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

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.

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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.

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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.

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Correspondence to Yasuyuki Fujita or Walter Birchmeier.

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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.

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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

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