Activation of protein kinase C in neutrophil cytoplasts: Localization of protein substrates and possible relationship with stimulus-response coupling

Treatment of enucleated, granule-free neutrophil cytoplasts with the protein kinase C activator phorbol 12O-myristate-13-acetate (PMA) causes an increased -³²P-incorporation into a variety of polypeptides. Permeabilization of PMA-stimulated, ³²P-labeled cytoplasts by 0.01% digitonin fully releases the majority of these phosphorylated proteins. A statistically significant correlation is found between the extent of PMA-induced activation of generation of Superoxide anion (O^?₂) and the phosphorylation of a cytosolic polypeptide with an apparent M_r, of 46000, whose -³²P-labeling is also enhanced by the treatment of cytoplasts with 1-oleyl-2-acetylglycerol, the Ca²⁺ ionophore ionomycin or latex beads. Furthermore, treatment of cytoplasts with the protein kinase C inhibitor trifluoperazine markedly inhibits the ³²P-labeling of proteins in the 40000 M_r range, including the 46 kDa polypeptide, and almost totally abolishes the activation of O^?₂ production by PMA.