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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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PI3Kγ-mediated membrane translocation of GFP-GRP1PH in vascular smooth muscle (VSM) cells. VSM cells were microinjected with plasmids for GFP-GRP1PH, fMLP-R, p101, and p110γ or p110γ-CAAX in different combinations. The localization of GFP-GRP1PH was monitored before and after the addition of 1 μM fMLP (picture taken after 4 min) and 10 ng/ml PDGF-BB (added 12 min later, picture taken after 8 min). White bars indicate a 10-μm scale.
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fig8: PI3Kγ-mediated membrane translocation of GFP-GRP1PH in vascular smooth muscle (VSM) cells. VSM cells were microinjected with plasmids for GFP-GRP1PH, fMLP-R, p101, and p110γ or p110γ-CAAX in different combinations. The localization of GFP-GRP1PH was monitored before and after the addition of 1 μM fMLP (picture taken after 4 min) and 10 ng/ml PDGF-BB (added 12 min later, picture taken after 8 min). White bars indicate a 10-μm scale.

Mentions: Next, we sought to confirm our principal findings using nontransformed cells in primary culture with a higher degree of differentiation than HEK cells. Vascular smooth muscle (VSM) cells were injected with plasmids encoding the fMLP receptor, p101, p110γ or p110γ-CAAX, and GFP-GRP1PH in different combinations, and redistribution of the fluorescent PtdIns-3,4,5-P3 sensor was monitored (Fig. 8). Again, this series of experiments confirmed that p101 is required for GPCR-mediated activation of PI3Kγ in living cells, whereas membrane-targeted p110γ can be stimulated even in the absence of p101.


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)

PI3Kγ-mediated membrane translocation of GFP-GRP1PH in vascular smooth muscle (VSM) cells. VSM cells were microinjected with plasmids for GFP-GRP1PH, fMLP-R, p101, and p110γ or p110γ-CAAX in different combinations. The localization of GFP-GRP1PH was monitored before and after the addition of 1 μM fMLP (picture taken after 4 min) and 10 ng/ml PDGF-BB (added 12 min later, picture taken after 8 min). White bars indicate a 10-μm scale.
© Copyright Policy
Related In: Results  -  Collection

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

fig8: PI3Kγ-mediated membrane translocation of GFP-GRP1PH in vascular smooth muscle (VSM) cells. VSM cells were microinjected with plasmids for GFP-GRP1PH, fMLP-R, p101, and p110γ or p110γ-CAAX in different combinations. The localization of GFP-GRP1PH was monitored before and after the addition of 1 μM fMLP (picture taken after 4 min) and 10 ng/ml PDGF-BB (added 12 min later, picture taken after 8 min). White bars indicate a 10-μm scale.
Mentions: Next, we sought to confirm our principal findings using nontransformed cells in primary culture with a higher degree of differentiation than HEK cells. Vascular smooth muscle (VSM) cells were injected with plasmids encoding the fMLP receptor, p101, p110γ or p110γ-CAAX, and GFP-GRP1PH in different combinations, and redistribution of the fluorescent PtdIns-3,4,5-P3 sensor was monitored (Fig. 8). Again, this series of experiments confirmed that p101 is required for GPCR-mediated activation of PI3Kγ in living cells, whereas membrane-targeted p110γ can be stimulated even in the absence of p101.

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