Dishevelled enables casein kinase 1-mediated phosphorylation of Frizzled 6 required for cell membrane localization.

Frizzleds (FZDs) are receptors for secreted lipoglycoproteins of the Wingless/Int-1 (WNT) family, initiating an important signal transduction network in multicellular organisms. FZDs are G protein-coupled receptors (GPCRs), which are well known to be regulated by phosphorylation, leading to specific downstream signaling or receptor desensitization. The role and underlying mechanisms of FZD phosphorylation remain largely unexplored. Here, we investigated the phosphorylation of human FZD Using MS analysis and a phospho-state- and -site-specific antibody, we found that Ser-648, located in the FZD C terminus, is efficiently phosphorylated by casein kinase 1 ϵ (CK1ϵ) and that this phosphorylation requires the scaffolding protein Dishevelled (DVL). In an overexpression system, DVL1, -2, and -3 promoted CK1ϵ-mediated FZD phosphorylation on Ser-648. This DVL activity required an intact DEP domain and FZD-mediated recruitment of this domain to the cell membrane. Substitution of the CK1ϵ-targeted phosphomotif reduced FZD surface expression, suggesting that Ser-648 phosphorylation controls membrane trafficking of FZD Phospho-Ser-648 FZD immunoreactivity in human fallopian tube epithelium was predominantly apical, associated with cilia in a subset of epithelial cells, compared with the total FZD protein expression, suggesting that FZD phosphorylation contributes to asymmetric localization of receptor function within the cell and to epithelial polarity. Given the key role of FZD in planar cell polarity, our results raise the possibility that asymmetric phosphorylation of FZD rather than asymmetric protein distribution accounts for polarized receptor signaling.