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

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 p67(phox) (pha gocyte oxidase), p47(phox), and p40(phox) which translocate to the mem brane upon activation, In a previous paper, we reported that p40(phox) undergoes phosphorylation on multiple sites upon stimulation of the N ADPH oxidase by either phorbol 12-myristate 13-acetate or by formyl pe ptide with a time course that is strongly correlated with that of supe roxide production (Fuchs, A, Bouin, A. P,, Rabilloud, T., and Vignais, P. V, (1997) Eur J, Biochem. 249, 531-5S9). In this study, through ph osphoamino acid and tryptic peptide maps of in vivo and in vitro phosp horylated p40(phox), we show that p40(phox) is phosphorylated on serin e and threonine residues during activation of the NADPH oxidase in dim ethyl sulfoxide-differentiated HL60 promyelocytes as well as in isolat ed human neutrophils, In vitro phosphorylation studies using casein ki nase II and protein kinase C (PKC) as well as the effect of various pr otein kinase inhibitors on the isoelectric focusing pattern of p40(pho x) whole cell lysates point to a role of a PKC type kinase in the phos phorylation of p40(phox) Directed mutagenesis of all PKC consensus sit es enable us to conclude that Thr(154) and Ser(315) in p40(phox) are p hosphorylated during activation of the NADPH oxidase.