Phosphorylation of the respiratory burst oxidase subunit p67(phox) during human neutrophil activation. Regulation by protein kinase C-dependent and independent pathways.
The respiratory burst oxidase of phagocytes and B lymphocytes catalyzes the reduction of oxygen to superoxide anion (O-2) at the expense of NADPH. This multicomponent enzyme is dormant in resting cells but is activated on exposure to an appropriate stimulus. The phosphorylation-dependent mechanisms regulating the activation of the respiratory burst oxidase are unclear, particularly the phosphorylation status of the cytosolic component p67(phox). In this study, we found that activation of human neutrophils with formyl-methionyl-leucyl-phenylalanine (fMLP), a chemotactic peptide, or phorbol myristate acetate (PMA), a stimulator of protein kinase C (PKC), resulted in the phosphorylation of p67(phox). Using an anti-p67(phox) antibody or an anti-p47(phox) antibody, we showed that phosphorylated p67(phox) and p47(phox) form a complex. Phosphoamino acid analysis of the phosphorylated p67(phox) revealed only 32P-labeled serine residues. Two-dimensional tryptic peptide mapping analysis showed that p67(phox) is phosphorylated at the same peptide whether fMLP or PMA is used as a stimulus. In addition, PKC induced the phosphorylation of recombinant GST-p67(phox) in vitro, at the same peptide as that phosphorylated in intact cells. PMA-induced phosphorylation of p67(phox) was strongly inhibited by the PKC inhibitor GF109203X. In contrast, fMLP-induced phosphorylation was minimally affected by this PKC inhibitor. Taken together, these results show that p67(phox) is phosphorylated in human neutrophils by different pathways, one of which involves protein kinase C.
Benna
J E
JE
INSERM U294, CHU Xavier Bichat, Service d'Hématologie et d'Immunologie Biologiques, 46 rue Henri Huchard, 75018 Paris, France. benna@bichat.inserm.fr
Dang
P M
PM
Gaudry
M
M
Fay
M
M
Morel
F
F
Hakim
J
J
Gougerot-Pocidalo
M A
MA
D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008213:Lymphocyte Activation; D008301:Maleimides / Q000494:pharmacology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D019255:NADPH Oxidases / Q000378:metabolism; D009504:Neutrophils / Q000378:metabolism*; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D004791:Enzyme Inhibitors; D007211:Indoles; D008301:Maleimides; D010750:Phosphoproteins; C079789:neutrophil cytosol factor 67K; D012694:Serine; D009240:N-Formylmethionine Leucyl-Phenylalanine; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; C022709:superoxide-forming enzyme; D011493:Protein Kinase C; C070515:bisindolylmaleimide I; D013755:Tetradecanoylphorbol Acetate