Roles of G beta gamma in membrane recruitment and activation of p110 gamma/p101 phosphoinositide 3-kinase gamma.
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.
Brock
Carsten
C
Institut für Physiologische Chemie II, Klinikum der Heinrich-Heine-Universität, 40225 Düsseldorf, Germany.
Schaefer
Michael
M
Reusch
H Peter
HP
Czupalla
Cornelia
C
Michalke
Manuela
M
Spicher
Karsten
K
Schultz
Günter
G
Nürnberg
Bernd
B
D000495:Allosteric Site; D001426:Bacterial Proteins / Q000378:metabolism; D020134:Catalytic Domain; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002850:Chromatography, Gel; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004735:Energy Transfer; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D049452:Green Fluorescent Proteins; D020962:Heterotrimeric GTP-Binding Proteins / Q000502:physiology*; D006801:Humans; D015151:Immunoblotting; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins; D018613:Microscopy, Confocal; D008954:Models, Biological; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014162:Transfection
D001426:Bacterial Proteins; C118981:G-protein Beta gamma; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D008164:Luminescent Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C067817:P101 protein, Mycoplasma hyorhinis; D049452:Green Fluorescent Proteins; D019869:Phosphatidylinositol 3-Kinases; D020962:Heterotrimeric GTP-Binding Proteins