WORTMANNIN INACTIVATES PHOSPHOINOSITIDE 3-KINASE BY COVALENT MODIFICATION OF LYS-802, A RESIDUE INVOLVED IN THE PHOSPHATE TRANSFER-REACTION

Wortmannin at nanomolar concentrations is a potent and specific inhibi tor of phosphoinositide (PI) 3-kinase and has been used extensively to demonstrate the role of this enzyme in diverse signal transduction pr ocesses, At higher concentrations, wortmannin inhibits the ataxia tela ngiectasia gene (ATM))-related DNA-dependent protein kinase (DNA-PKcs) . We report here the identification of the site of interaction of wort mannin on the catalytic subunit of PI 3-kinase, p110 alpha. At physiol ogical pH (6.5 to 8) wortmannin reacted specifically with p110 alpha. Phosphatidylinositol-4,5-diphosphate, ATP, and ATP analogs [adenine an d 5'-(4-fluorosulfonylbenzoyl)adenine] competed effectively with wortm annin, while substances containing nucleophilic amino acid side chain functions had no effect at the same concentrations, This suggests that the wortmannin target site is localized in proximity to the substrate -binding site and that residues involved in wortmannin binding have an increased nucleophilicity because of their protein environment, Prote olytic fragments of wortmannin-treated, recombinant p110 alpha were ma pped with anti-wortmannin and anti-p110 alpha peptide antibodies, thus limiting the target site within a 10-kDa fragment, colocalizing with the ATP-binding site. Site-directed mutagenesis of all candidate resid ues within this region showed that only the conservative Lys-802-to-Ar g mutation abolished wortmannin binding, Inhibition of PI 3-kinase occ urs, therefore, by the formation of an enamine following the attack of Lys-802 on the furan ring (at C-20) of wortmannin, The Lys-802-to-Arg mutant was also unable to bind FSBA and was catalytically inactive in lipid and protein kinase assays, indicating a crucial role for Lys-80 2 in the phosphotransfer reaction, In contrast, an Arg-916-to-Pro muta tion abolished the catalytic activity whereas covalent wortmannin bind ing remained intact, Our results provide the basis for the design of n ovel and specific inhibitors of an enzyme family, including PI kinases and ATM-related genes, that play a central role in many physiological processes.