The phosphorylation targets of p47^phox, a subunit of the respiratory burst oxidase: Functions of the individual target serines as evaluated by site-directed mutagenesis

The respiratory burst oxidase of phagocytes and B lymphocytes catalyzes the reduction of oxygen to O^-₂ at the expense of NADPH. Dormant in resting cells, the oxidase is activated by exposing the cells to appropriate stimuli. During activation, p47^phox, a cytosolic oxidase subunit, becomes extensively phosphorylated on a number of serines located between S303-S379. To determine whether this phosphorylation is necessary for oxidase activation, we examined phorbol-elicited oxidase activity in EBV-transformed B lymphoblasts deficient in p47^phox after transfection with plasmids expressing various S → A mutants of p47^phox. The mutant containing S → A mutations involving all serines between S303 and S379 [S(303-379) A] was not phosphorylated, did not translocate to plasma membrane during activation and was almost devoid of function. As to individual serines, S379 was of special interest because (a) p47^phox S379 was phosphorylated in phorbol-activated lymphoblasts expressing wild-type p47^phox, and (b) p47^phox S379A failed to translocate to the membrane, and was as functionless as p47^phox S(303-379)A; other single S → A mutations had little effect on oxidase activity. These findings suggest that the phosphorylation of S379 may be important for oxidase activation in whole cells.