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 catalyze s the reduction of oxygen to superoxide anion (O-2(radical anion) at t he expense of NADPH. This multicomponent enzyme is dormant in resting cells but is activated on exposure to an appropriate stimulus, The pho sphorylation-dependent mechanisms regulating the activation of the res piratory burst oxidase are unclear, particularly the phosphorylation s tatus of the cytosolic component p67(phox). In this study, we found th at activation of human neutrophils with formyl-methionyl-leucyl-phenyl alanine (fMLP), a chemotactic peptide, or phorbol myristate acetate (P MA), a stimulator of protein kinase C (PKC), resulted in the phosphory lation of p67(phox). Using an anti-p67(phox) antibody or all anti-p47( phox) antibody, we showed that phosphorylated p67(phox) and p47(phox) form a complex, Phosphoamino acid analysis of the phosphorylated p67(p hox) revealed only P-32-labeled serine residues, Two-dimensional trypt ic peptide mapping analysis showed that p67(phox) is phosphorylated at the same peptide whether fMLP or PMA is used as a stimulus, In additi on, PHC induced the phosphorylation of recombinant GST-p67(phox) in vi tro, at the same peptide as that phosphorylated in intact cells, PMA-i nduced phosphorylation of p67(phox) was strongly inhibited by the PKC inhibitor GF109203X. In contrast, fMLP-induced phosphorylation was min imally affected by this PKC inhibitor. Taken together, these results s how that p67(phox) is phosphorylated in human neutrophils by different pathways, one of which involves protein kinase C.