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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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Characterization of a constitutively membrane-associated p110γ-CAAX. HEK cells were transfected with the indicated plasmids and analyzed by confocal laser scanning microscopy. Images of typical cells are shown. White bars indicate a 10-μm scale. Top panel; Subcellular localization of YFP-p110γ-CAAX (left) or YFP-p101 expressed together with p110γ-CAAX (right). Bottom panel; Coexpression with Gβγ.
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fig6: Characterization of a constitutively membrane-associated p110γ-CAAX. HEK cells were transfected with the indicated plasmids and analyzed by confocal laser scanning microscopy. Images of typical cells are shown. White bars indicate a 10-μm scale. Top panel; Subcellular localization of YFP-p110γ-CAAX (left) or YFP-p101 expressed together with p110γ-CAAX (right). Bottom panel; Coexpression with Gβγ.

Mentions: The presented data suggest that in living cells, p101 is an indispensable adaptor for GPCR-induced translocation and activation of class IB PI3Kγ, which is equivalent to the role of p85 in RTK-induced class IA PI3K activation. Vice versa, Gβγ behaves as a membrane anchor recruiting PI3Kγ through association with p101. So far, these experiments did not clarify whether membrane recruitment itself is sufficient for activation of the enzyme or whether additional allosteric stimulation is required. To tackle this question, we generated p110γ mutants containing a COOH-terminal isoprenylation signal, i.e., a CAAX-box motif, which constitutively localizes p110γ to the plasma membrane. p110γ-CAAX accumulated at the plasma membrane of HEK cells, and was able to complex with p101 (Fig. 6, top). Coexpression of Gβγ together with YFP-p110γ-CAAX increased not only fluorescent staining of endomembranes, but also produced a wortmannin-sensitive rounding of the cells (Fig. 6, bottom) even in the absence of p101 (not depicted), which was similar to the effect after coexpression of Gβγ with p110γ/p101 dimers (see Fig. 4 C). These observations imply that membrane-bound PI3Kγ is not fully active, but can be further activated by 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)

Characterization of a constitutively membrane-associated p110γ-CAAX. HEK cells were transfected with the indicated plasmids and analyzed by confocal laser scanning microscopy. Images of typical cells are shown. White bars indicate a 10-μm scale. Top panel; Subcellular localization of YFP-p110γ-CAAX (left) or YFP-p101 expressed together with p110γ-CAAX (right). Bottom panel; Coexpression with Gβγ.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Characterization of a constitutively membrane-associated p110γ-CAAX. HEK cells were transfected with the indicated plasmids and analyzed by confocal laser scanning microscopy. Images of typical cells are shown. White bars indicate a 10-μm scale. Top panel; Subcellular localization of YFP-p110γ-CAAX (left) or YFP-p101 expressed together with p110γ-CAAX (right). Bottom panel; Coexpression with Gβγ.
Mentions: The presented data suggest that in living cells, p101 is an indispensable adaptor for GPCR-induced translocation and activation of class IB PI3Kγ, which is equivalent to the role of p85 in RTK-induced class IA PI3K activation. Vice versa, Gβγ behaves as a membrane anchor recruiting PI3Kγ through association with p101. So far, these experiments did not clarify whether membrane recruitment itself is sufficient for activation of the enzyme or whether additional allosteric stimulation is required. To tackle this question, we generated p110γ mutants containing a COOH-terminal isoprenylation signal, i.e., a CAAX-box motif, which constitutively localizes p110γ to the plasma membrane. p110γ-CAAX accumulated at the plasma membrane of HEK cells, and was able to complex with p101 (Fig. 6, top). Coexpression of Gβγ together with YFP-p110γ-CAAX increased not only fluorescent staining of endomembranes, but also produced a wortmannin-sensitive rounding of the cells (Fig. 6, bottom) even in the absence of p101 (not depicted), which was similar to the effect after coexpression of Gβγ with p110γ/p101 dimers (see Fig. 4 C). These observations imply that membrane-bound PI3Kγ is not fully active, but can be further activated by 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