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| CELL ADHESION: Destroying ties
A novel protein named Hakai has been identified that promotes the endocytosis of E-cadherin. Epithelial cells like being together. Through E-cadherin-mediated cell–cell adhesions, they interact closely to give rise to the 'cobblestone morphology' of epithelial colonies. There are occasions — for example, during development — when the cells must loosen ties with their neighbours, but dysregulation of this process can allow cells to become invasive. So what is known about the dynamic modulation of E-cadherin at cell–cell contacts? Much more now, thanks to the work of Walter Birchmeier, Yasuyuki Fujita and colleagues, who have identified Hakai, a protein which promotes the endocytosis of E-cadherin.
Activation of tyrosine kinases such as Src or Met is known to cause epithelial cells to develop a fibroblast-like morphology and scatter. E-cadherin is re-distributed into the intracellular compartment, indicating that it has been endocytosed. The authors first investigated whether E-cadherin was modified by these kinases, which it was. But not only was it phosphorylated (on tyrosines) but it was also ubiquitylated. Assuming that these modifications might alter the properties of E-cadherin, Birchmeier's group carried out a two-hybrid screen, using tyrosine-phosphorylated cytoplasmic E-cadherin as bait, to look for potential binding partners. They subsequently cloned Hakai — Japanese for 'destruction' — which interacted specifically with E-cadherin in a phosphorylation-dependent manner through tyrosines 756 and 757. In addition to its phosphotyrosine-binding domain, the predicted amino-acid sequence of Hakai contains a RING-finger at its amino terminus and a large percentage (35%) of proline residues in its carboxyl terminus. In this respect, it resembles c-Cbl, an E3 ubiquitin ligase that recognizes tyrosine-phosphorylated receptor kinases. Modelling studies showed that a Src-homology-2 (SH2) domain follows the combined RING and Zn-finger domain of Hakai. These occur in the reverse order in c-Cbl, but overall, the two domains seem to be arranged very similarly. On the basis of this structural homology, the authors investigated Hakai's ability to mediate an increase in E-cadherin ubiquitylation to see if Hakai was also functionally related to c-Cbl. Using in vitro assays, they showed that, in the presence of Hakai, active Src and E2 ubiquitin-conjugating enzymes, E-cadherin was strongly ubiquitylated. They could also immunoprecipitate ubiquitylated E-cadherin from Hakai-transfected MDCK cells that were stimulated with scatter factor, and showed that the E-cadherin tyrosine residues that interacted with Hakai were crucial for ubiquitylation. So what happens when E-cadherin is ubiquitylated? Cells transfected with Hakai were flatter and formed irregularly shaped colonies. The half-life of E-cadherin decreased and its localization at cell–cell contacts was less regular. The authors showed this to be the result of increased internalization of E-cadherin, and the net effect of this was to compromise cell aggregation and to increase cell scattering. The authors therefore propose that Hakai functions to regulate cell motility, by inducing ubiquitylation and endocytosis of E-cadherin. In response to tyrosine-kinase-mediated E-cadherin phosphorylation, Hakai is recruited — through SH2-domain interactions — to E-cadherin, where it functions as an E3 ubiquitin ligase to promote its ubiquitylation and subsequent endocytosis from cell–cell contacts. Katrin Bussell References
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