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 FZD6 Using MS analysis and a phospho-state- and -site-specific antibody, we found that Ser-648, located in the FZD6 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 FZD6 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 FZD6 surface expression, suggesting that Ser-648 phosphorylation controls membrane trafficking of FZD6 Phospho-Ser-648 FZD6 immunoreactivity in human fallopian tube epithelium was predominantly apical, associated with cilia in a subset of epithelial cells, compared with the total FZD6 protein expression, suggesting that FZD6 phosphorylation contributes to asymmetric localization of receptor function within the cell and to epithelial polarity. Given the key role of FZD6 in planar cell polarity, our results raise the possibility that asymmetric phosphorylation of FZD6 rather than asymmetric protein distribution accounts for polarized receptor signaling.

Central European Institute for Technology Masaryk University Kamenice 5 62500 Brno Czech Republic

Department of Functional Genomics Interfaculty Institute for Genetics and Functional Genomics Ernst Moritz Arndt University of Greifswald Friedrich Ludwig Jahn Strasse 15 17487 Greifswald Germany

Department of Immunology Genetics and Pathology Rudbeck Laboratory Science for Life Laboratory Uppsala University Dag Hammarskjölds väg 20 751 85 Uppsala Sweden

From the Laboratory of WNT Signaling Institute of Experimental Biology Faculty of Science Masaryk University Kotlarska 2 61137 Brno Czech Republic

Science for Life Laboratory Department of Neuroscience Karolinska Institute Tomtebodavägen 16 17165 Stockholm Sweden and

Section for Receptor Biology and Signaling Department of Physiology and Pharmacology Karolinska Institutet Biomedicum Tomtebodavägen 16 SE 17165 Stockholm Sweden

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