FIGURE 6.

GIV enhances STAT3 activation via its GEF motif. A–C, HeLa-GIV-WT, but not HeLa-GIV-FA, cells enhance STAT3 activation in response to EGF and scratch wounding. A, serum-starved HeLa WT and FA cell lines were stimulated with 50 nm EGF for the indicated time periods, and whole cell lysates were analyzed for total (t) and phospho (p)-Tyr-705 and -Ser-727 STAT3, Akt, ERK1/2, and tubulin by immunoblotting (IB). B, kinetics of EGF-initiated STAT3 signaling in HeLa WT and FA cell lines in A was determined by phospho-Tyr-705 STAT3:total STAT3 ratios at each time point after EGF stimulation and expressed as absolute values. The p value was calculated by comparing STAT3 activity in HeLa-GIV-WT and HeLa-GIV-FA cells at 6 h postwounding. C, monolayers of HeLa-GIV-WT and HeLa-GIV-FA cells were scratch-wounded and subsequently harvested at 6 h. Whole cell lysates were prepared and analyzed for Tyr(P)-705 and Ser(P)-727 STAT3 and tubulin by immunoblotting. D, upon activation, STAT3 localizes to the nucleus in HeLa-GIV-WT, but not in HeLa-GIV-FA, cells after ligand stimulation. HeLa cell lines were stimulated with 50 nm EGF for 5 min and costained for STAT3 (red) and the nucleus (DAPI; blue). Bar, 10 μm. E and F, HeLa-GIV-WT, but not HeLa-GIV-FA cells up-regulate GIV mRNA (E) and protein (F) levels after scratch wounding. Confluent monolayers of HeLa WT and FA cell lines were scratch-wounded, harvested at the indicated time points, and analyzed for GIV mRNA and protein by qPCR and immunoblotting, respectively. The p value in E was calculated by comparing GIV mRNA in HeLa-GIV-WT and HeLa-GIV-FA cells at 6 h postwounding. G–I, the GEF function of GIV is required for induction of STAT3 target genes Bcl-2, MMP2, and MMP9 after scratch wounding. Monolayers of HeLa-GIV-WT and HeLa-GIV-FA cells were scratch-wounded and subsequently harvested at 6 h. mRNA levels of Bcl-2 (G), MMP2 (H), and MMP9 (I) were analyzed by qPCR. Error bars represent S.D. *, p < 0.05; **, p < 0.01.