Phosphorylation of p47phoxSites by PKC α, βΙΙ, δ, and ζ: Effect on Binding to p22phoxand 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 α, β, δ, and ζ) 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 μM for PKC α, βΙΙ, δ, and ζ, respectively. Phosphopeptide mapping of p47^phox showed that, as opposed to PKC ζ, PKC α, βΙΙ, and δ 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 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 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 α, βΙΙ, δ, and ζ were able to induce production of superoxide anions in a cell-free system using recombinant cytosolic proteins. Surprisingly, PKC ζ, which phosphorylates a subset of selective p47^phox sites, induced stronger activation of the NADPH oxidase. Taken together, these results suggest that PKC α, βΙΙ, δ, and ζ 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.