Wortmannin phosphoinositide 3-kinase interaction site

摘要

The site of interaction of wortmannin on the catalytic subunit of PI3-kinase, p110 alpha is identified. At physiological pH (6.5-8) wortmannin reacted specifically with p110 alpha . PtdIns (4.5)P2, ATP and ATP-analogs (adenine and 5'(4-fluorosulfonylbenzoyl)-adenine, FSBA) completed effectively with wortmannin, while substances containing nucleophilic amino acid side chain functions had no effect at the same concentrations. Proteolytic fragments of wortmannin-treated, recombinant p110 alpha are mapped using anti-wortmannin and anti-p110 alpha peptide antibodies, thus limiting the target site within a 10 kD fragment, co-localizing with the ATP binding site. Site-directed mutagenesis of all candidate residues within this region shows that only the conservative Lys802 to Arg mutation abolished wortmannin binding. Inhibition of PI3-kinase occurs therefore by the formation of an enamine following the attack of Lys802 on the furan ring (at C20) of wortmannin. The Lys802Arg mutant was also unable to bind FSBA, and was catalytically inactive in lipid and protein kinase assays, indicating a crucial role for Lys802 in the phosphotransfer reaction. In contrast, an Arg916Pro mutation abolished the catalytic activity, while covalent wortmannin binding remained intact.