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

  • Katerina Strakova
    Footnotes
    Affiliations
    Laboratory of WNT Signaling, Institute of Experimental Biology, Faculty of Science, Masaryk University, Kotlarska 2, 61137 Brno, Czech Republic,

    Section for Receptor Biology and Signaling, Department of Physiology and Pharmacology, Karolinska Institutet, Biomedicum (6D), Tomtebodavägen 16, SE-17165 Stockholm, Sweden,
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  • Maria Kowalski-Jahn
    Footnotes
    Affiliations
    Section for Receptor Biology and Signaling, Department of Physiology and Pharmacology, Karolinska Institutet, Biomedicum (6D), Tomtebodavägen 16, SE-17165 Stockholm, Sweden,
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  • Tomas Gybel
    Affiliations
    Laboratory of WNT Signaling, Institute of Experimental Biology, Faculty of Science, Masaryk University, Kotlarska 2, 61137 Brno, Czech Republic,
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  • Jana Valnohova
    Affiliations
    Section for Receptor Biology and Signaling, Department of Physiology and Pharmacology, Karolinska Institutet, Biomedicum (6D), Tomtebodavägen 16, SE-17165 Stockholm, Sweden,
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  • Vishnu M. Dhople
    Affiliations
    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,
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  • Jakub Harnos
    Affiliations
    Laboratory of WNT Signaling, Institute of Experimental Biology, Faculty of Science, Masaryk University, Kotlarska 2, 61137 Brno, Czech Republic,
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  • Ondrej Bernatik
    Affiliations
    Laboratory of WNT Signaling, Institute of Experimental Biology, Faculty of Science, Masaryk University, Kotlarska 2, 61137 Brno, Czech Republic,
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  • Ranjani Sri Ganji
    Affiliations
    Laboratory of WNT Signaling, Institute of Experimental Biology, Faculty of Science, Masaryk University, Kotlarska 2, 61137 Brno, Czech Republic,

    Central European Institute for Technology, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic,
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  • Zbynek Zdrahal
    Affiliations
    Central European Institute for Technology, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic,
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  • Jan Mulder
    Affiliations
    Science for Life Laboratory, Department of Neuroscience, Karolinska Institute, Tomtebodavägen 16 17165 Stockholm, Sweden
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  • Cecilia Lindskog
    Affiliations
    Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Science for Life Laboratory, Uppsala University, Dag Hammarskjölds väg 20, 751 85 Uppsala, Sweden
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  • Vitezslav Bryja
    Correspondence
    To whom correspondence may be addressed: Laboratory of Cellular Communication, Dept. of Experimental Biology, Faculty of Science, Masaryk University, Kotlarska 2, 61137 Brno, Czech Republic. Tel.:420-549-49-3291
    Affiliations
    Laboratory of WNT Signaling, Institute of Experimental Biology, Faculty of Science, Masaryk University, Kotlarska 2, 61137 Brno, Czech Republic,
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  • Gunnar Schulte
    Correspondence
    To whom correspondence may be addressed: Karolinska Institutet, Dept. of Physiology and Pharmacology, Section for Receptor Biology and Signaling, S-171 77 Stockholm, Sweden. Tel.:46-852487933
    Affiliations
    Laboratory of WNT Signaling, Institute of Experimental Biology, Faculty of Science, Masaryk University, Kotlarska 2, 61137 Brno, Czech Republic,

    Section for Receptor Biology and Signaling, Department of Physiology and Pharmacology, Karolinska Institutet, Biomedicum (6D), Tomtebodavägen 16, SE-17165 Stockholm, Sweden,
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  • Author Footnotes
    5 Please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party-hosted site.
    1 Both authors contributed equally to this work.
    4 The abbreviations used are: GPCRG protein–coupled receptorCKcasein kinaseDEPDishevelled, Egl-10, and pleckstrin domainDIXDishevelled–AxinDVLDishevelledFZDFrizzledGRKGPCR kinasePCPplanar cell polarityWNTWingless/Int-1PKAcAMP-dependent protein kinaseGSK3glycogen synthase kinase 3APalkaline phosphataseKOknockoutRFPred fluorescent proteinIPimmunoprecipitationN-gFN-glycosidase FFAformic acidHPAHuman Protein AtlasANOVAanalysis of varianceGAPDHglyceraldehyde-3-phosphate dehydrogenase.
      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 δ (CK1δ) and that this phosphorylation requires the scaffolding protein Dishevelled (DVL). In an overexpression system, DVL1, -2, and -3 promoted CK1δ-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 CK1δ-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.

      Introduction

      Signal transduction through G protein–coupled receptors (GPCRs)
      The abbreviations used are: GPCR
      G protein–coupled receptor
      CK
      casein kinase
      DEP
      Dishevelled, Egl-10, and pleckstrin domain
      DIX
      Dishevelled–Axin
      DVL
      Dishevelled
      FZD
      Frizzled
      GRK
      GPCR kinase
      PCP
      planar cell polarity
      WNT
      Wingless/Int-1
      PKA
      cAMP-dependent protein kinase
      GSK3
      glycogen synthase kinase 3
      AP
      alkaline phosphatase
      KO
      knockout
      RFP
      red fluorescent protein
      IP
      immunoprecipitation
      N-gF
      N-glycosidase F
      FA
      formic acid
      HPA
      Human Protein Atlas
      ANOVA
      analysis of variance
      GAPDH
      glyceraldehyde-3-phosphate dehydrogenase.
      is fine-tuned by phosphorylation (
      • Tobin A.B.
      G-protein-coupled receptor phosphorylation: where, when and by whom.
      ,
      • Yang Z.
      • Yang F.
      • Zhang D.
      • Liu Z.
      • Lin A.
      • Liu C.
      • Xiao P.
      • Yu X.
      • Sun J.P.
      Phosphorylation of G protein-coupled receptors: from the barcode hypothesis to the flute model.
      ). Barcoding of receptor function through phosphorylation of intracellular domains of GPCRs is involved in receptor desensitization, recruitment of scaffold proteins such as β-arrestins, functional selectivity, and receptor internalization (
      • Reiter E.
      • Lefkowitz R.J.
      GRKs and β-arrestins: roles in receptor silencing, trafficking and signaling.
      ). FZDs functionally interact with the scaffold protein Dishevelled (DVL) to mediate WNT/β-catenin and β-catenin–independent signaling (
      • Gao C.
      • Chen Y.G.
      Dishevelled: the hub of Wnt signaling.
      ,
      • Mlodzik M.
      The Dishevelled protein family: still rather a mystery after over 20 years of molecular studies.
      ). Mammalian DVL exists in three isoforms, DVL1, -2, and -3, and interacts with FZDs, probably in a multimodal fashion involving DVL’s DEP domain and the PDZ domain binding the third intracellular loop and the conserved C-terminal KTXXXW motif of FZDs, respectively (
      • Mlodzik M.
      The Dishevelled protein family: still rather a mystery after over 20 years of molecular studies.
      • Gammons M.V.
      • Rutherford T.J.
      • Steinhart Z.
      • Angers S.
      • Bienz M.
      Essential role of the Dishevelled DEP domain in a Wnt-dependent human-cell-based complementation assay.
      ,
      • Gammons M.V.
      • Renko M.
      • Johnson C.M.
      • Rutherford T.J.
      • Bienz M.
      Wnt signalosome assembly by DEP domain swapping of Dishevelled.
      ,
      • Umbhauer M.
      • Djiane A.
      • Goisset C.
      • Penzo-Méndez A.
      • Riou J.F.
      • Boucaut J.C.
      • Shi D.L.
      The C-terminal cytoplasmic Lys-Thr-X-X-X-Trp motif in frizzled receptors mediates Wnt/β-catenin signalling.
      ,
      • Tauriello D.V.
      • Jordens I.
      • Kirchner K.
      • Slootstra J.W.
      • Kruitwagen T.
      • Bouwman B.A.
      • Noutsou M.
      • Rüdiger S.G.
      • Schwamborn K.
      • Schambony A.
      • Maurice M.M.
      Wnt/β-catenin signaling requires interaction of the Dishevelled DEP domain and C terminus with a discontinuous motif in Frizzled.
      ,
      • Pau M.S.
      • Gao S.
      • Malbon C.C.
      • Wang H.Y.
      • Bertalovitz A.C.
      The intracellular loop 2 F328S Frizzled-4 mutation implicated in familial exudative vitreoretinopathy impairs Dishevelled recruitment.
      • Strakova K.
      • Matricon P.
      • Yokota C.
      • Arthofer E.
      • Bernatik O.
      • Rodriguez D.
      • Arenas E.
      • Carlsson J.
      • Bryja V.
      • Schulte G.
      The tyrosine Y250(2.39) in Frizzled 4 defines a conserved motif important for structural integrity of the receptor and recruitment of Disheveled.
      ). Although our understanding of the coupling of Class F receptors to heterotrimeric G proteins is improving (
      • Dijksterhuis J.P.
      • Petersen J.
      • Schulte G.
      WNT/Frizzled signaling: receptor-ligand selectivity with focus on FZD-G protein signaling and its physiological relevance.
      • Schulte G.
      • Bryja V.
      The Frizzled family of unconventional G-protein-coupled receptors.
      ,
      • Schulte G.
      International Union of Basic and Clinical Pharmacology. LXXX. The class Frizzled receptors.
      • Koval A.
      • Purvanov V.
      • Egger-Adam D.
      • Katanaev V.L.
      Yellow submarine of the Wnt/Frizzled signaling: submerging from the G protein harbor to the targets.
      ), the role and underlying mechanisms of FZD phosphorylation remain largely obscure. Direct biochemical evidence for phosphorylation of FZDs is sparse, but phosphorylation of FZD1 and FZD3 is involved in negative feedback regulating FZD-mediated signaling along the planar cell polarity pathway, which was found to depend on DVL in the case of FZD3 (
      • Djiane A.
      • Yogev S.
      • Mlodzik M.
      The apical determinants aPKC and dPatj regulate Frizzled-dependent planar cell polarity in the Drosophila eye.
      • Shafer B.
      • Onishi K.
      • Lo C.
      • Colakoglu G.
      • Zou Y.
      Vangl2 promotes Wnt/planar cell polarity-like signaling by antagonizing Dvl1-mediated feedback inhibition in growth cone guidance.
      ,
      • Yanfeng W.A.
      • Tan C.
      • Fagan R.J.
      • Klein P.S.
      Phosphorylation of frizzled-3.
      • Onishi K.
      • Shafer B.
      • Lo C.
      • Tissir F.
      • Goffinet A.M.
      • Zou Y.
      Antagonistic functions of Dishevelleds regulate Frizzled3 endocytosis via filopodia tips in Wnt-mediated growth cone guidance.
      ).
      FZD6, the focus of this study, is closely related to FZD3 (
      • Schulte G.
      International Union of Basic and Clinical Pharmacology. LXXX. The class Frizzled receptors.
      ) and is expressed predominantly in the olfactory epithelium, thyroid and parathyroid glands, lung, and uterus in adult mice (Ref.
      • Regard J.B.
      • Sato I.T.
      • Coughlin S.R.
      Anatomical profiling of G protein-coupled receptor expression.
      ; for an overview of expression in human tissues, see https://www.proteinatlas.org/ENSG00000164930-FZD6/tissue).
      Please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party-hosted site.
      It has been found amplified in many different forms of cancer, including breast, ovarian, and prostate cancer (
      • Corda G.
      • Sala A.
      Non-canonical WNT/PCP signalling in cancer: Fzd6 takes centre stage.
      ). FZD6 mediates exclusively β-catenin–independent signaling through DVL and as a Gi- and Gq-coupled receptor (
      • Kilander M.B.
      • Petersen J.
      • Andressen K.W.
      • Ganji R.S.
      • Levy F.O.
      • Schuster J.
      • Dahl N.
      • Bryja V.
      • Schulte G.
      Disheveled regulates precoupling of heterotrimeric G proteins to Frizzled 6.
      • Kilander M.B.
      • Dahlström J.
      • Schulte G.
      Assessment of Frizzled 6 membrane mobility by FRAP supports G protein coupling and reveals WNT-Frizzled selectivity.
      ,
      • Wang Y.
      • Guo N.
      • Nathans J.
      The role of Frizzled3 and Frizzled6 in neural tube closure and in the planar polarity of inner-ear sensory hair cells.
      ,
      • Guo N.
      • Hawkins C.
      • Nathans J.
      Frizzled6 controls hair patterning in mice.
      • Golan T.
      • Yaniv A.
      • Bafico A.
      • Liu G.
      • Gazit A.
      The human Frizzled 6 (HFz6) acts as a negative regulator of the canonical Wnt·β-catenin signaling cascade.
      ). When mutated, FZD6 can cause nail dysplasia in humans, and knockout mice lacking FZD6 show defects in claw formation, hair patterning, and tissue polarity, phenotypes reflecting dysfunctional planar cell polarity (PCP) signaling (
      • Wang Y.
      • Guo N.
      • Nathans J.
      The role of Frizzled3 and Frizzled6 in neural tube closure and in the planar polarity of inner-ear sensory hair cells.
      ,
      • Guo N.
      • Hawkins C.
      • Nathans J.
      Frizzled6 controls hair patterning in mice.
      ,
      • Hua Z.L.
      • Chang H.
      • Wang Y.
      • Smallwood P.M.
      • Nathans J.
      Partial interchangeability of Fz3 and Fz6 in tissue polarity signaling for epithelial orientation and axon growth and guidance.
      • Cui C.Y.
      • Klar J.
      • Georgii-Heming P.
      • Fröjmark A.S.
      • Baig S.M.
      • Schlessinger D.
      • Dahl N.
      Frizzled6 deficiency disrupts the differentiation process of nail development.
      ,
      • Naz G.
      • Pasternack S.M.
      • Perrin C.
      • Mattheisen M.
      • Refke M.
      • Khan S.
      • Gul A.
      • Simons M.
      • Ahmad W.
      • Betz R.C.
      FZD6 encoding the Wnt receptor frizzled 6 is mutated in autosomal-recessive nail dysplasia.
      • Fröjmark A.S.
      • Schuster J.
      • Sobol M.
      • Entesarian M.
      • Kilander M.B.C.
      • Gabrikova D.
      • Nawaz S.
      • Baig S.M.
      • Schulte G.
      • Klar J.
      • Dahl N.
      Mutations in Frizzled 6 cause isolated autosomal-recessive nail dysplasia.
      ). Polarity of epithelial tissues is defined by asymmetry of epithelial cells perpendicular to the apical-basal axis. WNT/FZD signaling and in particular the closely related FZD3 and FZD6 are crucial for the polarity and PCP signaling in cells and tissues (
      • Wang Y.
      • Guo N.
      • Nathans J.
      The role of Frizzled3 and Frizzled6 in neural tube closure and in the planar polarity of inner-ear sensory hair cells.
      ,
      • Guo N.
      • Hawkins C.
      • Nathans J.
      Frizzled6 controls hair patterning in mice.
      ,
      • Hua Z.L.
      • Chang H.
      • Wang Y.
      • Smallwood P.M.
      • Nathans J.
      Partial interchangeability of Fz3 and Fz6 in tissue polarity signaling for epithelial orientation and axon growth and guidance.
      ,
      • Wang Y.
      • Thekdi N.
      • Smallwood P.M.
      • Macke J.P.
      • Nathans J.
      Frizzled-3 is required for the development of major fiber tracts in the rostral CNS.
      ). However, it remains a conundrum how asymmetry is achieved by a protein that does not necessarily show polarized cellular distribution (
      • Wang Y.
      • Chang H.
      • Rattner A.
      • Nathans J.
      Frizzled receptors in development and disease.
      ).
      In the present study, we investigated the mechanisms and role of C-terminal FZD6 phosphorylation. Our results identify serine 648 (Ser-648) as a CK1δ target site, and we propose a mechanism where DVL and CK1δ cooperate to achieve FZD6 phosphorylation at the plasma membrane. The development of a phospho-Ser-648–selective polyclonal FZD6 antibody enabled us to depict polarization of phosphorylated FZD6 in epithelial cells of the human fallopian tube, suggesting that phosphorylation of FZD6 rather than asymmetric localization could code for polarized signaling.

      Results

       Mass spectrometry analysis of FZD6 phosphorylation

      The C terminus of human FZD6 is 211 amino acids long and contains more than 50 phosphorylatable amino acids (Fig. 1A). Previous analysis with the MiniMotif Miner predicted the presence of two cAMP-dependent protein kinase (PKA), eight Ca2+-dependent protein kinase, eight casein kinase 1 (CK1), seven casein kinase 2 (CK2), one Ca2+/calmodulin-dependent protein kinase II, five ribosomal S6 kinase, one extracellular signal-regulated kinase 1/2, seven glycogen synthase kinase 3 (GSK3), one polo-like kinase, and one epidermal growth factor receptor sites (
      • Schulte G.
      Molecular pharmacology of Frizzleds—with implications for possible therapy.
      ). To assess FZD6 phosphorylation experimentally, we used MS. HEK293 cells expressing FZD6-GFP were treated with either control, WNT-3A–, or WNT-5A–containing conditioned medium for 30 min and processed for LC-MS/MS analysis. Six phosphorylated serine residues were detected in the C terminus of FZD6 (Fig. 1, B and C). Despite stimulation with conditioned medium, the detected phosphorylation events were rare compared with the amount of predicted kinase sites and phosphorylatable residues in FZD6. In total, phosphorylation in six positions was detected on Ser-592, Ser-620, Ser-629, Ser-648, Ser-653, and Ser-656 (Fig. 1C). Although Ser-592, Ser-620, and Ser-629 appeared to be constitutively phosphorylated and we could detect tendencies for agonist-induced phosphorylation in Ser-648, Ser-653, and Ser-656, the total number of detected peptides did not allow firm conclusions on WNT-induced changes.
      Figure thumbnail gr1
      Figure 1Mass spectrometry analysis of FZD6. A, amino acid sequence of the FZD6 C terminus. Putative phosphorylation sites are underlined (determined with the MiniMotif Miner software). The KTXXXW motif is shown in orange. Black/red residues correspond to phosphorylation sites detected by LC-MS/MS analysis presented in B. B, LC-MS/MS data are presented as relative peak areas of all peptides (percentage of maximum) in a sample containing a specific phosphorylated amino acid. Cells lysed for MS were treated with control (ctrl), WNT-3A–, or WNT-5A–conditioned medium. C, schematic depiction of detected phosphorylation sites (symbolized by red dots).

       FZD6 phosphorylation at Ser-648 is detectable using a phospho-specific antibody

      Despite our initial interest in several phosphorylatable residues in the C terminus of FZD6, we became particularly interested in the phosphorylation of Ser-648 because we were able to raise a phospho-specific antibody against this residue (SESARpS*EGRISP peptide where pS* is Ser(P)-648; Fig. 2A). The anti-Ser(P)-648 rabbit polyclonal antibody displayed a weak signal when detecting overexpressed FZD6 (V5-FZD6-mCherry; Fig. 2B) in HEK293 cells; however, the signal was specific as it did not detect the FZD6-S648A mutant where serine 648 was mutated to alanine (Fig. 2C). The antibody is phospho-selective because alkaline phosphatase (AP) treatment of the lysate abolished the signal (Fig. 2D). Concurrently, we validated the FZD6-S648A mutant with regard to its membrane localization (Fig. 2E) and glycosylation (Fig. 2F) compared with WT FZD6, indicating that the point mutation does not affect overall maturation of the receptor protein. In contrast, cell ELISA–based assessment of receptor surface expression indicated that significantly lower levels of FZD6-S648A compared with WT FZD6 are present in the plasma membrane (Fig. 2E).
      Figure thumbnail gr2
      Figure 2FZD6 is phosphorylated on Ser-648. A, schematic depiction of the phosphorylated (red, asterisk) human FZD6-based (UniProt ID O60353) peptide used for raising a phospho-specific polyclonal (rabbit) antibody (anti-Ser(P)-648 FZD6). B, schematic depiction of the WT FZD6 construct consisting of signal sequence (SS), V5 tag, FZD6, and mCherry. C, HEK293 cells transfected with pcDNA3.1 (control), WT V5-FZD6-mCherry, or V5-FZD6-S648A-mCherry were lysed, and immunoprecipitation was performed using an anti-RFP (mCherry) antibody. Precipitate (IP) was probed with anti-V5 and the Ser(P)-648 FZD6 antibodies. Total cell lysate (TCL) was probed with anti-V5 and anti-β-actin as a loading control. The density of Ser(P)-648–stained bands, measured using ImageJ, was normalized by dividing the value by the average of the whole experiment. Data are presented as a scatter plot; bars represent mean and error bars represent S.D. of three individual experiments. Selected results of the multiple comparison analysis of all data are presented. Results were analyzed with one-way ANOVA and Tukey’s multiple comparisons post hoc test. Significance levels are given as *** (p < 0.001) and ns (not significant). D, cell lysates were treated with AP. E, subcellular distribution of WT V5-FZD6-mCherry or V5-FZD6-S648A-mCherry was analyzed by confocal microscopy in living HEK293 cells. Scale bars, 10 μm. Cell ELISA was used to quantify surface expression of WT V5-FZD6-mCherry or V5-FZD6-S648A-mCherry using an antibody against the N-terminal V5 tag. Data are presented as a scatter plot; bars represent mean and error bars represent S.D. of five individual experiments performed in triplicates. Background fluorescence detected in pcDNA-transfected HEK293 cells was subtracted from all data, and mean values were normalized to WT FZD6 surface expression. Significance levels are given as * (p < 0.05). F, for detection of N-linked glycosylation, immunoprecipitates were treated with N-gF, analyzed by PAGE, and probed with anti-RFP. HEK293 lysates expressing WT V5-FZD6-mCheery or V5-FZD6-S648A-mCherry were divided into three equal parts, and FZD6 was pulled down using an anti-V5 antibody. The first sample was left untreated (−), the second was subjected to the deglycosylation treatment but without the enzyme (ctrl), and the third sample was subjected to the deglycosylation treatment including the enzyme (N-gF). WB, Western blot.

       FZD6 Ser-648 is phosphorylated in a CK1δ-dependent manner

      In silico phospho-prediction analysis of FZD6 using the MiniMotif Miner software (
      • Balla S.
      • Thapar V.
      • Verma S.
      • Luong T.
      • Faghri T.
      • Huang C.H.
      • Rajasekaran S.
      • del Campo J.J.
      • Shinn J.H.
      • Mohler W.A.
      • Maciejewski M.W.
      • Gryk M.R.
      • Piccirillo B.
      • Schiller S.R.
      • Schiller M.R.
      Minimotif Miner: a tool for investigating protein function.
      ) suggested that Ser-648 provides a consensus site for casein kinase 1 (Fig. 3A). Furthermore, Ser-645 was predicted to serve as a priming site enabling CK1-dependent phosphorylation of Ser-648.
      Figure thumbnail gr3
      Figure 3FZD6 Ser-648 is phosphorylated in a CK1δ-dependent manner. A, schematic depiction of a CK1 consensus site matching the sequence of human FZD6 (gray background). Ser-648, the phospho-acceptor serine in the CK1 consensus site, is highlighted in red and marked with an asterisk. The predicted CK1 priming site Ser-645 is underlined. B, HEK293 cells transfected with WT V5-FZD6-mCherry or V5-FZD6-S648A-mCherry in combination with CK1δ were treated with the CK1 inhibitor PF670462 (10 μm; overnight). Cells were lysed, and immunoprecipitation was performed using an anti-V5 antibody. Precipitate (IP) was probed with the anti-Ser(P)-648 FZD6, anti-CK1δ, and anti-RFP (mCherry) antibodies. To verify phosphate specificity of the anti-Ser(P)-648 antibody, lysates were treated with AP. The total cell lysate (TCL) was probed with anti-RFP and anti-CK1δ. Anti-β-actin served as a loading control. C, living HEK293 cells are shown expressing either WT V5-FZD6-mCherry, V5-FZD6-S645A-mCherry, or V5-FZD6-S645E-mCherry. Scale bars, 10 μm. Cell ELISA was performed to quantify the construct’s surface expression compared with the WT V5-FZD6-mCherry construct using an antibody against the N-terminal V5 tag. Data are presented as a scatter plot; bars represent mean and error bars represent S.D. of four individual experiments performed in triplicates. Background fluorescence detected in pcDNA-transfected HEK293 cells was subtracted, and mean values were normalized to FZD6 surface expression. ns, not significant. D, anti-RFP immunoprecipitation was done as in B. B and D, the density of Ser(P)-648–stained bands, measured using ImageJ, was normalized by dividing the value by the average of the whole experiment. Data are presented as a scatter plot; bars represent mean and error bars represent S.D. of three individual experiments. Selected results of the multiple comparison analysis of all data are presented. Results were analyzed with one-way ANOVA and Tukey’s multiple comparisons post hoc test. Significance levels are given as * (p < 0.05), *** (p < 0.001), and ns (not significant). WB, Western blot; ctrl, control.
      We therefore decided to test CK1δ as a candidate kinase and coexpressed it with FZD6. As shown in Fig. 3B, coexpression of CK1δ leads to a substantial increase in the Ser(P)-648 FZD6 signal accompanied by increased binding of CK1δ to FZD6. To verify that the Ser-648 phosphorylation depends on CK1δ activity, we treated the cells with the CK1δ/δ-selective inhibitor PF670462, which abrogated the Ser(P)-648 signal. Also, the Ser(P)-648 signal was sensitive to AP treatment of the lysates, and the S648A mutant was not phosphorylated upon CK1δ coexpression, which further confirmed the specificity of the detected Ser(P)-648 signal.
      To explore the priming properties of Ser-645, we created two additional mutants: Ser-645 was mutated to either alanine (S645A) to disable phosphorylation of Ser-645 or to glutamate (S645E), which was intended to mimic the priming phosphorylation event. Both mutants were localized in the membrane, albeit with slightly lower surface expression compared with the WT receptor (Fig. 3C). When coexpressed with CK1δ, the S645A mutant did not exhibit a Ser(P)-648 signal compared with the WT receptor (Fig. 3D). In contrast, when S645E was coexpressed with CK1δ, the observed Ser(P)-648 signal was stronger than that of the WT FZD6. Overexpressing S645E on its own led to a Ser(P)-648 signal comparable with the WT receptor in the presence of overexpressed CK1δ. These findings suggest that Ser-645 indeed serves as a priming site for CK1δ.
      The anti-Ser(P)-648 antibody enabled us to visualize phosphorylated FZD6 in relation to the FZD6-mCherry signal using indirect immunofluorescence in HEK293 cells coexpressing CK1δ and FZD6. FZD6-mCherry is widely distributed in the cell with a distinct fraction in the plasma membrane, similar to what we have reported before for this or similar constructs (
      • Kilander M.B.
      • Petersen J.
      • Andressen K.W.
      • Ganji R.S.
      • Levy F.O.
      • Schuster J.
      • Dahl N.
      • Bryja V.
      • Schulte G.
      Disheveled regulates precoupling of heterotrimeric G proteins to Frizzled 6.
      ,
      • Kilander M.B.
      • Dahlström J.
      • Schulte G.
      Assessment of Frizzled 6 membrane mobility by FRAP supports G protein coupling and reveals WNT-Frizzled selectivity.
      ,
      • Fröjmark A.S.
      • Schuster J.
      • Sobol M.
      • Entesarian M.
      • Kilander M.B.C.
      • Gabrikova D.
      • Nawaz S.
      • Baig S.M.
      • Schulte G.
      • Klar J.
      • Dahl N.
      Mutations in Frizzled 6 cause isolated autosomal-recessive nail dysplasia.
      ,
      • Petersen J.
      • Wright S.C.
      • Rodríguez D.
      • Matricon P.
      • Lahav N.
      • Vromen A.
      • Friedler A.
      • Strömqvist J.
      • Wennmalm S.
      • Carlsson J.
      • Schulte G.
      Agonist-induced dimer dissociation as a macromolecular step in G protein-coupled receptor signaling.
      ). However, the WT FZD6 phosphorylated at Ser-648 was predominantly present at the plasma membrane (Fig. 4A). In addition, phosphorylated FZD6 was detectable in intracellular puncta, most likely early endosomes or lysosomes. To confirm the localization of the phosphorylated FZD6 to the endosomal pathway, which would be in agreement with previous data (
      • Fröjmark A.S.
      • Schuster J.
      • Sobol M.
      • Entesarian M.
      • Kilander M.B.C.
      • Gabrikova D.
      • Nawaz S.
      • Baig S.M.
      • Schulte G.
      • Klar J.
      • Dahl N.
      Mutations in Frizzled 6 cause isolated autosomal-recessive nail dysplasia.
      ), we used the anti-Ser(P)-648 antibody in cells coexpressing CK1δ, DVL, V5-FZD6-mCherry, and GFP-tagged Rab5, a marker for early endosomes. Under these conditions, which allow efficient FZD6 phosphorylation at Ser-648, we observed punctate, intracellular structures that were positive for the FZD6 protein (mCherry), Ser(P)-648 (anti-Ser(P)-648 FZD6 antibody), and the endosomal marker Rab5-GFP (Fig. 4B).
      Figure thumbnail gr4
      Figure 4Phosphorylated FZD6 is predominantly localized in the plasma membrane and processed through the endosomal pathway. A, HEK293 cells transfected with WT V5-FZD6-mCherry or V5-FZD6-S648A-mCherry in combination with CK1δ were fixed in 4% paraformaldehyde, stained with anti-Ser(P)-648 FZD6 and anti-CK1δ, and visualized with a confocal microscope. Arrows point to Ser(P)-648 FZD6 detection in intracellular puncta. Scale bars, 10 μm. B, HEK293 cells cotransfected with WT V5-FZD6-mCherry, CK1δ, DVL2, and Rab5-GFP. Rab5-GFP serves as an early endosome marker. The cells were fixed in 4% paraformaldehyde, stained with anti-Ser(P)-648 and DAPI, and visualized with a confocal microscope. Arrows point to Ser(P)-648 FZD6– and Rab5-GFP–positive endosomes. Scale bar, 10 μm.
      As shown above, point mutations of phosphorylatable amino acids revealed phospho-specificity of the antibody and target-selective phosphorylation by CK1. However, we only observed slight reduction in surface expression of these receptor constructs (Fig. 3C). Based on a recent publication reporting on phosphorylation codes spanning three phosphorylatable residues mediating arrestin–GPCR interaction, we reasoned that functional consequences could become more apparent when mutating larger phosphorylation motifs (
      • Zhou X.E.
      • He Y.
      • de Waal P.W.
      • Gao X.
      • Kang Y.
      • Van Eps N.
      • Yin Y.
      • Pal K.
      • Goswami D.
      • White T.A.
      • Barty A.
      • Latorraca N.R.
      • Chapman H.N.
      • Hubbell W.L.
      • Dror R.O.
      • et al.
      Identification of phosphorylation codes for arrestin recruitment by G protein-coupled receptors.
      ). To evoke more serious malfunction, we therefore decided to systematically mutate the phosphorylatable motif, targeting several residues, including the Ser-648 and adjacent serines. We created two triple mutants, S643A/S645A/S648A and S648A/S653A/S656A, which were expressed well on the protein level. The S643A/S645A/S648A mutant, however, showed dramatically reduced surface expression (Fig. 5, A–C). This effect was phenocopied by the S645A/S648A double mutant, which is deficient in the CK1δ priming and target site.
      Figure thumbnail gr5
      Figure 5Characterization of the phosphorylatable serine motif by the help of Ser double and triple mutants. A, schematic depiction of Ser to Ala mutants, including Ser-648 and adjacent Ser residues. B, HEK293 cells cotransfected with pcDNA3.1 (control), WT V5-FZD6-mCherry, V5-FZD6-S645A/S648A-mCherry (double mut 1), V5-FZD6-S643A/S645A/S648A-mCherry (triple mut 1), or V5-FZD6-S648A/S653A/S656A-mCherry (triple mut 2); FLAG-DVL3; and CK1δ were lysed and analyzed by immunoblotting using anti-Ser(P)-648 and anti-FZD6 antibodies. Anti-GAPDH served as a loading control. The density of Ser(P)-648–stained bands, measured using ImageJ, was normalized by dividing each value by the average of the whole experiment. Data are presented as a scatter plot; bars represent mean and error bars represent S.D. of three individual experiments. Selected results of the multiple comparison analysis of all data are presented. Results were analyzed with one-way ANOVA and Tukey’s multiple comparisons post hoc test. Significance levels are given as *** (p < 0.001) and ns (not significant). C, receptor surface expression of HEK293 cells transiently transfected with pcDNA3.1, WT V5-FZD6-mCherry, V5-FZD6-S645A/S648A-mCherry (double mut 1), V5-FZD6-S643A/S645A/S648A-mCherry (triple mut 1), or V5-FZD6-S648A/S653A/S656A-mCherry (triple mut 2) was quantified by cell ELISA using an antibody against the N-terminal V5 tag. Data are presented as a scatter plot; bars represent mean and error bars represent S.D. of five individual experiments performed in triplicates. Background fluorescence detected in pcDNA-transfected HEK293 cells was subtracted from all data, and mean values were normalized to WT FZD6 surface expression. Significance levels are given as * (p < 0.05) and ns (not significant). WB, Western blot.

       DVL is required to induce phosphorylation of FZD6 in response to CK1δ

      CK1δ is a serine/threonine kinase with well defined function in WNT signaling. Its primary substrates in this pathway are the DVL proteins that strongly interact with CK1 and are phosphorylated in response to WNT proteins. Because DVL interacts with FZD6 and is phosphorylated and regulated by CK1δ (
      • Kilander M.B.
      • Petersen J.
      • Andressen K.W.
      • Ganji R.S.
      • Levy F.O.
      • Schuster J.
      • Dahl N.
      • Bryja V.
      • Schulte G.
      Disheveled regulates precoupling of heterotrimeric G proteins to Frizzled 6.
      ,
      • Cong F.
      • Schweizer L.
      • Varmus H.
      Casein kinase Iδ modulates the signaling specificities of dishevelled.
      ,
      • Bernatik O.
      • Ganji R.S.
      • Dijksterhuis J.P.
      • Konik P.
      • Cervenka I.
      • Polonio T.
      • Krejci P.
      • Schulte G.
      • Bryja V.
      Sequential activation and inactivation of Dishevelled in the Wnt/β-catenin pathway by casein kinases.
      • Bryja V.
      • Schulte G.
      • Rawal N.
      • Grahn A.
      • Arenas E.
      Wnt-5a induces Dishevelled phosphorylation and dopaminergic differentiation via a CK1-dependent mechanism.
      ), we asked whether DVL is required for phosphorylation of FZD6 at Ser-648. We overexpressed FZD6 and CK1δ in HEK293 cells depleted of all DVL proteins by CRISPR/Cas9 (DVL-KO-HEK293 cells; Ref.
      • Cervenka I.
      • Valnohova J.
      • Bernatik O.
      • Harnos J.
      • Radsetoulal M.
      • Sedova K.
      • Hanakova K.
      • Potesil D.
      • Sedlackova M.
      • Salasova A.
      • Steinhart Z.
      • Angers S.
      • Schulte G.
      • Hampl A.
      • Zdrahal Z.
      • et al.
      Dishevelled is a NEK2 kinase substrate controlling dynamics of centrosomal linker proteins.
      ) and observed no signal for Ser(P)-648 (Fig. 6A). Reconstitution with either of the three DVL isoforms rescued the Ser(P)-648 signal and resulted in an electrophoretic up-shift of FZD6 when FZD6 and CK1δ were coexpressed.
      Figure thumbnail gr6
      Figure 6DVL promotes CK1δ-dependent FZD6 phosphorylation. A, HEK293 cells lacking DVL1, -2, and -3 (DVL-KO-HEK293 (
      • Cervenka I.
      • Valnohova J.
      • Bernatik O.
      • Harnos J.
      • Radsetoulal M.
      • Sedova K.
      • Hanakova K.
      • Potesil D.
      • Sedlackova M.
      • Salasova A.
      • Steinhart Z.
      • Angers S.
      • Schulte G.
      • Hampl A.
      • Zdrahal Z.
      • et al.
      Dishevelled is a NEK2 kinase substrate controlling dynamics of centrosomal linker proteins.
      )) were transfected with WT V5-FZD6-mCherry and combinations of CK1δ with FLAG-DVL1, -DVL2, and -DVL3. Lysates were analyzed by immunoblotting and probed for Ser(P)-648, V5, FLAG, CK1δ, and β-actin as a loading control. The density of Ser(P)-648–stained bands, measured using ImageJ, was normalized by dividing each value by the average of the whole experiment. Data are presented as a scatter plot; bars represent mean and error bars represent S.D. of three individual experiments. Selected results of the multiple comparison analysis of all data are presented. Results were analyzed with one-way ANOVA and Tukey’s multiple comparisons post hoc test. Significance levels are given as ** (p < 0.01), *** (p < 0.001), and ns (not significant). B, DVL-KO-HEK293 lysates with overexpressed WT V5-FZD6-mCherry and combinations of CK1δ with FLAG-DVL3. Samples (numbered 1–4 according to the figure annotation) were divided into six equal fractions (denoted a–f), and V5-FZD6-mCherry was precipitated with an anti-V5 antibody in each fraction. Post-IP, fractions a/d were left untreated (−), fractions b/e were subjected to the dephosphorylation/deglycosylation treatment devoid of enzyme (ctrl), and fractions c/f were subjected to the dephosphorylation/deglycosylation treatment, including either AP or N-gF. Immunoblots show abundance of proteins in the pulldown (IP) or total cell lysate (TCL) detected by anti-V5, anti-FLAG, anti-CK1δ, and anti-RFP (mCherry) antibodies. Anti-β-actin served as a loading control. WB, Western blot.
      Furthermore, we wanted to find out whether the posttranslational modification of FZD6 induced by overexpression of DVL and resulting in the electrophoretic mobility shift of FZD6 is evoked by receptor phosphorylation and/or N-glycosylation. Therefore, we overexpressed FZD6 alone or together with CK1δ and DVL3 followed by either enzymatic dephosphorylation or deglycosylation. Only treatment with alkaline phosphatase resulted in a decrease of the FZD6 band at lower mobility, whereas treatment with N-glycosidase F did not (Fig. 6B), confirming the importance of FZD6 phosphorylation for the electrophoretic mobility shift of the receptor protein. Similar data were obtained when reconstituting the DVL-KO cells with FLAG-DVL1 or FLAG-DVL2 in combination with V5-FZD6-mCherry and CK1ε and subsequent treatment of the lysate with AP (Fig. S1).
      To investigate whether Ser-648 is phosphorylated as part of the DVL-induced FZD6 phosphorylation, the same two samples were loaded in parallel and analyzed by antibodies against RFP and Ser(P)-648 (Fig. 7A). We observed that the DVL-induced electrophoretic mobility shift of FZD6 (marked by a filled arrowhead) corresponds roughly to the Ser(P)-648 signal (marked by two stars), and both these signals migrate at ∼20 kDa higher molecular mass (“hyperphosphorylated” FZD6) compared with the two species of FZD6 (glycosylated and unglycosylated; marked by open arrowheads) detected by the anti-RFP antibody. In addition, we observed a weaker and faster migrating Ser(P)-648 FZD6 band (marked by one star), which could represent unspecific binding of anti-Ser(P)-648 to unphosphorylated FZD6. This band was only observed upon very high overexpression of the receptor and was insensitive to AP treatment (Fig. S2).
      Figure thumbnail gr7
      Figure 7DVL induces phosphorylation of FZD6. A, HEK293 cells were transfected with WT V5-FZD6-mCherry in the absence or presence of CK1δ and FLAG-DVL3. V5-FZD6-mCherry was precipitated using an anti-V5 antibody, and samples were probed with either anti-Ser(P)-648 FZD6 or anti-RFP (mCherry) antibody. The same two samples were loaded twice, separated by a marker lane. The filled arrowhead depicts hypershifted species of V5-FZD6-mCherry. Open arrowheads indicate glycosylated (upper) and unglycosylated (lower) forms of V5-FZD6-mCherry. ** marks the phosphorylated receptor positive for Ser(P)-648 FZD6. * depicts either an unspecific signal from anti-Ser(P)-648 antibody (unphosphorylated FZD6) or a weakly phosphorylated species. B, HEK293 cells overexpressing V5-FZD6-mCherry were cotransfected with combinations of CK1δ and FLAG-DVL3 and treated with the CK1δ inhibitor PF670462 (10 μm; overnight), and V5-FZD6-mCherry was precipitated using an anti-V5 antibody. Immunoblots show abundance of proteins in the pulldown (IP) or total cell lysate (TCL) detected by anti-Ser(P)-648, anti-RFP, anti-CK1δ, and anti-FLAG antibodies. Anti-β-actin served as a loading control. The density of Ser(P)-648–stained bands, measured using ImageJ, was normalized by dividing each value by the average of the whole experiment. Data are presented as a scatter plot; bars represent mean and error bars represent S.D. of three individual experiments. Selected results of the multiple comparison analysis of all data are presented. Results were analyzed with one-way ANOVA and Tukey’s multiple comparisons post hoc test. Significance levels are given as * (p < 0.05), ** (p < 0.01), *** (p < 0.001), and ns (not significant). WB, Western blot.
      We confirmed that the DVL-dependent shift of FZD6 is detectable in the WT HEK293 cells upon DVL overexpression (Fig. 7, A and B). In Fig. 7B, we show that overexpressing both CK1δ and DVL3 had an additive effect on the amount of Ser(P)-648 and the shifted form of FZD6 compared with expression of CK1δ or DVL3 alone. Also, the DVL-induced shift of FZD6 and phosphorylation of Ser-648 were to a great extent (although not exclusively) dependent on the activity of CK1δ (Fig. 7, A and B). Thus, CK1δ is crucial for phosphorylation of FZD6 in the presence of DVL even though we cannot exclude that other kinases participate in this process.
      The DVL-dependent electrophoretic mobility shift of FZD is not limited to FZD6. We were able to detect a DVL-dependent electrophoretic mobility up-shift of FZD4 when coexpressed with DVL2 (
      • Strakova K.
      • Matricon P.
      • Yokota C.
      • Arthofer E.
      • Bernatik O.
      • Rodriguez D.
      • Arenas E.
      • Carlsson J.
      • Bryja V.
      • Schulte G.
      The tyrosine Y250(2.39) in Frizzled 4 defines a conserved motif important for structural integrity of the receptor and recruitment of Disheveled.
      ). In DVL-KO-HEK293 cells, a DVL2-dependent shift of FZD4 was observed when WT receptor was expressed. However, when FZD4-Y250F, a mutant that is impaired in DVL2 binding (
      • Strakova K.
      • Matricon P.
      • Yokota C.
      • Arthofer E.
      • Bernatik O.
      • Rodriguez D.
      • Arenas E.
      • Carlsson J.
      • Bryja V.
      • Schulte G.
      The tyrosine Y250(2.39) in Frizzled 4 defines a conserved motif important for structural integrity of the receptor and recruitment of Disheveled.
      ), was expressed, the DVL2-dependent shift was reduced (Fig. S3), suggesting that FZD–DVL interaction and receptor phosphorylation go hand in hand. Furthermore, a similar DVL-induced, phosphorylation-dependent electrophoretic mobility shift was reported for FZD3 (
      • Shafer B.
      • Onishi K.
      • Lo C.
      • Colakoglu G.
      • Zou Y.
      Vangl2 promotes Wnt/planar cell polarity-like signaling by antagonizing Dvl1-mediated feedback inhibition in growth cone guidance.
      ,
      • Onishi K.
      • Shafer B.
      • Lo C.
      • Tissir F.
      • Goffinet A.M.
      • Zou Y.
      Antagonistic functions of Dishevelleds regulate Frizzled3 endocytosis via filopodia tips in Wnt-mediated growth cone guidance.
      ).

       FZD6 is phosphorylated in a GRK-dependent manner

      The findings that DVL-dependent FZD6 phosphorylation was not fully inhibited by pharmacologically blocking CK1δ/δ and that FZD4, which lacks a residue homologous to Ser-648 in FZD6, was also shifted by DVL raises the question whether there are other kinases involved in FZD6 phosphorylation. To investigate other candidate kinases potentially mediating the DVL-induced phosphorylation of FZD6, we used inhibitors of GSK3 (CHIR99021), PKA (H89), or GRK2 (paroxetine) (Fig. 8A). The CK1δ/δ inhibitor PF670462 was used as a positive control. Although inhibition of PKA or GSK3 did not reduce the Ser(P)-648 FZD6 signal, paroxetine reduced Ser(P)-648 to a similar extent as PF670462, pinpointing GRK2 as an FZD6 kinase. To investigate the role of GRK2 and the paroxetine-insensitive GRK5 as FZD6 kinases in more detail, we overexpressed either CK1δ, GRK2, or GRK5 with FZD6 in HEK293 cells endogenously expressing DVL. As shown in Fig. 8B, both GRK2 and GRK5 induced FZD6 phosphorylation effectively under these conditions.
      Figure thumbnail gr8
      Figure 8CK1δ, GRK2, and GRK5 efficiently phosphorylate Ser(P)-648 FZD6. A, HEK293 cells transfected with WT V5-FZD6-mCherry in the absence or presence of FLAG-DVL3. Cells were treated with the CK1 inhibitor PF670462 (10 μm; overnight), the GSK3 inhibitor CHIR 99021 (10 μm; overnight), the PKA inhibitor H89 (10 μm; overnight), or the GRK2 inhibitor paroxetine (10 μm; overnight). V5-FZD6-mCherry was precipitated (IP) with the anti-V5 antibody. IP samples and total cell lysates (TCL) were analyzed using anti-RFP (mCherry), anti-Ser(P)-648, anti-FLAG, and anti-β-actin as a loading control. B, HEK293 cells were cotransfected with WT V5-FZD6-mCherry and either no kinase, CK1δ, GRK2, or GRK5. Treatment with paroxetine or PF670642 was overnight at 10 μm. Anti-V5 immunoprecipitates were analyzed by immunoblotting with anti-RFP and anti-Ser(P)-648 antibodies. Total cell lysates (TCL) were probed for RFP, CK1δ, GRK2, GRK5, and β-actin as a loading control. A and B, the density of Ser(P)-648–stained bands, measured using ImageJ, was normalized by dividing each value by the average of the whole experiment. Data are presented as a scatter plot; bars represent mean and error bars represent S.D. of three individual experiments. Selected results of the multiple comparison analysis of all data are presented. Results were analyzed with one-way ANOVA and Tukey’s multiple comparisons post hoc test. Significance levels are given as ** (p < 0.01), *** (p < 0.001), and ns (not significant). C, HEK293 cells transfected with WT FZD6-GFP or the nail dysplasia mutant FZD6-GFP-R511C in the absence or presence of FLAG-DVL3 and CK1δ were lysed and analyzed by immunoblotting using anti-Ser(P)-648 and anti-FZD6 antibodies. Anti-α-tubulin served as a loading control. WB, Western blot.
      To investigate the relationship between the CK1-induced and DVL-dependent Ser-648 FZD6 phosphorylation and membranous expression of FZD6, we used a previously characterized nail dysplasia mutant of this receptor, the FZD6-R511C mutant (
      • Kilander M.B.
      • Petersen J.
      • Andressen K.W.
      • Ganji R.S.
      • Levy F.O.
      • Schuster J.
      • Dahl N.
      • Bryja V.
      • Schulte G.
      Disheveled regulates precoupling of heterotrimeric G proteins to Frizzled 6.
      ,
      • Fröjmark A.S.
      • Schuster J.
      • Sobol M.
      • Entesarian M.
      • Kilander M.B.C.
      • Gabrikova D.
      • Nawaz S.
      • Baig S.M.
      • Schulte G.
      • Klar J.
      • Dahl N.
      Mutations in Frizzled 6 cause isolated autosomal-recessive nail dysplasia.
      ). The R511C mutant results in decreased surface expression and prominent localization of FZD6-R511C to the endosomal/lysosomal pathway (
      • Fröjmark A.S.
      • Schuster J.
      • Sobol M.
      • Entesarian M.
      • Kilander M.B.C.
      • Gabrikova D.
      • Nawaz S.
      • Baig S.M.
      • Schulte G.
      • Klar J.
      • Dahl N.
      Mutations in Frizzled 6 cause isolated autosomal-recessive nail dysplasia.
      ). The comparison of WT FZD6 and FZD6-R511C in the absence and presence of coexpressed CK1δ and DVL3 indicates that WT FZD6 is more efficiently phosphorylated than FZD6-R511C at similar total expression levels of the two receptor constructs (Fig. 8C).

       The DEP domain of DVL is required for Ser-648 phosphorylation and the shift of FZD6

      To better understand underlying mechanisms of DVL-mediated phosphorylation of FZD6, we overexpressed FZD6 in DVL-KO-HEK293 cells in combination with either DVL3 or various deletion or point mutants of DVL3 (Fig. 9A). The DVL3 ΔDIX mutant is missing the N-terminal DIX domain important for multimerization and β-catenin signaling (
      • Schwarz-Romond T.
      • Fiedler M.
      • Shibata N.
      • Butler P.J.
      • Kikuchi A.
      • Higuchi Y.
      • Bienz M.
      The DIX domain of Dishevelled confers Wnt signaling by dynamic polymerization.
      ,
      • Fiedler M.
      • Mendoza-Topaz C.
      • Rutherford T.J.
      • Mieszczanek J.
      • Bienz M.
      Dishevelled interacts with the DIX domain polymerization interface of Axin to interfere with its function in down-regulating β-catenin.
      ). DVL3 ΔCterm lacks the very C terminus of DVL. Phosphorylation of this region disrupts the signalosomes observed as puncta and is inhibitory for β-catenin–dependent signaling (
      • Bernatik O.
      • Ganji R.S.
      • Dijksterhuis J.P.
      • Konik P.
      • Cervenka I.
      • Polonio T.
      • Krejci P.
      • Schulte G.
      • Bryja V.
      Sequential activation and inactivation of Dishevelled in the Wnt/β-catenin pathway by casein kinases.
      ). DVL3 Δ(DEP+C) is missing the DEP domain in addition to missing the C terminus, whereas DVL3 DEP represents the DEP domain of DVL3. In the DVL3-K435M mutant, the binding interface of FZD–DVL is disrupted, and thus, this mutant is not recruited to the membrane by FZD (
      • Axelrod J.D.
      • Miller J.R.
      • Shulman J.M.
      • Moon R.T.
      • Perrimon N.
      Differential recruitment of Dishevelled provides signaling specificity in the planar cell polarity and Wingless signaling pathways.
      ,
      • Wong H.C.
      • Mao J.
      • Nguyen J.T.
      • Srinivas S.
      • Zhang W.
      • Liu B.
      • Li L.
      • Wu D.
      • Zheng J.
      Structural basis of the recognition of the dishevelled DEP domain in the Wnt signaling pathway.
      ).
      Figure thumbnail gr9
      Figure 9The DEP domain and DVL–FZD interaction are essential to mediate phosphorylation of FZD6 at Ser-648. A, schematic depiction of DVL3 constructs used in B–F. For details, see the text. B–F, DVL-KO-HEK293 cells expressing WT V5-FZD6-mCherry either alone or together with FLAG-DVL3 or one of the FLAG-DVL3 mutants listed above. Cell lysates were precipitated using anti-V5 or anti-RFP. IP and total cell lysate (TCL) were analyzed by immunoblotting and probed with anti-Ser(P)-648, anti-RFP, anti-FLAG, and anti-V5 antibodies. Anti-β-actin served as a loading control. Quantification of the Ser(P)-648 FZD6 signal from three independent experiments for each condition is shown in Fig. S4. WB, Western blot.
      The DIX domain and the C terminus of DVL are dispensable for the induction of both Ser(P)-648 and the electrophoretic mobility shift of FZD6 (Fig. 9, B and C). However, deleting the C terminus and the DEP domain abolished the effects on FZD6 (Fig. 9D). In contrast, overexpression of the DEP domain of DVL3 was neither sufficient to induce Ser(P)-648 nor the shift of FZD6 (Fig. 9E), indicating that the DEP domain is required, but not sufficient, to induce Ser(P)-648 and the FZD6 shift. It should be noted, however, that both DVL3 Δ(DEP+C) and the minimal DVL3 DEP displayed lower expression compared with the WT DVL3. The DVL3-K435M mutant neither induced Ser(P)-648 nor the shift of FZD6 (Fig. 9F), consistent with the importance of the DEP domain found in Fig. 9D and indicating that the ability of DVL to bind FZD is crucial for its activity toward FZD phosphorylation.
      Furthermore, we asked which properties of the DVL DEP domain were required for induction of Ser(P)-648 and the FZD6 shift. Several mutants of the DVL1 DEP domain (described in the table in Fig. 10A and validated by Paclikova et al. (
      • Paclikova P.
      • Bernatik O.
      • Radaszkiewicz T.W.
      • Bryja V.
      N-terminal part of Dishevelled DEP domain is required for Wnt/β-catenin signaling in mammalian cells.
      )) were used. Instead of WT DVL3 as in Fig. 9, we used DVL1(1–502), a mutant of DVL1 lacking the C terminus, as a positive control for Fig. 10. All the other DVL1 mutants were created in the background of the DVL1(1–502) mutant. In Fig. 10B, we show that the K438M mutation in DVL1 (corresponding to K435M in DVL3) does not promote phosphorylation of Ser(P)-648 and FZD6 shift. Fig. 10C shows that Abl-mediated phosphorylation of tyrosine 494 in DVL1, implicated to have a positive effect on DVL function in the PCP pathway, was dispensable for the induction of Ser-648 phosphorylation and the overall FZD6 electrophoretic mobility shift. Mutating clusters of positively charged residues important for DVL binding to the phospholipids in the membrane (mutants RRRKA and HKA) neither affected the DVL1-mediated Ser(P)-648 nor the FZD6 shift (Fig. 10, D and E). Lastly, the deletion of the N-terminally positioned LPDSG motif in the DEP domain (mutant ΔLPDSG) rendered DVL1 unable to induce Ser(P)-648 or FZD6 shift (Fig. 10F). In all our experiments, the electrophoretic mobility shift of FZD6 was accompanied by the phosphorylation of FZD6 at Ser-648 (and vice versa), suggesting that the two events are interconnected.
      Figure thumbnail gr10
      Figure 10DVL1 DEP mutations differentially affect FZD6 phosphorylation on Ser-648. A, table summarizing properties of DVL1 constructs used in B–F. B–F, WT V5-FZD6-mCherry was overexpressed in DVL-KO-HEK293 cells together with FLAG-DVL1 constructs listed above and immunoprecipitated using an anti-V5 or anti-RFP antibody. Immunoblots show abundance of proteins in the pulldown (IP) or total cell lysate (TCL) detected by anti-Ser(P)-648, anti-V5, anti-RFP, and anti-FLAG antibodies. Anti-β-actin served as a loading control. Quantification of the Ser(P)-648 FZD6 signal from three independent experiments for each condition is shown in Fig. S5. WB, Western blot.
      In summary, our data using DVL mutants show that the DVL-induced hyperphosphorylation of FZD6, including Ser(P)-648, requires (i) the recruitment of DVL to FZD, (ii) DVL-CK1 binding, and (iii) an intact N-terminal LPDSG motif of the DEP domain. Conversely, FZD hyperphosphorylation is neither dependent on the DIX domain of DVL nor on the ability of DVL DEP to be attached to the membrane or to be phosphorylated by Abl.

       Localization of Ser(P)-648 FZD6 in human fallopian tube epithelium

      Despite the detailed mechanisms that we have identified leading to the DVL- and CK1-dependent phosphorylation of FZD6, its role for cell surface expression of the receptor and the full functional role of Ser(P)-648 remain obscure. Although deletion of the CK1δ phosphomotif impairs surface expression, we asked now where Ser(P)-648 FZD6 is localized compared with the total FZD6 pool in human epithelial tissue. According to the protein expression mapping efforts of the Human Protein Atlas (
      • Uhlén M.
      • Fagerberg L.
      • Hallström B.M.
      • Lindskog C.
      • Oksvold P.
      • Mardinoglu A.
      • Sivertsson Å.
      • Kampf C.
      • Sjöstedt E.
      • Asplund A.
      • Olsson I.
      • Edlund K.
      • Lundberg E.
      • Navani S.
      • Szigyarto C.A.
      • et al.
      Proteomics. Tissue-based map of the human proteome.
      ), FZD6 is widely expressed in epithelial tissues. We focused here on human fallopian tube tissue, which showed a distinct membranous FZD6 immunoreactivity using a polyclonal antibody with a large C-terminal epitope. Parallel staining of adjacent paraffin sections of the same fallopian tube tissue with the anti-Ser(P)-648 FZD6 antiserum revealed a more restricted and polarized immunoreactivity (Fig. 11A). Although total FZD6 immunoreactivity was evenly distributed in the plasma membrane of all epithelial cells both on the apical and basal side of the cells, Ser(P)-648 FZD6 immunoreactivity was predominantly apical, associated with cilia in a subset of epithelial cells (Fig. 11B). Also, double labeling and confocal imaging supported the polarized localization of the phosphorylated FZD6 in fallopian tube epithelial cells (Fig. 11, B and C).
      Figure thumbnail gr11
      Figure 11Localization of Ser(P)-648 FZD6 immunoreactivity in human fallopian tube epithelium. A, immunohistochemical staining of human fallopian tube tissue with anti-FZD6 and anti-Ser(P)-648 FZD6 antibodies. Arrows indicating chromogenic (brown) detection of the Ser(P)-648 FZD6 in polarized endothelial cells. Scale bar, 60 μm. B, immunocytochemical staining of human fallopian tube tissue with anti-FZD6 and anti-Ser(P)-648 FZD6 antibodies indicating a Ser(P)-648 FZD6 distribution in apical parts of endothelial cells and cilia. Nuclei were visualized using Hoechst stain. Scale bar, 10 μm. C, immunocytochemical staining of human fallopian tube tissue with anti-FZD6 and anti-Ser(P)-648 FZD6 antibodies. Arrows point to Ser(P)-648 FZD6 detection in cilia. Scale bar, 10 μm.

      Discussion

      FZDs interact with DVL phosphoproteins in a multimodal fashion, and the interplay has functional but mechanistically poorly understood implications for WNT signaling (
      • Gao C.
      • Chen Y.G.
      Dishevelled: the hub of Wnt signaling.
      ,
      • Mlodzik M.
      The Dishevelled protein family: still rather a mystery after over 20 years of molecular studies.
      ). In addition to DVL, CK1δ is a central player of WNT signaling both in β-catenin–dependent and –independent pathways (
      • Bernatik O.
      • Ganji R.S.
      • Dijksterhuis J.P.
      • Konik P.
      • Cervenka I.
      • Polonio T.
      • Krejci P.
      • Schulte G.
      • Bryja V.
      Sequential activation and inactivation of Dishevelled in the Wnt/β-catenin pathway by casein kinases.
      ,
      • Bryja V.
      • Schulte G.
      • Rawal N.
      • Grahn A.
      • Arenas E.
      Wnt-5a induces Dishevelled phosphorylation and dopaminergic differentiation via a CK1-dependent mechanism.
      ,
      • Strutt H.
      • Price M.A.
      • Strutt D.
      Planar polarity is positively regulated by casein kinase Iδ in Drosophila.
      • Bernatík O.
      • Šedová K.
      • Schille C.
      • Ganji R.S.
      • Červenka I.
      • Trantírek L.
      • Schambony A.
      • Zdráhal Z.
      • Bryja V.
      Functional analysis of dishevelled-3 phosphorylation identifies distinct mechanisms driven by casein kinase 1 and frizzled5.
      ,
      • Bryja V.
      • Gradl D.
      • Schambony A.
      • Arenas E.
      • Schulte G.
      β-Arrestin is a necessary component of Wnt/β-catenin signaling in vitro and in vivo.
      ,
      • Bryja V.
      • Schambony A.
      • Cajánek L.
      • Dominguez I.
      • Arenas E.
      • Schulte G.
      β-Arrestin and casein kinase 1/2 define distinct branches of non-canonical WNT signalling pathways.
      ,
      • Bryja V.
      • Schulte G.
      • Arenas E.
      Wnt-3a utilizes a novel low dose and rapid pathway that does not require casein kinase 1-mediated phosphorylation of Dvl to activate β-catenin.
      ,
      • Kaucká M.
      • Plevová K.
      • Pavlová S.
      • Janovská P.
      • Mishra A.
      • Verner J.
      • Procházková J.
      • Krejcí P.
      • Kotasková J.
      • Ovesná P.
      • Tichy B.
      • Brychtová Y.
      • Doubek M.
      • Kozubík A.
      • Mayer J.
      • et al.
      The planar cell polarity pathway drives pathogenesis of chronic lymphocytic leukemia by the regulation of B-lymphocyte migration.
      • Witte F.
      • Bernatik O.
      • Kirchner K.
      • Masek J.
      • Mahl A.
      • Krejci P.
      • Mundlos S.
      • Schambony A.
      • Bryja V.
      • Stricker S.
      Negative regulation of Wnt signaling mediated by CK1-phosphorylated Dishevelled via Ror2.
      ). Here, we provide evidence that FZD6, CK1δ, and DVL cooperate to maintain receptor surface expression and potentially FZD6 polarity in epithelial cells.
      Initially, we identified phosphorylation sites in the C terminus of FZD6 by MS/MS in cell lysates exposed to WNT-conditioned medium. Despite enrichment of phosphorylated peptides in cells exposed to WNT-conditioned medium compared with control stimulation, we were at a later time point not able to verify the WNT dependence of the Ser-648 phosphorylation. Because the conditions for the MS/MS experiment did not include overexpression of DVL and CK1 to efficiently enhance FZD phosphorylation, we speculate that Ser-648 FZD6 phosphorylation was a rare event and therefore not efficiently detectable by MS/MS under all conditions.
      GRKs represent a family of serine/threonine kinases that regulate GPCR function by direct phosphorylation and by the formation of a ternary complex (
      • Reiter E.
      • Lefkowitz R.J.
      GRKs and β-arrestins: roles in receptor silencing, trafficking and signaling.
      ,
      • Komolov K.E.
      • Du Y.
      • Duc N.M.
      • Betz R.M.
      • Rodrigues J.P.G.L.M.
      • Leib R.D.
      • Patra D.
      • Skiniotis G.
      • Adams C.M.
      • Dror R.O.
      • Chung K.Y.
      • Kobilka B.K.
      • Benovic J.L.
      Structural and functional analysis of a β2-adrenergic receptor complex with GRK5.
      ). Here, we have identified GRK2 and GRK5 as two additional kinases able to efficiently phosphorylate FZD6 in the presence of endogenous levels of DVL. Further studies are now required to shed light on the interaction of GRKs and DVL, the requirement of DVL for GRK-mediated FZD phosphorylation, GRK-mediated receptor barcoding regulating desensitization, internalization, and functional selectivity in resemblance to what is known for other GPCRs (
      • Tobin A.B.
      G-protein-coupled receptor phosphorylation: where, when and by whom.
      ,
      • Reiter E.
      • Lefkowitz R.J.
      GRKs and β-arrestins: roles in receptor silencing, trafficking and signaling.
      ). Interestingly, GRK5 and GRK6 are involved in β-catenin–dependent WNT signaling through phosphorylation of LRP6 (
      • Chen M.
      • Philipp M.
      • Wang J.
      • Premont R.T.
      • Garrison T.R.
      • Caron M.G.
      • Lefkowitz R.J.
      • Chen W.
      G Protein-coupled receptor kinases phosphorylate LRP6 in the Wnt pathway.
      ).
      The central tool that we present here is the polyclonal antiserum for detection of Ser-648 phosphorylation of FZD6. We have validated the antibody with regard to phosphorylation state and target selectivity using point mutations of FZD6 and phosphatase treatment of the cell lysates. Furthermore, an efficient and specific signal of the Ser(P)-648 antibody in HEK293 cell lysates is only visible when DVL and kinase are coexpressed, further underlining antibody selectivity. The phosphorylation event on Ser-648 can be seen as a consequence of FZD–DVL interaction. This interaction is intrinsically labile and short-lived and can experimentally be stabilized by chemical cross-linking (
      • Strakova K.
      • Matricon P.
      • Yokota C.
      • Arthofer E.
      • Bernatik O.
      • Rodriguez D.
      • Arenas E.
      • Carlsson J.
      • Bryja V.
      • Schulte G.
      The tyrosine Y250(2.39) in Frizzled 4 defines a conserved motif important for structural integrity of the receptor and recruitment of Disheveled.
      ,
      • Simons M.
      • Gault W.J.
      • Gotthardt D.
      • Rohatgi R.
      • Klein T.J.
      • Shao Y.
      • Lee H.J.
      • Wu A.L.
      • Fang Y.
      • Satlin L.M.
      • Dow J.T.
      • Chen J.
      • Zheng J.
      • Boutros M.
      • Mlodzik M.
      Electrochemical cues regulate assembly of the Frizzled/Dishevelled complex at the plasma membrane during planar epithelial polarization.
      ). The antibody provides a new tool to detect the pool of FZD6 that has been in functional contact with DVL and to monitor this transient interaction, which results in Ser-648 phosphorylation. Furthermore, the suitability of the Ser(P)-648 FZD6 antibody for immunohistochemistry could open new possibilities. It remains to be thoroughly validated whether detection of phosphorylated FZDs could present a useful diagnostic tool by screening patient-derived cancer tissue microarrays.
      Using the anti-Ser(P)-648 FZD6 antibody, we provide evidence in human fallopian tube epithelium that receptor phosphorylation can contribute to or mirrors cellular asymmetry and polarization of receptor pools. This notion, however, should be taken with a grain of salt because our findings do not distinguish between the possibility that differential localization of Ser(P)-648 FZD6 is the cause or consequence of the phosphorylation event at Ser-648. The experiments with the nail dysplasia mutant FZD6-R511C, a mutant that has an intact CK1 phosphorylation motif but shows a mislocalization phenotype with decreased presence at the cell surface (
      • Fröjmark A.S.
      • Schuster J.
      • Sobol M.
      • Entesarian M.
      • Kilander M.B.C.
      • Gabrikova D.
      • Nawaz S.
      • Baig S.M.
      • Schulte G.
      • Klar J.
      • Dahl N.
      Mutations in Frizzled 6 cause isolated autosomal-recessive nail dysplasia.
      ), suggest that Ser(P)-648 on FZD6 could mark FZD6 at the cell surface.
      It should also be underlined that receptor-overexpressing HEK293 cells apparently are not a suitable model to mimic receptor distribution in “real” cells such as fallopian epithelial cells. Yet undefined mechanisms appear to be in action to sort subpopulations of receptor pools in a delicate manner, mechanisms that are most likely not present or overridden by receptor overexpression in HEK293 cells. In contrast, the observed changes in receptor surface expression upon single, dual, and triple serine mutation of the CK1-targetd FZD6 motif underline that FZD6 phosphorylation is important for efficient membrane embedding or for stabilization of the membrane-expressed receptor pool. In the human fallopian epithelium, it was remarkable that distinct Ser(P) FZD6 expression was observed only in ciliated cells.
      Previously, it was suggested that differential sorting of PCP proteins such as Vangl2 and FZD6 could contribute to a polarized expression pattern in asymmetric cells (
      • Ma T.
      • Li B.
      • Wang R.
      • Lau P.K.
      • Huang Y.
      • Jiang L.
      • Schekman R.
      • Guo Y.
      A mechanism for differential sorting of the planar cell polarity proteins Frizzled6 and Vangl2 at the trans-Golgi network.
      ). Our findings indicate now that receptor phosphorylation could play a complementary role in sorting proteins in a polarized manner in asymmetric cells, providing novel means of cellular polarization by posttranslational modification.
      The overall importance of FZD phosphorylation is not yet understood. We have pinpointed a novel mechanism of FZD6 phosphorylation through CK1 and DVL or GRK2/5 with implications for receptor polarization and cell surface expression. Despite the fact that the C termini across the Class F receptors are poorly conserved, consensus sites of various Ser/Thr kinases are frequent (
      • Schulte G.
      Molecular pharmacology of Frizzleds—with implications for possible therapy.
      ), especially when it comes to the closely related FZD3 and FZD6, which present the longest C termini in Class F receptors. Thus, CK1-mediated or, more generally speaking, Ser/Thr kinase-mediated phosphorylation of the C termini of FZDs could represent a new concept for FZD regulation and cellular polarization. In support of our findings, previous studies have identified a role of DVL in phosphorylation of FZD3 with functional implications for receptor hyperphosphorylation, surface expression, and signaling (
      • Shafer B.
      • Onishi K.
      • Lo C.
      • Colakoglu G.
      • Zou Y.
      Vangl2 promotes Wnt/planar cell polarity-like signaling by antagonizing Dvl1-mediated feedback inhibition in growth cone guidance.
      ,
      • Onishi K.
      • Shafer B.
      • Lo C.
      • Tissir F.
      • Goffinet A.M.
      • Zou Y.
      Antagonistic functions of Dishevelleds regulate Frizzled3 endocytosis via filopodia tips in Wnt-mediated growth cone guidance.
      ). In those studies, the DVL-mediated phosphorylation of FZD3 evoked a negative feedback on receptor function.
      Given the recent information of arrestin barcodes of phosphorylated GPCRs (
      • Zhou X.E.
      • He Y.
      • de Waal P.W.
      • Gao X.
      • Kang Y.
      • Van Eps N.
      • Yin Y.
      • Pal K.
      • Goswami D.
      • White T.A.
      • Barty A.
      • Latorraca N.R.
      • Chapman H.N.
      • Hubbell W.L.
      • Dror R.O.
      • et al.
      Identification of phosphorylation codes for arrestin recruitment by G protein-coupled receptors.
      ), it will be interesting to follow up on our findings by investigating putative arrestin interaction with phosphorylated FZD6. Although previous findings suggest that DVL bridges FZD–arrestin interaction (
      • Chen W.
      • ten Berge D.
      • Brown J.
      • Ahn S.
      • Hu L.A.
      • Miller W.E.
      • Caron M.G.
      • Barak L.S.
      • Nusse R.
      • Lefkowitz R.J.
      Dishevelled 2 recruits β-arrestin 2 to mediate Wnt5A-stimulated endocytosis of Frizzled 4.
      ), the DVL-assisted phosphorylation of FZD could play a role in direct arrestin binding to FZDs, resembling the well established GPCR–arrestin interaction (
      • Reiter E.
      • Lefkowitz R.J.
      GRKs and β-arrestins: roles in receptor silencing, trafficking and signaling.
      ). Functionally, previous publications point at a central role of arrestins in the regulation of β-catenin–dependent and –independent signaling, and thus, Ser/Thr phosphorylation of FZDs could play an important part in the modulation of WNT signaling (
      • Bryja V.
      • Gradl D.
      • Schambony A.
      • Arenas E.
      • Schulte G.
      β-Arrestin is a necessary component of Wnt/β-catenin signaling in vitro and in vivo.
      ,
      • Bryja V.
      • Schambony A.
      • Cajánek L.
      • Dominguez I.
      • Arenas E.
      • Schulte G.
      β-Arrestin and casein kinase 1/2 define distinct branches of non-canonical WNT signalling pathways.
      ,
      • Chen W.
      • ten Berge D.
      • Brown J.
      • Ahn S.
      • Hu L.A.
      • Miller W.E.
      • Caron M.G.
      • Barak L.S.
      • Nusse R.
      • Lefkowitz R.J.
      Dishevelled 2 recruits β-arrestin 2 to mediate Wnt5A-stimulated endocytosis of Frizzled 4.
      ,
      • Schulte G.
      • Schambony A.
      • Bryja V.
      β-Arrestins—scaffolds and signalling elements essential for WNT/Frizzled signalling pathways?.
      ). Further experiments are needed to better understand the divergent functions of FZD and particularly FZD6 phosphorylation with regard to polarity, signal specification, desensitization, and arrestin and DVL interaction.

      Experimental procedures

       Cell culture, transfection, and treatments

      WT HEK293 or DVL-KO-HEK293 cells (ATCC) were cultured and transfected as described previously (
      • Arthofer E.
      • Hot B.
      • Petersen J.
      • Strakova K.
      • Jäger S.
      • Grundmann M.
      • Kostenis E.
      • Gutkind J.S.
      • Schulte G.
      WNT stimulation dissociates a Frizzled 4 inactive-state complex with Gα12/13.
      ). Treatments were done 24 h after transfection using the following agents: PF670462 (sc-204180A, Santa Cruz Biotechnology), CHIR 99021 (1386, Axon Medchem), H89 (B1427, Sigma-Aldrich), paroxetine (PHR1804, Sigma-Aldrich), phorbol 12,13-dibutyrate (P1269, Sigma-Aldrich), dithiobis(succinimidyl propionate) (D3669, Sigma-Aldrich), or the diluent (DMSO) in nontreated samples. Treatment with control, WNT-3A–, or WNT-5A–containing conditioned medium produced in L cells (ATCC) was done at a ratio 1:4 in the standard Dulbecco’s modified Eagle’s medium used for propagating the HEK293 cells.

       DNA constructs

      Human FZD6-GFP (pAcGFP1-N1); human CK1δ (pcDNA3); FLAG-DVL1 (pcDNA3); FLAG-DVL3 (pcDNA3.1); and DVL3 ΔDIX, DVL3 ΔC-term, DVL3 Δ(DEP+C), GRK2, and GRK5 (pRK5) were kind gifts from Niklas Dahl, Lukas Trantirek, Madelon Maurice, Randall Moon, and Robert J. Lefkowitz, respectively. FLAG-DVL2 was from Addgene (24802). All FZD6 mutants used in this study were created in the background of the human V5-FZD6-mCherry construct (N terminus, V5; C terminus, mCherry; Fig. 2B) using the QuikChange site-directed mutagenesis kit (Agilent) according to the manufacturer’s instruction, verified by sequencing, and characterized with regard to expression and localization. Primers were as follows: FZD6-S645A forward primer, 5′-GCAGGCAGTGTATCTGAAGCTGCGCGGAGTGAAGGAAGG-3′; FZD6-S645A reverse primer, 5′-CCTTCCTTCACTCCGCGCAGCTTCAGATACACTGCCTGC-3′; FZD6-S645E forward primer, 5′-GCAGGCAGTGTATCTGAAGAGGCGCGGAGTGAAGGAAGG-3′; FZD6-S645E reverse primer, 5′-CCTTCCTTCACTCCGCGCCTCTTCAGATACACTGCCTGC-3′; FZD6-S648A forward primer, 5′-GTATCTGAAAGTGCGCGGGCTGAAGGAAGGATTAGTCC-3′; and FZD6-S648A reverse primer, 5′-GGACTAATCCTTCCTTCAGCCCGCGCACTTTCAGATAC-3′. The FZD6-S645A/S648A double mutant and the FZD6-S643A/S645A/S648A and FZD6-S648A/S653A/S656A triple mutants were created in the background of the V5-FZD6-S648A-mCherry construct using the following primers: FZD6-S645A/S648A forward primer, 5′-GGCAGTGTATCTGAAGCTGCGCGGGCTGAAGG-3′; FZD6-S645A/S648A reverse primer, 5′-CCTTCAGCCCGCGCAGCTTCAGATACACTGCC-3′; FZD6-S643S/S645A/S648A forward primer, 5′-GGCCAGGCAGTGTAGCTGAAGCTGCGCGGGCTGAAGG-3′; FZD6-S643S/S645A/S648A reverse primer, 5′-CCTTCAGCCCGCGCAGCTTCAGCTACACTGCCTGGCC-3′; FZD6-S648A/S653A/S656A forward primer, 5′-TGCGCGGGCTGAAGGAAGGATTGCTCCAAAGGCTGATATTACTGACACTGGCC-3′; and FZD6-S648A/S653A/S656A reverse primer, 5′-GGCCAGTGTCAGTAATATCAGCCTTTGGAGCAATCCTTCCTTCAGCCCGCGCA-3′. In the same way, the mutant DVL3-K435M was created in the background of the FLAG-DVL3 construct (pcDNA3.1; N terminus, FLAG) using the following primers: DVL3-K435M forward primer, 5′-GTCCGTGACCGCATGTGGCTCATGATTACCATCCCTAATGCTTTC-3′; and DVL3-K435M reverse primer, 5′-GAAAGCATTAGGGATGGTAATCATGAGCCACATGCGGTCACGGAC-3′. FZD4 and FLAG-DVL1 constructs are described elsewhere (
      • Strakova K.
      • Matricon P.
      • Yokota C.
      • Arthofer E.
      • Bernatik O.
      • Rodriguez D.
      • Arenas E.
      • Carlsson J.
      • Bryja V.
      • Schulte G.
      The tyrosine Y250(2.39) in Frizzled 4 defines a conserved motif important for structural integrity of the receptor and recruitment of Disheveled.
      ,
      • Paclikova P.
      • Bernatik O.
      • Radaszkiewicz T.W.
      • Bryja V.
      N-terminal part of Dishevelled DEP domain is required for Wnt/β-catenin signaling in mammalian cells.
      ).

       Immunoprecipitation (IP), deglycosylation, and dephosphorylation assay

      The immunoprecipitation assay was carried out as described previously (
      • Bernatik O.
      • Ganji R.S.
      • Dijksterhuis J.P.
      • Konik P.
      • Cervenka I.
      • Polonio T.
      • Krejci P.
      • Schulte G.
      • Bryja V.
      Sequential activation and inactivation of Dishevelled in the Wnt/β-catenin pathway by casein kinases.
      ) using anti-V5 (mouse; R960-CUS, Thermo Fisher Scientific) or anti-RFP (rat; 5F8, Chromotek) antibody for pulldown. For the deglycosylation assay, immunoprecipitated samples were incubated overnight at 37 °C in lysis buffer with 1% β-mercaptoethanol and 2 units of N-glycosidase F (N-gF; 11365169001, Roche Applied Science). For the dephosphorylation assay, immunoprecipitated samples were incubated for 1 h at 37 °C in CIP buffer (100 mm NaCl, 50 mm Tris-Cl, 10 mm MgCl2, 1 mm DTT, pH 7.9) with EDTA-free protease inhibitor mixture (04693132001, Roche Applied Science) and 10 units of AP (P8361-5000u, Sigma-Aldrich).

       Immunoblotting, immunocytochemistry, and live-cell imaging

      Lysates in Laemmli buffer were separated by SDS-PAGE/immunoblotting following standard procedures (
      • Bernatik O.
      • Ganji R.S.
      • Dijksterhuis J.P.
      • Konik P.
      • Cervenka I.
      • Polonio T.
      • Krejci P.
      • Schulte G.
      • Bryja V.
      Sequential activation and inactivation of Dishevelled in the Wnt/β-catenin pathway by casein kinases.
      ). Primary antibodies used were anti-V5 (mouse; 1:5000; R960-CUS, Thermo Fisher Scientific), anti-FLAG M2 (mouse; 1:2000; F1804, Sigma-Aldrich), anti-CK1δ (goat; 1:500; sc-6471, Santa Cruz Biotechnology), anti-RFP (rat; 1:2000; 5F8, Chromotek), anti-GRK2 (rabbit; 1:500; sc-562, Santa Cruz Biotechnology), anti-GRK5 (rabbit; 1:500; sc-565, Santa Cruz Biotechnology), anti-DVL2 (rabbit; 1:1000; cs-3224, Cell Signaling Technology), anti-FZD6 (rabbit; 1:1000; NBP1-89702, NovusBio), anti-α-tubulin (mouse; 1:1000; T6199, Sigma-Aldrich), anti-GAPDH (rabbit; 1:8000; 2118S, Cell Signaling Technology), and anti-β-actin (rabbit; 1:2000; 4970, Cell Signaling Technology). The anti-Ser(P)-648 antibody was raised on a service basis by Moravian Biotechnology: the phosphorylated SESARpSEGRISP peptide was injected into rabbit, serum was affinity-purified, and a fraction binding the aforementioned phosphopeptide was used (dilution for both immunoblotting and immunocytochemistry, 1:500).
      For immunocytochemistry, transfected cells were fixed on glass coverslips coated with porcine gelatin and processed as described previously (
      • Bernatik O.
      • Ganji R.S.
      • Dijksterhuis J.P.
      • Konik P.
      • Cervenka I.
      • Polonio T.
      • Krejci P.
      • Schulte G.
      • Bryja V.
      Sequential activation and inactivation of Dishevelled in the Wnt/β-catenin pathway by casein kinases.
      ) using anti-CK1δ (goat; 1:1000; sc-6471, Santa Cruz Biotechnology) and anti-Ser(P)-648 FZD6 (rabbit; 1:500; Moravian Biotechnology) primary antibodies. For live-cell imaging, cells were seeded in glass-bottom dishes and imaged 24 h after transfection using a Leica TCS SP8 confocal microscope.

       LC-MS/MS analysis

      After the pulldown using the anti-GFP antibody (rabbit; 20R-GR-011, Fitzgerald), samples were incubated with 10 mm DTT at 56 °C for 45 min. After removal of excess DTT, samples were incubated with 55 mm iodoacetamide at room temperature in darkness for 30 min. The trypsin digestion was performed for 2 h at 40 °C on a Thermomixer (750 rpm; Eppendorf). Digested peptides were extracted using 50% acetonitrile solution with 2.5% formic acid (FA) and concentrated in a SpeedVac concentrator (Eppendorf). An aliquot of a concentrated sample was directly analyzed by LC-MS/MS for protein identification. The rest of the sample was used for phosphopeptide analysis. The sample was diluted with acidified acetonitrile solution (80% acetonitrile and 2% FA). Phosphopeptides were enriched using the Pierce Magnetic Titanium Dioxide Phosphopeptide Enrichment kit (Thermo Fisher Scientific) according to the manufacturer’s protocol with slight modifications (samples were mixed with binding solution at a 1:2 ratio before loading, and one additional washing step with binding solution after phosphopeptide capture was implemented). Eluates were concentrated under vacuum and then diluted in 10 μl of 0.1% FA solution before LC-MS/MS analysis. The LC-MS analysis was performed on a nanoACQUITY UPLC (Waters Corp.) coupled to a LTQ-Orbitrap Velos mass spectrometer (Thermo Electron Corporation) equipped with a nanoelectrospray ionization source and installed with a Picotip Emitter (New Objective).
      For LC separation, the digested peptides were first enriched on a nanoACQUITY UPLC 2G-V/Mtrap Symmetry C18 precolumn (2-cm length, 180-μm inner diameter, and 5-μm particle size) from Waters Corp. and separated using a nanoACQUITY BEH130 C18 column (10-cm length, 100-μm inner diameter, and 1.7-μm particle size from Waters Corp.). The separation was achieved with the formation of 92-min gradient containing buffer A (2% acetonitrile in water with 0.1% acetic acid) and buffer B (acetonitrile with 0.1% acetic acid; gradient, 2–5% buffer B in 2 min, 5–25% buffer B in 63 min, 25–60% buffer B in 25 min, 60–99% buffer B in 1 min). The peptides were eluted at a flow rate of 400 nl/min. The eluted peptides were analyzed first in the Fourier transform MS operated in positive and profile mode. The second scan event is an MS/MS scan performed in data-dependent mode to fragment peptides and acquire data in positive and centroid mode. The MS automatically switches between Orbitrap-MS and LTQ-MS/MS acquisition to carry out the MS and MS/MS events. Survey full-scan MS spectra (from m/z 300 to 1700) were acquired in the Orbitrap with resolution R = 30 000 with a target value of 1 × E6. The method allowed sequential isolation of a maximum of the 20 most intense ions (depending on signal intensity), which were subjected to collision-induced dissociation fragmentation with an isolation width of 2 Da and a target value of 1 × E4 or with a maximum ion time of 100 ms. Target ions already selected for MS/MS were dynamically excluded for 60 s. General MS conditions were electrospray voltage of 1.65 kV, no sheath and auxiliary gas flow, and capillary temperature of 300 °C. Ion selection threshold was 1500 counts for MS/MS with an activation time of 10 ms and activation energy of 35% normalized (also applied for MS/MS). Only doubly and triply charged ions were triggered for tandem MS analysis.
      The data were analyzed by Thermo Proteome Discoverer 1.4 and searched against the human UniProt fasta database using Sequest HT. The search parameter settings included trypsin as protease with two miscleavages; oxidation of methionine and phosphorylation of serine, threonine, and tyrosine as dynamic modifications; and carbamidomethylation of cysteine as a static modification. A peptide error tolerance of 10 ppm and fragment tolerance of 0.8 Da were used for peptide identifications with a false discovery rate of 1%. Peptides were validated by node Percolator, and the phosphopeptides were validated by node PhosphoRs 3.0. The output results were later filtered to yield high-confidence peptides and enable protein grouping. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (
      • Vizcaíno J.A.
      • Csordas A.
      • del-Toro N.
      • Dianes J.A.
      • Griss J.
      • Lavidas I.
      • Mayer G.
      • Perez-Riverol Y.
      • Reisinger F.
      • Ternent T.
      • Xu Q.W.
      • Wang R.
      • Hermjakob H.
      2016 update of the PRIDE database and its related tools.
      ) with the data set identifier PXD011173.

       Cell ELISA

      For quantification of cell surface receptor expression, 5 × 104 HEK293 cells were plated in 96-well plates coated with 0.1 mg/ml poly-d-lysine. Cells were transfected with 0.1 μg of the indicated constructs and maintained for an additional 24 h. Cells were then incubated with an anti-V5 antibody (mouse; 1:1000; R960-CUS, Thermo Fisher Scientific) in 1% BSA in PBS for 1 h at 4 °C. Following incubation, cells were washed five times with 0.5% BSA in PBS and probed with an horseradish peroxidase–conjugated goat anti-mouse antibody (1:4000; 31430, Thermo Fisher Scientific) in 1% BSA in PBS for 1 h at 4 °C. The cells were washed five times with 0.5% BSA in PBS, and 100 μl of the peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (T8665, Sigma-Aldrich) was added (30 min at room temperature). After acidification with 100 μl of 2 m HCl, the yellow color was read at 450 nm using a POLARstar Omega plate reader (BMG LABTECH).

       Human tissue samples and generation of tissue microarrays

      The use of Human Protein Atlas (HPA) tissue was covered by the HPA ethical permit (EPN Uppsala, Sweden, 2002/577, 2005/338, 2011/473) following the Declaration of Helsinki principles. Tissue was acquired from the archives at the Department of Pathology of Uppsala University Hospital. In brief, formalin-fixed and paraffin-embedded tissue samples were collected based on hematoxylin- and eosin-stained tissue sections showing representative normal histology, quality-controlled by a certified pathologist. Tissue was sectioned in 4-μm-thick sections using waterfall microtomes (Microm HM 355S, Thermo Fisher Scientific), dried at room temperature overnight, and baked at 50 °C for 12–24 h prior to immunohistochemical staining.

       Immunohistochemistry

      Tissue sections were deparaffinized in xylene, hydrated in graded alcohols, and blocked for endogenous peroxidase in 0.3% hydrogen peroxide diluted in 95% ethanol. For antigen retrieval, a Decloaking Chamber® (Biocare Medical) was used. Slides were immersed and boiled in citrate buffer, pH 6 (Lab Vision), for 4 min at 125 °C and then allowed to cool to 90 °C (the whole cycle was ∼40 min). Automated immunohistochemistry was performed using an Autostainer® 480 instrument (Thermo Fischer Scientific). The primary anti-FZD6 (rabbit; 1:300; HPA017991, Human Protein Atlas project–Atlas Antibodies AB) and the anti-Ser(P)-648 FZD6 (rabbit; 1:200; Moravian Biotechnology) were diluted in UltraAb Diluent (Thermo Fisher Scientific) followed by incubation for 30 min at room temperature. The slides were further incubated with the secondary reagent anti-rabbit/mouse horseradish peroxidase-conjugated UltraVision (Thermo Fischer Scientific) for 30 min at room temperature and developed for 10 min using diaminobenzidine Quanto (Thermo Fisher Scientific) as chromogen. All incubations were followed by a rinse in Wash Buffer (Thermo Fisher Scientific) two times for 5 min. Slides were counterstained in Mayer’s hematoxylin (Histolab) and coverslipped using Pertex® (Histolab) as mounting medium. The stained slides were dried in oven overnight and digitalized using Scanscope AT2 (Aperio) using a 20× objective.

       Immunofluorescence

      Anti-FZD6 and anti-Ser(P)-648 FZD6 immunoreactivity were visualized using a sequential tyramide signal amplification (PerkinElmer Life Sciences) protocol with an intermediate antibody elution step. Staining was performed in a BOND-RX automated stainer (Leica Biosystems). Slides were dewaxed (Bond Dewax Solution; 72 °C; Leica Biosystems) followed by a heat-induced epitope retrieval step (HIER1 citrate buffer, pH 6.0; 20 min at 100 °C; Leica Biosystems). Endogenous peroxidase activity was blocked with Novocastra Peroxidase Block (RE7171, Leica Biosystems). Samples were sequentially stained with anti-FZD6 (rabbit; 1:250; HPA017991, Human Protein Atlas project–Atlas Antibodies AB) followed by anti-Ser(P)-648 FZD6 (rabbit; 1:500; Moravian Biotechnology) and vice versa. Samples were incubated with the first primary antibody for 4 h (2 × 2 h at room temperature), washed, treated with Post Primary Block before incubation with the horseradish peroxidase polymer detection system (Novacastra Novolink, Leica Biosystems). After several washes, fluorescein-labeled tyramide in amplification diluent (1:100; PerkinElmer Life Sciences) was applied. Tissue-bound primary antibodies and polymer were eluted using a short antigen retrieval step (HIER1; 10 min at 100 °C; Leica Biosystems). For the second primary antibody, the same protocol based on Cy5-labeled tyramide was used. Nuclei were visualized using Hoechst (1:10,000), and lipofuscin autofluorescence in the tissue was quenched with lipophilic Sudan Black B solution (1% (w/v) in 70% ethanol; Sigma-Aldrich) for 5 min. All pictures were taken with a Zeiss LSM880 confocal microscope.

       Statistical analysis

      All immunoblot experiments are representative of n = 3. Statistical and graphical analyses were done using GraphPad Prism 6 software. Quantification of the Western blots was performed by ImageJ software. Briefly, the density of each Ser(P)-648–stained band (Figs. 2C; 3, B and D; 5B; 6A; 7B; 8, A and B; 9, B–F; and 10, B–F) was measured and normalized by dividing the value by the average of the whole experiment. Normalized values were plotted as a scatter plot, and bars represent mean ± S.D. of three independent experiments. Differences among the groups were analyzed by one-way ANOVA with Tukey’s multiple comparisons post hoc test. Significance levels are given as follows: *, p < 0.05; **, p < 0.01; and ***, p < 0.001. For analyzing cell ELISA data, the basal fluorescence detected in pcDNA-transfected HEK293 cells was subtracted from all data, and mean values were normalized to WT FZD6 surface expression. Differences among the groups were analyzed by two-tailed t test or one-way ANOVA with Dunnett’s multiple comparisons post hoc test. Significance levels are given as follows: *, p < 0.05; **, p < 0.01; and ***, p < 0.001. All data points represent normalized values, each performed in triplicates, and values were plotted as a scatter plot. Bars represent mean ± S.D. of four to five independent experiments.

      Author contributions

      K. S., J. V., O. B., V. B., and G. S. conceptualization; K. S., J. V., O. B., V. B., and G. S. data curation; K. S., M. K.-J., T. G., V. M. D., R. S. G., Z. Z., and J. M. formal analysis; K. S., M. K.-J., O. B., and V. B. validation; K. S., M. K.-J., T. G., J. V., V. M. D., O. B., R. S. G., Z. Z., J. M., C. L., V. B., and G. S. investigation; K. S., M. K.-J., J. V., O. B., V. B., and G. S. visualization; K. S., M. K.-J., V. M. D., J. H., O. B., Z. Z., J. M., C. L., V. B., and G. S. methodology; K. S., M. K.-J., J. V., O. B., V. B., and G. S. writing-original draft; K. S., M. K.-J., J. V., O. B., V. B., and G. S. writing-review and editing; M. K.-J., J. V., O. B., V. B., and G. S. funding acquisition; J. V., V. M. D., O. B., J. M., C. L., V. B., and G. S. resources; J. V., J. H., O. B., V. B., and G. S. supervision; J. V., O. B., V. B., and G. S. project administration.

      Acknowledgments

      We thank Lukas Trantirek, Niklas Dahl, Madelon Maurice, Randall Moon, and Robert J Lefkowitz for providing plasmids. Czech Infrastructure for Integrative Structural Biology Research Infrastructure Project LM2015043 funded by the Ministry of Education, Youth and Sports of the Czech Republic is gratefully acknowledged for the financial support of the LC-MS/MS measurements at the Central European Institute of Technology Proteomics Core Facility.

      Supplementary Material

      References

        • Tobin A.B.
        G-protein-coupled receptor phosphorylation: where, when and by whom.
        Br. J. Pharmacol. 2008; 153 (18193069): S167-S176
        • Yang Z.
        • Yang F.
        • Zhang D.
        • Liu Z.
        • Lin A.
        • Liu C.
        • Xiao P.
        • Yu X.
        • Sun J.P.
        Phosphorylation of G protein-coupled receptors: from the barcode hypothesis to the flute model.
        Mol. Pharmacol. 2017; 92 (28246190): 201-210
        • Reiter E.
        • Lefkowitz R.J.
        GRKs and β-arrestins: roles in receptor silencing, trafficking and signaling.
        Trends Endocrinol. Metab. 2006; 17 (16595179): 159-165
        • Gao C.
        • Chen Y.G.
        Dishevelled: the hub of Wnt signaling.
        Cell. Signal. 2010; 22 (20006983): 717-727
        • Mlodzik M.
        The Dishevelled protein family: still rather a mystery after over 20 years of molecular studies.
        Curr. Top. Dev. Biol. 2016; 117 (26969973): 75-91
        • Gammons M.V.
        • Rutherford T.J.
        • Steinhart Z.
        • Angers S.
        • Bienz M.
        Essential role of the Dishevelled DEP domain in a Wnt-dependent human-cell-based complementation assay.
        J. Cell Sci. 2016; 129 (27744318): 3892-3902
        • Gammons M.V.
        • Renko M.
        • Johnson C.M.
        • Rutherford T.J.
        • Bienz M.
        Wnt signalosome assembly by DEP domain swapping of Dishevelled.
        Mol. Cell. 2016; 64 (27692984): 92-104
        • Umbhauer M.
        • Djiane A.
        • Goisset C.
        • Penzo-Méndez A.
        • Riou J.F.
        • Boucaut J.C.
        • Shi D.L.
        The C-terminal cytoplasmic Lys-Thr-X-X-X-Trp motif in frizzled receptors mediates Wnt/β-catenin signalling.
        EMBO J. 2000; 19 (10990458): 4944-4954
        • Tauriello D.V.
        • Jordens I.
        • Kirchner K.
        • Slootstra J.W.
        • Kruitwagen T.
        • Bouwman B.A.
        • Noutsou M.
        • Rüdiger S.G.
        • Schwamborn K.
        • Schambony A.
        • Maurice M.M.
        Wnt/β-catenin signaling requires interaction of the Dishevelled DEP domain and C terminus with a discontinuous motif in Frizzled.
        Proc. Natl. Acad. Sci. U.S.A. 2012; 109 (22411803): E812-E820
        • Pau M.S.
        • Gao S.
        • Malbon C.C.
        • Wang H.Y.
        • Bertalovitz A.C.
        The intracellular loop 2 F328S Frizzled-4 mutation implicated in familial exudative vitreoretinopathy impairs Dishevelled recruitment.
        J. Mol. Signal. 2015; 10 (27096003): 5
        • Strakova K.
        • Matricon P.
        • Yokota C.
        • Arthofer E.
        • Bernatik O.
        • Rodriguez D.
        • Arenas E.
        • Carlsson J.
        • Bryja V.
        • Schulte G.
        The tyrosine Y250(2.39) in Frizzled 4 defines a conserved motif important for structural integrity of the receptor and recruitment of Disheveled.
        Cell. Signal. 2017; 38 (28668722): 85-96
        • Dijksterhuis J.P.
        • Petersen J.
        • Schulte G.
        WNT/Frizzled signaling: receptor-ligand selectivity with focus on FZD-G protein signaling and its physiological relevance.
        Br. J. Pharmacol. 2014; 171 (24032637): 1195-1209
        • Schulte G.
        • Bryja V.
        The Frizzled family of unconventional G-protein-coupled receptors.
        Trends Pharmacol. Sci. 2007; 28 (17884187): 518-525
        • Schulte G.
        International Union of Basic and Clinical Pharmacology. LXXX. The class Frizzled receptors.
        Pharmacol. Rev. 2010; 62 (21079039): 632-667
        • Koval A.
        • Purvanov V.
        • Egger-Adam D.
        • Katanaev V.L.
        Yellow submarine of the Wnt/Frizzled signaling: submerging from the G protein harbor to the targets.
        Biochem. Pharmacol. 2011; 82 (21689640): 1311-1319
        • Djiane A.
        • Yogev S.
        • Mlodzik M.
        The apical determinants aPKC and dPatj regulate Frizzled-dependent planar cell polarity in the Drosophila eye.
        Cell. 2005; 121 (15907474): 621-631
        • Shafer B.
        • Onishi K.
        • Lo C.
        • Colakoglu G.
        • Zou Y.
        Vangl2 promotes Wnt/planar cell polarity-like signaling by antagonizing Dvl1-mediated feedback inhibition in growth cone guidance.
        Dev. Cell. 2011; 20 (21316586): 177-191
        • Yanfeng W.A.
        • Tan C.
        • Fagan R.J.
        • Klein P.S.
        Phosphorylation of frizzled-3.
        J. Biol. Chem. 2006; 281 (16513654): 11603-11609
        • Onishi K.
        • Shafer B.
        • Lo C.
        • Tissir F.
        • Goffinet A.M.
        • Zou Y.
        Antagonistic functions of Dishevelleds regulate Frizzled3 endocytosis via filopodia tips in Wnt-mediated growth cone guidance.
        J. Neurosci. 2013; 33 (24305805): 19071-19085
        • Regard J.B.
        • Sato I.T.
        • Coughlin S.R.
        Anatomical profiling of G protein-coupled receptor expression.
        Cell. 2008; 135 (18984166): 561-571
        • Corda G.
        • Sala A.
        Non-canonical WNT/PCP signalling in cancer: Fzd6 takes centre stage.
        Oncogenesis. 2017; 6 (28737757): e364
        • Kilander M.B.
        • Petersen J.
        • Andressen K.W.
        • Ganji R.S.
        • Levy F.O.
        • Schuster J.
        • Dahl N.
        • Bryja V.
        • Schulte G.
        Disheveled regulates precoupling of heterotrimeric G proteins to Frizzled 6.
        FASEB J. 2014; 28 (24500924): 2293-2305
        • Kilander M.B.
        • Dahlström J.
        • Schulte G.
        Assessment of Frizzled 6 membrane mobility by FRAP supports G protein coupling and reveals WNT-Frizzled selectivity.
        Cell. Signal. 2014; 26 (24873871): 1943-1949
        • Wang Y.
        • Guo N.
        • Nathans J.
        The role of Frizzled3 and Frizzled6 in neural tube closure and in the planar polarity of inner-ear sensory hair cells.
        J. Neurosci. 2006; 26 (16495441): 2147-2156
        • Guo N.
        • Hawkins C.
        • Nathans J.
        Frizzled6 controls hair patterning in mice.
        Proc. Natl. Acad. Sci. U.S.A. 2004; 101 (15169958): 9277-9281
        • Golan T.
        • Yaniv A.
        • Bafico A.
        • Liu G.
        • Gazit A.
        The human Frizzled 6 (HFz6) acts as a negative regulator of the canonical Wnt·β-catenin signaling cascade.
        J. Biol. Chem. 2004; 279 (14747478): 14879-14888
        • Hua Z.L.
        • Chang H.
        • Wang Y.
        • Smallwood P.M.
        • Nathans J.
        Partial interchangeability of Fz3 and Fz6 in tissue polarity signaling for epithelial orientation and axon growth and guidance.
        Development. 2014; 141 (25294940): 3944-3954
        • Cui C.Y.
        • Klar J.
        • Georgii-Heming P.
        • Fröjmark A.S.
        • Baig S.M.
        • Schlessinger D.
        • Dahl N.
        Frizzled6 deficiency disrupts the differentiation process of nail development.
        J. Invest. Dermatol. 2013; 133 (23439395): 1990-1997
        • Naz G.
        • Pasternack S.M.
        • Perrin C.
        • Mattheisen M.
        • Refke M.
        • Khan S.
        • Gul A.
        • Simons M.
        • Ahmad W.
        • Betz R.C.
        FZD6 encoding the Wnt receptor frizzled 6 is mutated in autosomal-recessive nail dysplasia.
        Br. J. Dermatol. 2012; 166 (22211385): 1088-1094
        • Fröjmark A.S.
        • Schuster J.
        • Sobol M.
        • Entesarian M.
        • Kilander M.B.C.
        • Gabrikova D.
        • Nawaz S.
        • Baig S.M.
        • Schulte G.
        • Klar J.
        • Dahl N.
        Mutations in Frizzled 6 cause isolated autosomal-recessive nail dysplasia.
        Am. J. Hum. Genet. 2011; 88 (21665003): 852-860
        • Wang Y.
        • Thekdi N.
        • Smallwood P.M.
        • Macke J.P.
        • Nathans J.
        Frizzled-3 is required for the development of major fiber tracts in the rostral CNS.
        J. Neurosci. 2002; 22 (12351730): 8563-8573
        • Wang Y.
        • Chang H.
        • Rattner A.
        • Nathans J.
        Frizzled receptors in development and disease.
        Curr. Top. Dev. Biol. 2016; 117 (26969975): 113-139
        • Schulte G.
        Molecular pharmacology of Frizzleds—with implications for possible therapy.
        in: Gilchrist A. GPCR Molecular Pharmacology and Drug Targeting. John Wiley and Sons, Hoboken, NJ2010
        • Balla S.
        • Thapar V.
        • Verma S.
        • Luong T.
        • Faghri T.
        • Huang C.H.
        • Rajasekaran S.
        • del Campo J.J.
        • Shinn J.H.
        • Mohler W.A.
        • Maciejewski M.W.
        • Gryk M.R.
        • Piccirillo B.
        • Schiller S.R.
        • Schiller M.R.
        Minimotif Miner: a tool for investigating protein function.
        Nat. Methods. 2006; 3 (16489333): 175-177
        • Petersen J.
        • Wright S.C.
        • Rodríguez D.
        • Matricon P.
        • Lahav N.
        • Vromen A.
        • Friedler A.
        • Strömqvist J.
        • Wennmalm S.
        • Carlsson J.
        • Schulte G.
        Agonist-induced dimer dissociation as a macromolecular step in G protein-coupled receptor signaling.
        Nat. Commun. 2017; 8 (28790300): 226
        • Zhou X.E.
        • He Y.
        • de Waal P.W.
        • Gao X.
        • Kang Y.
        • Van Eps N.
        • Yin Y.
        • Pal K.
        • Goswami D.
        • White T.A.
        • Barty A.
        • Latorraca N.R.
        • Chapman H.N.
        • Hubbell W.L.
        • Dror R.O.
        • et al.
        Identification of phosphorylation codes for arrestin recruitment by G protein-coupled receptors.
        Cell. 2017; 170 (28753425): 457-469.e13
        • Cong F.
        • Schweizer L.
        • Varmus H.
        Casein kinase Iδ modulates the signaling specificities of dishevelled.
        Mol. Cell. Biol. 2004; 24 (14966280): 2000-2011
        • Bernatik O.
        • Ganji R.S.
        • Dijksterhuis J.P.
        • Konik P.
        • Cervenka I.
        • Polonio T.
        • Krejci P.
        • Schulte G.
        • Bryja V.
        Sequential activation and inactivation of Dishevelled in the Wnt/β-catenin pathway by casein kinases.
        J. Biol. Chem. 2011; 286 (21285348): 10396-10410
        • Bryja V.
        • Schulte G.
        • Rawal N.
        • Grahn A.
        • Arenas E.
        Wnt-5a induces Dishevelled phosphorylation and dopaminergic differentiation via a CK1-dependent mechanism.
        J. Cell Sci. 2007; 120 (17244647): 586-595
        • Cervenka I.
        • Valnohova J.
        • Bernatik O.
        • Harnos J.
        • Radsetoulal M.
        • Sedova K.
        • Hanakova K.
        • Potesil D.
        • Sedlackova M.
        • Salasova A.
        • Steinhart Z.
        • Angers S.
        • Schulte G.
        • Hampl A.
        • Zdrahal Z.
        • et al.
        Dishevelled is a NEK2 kinase substrate controlling dynamics of centrosomal linker proteins.
        Proc. Natl. Acad. Sci. U.S.A. 2016; 113 (27486244): 9304-9309
        • Schwarz-Romond T.
        • Fiedler M.
        • Shibata N.
        • Butler P.J.
        • Kikuchi A.
        • Higuchi Y.
        • Bienz M.
        The DIX domain of Dishevelled confers Wnt signaling by dynamic polymerization.
        Nat. Struct. Mol. Biol. 2007; 14 (17529994): 484-492
        • Fiedler M.
        • Mendoza-Topaz C.
        • Rutherford T.J.
        • Mieszczanek J.
        • Bienz M.
        Dishevelled interacts with the DIX domain polymerization interface of Axin to interfere with its function in down-regulating β-catenin.
        Proc. Natl. Acad. Sci. U.S.A. 2011; 108 (21245303): 1937-1942
        • Axelrod J.D.
        • Miller J.R.
        • Shulman J.M.
        • Moon R.T.
        • Perrimon N.
        Differential recruitment of Dishevelled provides signaling specificity in the planar cell polarity and Wingless signaling pathways.
        Genes Dev. 1998; 12 (9716412): 2610-2622
        • Wong H.C.
        • Mao J.
        • Nguyen J.T.
        • Srinivas S.
        • Zhang W.
        • Liu B.
        • Li L.
        • Wu D.
        • Zheng J.
        Structural basis of the recognition of the dishevelled DEP domain in the Wnt signaling pathway.
        Nat. Struct. Biol. 2000; 7 (11101902): 1178-1184
        • Paclikova P.
        • Bernatik O.
        • Radaszkiewicz T.W.
        • Bryja V.
        N-terminal part of Dishevelled DEP domain is required for Wnt/β-catenin signaling in mammalian cells.
        Mol. Cell. Biol. 2017; 37 (28674183): e00117-e00145
        • Uhlén M.
        • Fagerberg L.
        • Hallström B.M.
        • Lindskog C.
        • Oksvold P.
        • Mardinoglu A.
        • Sivertsson Å.
        • Kampf C.
        • Sjöstedt E.
        • Asplund A.
        • Olsson I.
        • Edlund K.
        • Lundberg E.
        • Navani S.
        • Szigyarto C.A.
        • et al.
        Proteomics. Tissue-based map of the human proteome.
        Science. 2015; 347 (25613900)1260419
        • Strutt H.
        • Price M.A.
        • Strutt D.
        Planar polarity is positively regulated by casein kinase Iδ in Drosophila.
        Curr. Biol. 2006; 16 (16824921): 1329-1336
        • Bernatík O.
        • Šedová K.
        • Schille C.
        • Ganji R.S.
        • Červenka I.
        • Trantírek L.
        • Schambony A.
        • Zdráhal Z.
        • Bryja V.
        Functional analysis of dishevelled-3 phosphorylation identifies distinct mechanisms driven by casein kinase 1 and frizzled5.
        J. Biol. Chem. 2014; 289 (24993822): 23520-23533
        • Bryja V.
        • Gradl D.
        • Schambony A.
        • Arenas E.
        • Schulte G.
        β-Arrestin is a necessary component of Wnt/β-catenin signaling in vitro and in vivo.
        Proc. Natl. Acad. Sci. U.S.A. 2007; 104 (17426148): 6690-6695
        • Bryja V.
        • Schambony A.
        • Cajánek L.
        • Dominguez I.
        • Arenas E.
        • Schulte G.
        β-Arrestin and casein kinase 1/2 define distinct branches of non-canonical WNT signalling pathways.
        EMBO Rep. 2008; 9 (18953287): 1244-1250
        • Bryja V.
        • Schulte G.
        • Arenas E.
        Wnt-3a utilizes a novel low dose and rapid pathway that does not require casein kinase 1-mediated phosphorylation of Dvl to activate β-catenin.
        Cell. Signal. 2007; 19 (17027228): 610-616
        • Kaucká M.
        • Plevová K.
        • Pavlová S.
        • Janovská P.
        • Mishra A.
        • Verner J.
        • Procházková J.
        • Krejcí P.
        • Kotasková J.
        • Ovesná P.
        • Tichy B.
        • Brychtová Y.
        • Doubek M.
        • Kozubík A.
        • Mayer J.
        • et al.
        The planar cell polarity pathway drives pathogenesis of chronic lymphocytic leukemia by the regulation of B-lymphocyte migration.
        Cancer Res. 2013; 73 (23338609): 1491-1501
        • Witte F.
        • Bernatik O.
        • Kirchner K.
        • Masek J.
        • Mahl A.
        • Krejci P.
        • Mundlos S.
        • Schambony A.
        • Bryja V.
        • Stricker S.
        Negative regulation of Wnt signaling mediated by CK1-phosphorylated Dishevelled via Ror2.
        FASEB J. 2010; 24 (20215527): 2417-2426
        • Komolov K.E.
        • Du Y.
        • Duc N.M.
        • Betz R.M.
        • Rodrigues J.P.G.L.M.
        • Leib R.D.
        • Patra D.
        • Skiniotis G.
        • Adams C.M.
        • Dror R.O.
        • Chung K.Y.
        • Kobilka B.K.
        • Benovic J.L.
        Structural and functional analysis of a β2-adrenergic receptor complex with GRK5.
        Cell. 2017; 169 (28431242): 407-421.e16
        • Chen M.
        • Philipp M.
        • Wang J.
        • Premont R.T.
        • Garrison T.R.
        • Caron M.G.
        • Lefkowitz R.J.
        • Chen W.
        G Protein-coupled receptor kinases phosphorylate LRP6 in the Wnt pathway.
        J. Biol. Chem. 2009; 284 (19801552): 35040-35048
        • Simons M.
        • Gault W.J.
        • Gotthardt D.
        • Rohatgi R.
        • Klein T.J.
        • Shao Y.
        • Lee H.J.
        • Wu A.L.
        • Fang Y.
        • Satlin L.M.
        • Dow J.T.
        • Chen J.
        • Zheng J.
        • Boutros M.
        • Mlodzik M.
        Electrochemical cues regulate assembly of the Frizzled/Dishevelled complex at the plasma membrane during planar epithelial polarization.
        Nat. Cell Biol. 2009; 11 (19234454): 286-294
        • Ma T.
        • Li B.
        • Wang R.
        • Lau P.K.
        • Huang Y.
        • Jiang L.
        • Schekman R.
        • Guo Y.
        A mechanism for differential sorting of the planar cell polarity proteins Frizzled6 and Vangl2 at the trans-Golgi network.
        J. Biol. Chem. 2018; 293 (29666182): 8410-8427
        • Chen W.
        • ten Berge D.
        • Brown J.
        • Ahn S.
        • Hu L.A.
        • Miller W.E.
        • Caron M.G.
        • Barak L.S.
        • Nusse R.
        • Lefkowitz R.J.
        Dishevelled 2 recruits β-arrestin 2 to mediate Wnt5A-stimulated endocytosis of Frizzled 4.
        Science. 2003; 301 (12958364): 1391-1394
        • Schulte G.
        • Schambony A.
        • Bryja V.
        β-Arrestins—scaffolds and signalling elements essential for WNT/Frizzled signalling pathways?.
        Br. J. Pharmacol. 2010; 159 (19888962): 1051-1058
        • Arthofer E.
        • Hot B.
        • Petersen J.
        • Strakova K.
        • Jäger S.
        • Grundmann M.
        • Kostenis E.
        • Gutkind J.S.
        • Schulte G.
        WNT stimulation dissociates a Frizzled 4 inactive-state complex with Gα12/13.
        Mol. Pharmacol. 2016; 90 (27458145): 447-459
        • Vizcaíno J.A.
        • Csordas A.
        • del-Toro N.
        • Dianes J.A.
        • Griss J.
        • Lavidas I.
        • Mayer G.
        • Perez-Riverol Y.
        • Reisinger F.
        • Ternent T.
        • Xu Q.W.
        • Wang R.
        • Hermjakob H.
        2016 update of the PRIDE database and its related tools.
        Nucleic Acids Res. 2016; 44 (26527722): D447-D456