Phosphorylation of p47phox Sites by PKC α, βΙΙ, δ, and ζ: Effect on Binding to p22phox and on NADPH Oxidase Activation

Production of superoxide anions by the multicomponent enzyme of human neutrophil NADPH oxidase is accompanied by extensive phosphorylation of p47phox, one of its cytosolic components. p47phox 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 α, β, δ, and ζ) phosphorylate p47phox in a time- and concentration-dependent manner, with apparent Km values of 10.33, 3.37, 2.37, and 2.13 μM for PKC α, βΙΙ, δ, and ζ, respectively. Phosphopeptide mapping of p47phox showed that, as opposed to PKC ζ, PKC α, βΙΙ, and δ are able to phosphorylate all the major PKC sites. The use of p47phox mutants identified serines 303, 304, 315, 320, 328, 359, 370, and 379 as targets of PKC α, βΙΙ, and δ. Comparison of the intensity of phosphopeptides suggests that Ser 328 is the most phosphorylated serine. The ability of each PKC isoform to induce p47pho...