Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Hakai, a c-Cbl-like protein, ubiquitinates and induces endocytosis of the E-cadherin complex

Nature Cell Biology volume 4, pages 222–231 (2002)Cite this article

Abstract

In epithelial cells, tyrosine kinases induce the tyrosine phosphorylation and ubiquitination of the E-cadherin complex, which induces endocytosis of E-cadherin. With a modified yeast 2-hybrid system, we isolated Hakai, an E-cadherin binding protein, which we have identified as an E3 ubiquitin-ligase. Hakai contains SH2, RING, zinc-finger and proline-rich domains, and interacts with E-cadherin in a tyrosine phosphorylation-dependent manner, inducing ubiquitination of the E-cadherin complex. Expression of Hakai in epithelial cells disrupts cell–cell contacts and enhances endocytosis of E-cadherin and cell motility. Through dynamic recycling of E-cadherin, Hakai can thus modulate cell adhesion, and could participate in the regulation of epithelial–mesenchymal transitions in development or metastasis.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Localization, tyrosine phosphorylation, and ubiquitination of the E-cadherin complex in ts-Src MDCK cells.
Figure 2: Characterization of the interaction between E-cadherin and Hakai with the yeast 2-hybrid system.
Figure 3: Characteristics of the novel protein Hakai.
Figure 4: The interaction between Hakai and E-cadherin in cell culture.
Figure 5: Effect of Hakai on the ubiquitination of the E-cadherin complex.
Figure 6: Biological effects of Hakai overexpression in MDCK cells.
Figure 7: Effect of Hakai on endocytosis of E-cadherin in MDCK cells.
Figure 8: A model of the suggested function of Hakai.

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  1. Takeichi, M. Morphogenetic roles of classic cadherins. Curr. Opin. Cell Biol. 7, 619–627 (1995).

    Article  CAS  Google Scholar 

  2. Kemler, R. Classical cadherins. Semin. Cell Biol. 3, 149–155 (1992).

    Article  CAS  Google Scholar 

  3. Behrens, J., Mareel, M. M., Van Roy, F. M. & Birchmeier, W. Dissecting tumour cell invasion: epithelial cells acquire invasive properties after the loss of uvomorulin-mediated cell–cell adhesion. J. Cell Biol. 108, 2435–2447 (1989).

    Article  CAS  Google Scholar 

  4. Larue, L., Ohsugi, M., Hirchenhain, J. & Kemler, R. E-cadherin null mutant embryos fail to form a trophectoderm epithelium. Proc. Natl Acad. Sci. USA 91, 8263–8267 (1994).

    Article  CAS  Google Scholar 

  5. Riethmacher, D., Brinkmann, V. & Birchmeier, C. A targeted mutation in the mouse E-cadherin gene results in defective preimplantation development. Proc. Natl Acad. Sci. USA 92, 855–859 (1995).

    Article  CAS  Google Scholar 

  6. Perl, A. K., Wilgenbus, P., Dahl, U., Semb, H. & Christofori, G. A causal role for E-cadherin in the transition from adenoma to carcinoma. Nature 392, 190–193 (1998).

    Article  CAS  Google Scholar 

  7. Weidner, K. M., Behrens, J., Vandekerckhove, J. & Birchmeier, W. Scatter factor: molecular characteristics and effect on the invasiveness of epithelial cells. J. Cell Biol. 111, 2097–2108 (1990).

    Article  CAS  Google Scholar 

  8. Stoker, M. & Gherardi, E. Regulation of cell movement: the motogenic cytokines. Biochim. Biophys. Acta 1072, 81–102 (1991).

    CAS  PubMed  Google Scholar 

  9. Behrens, J. et al. Loss of epithelial differentiation and gain of invasiveness correlates with tyrosine phosphorylation of the E-cadherin/β-catenin complex in cells transformed with a temperature-sensitive v-SRC gene. J. Cell Biol. 120, 757–766 (1993).

    Article  CAS  Google Scholar 

  10. Hamaguchi, M. et al. p60v-src causes tyrosine phosphorylation and inactivation of the N-cadherin-catenin cell adhesion system. EMBO J. 12, 307–314 (1993).

    Article  CAS  Google Scholar 

  11. Bauer, A., Lickert, H., Kemler, R. & Stappert, J. Modification of the E-cadherin-catenin complex in mitotic Madin-Darby canine kidney epithelial cells. J. Biol. Chem. 273, 28314–28321 (1998).

    Article  CAS  Google Scholar 

  12. Kamei, T. et al. Coendocytosis of cadherin and c-Met coupled to disruption of cell–cell adhesion in MDCK cells — regulation by Rho, Rac and Rab small G proteins. Oncogene 18, 6776–6784 (1999).

    Article  CAS  Google Scholar 

  13. Le, T. L., Yap, A. S. & Stow, J. L. Recycling of E-cadherin: a potential mechanism for regulating cadherin dynamics. J. Cell Biol. 146, 219–232 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Hershko, A. & Ciechanover, A. The ubiquitin system. Annu. Rev. Biochem. 67, 425–479 (1998).

    Article  CAS  Google Scholar 

  15. Hicke, L. Gettin' down with ubiquitin: turning off cell-surface receptors, transporters and channels. Trends Cell Biol. 9, 107–112 (1999).

    Article  CAS  Google Scholar 

  16. Strous, G. J. & Govers, R. The ubiquitin-proteasome system and endocytosis. J. Cell Sci. 112, 1417–1423 (1999).

    CAS  PubMed  Google Scholar 

  17. Hicke, L., Zanolari, B. & Riezman, H. Cytoplasmic tail phosphorylation of the α-factor receptor is required for its ubiquitination and internalization. J. Cell Biol. 141, 349–358 (1998).

    Article  CAS  Google Scholar 

  18. Joazeiro, C. A. et al. The tyrosine kinase negative regulator c-Cbl as a RING-type, E2-dependent ubiquitin-protein ligase. Science 286, 309–312 (1999).

    Article  CAS  Google Scholar 

  19. Levkowitz, G. et al. Ubiquitin ligase activity and tyrosine phosphorylation underlie suppression of growth factor signaling by c-Cbl/Sli-1. Mol. Cell 4, 1029–1040 (1999).

    Article  CAS  Google Scholar 

  20. Deveraux, Q., Ustrell, V., Pickart, C. & Rechsteiner, M. A 26 S protease subunit that binds ubiquitin conjugates. J. Biol. Chem. 269, 7059–7061 (1994).

    CAS  PubMed  Google Scholar 

  21. Roth, A. F. & Davis, N. G. Ubiquitination of the yeast α-factor receptor. J. Cell Biol. 134, 661–674 (1996).

    Article  CAS  Google Scholar 

  22. Terrell, J., Shih, S., Dunn, R. & Hicke, L. A function for monoubiquitination in the internalization of a G protein-coupled receptor. Mol. Cell 1, 193–202 (1998).

    Article  CAS  Google Scholar 

  23. Chau, V. et al. A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein. Science 243, 1576–1583 (1989).

    Article  CAS  Google Scholar 

  24. Galan, J. & Haguenauer, T. R. Ubiquitin lys63 is involved in ubiquitination of a yeast plasma membrane protein. EMBO J. 16, 5847–5854 (1997).

    Article  CAS  Google Scholar 

  25. Horak, J. & Wolf, D. H. Catabolite inactivation of the galactose transporter in the yeast Saccharomyces cerevisiae: ubiquitination, endocytosis, and degradation in the vacuole. J. Bacteriol. 179, 1541–1549 (1997).

    Article  CAS  Google Scholar 

  26. Schaeper, U. et al. Coupling of Gab1 to c-Met, Grb2, and Shp2 mediates biological responses. J. Cell Biol. 149, 1419–1432 (2000).

    Article  CAS  Google Scholar 

  27. Gumbiner, B. M. Regulation of cadherin adhesive activity. J. Cell Biol. 148, 399–404 (2000).

    Article  CAS  Google Scholar 

  28. Xu, Y., Guo, D. F., Davidson, M., Inagami, T. & Carpenter, G. Interaction of the adaptor protein Shc and the adhesion molecule cadherin. J. Biol. Chem. 272, 13463–13466 (1997).

    Article  CAS  Google Scholar 

  29. Kozak, M. An analysis of 5′-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15, 8125–8148 (1987).

    Article  CAS  Google Scholar 

  30. Lorick, K. L. et al. RING fingers mediate ubiquitin-conjugating enzyme (E2)-dependent ubiquitination. Proc. Natl Acad. Sci. USA 96, 11364–11369 (1999).

    Article  CAS  Google Scholar 

  31. Thoreson, M. A. et al. Selective uncoupling of p120(ctn) from E-cadherin disrupts strong adhesion. J. Cell Biol. 148, 189–202 (2000).

    Article  CAS  Google Scholar 

  32. Wang, Y., Yeung, Y. G., Langdon, W. Y. & Stanley, E. R. c-Cbl Is Transiently Tyrosine-phosphorylated, Ubiquitinated, and Membrane-targeted following CSF-1 Stimulation of Macrophages. J. Biol. Chem. 271, 17–20 (1996).

    Article  CAS  Google Scholar 

  33. Tagawa, M. et al. Expression of novel DNA-binding protein with zinc finger structure in various tumour cells. J. Biol. Chem. 265, 20021–20026 (1990).

    CAS  PubMed  Google Scholar 

  34. Weidner, K. M. et al. Interaction between Gab1 and the c-Met receptor tyrosine kinase is responsible for epithelial morphogenesis. Nature 384, 173–176 (1996).

    Article  CAS  Google Scholar 

  35. Nagafuchi, A., Ishihara, S. & Tsukita, S. The roles of catenins in the cadherin-mediated cell adhesion: functional analysis of E-cadherin-α-catenin fusion molecules. J. Cell Biol. 127, 235–245 (1994).

    Article  CAS  Google Scholar 

  36. Meng, W., Sawasdikosol, S., Burakoff, S. J. & Eck, M. J. Structure of the amino-terminal domain of Cbl complexed to its binding site on ZAP-70 kinase. Nature 398, 84–90 (1999).

    Article  CAS  Google Scholar 

  37. Takata, K. & Singer, S. J. Phosphotyrosine-modified proteins are concentrated at the membranes of epithelial and endothelial cells during tissue development in chick embryos. J. Cell Biol. 106, 1757–1764 (1988).

    Article  CAS  Google Scholar 

  38. Maher, P. A. & Pasquale, E. B. Heat shock induces protein tyrosine phosphorylation in cultured cells. J. Cell Biol. 108, 2029–2035 (1989).

    Article  CAS  Google Scholar 

  39. Tsukita, S. et al. Specific proto-oncogenic tyrosine kinases of src family are enriched in cell-to-cell adherens junctions where the level of tyrosine phosphorylation is elevated. J. Cell Biol. 113, 867–879 (1991).

    Article  CAS  Google Scholar 

  40. Ukropec, J. A., Hollinger, M. K., Salva, S. M. & Woolkalis, M. J. SHP2 association with VE-cadherin complexes in human endothelial cells is regulated by thrombin. J. Biol. Chem. 275, 5983–5986 (2000).

    Article  CAS  Google Scholar 

  41. Tsukamoto, T. & Nigam, S. K. Cell–cell dissociation upon epithelial cell scattering requires a step mediated by the proteasome. J. Biol. Chem. 274, 24579–24584 (1999).

    Article  CAS  Google Scholar 

  42. Taya, S., Yamamoto, T., Kanai-Azuma, M., Wood, S. A. & Kaibuchi, K. The deubiquitinating enzyme Fam interacts with and stabilizes β-catenin. Genes Cells 4, 757–767 (1999).

    Article  CAS  Google Scholar 

  43. Pece, S., Chiariello, M., Murga, C. & Gutkind, J. S. Activation of the protein kinase Akt/PKB by the formation of E-cadherin-mediated cell–cell junctions. Evidence for the association of phosphatidylinositol 3-kinase with the E-cadherin adhesion complex. J. Biol. Chem. 274, 19347–19351 (1999).

    Article  CAS  Google Scholar 

  44. Birchmeier, W. & Behrens, J. Cadherin expression in carcinomas: role in the formation of cell junctions and the prevention of invasiveness. Biochim. Biophys. Acta 1198, 11–26 (1994).

    CAS  PubMed  Google Scholar 

  45. Behrens, J. et al. Functional interaction of β-catenin with the transcription factor LEF-1. Nature 382, 638–642 (1996).

    Article  CAS  Google Scholar 

  46. Nuber, U., Schwarz, S., Kaiser, P., Schneider, R. & Scheffner, M. Cloning of human ubiquitin-conjugating enzymes UbcH6 and UbcH7(E2-F1) and characterization of their interaction with E6-AP and RSP5. J. Biol. Chem. 271, 2795–2800 (1996).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank C. Birchmeier and U. Schaeper for critical reading of the manuscript. A. Dell'Oro is acknowledged for technical help. We also thank U. Lenk for help with the in vitro ubiquitination assays, A. Ciechanover for technical advice on pulse–chase assays, and D. Bohmann for providing the pBSSR-HA–ubiquitin construct. This work was supported by a grant from the Deutsche Forschungsgemeinshaft (DFG). Y.F. received support from the Japan-Europe Scientists Exchange Program from the Ciba-Geigy Foundation of Japan and a Postdoctoral Fellowship for Research Abroad from the Japan Society for the Promotion of Science.

Author information

Author notes

  1. Jürgen Behrens

    Present address: University of Erlangen, Nikolaus-Fiebiger-Center, Glueckstr. 6, 91054, Erlangen, Germany

Authors and Affiliations

  1. Max-Delbrück-Center for Molecular Medicine (MDC), Robert-Rössle-Str. 10, Berlin, 13125, Germany

    Yasuyuki Fujita, Dietmar Zechner, Hugo E. Molina Leddy, Jürgen Behrens, Thomas Sommer & Walter Birchmeier

  2. Forschungsinstitut fuer Molekulare Pharmakologie (FMP), Robert-Rössle-Str. 10, Berlin, 13125, Germany

    Gerd Krause

  3. University of Köln, Institute of Biochemistry I, Joseph-Stelzmann-Str. 52, Köln, 50931, Germany

    Martin Scheffner

Authors
  1. Yasuyuki Fujita
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gerd Krause
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Martin Scheffner
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dietmar Zechner
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hugo E. Molina Leddy
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jürgen Behrens
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Thomas Sommer
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Walter Birchmeier
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Yasuyuki Fujita or Walter Birchmeier.

Supplementary information

Supplementary figures

Figure S1 Northern blotting of Hakai using RNAs from adult mouse tissues and from mouse embryos. (PDF 271 kb)

Figure S2 Effect of Hakai on the MAPK pathway.

Figure S3 A suggested molecular structure of Hakai.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Fujita, Y., Krause, G., Scheffner, M. et al. Hakai, a c-Cbl-like protein, ubiquitinates and induces endocytosis of the E-cadherin complex. Nat Cell Biol 4, 222–231 (2002). https://doi.org/10.1038/ncb758

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb758

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing