Regulation mechanism of E-cadherin [electronic resource] : competitive regulation of E-cadherin juxtamembrane domain degradation by p120-catenin binding and hakai mediated ubiquitination
Keywords: Molecular and Cellular Physiology.

Throughout development cells must go through numerous changes in cell-cell adhesiveness. Within an epithelial layer, cell-cell contacts form and maintenance junctional complexes such as Adherens Junctions, Tight Junctions, and Desmosomes. The Adherens junction (AJ) and Tight junction (TJ) provide important adhesive contacts between neighboring epithelial cells. Although these junctions comprise different proteins, there are similarities in the roles of specialized transmembrane proteins in forming extracellular adhesive contacts between cells, and intracellular links to the actin cytoskeleton and signaling pathways including the regulation of gene transcription. The Adherens junction performs multiple functions including initiation and stabilization of cell--cell adhesion, regulation of the actin cytoskeleton, intracellular signaling and transcriptional regulation. The core of the Adherens junction includes interactions among transmembrane glycoproteins of the classical cadherin superfamily, such as E-cadherin, and the catenin family members including p120-catenin, [Beta]-catenin, and [Alpha]-catenin. Together, these proteins control the formation, maintenance and function of adherens junctions. Tight junctions have been proposed to have two mutually exclusive functions: a fence function which prevents the mixing of membrane proteins between the apical and basolateral membranes; and a gate function which controls the paracellular passage of ions and solutes in-between cells. Tight junctions contain two types of transmembrane proteins, occludins and claudins, which confer these functions, and associated cytoplasmic proteins that may link tight junctions to the actin-cytoskeleton and the adherens junction. Adherens Junction mediated cell--cell adhesion is highly dynamic enabling the reorganization and dispersal of cells, for example, during epithelial-to-mesenchymal transition in normal development and carcinogenesis. p120-Catenin binding to, and Hakai (an E3 ubiquitin ligase)-mediated ubiquitination of the juxtamembrane domain (JMD) of E-cadherin are thought to be involved in regulating E-cadherin internalization and degradation. However, the relationship between these two pathways is not known. The aim of my project was to determine if p120-catenin/E-cadherin and Hakai dependent ubiquitination of E-cadherin are mechanistically linked. We targeted the E-cadherin JMD to mitochondria (WT-JMD) to isolate this domain from the plasma membrane and internalization, and to examine protein modifications and degradation. WT-JMD was targeted to mitochondria, but it did not accumulate there except when proteasome activity was inhibited. We found that WT-JMD was ubiquitinated, and arginine substitution of lysines at position 5 (K5R) and 83 (K83R) resulted in the stable accumulation of mutant JMD at mitochondria. p120-Catenin did not localize, or bind to WT-JMD even upon proteasome inhibition, whereas the K5,83R JMD mutant bound p120-catenin and localized it to mitochondria. Mutation of the p120-catenin binding site in combination with these lysine mutations inhibited p120-catenin bind-ing, but did not decrease JMD stability or its accumulation at mitochondria. Further-more, over-expression of Hakai resulted in inhibition of p120-catenin binding to WT-JMD. Thus, increased stability of JMD lysine mutants was due to inhibition of ubiquitination and not to p120-catenin binding. Finally, mutation of these critical lysines in full length E-cadherin had the same effects on protein stability as mitochondria-targeted E-cadherin JMD. Our results indicate that ubiquitination of the JMD inhibits p120-catenin binding, and targets E-cadherin for degradation via the proteasome. Further work needs to be done to test our hypothesis that p120-catenin stabilizes E-cadherin by masking ubiquitination sites within E-cadherin-JMD.