Figure 6. Wnt-induced Vangl2 phosphorylation is required for its function.

(A) CHO cells expressing wild type or the indicated mutant Wnt5a were cocultured with Vangl2- and Ror2- expressing CHO cells at a ratio of 4:1. (B) Ror2-BDB (W749X) and Ror2-RS (Q502X) mutants failed to enhance Wnt5a-induced Vangl2 phosphorylation in Ror2^−/− MEFs. The kinase-dead Ror2 (Ror2-KD) showed similar activity as the wild type Ror2. (C) Effects of Vangl2 Lp mutations (D255E and S464N) and the control S464A mutation on Vangl2 phosphorylation were analyzed by immunoblot in CHO cells. (D) Analysis of endogenous Vangl2 protein levels and phosphorylation by immunoblot in the brain of the Vangl2^Lp/Lp embryo. (E) Analysis of wild type Vangl2 phosphorylation in CHO cells when co-expressed with the D255E or S464N Lp mutant Vangl2. (F) Wild type, S84A,S5~17A∷S76~84A (all phospho-mutant) or S5,82,84E (phospho-mimicking) mouse Vangl2 mRNA was injected to offsprings from the zebrafish tri^m209/+ × tri^m209/+ matings at different doses. The phenotypes of injected zebrafish embryos were classified into four groups (group A, B, C and D) as shown by the representative pictures. (G) Statistical analysis of results of wild type (a, d), S84A (b, e), S5~17A∷S76~84A (c, f) and S5,82,84E (g) mouse Vangl2 mRNA injection. Numbers of injected zebrafish embryos of the indicated genotypes are indicated above the bars. The percentage of rescued tri^m209/m209 emrbyos (Group A and B) was shown on the right of each panel. (H) Analysis of Vangl2 proteins in the injected fish embryos. HA-tagged wild type and mutant Vangl2 proteins synthesized from different amounts of injected mRNA in wild type embryos were shown in left panel. Quantified results by densitometer were shown in the right panel. Error bars are ± SD, n=3. See also Figure S6.