FIGURE 3.

Alteration in the signaling induced by adhesion to ECM in the clone 4–17 and the cells with the vinculin gene knocked down. A, the truncated vinculin derived from the trapped allele was unable to bind to paxillin. COS7 cells were transfected with the expression plasmid encoding Myc-tagged paxillin (paxillin-Myc) together with the plasmid encoding V5-tagged wild type (vinculin(WT)-V5), the truncated vinculin corresponding to the mutant derived from the trapped allele in clone 4-17 (vinculin(1–624)-V5), or the mutant encoding the carboxyl fragment of vinculin (vinculin(625–1066)-V5), and lysates were harvested for immunoprecipitation (IP) using anti-V5 antibody. The immunoprecipitates and the input protein (10 μg) were subjected to Western blotting using the indicated antibodies. B, the binding to talin was enhanced in the truncated vinculin derived from the trapped allele. COS7 cells were transfected with the plasmid encoding vinculin(WT)-V5, vinculin(1–624)-V5, or vinculin(625–1066)-V5, and lysates were used for immunoprecipitation with anti-talin antibody. The immunoprecipitates and the input protein (10 μg) were subjected to Western blotting with the indicated antibodies. C, increase in phosphorylation of FAK at Tyr-397 and ERK1/2 induced by adhesion to FN in clone 4-17. Parental ATDC5 cells or clone 4-17 were plated onto FN. Thirty minutes later, lysates were harvested for Western blot analyses. D and E, increase in phosphorylation of FAK at Tyr-397 and ERK1/2 induced by adhesion to Col II in clone 4-17. Cells were plated onto Col II, and lysates were harvested at the indicated time points. The densitometric ratio of the intensity of the signal corresponding to phosphorylated FAK to that of total FAK is shown in E. The data are shown as the mean ± S.D. (error bars) (n = 3). *, p < 0.05 versus 0 h; #, p < 0.05 versus parental cells. F, knockdown of vinculin in parental ATDC5 cells increased the adhesion-induced phosphorylation of FAK and ERK1/2, which was cancelled by simultaneous introduction of the expression plasmid encoding vinculin(WT). Lysates were harvested for Western blotting from the parental ATDC5 cells transfected with control siRNA, vinculin-specific siRNA with or without the expression plasmids encoding vinculin(WT) or lacZ, the non-transfection control, and clone 4-17 with the vinculin gene trapped 30 min after the plating onto FN. G, exogenous expression of the truncated vinculin(1–624) in parental ATDC5 cells increased the adhesion-induced phosphorylation of FAK, which was not abrogated by the simultaneous expression of exogenous wild-type vinculin. Lysates were harvested from the parental ATDC5 cells infected with the adenoviral vector for vinculin(1–624)-V5 together with that for lacZ or vinculin(WT)-V5 and the cells expressing lacZ alone 30 min after the plating onto FN. H, exogenous expression of the truncated vinculin(625–1066) in parental ATDC5 cells had no effects on the adhesion-induced phosphorylation of FAK. ATDC5 cells were infected with the vector for vinculin(625–1066)-V5 together with that for lacZ or vinculin(WT)-V5 and the cells expressing lacZ alone, and lysates were harvested 30 min after the plating onto FN. I, the truncated vinculin(1–624) interfered with the interaction between the wild-type vinculin and talin. COS7 cells were transfected with Myc-tagged wild-type vinculin (vinculin(WT)-Myc) together with vinculin(WT)-V5 or the mutant vinculin(1–624)-V5, and the lysates were subjected to immunoprecipitation using anti-talin antibody. The immunoprecipitates and the input protein (10 μg) were subjected to Western blotting using the indicated antibodies. In the immunoblot using anti-Myc antibody, the co-transfection of vinculin(1–624)-V5 reduced the signal for the co-immunoprecipitation of vinculin(WT)-Myc with talin. p-FAK, phosphorylated FAK; t-FAK, total FAK.