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Structural basis for activation of trimeric Gi proteins by multiple growth factor receptors via GIV/Girdin.

Lin C, Ear J, Midde K, Lopez-Sanchez I, Aznar N, Garcia-Marcos M, Kufareva I, Abagyan R, Ghosh P - Mol. Biol. Cell (2014)

Bottom Line: We discovered a unifying mechanism that allows GIV/Girdin, a bona fide metastasis-related protein and a guanine-nucleotide exchange factor (GEF) for Gαi, to serve as a direct platform for multiple RTKs to activate Gαi proteins.Using a combination of homology modeling, protein-protein interaction, and kinase assays, we demonstrate that a stretch of ∼110 amino acids within GIV C-terminus displays structural plasticity that allows folding into a SH2-like domain in the presence of phosphotyrosine ligands.Expression of a SH2-deficient GIV mutant (Arg 1745→Leu) that cannot bind RTKs impaired all previously demonstrated functions of GIV-Akt enhancement, actin remodeling, and cell migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California, San Diego, School of Medicine, CA 92093.

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The SH2-like domain of GIV is required for enhanced tyrosine phosphorylation of GIV and subsequent binding to RTKs. (a) Homology model of GIV's SH2 domain is depicted as a ribbon as in Figure 2d. The positions and the orientations of the two tyrosines are displayed in two views: from the “front” (pTyr-binding interface) and “back.” Both tyrosines are well exposed to solvent, consistent with the fact that multiple receptor and nonreceptor tyrosine kinases can readily access and phosphorylate them and that they can directly bind and activate PI3K (Lin et al., 2011). (b) Cos7 cells expressing FLAG-tagged wild-type GIV (GIV-WT FLAG), SH2-deficient R1745L mutant GIV (GIV-RL FLAG), a tyrosine phosphorylation–deficient mutant (GIV-YF FLAG; Lin et al., 2011), or vector alone were serum starved, pretreated with Src inhibitor (PP2), and subsequently stimulated with EGF before lysis. GIV was immunoprecipitated from equal aliquots of lysates (right) with FLAG mAb, and immunoprecipitates (left) were analyzed for GIV (red) and pTyr (green) by IB and dual-color imaging. The merge confirms that tyrosine-phosphorylated GIV (yellow) was immunoprecipitated exclusively from EGF-treated cells expressing GIV-WT (lane 3) but not from cells expressing GIV-RL (lane 4). As shown previously (Lin et al., 2011), the negative control GIV-YF was not phosphorylated (lane 5). The lysates (right) were analyzed for FLAG (GIV-FLAG), phospho-Akt (pAkt), and tubulin by IB. (c) Equal aliquots of His-GIV-CT proteins were first incubated with 10-fold molar excess of either phosphorylated (left lane) or unphosphorylated (right lane) EGFR tail peptides (sequence flanking Y1173) before in vitro kinase assays with recombinant EGFR kinase. GIV-CT is phosphorylated only in the presence of dephosphorylated EGFR peptide (which it cannot bind) but not in the presence of phospho-EGFR ligand. (d) EGFR was immunoprecipitated from lysates of HeLa cells starved and stimulated with EGF with anti-EGFR (528) mAb or control IgG. Immune complexes were analyzed for the presence of tyrosine-phosphorylated GIV using anti-pY1764GIV and ligand-activated EGFR (pY1173 EGFR) by IB. (e) InsR was immunoprecipitated from lysates of Cos7 cells starved and stimulated with insulin with anti-pYInsR mAb. Immune complexes were analyzed for the presence of tyrosine phosphorylated GIV using anti-pY1764GIV and ligand-activated InsR (pYInsR) by IB.
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Figure 5: The SH2-like domain of GIV is required for enhanced tyrosine phosphorylation of GIV and subsequent binding to RTKs. (a) Homology model of GIV's SH2 domain is depicted as a ribbon as in Figure 2d. The positions and the orientations of the two tyrosines are displayed in two views: from the “front” (pTyr-binding interface) and “back.” Both tyrosines are well exposed to solvent, consistent with the fact that multiple receptor and nonreceptor tyrosine kinases can readily access and phosphorylate them and that they can directly bind and activate PI3K (Lin et al., 2011). (b) Cos7 cells expressing FLAG-tagged wild-type GIV (GIV-WT FLAG), SH2-deficient R1745L mutant GIV (GIV-RL FLAG), a tyrosine phosphorylation–deficient mutant (GIV-YF FLAG; Lin et al., 2011), or vector alone were serum starved, pretreated with Src inhibitor (PP2), and subsequently stimulated with EGF before lysis. GIV was immunoprecipitated from equal aliquots of lysates (right) with FLAG mAb, and immunoprecipitates (left) were analyzed for GIV (red) and pTyr (green) by IB and dual-color imaging. The merge confirms that tyrosine-phosphorylated GIV (yellow) was immunoprecipitated exclusively from EGF-treated cells expressing GIV-WT (lane 3) but not from cells expressing GIV-RL (lane 4). As shown previously (Lin et al., 2011), the negative control GIV-YF was not phosphorylated (lane 5). The lysates (right) were analyzed for FLAG (GIV-FLAG), phospho-Akt (pAkt), and tubulin by IB. (c) Equal aliquots of His-GIV-CT proteins were first incubated with 10-fold molar excess of either phosphorylated (left lane) or unphosphorylated (right lane) EGFR tail peptides (sequence flanking Y1173) before in vitro kinase assays with recombinant EGFR kinase. GIV-CT is phosphorylated only in the presence of dephosphorylated EGFR peptide (which it cannot bind) but not in the presence of phospho-EGFR ligand. (d) EGFR was immunoprecipitated from lysates of HeLa cells starved and stimulated with EGF with anti-EGFR (528) mAb or control IgG. Immune complexes were analyzed for the presence of tyrosine-phosphorylated GIV using anti-pY1764GIV and ligand-activated EGFR (pY1173 EGFR) by IB. (e) InsR was immunoprecipitated from lysates of Cos7 cells starved and stimulated with insulin with anti-pYInsR mAb. Immune complexes were analyzed for the presence of tyrosine phosphorylated GIV using anti-pY1764GIV and ligand-activated InsR (pYInsR) by IB.

Mentions: We previously demonstrated that upon ligand stimulation, multiple RTKs phosphorylate GIV at two critical tyrosines, Y1764 and Y1798, and that both sites are capable of binding and activating class 1A PI3Ks (Lin et al., 2011). Although these two tyrosines are located within the boundaries of the SH2-like domain of GIV (Figure 5a and Supplemental Figure S6a), the homology model predicts that they are exposed and accessible to RTKs (Figure 5a). We asked whether receptor-mediated phosphorylation of GIV in cells requires an intact SH2-like domain of GIV, thereby bringing the substrate (GIV) in proximity to the kinase (EGFR). To answer this, we compared the extent of EGF-stimulated tyrosine phosphorylation of wild-type GIV to the SH2-deficient RL mutant and a previously described phosphorylation-deficient GIV-YF mutant (Lin et al., 2011); the latter is a negative control in which both tyrosines were replaced by Phe (F). Because nonreceptor TKs of the Src family can also phosphorylate GIV on those two tyrosines (Lin et al., 2011), we used the kinase inhibitor PP2 to abolish any contribution to GIV phosphorylation via the Src family of kinases, exactly as we used previously (Lin et al., 2011; Mittal et al., 2011). On EGF stimulation, wild-type GIV, but not the RL and the YF mutants, was tyrosine phosphorylated (Figure 5b), indicating that an intact SH2-like domain is essential for enhanced tyrosine phosphorylation in GIV. Because EGFR kinase phosphorylated the RL mutant just as efficiently as WT GIV-CT in vitro (Supplemental Figure S6b), its failure to be phosphorylated in cells (Figure 5b) indicates that the SH2-like function of GIV is required for enhanced phosphorylation of GIV by EGFR in cells.


Structural basis for activation of trimeric Gi proteins by multiple growth factor receptors via GIV/Girdin.

Lin C, Ear J, Midde K, Lopez-Sanchez I, Aznar N, Garcia-Marcos M, Kufareva I, Abagyan R, Ghosh P - Mol. Biol. Cell (2014)

The SH2-like domain of GIV is required for enhanced tyrosine phosphorylation of GIV and subsequent binding to RTKs. (a) Homology model of GIV's SH2 domain is depicted as a ribbon as in Figure 2d. The positions and the orientations of the two tyrosines are displayed in two views: from the “front” (pTyr-binding interface) and “back.” Both tyrosines are well exposed to solvent, consistent with the fact that multiple receptor and nonreceptor tyrosine kinases can readily access and phosphorylate them and that they can directly bind and activate PI3K (Lin et al., 2011). (b) Cos7 cells expressing FLAG-tagged wild-type GIV (GIV-WT FLAG), SH2-deficient R1745L mutant GIV (GIV-RL FLAG), a tyrosine phosphorylation–deficient mutant (GIV-YF FLAG; Lin et al., 2011), or vector alone were serum starved, pretreated with Src inhibitor (PP2), and subsequently stimulated with EGF before lysis. GIV was immunoprecipitated from equal aliquots of lysates (right) with FLAG mAb, and immunoprecipitates (left) were analyzed for GIV (red) and pTyr (green) by IB and dual-color imaging. The merge confirms that tyrosine-phosphorylated GIV (yellow) was immunoprecipitated exclusively from EGF-treated cells expressing GIV-WT (lane 3) but not from cells expressing GIV-RL (lane 4). As shown previously (Lin et al., 2011), the negative control GIV-YF was not phosphorylated (lane 5). The lysates (right) were analyzed for FLAG (GIV-FLAG), phospho-Akt (pAkt), and tubulin by IB. (c) Equal aliquots of His-GIV-CT proteins were first incubated with 10-fold molar excess of either phosphorylated (left lane) or unphosphorylated (right lane) EGFR tail peptides (sequence flanking Y1173) before in vitro kinase assays with recombinant EGFR kinase. GIV-CT is phosphorylated only in the presence of dephosphorylated EGFR peptide (which it cannot bind) but not in the presence of phospho-EGFR ligand. (d) EGFR was immunoprecipitated from lysates of HeLa cells starved and stimulated with EGF with anti-EGFR (528) mAb or control IgG. Immune complexes were analyzed for the presence of tyrosine-phosphorylated GIV using anti-pY1764GIV and ligand-activated EGFR (pY1173 EGFR) by IB. (e) InsR was immunoprecipitated from lysates of Cos7 cells starved and stimulated with insulin with anti-pYInsR mAb. Immune complexes were analyzed for the presence of tyrosine phosphorylated GIV using anti-pY1764GIV and ligand-activated InsR (pYInsR) by IB.
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Figure 5: The SH2-like domain of GIV is required for enhanced tyrosine phosphorylation of GIV and subsequent binding to RTKs. (a) Homology model of GIV's SH2 domain is depicted as a ribbon as in Figure 2d. The positions and the orientations of the two tyrosines are displayed in two views: from the “front” (pTyr-binding interface) and “back.” Both tyrosines are well exposed to solvent, consistent with the fact that multiple receptor and nonreceptor tyrosine kinases can readily access and phosphorylate them and that they can directly bind and activate PI3K (Lin et al., 2011). (b) Cos7 cells expressing FLAG-tagged wild-type GIV (GIV-WT FLAG), SH2-deficient R1745L mutant GIV (GIV-RL FLAG), a tyrosine phosphorylation–deficient mutant (GIV-YF FLAG; Lin et al., 2011), or vector alone were serum starved, pretreated with Src inhibitor (PP2), and subsequently stimulated with EGF before lysis. GIV was immunoprecipitated from equal aliquots of lysates (right) with FLAG mAb, and immunoprecipitates (left) were analyzed for GIV (red) and pTyr (green) by IB and dual-color imaging. The merge confirms that tyrosine-phosphorylated GIV (yellow) was immunoprecipitated exclusively from EGF-treated cells expressing GIV-WT (lane 3) but not from cells expressing GIV-RL (lane 4). As shown previously (Lin et al., 2011), the negative control GIV-YF was not phosphorylated (lane 5). The lysates (right) were analyzed for FLAG (GIV-FLAG), phospho-Akt (pAkt), and tubulin by IB. (c) Equal aliquots of His-GIV-CT proteins were first incubated with 10-fold molar excess of either phosphorylated (left lane) or unphosphorylated (right lane) EGFR tail peptides (sequence flanking Y1173) before in vitro kinase assays with recombinant EGFR kinase. GIV-CT is phosphorylated only in the presence of dephosphorylated EGFR peptide (which it cannot bind) but not in the presence of phospho-EGFR ligand. (d) EGFR was immunoprecipitated from lysates of HeLa cells starved and stimulated with EGF with anti-EGFR (528) mAb or control IgG. Immune complexes were analyzed for the presence of tyrosine-phosphorylated GIV using anti-pY1764GIV and ligand-activated EGFR (pY1173 EGFR) by IB. (e) InsR was immunoprecipitated from lysates of Cos7 cells starved and stimulated with insulin with anti-pYInsR mAb. Immune complexes were analyzed for the presence of tyrosine phosphorylated GIV using anti-pY1764GIV and ligand-activated InsR (pYInsR) by IB.
Mentions: We previously demonstrated that upon ligand stimulation, multiple RTKs phosphorylate GIV at two critical tyrosines, Y1764 and Y1798, and that both sites are capable of binding and activating class 1A PI3Ks (Lin et al., 2011). Although these two tyrosines are located within the boundaries of the SH2-like domain of GIV (Figure 5a and Supplemental Figure S6a), the homology model predicts that they are exposed and accessible to RTKs (Figure 5a). We asked whether receptor-mediated phosphorylation of GIV in cells requires an intact SH2-like domain of GIV, thereby bringing the substrate (GIV) in proximity to the kinase (EGFR). To answer this, we compared the extent of EGF-stimulated tyrosine phosphorylation of wild-type GIV to the SH2-deficient RL mutant and a previously described phosphorylation-deficient GIV-YF mutant (Lin et al., 2011); the latter is a negative control in which both tyrosines were replaced by Phe (F). Because nonreceptor TKs of the Src family can also phosphorylate GIV on those two tyrosines (Lin et al., 2011), we used the kinase inhibitor PP2 to abolish any contribution to GIV phosphorylation via the Src family of kinases, exactly as we used previously (Lin et al., 2011; Mittal et al., 2011). On EGF stimulation, wild-type GIV, but not the RL and the YF mutants, was tyrosine phosphorylated (Figure 5b), indicating that an intact SH2-like domain is essential for enhanced tyrosine phosphorylation in GIV. Because EGFR kinase phosphorylated the RL mutant just as efficiently as WT GIV-CT in vitro (Supplemental Figure S6b), its failure to be phosphorylated in cells (Figure 5b) indicates that the SH2-like function of GIV is required for enhanced phosphorylation of GIV by EGFR in cells.

Bottom Line: We discovered a unifying mechanism that allows GIV/Girdin, a bona fide metastasis-related protein and a guanine-nucleotide exchange factor (GEF) for Gαi, to serve as a direct platform for multiple RTKs to activate Gαi proteins.Using a combination of homology modeling, protein-protein interaction, and kinase assays, we demonstrate that a stretch of ∼110 amino acids within GIV C-terminus displays structural plasticity that allows folding into a SH2-like domain in the presence of phosphotyrosine ligands.Expression of a SH2-deficient GIV mutant (Arg 1745→Leu) that cannot bind RTKs impaired all previously demonstrated functions of GIV-Akt enhancement, actin remodeling, and cell migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California, San Diego, School of Medicine, CA 92093.

Show MeSH
Related in: MedlinePlus