INVOLVEMENT OF PROTEIN-KINASE-C AND OF PROTEIN PHOSPHATASE-1 AND OR PHOSPHATASE-2A IN P47 PHOX PHOSPHORYLATION IN FORMYLMET-LEU-PHE STIMULATED NEUTROPHILS - STUDIES WITH SELECTIVE INHIBITORS RO-31-8220 AND CALYCULIN-A/

Previously employed non-selective protein kinase inhibitors yielded in conclusive results regarding involvement of protein kinase C (PKC) in phosphorylation of 47 kDa protein (p47 phox) in intact neutrophils sti mulated with physiologic agonists of superoxide generation. In the pre sent study, phosphorylation of p47 phox in formylMet-Leu-Phe (fMLP) st imulated neutrophils was potently inhibited in the presence of 0.3 mu M RO 31-8220, a selective inhibitor of PKC. These results provide expe rimental evidence in support of the currently considered essential inv olvement of PKC in p47 phox phosphorylation in response to physiologic stimulation of neutrophil surface receptors. The fMLP-induced phospho rylation of p47 phox was enhanced and prolonged by calyculin A, a spec ific inhibitor of protein phosphatases of types 1 and 2A, and such enh anced phosphorylation was also effectively inhibited by RO 31-8220. Ou r results suggest that the extent and duration of p47 phox phosphoryla tion in intact fMLP-stimulated neutrophils is probably controlled by a balance between the activities of PKC, on the one hand, and of protei n phosphatase(s) of type(s) 1 and/or 2A, on the other. Effects of RO 3 1-8220 and of calyculin A on the fMLP-induced p47 phox phosphorylation were paralleled by similar effects on superoxide release. Calyculin A and RO 31-8220 were also used to study signal transduction by a post- receptor agonist of superoxide generation, a calcium ionophore A23187. The results of the latter study indicated that PKC was activated in A 23187-stimulated neutrophils and was essentially involved in superoxid e generation and p47 phox phosphorylation. Further, these results sugg ested that protein phosphatase(s) of type(s) 1 and/or 2A were also act ivated in A23187-signalling pathway, and limited the extent of superox ide release and p47 phox phosphorylation.