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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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Membrane recruitment of PI3Kγ by Gβγ. The effect of coexpression of Gβγ on the subcellular distribution of p110γ, p101, and heterodimeric PI3Kγ in HEK cells was analyzed by confocal laser scanning microscopy. Images of typical cells are shown (white bars, 10 μm). (A) Coexpression of monomeric PI3Kγ subunits with Gβγ. (B) Controls; coexpression of YFP-p101 with Gβ1, Gγ2, Gβ1γ2, or Gβ1γ2 and the Gβγ- scavenging Gαi2. (C) Coexpression of heterodimeric PI3Kγ with Gβγ. Note the round shape of the cells. Controls; coexpression of Gβγ with YFP or with kinase-deficient YFP-p110γ-K833R and p101; effect of 100 nM wortmannin. (D) Subcellular localization of Gβγ. Cells were transfected with a plasmid encoding CFP-tagged Gβ1 alone or together with the Gγ2 plasmid. (E) Immunoblot analysis of membrane fractions. HEK cells were transfected with the plasmids encoding PI3Kγ, Gβγ, or both together. To avoid rounding and detachment of the cells, the kinase-deficient mutant YFP-p110γ-K833R was used. Membrane fractions were prepared and analyzed by immunoblotting with anti-p110γ and anti-Gβ antibodies.
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fig4: Membrane recruitment of PI3Kγ by Gβγ. The effect of coexpression of Gβγ on the subcellular distribution of p110γ, p101, and heterodimeric PI3Kγ in HEK cells was analyzed by confocal laser scanning microscopy. Images of typical cells are shown (white bars, 10 μm). (A) Coexpression of monomeric PI3Kγ subunits with Gβγ. (B) Controls; coexpression of YFP-p101 with Gβ1, Gγ2, Gβ1γ2, or Gβ1γ2 and the Gβγ- scavenging Gαi2. (C) Coexpression of heterodimeric PI3Kγ with Gβγ. Note the round shape of the cells. Controls; coexpression of Gβγ with YFP or with kinase-deficient YFP-p110γ-K833R and p101; effect of 100 nM wortmannin. (D) Subcellular localization of Gβγ. Cells were transfected with a plasmid encoding CFP-tagged Gβ1 alone or together with the Gγ2 plasmid. (E) Immunoblot analysis of membrane fractions. HEK cells were transfected with the plasmids encoding PI3Kγ, Gβγ, or both together. To avoid rounding and detachment of the cells, the kinase-deficient mutant YFP-p110γ-K833R was used. Membrane fractions were prepared and analyzed by immunoblotting with anti-p110γ and anti-Gβ antibodies.

Mentions: Current concepts of PI3K activation are based on the recruitment of the cytosolic lipid kinase to the plasma membrane (Klippel et al., 1996). In the case of PI3Kγ, the major stimulus is assumed to be Gβγ, which is membrane-bound and has been shown to directly bind to both kinase subunits under in vitro conditions (Stephens et al., 1997; Maier et al., 1999). Therefore, we asked whether overexpression of free Gβγ directs YFP-fused PI3Kγ subunits to the cell membrane in vivo. Surprisingly, p110γ was not membrane-localized in the presence of coexpressed Gβγ in HEK cells (Fig. 4 A, top). In contrast, Gβγ recruited p101 to the membrane, resulting in accumulation of NH2- or COOH-terminally YFP-tagged p101 at both plasma- and endomembranes (Fig. 4 A, bottom, and Fig. 4 B). This corresponds to the subcellular distribution of overexpressed Gβγ dimers (see below), suggesting that p101 interacted with membrane-bound Gβγ.


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)

Membrane recruitment of PI3Kγ by Gβγ. The effect of coexpression of Gβγ on the subcellular distribution of p110γ, p101, and heterodimeric PI3Kγ in HEK cells was analyzed by confocal laser scanning microscopy. Images of typical cells are shown (white bars, 10 μm). (A) Coexpression of monomeric PI3Kγ subunits with Gβγ. (B) Controls; coexpression of YFP-p101 with Gβ1, Gγ2, Gβ1γ2, or Gβ1γ2 and the Gβγ- scavenging Gαi2. (C) Coexpression of heterodimeric PI3Kγ with Gβγ. Note the round shape of the cells. Controls; coexpression of Gβγ with YFP or with kinase-deficient YFP-p110γ-K833R and p101; effect of 100 nM wortmannin. (D) Subcellular localization of Gβγ. Cells were transfected with a plasmid encoding CFP-tagged Gβ1 alone or together with the Gγ2 plasmid. (E) Immunoblot analysis of membrane fractions. HEK cells were transfected with the plasmids encoding PI3Kγ, Gβγ, or both together. To avoid rounding and detachment of the cells, the kinase-deficient mutant YFP-p110γ-K833R was used. Membrane fractions were prepared and analyzed by immunoblotting with anti-p110γ and anti-Gβ antibodies.
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fig4: Membrane recruitment of PI3Kγ by Gβγ. The effect of coexpression of Gβγ on the subcellular distribution of p110γ, p101, and heterodimeric PI3Kγ in HEK cells was analyzed by confocal laser scanning microscopy. Images of typical cells are shown (white bars, 10 μm). (A) Coexpression of monomeric PI3Kγ subunits with Gβγ. (B) Controls; coexpression of YFP-p101 with Gβ1, Gγ2, Gβ1γ2, or Gβ1γ2 and the Gβγ- scavenging Gαi2. (C) Coexpression of heterodimeric PI3Kγ with Gβγ. Note the round shape of the cells. Controls; coexpression of Gβγ with YFP or with kinase-deficient YFP-p110γ-K833R and p101; effect of 100 nM wortmannin. (D) Subcellular localization of Gβγ. Cells were transfected with a plasmid encoding CFP-tagged Gβ1 alone or together with the Gγ2 plasmid. (E) Immunoblot analysis of membrane fractions. HEK cells were transfected with the plasmids encoding PI3Kγ, Gβγ, or both together. To avoid rounding and detachment of the cells, the kinase-deficient mutant YFP-p110γ-K833R was used. Membrane fractions were prepared and analyzed by immunoblotting with anti-p110γ and anti-Gβ antibodies.
Mentions: Current concepts of PI3K activation are based on the recruitment of the cytosolic lipid kinase to the plasma membrane (Klippel et al., 1996). In the case of PI3Kγ, the major stimulus is assumed to be Gβγ, which is membrane-bound and has been shown to directly bind to both kinase subunits under in vitro conditions (Stephens et al., 1997; Maier et al., 1999). Therefore, we asked whether overexpression of free Gβγ directs YFP-fused PI3Kγ subunits to the cell membrane in vivo. Surprisingly, p110γ was not membrane-localized in the presence of coexpressed Gβγ in HEK cells (Fig. 4 A, top). In contrast, Gβγ recruited p101 to the membrane, resulting in accumulation of NH2- or COOH-terminally YFP-tagged p101 at both plasma- and endomembranes (Fig. 4 A, bottom, and Fig. 4 B). This corresponds to the subcellular distribution of overexpressed Gβγ dimers (see below), suggesting that p101 interacted with membrane-bound Gβγ.

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