Competitive Regulation of E-Cadherin JuxtaMembrane Domain Degradation by p120-Catenin Binding and Hakai-Mediated Ubiquitination

p120-Catenin binding to, and Hakai-mediated ubiquitination of the E-cadherin juxtamembrane domain (JMD) are thought to be involved in regulating E-cadherin internalization and degradation. However, the relationship between these two pathways is not understood. 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 localized to mitochondria, but did not accumulate there except when proteasome activity was inhibited. We found 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 and localized p120-catenin to mitochondria. Mutation of the p120-catenin binding site in combination with these lysine mutations inhibited p120-catenin binding, but did not decrease JMD stability or its accumulation at mitochondria. 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 similar effects on protein stability as WT-JMD. Our results indicate that ubiquitination of the JMD inhibits p120-catenin binding, and targets E-cadherin for degradation

A potential new, stable state of the E-cadherin strand-swapped dimer in solution

E-cadherin is a transmembrane glycoprotein that facilitates inter-cellular adhesion in the epithelium. The ectodomain of the native structure is comprised of five repeated immunoglobulin-like domains. All E-cadherin crystal structures show the protein in one of three alternative conformations: a monomer, a strand-swapped trans homodimer and the so-called X-dimer, which is proposed to be a kinetic intermediate to forming the strand-swapped trans homodimer. However, previous studies have indicated that even once the trans strand-swapped dimer is formed, the complex is highly dynamic and the E-cadherin monomers may reorient relative to each other. Here, molecular dynamics simulations have been used to investigate the stability and conformational flexibility of the human E-cadherin trans strand-swapped dimer. In four independent, 100 ns simulations, the dimer moved away from the starting structure and converged to a previously unreported structure, which we call the Y-dimer. The Y-dimer was present for over 90% of the combined simulation time, suggesting that it represents a stable conformation of the E-cadherin dimer in solution. The Y-dimer conformation is stabilised by interactions present in both the trans strand-swapped dimer and X-dimer crystal structures, as well as additional interactions not found in any E-cadherin dimer crystal structures. The Y-dimer represents a previously unreported, stable conformation of the human E-cadherin trans strand-swapped dimer and suggests that the available crystal structures do not fully capture the conformations that the human E-cadherin trans homodimer adopts in solution

ReCLIP (Reversible Cross-Link Immuno-Precipitation): An Efficient Method for Interrogation of Labile Protein Complexes

The difficulty of maintaining intact protein complexes while minimizing non-specific background remains a significant limitation in proteomic studies. Labile interactions, such as the interaction between p120-catenin and the E-cadherin complex, are particularly challenging. Using the cadherin complex as a model-system, we have developed a procedure for efficient recovery of otherwise labile protein-protein interactions. We have named the procedure “ReCLIP” (Reversible Cross-Link Immuno-Precipitation) to reflect the primary elements of the method. Using cell-permeable, thiol-cleavable crosslinkers, normally labile interactions (i.e. p120 and E-cadherin) are stabilized in situ prior to isolation. After immunoprecipitation, crosslinked binding partners are selectively released and all other components of the procedure (i.e. beads, antibody, and p120 itself) are discarded. The end result is extremely efficient recovery with exceptionally low background. ReCLIP therefore appears to provide an excellent alternative to currently available affinity-purification approaches, particularly for studies of labile complexes

Protein kinase C activation disrupts epithelial apical junctions via ROCK-II dependent stimulation of actomyosin contractility

<p>Abstract</p> <p>Background</p> <p>Disruption of epithelial cell-cell adhesions represents an early and important stage in tumor metastasis. This process can be modeled <it>in vitro </it>by exposing cells to chemical tumor promoters, phorbol esters and octylindolactam-V (OI-V), known to activate protein kinase C (PKC). However, molecular events mediating PKC-dependent disruption of epithelial cell-cell contact remain poorly understood. In the present study we investigate mechanisms by which PKC activation induces disassembly of tight junctions (TJs) and adherens junctions (AJs) in a model pancreatic epithelium.</p> <p>Results</p> <p>Exposure of HPAF-II human pancreatic adenocarcinoma cell monolayers to either OI-V or 12-O-tetradecanoylphorbol-13-acetate caused rapid disruption and internalization of AJs and TJs. Activity of classical PKC isoenzymes was responsible for the loss of cell-cell contacts which was accompanied by cell rounding, phosphorylation and relocalization of the F-actin motor nonmuscle myosin (NM) II. The OI-V-induced disruption of AJs and TJs was prevented by either pharmacological inhibition of NM II with blebbistatin or by siRNA-mediated downregulation of NM IIA. Furthermore, AJ/TJ disassembly was attenuated by inhibition of Rho-associated kinase (ROCK) II, but was insensitive to blockage of MLCK, calmodulin, ERK1/2, caspases and RhoA GTPase.</p> <p>Conclusion</p> <p>Our data suggest that stimulation of PKC disrupts epithelial apical junctions via ROCK-II dependent activation of NM II, which increases contractility of perijunctional actin filaments. This mechanism is likely to be important for cancer cell dissociation and tumor metastasis.</p

A Membrane Fusion Protein αSNAP Is a Novel Regulator of Epithelial Apical Junctions

Tight junctions (TJs) and adherens junctions (AJs) are key determinants of the structure and permeability of epithelial barriers. Although exocytic delivery to the cell surface is crucial for junctional assembly, little is known about the mechanisms controlling TJ and AJ exocytosis. This study was aimed at investigating whether a key mediator of exocytosis, soluble N-ethylmaleimide sensitive factor (NSF) attachment protein alpha (αSNAP), regulates epithelial junctions. αSNAP was enriched at apical junctions in SK-CO15 and T84 colonic epithelial cells and in normal human intestinal mucosa. siRNA-mediated knockdown of αSNAP inhibited AJ/TJ assembly and establishment of the paracellular barrier in SK-CO15 cells, which was accompanied by a significant down-regulation of p120-catenin and E-cadherin expression. A selective depletion of p120 catenin effectively disrupted AJ and TJ structure and compromised the epithelial barrier. However, overexpression of p120 catenin did not rescue the defects of junctional structure and permeability caused by αSNAP knockdown thereby suggesting the involvement of additional mechanisms. Such mechanisms did not depend on NSF functions or induction of cell death, but were associated with disruption of the Golgi complex and down-regulation of a Golgi-associated guanidine nucleotide exchange factor, GBF1. These findings suggest novel roles for αSNAP in promoting the formation of epithelial AJs and TJs by controlling Golgi-dependent expression and trafficking of junctional proteins

The human keratins: biology and pathology

The keratins are the typical intermediate filament proteins of epithelia, showing an outstanding degree of molecular diversity. Heteropolymeric filaments are formed by pairing of type I and type II molecules. In humans 54 functional keratin genes exist. They are expressed in highly specific patterns related to the epithelial type and stage of cellular differentiation. About half of all keratins—including numerous keratins characterized only recently—are restricted to the various compartments of hair follicles. As part of the epithelial cytoskeleton, keratins are important for the mechanical stability and integrity of epithelial cells and tissues. Moreover, some keratins also have regulatory functions and are involved in intracellular signaling pathways, e.g. protection from stress, wound healing, and apoptosis. Applying the new consensus nomenclature, this article summarizes, for all human keratins, their cell type and tissue distribution and their functional significance in relation to transgenic mouse models and human hereditary keratin diseases. Furthermore, since keratins also exhibit characteristic expression patterns in human tumors, several of them (notably K5, K7, K8/K18, K19, and K20) have great importance in immunohistochemical tumor diagnosis of carcinomas, in particular of unclear metastases and in precise classification and subtyping. Future research might open further fields of clinical application for this remarkable protein family

The desmosome and pemphigus

Desmosomes are patch-like intercellular adhering junctions (“maculae adherentes”), which, in concert with the related adherens junctions, provide the mechanical strength to intercellular adhesion. Therefore, it is not surprising that desmosomes are abundant in tissues subjected to significant mechanical stress such as stratified epithelia and myocardium. Desmosomal adhesion is based on the Ca2+-dependent, homo- and heterophilic transinteraction of cadherin-type adhesion molecules. Desmosomal cadherins are anchored to the intermediate filament cytoskeleton by adaptor proteins of the armadillo and plakin families. Desmosomes are dynamic structures subjected to regulation and are therefore targets of signalling pathways, which control their molecular composition and adhesive properties. Moreover, evidence is emerging that desmosomal components themselves take part in outside-in signalling under physiologic and pathologic conditions. Disturbed desmosomal adhesion contributes to the pathogenesis of a number of diseases such as pemphigus, which is caused by autoantibodies against desmosomal cadherins. Beside pemphigus, desmosome-associated diseases are caused by other mechanisms such as genetic defects or bacterial toxins. Because most of these diseases affect the skin, desmosomes are interesting not only for cell biologists who are inspired by their complex structure and molecular composition, but also for clinical physicians who are confronted with patients suffering from severe blistering skin diseases such as pemphigus. To develop disease-specific therapeutic approaches, more insights into the molecular composition and regulation of desmosomes are required

Listeria monocytogenes Internalin B Activates Junctional Endocytosis to Accelerate Intestinal Invasion

Listeria monocytogenes (Lm) uses InlA to invade the tips of the intestinal villi, a location at which cell extrusion generates a transient defect in epithelial polarity that exposes the receptor for InlA, E-cadherin, on the cell surface. As the dying cell is removed from the epithelium, the surrounding cells reorganize to form a multicellular junction (MCJ) that Lm exploits to find its basolateral receptor and invade. By examining individual infected villi using 3D-confocal imaging, we uncovered a novel role for the second major invasin, InlB, during invasion of the intestine. We infected mice intragastrically with isogenic strains of Lm that express or lack InlB and that have a modified InlA capable of binding murine E-cadherin and found that Lm lacking InlB invade the same number of villi but have decreased numbers of bacteria within each infected villus tip. We studied the mechanism of InlB action at the MCJs of polarized MDCK monolayers and find that InlB does not act as an adhesin, but instead accelerates bacterial internalization after attachment. InlB locally activates its receptor, c-Met, and increases endocytosis of junctional components, including E-cadherin. We show that MCJs are naturally more endocytic than other sites of the apical membrane, that endocytosis and Lm invasion of MCJs depends on functional dynamin, and that c-Met activation by soluble InlB or hepatocyte growth factor (HGF) increases MCJ endocytosis. Also, in vivo, InlB applied through the intestinal lumen increases endocytosis at the villus tips. Our findings demonstrate a two-step mechanism of synergy between Lm's invasins: InlA provides the specificity of Lm adhesion to MCJs at the villus tips and InlB locally activates c-Met to accelerate junctional endocytosis and bacterial invasion of the intestine