Phosphorylation of neutrophil 47-kDa cytosolic oxidase factor. Translocation to membrane is associated with distinct phosphorylation events.

Activation of the phagocytic cell superoxide-generating NADPH oxidase requires interaction of cytosolic and membrane-associated components. With most stimuli activation of the oxidase is accompanied by multisite phosphorylation of the 47-kDa cytosolic oxidase factor (p47) which translocates from cytosol to membranes. Native p47 is a highly basic protein that undergoes stepwise charge shifts with successive phosphorylation events. Phosphorylation of p47 was studied by immunoprecipitation from neutrophil cytosol and membrane fractions followed by two-dimensional gel electrophoresis and autoradiography. In the resting cell p47 was not phosphorylated. In the cytosol of phorbol myristate acetate-activated neutrophils eight distinct p47 phosphoproteins were present. The membrane fraction from these activated cells contained a family of p47 phosphoproteins of electrophoretic mobilities identical to those seen in cytosol plus an additional, more acidic p47 phosphoprotein not present in cytosol. Very early after activation (30 s) only the four most acidic p47 phosphoproteins were present in the membrane fraction. Only at later times (5-15 min) was the full spectrum of p47 phosphoproteins present in the membrane fraction. In contrast, the full spectrum of p47 phosphoproteins was present in the cytosol over the entire time course we studied. In neutrophils from patients with cytochrome b558-deficient chronic granulomatous disease p47 phosphorylation was incomplete and p47 translocation to membrane did not occur. These studies demonstrated that the cytochrome was essential for formation of the three most acidic p47 phosphoproteins and greatly augmented formation of the fourth most acidic p47 phosphoprotein found in normal neutrophils. The temporal correlation between specific p47 phosphorylation events and p47 translocation to membrane is consistent with a model of oxidase activation in which a series of p47 phosphorylation events which occurs in cytosol precedes and may be required for p47 interaction with membrane.