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Published online 30 December 2002. doi:10.1083/jcb.200210115
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© The Rockefeller University Press, 0021-9525/2003/1/89 $5.00
The Journal of Cell Biology, Volume 160, Number 1, 89-99

Article

Roles of Gß{gamma} in membrane recruitment and activation of p110{gamma}/p101 phosphoinositide 3-kinase {gamma}



Carsten Brock1,2,4, Michael Schaefer2, H. Peter Reusch3, Cornelia Czupalla1,2, Manuela Michalke2, Karsten Spicher2, Günter Schultz2 and Bernd Nürnberg1,2

1 Institut für Physiologische Chemie II, Klinikum der Heinrich-Heine-Universität, 40225 Düsseldorf, Germany
2 Institut für Pharmakologie, Chemie, Pharmazie, Freie Universität Berlin, 14195 Berlin, Germany
3 Institut für Klinische Pharmakologie und Toxikologie, Chemie, Pharmazie, Freie Universität Berlin, 14195 Berlin, Germany
4 Fachbereich Biologie, Chemie, Pharmazie, Freie Universität Berlin, 14195 Berlin, Germany

Address correspondence to Bernd Nürnberg, Institut für Physiologische Chemie II, Klinikum der Heinrich-Heine-Universität, Universitätsstr. 1, Gebäude 22.03, 40 225 Düsseldorf. Tel.: 49-211-811-2724. Fax: 49-211-811-2726. E-mail: bernd.nuernberg{at}uni-duesseldorf.de

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 Gß{gamma} on stimulation of G protein–coupled receptors. It is currently unknown whether in living cells Gß{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 Gß{gamma}. Using GFP-tagged PI3K{gamma} subunits expressed in HEK cells, we show that Gß{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 Gß{gamma}, even in the absence of p101. Therefore, we conclude that in vivo, Gß{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 Gß{gamma} with p110{gamma} contributes to activation of PI3K{gamma}.

Key Words: G protein; fluorescence imaging; phosphoinositide 3-kinase; FRET; PH domain


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