ACTIVATION OF NADPH OXIDASE OF HUMAN NEUTROPHILS INVOLVES THE PHOSPHORYLATION AND THE TRANSLOCATION OF CYTOSOLIC P67PHOX

Activation of human neutrophil NADPH oxidase requires the interaction of cytosolic and membrane-associated components. Evidence has been acc umulated that in phorbol 12-myristate 13-acetate (PMA)-stimulated neut rophils, the translocation to the plasma membrane of the cytosolic com ponents p47phox and p67phox and the phosphorylation of p47phox are ess ential steps in activation of NADPH oxidase. No direct evidence has be en presented to date as to whether p67phox is also phosphorylated. To address this problem we have immunoprecipitated p67phox from neutrophi l cytosol and membrane fractions. The results indicate that, very soon after activation with PMA (20 s), p67phox was present in a phosphoryl ated form in the cytosol and in the membranes. At later times (1-3 min ) the extent of p67phox phosphorylation continuously increased both in the cytosol and in the membrane fraction, while oxygen consumption re ached the maximal rate within 40 s, and then remained linear. p67phox was also phosphorylated in formyl-methionyl-leucyl-phenylalanine-activ ated neutrophils. That the phosphorylated p67 protein we identified in immunoprecipitation experiments was p67phox was confirmed by the obse rvation that no phosphorylated band of 67 kDa was immunoprecipitated f rom the cytosol and membranes of PMA-stimulated neutrophils from a p67 phox-deficient chronic granulomatous disease patient. In this case, p4 7phox was normally phosphorylated. These data demonstrate that: (1) th e phosphorylation of p67phox is correlated with activation of NADPH ox idase, and (2) continuous phosphorylation of p67phox is required in or der to maintain the linearity of the respiratory burst.