Figure 1

Mutations in C-terminal domain of Dvl enhance level of K63-linked polyubiquitination. (a) Deletion of the RDU of Dvl increases polyubiquitination. Plasmids expressing HA-tagged ubiquitin were co-transfected with myc-tagged Dvl-WT, Dvl-ΔRDU or empty vector into HEK293T cells. Total cell lysates were subjected to immunoprecipitation with anti-myc antibody, followed by immunoblotting with anti-HA antibody. α-tubulin is a loading control. (b) Polyubiquitin chains of Dvl-ΔRDU mainly form through K63 linkages. Experiments were performed as described in (a) except for the usage of flag-tagged Ub-K48R or Ub-K63R mutants. After immunoprecipitation using anti-myc antibody, ubiquitinated fractions of Dvl were detected by immunoblotting with anti-flag antibody. (c) C-terminus of Dvl containing the RDU (Dvl-ct) directly interacts with K63-linked polyubiqutin chains, but not with monoubiquitin or K48-linked polyubiquitin chains. Recombinant GST-Dvl-ct, GST-Dvl-ct-VK2 or GST alone (empty) were incubated with various ubiquitin species, followed by GST-pull-down, SDS–PAGE and immunoblotting for Dvl-bound ubiquitin proteins (right, top). Input is 10% of total ubiquitins used in the pull-down assay (left). Coomassie Brilliant Blue staining of pulled-down GST species is shown in the bottom panels as a control (right, bottom). (d) Point mutations of putative ubiquitin recognition residues (V644 and V667) in the RDU of Dvl promote polyubiquitination. Flag-tagged K48R ubiquitin was coexpressed with wild-type Dvl or its mutant variants into HEK293T cells, followed by immunoprecipitation/immunoblotting for detection of polyubiquitinated forms of Dvl. (e) Polyubiquitin chain of mutant Dvl is preferentially K63-linked. As in (e), but endogenous ubiquitins and anti-K63-linkage-specific Ub antibody (Apu3) were used. (f) Dvl-ΔRDU, Dvl-V667K and Dvl-VK2 had significantly reduced reporter activity compared with wild-type Dvl. Result of the TOPFlash reporter analysis performed in HEK293T cells in triplicates is shown (top). P<0.00002. As transfection and loading control, total lysates subjected to immunoblotting for Dvl using anti-myc and β-actin antibodies are shown (bottom). (g) In contrast to wild-type Dvl, which mainly exists in phosphorylated forms, a significant fraction of Dvl-ΔRDU mutant is not phosphorylated. After transient expression of myc-tagged Dvl-WT and Dvl-ΔRDU mutants, lysates were collected, incubated with λ phosphatase and subjected to immunoblotting with anti-myc antibody. Band for endogenous levels of phosphorylated c-jun (p-c-jun) was abolished after treatment with λ phosphatase (marked with arrowhead). β-actin was used as a loading control.