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Fig. 2. Tyrosine-phosphorylated villin regulates intestinal cell migration. A: 2-wk-old postconfluent Caco-2 cells were infected with adenovirus-expressing vector alone (vector) or the villin phosphorylation site mutant VIL/AYFM. A quantitative Western blot performed with anti-hemagglutinin (HA) antibody and anti-villin monoclonal antibody shows the expression of VIL/AYFM relative to endogenous villin protein. A quantitative Western blot with anti-actin antibody was performed in parallel. Western blots are representative of 3 experiments with similar results. B: infection of Caco-2 cells with adenovirus expressing VIL/AYFM inhibited both basal and EGF-stimulated cell migration. Cells infected with vector alone migrated similarly to uninfected cells (data not shown). Data were normalized to control values (set as 100) and expressed as percent change in migration compared with control. Control refers to cell migration at 24 h postwounding in the absence of growth factors in cells expressing vector alone. Values are means ± SE. *P < 0.001, statistically significant compared with control cells.  P < 0.001, statistically significant compared with EGF-treated cells expressing vector alone. C: overexpression of VIL/AYFM decreased both tyrosine phosphorylation of villin and formation of phospho-villin-PLC- 1 complex. Quantitative immunoprecipitation shows a 50% decrease in tyrosine-phosphorylated villin levels and a 58% decrease in association of phospho-villin with PLC-1 in Caco-2 cells infected with adenovirus expressing VIL/AYFM. Bottom blot with anti-actin antibody was performed in parallel as a quantitative control. Western blots are representative of 3 experiments with similar results.
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