Wortmannin Inactivates Phosphoinositide 3-Kinase by Covalent Modification of Lys-802, a Residue Involved in the Phosphate Transfer Reaction

Abstract

Wortmannin at nanomolar concentrations is a potent and specific inhibitor of phosphoinositide (PI) 3-kinase and has been used extensively to demonstrate the role of this enzyme in diverse signal transduction processes. At higher concentrations, wortmannin inhibits the ataxia telangiectasia gene (ATM)-related DNAdependent protein kinase (DNA-PK cs ). We report here the identification of the site of interaction of wortmannin on the catalytic subunit of PI 3-kinase, p110␣. At physiological pH (6.5 to 8) wortmannin reacted specifically with p110␣. Phosphatidylinositol-4,5-diphosphate, ATP, and ATP analogs [adenine and 5-(4-fluorosulfonylbenzoyl)adenine] competed effectively with wortmannin, 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. Proteolytic fragments of wortmannin-treated, recombinant p110␣ were mapped with anti-wortmannin and anti-p110␣ peptide antibodies, thus limiting the target site within a 10-kDa fragment, colocalizing with the ATP-binding site. Site-directed mutagenesis of all candidate residues within this region showed that only the conservative Lys-802-to-Arg mutation abolished wortmannin binding. Inhibition of PI 3-kinase occurs, 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-802 in the phosphotransfer reaction. In contrast, an Arg-916-to-Pro mutation abolished the catalytic activity whereas covalent wortmannin binding remained intact. Our results provide the basis for the design of novel and specific inhibitors of an enzyme family, including PI kinases and ATM-related genes, that play a central role in many physiological processes.

CSF-1 stimulation induces the formation of a multiprotein complex including CSF-1 receptor, c-Cbl, PI 3-kinase, Crk-II and Grb2

“…Exposure of c-FmsCHO cells to CSF-1 induced an increase in a PI kinase activity associated to c-Cb1 immunopellets. This activity likely corresponded to a PI 3-kinase activity as evidenced by the inhibitory e ect of wortmannin (a speci®c PI 3-kinase inhibitor) when added to the PI-kinase assay (Wymann et al, 1996). This observation is in agreement with previous reports (Kim et al, 1995;.…”

“…Both PI 3-and PI 4-kinase activities have been reported to interact with tyrosine phosphorylated substrates (Prasad et al, 1993). To precise the nature of the PI-kinase associated to c-Cbl, the kinase assay was performed in presence of wortmannin, a PI 3-kinase speci®c covalent inhibitor (Wymann et al, 1996). Under these conditions, we observed a 90% decrease in the PI-kinase activity associated with c-Cbl, indicating that c-Cbl was essentially associated to PI 3-kinase.…”

A new approach to the molecular analysis of docking, priming, and regulated membrane fusion

“…It was shown that synapsin1-associated PI-3K regulates the size of the ready-releasable pool of synaptic vesicles [32]. Considering the essential role of PI-3K in ATP-dependent priming [59] and that PI-3K inhibitors can compete with PIP for the kinase ATP-binding site [81,95], inhibition of fusion extent seen here (Figs. 8a, b and 9a, b) is likely caused by CV depriming in the fully docked state.…”

Rapamycin and mTOR kinase inhibitors

“…First, wortmannin binds with high affinity to the ATP-binding site to block substrate binding. Second, the ε-amino group of a lysine in the active site forms a covalent bond with carbon-20 of the wortmannin furan ring to irreversibly inhibit the enzyme [58,59]. The active site lysine (Lys802 in p110α and Lys833 in p110γ) is critical for catalysis and is highly conserved in lipid and protein kinases.…”