p40 phox Is Phosphorylated on Threonine 154 and Serine 315 during Activation of the Phagocyte NADPH Oxidase IMPLICATION OF A PROTEIN KINASE C-TYPE KINASE IN THE PHOSPHORYLATION PROCESS

Abstract 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(phagocyte oxidase), p47phox, and p40phox, which translocate to the membrane upon activation. In a previous paper, we reported that p40phox undergoes phosphorylation on multiple sites upon stimulation of the NADPH oxidase by either phorbol 12-myristate 13-acetate or by formyl peptide with a time course that is strongly correlated with that of superoxide production (Fuchs, A., Bouin, A. P., Rabilloud, T., and Vignais, P. V. (1997) Eur. J. Biochem. 249, 531–539). In this study, through phosphoamino acid and tryptic peptide maps of in vivo and in vitro phosphorylated p40phox, we show that p40phox is phosphorylated on serine and threonine residues during activation of the NADPH oxidase in dimethyl sulfoxide-differentiated HL60 promyelocytes as well as in isolated human neutrophils. In vitro phosphorylation studies using casein kinase II and protein kinase C (PKC) as well as the effect of various protein kinase inhibitors on the isoelectric focusing pattern of p40phox in whole cell lysates point to a role of a PKC type kinase in the phosphorylation of p40phox. Directed mutagenesis of all PKC consensus sites enable us to conclude that Thr154 and Ser315 in p40phox are phosphorylated during activation of the NADPH oxidase.