Figure 4.

Cleaved forms of β-catenin are transcriptionally active. (A) Schematic of β-catenin constructs with caspase 3 cleavage sites denoted by black arrowheads. (B) Expression of β-catenin mutants. Lane 1, mock transfection; lane 2, β-cat^WT; lane 3, β-cat^116–781; lane 4, β-cat^116–764; lane 5, β-cat^116–751; lane 6, N-Adh Tsc2^−/− (8) cells; lane 7, N-Adh Tsc2^−/− (4a) cells. (C) Coimmunoprecipitation experiments show that truncated β-catenin proteins identified in N-Adh Tsc2^−/− cells do not associate with GSK3β (upper and lower panels, lanes 2 and 4). Left panels, GSK3β immunoprecipitates; right panels, β-catenin immunoprecipitates; upper panels, β-catenin immunoblots; lower panels, GSK3β immunoblots. (D) TOPFLASH reporter assays demonstrate that β-catenin mutants maintain significant transcriptional activity (compare lanes 4 and 5 with lane 2). (E) Cyclin D1 reporter assays demonstrate that β-catenin mutants maintain some activities (compare lanes 4 and 5 with lane 2). (F) MMP7 reporter assays show that β-catenin mutants are more active than wild-type β-catenin (compare lanes 4 and 5 with lane 2). Lane 1, mock; lane 2, β-cat^WT; lane 3, β-cat^116–781; lane 4, β-cat^116–764; lane 5, β-cat^116–751; lane 6, β-cat^WT and dominant negative TCF-4. Each reporter assay was performed in duplicate, and the results shown represent the mean values ± SEM of three independent experiments, presented as the relative fold increase (relative light units, RLU) over mock-transfected cells (set at a value of 1). *P < 0.05 as compared with mock samples. (G) RT-PCR analysis demonstrates the up-regulation of MMP7 mRNA in N-Adh Tsc2^−/− cells (upper panel). Expression of GAPDH serves as a control for RNA expression (lower panel). (H) β-catenin mutant overexpression induces MMP7 protein expression (lanes 4 and 5). HEK293 cells were transfected with the indicated constructs: lane 1, mock transfection; lane 2, β-cat^WT; lane 3, β-cat^116–781; lane 4, β-cat^116–764; lane 5, β-cat^116–751. Cell lysates were immunoprecipitated with MMP7 antibody to detect MMP7 protein (upper panel, lanes 4 and 5). Lower panel, Western immunoblot of IgG bands of MMP7 immunoprecipitated samples serves as a loading control.