FIGURE 6.

GSK-3β phosphorylation-defective p190A mutants fail to rescue the directional migration defects in p190A-deficient cells. A, stable cell lines derived from p190A^-/- fibroblasts, expressing either pEGFP-C1 vector alone or with pEGFP-C1-p190A-WT (full-length) or the various phosphorylation site mutants, were established. Immunoblots demonstrate expression of p190A. p120 RasGAP was also detected as a loading control. B, wound closure assays of the various stable cell lines as indicated. Photomicrographs were taken immediately after and 15 h after wounding. The dashed lines correspond to the boundaries of cell monolayers along the wound edge. Note that the wound closure defect can be rescued by wild-type p190A and the 1472A and 1483A mutants but not by the 1476A and 1480A mutants. C, Golgi reorientation 6 h post-wounding with the indicated p190A-expressing cell lines as indicated. Cells were seeded on coverslips and grown to confluence. A single wound was induced, and the cells were fixed at 6 h post-wounding. They were then double-stained with anti-GM130 and DAPI, and immunofluorescence microscopy was performed. For each genotype, 200 cells were counted for two individual lines. Graphs depict the mean from three independent experiments, and S.D. are indicated by the error bars. The dashed horizontal line corresponds to the 33% value, which would reflect random polarization. Note that the 1476A and 1480A mutants are defective for Golgi reorientation. D, F-actin staining (phalloidin) at 6 h post-wounding of the indicated p190A-expressing cell lines. White arrows highlight the oriented protrusions along the leading edge.