ACTIVATION OF THE LEUKOCYTE NADPH OXIDASE BY PHORBOL ESTER REQUIRES THE PHOSPHORYLATION OF P47(PHOX) ON SERINE 303 OR 304

The leukocyte NADPH oxidase is an enzyme in phagocytes and B lymphocyt es that when activated catalyzes the production of O-2((.) over bar) f rom oxygen and NADPH, During oxidase activation, serine residues in th e C-terminal quarter of the oxidase component p47(PHOX) become extensi vely phosphorylated, the protein acquiring as many as 9 phosphate resi dues. In a study of 11 p47(PHOX) mutants, each containing an alanine i nstead of a serine at a single potential phosphorylation site, we foun d that all but S379A corrected the defect in O-2((.) over bar) product ion in Epstein-Barr virus (EBV)-transformed p47(PHOX)-deficient B cell s (Faust, L. P., El Benna, J., Babior, B. M., and Chanock, S. J. (1995 ) J. Clin. Invest. 96, 1499-1505). In particular, O-2((.) over bar) pr oduction was restored to these cells by the mutants S303A and S304A. T herefore, apart from serine 379, whose state of phosphorylation in the activated oxidase is unclear, no single potential phosphorylation sit e appeared to be essential for oxidase activation. We now report that the double mutant p47(PHOX) S303A/S304A was almost completely inactive when ex pressed in EBV-transformed p47(PHOX)-deficient B cells, even though it was expressed in normal amounts in the transfected cells and was able to translocate to the plasma membrane when the cells were st imulated, In contrast, the double mutant p47(PHOX) S303E/S304E was abl e to support high levels of O-2((.) over bar) production by EBV-transf ormed p47(PHOX)-deficient B cells. The surprising discovery that the d ouble mutant S303K/S304K was also able to support considerable O-2((.) over bar) production suggests either that the effect of phosphorylati on is related to the increase in hydrophilicity around serines 303 and 304 or that activation involves the formation of a metal bridge betwe en the phosphorylated serines and another region of the protein.