Ubiquitination and Degradation of the Hominoid-Specific Oncoprotein TBC1D3 Is Mediated by CUL7 E3 Ligase
Figure 6
TBC1D3 interacts with Fbw8 in a phosphorylation-dependent manner.
(A) In vitro interaction between TBC1D3 and Fbw8. Separate sets of HeLa cells were transfected with GFP-TBC1D3 or Myc-Fbw8. At 18 h after transfection, the GFP-TBC1D3-expressing cells were starved and stimulated with 10% FCS. To prepare TBC1D3-beads, GFP-TBC1D3 was immunoprecipitated with mouse anti-GFP antibody on Protein G beads. The TBC1D3-beads were treated with or without alkaline phosphatase (AP) for 1 h at 37°C. TBC1D3-beads were incubated with Fbw8-cytosol prepared from HeLa cells expressing Myc-Fbw8. Following incubation at 4°C, the beads were washed and the bound proteins were separated and analyzed by immunoblot using anti-Myc antibody. TBC1D3-beads pulled down Fbw8 and alkaline phosphatase treatment blocked TBC1D3-Fbw8 interaction. (B) In vivo interactions between TBC1D3 and Fbw8. HeLa cells were co-transfected with GFP-TBC1D3 and Myc-Fbw8. After 18 h, the cells were starved and stimulated with 10% FCS. Cell lysates were treated with and without AP and immunoprecipitated with anti-GFP antibody. The precipitates were separated by SDS-PAGE followed by immunoblot analysis. (C) GST-Fbw8-Skp1 pull-down of TBC1D3 is phosphorylation dependent. Glutathione beads with bound GST-Fbw8-Skp1 complex or GST alone were incubated with GFP-TBC1D3 expressing lysates, treated with or without AP for 1 hour at 37°C. The beads were washed and eluted proteins were separated by SDS-PAGE. Immunoblotting with monoclonal anti-TBC1D3 antibody (2C7) showed that GST-Fbw8-Skp1 pull-down of TBC1D3 was nearly abolished by prior alkaline phosphatase treatment (right panel). The right panel shows the Ponceau S staining of the transferred membrane. The experiments were repeated three times.
