Dishevelled (DVL) is the key component of the Wnt signaling pathway. Currently, DVL conformational dynamics under native conditions is unknown. To overcome this limitation, we develop the Fluorescein Arsenical Hairpin Binder- (FlAsH-) based FRET in vivo approach to study DVL conformation in living cells. Using this single-cell FRET approach, we demonstrate that (i) Wnt ligands induce open DVL conformation, (ii) DVL variants that are predominantly open, show more even subcellular localization and more efficient membrane recruitment by Frizzled (FZD) and (iii) Casein kinase 1 ɛ (CK1ɛ) has a key regulatory function in DVL conformational dynamics. In silico modeling and in vitro biophysical methods explain how CK1ɛ-specific phosphorylation events control DVL conformations via modulation of the PDZ domain and its interaction with DVL C-terminus. In summary, our study describes an experimental tool for DVL conformational sampling in living cells and elucidates the essential regulatory role of CK1ɛ in DVL conformational dynamics.

Biology Department Developmental Biology Friedrich Alexander University Erlangen Nüremberg Erlangen 91058 Germany

CEITEC Central European Institute of Technology Masaryk University Brno 62500 Czech Republic

Department of Cell Developmental and Regenerative Biology Icahn School of Medicine at Mount Sinai New York NY 10029 USA

Department of Experimental Biology Faculty of Science Masaryk University Brno 62500 Czech Republic

Department of Pharmacology and Toxicology University of Würzburg Würzburg 97078 Germany

Institute for Molecular Cell Biology CMB Center for Molecular Biomedicine University Hospital Jena Friedrich Schiller University Jena Jena 07745 Germany

Institute of Biophysics Academy of Sciences of the Czech Republic v v i Brno 612 65 Czech Republic

Max Planck Institute for the Science of Light Erlangen 91058 Germany

National Centre for Biomolecular Research Faculty of Science Masaryk University Brno 62500 Czech Republic

Pepscan Therapeutics B 5 Lelystad 8243 The Netherlands

Rudolf Virchow Center for Experimental Biomedicine University of Würzburg Würzburg 97078 Germany

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Clevers H. Wnt/beta-catenin signaling in development and disease. Cell. 2006;127:469–480. doi: 10.1016/j.cell.2006.10.018. PubMed DOI

Gao C, Chen YG. Dishevelled: the hub of Wnt signaling. Cell. Signal. 2010;22:717–727. doi: 10.1016/j.cellsig.2009.11.021. PubMed DOI

Wallingford JB, Habas R. The developmental biology of Dishevelled: an enigmatic protein governing cell fate and cell polarity. Development. 2005;132:4421–4436. doi: 10.1242/dev.02068. PubMed DOI

Mlodzik M. The Dishevelled protein family: still rather a mystery after over 20 years of molecular studies. Curr. Top. Dev. Biol. 2016;117:75–91. doi: 10.1016/bs.ctdb.2015.11.027. PubMed DOI PMC

Gentzel M, Schambony A. Dishevelled paralogs in vertebrate development: redundant or distinct? Front. Cell Dev. Biol. 2017;5:59. doi: 10.3389/fcell.2017.00059. PubMed DOI PMC

Wynshaw-Boris A. Dishevelled: in vivo roles of a multifunctional gene family during development. Curr. Top. Dev. Biol. 2012;101:213–235. doi: 10.1016/B978-0-12-394592-1.00007-7. PubMed DOI

Madrzak J, et al. Ubiquitination of the Dishevelled DIX domain blocks its head-to-tail polymerization. Nat. Commun. 2015;6:6718. doi: 10.1038/ncomms7718. PubMed DOI PMC

Cheyette BN, et al. Dapper, a Dishevelled-associated antagonist of beta-catenin and JNK signaling, is required for notochord formation. Dev. Cell. 2002;2:449–461. doi: 10.1016/S1534-5807(02)00140-5. PubMed DOI

Wong HC, et al. Structural basis of the recognition of the dishevelled DEP domain in the Wnt signaling pathway. Nat. Struct. Biol. 2000;7:1178–1184. doi: 10.1038/82047. PubMed DOI PMC

Schwarz-Romond T, et al. The DIX domain of Dishevelled confers Wnt signaling by dynamic polymerization. Nat. Struct. Mol. Biol. 2007;14:484–492. doi: 10.1038/nsmb1247. PubMed DOI

Fiedler M, Mendoza-Topaz C, Rutherford TJ, Mieszczanek J, Bienz M. Dishevelled interacts with the DIX domain polymerization interface of Axin to interfere with its function in down-regulating beta-catenin. Proc. Natl. Acad. Sci. USA. 2011;108:1937–1942. doi: 10.1073/pnas.1017063108. PubMed DOI PMC

Schwarz-Romond T, Metcalfe C, Bienz M. Dynamic recruitment of axin by Dishevelled protein assemblies. J. Cell Sci. 2007;120:2402–2412. doi: 10.1242/jcs.002956. PubMed DOI

Bilic J, et al. Wnt induces LRP6 signalosomes and promotes dishevelled-dependent LRP6 phosphorylation. Science. 2007;316:1619–1622. doi: 10.1126/science.1137065. PubMed DOI

Gammons MV, Renko M, Johnson CM, Rutherford TJ, Bienz M. Wnt signalosome assembly by DEP domain swapping of Dishevelled. Mol. Cell. 2016;64:92–104. doi: 10.1016/j.molcel.2016.08.026. PubMed DOI PMC

Smietana K, Mateja A, Krezel A, Otlewski J. PDZ domain from Dishevelled—a specificity study. Acta Biochim. Pol. 2011;58:243–249. doi: 10.18388/abp.2011_2272. PubMed DOI

Lee HJ, Shi DL, Zheng JJ. Conformational change of Dishevelled plays a key regulatory role in the Wnt signaling pathways. eLife. 2015;4:e08142. doi: 10.7554/eLife.08142. PubMed DOI PMC

Qi J, et al. Autoinhibition of Dishevelled protein regulated by its extreme C terminus plays a distinct role in Wnt/beta-catenin and Wnt/planar cell polarity (PCP) signaling pathways. J. Biol. Chem. 2017;292:5898–5908. doi: 10.1074/jbc.M116.772509. PubMed DOI PMC

Hoffmann C, et al. A FlAsH-based FRET approach to determine G protein-coupled receptor activation in living cells. Nat. Methods. 2005;2:171–176. doi: 10.1038/nmeth742. PubMed DOI

Hoffmann C, et al. Fluorescent labeling of tetracysteine-tagged proteins in intact cells. Nat. Protoc. 2010;5:1666–1677. doi: 10.1038/nprot.2010.129. PubMed DOI PMC

Nuber S, et al. beta-Arrestin biosensors reveal a rapid, receptor-dependent activation/deactivation cycle. Nature. 2016;531:661–664. doi: 10.1038/nature17198. PubMed DOI PMC

Xue B, Dunbrack RL, Williams RW, Dunker AK, Uversky VN. PONDR-FIT: a meta-predictor of intrinsically disordered amino acids. Biochim. Biophys. Acta. 2010;1804:996–1010. doi: 10.1016/j.bbapap.2010.01.011. PubMed DOI PMC

Paclikova, P., Bernatik, O., Radaszkiewicz, T. W. & Bryja, V. N-terminal part of Dishevelled DEP domain is required for Wnt/beta-catenin signaling in mammalian cells. Mol. Cell Biol.10.1128/MCB.00145-17 (2017). PubMed PMC

Yanagawa S, van Leeuwen F, Wodarz A, Klingensmith J, Nusse R. The dishevelled protein is modified by wingless signaling in Drosophila. Gene Dev. 1995;9:1087–1097. doi: 10.1101/gad.9.9.1087. PubMed DOI

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;120:586–595. doi: 10.1242/jcs.03368. PubMed DOI

Rothbacher U, et al. Dishevelled phosphorylation, subcellular localization and multimerization regulate its role in early embryogenesis. EMBO J. 2000;19:1010–1022. doi: 10.1093/emboj/19.5.1010. PubMed DOI PMC

Gonzalez-Sancho JM, et al. Functional consequences of Wnt-induced Dishevelled 2 phosphorylation in canonical and noncanonical Wnt signaling. J. Biol. Chem. 2013;288:9428–9437. doi: 10.1074/jbc.M112.448480. PubMed DOI PMC

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 beta-catenin. Cell. Signal. 2007;19:610–616. doi: 10.1016/j.cellsig.2006.08.011. PubMed DOI

Bernatik O, et al. Sequential activation and inactivation of Dishevelled in the Wnt/beta-catenin pathway by casein kinases. J. Biol. Chem. 2011;286:10396–10410. doi: 10.1074/jbc.M110.169870. PubMed DOI PMC

Bernatik O, et al. Functional analysis of dishevelled-3 phosphorylation identifies distinct mechanisms driven by casein kinase 1 and frizzled5. J. Biol. Chem. 2014;289:23520–23533. doi: 10.1074/jbc.M114.590638. PubMed DOI PMC

Kaucka M, et al. Asymmetry of VANGL2 in migrating lymphocytes as a tool to monitor activity of the mammalian WNT/planar cell polarity pathway. Cell Commun. Signal. 2015;13:2. doi: 10.1186/s12964-014-0079-1. PubMed DOI PMC

Cong F, Schweizer L, Varmus H. Casein kinase Iepsilon modulates the signaling specificities of dishevelled. Mol. Cell. Biol. 2004;24:2000–2011. doi: 10.1128/MCB.24.5.2000-2011.2004. PubMed DOI PMC

Xue B, Dunker AK, Uversky VN. Retro-MoRFs: identifying protein binding sites by normal and reverse alignment and intrinsic disorder prediction. Int. J. Mol. Sci. 2010;11:3725–3747. doi: 10.3390/ijms11103725. PubMed DOI PMC

Foldynova-Trantirkova S, et al. Breast cancer-specific mutations in CK1epsilon inhibit Wnt/beta-catenin and activate the Wnt/Rac1/JNK and NFAT pathways to decrease cell adhesion and promote cell migration. Breast Cancer Res. 2010;12:R30. doi: 10.1186/bcr2581. PubMed DOI PMC

Luck K, Charbonnier S, Trave G. The emerging contribution of sequence context to the specificity of protein interactions mediated by PDZ domains. FEBS Lett. 2012;586:2648–2661. doi: 10.1016/j.febslet.2012.03.056. PubMed DOI

Mostarda S, Gfeller D, Rao F. Beyond the binding site: the role of the beta(2)-beta(3) loop and extra-domain structures in PDZ domains. PLoS Comput. Biol. 2012;8:e1002429. doi: 10.1371/journal.pcbi.1002429. PubMed DOI PMC

Zhang J, et al. Structural basis of beta-catenin recognition by Tax-interacting protein-1. J. Mol. Biol. 2008;384:255–263. doi: 10.1016/j.jmb.2008.09.034. PubMed DOI

Strakova Katerina, Kowalski-Jahn Maria, Gybel Tomas, Valnohova Jana, Dhople Vishnu M., Harnos Jakub, Bernatik Ondrej, Ganji Ranjani Sri, Zdrahal Zbynek, Mulder Jan, Lindskog Cecilia, Bryja Vitezslav, Schulte Gunnar. Dishevelled enables casein kinase 1–mediated phosphorylation of Frizzled 6 required for cell membrane localization. Journal of Biological Chemistry. 2018;293(48):18477–18493. doi: 10.1074/jbc.RA118.004656. PubMed DOI PMC

Jung H, et al. Deubiquitination of Dishevelled by Usp14 is required for Wnt signaling. Oncogenesis. 2013;2:e64. doi: 10.1038/oncsis.2013.28. PubMed DOI PMC

Kishida M, et al. Synergistic activation of the Wnt signaling pathway by Dvl and casein kinase Iepsilon. J. Biol. Chem. 2001;276:33147–33155. doi: 10.1074/jbc.M103555200. PubMed DOI

Rauch J, Volinsky N, Romano D, Kolch W. The secret life of kinases: functions beyond catalysis. Cell Commun. Signal. 2011;9:23. doi: 10.1186/1478-811X-9-23. PubMed DOI PMC

Klein TJ, Jenny A, Djiane A, Mlodzik M. CKIepsilon/discs overgrown promotes both Wnt-Fz/beta-catenin and Fz/PCP signaling in Drosophila. Curr. Biol. 2006;16:1337–1343. doi: 10.1016/j.cub.2006.06.030. PubMed DOI

Wong HC, et al. Direct binding of the PDZ domain of Dishevelled to a conserved internal sequence in the C-terminal region of frizzled. Mol. Cell. 2003;12:1251–1260. doi: 10.1016/S1097-2765(03)00427-1. PubMed DOI PMC

Punchihewa C, Ferreira AM, Cassell R, Rodrigues P, Fujii N. Sequence requirement and subtype specificity in the high-affinity interaction between human frizzled and dishevelled proteins. Protein Sci. 2009;18:994–1002. doi: 10.1002/pro.109. PubMed DOI PMC

Witte F, et al. Negative regulation of Wnt signaling mediated by CK1-phosphorylated Dishevelled via Ror2. FASEB J. 2010;24:2417–2426. doi: 10.1096/fj.09-150615. PubMed DOI

Funato Y, et al. Nucleoredoxin sustains Wnt/beta-catenin signaling by retaining a pool of inactive dishevelled protein. Curr. Biol. 2010;20:1945–1952. doi: 10.1016/j.cub.2010.09.065. PubMed DOI

Funato Y, Michiue T, Asashima M, Miki H. The thioredoxin-related redox-regulating protein nucleoredoxin inhibits Wnt-beta-catenin signalling through dishevelled. Nat. Cell Biol. 2006;8:501–U135. doi: 10.1038/ncb1405. PubMed DOI

Angers S, et al. The KLHL12-Cullin-3 ubiquitin ligase negatively regulates the Wnt-beta-catenin pathway by targeting Dishevelled for degradation. Nat. Cell Biol. 2006;8:348–357. doi: 10.1038/ncb1381. PubMed DOI

McKay RM, Peters JM, Graff JM. The casein kinase I family in Wnt signaling. Dev. Biol. 2001;235:388–396. doi: 10.1006/dbio.2001.0308. PubMed DOI

Ran FA, et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 2013;8:2281–2308. doi: 10.1038/nprot.2013.143. PubMed DOI PMC

Chu VT, et al. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat. Biotechnol. 2015;33:543–548. doi: 10.1038/nbt.3198. PubMed DOI

Bryja V, et al. Increased apoptosis in differentiating p27-deficient mouse embryonic stem cells. Cell. Mol. Life Sci. 2004;61:1384–1400. doi: 10.1007/s00018-004-4081-4. PubMed DOI PMC

Cervenka I, et al. Dishevelled is a NEK2 kinase substrate controlling dynamics of centrosomal linker proteins. Proc. Natl. Acad. Sci. USA. 2016;113:9304–9309. doi: 10.1073/pnas.1608783113. PubMed DOI PMC

Nieuwkoop, P. D. & Faber Garland, J. Normal table of Xenopus laevis (Garland Publishing Inc, New York, 1994).

Gentzel M, Schille C, Rauschenberger V, Schambony A. Distinct functionality of dishevelled isoforms on Ca2+/calmodulin-dependent protein kinase 2 (CamKII) in Xenopus gastrulation. Mol. Biol. Cell. 2015;26:966–977. doi: 10.1091/mbc.E14-06-1089. PubMed DOI PMC

Bryja V, et al. Beta-arrestin and casein kinase 1/2 define distinct branches of non-canonical WNT signalling pathways. EMBO Rep. 2008;9:1244–1250. doi: 10.1038/embor.2008.193. PubMed DOI PMC

Vilardaga JP, Bunemann M, Krasel C, Castro M, Lohse MJ. Measurement of the millisecond activation switch of G protein-coupled receptors in living cells. Nat. Biotechnol. 2003;21:807–812. doi: 10.1038/nbt838. PubMed DOI

Jost CA, Reither G, Hoffmann C, Schultz C. Contribution of fluorophores to protein kinase C FRET probe performance. Chembiochem. 2008;9:1379–1384. doi: 10.1002/cbic.200700728. PubMed DOI

Stejskal K, Potesil D, Zdrahal Z. Suppression of peptide sample losses in autosampler vials. J. Proteome Res. 2013;12:3057–3062. doi: 10.1021/pr400183v. PubMed DOI

Stanek J, Augustyniak R, Kozminski W. Suppression of sampling artefacts in high-resolution four-dimensional NMR spectra using signal separation algorithm. J. Magn. Reson. 2012;214:91–102. doi: 10.1016/j.jmr.2011.10.009. PubMed DOI

Evangelidis T, et al. Automated NMR resonance assignments and structure determination using a minimal set of 4D spectra. Nat. Commun. 2018;9:384. doi: 10.1038/s41467-017-02592-z. PubMed DOI PMC

Pronk S, et al. GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit. Bioinformatics. 2013;29:845–854. doi: 10.1093/bioinformatics/btt055. PubMed DOI PMC

Sali A, Blundell TL. Comparative protein modeling by satisfaction of spatial restraints. J. Mol. Biol. 1993;234:779–815. doi: 10.1006/jmbi.1993.1626. PubMed DOI

Fiser A, Sali A. MODELLER: Generation and refinement of homology-based protein structure models. Methods Enzymol. 2003;374:461–491. doi: 10.1016/S0076-6879(03)74020-8. PubMed DOI

Fiser A, Do RKG, Sali A. Modeling of loops in protein structures. Protein Sci. 2000;9:1753–1773. doi: 10.1110/ps.9.9.1753. PubMed DOI PMC

Bereau T, Deserno M. Generic coarse-grained model for protein folding and aggregation. J. Chem. Phys. 2009;130:235106. doi: 10.1063/1.3152842. PubMed DOI PMC

Lindorff-Larsen K, et al. Improved side-chain torsion potentials for the Amber ff99SB protein force field. Proteins. 2010;78:1950–1958. PubMed PMC

Homeyer N, Horn AH, Lanig H, Sticht H. AMBER force-field parameters for phosphorylated amino acids in different protonation states: phosphoserine, phosphothreonine, phosphotyrosine, and phosphohistidine. J. Mol. Model. 2006;12:281–289. doi: 10.1007/s00894-005-0028-4. PubMed DOI

Jorgensen WL, Chandrasekhar J, Madura JD, Impey RW, Klein ML. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 1983;79:926–935. doi: 10.1063/1.445869. DOI

Best RB, Zheng WW, Mittal J. Balanced protein-water interactions improve properties of disordered proteins and non-specific protein association. J. Chem. Theory Comput. 2014;10:5113–5124. doi: 10.1021/ct500569b. PubMed DOI PMC

Bussi, G., Donadio, D. & Parrinello, M. Canonical sampling through velocity rescaling. J. Chem. Phys.126, 014101 (2007). PubMed

Parrinello M, Rahman A. Polymorphic transitions in single-crystals—a new molecular-dynamics method. J. Appl. Phys. 1981;52:7182–7190. doi: 10.1063/1.328693. DOI

Nose S, Klein ML. Constant pressure molecular-dynamics for molecular-systems. Mol. Phys. 1983;50:1055–1076. doi: 10.1080/00268978300102851. DOI

Darden T, York D, Pedersen L. Particle Mesh Ewald—an N.Log(N) method for Ewald sums in large systems. J. Chem. Phys. 1993;98:10089–10092. doi: 10.1063/1.464397. DOI

Essmann U, et al. A smooth particle Mesh Ewald Method. J. Chem. Phys. 1995;103:8577–8593. doi: 10.1063/1.470117. DOI

Hess B, Bekker H, Berendsen HJC, Fraaije JGEM. LINCS: a linear constraint solver for molecular simulations. J. Comput. Chem. 1997;18:1463–1472. doi: 10.1002/(SICI)1096-987X(199709)18:12<1463::AID-JCC4>3.0.CO;2-H. DOI

Perez-Riverol Y, et al. The PRIDE database and related tools and resources in 2019: improving support for quantification data. Nucleic Acids Res. 2019;47:D442–D450. doi: 10.1093/nar/gky1106. PubMed DOI PMC

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