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 catalyze s the reduction of oxygen to O-2(-) at the expense of NADPH. Dormant i n resting cells, the oxidase is activated by exposing the cells to app ropriate stimuli. During activation, p47(phox), a cytosolic oxidase su bunit, becomes extensively phosphorylated on a number of serines locat ed between S303-S379. To determine whether this phosphorylation is nec essary for oxidase activation, we examined phorbol-elicited oxidase ac tivity in EBV-transformed B lymphoblasts deficient in p47(phox) after transfection with plasmids expressing various S --> A mutants of p47(p hox). The mutant containing S --> A mutations involving all serines be tween S303 and S379 [S(303-379) A] was not phosphorylated, did not tra nslocate to plasma membrane during activation and was almost devoid of function, As to individual serines, S379 was of special interest beca use (a) p47(phox) S379 was phosphorylated in phorbol-activated lymphob lasts expressing wild-type p47(phox), and (b) p47(phox) S379A failed t o translocate to the membrane, and was as functionless as p47(phox) S( 303-379) A; other single S --> A mutations had little effect on oxidas e activity. These findings suggest that the phosphorylation of S379 ma y be important for oxidase activation in whole cells.