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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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Expression and subcellular distribution of fluorescent p110γ and p101 in HEK cells. (A) Cells were transfected with plasmids for YFP-tagged single PI3Kγ subunits as indicated. Images were taken by confocal laser scanning microscopy. Images of typical cells are shown. White bars indicate a 10-μm scale. (B) Cells were cotransfected with both p110γ and p101. Only one subunit was fused to YFP as indicated. (C) Cells were transfected with plasmids encoding fluorescent p110γ and p101 at different ratios. The total amount of transfected cDNA was kept constant by the addition of pcDNA3. Equal amounts of whole-cell lysates were subjected to SDS-PAGE followed by immunoblotting with an anti-GFP antibody.
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fig2: Expression and subcellular distribution of fluorescent p110γ and p101 in HEK cells. (A) Cells were transfected with plasmids for YFP-tagged single PI3Kγ subunits as indicated. Images were taken by confocal laser scanning microscopy. Images of typical cells are shown. White bars indicate a 10-μm scale. (B) Cells were cotransfected with both p110γ and p101. Only one subunit was fused to YFP as indicated. (C) Cells were transfected with plasmids encoding fluorescent p110γ and p101 at different ratios. The total amount of transfected cDNA was kept constant by the addition of pcDNA3. Equal amounts of whole-cell lysates were subjected to SDS-PAGE followed by immunoblotting with an anti-GFP antibody.

Mentions: Next, we examined the subcellular distribution of the YFP-tagged PI3Kγ subunits in living cells by confocal laser scanning microscopy. Although YFP was uniformly distributed inside HEK cells (unpublished data), fluorescence signals from YFP fused to the NH2 or COOH termini of p110γ were only detected in the cytosolic compartment (Fig. 2 A, top). However, a small fraction of the YFP-fused p110γ was also detected in the nucleus when coexpressed with wild-type p101 (Fig. 2 B, top). The same distribution was seen for YFP-tagged p101 coexpressed with wild-type p110γ (Fig. 2 B, bottom). Thus, fluorescent PI3Kγ subunits form heterodimers (see next section), present in the cytosol and nucleus of HEK cells, whereas distribution of the p110γ subunit alone seems restricted to the cytosol. Interestingly, YFP-fused p101 alone showed a predominant nuclear localization with a significantly lower overall fluorescence intensity (Fig. 2 A, bottom).


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)

Expression and subcellular distribution of fluorescent p110γ and p101 in HEK cells. (A) Cells were transfected with plasmids for YFP-tagged single PI3Kγ subunits as indicated. Images were taken by confocal laser scanning microscopy. Images of typical cells are shown. White bars indicate a 10-μm scale. (B) Cells were cotransfected with both p110γ and p101. Only one subunit was fused to YFP as indicated. (C) Cells were transfected with plasmids encoding fluorescent p110γ and p101 at different ratios. The total amount of transfected cDNA was kept constant by the addition of pcDNA3. Equal amounts of whole-cell lysates were subjected to SDS-PAGE followed by immunoblotting with an anti-GFP antibody.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Expression and subcellular distribution of fluorescent p110γ and p101 in HEK cells. (A) Cells were transfected with plasmids for YFP-tagged single PI3Kγ subunits as indicated. Images were taken by confocal laser scanning microscopy. Images of typical cells are shown. White bars indicate a 10-μm scale. (B) Cells were cotransfected with both p110γ and p101. Only one subunit was fused to YFP as indicated. (C) Cells were transfected with plasmids encoding fluorescent p110γ and p101 at different ratios. The total amount of transfected cDNA was kept constant by the addition of pcDNA3. Equal amounts of whole-cell lysates were subjected to SDS-PAGE followed by immunoblotting with an anti-GFP antibody.
Mentions: Next, we examined the subcellular distribution of the YFP-tagged PI3Kγ subunits in living cells by confocal laser scanning microscopy. Although YFP was uniformly distributed inside HEK cells (unpublished data), fluorescence signals from YFP fused to the NH2 or COOH termini of p110γ were only detected in the cytosolic compartment (Fig. 2 A, top). However, a small fraction of the YFP-fused p110γ was also detected in the nucleus when coexpressed with wild-type p101 (Fig. 2 B, top). The same distribution was seen for YFP-tagged p101 coexpressed with wild-type p110γ (Fig. 2 B, bottom). Thus, fluorescent PI3Kγ subunits form heterodimers (see next section), present in the cytosol and nucleus of HEK cells, whereas distribution of the p110γ subunit alone seems restricted to the cytosol. Interestingly, YFP-fused p101 alone showed a predominant nuclear localization with a significantly lower overall fluorescence intensity (Fig. 2 A, bottom).

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