Ferber, E.C.; (2008) The role of the cleaved cytoplasmic domain of E-cadherin in signalling. Doctoral thesis , University of London.

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Abstract

E-cadherin is the most crucial membrane protein for the formation of tight and compact cell-cell adhesions in epithelial cells. E-cadherin based cell-cell junctions (adherens junctions) are highly dynamically regulated, for example during epithelial to mesenchymal transition (EMT) when E-cadherin at cell-cell contacts is down-regulated, leading to a loss of cell-cell adhesion. In addition to its structural role, E-cadherin has been shown to be involved in several signalling pathways during cell proliferation, differentiation and cell survival. However the molecular mechanisms of E-cadherin-mediated signalling are largely unknown. E-cadherin binding proteins, (3-catenin and pl20-catenin (pi20), can translocate to the nucleus, and bind and regulate the activity of transcription factors TCF/LEF-1 and Kaiso respectively. The influence of E-cadherin in these pathways is not fully understood. E-cadherin has been shown to be cleaved by metalloproteases and also by gamma-secretase. In this thesis I have examined E- cadherin cleavage and the role of cleaved E-cadherin in signalling. The conditions inducing the cleavage of E-cadherin in epithelial cell lines were investigated. It was found that the gamma-secretase cleavage product of E-cadherin (E- cad/CTF2) was produced in MDCK cells in response to stimuli leading to EMT: treatment with hepatacyte growth factor and activation of Ras. pi20 was shown to specifically enhance the nuclear localisation of E-cad/CTF2. pi20 was also found to induce the ubiquitination of E-cad/CTF2. Biochemical assays were used to show that pi20 binds to a RING-fmger containing protein, ret finger protein (RFP), which also enhances ubiquitination and nuclear translocation of E- cad/CTF2. The role of ubiquitination and phosphorylation of E-cad/CTF2 in its nuclear translocation was investigated. In the nucleus, E-cad/CTF2 was shown to bind specifically to DNA. Its association with DNA is enhanced by both pi20 and RFP. The transactivation potential of E-cad/CTF2 was investigated. It was shown that nuclear E-cad/CTF2 regulates 3-catenin-TCF/LEF-l- and pi 20- Kaiso-mediated signalling pathways.

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