Figure 2

Inhibition of Usp14 increases level of polyubiquitinated Dvl. (a) Knockdown of DUB6 (Usp14) increases polyubiquitination of endogenous Dvl. siRNAs for different DUBs or control siGFP were co-transfected with plasmids expressing flag-tagged Ub-K48R into HEK293T cells. Total cell lysates were subjected to immunoprecipitation with anti-Dvl2 antibody or control immunoglobulin G, followed by immunoblotting with anti-flag antibody to detect the ubiquitinated forms of Dvl2. siRNA targeting Usp14 yielded ∼90% knockdown efficiency. Note that the data for DUB9/Usp36 knockdown are excluded in the figure due to severe cell death as previously reported.³⁴ (b) As in a, except that cells were also treated with IU1, an inhibitor of Usp14, at 40 μm for 2 days. Effect of IU1 on Dvl2 polyubiquitination was comparable to that of Usp14 knockdown by siRNA. Dvl2 and Usp14 levels were measured, and endogenous β-actin was used as a loading control. (c) Expression of catalytically inactive Usp14-CA promotes Dvl polyubiquitination. Wild-type and mutant variants of Usp14 were co-transfected with flag-tagged Ub-K48R into HEK293T cells. Protein samples were analyzed by immunoprecipitation/immunoblotting as indicated. (d) As in c, except that Usp14 siRNA was simultaneously introduced along with transient co-expression of Usp14 variants and flag-Ub-K48R. Ectopic expression of wild-type Usp14 and Usp14-ΔUBL was sufficient to reverse Dvl2 ubiquitination induced by Usp14 knockdown. (e) Usp14 is essential for transduction of Wnt signaling. TOPFlash assay was performed in HEK293T cells introduced with either siGFP or siUsp14. At 2 days post transfection of siRNA and reporter plasmids, Wnt3a CM were treated for 0, 4, 8 and 12 h. Data represent average values of relative TOPFlash over FOPFlash ratios from one representative experiment performed in triplicate. Error bars indicate standard deviations in triplicate (*P<0.002). (f) The Usp14-CA mutant inhibits reporter activity induced by Dvl, but not β-catenin. Catalytically inactive Usp14-CA mutant was coexpressed with β-catenin or Dvl, and reporter assays were performed as described in (e) (*P<0.0003). (g) Ubiquitin chain trimming assay. HEK293T cells under Usp14 siRNA conditions were transfected with Ub-K48R only (left) or Ub-K48R and Dvl-ΔRDU (right). At 2 days post transfection, cells were treated with Wnt3a CM for 1 h and whole-cell lysates were collected. Polyubiquitinated species of endogenous and transfected Dvl were enriched by immunoprecipitation using anti-Dvl2 (left) and anti-myc antibodies (right), respectively. Immunoprecipitates were incubated with BSA (marked with ‘-’), recombinant GST-Usp14-WT, or GST-Usp14-CA protein for an additional 1 h. Levels of Dvl ubiquitination were analyzed by SDS–PAGE and immunoblotting with anti-Flag antibody. Levels of GST-Usp14-WT and GST-Usp14-CA were determined by Coomassie Brilliant Blue staining.