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 (PI3Kγ). We show here that PI3Kγ is a bifunctional lipid kinase and protein kinase, and that both activities are inhibited by wortmannin at concentrations equal to those affecting the p85/p110α heterodimeric PI3K (IC50 approx. 2 nM). The binding of wortmannin to PI3Kγ, 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 PI3Kγ 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 PI3Kγ was prevented by preincubation with phosphoinositides, ATP and its analogues adenine and 5′-(4-fluorosulphonylbenzoyl)adenine. Proteolytic analysis of wortmannin-prelabelled PI3Kγ 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 PI3Kγ-associated protein kinase and lipid kinase by wortmannin and by the Lys-799 → Arg mutation reveals that both activities are inherent in the PI3Kγ polypeptide.

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