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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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Activation of PI3Kγ by a G protein–coupled receptor. (A) Akt phosphorylation. Whole-cell lysates were analyzed by immunoblotting using an antibody that specifically recognizes the phosphorylated form of Akt. Equal loading was shown by using an anti-ERK antibody. Left; FCS-induced Akt phosphorylation in untransfected HEK cells. Right; fMLP-induced Akt phosphorylation in cells expressing the fMLP receptor and only the catalytic (p110γ) or both PI3Kγ subunits. (B) Membrane recruitment of the PtdIns-3,4,5-P3–binding PH domain of GRP1. HEK 293 cells were transfected with plasmids encoding the PH domain of GRP1 fused to GFP (GFP-GRP1PH), and the human fMLP receptor (fMLP-R), p110γ, and p101 in different combinations. The localization of the GFP-GRP1PH was monitored before and after the addition of 1 μM fMLP and 100 ng/ml EGF (added 8 min later) by confocal laser scanning microscopy. Pictures were taken 4 min after addition of either agonist. Images of typical experiments are shown (white bars, 10 μm). (C) Membrane recruitment of the PtdIns-3,4,5-P3–binding PH domain of Btk. Instead of GFP-GRP1PH, the PH domain of Btk fused to CFP (BtkPH-CFP) was used as a PtdIns-3,4,5-P3 sensor.
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fig5: Activation of PI3Kγ by a G protein–coupled receptor. (A) Akt phosphorylation. Whole-cell lysates were analyzed by immunoblotting using an antibody that specifically recognizes the phosphorylated form of Akt. Equal loading was shown by using an anti-ERK antibody. Left; FCS-induced Akt phosphorylation in untransfected HEK cells. Right; fMLP-induced Akt phosphorylation in cells expressing the fMLP receptor and only the catalytic (p110γ) or both PI3Kγ subunits. (B) Membrane recruitment of the PtdIns-3,4,5-P3–binding PH domain of GRP1. HEK 293 cells were transfected with plasmids encoding the PH domain of GRP1 fused to GFP (GFP-GRP1PH), and the human fMLP receptor (fMLP-R), p110γ, and p101 in different combinations. The localization of the GFP-GRP1PH was monitored before and after the addition of 1 μM fMLP and 100 ng/ml EGF (added 8 min later) by confocal laser scanning microscopy. Pictures were taken 4 min after addition of either agonist. Images of typical experiments are shown (white bars, 10 μm). (C) Membrane recruitment of the PtdIns-3,4,5-P3–binding PH domain of Btk. Instead of GFP-GRP1PH, the PH domain of Btk fused to CFP (BtkPH-CFP) was used as a PtdIns-3,4,5-P3 sensor.

Mentions: Because changes in morphology are not a very reliable measure of PI3K activity, we examined phosphorylation of endogenous protein kinase B (PKB or Akt) as an established PI3K-specific read-out system. To avoid detachment of the cells as a consequence of constitutive PI3Kγ stimulation by overexpressed Gβγ, we transiently stimulated the heterologously expressed Gi-coupled formyl-methionyl-leucyl-phenylalanine (fMLP) receptor, which is known to activate PI3Kγ (Stephens et al., 1993). Stimulation with fMLP induced Akt phosphorylation in HEK cells in the same range as with FCS (Fig. 5 A). However, Akt phosphorylation was only seen when the receptor was coexpressed with both PI3Kγ subunits, and not with p110γ alone. These data imply that p101 is required for GPCR-induced activation of PI3Kγ in vivo.


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)

Activation of PI3Kγ by a G protein–coupled receptor. (A) Akt phosphorylation. Whole-cell lysates were analyzed by immunoblotting using an antibody that specifically recognizes the phosphorylated form of Akt. Equal loading was shown by using an anti-ERK antibody. Left; FCS-induced Akt phosphorylation in untransfected HEK cells. Right; fMLP-induced Akt phosphorylation in cells expressing the fMLP receptor and only the catalytic (p110γ) or both PI3Kγ subunits. (B) Membrane recruitment of the PtdIns-3,4,5-P3–binding PH domain of GRP1. HEK 293 cells were transfected with plasmids encoding the PH domain of GRP1 fused to GFP (GFP-GRP1PH), and the human fMLP receptor (fMLP-R), p110γ, and p101 in different combinations. The localization of the GFP-GRP1PH was monitored before and after the addition of 1 μM fMLP and 100 ng/ml EGF (added 8 min later) by confocal laser scanning microscopy. Pictures were taken 4 min after addition of either agonist. Images of typical experiments are shown (white bars, 10 μm). (C) Membrane recruitment of the PtdIns-3,4,5-P3–binding PH domain of Btk. Instead of GFP-GRP1PH, the PH domain of Btk fused to CFP (BtkPH-CFP) was used as a PtdIns-3,4,5-P3 sensor.
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Related In: Results  -  Collection

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fig5: Activation of PI3Kγ by a G protein–coupled receptor. (A) Akt phosphorylation. Whole-cell lysates were analyzed by immunoblotting using an antibody that specifically recognizes the phosphorylated form of Akt. Equal loading was shown by using an anti-ERK antibody. Left; FCS-induced Akt phosphorylation in untransfected HEK cells. Right; fMLP-induced Akt phosphorylation in cells expressing the fMLP receptor and only the catalytic (p110γ) or both PI3Kγ subunits. (B) Membrane recruitment of the PtdIns-3,4,5-P3–binding PH domain of GRP1. HEK 293 cells were transfected with plasmids encoding the PH domain of GRP1 fused to GFP (GFP-GRP1PH), and the human fMLP receptor (fMLP-R), p110γ, and p101 in different combinations. The localization of the GFP-GRP1PH was monitored before and after the addition of 1 μM fMLP and 100 ng/ml EGF (added 8 min later) by confocal laser scanning microscopy. Pictures were taken 4 min after addition of either agonist. Images of typical experiments are shown (white bars, 10 μm). (C) Membrane recruitment of the PtdIns-3,4,5-P3–binding PH domain of Btk. Instead of GFP-GRP1PH, the PH domain of Btk fused to CFP (BtkPH-CFP) was used as a PtdIns-3,4,5-P3 sensor.
Mentions: Because changes in morphology are not a very reliable measure of PI3K activity, we examined phosphorylation of endogenous protein kinase B (PKB or Akt) as an established PI3K-specific read-out system. To avoid detachment of the cells as a consequence of constitutive PI3Kγ stimulation by overexpressed Gβγ, we transiently stimulated the heterologously expressed Gi-coupled formyl-methionyl-leucyl-phenylalanine (fMLP) receptor, which is known to activate PI3Kγ (Stephens et al., 1993). Stimulation with fMLP induced Akt phosphorylation in HEK cells in the same range as with FCS (Fig. 5 A). However, Akt phosphorylation was only seen when the receptor was coexpressed with both PI3Kγ subunits, and not with p110γ alone. These data imply that p101 is required for GPCR-induced activation of PI3Kγ in vivo.

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