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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 a constitutively membrane-associated p110γ-CAAX. (A) GFP-GRP1PH translocation. HEK cells were transfected with plasmids for GFP-GRP1PH and p110γ-CAAX, p101, fMLP-R, and dominant-negative Ras N17 in different combinations. Translocation of the PtdIns-3,4,5-P3–binding GFP-GRP1PH in response to agonist stimulation was monitored as described above (see Fig. 5). White bars indicate a 10-μm scale. As a positive control for H-Ras N17, cells were transfected with the H-Ras N17 plasmid (same amount as for the GFP-GRP1PH translocation experiment) or empty vector. Cells were stimulated with 10 ng/ml EGF, and whole-cell lysates were analyzed by immunoblotting with an antibody that specifically recognizes the phosphorylated form of ERK (p-ERK). Equal loading was shown using the anti-ERK-antibody. (B) BtkPH-CFP translocation. (C) Akt phosphorylation. The experiment shown in Fig. 5 was repeated. In addition, cells were transfected with the plasmid for the membrane-targeted p110γ-CAAX instead of wild-type p110γ.
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fig7: Activation of a constitutively membrane-associated p110γ-CAAX. (A) GFP-GRP1PH translocation. HEK cells were transfected with plasmids for GFP-GRP1PH and p110γ-CAAX, p101, fMLP-R, and dominant-negative Ras N17 in different combinations. Translocation of the PtdIns-3,4,5-P3–binding GFP-GRP1PH in response to agonist stimulation was monitored as described above (see Fig. 5). White bars indicate a 10-μm scale. As a positive control for H-Ras N17, cells were transfected with the H-Ras N17 plasmid (same amount as for the GFP-GRP1PH translocation experiment) or empty vector. Cells were stimulated with 10 ng/ml EGF, and whole-cell lysates were analyzed by immunoblotting with an antibody that specifically recognizes the phosphorylated form of ERK (p-ERK). Equal loading was shown using the anti-ERK-antibody. (B) BtkPH-CFP translocation. (C) Akt phosphorylation. The experiment shown in Fig. 5 was repeated. In addition, cells were transfected with the plasmid for the membrane-targeted p110γ-CAAX instead of wild-type p110γ.

Mentions: To substantiate this assumption, we analyzed the subcellular redistribution of GFP-GRP1PH in HEK cells coexpressing membrane-targeted p110γ-CAAX, p101 and the fMLP receptor in various combinations (Fig. 7 A). A slight basal membrane localization of the fluorescent PH domain was evident on coexpression with p110γ-CAAX (Fig. 7 A, top panel) indicating an elevated PtdIns-3,4,5-P3 level. The same picture emerged when p101 was present in the panel of transfected plasmids (Fig. 7 A, second panel). On coexpression of the receptor, fMLP induced a significant additional translocation of GFP-GRP1PH from the cytosol to the membrane (Fig. 7 A, third and fourth panel) indicating additional stimulation of the membrane-bound lipid kinase. Interestingly, the effect was similar irrespective of whether p101 was present or not, suggesting direct activation of monomeric p110γ. PTX sensitivity confirmed that fMLP-induced activation of p110γ-CAAX was mediated by Gi proteins (unpublished data). Because Gβγ can activate Ras, which in turn may activate p110γ, one may imagine that fMLP-induced, p101-independent stimulation of p110γ-CAAX was mediated by Ras. However, coexpression of dominant-negative Ras N17 had no effect on fMLP-induced p110γ-CAAX stimulation, whereas its inhibitory effect was evident when EGF-induced MAP kinase activation was assessed (Fig. 7 A, bottom panel). In addition, an indirect autocrine stimulatory effect was excluded because supernatants of fMLP-stimulated cells failed to induce GFP-GRP1PH translocation in cells omitting fMLP receptors (unpublished data).


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 a constitutively membrane-associated p110γ-CAAX. (A) GFP-GRP1PH translocation. HEK cells were transfected with plasmids for GFP-GRP1PH and p110γ-CAAX, p101, fMLP-R, and dominant-negative Ras N17 in different combinations. Translocation of the PtdIns-3,4,5-P3–binding GFP-GRP1PH in response to agonist stimulation was monitored as described above (see Fig. 5). White bars indicate a 10-μm scale. As a positive control for H-Ras N17, cells were transfected with the H-Ras N17 plasmid (same amount as for the GFP-GRP1PH translocation experiment) or empty vector. Cells were stimulated with 10 ng/ml EGF, and whole-cell lysates were analyzed by immunoblotting with an antibody that specifically recognizes the phosphorylated form of ERK (p-ERK). Equal loading was shown using the anti-ERK-antibody. (B) BtkPH-CFP translocation. (C) Akt phosphorylation. The experiment shown in Fig. 5 was repeated. In addition, cells were transfected with the plasmid for the membrane-targeted p110γ-CAAX instead of wild-type p110γ.
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fig7: Activation of a constitutively membrane-associated p110γ-CAAX. (A) GFP-GRP1PH translocation. HEK cells were transfected with plasmids for GFP-GRP1PH and p110γ-CAAX, p101, fMLP-R, and dominant-negative Ras N17 in different combinations. Translocation of the PtdIns-3,4,5-P3–binding GFP-GRP1PH in response to agonist stimulation was monitored as described above (see Fig. 5). White bars indicate a 10-μm scale. As a positive control for H-Ras N17, cells were transfected with the H-Ras N17 plasmid (same amount as for the GFP-GRP1PH translocation experiment) or empty vector. Cells were stimulated with 10 ng/ml EGF, and whole-cell lysates were analyzed by immunoblotting with an antibody that specifically recognizes the phosphorylated form of ERK (p-ERK). Equal loading was shown using the anti-ERK-antibody. (B) BtkPH-CFP translocation. (C) Akt phosphorylation. The experiment shown in Fig. 5 was repeated. In addition, cells were transfected with the plasmid for the membrane-targeted p110γ-CAAX instead of wild-type p110γ.
Mentions: To substantiate this assumption, we analyzed the subcellular redistribution of GFP-GRP1PH in HEK cells coexpressing membrane-targeted p110γ-CAAX, p101 and the fMLP receptor in various combinations (Fig. 7 A). A slight basal membrane localization of the fluorescent PH domain was evident on coexpression with p110γ-CAAX (Fig. 7 A, top panel) indicating an elevated PtdIns-3,4,5-P3 level. The same picture emerged when p101 was present in the panel of transfected plasmids (Fig. 7 A, second panel). On coexpression of the receptor, fMLP induced a significant additional translocation of GFP-GRP1PH from the cytosol to the membrane (Fig. 7 A, third and fourth panel) indicating additional stimulation of the membrane-bound lipid kinase. Interestingly, the effect was similar irrespective of whether p101 was present or not, suggesting direct activation of monomeric p110γ. PTX sensitivity confirmed that fMLP-induced activation of p110γ-CAAX was mediated by Gi proteins (unpublished data). Because Gβγ can activate Ras, which in turn may activate p110γ, one may imagine that fMLP-induced, p101-independent stimulation of p110γ-CAAX was mediated by Ras. However, coexpression of dominant-negative Ras N17 had no effect on fMLP-induced p110γ-CAAX stimulation, whereas its inhibitory effect was evident when EGF-induced MAP kinase activation was assessed (Fig. 7 A, bottom panel). In addition, an indirect autocrine stimulatory effect was excluded because supernatants of fMLP-stimulated cells failed to induce GFP-GRP1PH translocation in cells omitting fMLP receptors (unpublished data).

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