WNT-dependent phosphorylation of the DVL2 mutant protein is reduced.

(A) Lysates from NIH/3T3 stable cell lines expressing the dog, Myc-tagged wild-type (Wt) or mutant variant (Mut) DVL2, which is 23 aa shorter than wild-type exogenous DVL2, were analyzed by western blotting using an anti-c-Myc antibody. To assess the ability of the wild-type and mutant proteins to respond to WNT stimulation, cells were treated with WNT5A or WNT3A for 6 hours. Both treatments resulted in increased gel mobility shifts of the wild-type DVL2 protein, indicative of increased phosphorylation; this effect was reduced on the mutant DVL2 protein. (B) To confirm that the DVL2 gel mobility shifts observed in (A) were due to phosphorylation, cell lysates were subjected to mock treatment (30 min incubation at 37 C), or calf intestinal phosphatase (CIP) treatment (30 min incubation at 37 C in the presence of CIP) before separation by SDS-PAGE. The DVL2 gel mobility shifts above wild-type and mutant proteins were lost after CIP treatment, confirming that they are caused by phosphorylation. (C) To test whether the DVL2 gel mobility shifts observed in (A) were driven by casein kinase 1 (CK1), cells were treated with D4476, a CK1 inhibitor, for 1 hour prior to and concurrently during the Wnt stimulation for 6 hours. The DVL2 gel mobility shifts were lost after D4476 treatment, further indicating that they are caused by CK1-dependent phosphorylation. α-tubulin was used for loading controls. Cell lysates were normalized by BCA assays for total protein.