THE 40-KDA COMPONENT OF THE PHAGOCYTE NADPH OXIDASE (P40PHOX) IS PHOSPHORYLATED DURING ACTIVATION IN DIFFERENTIATED HL60 CELLS

The superoxide-generating NADPH oxidase complex of phagocytic cells is a multicomponent system containing a membrane-bound flavocytochrome b and a small G protein Rac as well as cytosolic factors p67phox, p47ph ox and p40phox which translocate to the membrane upon activation. Know n mechanisms underlying the translocation of these proteins include po lyphosphorylation of p47phox and specific Src homology 3/polyproline m otif interactions. In this study, through two-dimensionnal electrophor esis and immunoprecipitation experiments, we show using dimethylsulfox ide-differentiated HL60 promyelocytes that p40phox is in a basal phosp horylated state in resting cells and undergoes further phosphorylation on multiple sites upon stimulation of the NADPH oxidase by either pho rbol myristate acetate or by the formyl peptide fMet-Leu-Phe-Lys. More over, the extent of phosphorylation is strongly correlated with the le vel of superoxide production. Typically, in cells transiently activate d by fMet-Leu-Phe-Lys, onset of superoxide production coincides with t he appearance of new phosphorylated species of p40phox and, at the end of the respiratory burst, dephosphorylation of p40phox is observed, I n vitro assays show that the kinase(s) involved in the phosphorylation of p40phox differ from those which participate in the phosphorylation of p47phox. This suggests that, in the cell, the phosphorylation of p 40phox and of p47phox are under the control of two different kinase pa thways.