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

Brock C, Schaefer M, Reusch HP, Czupalla C, Michalke M, Spicher K, Schultz G, Nürnberg B - J. Cell Biol. (2002)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institut für Physiologische Chemie II, Klinikum der Heinrich-Heine-Universität, 40225 Düsseldorf, Germany.

ABSTRACT
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.

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Construction and characterization of fluorescent PI3Kγ fusion proteins. (A) Schematic representation of the wild-type, fluorescent, and membrane-targeted PI3Kγ subunits. A YFP (or CFP) tag was fused to either the NH2- or the COOH terminus of p110γ and p101. The p110γ-CAAX fusion protein contains an isoprenylation motif (bars for lipid modifications). (B) Differently fluorescence-tagged PI3Kγ subunits were coexpressed in HEK cells, and cell lysates were subjected to SDS-PAGE followed by immunoblotting with a GFP-specific antibody. (C) Catalytic activity of fluorescent PI3Kγ fusion proteins. HEK cells were transfected with the indicated plasmids. Lysates were subjected to immunoprecipitation (IP) with (+) or without (−) an anti-p110γ antibody. immunoprecipitation was controlled by immunoblotting (IB) with another anti-p110γ antibody. Shown are chemiluminescence images (top panels). Immunoprecipitates were assayed for in vitro PI3K activity in the absence or presence of 120 nM Gβγ using PtdIns-4,5-P2– containing lipid vesicles and γ[32P]ATP as substrates. Depicted are autoradiographs of the generated 32P-PtdIns-3,4,5-P3 (bottom panels).
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fig1: Construction and characterization of fluorescent PI3Kγ fusion proteins. (A) Schematic representation of the wild-type, fluorescent, and membrane-targeted PI3Kγ subunits. A YFP (or CFP) tag was fused to either the NH2- or the COOH terminus of p110γ and p101. The p110γ-CAAX fusion protein contains an isoprenylation motif (bars for lipid modifications). (B) Differently fluorescence-tagged PI3Kγ subunits were coexpressed in HEK cells, and cell lysates were subjected to SDS-PAGE followed by immunoblotting with a GFP-specific antibody. (C) Catalytic activity of fluorescent PI3Kγ fusion proteins. HEK cells were transfected with the indicated plasmids. Lysates were subjected to immunoprecipitation (IP) with (+) or without (−) an anti-p110γ antibody. immunoprecipitation was controlled by immunoblotting (IB) with another anti-p110γ antibody. Shown are chemiluminescence images (top panels). Immunoprecipitates were assayed for in vitro PI3K activity in the absence or presence of 120 nM Gβγ using PtdIns-4,5-P2– containing lipid vesicles and γ[32P]ATP as substrates. Depicted are autoradiographs of the generated 32P-PtdIns-3,4,5-P3 (bottom panels).

Mentions: To visualize PI3Kγ subunits in vivo, we generated constructs encoding p110γ and p101 fused to YFP or CFP (Fig. 1 A). Their expression in human embryonic kidney (HEK) 293 cells was verified by immunoblot analysis using an antibody against GFP (Fig. 1 B). The catalytic activity and Gβγ sensitivity of PI3Kγ fusion proteins was confirmed by in vitro lipid kinase assays after immunoprecipitation with an mAb recognizing only intact p110γ (Fig. 1 C). Precipitates from vector-transfected control cells neither exhibited p110γ immunoreactivity nor lipid kinase activity. These results demonstrate that YFP-fused p110γ or p101 retain essential functions of the wild-type PI3Kγ.


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

Brock C, Schaefer M, Reusch HP, Czupalla C, Michalke M, Spicher K, Schultz G, Nürnberg B - J. Cell Biol. (2002)

Construction and characterization of fluorescent PI3Kγ fusion proteins. (A) Schematic representation of the wild-type, fluorescent, and membrane-targeted PI3Kγ subunits. A YFP (or CFP) tag was fused to either the NH2- or the COOH terminus of p110γ and p101. The p110γ-CAAX fusion protein contains an isoprenylation motif (bars for lipid modifications). (B) Differently fluorescence-tagged PI3Kγ subunits were coexpressed in HEK cells, and cell lysates were subjected to SDS-PAGE followed by immunoblotting with a GFP-specific antibody. (C) Catalytic activity of fluorescent PI3Kγ fusion proteins. HEK cells were transfected with the indicated plasmids. Lysates were subjected to immunoprecipitation (IP) with (+) or without (−) an anti-p110γ antibody. immunoprecipitation was controlled by immunoblotting (IB) with another anti-p110γ antibody. Shown are chemiluminescence images (top panels). Immunoprecipitates were assayed for in vitro PI3K activity in the absence or presence of 120 nM Gβγ using PtdIns-4,5-P2– containing lipid vesicles and γ[32P]ATP as substrates. Depicted are autoradiographs of the generated 32P-PtdIns-3,4,5-P3 (bottom panels).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172741&req=5

fig1: Construction and characterization of fluorescent PI3Kγ fusion proteins. (A) Schematic representation of the wild-type, fluorescent, and membrane-targeted PI3Kγ subunits. A YFP (or CFP) tag was fused to either the NH2- or the COOH terminus of p110γ and p101. The p110γ-CAAX fusion protein contains an isoprenylation motif (bars for lipid modifications). (B) Differently fluorescence-tagged PI3Kγ subunits were coexpressed in HEK cells, and cell lysates were subjected to SDS-PAGE followed by immunoblotting with a GFP-specific antibody. (C) Catalytic activity of fluorescent PI3Kγ fusion proteins. HEK cells were transfected with the indicated plasmids. Lysates were subjected to immunoprecipitation (IP) with (+) or without (−) an anti-p110γ antibody. immunoprecipitation was controlled by immunoblotting (IB) with another anti-p110γ antibody. Shown are chemiluminescence images (top panels). Immunoprecipitates were assayed for in vitro PI3K activity in the absence or presence of 120 nM Gβγ using PtdIns-4,5-P2– containing lipid vesicles and γ[32P]ATP as substrates. Depicted are autoradiographs of the generated 32P-PtdIns-3,4,5-P3 (bottom panels).
Mentions: To visualize PI3Kγ subunits in vivo, we generated constructs encoding p110γ and p101 fused to YFP or CFP (Fig. 1 A). Their expression in human embryonic kidney (HEK) 293 cells was verified by immunoblot analysis using an antibody against GFP (Fig. 1 B). The catalytic activity and Gβγ sensitivity of PI3Kγ fusion proteins was confirmed by in vitro lipid kinase assays after immunoprecipitation with an mAb recognizing only intact p110γ (Fig. 1 C). Precipitates from vector-transfected control cells neither exhibited p110γ immunoreactivity nor lipid kinase activity. These results demonstrate that YFP-fused p110γ or p101 retain essential functions of the wild-type PI3Kγ.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institut für Physiologische Chemie II, Klinikum der Heinrich-Heine-Universität, 40225 Düsseldorf, Germany.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus