Protein kinase C xi phosphorylates a subset of selective sites of the NADPH oxidase component p47(phox) 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 c omponent, p47(phox), a major cytosolic component of this oxidase. Protein k inase C zeta (PKC zeta), an atypical PKC isoform expressed abundantly in hu man polymorphonuclear leukocytes (PMN), translocates to the PMN plasma memb rane upon stimulation by the chemoattractant fMLP. We investigated the role of PKC zeta in p47(phox) phosphorylation and in superoxide anion productio n by human PMN. In vitro incubation of recombinant p47(phox) with recombina nt PKC zeta induced a time- and concentration-dependent phosphorylation of p47(phox) with an apparent K-m value of 2 muM. Phosphopeptide mapping analy sis of p47(phox) showed that PKC zeta phosphorylated fewer selective sites in comparison to "conventional" PKCs. Serine 303/304 and serine 315 were id entified as targets of PKC zeta by site-directed mutagenesis. Stimulation o f PMN by fMLP induced a rapid and sustained plasma membrane translocation o f PKC zeta that correlated to that of p47(phox). A cell-permeant-specific p eptide antagonist of PKC zeta inhibited both fMLP-induced phosphorylation o f p47(phox) and its membrane translocation. The antagonist also inhibited t he fMLP-induced production of oxidant (IC50 of 10 muM), but not that induce d by PMA. The inhibition of PKC zeta expression in HL-60 neutrophil-like ce lls using antisense oligonucleotides (5 and 10 muM) inhibited fMLP-promoted oxidant production (27 and 50%, respectively), but not that induced by PMA . In conclusion, p47(phox) is a substrate for PKC zeta and participates in the signaling cascade between fMLP receptors and NADPH oxidase activation.