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Activation of Galphai3 triggers cell migration via regulation of GIV.

Ghosh P, Garcia-Marcos M, Bornheimer SJ, Farquhar MG - J. Cell Biol. (2008)

Bottom Line: We find that Galphai3 preferentially localizes to the leading edge and that cells lacking Galphai3 fail to polarize or migrate.A conformational change induced by association of GIV with Galphai3 promotes Akt-mediated phosphorylation of GIV, resulting in its redistribution to the plasma membrane.Galphai3-GIV coupling is essential for cell migration during wound healing, macrophage chemotaxis, and tumor cell migration, indicating that the Galphai3-GIV switch serves to link direction sensing from different families of chemotactic receptors to formation of the leading edge during cell migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA.

ABSTRACT
During migration, cells must couple direction sensing to signal transduction and actin remodeling. We previously identified GIV/Girdin as a Galphai3 binding partner. We demonstrate that in mammalian cells Galphai3 controls the functions of GIV during cell migration. We find that Galphai3 preferentially localizes to the leading edge and that cells lacking Galphai3 fail to polarize or migrate. A conformational change induced by association of GIV with Galphai3 promotes Akt-mediated phosphorylation of GIV, resulting in its redistribution to the plasma membrane. Activation of Galphai3 serves as a molecular switch that triggers dissociation of Gbetagamma and GIV from the Gi3-GIV complex, thereby promoting cell migration by enhancing Akt signaling and actin remodeling. Galphai3-GIV coupling is essential for cell migration during wound healing, macrophage chemotaxis, and tumor cell migration, indicating that the Galphai3-GIV switch serves to link direction sensing from different families of chemotactic receptors to formation of the leading edge during cell migration.

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Activation status of Gαi3 determines its role in cell migration. (A) Cell migratory behavior is restored by wild-type (a and b) and active (Q204L; c and d), but not the inactive (G203A; e and f), Gαi3 mutant. (Top) HeLa cells expressing rGαi3wt, active, or inactive mutants were subjected to scratch wounding. (Bottom) Equal overexpression of Gαi3 constructs was confirmed by immunoblotting. In assays in which siRNA was followed by plasmid overexpression, the efficiency of transfection (∼45–55%) was similar for wt, active, or inactive Gαi3 constructs by IF (n = 3). (B) Insulin-stimulated Akt activation is restored by wt and active (Q204L) but not the inactive (G203A) Gαi3 mutant. (Top) Assays were performed as in Fig. 4 (A and B) and samples were immunoblotted for tAkt and pAkt 5 min after stimulation. (Bottom) Bar graph showing percentage of Akt activation in cells treated as in top. Results are shown as mean ± SEM (n = 3). (C) Transfection of active (Q204L; a–c), but not inactive (G203A; d–f), mutant rGαi3-YFP restores normal actin organization (Phalloidin, red) in Gαi3-depleted cells. *, cell expressing rGαi3-YFP visualized with anti-GFP (green). Bar, 10 μm. (D) Transfection of active (Q204L; a–c), but not inactive (G203A; d–f), rGαi3-YFP restores the normal (Fig. 4D, a) scattered peripheral and Golgi-associated (arrowheads) punctate distribution of GIV (red). *, cell expressing rGαi3-YFP visualized with anti-GFP (green). Bar, 10 μm.
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fig5: Activation status of Gαi3 determines its role in cell migration. (A) Cell migratory behavior is restored by wild-type (a and b) and active (Q204L; c and d), but not the inactive (G203A; e and f), Gαi3 mutant. (Top) HeLa cells expressing rGαi3wt, active, or inactive mutants were subjected to scratch wounding. (Bottom) Equal overexpression of Gαi3 constructs was confirmed by immunoblotting. In assays in which siRNA was followed by plasmid overexpression, the efficiency of transfection (∼45–55%) was similar for wt, active, or inactive Gαi3 constructs by IF (n = 3). (B) Insulin-stimulated Akt activation is restored by wt and active (Q204L) but not the inactive (G203A) Gαi3 mutant. (Top) Assays were performed as in Fig. 4 (A and B) and samples were immunoblotted for tAkt and pAkt 5 min after stimulation. (Bottom) Bar graph showing percentage of Akt activation in cells treated as in top. Results are shown as mean ± SEM (n = 3). (C) Transfection of active (Q204L; a–c), but not inactive (G203A; d–f), mutant rGαi3-YFP restores normal actin organization (Phalloidin, red) in Gαi3-depleted cells. *, cell expressing rGαi3-YFP visualized with anti-GFP (green). Bar, 10 μm. (D) Transfection of active (Q204L; a–c), but not inactive (G203A; d–f), rGαi3-YFP restores the normal (Fig. 4D, a) scattered peripheral and Golgi-associated (arrowheads) punctate distribution of GIV (red). *, cell expressing rGαi3-YFP visualized with anti-GFP (green). Bar, 10 μm.

Mentions: Next, we asked whether Gαi3 regulates GIV's functions in activating Akt and remodeling actin after growth factor stimulation, an approach which mimics scratch wound–induced Akt signaling in a more synchronized fashion (Enomoto et al., 2005). When serum-starved HeLa cells were stimulated with insulin, Akt activity peaked at 5 min and was rapidly down-regulated within 15–30 min in controls (Fig. 4 A). In Gαi3-silenced cells, the peak activation was reduced by ∼60% (Fig. 4, A and B), which is similar to the effect observed after GIV depletion in HeLa (Fig. 4 B) or HepG2 cells (Anai et al., 2005). The effect was Gαi specific because Gαs depletion did not significantly affect Akt activation (Fig. 4 A) and was reversed when rGαi3wt was restored in Gαi3 siRNA-treated cells (see Fig. 5 B). Therefore, Gαi3 links Akt activation and cell migration in a manner similar to that reported for GIV (Anai et al., 2005). To distinguish whether Gαi3 and GIV function in a common pathway or in independent parallel pathways mediating enhancement of Akt signaling, we investigated the effect of silencing both proteins. Silencing of Gαi3 or GIV alone reduced Akt activation by ∼60 and 80%, respectively (Fig. 4 B). When both were silenced (Fig. 4 B), no significant difference was observed from GIV-depleted cells, indicating that the effect on Akt was not additive. The fact that depletion of Gαi3 promoted weaker inhibition of Akt than GIV suggests that other Gi3-independent pathways might exist in which GIV is a common effector. Akt activation was also impaired when Gαi3-depleted HeLa cells were stimulated with EGF (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200712066/DC1), which implicates Gαi3 and GIV in a common pathway mediating Akt activation upon RTK stimulation.


Activation of Galphai3 triggers cell migration via regulation of GIV.

Ghosh P, Garcia-Marcos M, Bornheimer SJ, Farquhar MG - J. Cell Biol. (2008)

Activation status of Gαi3 determines its role in cell migration. (A) Cell migratory behavior is restored by wild-type (a and b) and active (Q204L; c and d), but not the inactive (G203A; e and f), Gαi3 mutant. (Top) HeLa cells expressing rGαi3wt, active, or inactive mutants were subjected to scratch wounding. (Bottom) Equal overexpression of Gαi3 constructs was confirmed by immunoblotting. In assays in which siRNA was followed by plasmid overexpression, the efficiency of transfection (∼45–55%) was similar for wt, active, or inactive Gαi3 constructs by IF (n = 3). (B) Insulin-stimulated Akt activation is restored by wt and active (Q204L) but not the inactive (G203A) Gαi3 mutant. (Top) Assays were performed as in Fig. 4 (A and B) and samples were immunoblotted for tAkt and pAkt 5 min after stimulation. (Bottom) Bar graph showing percentage of Akt activation in cells treated as in top. Results are shown as mean ± SEM (n = 3). (C) Transfection of active (Q204L; a–c), but not inactive (G203A; d–f), mutant rGαi3-YFP restores normal actin organization (Phalloidin, red) in Gαi3-depleted cells. *, cell expressing rGαi3-YFP visualized with anti-GFP (green). Bar, 10 μm. (D) Transfection of active (Q204L; a–c), but not inactive (G203A; d–f), rGαi3-YFP restores the normal (Fig. 4D, a) scattered peripheral and Golgi-associated (arrowheads) punctate distribution of GIV (red). *, cell expressing rGαi3-YFP visualized with anti-GFP (green). Bar, 10 μm.
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fig5: Activation status of Gαi3 determines its role in cell migration. (A) Cell migratory behavior is restored by wild-type (a and b) and active (Q204L; c and d), but not the inactive (G203A; e and f), Gαi3 mutant. (Top) HeLa cells expressing rGαi3wt, active, or inactive mutants were subjected to scratch wounding. (Bottom) Equal overexpression of Gαi3 constructs was confirmed by immunoblotting. In assays in which siRNA was followed by plasmid overexpression, the efficiency of transfection (∼45–55%) was similar for wt, active, or inactive Gαi3 constructs by IF (n = 3). (B) Insulin-stimulated Akt activation is restored by wt and active (Q204L) but not the inactive (G203A) Gαi3 mutant. (Top) Assays were performed as in Fig. 4 (A and B) and samples were immunoblotted for tAkt and pAkt 5 min after stimulation. (Bottom) Bar graph showing percentage of Akt activation in cells treated as in top. Results are shown as mean ± SEM (n = 3). (C) Transfection of active (Q204L; a–c), but not inactive (G203A; d–f), mutant rGαi3-YFP restores normal actin organization (Phalloidin, red) in Gαi3-depleted cells. *, cell expressing rGαi3-YFP visualized with anti-GFP (green). Bar, 10 μm. (D) Transfection of active (Q204L; a–c), but not inactive (G203A; d–f), rGαi3-YFP restores the normal (Fig. 4D, a) scattered peripheral and Golgi-associated (arrowheads) punctate distribution of GIV (red). *, cell expressing rGαi3-YFP visualized with anti-GFP (green). Bar, 10 μm.
Mentions: Next, we asked whether Gαi3 regulates GIV's functions in activating Akt and remodeling actin after growth factor stimulation, an approach which mimics scratch wound–induced Akt signaling in a more synchronized fashion (Enomoto et al., 2005). When serum-starved HeLa cells were stimulated with insulin, Akt activity peaked at 5 min and was rapidly down-regulated within 15–30 min in controls (Fig. 4 A). In Gαi3-silenced cells, the peak activation was reduced by ∼60% (Fig. 4, A and B), which is similar to the effect observed after GIV depletion in HeLa (Fig. 4 B) or HepG2 cells (Anai et al., 2005). The effect was Gαi specific because Gαs depletion did not significantly affect Akt activation (Fig. 4 A) and was reversed when rGαi3wt was restored in Gαi3 siRNA-treated cells (see Fig. 5 B). Therefore, Gαi3 links Akt activation and cell migration in a manner similar to that reported for GIV (Anai et al., 2005). To distinguish whether Gαi3 and GIV function in a common pathway or in independent parallel pathways mediating enhancement of Akt signaling, we investigated the effect of silencing both proteins. Silencing of Gαi3 or GIV alone reduced Akt activation by ∼60 and 80%, respectively (Fig. 4 B). When both were silenced (Fig. 4 B), no significant difference was observed from GIV-depleted cells, indicating that the effect on Akt was not additive. The fact that depletion of Gαi3 promoted weaker inhibition of Akt than GIV suggests that other Gi3-independent pathways might exist in which GIV is a common effector. Akt activation was also impaired when Gαi3-depleted HeLa cells were stimulated with EGF (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200712066/DC1), which implicates Gαi3 and GIV in a common pathway mediating Akt activation upon RTK stimulation.

Bottom Line: We find that Galphai3 preferentially localizes to the leading edge and that cells lacking Galphai3 fail to polarize or migrate.A conformational change induced by association of GIV with Galphai3 promotes Akt-mediated phosphorylation of GIV, resulting in its redistribution to the plasma membrane.Galphai3-GIV coupling is essential for cell migration during wound healing, macrophage chemotaxis, and tumor cell migration, indicating that the Galphai3-GIV switch serves to link direction sensing from different families of chemotactic receptors to formation of the leading edge during cell migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA.

ABSTRACT
During migration, cells must couple direction sensing to signal transduction and actin remodeling. We previously identified GIV/Girdin as a Galphai3 binding partner. We demonstrate that in mammalian cells Galphai3 controls the functions of GIV during cell migration. We find that Galphai3 preferentially localizes to the leading edge and that cells lacking Galphai3 fail to polarize or migrate. A conformational change induced by association of GIV with Galphai3 promotes Akt-mediated phosphorylation of GIV, resulting in its redistribution to the plasma membrane. Activation of Galphai3 serves as a molecular switch that triggers dissociation of Gbetagamma and GIV from the Gi3-GIV complex, thereby promoting cell migration by enhancing Akt signaling and actin remodeling. Galphai3-GIV coupling is essential for cell migration during wound healing, macrophage chemotaxis, and tumor cell migration, indicating that the Galphai3-GIV switch serves to link direction sensing from different families of chemotactic receptors to formation of the leading edge during cell migration.

Show MeSH
Related in: MedlinePlus