Modular mechanism of Wnt signaling inhibition by Wnt inhibitory factor 1

Abstract

Wnt morphogens control embryonic development and homeostasis in adult tissues. In vertebrates the N-terminal WIF domain (WIF-1WD) of Wnt inhibitory factor 1 (WIF-1) binds Wnt ligands. Our crystal structure of WIF-1WD reveals a previously unidentified binding site for phospholipid; two acyl chains extend deep into the domain, and the head group is exposed to the surface. Biophysical and cellular assays indicate that there is a WIF-1WD Wnt-binding surface proximal to the lipid head group but also implicate the five epidermal growth factor (EGF)-like domains (EGFs I–V) in Wnt binding. The six-domain WIF-1 crystal structure shows that EGFs I–V are wrapped back, interfacing with WIF-1WD at EGF III. EGFs II–V contain a heparan sulfate proteoglycan (HSPG)-binding site, consistent with conserved positively charged residues on EGF IV. This combination of HSPG- and Wnt-binding properties suggests a modular model for the localization of WIF-1 and for signal inhibition within morphogen gradients.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: The WIF-1WD-EGF-I structure and lipid-binding cavity.
Figure 2: The contributions of the WIF and EGF-like domains to Wnt inhibition.
Figure 3: A discontinuous Wnt-binding site on the WIF domain.
Figure 4: The structure of WIF-1ΔC.
Figure 5: HSPG binding contributes to a modular mechanism for WIF-1 function.

Accession codes

Primary accessions

Protein Data Bank

References

  1. 1

    Logan, C.Y. & Nusse, R. The Wnt signaling pathway in development and disease. Annu. Rev. Cell Dev. Biol. 20, 781–810 (2004).

  2. 2

    MacDonald, B.T., Tamai, K. & He, X. Wnt/beta-catenin signaling: components, mechanisms, and diseases. Dev. Cell 17, 9–26 (2009).

  3. 3

    Bhanot, P. et al. A new member of the frizzled family from Drosophila functions as a Wingless receptor. Nature 382, 225–230 (1996).

  4. 4

    Tamai, K. et al. LDL-receptor-related proteins in Wnt signal transduction. Nature 407, 530–535 (2000).

  5. 5

    Kawano, Y. & Kypta, R. Secreted antagonists of the Wnt signalling pathway. J. Cell Sci. 116, 2627–2634 (2003).

  6. 6

    Surmann-Schmitt, C. et al. Wif-1 is expressed at cartilage-mesenchyme interfaces and impedes Wnt3a-mediated inhibition of chondrogenesis. J. Cell Sci. 122, 3627–3637 (2009).

  7. 7

    Hsieh, J.C. et al. A new secreted protein that binds to Wnt proteins and inhibits their activities. Nature 398, 431–436 (1999).

  8. 8

    Nakaya, N., Lee, H.S., Takada, Y., Tzchori, I. & Tomarev, S.I. Zebrafish olfactomedin 1 regulates retinal axon elongation in vivo and is a modulator of Wnt signaling pathway. J. Neurosci. 28, 7900–7910 (2008).

  9. 9

    Liepinsh, E., Bányai, L., Patthy, L. & Otting, G. NMR structure of the WIF domain of the human Wnt-inhibitory factor-1. J. Mol. Biol. 357, 942–950 (2006).

  10. 10

    Malinauskas, T. Docking of fatty acids into the WIF domain of the human Wnt inhibitory factor-1. Lipids 43, 227–230 (2008).

  11. 11

    Bazan, J.F. & de Sauvage, F.J. Structural ties between cholesterol transport and morphogen signaling. Cell 138, 1055–1056 (2009).

  12. 12

    Kansara, M. et al. Wnt inhibitory factor 1 is epigenetically silenced in human osteosarcoma, and targeted disruption accelerates osteosarcomagenesis in mice. J. Clin. Invest. 119, 837–851 (2009).

  13. 13

    Mazieres, J. et al. Wnt inhibitory factor-1 is silenced by promoter hypermethylation in human lung cancer. Cancer Res. 64, 4717–4720 (2004).

  14. 14

    Kim, J. et al. Wnt inhibitory factor inhibits lung cancer cell growth. J. Thorac. Cardiovasc. Surg. 133, 733–737 (2007).

  15. 15

    Tang, Y. et al. WIF1, a Wnt pathway inhibitor, regulates SKP2 and c-myc expression leading to G1 arrest and growth inhibition of human invasive urinary bladder cancer cells. Mol. Cancer Ther. 8, 458–468 (2009).

  16. 16

    Tabata, T. & Takei, Y. Morphogens, their identification and regulation. Development 131, 703–712 (2004).

  17. 17

    Panáková, D., Sprong, H., Marois, E., Thiele, C. & Eaton, S. Lipoprotein particles are required for Hedgehog and Wingless signalling. Nature 435, 58–65 (2005).

  18. 18

    Yan, D. & Lin, X. Shaping morphogen gradients by proteoglycans. Cold Spring Harb. Perspect. Biol. 1, a002493 (2009).

  19. 19

    Willert, K. et al. Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 423, 448–452 (2003).

  20. 20

    Dundas, J. et al. CASTp: computed atlas of surface topography of proteins with structural and topographical mapping of functionally annotated residues. Nucleic Acids Res. 34, W116–W118 (2006).

  21. 21

    DasGupta, R., Kaykas, A., Moon, R.T. & Perrimon, N. Functional genomic analysis of the Wnt-wingless signaling pathway. Science 308, 826–833 (2005).

  22. 22

    Walter, T.S. et al. Lysine methylation as a routine rescue strategy for protein crystallization. Structure 14, 1617–1622 (2006).

  23. 23

    Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007).

  24. 24

    Inoue, T. et al. C. elegans LIN-18 is a Ryk ortholog and functions in parallel to LIN-17/Frizzled in Wnt signaling. Cell 118, 795–806 (2004).

  25. 25

    Glise, B. et al. Shifted, the Drosophila ortholog of Wnt inhibitory factor-1, controls the distribution and movement of Hedgehog. Dev. Cell 8, 255–266 (2005).

  26. 26

    Gorfinkiel, N., Sierra, J., Callejo, A., Ibañez, C. & Guerrero, I. The Drosophila ortholog of the human Wnt inhibitor factor Shifted controls the diffusion of lipid-modified Hedgehog. Dev. Cell 8, 241–253 (2005).

  27. 27

    Takei, Y., Ozawa, Y., Sato, M., Watanabe, A. & Tabata, T. Three Drosophila EXT genes shape morphogen gradients through synthesis of heparan sulfate proteoglycans. Development 131, 73–82 (2004).

  28. 28

    Ahn, J. et al. Cloning of the putative tumour suppressor gene for hereditary multiple exostoses (EXT1). Nat. Genet. 11, 137–143 (1995).

  29. 29

    Binari, R.C. et al. Genetic evidence that heparin-like glycosaminoglycans are involved in wingless signaling. Development 124, 2623–2632 (1997).

  30. 30

    Veverka, V. et al. Characterization of the structural features and interactions of sclerostin: molecular insight into a key regulator of Wnt-mediated bone formation. J. Biol. Chem. 284, 10890–10900 (2009).

  31. 31

    Fedi, P. et al. Isolation and biochemical characterization of the human Dkk-1 homologue, a novel inhibitor of mammalian Wnt signaling. J. Biol. Chem. 274, 19465–19472 (1999).

  32. 32

    Finch, P.W. et al. Purification and molecular cloning of a secreted, Frizzled-related antagonist of Wnt action. Proc. Natl. Acad. Sci. USA 94, 6770–6775 (1997).

  33. 33

    Bafico, A. et al. Interaction of frizzled related protein (FRP) with Wnt ligands and the frizzled receptor suggests alternative mechanisms for FRP inhibition of Wnt signaling. J. Biol. Chem. 274, 16180–16187 (1999).

  34. 34

    Üren, A. et al. Secreted frizzled-related protein-1 binds directly to Wingless and is a biphasic modulator of Wnt signaling. J. Biol. Chem. 275, 4374–4382 (2000).

  35. 35

    Aricescu, A.R., Lu, W. & Jones, E.Y. A time- and cost-efficient system for high-level protein production in mammalian cells. Acta Crystallogr. D Biol. Crystallogr. 62, 1243–1250 (2006).

  36. 36

    Reeves, P.J., Callewaert, N., Contreras, R. & Khorana, H.G. Structure and function in rhodopsin: high-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N-acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line. Proc. Natl. Acad. Sci. USA 99, 13419–13424 (2002).

  37. 37

    Chang, V.T. et al. Glycoprotein structural genomics: solving the glycosylation problem. Structure 15, 267–273 (2007).

  38. 38

    Walter, T.S. et al. A procedure for setting up high-throughput nanolitre crystallization experiments. Crystallization workflow for initial screening, automated storage, imaging and optimization. Acta Crystallogr. D Biol. Crystallogr. 61, 651–657 (2005).

Download references

Acknowledgements

We thank the staff of the European Synchrotron Radiation Facility and Diamond Light Source for assistance with data collection; T.S. Walter and K. Harlos for help with crystallization; B.M. Kessler and K. di Gleria for MS analysis; B.J.C. Janssen for help with solvent flattening; and E. Seiradake, Y. Zhao and M.A. Jones for help with tissue culture. T.M. and E.Y.J. are funded by Cancer Research UK, C.S. by the Wellcome Trust and A.R.A. by the UK Medical Research Council.

Author information

Affiliations

Authors

Contributions

T.M., A.R.A., C.S. and E.Y.J. designed the project. T.M. performed all the experiments. W.L. contributed to WIF-1 protein expression. A.R.A. and C.S. contributed to X-ray data collection and analysis. T.M., A.R.A., C.S. and E.Y.J. analyzed the data and wrote the manuscript.

Corresponding authors

Correspondence to Christian Siebold or E Yvonne Jones.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Methods and Supplementary Figures 1–9 (PDF 1879 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Malinauskas, T., Aricescu, A., Lu, W. et al. Modular mechanism of Wnt signaling inhibition by Wnt inhibitory factor 1. Nat Struct Mol Biol 18, 886–893 (2011). https://doi.org/10.1038/nsmb.2081

Download citation

Further reading