CONTINUOUS PHOSPHORYLATION OF BOTH THE 47 AND THE 49 KDA PROTEINS OCCURS DURING SUPEROXIDE PRODUCTION BY NEUTROPHILS

Neutrophils stimulated with 4β-phorbol 12-myristate 13-acetate release large quantities of superoxide (O2-) and exhibit an intense phosphorylation of two proteins with molecular masses of approx. 47 and 49 kDa. Treatment of unstimulated cells with antagonists of protein kinase C (e.g., staurosporine; 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7)) is known to inhibit both of these phenomena upon stimulation. These antagonists of PKC also cause a rapid cessation of O2- release when added to cells that are already stimulated. In this paper, we report that the addition of staurosporine or H-7 to stimulated neutrophils resulted in a rapid loss of 32P from both the 47 and the 49 kDa phosphoprotein bands, as detected by autoradiography. This suggests that these two proteins may be regulated by a continual cycle of phosphorylation and dephosphorylation in the stimulated cell, with the phosphorylation reactions predominating, or undergo a rapid degradation subsequent to phosphorylation. Either explanation is consistent with the view that protein kinase C activity is necessary to both innitiate and maintain O2- production in neutrophils stimulated with tumor promoters. © 1990.