Dishevelled (DVL) proteins are key mediators of the Wnt/β-catenin signaling pathway. All DVL proteins contain three conserved domains: DIX, PDZ, and DEP. There is a consensus in the field that the DIX domain is critical for Wnt/β-catenin signaling, but contradictory evidence regarding the function of the DEP domain exists. It has been difficult, until recently, to test the importance of the DEP domain rigorously because of the interference with endogenous DVL, expressed in all Wnt-responsive cell lines. In this study, we took advantage of DVL knockout (DVL1/DVL2/DVL3 triple knockout) cells fully deficient in Wnt3a-induced signaling events and performed a series of rescue experiments. Using these complementation assays, we analyzed the role of individual DVL isoforms. Further domain mapping of DVL1 showed that both the DVL1 DEP domain and especially its N-terminal region are required and sufficient for Wnt3a-induced phosphorylation of LRP6 and TopFlash reporter activation. On the contrary, multiple DEP domain mutants deficient in the planar cell polarity (PCP) pathway could fully rescue the Wnt3a response. This study provides conclusive evidence that the DVL DEP domain is essential for Wnt/β-catenin signaling in mammalian cells and establishes an experimental system suitable for further functional testing of DVL.

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

Institute of Biophysics The Czech Academy of Sciences Brno Czech Republic

Zobrazit více v PubMed

MacDonald BT, Tamai K, He X. 2009. Wnt/beta-catenin signaling: components, mechanisms, and diseases. Dev Cell 17:9–26. doi:10.1016/j.devcel.2009.06.016. PubMed DOI PMC

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

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

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

Hart MJ, de los Santos R, Albert IN, Rubinfeld B, Polakis P. 1998. Downregulation of beta-catenin by human Axin and its association with the APC tumor suppressor, beta-catenin and GSK3 beta. Curr Biol 8:573–581. doi:10.1016/S0960-9822(98)70226-X. PubMed DOI

Yost C, Torres M, Miller JR, Huang E, Kimelman D, Moon RT. 1996. The axis-inducing activity, stability, and subcellular distribution of beta-catenin is regulated in Xenopus embryos by glycogen synthase kinase 3. Genes Dev 10:1443–1454. doi:10.1101/gad.10.12.1443. PubMed DOI

Schwarz-Romond T, Fiedler M, Shibata N, Butler PJ, Kikuchi A, Higuchi Y, Bienz M. 2007. The DIX domain of Dishevelled confers Wnt signaling by dynamic polymerization. Nat Struct Mol Biol 14:484–492. doi:10.1038/nsmb1247. PubMed DOI

Bilic J, Huang YL, Davidson G, Zimmermann T, Cruciat CM, Bienz M, Niehrs C. 2007. Wnt induces LRP6 signalosomes and promotes Dishevelled-dependent LRP6 phosphorylation. Science 316:1619–1622. doi:10.1126/science.1137065. PubMed DOI

Axelrod JD, Miller JR, Shulman JM, Moon RT, Perrimon N. 1998. Differential recruitment of Dishevelled provides signaling specificity in the planar cell polarity and Wingless signaling pathways. Genes Dev 12:2610–2622. doi:10.1101/gad.12.16.2610. PubMed DOI PMC

Rothbacher U, Laurent MN, Deardorff MA, Klein PS, Cho KW, Fraser SE. 2000. Dishevelled phosphorylation, subcellular localization and multimerization regulate its role in early embryogenesis. EMBO J 19:1010–1022. PubMed PMC

Collu GM, Mlodzik M. 2015. Planar polarity: converting a morphogen gradient into cellular polarity. Curr Biol 25:R372–R374. doi:10.1016/j.cub.2015.03.011. PubMed DOI PMC

Boutros M, Paricio N, Strutt DI, Mlodzik M. 1998. Dishevelled activates JNK and discriminates between JNK pathways in planar polarity and Wingless signaling. Cell 94:109–118. doi:10.1016/S0092-8674(00)81226-X. PubMed DOI

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, Bryja V. 2016. Dishevelled is a NEK2 kinase substrate controlling dynamics of centrosomal linker proteins. Proc Natl Acad Sci U S A 113:9304–9309. doi:10.1073/pnas.1608783113. PubMed DOI PMC

Gammons MV, Rutherford TJ, Steinhart Z, Angers S, Bienz M. 2016. Essential role of the Dishevelled DEP domain in a Wnt-dependent human-cell-based complementation assay. J Cell Sci 129:3892–3902. doi:10.1242/jcs.195685. PubMed DOI PMC

Liu J, Pan S, Hsieh MH, Ng N, Sun F, Wang T, Kasibhatla S, Schuller AG, Li AG, Cheng D, Li J, Tompkins C, Pferdekamper A, Steffy A, Cheng J, Kowal C, Phung V, Guo G, Wang Y, Graham MP, Flynn S, Brenner JC, Li C, Villarroel MC, Schultz PG, Wu X, McNamara P, Sellers WR, Petruzzelli L, Boral AL, Seidel HM, McLaughlin ME, Che J, Carey TE, Vanasse G, Harris JL. 2013. Targeting Wnt-driven cancer through the inhibition of Porcupine by LGK974. Proc Natl Acad Sci U S A 110:20224–20229. doi:10.1073/pnas.1314239110. PubMed DOI PMC

Korinek V, Barker N, Morin PJ, van Wichen D, de Weger R, Kinzler KW, Vogelstein B, Clevers H. 1997. Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC−/− colon carcinoma. Science 275:1784–1787. doi:10.1126/science.275.5307.1784. PubMed DOI

Tauriello DV, Haegebarth A, Kuper I, Edelmann MJ, Henraat M, Canninga-van Dijk MR, Kessler BM, Clevers H, Maurice MM. 2010. Loss of the tumor suppressor CYLD enhances Wnt/beta-catenin signaling through K63-linked ubiquitination of Dvl. Mol Cell 37:607–619. doi:10.1016/j.molcel.2010.01.035. PubMed DOI

Hao HX, Xie Y, Zhang Y, Charlat O, Oster E, Avello M, Lei H, Mickanin C, Liu D, Ruffner H, Mao X, Ma Q, Zamponi R, Bouwmeester T, Finan PM, Kirschner MW, Porter JA, Serluca FC, Cong F. 2012. ZNRF3 promotes Wnt receptor turnover in an R-spondin-sensitive manner. Nature 485:195–200. doi:10.1038/nature11019. PubMed DOI

Simons M, Gault WJ, Gotthardt D, Rohatgi R, Klein TJ, Shao Y, Lee HJ, Wu AL, Fang Y, Satlin LM, Dow JT, Chen J, Zheng J, Boutros M, Mlodzik M. 2009. Electrochemical cues regulate assembly of the Frizzled/Dishevelled complex at the plasma membrane during planar epithelial polarization. Nat Cell Biol 11:286–294. doi:10.1038/ncb1836. PubMed DOI PMC

Singh J, Yanfeng WA, Grumolato L, Aaronson SA, Mlodzik M. 2010. Abelson family kinases regulate Frizzled planar cell polarity signaling via Dsh phosphorylation. Genes Dev 24:2157–2168. doi:10.1101/gad.1961010. PubMed DOI PMC

Tauriello DV, Jordens I, Kirchner K, Slootstra JW, Kruitwagen T, Bouwman BA, Noutsou M, Rudiger SG, Schwamborn K, Schambony A, Maurice MM. 2012. Wnt/beta-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 109:E812–E820. doi:10.1073/pnas.1114802109. PubMed DOI PMC

Zeng X, Huang H, Tamai K, Zhang X, Harada Y, Yokota C, Almeida K, Wang J, Doble B, Woodgett J, Wynshaw-Boris A, Hsieh JC, He X. 2008. Initiation of Wnt signaling: control of Wnt coreceptor Lrp6 phosphorylation/activation via Frizzled, Dishevelled and Axin functions. Development 135:367–375. PubMed PMC

Etheridge SL, Ray S, Li S, Hamblet NS, Lijam N, Tsang M, Greer J, Kardos N, Wang J, Sussman DJ, Chen P, Wynshaw-Boris A. 2008. Murine Dishevelled 3 functions in redundant pathways with Dishevelled 1 and 2 in normal cardiac outflow tract, cochlea, and neural tube development. PLoS Genet 4:e1000259. doi:10.1371/journal.pgen.1000259. PubMed DOI PMC

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

Penton A, Wodarz A, Nusse R. 2002. A mutational analysis of Dishevelled in Drosophila defines novel domains in the Dishevelled protein as well as novel suppressing alleles of Axin. Genetics 161:747–762. PubMed PMC

Bernatik O, Sedova K, Schille C, Ganji RS, Cervenka I, Trantirek L, Schambony A, Zdrahal Z, Bryja V. 2014. Functional analysis of Dishevelled-3 phosphorylation identifies distinct mechanisms driven by casein kinase 1 and Frizzled5. J Biol Chem 289:23520–23533. doi:10.1074/jbc.M114.590638. PubMed DOI PMC

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

Capelluto DG, Zhao X, Lucas A, Lemkul JA, Xiao S, Fu X, Sun F, Bevan DR, Finkielstein CV. 2014. Biophysical and molecular-dynamics studies of phosphatidic acid binding by the Dvl-2 DEP domain. Biophys J 106:1101–1111. doi:10.1016/j.bpj.2014.01.032. PubMed DOI PMC

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

Wong HC, Mao J, Nguyen JT, Srinivas S, Zhang W, Liu B, Li L, Wu D, Zheng J. 2000. Structural basis of the recognition of the Dishevelled DEP domain in the Wnt signaling pathway. Nat Struct Biol 7:1178–1184. doi:10.1038/82047. PubMed DOI PMC

Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM. 2013. RNA-guided human genome engineering via Cas9. Science 339:823–826. doi:10.1126/science.1232033. PubMed DOI PMC

Li X, Burnight ER, Cooney AL, Malani N, Brady T, Sander JD, Staber J, Wheelan SJ, Joung JK, McCray PB Jr, Bushman FD, Sinn PL, Craig NL. 2013. piggyBac transposase tools for genome engineering. Proc Natl Acad Sci U S A 110:E2279–E2287. doi:10.1073/pnas.1305987110. PubMed DOI PMC

Bryja V, Schulte G, Rawal N, Grahn A, Arenas E. 2007. Wnt-5a induces Dishevelled phosphorylation and dopaminergic differentiation via a CK1-dependent mechanism. J Cell Sci 120:586–595. doi:10.1242/jcs.03368. PubMed DOI

Angers S, Thorpe CJ, Biechele TL, Goldenberg SJ, Zheng N, MacCoss MJ, Moon RT. 2006. The KLHL12-Cullin-3 ubiquitin ligase negatively regulates the Wnt-beta-catenin pathway by targeting Dishevelled for degradation. Nat Cell Biol 8:348–357. doi:10.1038/ncb1381. PubMed DOI

Veeman MT, Axelrod JD, Moon RT. 2003. A second canon. Functions and mechanisms of beta-catenin-independent Wnt signaling. Dev Cell 5:367–377. PubMed

Kilander MB, Petersen J, Andressen KW, Ganji RS, Levy FO, Schuster J, Dahl N, Bryja V, Schulte G. 2014. Disheveled regulates precoupling of heterotrimeric G proteins to Frizzled 6. FASEB J 28:2293–2305. doi:10.1096/fj.13-246363. PubMed DOI

Narimatsu M, Bose R, Pye M, Zhang L, Miller B, Ching P, Sakuma R, Luga V, Roncari L, Attisano L, Wrana JL. 2009. Regulation of planar cell polarity by Smurf ubiquitin ligases. Cell 137:295–307. doi:10.1016/j.cell.2009.02.025. PubMed DOI

Ma L, Wang Y, Malbon CC, Wang HY. 2010. Dishevelled-3 C-terminal His single amino acid repeats are obligate for Wnt5a activation of non-canonical signaling. J Mol Signal 5:19. doi:10.1186/1750-2187-5-19. PubMed DOI PMC

Endogenous oligomer formation underlies DVL2 condensates and promotes Wnt/β-catenin signaling

Carboxy-terminal polyglutamylation regulates signaling and phase separation of the Dishevelled protein

Intestinal Paneth cell differentiation relies on asymmetric regulation of Wnt signaling by Daam1/2

RNF43 inhibits WNT5A-driven signaling and suppresses melanoma invasion and resistance to the targeted therapy

Primary Cilia Formation Does Not Rely on WNT/β-Catenin Signaling

Phosphorylation-induced changes in the PDZ domain of Dishevelled 3

12-O-Tetradecanoylphorbol-13-acetate increases cardiomyogenesis through PKC/ERK signaling

Protease associated domain of RNF43 is not necessary for the suppression of Wnt/β-catenin signaling in human cells

Activity of Smurf2 Ubiquitin Ligase Is Regulated by the Wnt Pathway Protein Dishevelled

Comparative phosphorylation map of Dishevelled 3 links phospho-signatures to biological outputs

Dishevelled-3 conformation dynamics analyzed by FRET-based biosensors reveals a key role of casein kinase 1

The planar cell polarity protein VANG-1/Vangl negatively regulates Wnt/β-catenin signaling through a Dvl dependent mechanism

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

Dishevelled has a YAP nuclear export function in a tumor suppressor context-dependent manner

The connections of Wnt pathway components with cell cycle and centrosome: side effects or a hidden logic?