ANTAGONISTS OF PHOSPHATIDYLINOSITOL 3-KINASE BLOCK ACTIVATION OF SEVERAL NOVEL PROTEIN-KINASES IN NEUTROPHILS

Several novel protein kinases are known to be rapidly activated in neutrophils stimulated with the chemoattractant fMet-Leu-Phe (fMLP), These kinases include a histone H4 protein kinase and several renaturable kinases with molecular masses of about 69, 63, 49, and 40 kDa, The renaturable kinases can catalyze the phosphorylation of a peptide that corresponds to residues 297-331 of the 47-kDa subunit of the NADPH-oxidase system (p47-phox). Previous studies have indicated that the activation of all of these protein kinases involves an uncharacterized stimulatory pathway and/or novel second messenger, The studies reported herein were undertaken to determine if phosphatidylinositol 3-kinase (PI3-K) is a component of this pathway, We report that certain chromone derivatives (e.g. 2-(4-morpholinyl)-8-phenylchromone (LY294002)) and wortmannin, which inhibit PIS-K by distinct mechanisms, blocked activation of all of these novel kinases, These antagonists also inhibited the phosphorylation of p47-phox (about 50%) and O-2(radical anion) release (about 80%) in cells stimulated with fMLP, but not with 4 beta-phorbol 12-myristate 13-acetate. A strong correlation exists between the amounts of these antagonists required to produce 50% inhibition of PB-K in vitro and O-2(radical anion) release in vivo. In contrast, a single atom substitution of LY294002 produced a compound (LY303511) that did not inhibit PI3-K, Compound LY303511 did not appreciably inhibit the activation of the novel protein kinases or O-2(radical anion) generation, These data strongly suggest that PI3-K is involved in the activation of several novel protein kinases in neutrophils, one or more of which may be involved in O-2(radical anion) release.