GSK-3 negatively affects δ-catenin levels. A, CWR22Rv-1 cells were treated with different concentrations of LiCl, a specific inhibitor of GSK-3 (10, 20, and 30 mm) for 12 h. The cells were then harvested, and the endogenous δ-catenin was detected using anti-δ-catenin antibody. β-Catenin was used as positive control (Ctrl) to determine the effect of LiCl, and α-actin was used as a loading control. B, primary cortical neurons were treated with 1 mm of LiCl for 12 h. Endogenous δ-catenin was detected using anti-δ-catenin antibody. Actin was used as a loading control. C, CWR22Rv-1 cells were treated with GSK-3 inhibitor XV (0.6 nm; Calbiochem) for 16 h. β-Catenin was used as a positive control, and actin was used as a loading control. D, CWR22Rv-1 cells were treated with Wnt-3a or control conditioned media for 8 h. Endogenous δ-catenin was detected using anti-δ-catenin antibody. β-Catenin was used as a positive control, and actin was used as a loading control. E, CWR22Rv-1 cells were transfected with two different doses of the GSK-3α and -3β KD tagged with HA. GSK-3α and -3β KD were detected with anti-HA and actin as a loading control. F, CWR22Rv-1 cells were transfected with either siRNA for GSK-3α/β or GSK-3α (panel i) and GSK-3β^+/+ cells were transfected with δ-catenin together with either siRNA for GSK-3α/β or GSK-3α (panel ii). Control siRNA was also transfected and used as a negative control. Endogenous (panel i) or exogenous δ-catenin (panel ii) was detected with anti-δ-catenin antibody, and GSK-3α/β was detected with anti-GSK-3α/β antibody.

GSK-3 phosphorylates δ-catenin, and the Thr¹⁰⁷⁸ residue of δ-catenin is one of multiple phosphorylation sites by GSK-3. In vitro kinase assays were performed as described under “Experimental Procedures.” A and E, GFP protein was used as a negative control. Full-length (A) or deletion constructs of δ-catenin (ΔC207, ΔN85–325, ΔN85–325/C207, and ΔN19–1153) (E) were transiently transfected into HEK293 cells. The level of each protein used for kinase assays was represented by Western blot (D). B, GSK-3α WT/KD, GSK-3β WT/KD, or δ-catenin were transfected into HEK293 cells. Each immunocomplex was formed by immunoprecipitating with anti-HA antibody or anti-δ-catenin antibody and protein G-Sepharose. HEK293 cell lysate (indicated as none) was used as a negative control. After mixing δ-catenin immunocomplex and the immunocomplexes of GSK-3α WT/KD or GSK-3β WT/KD, the mixed immunocomplexes were incubated for 30 min at 30 °C in a 20-μl reaction mixture with 10 μCi of [γ-³²P]ATP. C, purified immunocomplex of 1 day post-natal mouse brain lysate with anti-δ-catenin antibody was used as substrates for in vitro kinase assays. D, a schematic diagram of δ-catenin constructs. F, T1078A-δ-catenin, T337A-δ-catenin, or δ-catenin WT were transiently transfected into HEK293 cells, and δ-catenin immunocomplexes were used for GSK-3 kinase assays as described under “Experimental Procedures.”

δ-Catenin undergoes GSK-3-mediated ubiquitination and proteosome-dependent degradation. A, MEF cells were transfected with Ub-HA alone, δ-catenin alone, or Ub-HA+δ-catenin as indicated. The cells were treated with MG132 for 4 h prior to lysis and then anti-δ-catenin immunoprecipitations (IP) followed by anti-HA or anti-δ-catenin Western analyses were carried out on the extracts (top and middle panels). Anti-δ-catenin Western blot (WB) analysis was also performed on cell extracts (bottom panel). B, MEF cells were transfected with δ-catenin and Ub-HA. The cells were treated with MG132 for different times, as indicated. The extracts were subjected to immunoprecipitation and Western analysis with anti-HA or anti-δ-catenin antibody (top and middle panels). For lysates Western analysis was also performed with anti-δ-catenin (bottom panel). C, MEF cells were transfected Ub-HA alone, δ-catenin alone, and co-transfected with δ-catenin, Ub-HA, and with or without GSK-3β WT. Extracts of adjusted amounts of δ-catenin in these two groups to be equal were immunoprecipitated by anti-δ-catenin antibody, and Western analyses were performed by anti-HA or anti-δ-catenin antibody (top and second panels). Anti-δ-catenin antibody was used for detecting δ-catenin and ubiquitinated δ-catenin on cell extracts (third panel). Anti-HA antibody was used for detecting GSK-3β WT (bottom panel). D, MEF cells were co-transfected with δ-catenin and Ub-HA. The cells were treated with or without LiCl for 4 h and then treated with MG132 for 2 h. Immunoprecipitations were performed with anti-δ-catenin, and Western analyses were performed with anti-HA or anti-δ-catenin (top and second panels). Anti-δ-catenin Western blot analysis was also performed on cell extracts (third panel). β-Catenin was used for confirming effects of LiCl on cells (bottom panel). E, MEF cells were transfected with Ub-HA and with either T1078-δ-catenin or δ-catenin WT. The cells were treated with MG132 for 4 h prior to lysis. Immunoprecipitations were performed with anti-δ-catenin, and Western analyses were performed with anti-HA (top panel). Anti-δ-catenin Western blot analysis was also performed on cell extracts (bottom panel). ◀ indicates δ-catenin and ◀* indicates ubiquitinated δ-catenin. F, GSK-3β^+/+ fibroblasts were transfected with either wild type or T1078A δ-catenin. Pulse-chase experiments were performed as described in the legend to Fig. 1. F, graph represented the relative intensities of δ-catenin. Band intensities at the various time points were measured using TINA 2.09 software program (Raytest).

GSK-3β affects the level of δ-catenin. A, δ-catenin was transfected into GSK-3β^+/+ fibroblast cells and GSK-3β^−/− fibroblast cells. δ-Catenin, GSK-3α and -3β, and actin proteins were detected using its specific antibody. B, GSK-3β^+/+ and GSK-3β^−/− fibroblast cells were transfected with δ-catenin. The cells were treated with methionine/cysteine-free DMEM (Sigma) containing 1% fetal bovine serum and 0.04 m l-glutamine (Sigma) overnight. After cells were treated with new conditioned medium containing 150 μCi of ³⁵S for 1 h, the cells were harvested at indicated time. The extracts were immunoprecipitated with anti-δ-catenin. C, graph represented the relative intensities of δ-catenin. Band intensities at the various time points in B were measured using TINA 2.09 software program (Raytest). D, after GSK-3β^+/+ fibroblast cells and GSK-3β^−/− fibroblast cells had been transfected with δ-catenin, the cells were treated with cycloheximide (40 μg/ml) and harvested at the times indicated. β-Tubulin was used as a loading control.

δ-Catenin interacts with GSK-3α or GSK-3β. A, purified immunocomplex of 1 day post-natal mouse brain lysate with anti-δ-catenin antibody was used for immunoprecipitation (IP). Endogenous δ-Catenin was detected using anti-δ-catenin antibody, and endogenous GSK-3α and -3β proteins were detected using anti-GSK-3 (α + β) antibody. HA antibody was used as a negative control. B, δ-catenin-GFP was co-transfected with GSK-3α, GSK-3β, or 14-3-3 into HEK293 cells to determine whether it interacts with GSK-3α and GSK-3β. The interaction between δ-catenin and 14-3-3 was used as a positive control. Immunoprecipitations were performed using HA antibody, and immunoprecipitated proteins were immunoblotted with anti-GFP antibody or HA antibody. WB, Western blot.

δ-Catenin undergoes the proteosomal degradation. A, to determine the effects of MG132 or ALLN on exogenous δ-catenin, MEF cells were transfected with δ-catenin and treated with different concentrations of MG132 or ALLN for 4 h prior to lysis (upper panels). δ-Catenin-GFP proteins were detected by anti-δ-catenin (panel i) or anti-GFP (panel ii) antibody. Actin (panel i) and nonspecific band (indicated as ns) of anti-GFP (panel ii) were used as a loading control. B, to determine the effects of MG132 on endogenous δ-catenin, CWR22Rv-1 cells were treated with CHX for 8 h (first lane) or with MG132 for 2 h followed by CHX for 8 h (second lane) left with no treatments (third lane) or with MG132 alone for 2 h (fourth lane) prior to lysis. Proteins of δ-catenin were detected by anti-δ-catenin antibody, and actin was used as a loading control. C, to determine the effects of ALLN on endogenous δ-catenin, CWR22Rv-1 cells were treated with ALLN for 4 h prior to lysis. Actin was used as a loading control.