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.

Phosphoinositide 3-kinases (PI3Ks) activate protein kinase PKB (also termed Akt), and PI3Kgamma activated by heterotrimeric guanosine triphosphate-binding protein can stimulate mitogen-activated protein kinase (MAPK). Exchange of a putative lipid substrate-binding site generated PI3Kgamma proteins with altered or aborted lipid but retained protein kinase activity. Transiently expressed, PI3Kgamma hybrids exhibited wortmannin-sensitive activation of MAPK, whereas a catalytically inactive PI3Kgamma did not. Membrane-targeted PI3Kgamma constitutively produced phosphatidylinositol 3,4, 3,4,5-trisphosphate and activated PKB but not MAPK. Moreover, stimulation of MAPK in response to lysophosphatidic acid was blocked by catalytically inactive PI3Kgamma but not by hybrid PI3Kgammas. Thus, two major signals emerge from PI3Kgamma: phosphoinositides that target PKB and protein phosphorylation that activates MAPK.

Signalling via seven transmembrane helix receptors can lead to a massive increase in cellular PtdIns(3,4,5)P3, which is critical for the induction of various cell responses and is likely to be produced by a trimeric G-protein-sensitive phosphoinositide 3-kinase (PI3Kgamma). We show here that PI3Kgamma is a bifunctional lipid kinase and protein kinase, and that both activities are inhibited by wortmannin at concentrations equal to those affecting the p85/p110alpha heterodimeric PI3K (IC50 approx. 2 nM). The binding of wortmannin to PI3Kgamma, as detected by anti-wortmannin antisera, closely followed the inhibition of the kinase activities. Truncation of more than the 98 N-terminal amino acid residues from PI3Kgamma produced proteins that were inactive in wortmannin binding and kinase assays. This suggests that regions apart from the core catalytic domain are important in catalysis and inhibitor interaction. The covalent reaction of wortmannin with PI3Kgamma was prevented by preincubation with phosphoinositides, ATP and its analogues adenine and 5'-(4-fluorosulphonylbenzoyl)adenine. Proteolytic analysis of wortmannin-prelabelled PI3Kgamma revealed candidate wortmannin-binding peptides around Lys-799. Replacement of Lys-799 by Arg through site-directed mutagenesis aborted the covalent reaction with wortmannin and the lipid kinase and protein kinase activities completely. The above illustrates that Lys-799 is crucial to the phosphate transfer reaction and wortmannin reactivity. Parallel inhibition of the PI3Kgamma-associated protein kinase and lipid kinase by wortmannin and by the Lys-799-->Arg mutation reveals that both activities are inherent in the PI3Kgamma polypeptide.

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