Effects of antagonists of protein phosphatases on superoxide release by neutrophils

Neutrophils stimulated with 4 beta-phorbol 12-myristate 13-acetate (PMA) release large quantities of superoxide (O2-) and exhibit phosphorylation of two proteins with molecular masses of 47(p47) and 49 kDa (p49). Addition of inhibitors of protein kinases (e.g. 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7)) to these cells after stimulation with PMA results in the loss of 32P from these proteins and a rapid cessation of O2- release (e.g. Heyworth, P. G., and Badwey, J. A. (1990) Biochim. Biophys. Acta 1052, 299-305). In this paper we report that antagonists of type 1 and 2A protein phosphatases (okadaic acid, calyculin A) prevented both the loss of 32P from p47 and the termination of O2- release in stimulated neutrophils treated with H-7. Calyculin A also caused a remarkable hyperphosphorylation of a number of proteins in neutrophils and increased O2- release from these cells in response to a suboptimal amount of PMA. Enzymes present in both the soluble and particulate fractions of neutrophils catalyzed the near complete dephosphorylation of 32P-labeled p47 and p49 bound to Immobilon-P membranes. Dephosphorylation of these blotted phosphoproteins occurred at physiological rates and was inhibited by okadaic acid and calyculin A. These data strongly suggest that p47 undergoes a continual cycle of phosphorylation and dephosphorylation throughout the period of O2- release when PMA is the stimulus. Moreover, we show that antagonists of type 1 and 2A protein phosphatases block dephosphorylation of p47 both in vivo and in vitro, indicating that these enzymes may modulate O2- release under certain circumstances.