Wnt and BMP signaling pathways are two key molecular machineries regulating development and homeostasis. The efficient coordination of Wnt and BMP is essential in many developmental processes such as establishment of antero-posterior and dorso-ventral body axis, regulation of convergent extension, or development of various organ systems. SMAD ubiquitination regulatory factor (Smurf) family of E3 ubiquitin ligases are important and evolutionary conserved regulators of TGF-β/BMP signaling pathways. Smurf2 has been previously shown to regulate Wnt/planar cell polarity (PCP) signaling pathway by ubiquitinating Prickle1, one of the key components of PCP. We explored the role of Smurf2 in Wnt pathways in further detail and identified that Smurf2 is also a ubiquitin ligase of Dishevelled (DVL), the key cytoplasmic signal transducer in the Wnt pathway. Interestingly, the Smurf2 and DVL relationship expands beyond substrate-E3 ligase. We can show that DVL activates Smurf2, which allows Smurf2 to ubiquitinate its substrates from Wnt/PCP (Prickle1) as well as TGF-β/BMP (Smad2) pathways more efficiently. Using SMAD7 as an example of Smurf2 activator we show that DVL and SMAD7 both activates Smurf2 activity. In HEK293 cells the deficiency of DVL phenocopies absence of Smurf2 and leads to the increased phosphorylation of R-Smads. Smurf2-DVL connection provides a novel and intriguing point of crosstalk for Wnt and BMP pathways.

Department of Cytokinetics Institute of Biophysics Academy of Sciences of the Czech Republic 61200 Brno Czech Republic

Institute of Experimental Biology Faculty of Science Masaryk University 61137 Brno Czech Republic

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Carron C., Shi D.L. Specification of anteroposterior axis by combinatorial signaling during Xenopus development. Wiley Interdiscip. Rev. Dev. Biol. 2016;5:150–168. doi: 10.1002/wdev.217. PubMed DOI

Mayor R., Theveneau E. The role of the non-canonical Wnt–planar cell polarity pathway in neural crest migration. Biochem. J. 2013 doi: 10.1042/BJ20131182. PubMed DOI

Zuniga A. Next generation limb development and evolution: Old questions, new perspectives. Development. 2015;142:3810–3820. doi: 10.1242/dev.125757. PubMed DOI

Bernassola F., Karin M., Ciechanover A., Melino G. The HECT Family of E3 Ubiquitin Ligases: Multiple Players in Cancer Development. Cancer Cell. 2008;14:10–21. doi: 10.1016/j.ccr.2008.06.001. PubMed DOI

Zhu H., Kavsak P., Abdollah S., Wrana J.L., Thomsen G.H. A SMAD ubiquitin ligase targets the BMP pathway and affects embryonic pattern formation. Nature. 1999;400:687. doi: 10.1038/23293. PubMed DOI

Zhang Y., Chang C., Gehling D.J., Hemmati-Brivanlou A., Derynck R. Regulation of Smad degradation and activity by Smurf2, an E3 ubiquitin ligase. Proc. Natl. Acad. Sci. USA. 2002;98:974–979. doi: 10.1073/pnas.98.3.974. PubMed DOI PMC

Kavsak P., Rasmussen R.K., Causing C.G., Bonni S., Zhu H., Thomsen G.H., Wrana J.L. Smad7 binds to Smurf2 to form an E3 ubiquitin ligase that targets the TGFβ receptor for degradation. Mol. Cell. 2000;6:1365–1375. doi: 10.1016/S1097-2765(00)00134-9. PubMed DOI

Osmundson E.C., Ray D., Moore F.E., Gao Q., Thomsen G.H., Kiyokawa H. The HECT E3 ligase Smurf2 is required for Mad2-dependent spindle assembly checkpoint. J. Cell Biol. 2008;183:267–277. doi: 10.1083/jcb.200801049. PubMed DOI PMC

Moore F.E., Osmundson E.C., Koblinski J., Pugacheva E., Golemis E.A., Ray D., Kiyokawa H. The WW-HECT protein Smurf2 interacts with the Docking Protein NEDD9/HEF1 for Aurora A activation. Cell Div. 2010;5:22. doi: 10.1186/1747-1028-5-22. PubMed DOI PMC

Wang H.R., Ogunjimi A.A., Zhang Y., Ozdamar B., Bose R., Wrana J.L. Degradation of RhoA by Smurf1 ubiquitin ligase. Methods Enzymol. 2006;406:437–447. doi: 10.1016/S0076-6879(06)06032-0. PubMed DOI

Huang C., Rajfur Z., Yousefi N., Chen Z., Jacobson K., Ginsberg M.H. Talin phosphorylation by Cdk5 regulates Smurf1-mediated talin head ubiquitylation and cell migration. Nat. Cell Biol. 2009;11:624–630. doi: 10.1038/ncb1868. PubMed DOI PMC

Narimatsu M., Bose R., Pye M., Zhang L., Miller B., Ching P., Sakuma R., Luga V., Roncari L., Attisano L., et al. Regulation of Planar Cell Polarity by Smurf Ubiquitin Ligases. Cell. 2009;137:295–307. doi: 10.1016/j.cell.2009.02.025. PubMed DOI

Yang Y., Mlodzik M. Wnt-Frizzled/Planar Cell Polarity Signaling: Cellular Orientation by Facing the Wind (Wnt) Annu. Rev. Cell Dev. Biol. 2015;31:623–646. doi: 10.1146/annurev-cellbio-100814-125315. PubMed DOI PMC

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

Angers S., Thorpe C.J., Biechele T.L., Goldenberg S.J., Zheng N., MacCoss M.J., Moon R.T. The KLHL12-Cullin-3 ubiquitin ligase negatively regulates the Wnt-β-catenin pathway by targeting dishevelled for degradation. Nat. Cell Biol. 2006;8:348–357. doi: 10.1038/ncb1381. PubMed DOI

Tauriello D.V.F., Haegebarth A., Kuper I., Edelmann M.J., Henraat M., Canninga-van Dijk M.R., Kessler B.M., Clevers H., Maurice M.M. Loss of the Tumor Suppressor CYLD Enhances Wnt/β-Catenin Signaling through K63-Linked Ubiquitination of Dvl. Mol. Cell. 2010;37:607–619. doi: 10.1016/j.molcel.2010.01.035. PubMed DOI

Yamashita M., Ying S.-X., Zhang G.-M., Li C., Cheng S.Y., Deng C.-X., Zhang Y.E. Ubiquitin ligase Smurf1 controls osteoblast activity and bone homeostasis by targeting MEKK2 for degradation. Cell. 2005;121:101–113. doi: 10.1016/j.cell.2005.01.035. PubMed DOI PMC

Schwarz-Romond T., Fiedler M., Shibata N., Butler P.J.G., Kikuchi A., Higuchi Y., Bienz M. 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

Tauriello D.V.F., Jordens I., Kirchner K., Slootstra J.W., Kruitwagen T., Bouwman B.A.M., Noutsou M., Rudiger S.G.D., Schwamborn K., Schambony A., et al. Wnt/-catenin signaling requires interaction of the Dishevelled DEP domain and C terminus with a discontinuous motif in Frizzled. Proc. Natl. Acad. Sci. USA. 2012;109:E812–E820. doi: 10.1073/pnas.1114802109. PubMed DOI PMC

Sammar M., Stricker S., Schwabe G.C., Sieber C., Hartung A., Hanke M., Oishi I., Pohl J., Minami Y., Sebald W., et al. Modulation of GDF5/BRI-b signalling through interaction with the tyrosine kinase receptor Ror2. Genes Cells. 2004;9:1227–1238. doi: 10.1111/j.1365-2443.2004.00799.x. PubMed DOI

Kallay L.M., Mc Nickle A., Brennwald P.J., Hubbard A.L., Braiterman L.T. Scribble associates with two polarity proteins, Lgl2 and Vangl2, via distinct molecular domains. J. Cell. Biochem. 2006;99:647–664. doi: 10.1002/jcb.20992. PubMed DOI

Massagué J., Hata A., Lo R.S., Wotton D., Lagna G. Mutations increasing autoinhibition inactivate tumour suppressors Smad2 and Smad4. Nature. 1997;388:82–87. doi: 10.1038/40424. PubMed DOI

Hayashi H., Abdollah S., Qiu Y., Cai J., Xu Y.Y., Grinnell B.W., Richardson M.A., Topper J.N., Gimbrone M.A., Wrana J.L., et al. The MAD-related protein Smad7 associates with the TGFβ receptor and functions as an antagonist of TGFβ signaling. Cell. 1997;89:1165–1173. doi: 10.1016/S0092-8674(00)80303-7. PubMed DOI

Ann Ran F., Hsu P.D., Wright J., Agarwala V., Scott D.A., Zhang F. Genome engineering using the CRIPR-Cas9 system. Nature. 2013;8:2281. PubMed PMC

Wiesner S., Ogunjimi A.A., Wang H.R., Rotin D., Sicheri F., Wrana J.L., Forman-Kay J.D. Autoinhibition of the HECT-Type Ubiquitin Ligase Smurf2 through Its C2 Domain. Cell. 2007;130:651–662. doi: 10.1016/j.cell.2007.06.050. PubMed DOI

Kee Y., Huibregtse J.M. Regulation of catalytic activities of HECT ubiquitin ligases. Biochem. Biophys. Res. Commun. 2007;354:329–333. doi: 10.1016/j.bbrc.2007.01.025. PubMed DOI PMC

Mund T., Graeb M., Mieszczanek J., Gammons M., Pelham H.R.B., Bienz M. Disinhibition of the HECT E3 ubiquitin ligase WWP2 by polymerized Dishevelled. Open Biol. 2015;5:150185. doi: 10.1098/rsob.150185. PubMed DOI PMC

Paclíková P., Bernatík O., Radaszkiewicz T.W., Bryja V. The N-Terminal Part of the Dishevelled DEP Domain Is Required for Wnt/β-Catenin Signaling in Mammalian Cells. Mol. Cell. Biol. 2017;37:e00145-17. doi: 10.1128/mcb.00145-17. PubMed DOI PMC

Bonni S., Wang H.R., Causing C.G., Kavsak P., Stroschein S.L., Luo K., Wrana J.L. TGF-β induces assembly of a Smad2-Smurf2 ubiquitin ligase complex that targets SnoN for degradation. Nat. Cell Biol. 2001;3:587–595. doi: 10.1038/35078562. PubMed DOI

Podos S.D., Hanson K.K., Wang Y.C., Ferguson E.L. The DSmurf Ubiquitin-Protein Ligase Restricts BMP Signaling Spatially and Temporally during Drosophila Embryogenesis. Dev. Cell. 2001;1:567–578. doi: 10.1016/S1534-5807(01)00057-0. PubMed DOI

David D., Nair S.A., Pillai M.R. Smurf E3 ubiquitin ligases at the cross roads of oncogenesis and tumor suppression. Biochim. Biophys. Acta Rev. Cancer. 2013;1835:119–128. doi: 10.1016/j.bbcan.2012.11.003. PubMed DOI

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., et al. Electrochemical cues regulate assembly of the Frizzled/Dishevelled complex at the plasma membrane during planar epithelial polarization. Nat. Cell Biol. 2009;11:286–294. doi: 10.1038/ncb1836. PubMed DOI PMC

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

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

Yanfeng W.A., Berhane H., Mola M., Singh J., Jenny A., Mlodzik M. Functional dissection of phosphorylation of Disheveled in Drosophila. Dev. Biol. 2011;360:132–142. doi: 10.1016/j.ydbio.2011.09.017. PubMed DOI PMC

Bernatík O., Šedová K., Schille C., Ganji R.S., Ćervenka I., Trantírek L., Schambony A., Zdráhal Z., Bryja V. Functional analysis of dishevelled-3 phosphorylation identifies distinct mechanisms driven by casein kinase 1ε and Frizzled5. J. Biol. Chem. 2014;289:23520–23533. doi: 10.1074/jbc.M114.590638. PubMed DOI PMC

Wei W., Li M., Wang J., Nie F., Li L. The E3 Ubiquitin Ligase ITCH Negatively Regulates Canonical Wnt Signaling by Targeting Dishevelled Protein. Mol. Cell. Biol. 2012;32:3903–3912. doi: 10.1128/MCB.00251-12. PubMed DOI PMC

Ding Y., Zhang Y., Xu C., Tao Q.H., Chen Y.G. HECT domain-containing E3 ubiquitin ligase NEDD4L negatively regulates Wnt signaling by targeting dishevelled for proteasomal degradation. J. Biol. Chem. 2013;288:8289–8298. doi: 10.1074/jbc.M112.433185. PubMed DOI PMC

De Groot R.E.A., Ganji R.S., Bernatik O., Lloyd-Lewis B., Seipel K., Šedová K., Zdráhal Z., Dhople V.M., Dale T.C., Korswagen H.C., et al. Huwe1-mediated ubiquitylation of dishevelled defines a negative feedback loop in the Wnt signaling pathway. Sci. Signal. 2014;7:ra26. doi: 10.1126/scisignal.2004985. PubMed DOI

Madrzak J., Fiedler M., Johnson C.M., Ewan R., Knebel A., Bienz M., Chin J.W. Ubiquitination of the Dishevelled DIX domain blocks its head-to-tail polymerization. Nat. Commun. 2015;6:1–11. doi: 10.1038/ncomms7718. PubMed DOI PMC

Mund T., Pelham H.R.B. Control of the activity of WW-HECT domain E3 ubiquitin ligases by NDFIP proteins. EMBO Rep. 2009;10:501–507. doi: 10.1038/embor.2009.30. PubMed DOI PMC

Malonis R.J., Fu W., Jelcic M.J., Thompson M., Canter B.S., Tsikitis M., Esteva F.J., Sánchez I. RNF11 sequestration of the E3 ligase SMURF2 on membranes antagonizes SMAD7 down-regulation of transforming growth factor β signaling. J. Biol. Chem. 2017;292:7435–7451. doi: 10.1074/jbc.M117.783662. PubMed DOI PMC

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

Lin X., Liang M., Feng X.H. Smurf2 is a ubiquitin E3 ligase mediating proteasome-dependent degradation of Smad2 in transforming growth factor-β signaling. J. Biol. Chem. 2000;275:36818–36822. doi: 10.1074/jbc.C000580200. PubMed DOI