cell-Free System / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D026901:Membrane Transport Proteins; D016297:Mutagenesis, Site-Directed; D009252:NADPH Dehydrogenase / Q000378:metabolism; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding / Q000235:genetics; D011493:Protein Kinase C / Q000378:metabolism*; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000494:pharmacology; D012694:Serine / Q000235:genetics; D000099221:Protein Kinase C zeta
chemical_list: D000409:Alanine; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D026901:Membrane Transport Proteins; D009252:NADPH Dehydrogenase; D019255:NADPH Oxidases; D010748:Phosphopeptides; D010750:Phosphoproteins; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D011993:Recombinant Fusion Proteins; D012694:Serine; D000099221:Protein Kinase C zeta; C086986:CYBA protein, human; C060505:neutrophil cytosolic factor 1; C496628:PRKCA protein, human; C497258:PRKCD protein, human