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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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Gαi3 is necessary for phosphorylation of GIV by Akt. (A) Phosphorylation of GIV is decreased upon depletion of Gαi3 (Gαi3 siRNA) and restored upon transfecting rGαi3 (Gαi3 siRNA and rGαi3). GIV was immunoprecipitated from HeLa cells treated as indicated and phosphorylation determined by immunoblotting with anti-pSer/Thr IgG. (B, top) Gαi3 binding to GIV promotes phosphorylation of GIV at S1416 by Akt. Immunoprecipitated GIV was preincubated in the presence or absence of ∼5 μg GST (lane 3), GST-Gαi3wt (lane 4), Gαi3 Q204L (lane 5), or Gαi3 G203A (lane 6) and subsequently phosphorylated in vitro with recombinant Akt1, followed by SDS-PAGE. Phosphorylation was determined by immunoblotting with anti-pSer IgG. (B, bottom) Equal loading of preimmune or anti-GIV IgG and GST proteins was confirmed by Ponceau S staining. (C) Gαi3 binding to GIV increases its susceptibility to trypsin-mediated proteolysis. Immunoprecipitated GIV was incubated in the presence or absence of GST-Gαi3 proteins as in B and digested with increasing concentrations of trypsin at 37°C for 8 min. The amount of uncleaved (trypsin resistant) GIV was determined by immunoblotting (inset) and displayed as percentage of starting amount (y axis) plotted against trypsin concentration (x axis). (D) GST-Gαi3 interacts similarly with in vitro–translated GIV wild-type and phosphorylation mimic (S1416D) or nonphosphorylatable (S1416A) GIV mutants. Semiquantitative densitometry revealed that 14 ± 2, 13 ± 1, and 14 ± 3% of wild-type, S1416A, and S1416D, respectively, bound to GST-Gαi3 (n = 5). (E) Schematic representation of how binding of Gαi3 to GIV may mediate phosphorylation of GIV by bringing the C-terminal Akt binding site closer to the Akt phosphorylation site.
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fig7: Gαi3 is necessary for phosphorylation of GIV by Akt. (A) Phosphorylation of GIV is decreased upon depletion of Gαi3 (Gαi3 siRNA) and restored upon transfecting rGαi3 (Gαi3 siRNA and rGαi3). GIV was immunoprecipitated from HeLa cells treated as indicated and phosphorylation determined by immunoblotting with anti-pSer/Thr IgG. (B, top) Gαi3 binding to GIV promotes phosphorylation of GIV at S1416 by Akt. Immunoprecipitated GIV was preincubated in the presence or absence of ∼5 μg GST (lane 3), GST-Gαi3wt (lane 4), Gαi3 Q204L (lane 5), or Gαi3 G203A (lane 6) and subsequently phosphorylated in vitro with recombinant Akt1, followed by SDS-PAGE. Phosphorylation was determined by immunoblotting with anti-pSer IgG. (B, bottom) Equal loading of preimmune or anti-GIV IgG and GST proteins was confirmed by Ponceau S staining. (C) Gαi3 binding to GIV increases its susceptibility to trypsin-mediated proteolysis. Immunoprecipitated GIV was incubated in the presence or absence of GST-Gαi3 proteins as in B and digested with increasing concentrations of trypsin at 37°C for 8 min. The amount of uncleaved (trypsin resistant) GIV was determined by immunoblotting (inset) and displayed as percentage of starting amount (y axis) plotted against trypsin concentration (x axis). (D) GST-Gαi3 interacts similarly with in vitro–translated GIV wild-type and phosphorylation mimic (S1416D) or nonphosphorylatable (S1416A) GIV mutants. Semiquantitative densitometry revealed that 14 ± 2, 13 ± 1, and 14 ± 3% of wild-type, S1416A, and S1416D, respectively, bound to GST-Gαi3 (n = 5). (E) Schematic representation of how binding of Gαi3 to GIV may mediate phosphorylation of GIV by bringing the C-terminal Akt binding site closer to the Akt phosphorylation site.

Mentions: In addition to being an enhancer of Akt activity, GIV is a substrate for Akt at S1416, and this phosphorylation is critical for the function of GIV in cell migration (Enomoto et al., 2005). Interestingly, S1416 lies within the G binding domain of GIV (Le-Niculescu et al., 2005), raising the question of whether the Gαi3–GIV interaction modulates ser/thr phosphorylation of GIV. We found that in Gαi3-depleted cells GIV was poorly phosphorylated compared with controls (Fig. 7 A). Repletion of Gαi3 with rGαi3wt restored phosphorylation of GIV to control levels (Fig. 7 A). To determine if the Gαi3–GIV interaction enhanced or interfered with Akt mediated phosphorylation of GIV on S1416, we performed in vitro phosphorylation reactions with immunopurified GIV, GST-Gαi3, and recombinant Akt. Because Akt phosphorylates immunopurified GIV at a single site, i.e., S1416 (Enomoto et al., 2005), p-ser/thr antibody was used to estimate the amount of phosphorylation that occurred. We found that preincubation of GIV with GST-Gαi3G203A, the inactive mutant that binds GIV most avidly, increased the phosphorylation of GIV ∼2.5 fold (Fig. 7 B, lane 6), whereas GST-Gαi3Q204L and GST-Gαi3wt were not significantly different (Fig. 7 B, lanes 4 and 5). We conclude that binding of Gαi3 to GIV promotes phosphorylation of GIV at S1416 by Akt. We reasoned that this is likely via a change in the conformation of GIV that might facilitate access of Akt to S1416. To investigate whether such a change in confirmation takes place, we performed limited proteolysis using increasing amounts of trypsin on immunopurified GIV (Fig. 7 C). GIV preincubated with GST, GST-Gαi3wt, or GST-Gαi3Q204L was relatively resistant to proteolysis, whereas GIV preincubated with Gαi3G203A was more susceptible to proteolysis, indicating a change in the conformation of GIV upon binding the inactive mutant.


Activation of Galphai3 triggers cell migration via regulation of GIV.

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

Gαi3 is necessary for phosphorylation of GIV by Akt. (A) Phosphorylation of GIV is decreased upon depletion of Gαi3 (Gαi3 siRNA) and restored upon transfecting rGαi3 (Gαi3 siRNA and rGαi3). GIV was immunoprecipitated from HeLa cells treated as indicated and phosphorylation determined by immunoblotting with anti-pSer/Thr IgG. (B, top) Gαi3 binding to GIV promotes phosphorylation of GIV at S1416 by Akt. Immunoprecipitated GIV was preincubated in the presence or absence of ∼5 μg GST (lane 3), GST-Gαi3wt (lane 4), Gαi3 Q204L (lane 5), or Gαi3 G203A (lane 6) and subsequently phosphorylated in vitro with recombinant Akt1, followed by SDS-PAGE. Phosphorylation was determined by immunoblotting with anti-pSer IgG. (B, bottom) Equal loading of preimmune or anti-GIV IgG and GST proteins was confirmed by Ponceau S staining. (C) Gαi3 binding to GIV increases its susceptibility to trypsin-mediated proteolysis. Immunoprecipitated GIV was incubated in the presence or absence of GST-Gαi3 proteins as in B and digested with increasing concentrations of trypsin at 37°C for 8 min. The amount of uncleaved (trypsin resistant) GIV was determined by immunoblotting (inset) and displayed as percentage of starting amount (y axis) plotted against trypsin concentration (x axis). (D) GST-Gαi3 interacts similarly with in vitro–translated GIV wild-type and phosphorylation mimic (S1416D) or nonphosphorylatable (S1416A) GIV mutants. Semiquantitative densitometry revealed that 14 ± 2, 13 ± 1, and 14 ± 3% of wild-type, S1416A, and S1416D, respectively, bound to GST-Gαi3 (n = 5). (E) Schematic representation of how binding of Gαi3 to GIV may mediate phosphorylation of GIV by bringing the C-terminal Akt binding site closer to the Akt phosphorylation site.
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fig7: Gαi3 is necessary for phosphorylation of GIV by Akt. (A) Phosphorylation of GIV is decreased upon depletion of Gαi3 (Gαi3 siRNA) and restored upon transfecting rGαi3 (Gαi3 siRNA and rGαi3). GIV was immunoprecipitated from HeLa cells treated as indicated and phosphorylation determined by immunoblotting with anti-pSer/Thr IgG. (B, top) Gαi3 binding to GIV promotes phosphorylation of GIV at S1416 by Akt. Immunoprecipitated GIV was preincubated in the presence or absence of ∼5 μg GST (lane 3), GST-Gαi3wt (lane 4), Gαi3 Q204L (lane 5), or Gαi3 G203A (lane 6) and subsequently phosphorylated in vitro with recombinant Akt1, followed by SDS-PAGE. Phosphorylation was determined by immunoblotting with anti-pSer IgG. (B, bottom) Equal loading of preimmune or anti-GIV IgG and GST proteins was confirmed by Ponceau S staining. (C) Gαi3 binding to GIV increases its susceptibility to trypsin-mediated proteolysis. Immunoprecipitated GIV was incubated in the presence or absence of GST-Gαi3 proteins as in B and digested with increasing concentrations of trypsin at 37°C for 8 min. The amount of uncleaved (trypsin resistant) GIV was determined by immunoblotting (inset) and displayed as percentage of starting amount (y axis) plotted against trypsin concentration (x axis). (D) GST-Gαi3 interacts similarly with in vitro–translated GIV wild-type and phosphorylation mimic (S1416D) or nonphosphorylatable (S1416A) GIV mutants. Semiquantitative densitometry revealed that 14 ± 2, 13 ± 1, and 14 ± 3% of wild-type, S1416A, and S1416D, respectively, bound to GST-Gαi3 (n = 5). (E) Schematic representation of how binding of Gαi3 to GIV may mediate phosphorylation of GIV by bringing the C-terminal Akt binding site closer to the Akt phosphorylation site.
Mentions: In addition to being an enhancer of Akt activity, GIV is a substrate for Akt at S1416, and this phosphorylation is critical for the function of GIV in cell migration (Enomoto et al., 2005). Interestingly, S1416 lies within the G binding domain of GIV (Le-Niculescu et al., 2005), raising the question of whether the Gαi3–GIV interaction modulates ser/thr phosphorylation of GIV. We found that in Gαi3-depleted cells GIV was poorly phosphorylated compared with controls (Fig. 7 A). Repletion of Gαi3 with rGαi3wt restored phosphorylation of GIV to control levels (Fig. 7 A). To determine if the Gαi3–GIV interaction enhanced or interfered with Akt mediated phosphorylation of GIV on S1416, we performed in vitro phosphorylation reactions with immunopurified GIV, GST-Gαi3, and recombinant Akt. Because Akt phosphorylates immunopurified GIV at a single site, i.e., S1416 (Enomoto et al., 2005), p-ser/thr antibody was used to estimate the amount of phosphorylation that occurred. We found that preincubation of GIV with GST-Gαi3G203A, the inactive mutant that binds GIV most avidly, increased the phosphorylation of GIV ∼2.5 fold (Fig. 7 B, lane 6), whereas GST-Gαi3Q204L and GST-Gαi3wt were not significantly different (Fig. 7 B, lanes 4 and 5). We conclude that binding of Gαi3 to GIV promotes phosphorylation of GIV at S1416 by Akt. We reasoned that this is likely via a change in the conformation of GIV that might facilitate access of Akt to S1416. To investigate whether such a change in confirmation takes place, we performed limited proteolysis using increasing amounts of trypsin on immunopurified GIV (Fig. 7 C). GIV preincubated with GST, GST-Gαi3wt, or GST-Gαi3Q204L was relatively resistant to proteolysis, whereas GIV preincubated with Gαi3G203A was more susceptible to proteolysis, indicating a change in the conformation of GIV upon binding the inactive mutant.

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