Oxidant-dependent phosphorylation of p40phox in B lymphocytes

As with the neutrophil NADPH oxidase, the B lymphocyte NADPH oxidase consis ts of a membrane-bound flavocytochrome b and regulatory factors including R ac and the cytosolic phox protein triad p67phox, p47phox, and p40phox. Here we demonstrate by phosphoamino acid analysis and the use of the potent PKC inhibitor GFX that, in response to stimulation of B lymphocytes with sodiu m orthovanadate and H2O2, the p40phox component of the cytosolic phox triad is selectively phosphorylated on serine and threonine residues by a PKC-ty pe protein kinase. The pattern of p40phox phosphorylation was closely relat ed to the kinetics of tyrosine phosphorylation of PKC-delta, the main PKC i sotype of B lymphocytes. Blocking H2O2-dependent tyrosine phosphorylation o f PKC by genistein resulted in inhibition of p40phox phosphorylation. The c orrelation between the tyrosine phosphorylation of PKC-delta and the serine /threonine phosphorylation of p40phox, together with the inhibition of p40p hox phosphorylation by rottlerin, a selective inhibitor of PKC-delta, makes the activated PKC-delta a likely candidate in the process of the oxidant-d ependent phosphorylation of p40phox in B cells. (C) 2001 Academic Press.