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Phosphoinositide-3 kinase activity is implicated in diverse cellular responses triggered by mammalian cell surface receptors and in the regulation of protein sorting in yeast. Receptors with intrinsic and associated tyrosine kinase activity recruit heterodimeric phosphoinositide-3 kinases that consist of p110 catalytic subunits and p85 adaptor molecules containing Src homology 2 (SH2) domains. A phosphoinositide-3 kinase isotype, p110γ, was cloned and characterized. The p110γ enzyme was activated in vitro by both the α and βγ subunits of heterotrimeric guanosine triphosphate (GTP)-binding proteins (G proteins) and did not interact with p85. A potential pleckstrin homology domain is located near its amino terminus. The p110γ isotype may link signaling through G protein-coupled receptors to the generation of phosphoinositide second messengers phosphorylated in the D-3 position.

Infections with influenza A viruses result in the activation of a variety of intracellular signalling pathways. Recent findings suggest that in response to double-stranded RNA (dsRNA), which is commonly used as a mimic for accumulating viral RNA, the phosphatidylinositol-3-kinase (PI3K) is activated and mediates activation of the transcription factor interferon regulatory factor 3 (IRF-3). Thus, we investigated the function of PI3K during influenza virus infection. The pathway was activated upon infection and consistent with earlier findings using dsRNA, inhibition of PI3K itself or block of signalling by the PI3K product, the second messenger phosphatidylinositol-3,4,5-trisphosphate (PIP3), results in misphosphorylation and impaired dimerization of IRF-3 as well as reduced IRF-3-dependent promoter activity. This would imply an antiviral function of the kinase in influenza virus-infected cells. However, upon inhibition of PI3K, titers of progeny virus were reduced rather than enhanced. This was coincident with a strong decrease of viral protein accumulation that was not due to a block of protein synthesis or inhibition of the viral polymerase complex. Immunofluorescence studies revealed that PI3K rather appears to regulate a very early step during viral entry. Thus PI3K is a perfect example of a seemingly antiviral signalling component that is misused by the virus to support effective replication.

Receptor-regulated class I phosphoinositide 3-kinases (PI3K) phosphorylate the membrane lipid phosphatidylinositol (PtdIns)-4,5-P₂ to PtdIns-3,4,5-P₃. This, in turn, recruits and activates cytosolic effectors with PtdIns-3,4,5-P₃–binding pleckstrin homology (PH) domains, thereby controlling important cellular functions such as proliferation, survival, or chemotaxis. The class I_B p110γ/p101 PI3Kγ is activated by Gβγ on stimulation of G protein–coupled receptors. It is currently unknown whether in living cells Gβγ acts as a membrane anchor or an allosteric activator of PI3Kγ, and which role its noncatalytic p101 subunit plays in its activation by Gβγ. Using GFP-tagged PI3Kγ subunits expressed in HEK cells, we show that Gβγ recruits the enzyme from the cytosol to the membrane by interaction with its p101 subunit. Accordingly, p101 was found to be required for G protein–mediated activation of PI3Kγ in living cells, as assessed by use of GFP-tagged PtdIns-3,4,5-P₃–binding PH domains. Furthermore, membrane-targeted p110γ displayed basal enzymatic activity, but was further stimulated by Gβγ, even in the absence of p101. Therefore, we conclude that in vivo, Gβγ activates PI3Kγ by a mechanism assigning specific roles for both PI3Kγ subunits, i.e., membrane recruitment is mediated via the noncatalytic p101 subunit, and direct stimulation of Gβγ with p110γ contributes to activation of PI3Kγ.

By using purified preparations we show that nanomolar concentrations of Gβγ significantly stimulated lipid kinase activity of phosphatidylinositol 3-kinase (PI3K) β and PI3Kγ in the presence as well as in the absence of non-catalytic subunits such as p85α or p101. Concomitantly, Gβγ stimulated autophosphorylation of the catalytic subunit of PI3Kγ (EC₅₀, 30 nm; stoichiometry ≥0.6 mol of P_i/mol of p110γ), which also occurred in the absence of p101. Surprisingly, we found that p101 affected the lipid substrate preference of PI3Kγ in its Gβγ-stimulated state. With phosphatidylinositol as substrate, p110γ but not p101/p110γ was significantly stimulated by Gβγ to form PI-3-phosphate (EC₅₀, 20 nm). The opposite situation was found when PI-4,5-bisphosphate served as substrate. Gβγ efficiently and potently (EC₅₀, 5 nm) activated the p101/p110γ heterodimer but negligibly stimulated the p110γ monomer to form PI-3,4,5-trisphosphate. However, this weak stimulatory effect on p110γ was overcome by excess concentrations of Gβγ (EC₅₀, 100 nm). This finding is in accordance with the in vivo situation, where activated PI3K catalyzes the formation of PI-3,4,5-trisphosphate but not PI-3-phosphate. We conclude that p101 is responsible for PI-4,5-bisphosphate substrate selectivity of PI3Kγ by sensitizing p110γ toward Gβγ in the presence of PI-4,5-P₂.