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Focal adhesions are foci for tyrosine-based signal transduction via GIV/Girdin and G proteins.

Lopez-Sanchez I, Kalogriopoulos N, Lo IC, Kabir F, Midde KK, Wang H, Ghosh P - Mol. Biol. Cell (2015)

Bottom Line: As a guanidine exchange factor (GEF), GIV modulates signals initiated by growth factors (chemical signals) by activating the G protein Gαi.Activation of Gαi by GIV-GEF further potentiates FAK-GIV-PI3K-Akt signaling at the FAs.Spatially restricted signaling via tyrosine phosphorylated GIV at the FAs is enhanced during cancer metastasis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California, San Diego, School of Medicine, La Jolla, CA 92093 inmalopezsanchez@hotmail.com prghosh@ucsd.edu.

No MeSH data available.


Related in: MedlinePlus

GIV is essential for the integrity of FAs and ECM-induced chemotaxis in metastatic cancer cells. Control (sh Control) or GIV-depleted (sh GIV) MDA MB-231 (left), Hs578T (middle), and HeLa (right) cells were fixed and stained for vinculin (A) or pYFAK (B) and analyzed by confocal microscopy. Bar, 25 μm. Depletion of GIV was confirmed by immunoblotting (Supplemental Figure S4, B–D). (C) Cell lines in A were analyzed for collagen-induced haptotactic cell motility using Transwell assays as in Figure 1H. Bar graphs show quantification of the number of migrating cells per high-power field (HPF). Data are presented as mean ± SEM; n = 3. Representative fields of the Transwell membrane are shown in Supplemental Figure S4E. *p < 0.05, ***p < 0.001. (D) Parental H2030 (left) and its corresponding metastatic BrM subclone (right) were fixed and stained for phospho–Y1764-GIV (pYGIV; red), vinculin (green), and DAPI (nuclei; blue) and analyzed by confocal microscopy. Representative fields are shown. Bar, 25 μm. (E) Bar graphs show quantification of the number of pYGIV-stained, vinculin-positive FA structures per cell in D (y-axis) determined using ImageJ. Error bars represent mean ± SD. ****p < 0.0001. (F) Immunoblotting of whole-cell lysates of cells in D showed similar levels of GIV, vinculin, and tubulin.
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Figure 5: GIV is essential for the integrity of FAs and ECM-induced chemotaxis in metastatic cancer cells. Control (sh Control) or GIV-depleted (sh GIV) MDA MB-231 (left), Hs578T (middle), and HeLa (right) cells were fixed and stained for vinculin (A) or pYFAK (B) and analyzed by confocal microscopy. Bar, 25 μm. Depletion of GIV was confirmed by immunoblotting (Supplemental Figure S4, B–D). (C) Cell lines in A were analyzed for collagen-induced haptotactic cell motility using Transwell assays as in Figure 1H. Bar graphs show quantification of the number of migrating cells per high-power field (HPF). Data are presented as mean ± SEM; n = 3. Representative fields of the Transwell membrane are shown in Supplemental Figure S4E. *p < 0.05, ***p < 0.001. (D) Parental H2030 (left) and its corresponding metastatic BrM subclone (right) were fixed and stained for phospho–Y1764-GIV (pYGIV; red), vinculin (green), and DAPI (nuclei; blue) and analyzed by confocal microscopy. Representative fields are shown. Bar, 25 μm. (E) Bar graphs show quantification of the number of pYGIV-stained, vinculin-positive FA structures per cell in D (y-axis) determined using ImageJ. Error bars represent mean ± SD. ****p < 0.0001. (F) Immunoblotting of whole-cell lysates of cells in D showed similar levels of GIV, vinculin, and tubulin.

Mentions: Because both GIV and FAK facilitate cancer progression (Ghosh et al., 2011; Sulzmaier et al., 2014), next we examined the distribution and function of GIV in multiple cancer cells. Tyrosine-phosphorylated GIV specifically localized to FAs across all cancer cell lines examined (Figure 5A and Supplemental Figure S4A). Depletion of GIV (by shRNA, ∼75-85% efficacy; Supplemental Figure S4, B–D) resulted in a decrease of FAs, as determined by a shift of vinculin (Figure 5A) and paxillin (Supplemental Figure S4A) from FAs to the cytosol, reduced FAK activity (Figure 5B), and impaired ECM-induced cell motility (Figure 5C and Supplemental Figure S4E), indicating that GIV is an essential functional component of FAs in multiple cancer cells. To determine how phosphorylation of GIV and its localization at FAs changes during cancer invasion/metastasis, we used a well-characterized metastatic H2030 lung adenocarcinoma cell line and its corresponding highly metastatic subclone 3 (BrM3) that were selected in mice and display ∼10-fold enhanced ability to metastasize to the bones and brain (Nguyen et al., 2009). Tyrosine-phosphorylated GIV showed a heterogeneous staining pattern (intensity and distribution). Despite heterogeneity, tyrosine-phosphorylated GIV colocalized with vinculin-positive FAs exclusively in the BrM3 clone (Figure 5, D–F). These findings indicate that metastatic progression is associated with increased association of active pYGIV with FAs.


Focal adhesions are foci for tyrosine-based signal transduction via GIV/Girdin and G proteins.

Lopez-Sanchez I, Kalogriopoulos N, Lo IC, Kabir F, Midde KK, Wang H, Ghosh P - Mol. Biol. Cell (2015)

GIV is essential for the integrity of FAs and ECM-induced chemotaxis in metastatic cancer cells. Control (sh Control) or GIV-depleted (sh GIV) MDA MB-231 (left), Hs578T (middle), and HeLa (right) cells were fixed and stained for vinculin (A) or pYFAK (B) and analyzed by confocal microscopy. Bar, 25 μm. Depletion of GIV was confirmed by immunoblotting (Supplemental Figure S4, B–D). (C) Cell lines in A were analyzed for collagen-induced haptotactic cell motility using Transwell assays as in Figure 1H. Bar graphs show quantification of the number of migrating cells per high-power field (HPF). Data are presented as mean ± SEM; n = 3. Representative fields of the Transwell membrane are shown in Supplemental Figure S4E. *p < 0.05, ***p < 0.001. (D) Parental H2030 (left) and its corresponding metastatic BrM subclone (right) were fixed and stained for phospho–Y1764-GIV (pYGIV; red), vinculin (green), and DAPI (nuclei; blue) and analyzed by confocal microscopy. Representative fields are shown. Bar, 25 μm. (E) Bar graphs show quantification of the number of pYGIV-stained, vinculin-positive FA structures per cell in D (y-axis) determined using ImageJ. Error bars represent mean ± SD. ****p < 0.0001. (F) Immunoblotting of whole-cell lysates of cells in D showed similar levels of GIV, vinculin, and tubulin.
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Figure 5: GIV is essential for the integrity of FAs and ECM-induced chemotaxis in metastatic cancer cells. Control (sh Control) or GIV-depleted (sh GIV) MDA MB-231 (left), Hs578T (middle), and HeLa (right) cells were fixed and stained for vinculin (A) or pYFAK (B) and analyzed by confocal microscopy. Bar, 25 μm. Depletion of GIV was confirmed by immunoblotting (Supplemental Figure S4, B–D). (C) Cell lines in A were analyzed for collagen-induced haptotactic cell motility using Transwell assays as in Figure 1H. Bar graphs show quantification of the number of migrating cells per high-power field (HPF). Data are presented as mean ± SEM; n = 3. Representative fields of the Transwell membrane are shown in Supplemental Figure S4E. *p < 0.05, ***p < 0.001. (D) Parental H2030 (left) and its corresponding metastatic BrM subclone (right) were fixed and stained for phospho–Y1764-GIV (pYGIV; red), vinculin (green), and DAPI (nuclei; blue) and analyzed by confocal microscopy. Representative fields are shown. Bar, 25 μm. (E) Bar graphs show quantification of the number of pYGIV-stained, vinculin-positive FA structures per cell in D (y-axis) determined using ImageJ. Error bars represent mean ± SD. ****p < 0.0001. (F) Immunoblotting of whole-cell lysates of cells in D showed similar levels of GIV, vinculin, and tubulin.
Mentions: Because both GIV and FAK facilitate cancer progression (Ghosh et al., 2011; Sulzmaier et al., 2014), next we examined the distribution and function of GIV in multiple cancer cells. Tyrosine-phosphorylated GIV specifically localized to FAs across all cancer cell lines examined (Figure 5A and Supplemental Figure S4A). Depletion of GIV (by shRNA, ∼75-85% efficacy; Supplemental Figure S4, B–D) resulted in a decrease of FAs, as determined by a shift of vinculin (Figure 5A) and paxillin (Supplemental Figure S4A) from FAs to the cytosol, reduced FAK activity (Figure 5B), and impaired ECM-induced cell motility (Figure 5C and Supplemental Figure S4E), indicating that GIV is an essential functional component of FAs in multiple cancer cells. To determine how phosphorylation of GIV and its localization at FAs changes during cancer invasion/metastasis, we used a well-characterized metastatic H2030 lung adenocarcinoma cell line and its corresponding highly metastatic subclone 3 (BrM3) that were selected in mice and display ∼10-fold enhanced ability to metastasize to the bones and brain (Nguyen et al., 2009). Tyrosine-phosphorylated GIV showed a heterogeneous staining pattern (intensity and distribution). Despite heterogeneity, tyrosine-phosphorylated GIV colocalized with vinculin-positive FAs exclusively in the BrM3 clone (Figure 5, D–F). These findings indicate that metastatic progression is associated with increased association of active pYGIV with FAs.

Bottom Line: As a guanidine exchange factor (GEF), GIV modulates signals initiated by growth factors (chemical signals) by activating the G protein Gαi.Activation of Gαi by GIV-GEF further potentiates FAK-GIV-PI3K-Akt signaling at the FAs.Spatially restricted signaling via tyrosine phosphorylated GIV at the FAs is enhanced during cancer metastasis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California, San Diego, School of Medicine, La Jolla, CA 92093 inmalopezsanchez@hotmail.com prghosh@ucsd.edu.

No MeSH data available.


Related in: MedlinePlus