Roles of G beta gamma in membrane recruitment and activation of p110 gamma/p101 phosphoinositide 3-kinase gamma

• Published in: The Journal of cell biology Vol. 160; no. 1; pp. 89 - 99
• Main Authors: Brock, Carsten, Schaefer, Michael, Reusch, H Peter, Czupalla, Cornelia, Michalke, Manuela, Spicher, Karsten, Schultz, Günter, Nürnberg, Bernd
• Format: Journal Article
• Language: English
• Published: United States 06.01.2003
• Subjects: Allosteric Site; Bacterial Proteins - metabolism; Catalytic Domain; Cell Line; Cell Membrane - metabolism; Chromatography, Gel; Dimerization; Dose-Response Relationship, Drug; Energy Transfer; Green Fluorescent Proteins; GTP-Binding Protein beta Subunits; GTP-Binding Protein gamma Subunits; Heterotrimeric GTP-Binding Proteins - physiology; Humans; Immunoblotting; Luminescent Proteins - metabolism; Membrane Proteins; Microscopy, Confocal; Models, Biological; Phosphatidylinositol 3-Kinases - metabolism; Phosphorylation; Plasmids - metabolism; Protein Binding; Protein Structure, Tertiary; Recombinant Fusion Proteins - metabolism; Spectrometry, Fluorescence; Transfection
• ISSN: 0021-9525
• DOI: 10.1083/jcb.200210115

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 IB p110 gamma/p101 PI3K gamma is activated by G beta gamma on stimulation of G protein-coupled receptors. It is currently unknown whether in living cells G beta gamma acts as a membrane anchor or an allosteric activator of PI3K gamma, and which role its noncatalytic p101 subunit plays in its activation by G beta gamma. Using GFP-tagged PI3K gamma subunits expressed in HEK cells, we show that G beta gamma 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 PI3K gamma in living cells, as assessed by use of GFP-tagged PtdIns-3,4,5-P3-binding PH domains. Furthermore, membrane-targeted p110 gamma displayed basal enzymatic activity, but was further stimulated by G beta gamma, even in the absence of p101. Therefore, we conclude that in vivo, G beta gamma activates PI3K gamma by a mechanism assigning specific roles for both PI3K gamma subunits, i.e., membrane recruitment is mediated via the noncatalytic p101 subunit, and direct stimulation of G beta gamma with p110 gamma contributes to activation of PI3K gamma.