Figure 1

Engineering a βTrCP-E-cadherin chimera that targets oncogenic β-catenin for degradation. A. Schematic diagrams of F-TrCP-Ecad chimeras used in this study. The β-catenin binding domain (amino acids 794–883) of E-cadherin (Ecad) was fused to the C-terminus of FLAG-tagged βTrCP to form F-TrCP-Ecad. The N-terminus of βTrCP (amino acid 1–297) was deleted from F-TrCP-Ecad to make F-TrCP(ΔN)-Ecad. The transmembrane (TM) region and the β-catenin binding domain in E-cadherin are indicated. The F box of βTrCP is hatched. The FLAG epitope fused at the N-termini of βTrCP, βTrCP-Ecad, or βTrCP(ΔN)-Ecad is indicated by a solid rectangle. B. F-TrCP-Ecad binds to β-catenin S37A in vivo. HA-tagged β-catenin S37A and FLAG-tagged βTrCP derivatives were co-expressed in 293 cells. Cells were treated with the proteasome inhibitor MG132 for 4 hours before harvesting. Cell lysates were immunoprecipitated with the anti-FLAG antibody, precipitates were resolved by a 10% SDS-PAGE gel, transferred to a nitrocellulose membrane, and immunoblotted with the anti-HA antibody (top panel). Note that a nonspecific band that cross-reacted with the anti-HA antibody migrated slightly above β-catenin S37A in all lanes of this panel. The membrane was stripped and blotted with the anti-FLAG antibody (middle panel). The expression of β-catenin S37A in total cell lysates was examined by immunoblotting with the anti-HA antibody (bottom panel). C. F-TrCP-Ecad induces ubiquitination of β-catenin S37A in vivo. MYC-tagged β-catenin S37A, HA-tagged ubiquitin, and indicated βTrCP derivatives were co-expressed in 293 cells. Cells were treated with the proteasome inhibitor ALLN for 4 hours, and lysed in the denaturing buffer by boiling to disrupt non-covalent protein-protein interactions. Cell lysates were immunoprecipitated with the anti-HA antibody, and immunoprecipitates were resolved in SDS-PAGE and blotted with the anti-MYC antibody (top panel). The expression of β-catenin S37A in total cell lysates was determined by immunoblotting with the anti-MYC antibody (bottom panel). D. F-TrCP-Ecad reduces the steady state levels of β-catenin. HA-tagged β-catenin and CDK2 were co-expressed with indicated βTrCP derivatives in 293 cells. Total cell lysates were resolved by SDS-PAGE, and immunoblotted with the anti-HA antibody.