Fig. 2.

Fz3 reduces the interaction between Vangl2 and Pk3 proteins in Xenopus embryos. (A) Vangl2 biotinylation by BLN–Pk3 is decreased in the presence of Fz3. Biotin and mRNAs encoding FLAG–BLN–Pk3 (400 pg), HA–Vangl2 or ΔPBD RNAs (100 pg each) and Fz3–FLAG (100 or 500 pg) as indicated, were injected into the animal region of 4- to 8-cell embryos. Protein lysates were immunoprecipitated (IP) with anti-HA antibody from stage 13 Xenopus embryos. Proteins were detected by immunoblotting (IB) with anti-biotin, anti-HA and anti-FLAG antibodies as indicated. Black arrowheads point to Vangl2 and Pk3, white arrowhead points to the slower migrating Vangl2 in pulldowns and lysates. Asterisks indicate endogenous proteins labeled by the anti-biotin antibody. (B) Lysates of embryos overexpressing Pk3 and Fz3 show that Pk3 increases Vangl2 mobility, whereas Fz3 reduces it. (C) Physical interaction of HA–Vangl2 and FLAG–Pk3 in transfected HEK293T cells. HA–Vangl2, but not the construct lacking the presumed Pk3-binding domain (ΔPBD, residues 298–382), is detected in pulldowns of FLAG–Pk3 from lysates of the transfected HEK293T cells. (D) HA-tagged Vangl2 but not ΔPBD is biotinylated by FLAG–BLN–Pk3. Embryo microinjection details and abbreviations are as in A. (E) Model. The formation of a complex between Vangl2 and Pk3 results in the biotinylation of Vangl2 by BLN–Pk3, green circles depict biotin. Fz3 causes an upshift of Vangl2 due to phosphorylation (circled red P) and inhibits the Vangl2–Pk3 interaction. Data is representative of three experiments.