Focal adhesions are foci for tyrosine-based signal transduction via GIV/Girdin and G proteins.
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.
Affiliation: Department of Medicine, University of California, San Diego, School of Medicine, La Jolla, CA 92093 email@example.com firstname.lastname@example.org.
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Figure 2: Phosphorylation of GIV by FAK is required for cell–ECM adhesion and collagen-induced haptotaxis. (A) In vitro kinase assays were carried out with recombinant FAK and equal aliquots of either WT or mutant His-GIV-CT (aa 1660–1870) proteins. Phosphorylated GIV was detected by immunoblotting using anti–phospho-Tyr antibody (top; green). His-GIV-CT proteins were visualized using GIV-CT antibody (middle; red). Yellow pixels in the merged panel represent tyrosine-phosphorylated GIV-CT. (B) Cos7 cells expressing control vector, wild-type FAK (myc-FAK-WT), or a kinase-dead mutant FAK (myc-FAK-KD) were analyzed by immunoblotting for phosphorylation of endogenous GIV using anti–phospho-Tyr-1764-GIV (pYGIV) antibody. Expression of FAK (myc), Gαi3, and tubulin was analyzed by immunoblotting. (C) Cos7 cells were grown on culture dishes coated with poly-d-lysine, treated with FAK inhibitor 14 (FAK-Inh-14) or vehicle (dimethyl sulfoxide [DMSO]) for 24 h, and then seeded on collagen-coated dishes for 1 h before lysis and analyzed for total (t) and phosphoproteins (p) by immunoblotting (IB). (D) Cos7 cells were acutely stimulated with collagen before fixation (see Materials and Methods) and stained with phospho–Tyr-1764-GIV (pYGIV; green), phospho–Tyr-397-FAK (pYFAK; red), and DAPI (nuclei; blue) and analyzed by confocal microscopy. Bar, 25 μm. (E) Cos7 cells were analyzed for interaction between active pYGIV and active pYFAK by in situ PLA using rabbit anti-pYGIV and mouse anti-pYFAK antibodies. Red dots indicate sites of interaction. Incubation with secondary antibodies alone or with paxillin antibody showed no signal (negative controls; Supplemental Figure S2A). Bar, 25 μm. (F) GIV-depleted Cos7 cells stably expressing FLAG-tagged WT or mutant GIV constructs were grown on culture dishes coated with poly-d-Lysine (P) and then seeded on collagen-coated (C) dishes for 1 h before lysis and analyzed for phospho (p-) and total (t-) Akt by immunoblotting (IB). Compared to GIV-WT cells, percentage phosphorylation of Akt was suppressed by 65% in GIV-YF cells, 82% in GIV-FA cells, and 73% in GIV-YF/FA cells (p < 0.001), as determined by band densitometry using LiCOR Odyssey. (G, H) GIV-depleted (sh GIV) Cos7 cells stably expressing GIV-WT or GIV-YF mutant were fixed and stained for phospho–Tyr-1764-GIV (pYGIV; green), vinculin (red), and DAPI (nuclei; blue; G) or for paxillin (red, shown in grayscale; H) and analyzed by confocal microscopy. Bar, 25 μm. (I) Colorimetric adhesion assays were carried out using control (sh Control), GIV-depleted (sh GIV), or GIV-depleted cells stably expressing various GIV constructs in media with low serum. Error bars represent mean ± SD; n = 3; **p < 0.01; ***p < 0.001; n.s, not significant. See also Supplemental Figure S2, B and C, for immunoblots showing the levels of GIV expression and other FA proteins in these cell lines. (J) Cells in I were analyzed for collagen-induced haptotactic cell motility using Transwell assays. Images of representative fields are displayed in Supplemental Figure S2D. Bar graphs show quantification of the number of cells that migrated averaged from ∼ 20 high-power field-of-view images per experiment. Error bars represent mean ± SD; n = 3; *p < 0.05, **p < 0.01, ***p < 0.001.
Because tyrosine-phosphorylated GIV is primarily restricted to FAs (Figure 1), we asked whether GIV is a substrate of the non-RTK focal adhesion kinase (FAK), a key component of the signal transduction pathways triggered by integrins whose activity is restricted to the FAs (Sulzmaier et al., 2014). In vitro kinase assays on the histidine (His)-tagged C-terminus of GIV (GIV-CT) showed that such is indeed the case; GIV was phosphorylated at two critical sites, Tyr (Y)-1764 and -1798, by recombinant FAK, and no phosphorylation was observed using a mutant in which both tyrosines were replaced with Phe (YF; Figure 2A). Because the commercially obtained FAK used in these assays was purified from insect cells to >95% purity, we conclude that FAK phosphorylates GIV; it targets the two key tyrosines on GIV that were previously shown to directly bind Src-homology 2 domains of p85α (PI3K) and activate the p110 (PI3K) catalytic subunit (Lin et al., 2011). FAK also phosphorylated GIV in cells because expression of the wild type but not the kinase-dead mutant FAK(K454R) in Cos7 cells triggered tyrosine phosphorylation of GIV (Figure 2B) and pharmacologic inhibition of FAK abolished tyrosine phosphorylation of GIV (Figure 2C). Immunofluorescence on Cos7 cells undergoing adhesion on collagen-coated surface showed that by 30 min, active FAK, as determined by phosphorylation of Y397 (Chen et al., 1996), and active pYGIV colocalized extensively within nascent FAs at the cell periphery (Figure 2D). By 4 h, extensive colocalization was maintained but shifted to mature FAs (Figure 2D). Whereas ∼50% of the cells showed pYGIV labeling of nascent FAs at the cell periphery without paxillin in those structures, the reverse (i.e., paxillin-labeled structures devoid of pYGIV) was never encountered (Supplemental Figure S1F). Thus the localization of active pYGIV to FAs coincided temporospatially with activated FAK and preceded the localization of paxillin to FAs.