Protein kinase C zeta phosphorylates a subset of selective sites of the NADPH oxidase component p47phox 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(phox), a major cytosolic component of this oxidase. Protein kinase C zeta (PKC zeta), 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 zeta in p47(phox) phosphorylation and in superoxide anion production by human PMN. In vitro incubation of recombinant p47(phox) with recombinant PKC zeta induced a time- and concentration-dependent phosphorylation of p47(phox) with an apparent K(m) value of 2 microM. Phosphopeptide mapping analysis of p47(phox) showed that PKC zeta phosphorylated fewer selective sites in comparison to "conventional" PKCs. Serine 303/304 and serine 315 were identified as targets of PKC zeta by site-directed mutagenesis. Stimulation of PMN by fMLP induced a rapid and sustained plasma membrane translocation of PKC zeta that correlated to that of p47(phox). A cell-permeant-specific peptide antagonist of PKC zeta inhibited both fMLP-induced phosphorylation of p47(phox) and its membrane translocation. The antagonist also inhibited the fMLP-induced production of oxidant (IC(50) of 10 microM), but not that induced by PMA. The inhibition of PKC zeta expression in HL-60 neutrophil-like cells using antisense oligonucleotides (5 and 10 microM) 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.
Dang
P M
PM
Institut National de la Santé et de la Recherche Médicale Unité 479, Centre Hospitalier Universitaire Xavier Bichat, Paris, France.
Fontayne
A
A
Hakim
J
J
El Benna
J
J
Périanin
A
A
D000595:Amino Acid Sequence; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics / Q000276:immunology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018922:HL-60 Cells; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000097:blood / Q000235:genetics; D008969:Molecular Sequence Data; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000097:blood / Q000494:pharmacology*; D019255:NADPH Oxidases / Q000097:blood*; D018375:Neutrophil Activation / Q000276:immunology; D009504:Neutrophils / Q000201:enzymology* / Q000378:metabolism; D010449:Peptide Mapping; D010455:Peptides / Q000494:pharmacology; D010748:Phosphopeptides / Q000097:blood; D010750:Phosphoproteins / Q000097:blood*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000097:blood* / Q000235:genetics; D016372:RNA, Antisense / Q000494:pharmacology; D016897:Respiratory Burst / Q000276:immunology; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology; D000099221:Protein Kinase C zeta
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019255:NADPH Oxidases; D010455:Peptides; D010748:Phosphopeptides; D010750:Phosphoproteins; D011493:Protein Kinase C; D016372:RNA, Antisense; D000099221:Protein Kinase C zeta; C060505:neutrophil cytosolic factor 1