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. Author manuscript; available in PMC: 2014 Nov 10.
Published in final edited form as: Sci Signal. 2011 Sep 27;4(192):ra64. doi: 10.1126/scisignal.2002049

Figure 2. Tyrosine phosphorylation of GIV does not affect its ability to bind or activate Gαi, but is required for phosphorylation of Akt, actin remodeling, and cell migration.

Figure 2

A. HeLa cells stably expressing control vector or siRNA resistant GIV-WT-FLAG or GIV-YF-FLAG plasmids were transfected with GIV siRNA. Lysates were immunoblotted for GIV, phospho-Akt (pAkt), Gαi3, and tubulin. Phosphorylation of Akt (pAkt) was significantly reduced in GIV-depleted control cells (lane 2) and GIV-YF cells (lane 3) compared to GIV-WT cells (lane 1). p < 0.001 for both sets of comparisons; N = 3 experiments. B. HeLa cells stably expressing vector (control), siRNA resistant GIV-WT-FLAG, GIV-FA-FLAG (the GEF-deficient mutant), or GIV-YF-FLAG plasmids were treated with scrambled or GIV siRNA as indicated. Cells were costained with phalliodin-Texas red (F-actin, red) and DAPI (DNA, blue). Depletion of GIV in control cells resulted in loss of stress fibers, which were restored by expression of siRNA resistant GIV-WT-FLAG, but not GIV-FA-FLAG or GIV-YF-FLAG. Bar = 10 μM. C. Untransfected HeLa cells (control; Ctr), HeLa-GIV-WT, HeLa-GIV-FA, and HeLa-GIV-YF cells were transfected with scrambled or GIV siRNA. Results are shown as mean ± S.D. of 8-12 randomly chosen fields from n=3 experiments. p < 0.001 for either sets of comparisons between Ctr and GIV-depleted cells and between GIV-WT-FLAG and GIV-YF-FLAG cells. D. Control HeLa cells and HeLa cells stably expressing siRNA resistant GIV-WT-FLAG, GIV-YF-FLAG, and GIV-FA-FLAG plasmids were transfected with scrambled and GIV siRNA and immunoblotted for GIV, phospho-Akt (pAkt), and tubulin. p -values for both comparisons between GIV-WT-FLAG and GIV-FA-FLAG and between GIV-WT-FLAG and GIVYF-FLAG are < 0.001; N = 3 experiments. (E) Mock-treated His-GIV-CT and in vitro EGFR-phosphorylated His-GIV-CT were incubated with GST-Gαi3 or GST preloaded with GDP immobilized on glutathione beads. Bound proteins were analyzed by two-color immunoblotting (IB) for GIV CT (His) and pTyr. (F) The amount of GTP hydrolyzed in 10 min by His-Gαi3 was determined in the presence of the indicated amounts of sham-treated and in vitro EGFR-phosphorylated His-GIV-CT.