Antagonists of Phosphatidylinositol 3-Kinase Block Activation of Several Novel Protein Kinases in Neutrophils

• Published in: The Journal of biological chemistry 1995-05, Vol.270 (19), p.11684-11691
• Main Authors: Ding, Jiabing, Vlahos, Chris J., Liu, Ruichun, Brown, Raymond F., Badweyt, John A.
• Format: Article
• Language: English
• Subjects: Androstadienes - pharmacology; Animals; Chromones - pharmacology; Enzyme Activation - drug effects; Enzyme Inhibitors - pharmacology; Guinea Pigs; In Vitro Techniques; Kinetics; Morpholines - pharmacology; N-Formylmethionine Leucyl-Phenylalanine - pharmacology; NADH, NADPH Oxidoreductases - metabolism; NADPH Oxidases; Neutrophils - drug effects; Neutrophils - enzymology; Neutrophils - physiology; Phosphatidylinositol 3-Kinases; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor) - antagonists & inhibitors; Piperazines - pharmacology; Protamine Kinase - metabolism; Protein Denaturation; Protein Kinase Inhibitors; Protein Kinases - metabolism; Superoxides - metabolism; Tetradecanoylphorbol Acetate - pharmacology; Wortmannin
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.270.19.11684

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 PI3-K by distinct mechanisms, blocked activation of all of these novel kinases. These antagonists also inhibited the phosphorylation of p47-phox (about 50%) and O2⨪ release (about 80%) in cells stimulated with fMLP, but not with 4β3-phorbol 12-myristate 13-acetate. A strong correlation exists between the amounts of these antagonists required to produce 50% inhibition of PI3-K in vitro and O2⨪ 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 OT2 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 O2⨪ release.