Protein kinase C ζ phosphorylates a subset of selective sites of the NADPH oxidase component p47 and participates in formyl peptide-mediated neutrophil respiratory burst

Generation of superoxide anion by the multiprotein complex NADPH phagocyte oxidase is accompanied by extensive phosphorylation of its 47-kDa protein component, p47, a major cytosolic component of this oxidase. Protein kinase C ζ (PKC ζ), an atypical PKC isoform expressed abundantly in human polymorphonuclear leukocytes (PMN), translocates to the PMN plasma membrane upon stimulation by the chemoattractant fMLP. We investigated the role of PKC ζ in p47 phosphorylation and in superoxide anion production by human PMN. In vitro incubation of recombinant p47 with recombinant PKC ζ induced a time-and concentration-dependent phosphorylation of p47 with an apparent K value of 2 μ M. Phosphopeptide mapping analysis of p47 showed that PKC ζ phosphorylated fewer selective sites in comparison to "conventional" PKCs. Serine 303/304 and serine 315 were identified as targets of PKC ζ by site-directed mutagenesis. Stimulation of PMN by fMLP induced a rapid and sustained plasma membrane translocation of PKC ζ that correlated to that of p47. A cell-permeant-specifiC peptide antagonist of PKC ζ inhibited both fMLP-induced phosphorylation of p47 and its membrane translocation. The antagonist also inhibited the fMLP-induced production of oxidant (IC of 10 μM), but not that induced by PMA. The inhibition of PKC ζ expression in HL-60 neutrophil-like cells using antisense oligonucleotides (5 and 10 μM) inhibited fMLP-promoted oxidant production (27 and 50%, respectively), but not that induced by PMA. In conclusion, p47 is a substrate for PKC ζ and participates in the signaling cascade between fMLP receptors and NADPH oxidase activation.