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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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Dimerization of p110γ and p101. (A) HEK cells were cotransfected with plasmids encoding NH2- or COOH-terminally YFP-tagged p110γ and NH2- or COOH-terminally CFP-tagged p101 in different combinations. FRET was measured in vivo. An increase in CFP (donor) fluorescence during YFP (acceptor) bleach indicates FRET between fluorescent PI3Kγ subunits. The depicted data represent means ± SEM of at least 18 single cells in three independent transfection experiments. (B) HEK cells were transfected with the indicated plasmids. Cytosols were prepared and subjected to gel filtration. The elution profiles were analyzed by immunoblotting with an anti-GFP antibody (L, load diluted 1:5; V, void volume; 20–26, fraction numbers).
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fig3: Dimerization of p110γ and p101. (A) HEK cells were cotransfected with plasmids encoding NH2- or COOH-terminally YFP-tagged p110γ and NH2- or COOH-terminally CFP-tagged p101 in different combinations. FRET was measured in vivo. An increase in CFP (donor) fluorescence during YFP (acceptor) bleach indicates FRET between fluorescent PI3Kγ subunits. The depicted data represent means ± SEM of at least 18 single cells in three independent transfection experiments. (B) HEK cells were transfected with the indicated plasmids. Cytosols were prepared and subjected to gel filtration. The elution profiles were analyzed by immunoblotting with an anti-GFP antibody (L, load diluted 1:5; V, void volume; 20–26, fraction numbers).

Mentions: To directly demonstrate heterodimerization of PI3Kγ subunits in living cells, we used fluorescence resonance energy transfer (FRET). FRET between two fluorophores, e.g., CFP and YFP, is restricted to distances of <100 Å, and therefore, provides direct evidence for a protein–protein interaction (Teruel and Meyer, 2000). We coexpressed CFP- and YFP-tagged PI3Kγ subunits and determined FRET by following the donor (CFP) recovery during acceptor (YFP) bleach (Fig. 3 A). All combinations showed significant FRET, although quantitative differences were evident.


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)

Dimerization of p110γ and p101. (A) HEK cells were cotransfected with plasmids encoding NH2- or COOH-terminally YFP-tagged p110γ and NH2- or COOH-terminally CFP-tagged p101 in different combinations. FRET was measured in vivo. An increase in CFP (donor) fluorescence during YFP (acceptor) bleach indicates FRET between fluorescent PI3Kγ subunits. The depicted data represent means ± SEM of at least 18 single cells in three independent transfection experiments. (B) HEK cells were transfected with the indicated plasmids. Cytosols were prepared and subjected to gel filtration. The elution profiles were analyzed by immunoblotting with an anti-GFP antibody (L, load diluted 1:5; V, void volume; 20–26, fraction numbers).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172741&req=5

fig3: Dimerization of p110γ and p101. (A) HEK cells were cotransfected with plasmids encoding NH2- or COOH-terminally YFP-tagged p110γ and NH2- or COOH-terminally CFP-tagged p101 in different combinations. FRET was measured in vivo. An increase in CFP (donor) fluorescence during YFP (acceptor) bleach indicates FRET between fluorescent PI3Kγ subunits. The depicted data represent means ± SEM of at least 18 single cells in three independent transfection experiments. (B) HEK cells were transfected with the indicated plasmids. Cytosols were prepared and subjected to gel filtration. The elution profiles were analyzed by immunoblotting with an anti-GFP antibody (L, load diluted 1:5; V, void volume; 20–26, fraction numbers).
Mentions: To directly demonstrate heterodimerization of PI3Kγ subunits in living cells, we used fluorescence resonance energy transfer (FRET). FRET between two fluorophores, e.g., CFP and YFP, is restricted to distances of <100 Å, and therefore, provides direct evidence for a protein–protein interaction (Teruel and Meyer, 2000). We coexpressed CFP- and YFP-tagged PI3Kγ subunits and determined FRET by following the donor (CFP) recovery during acceptor (YFP) bleach (Fig. 3 A). All combinations showed significant FRET, although quantitative differences were evident.

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