Successive post-translational modifications of E-cadherin are required for InlA-mediated internalization of Listeria monocytogenes

Listeria monocytogenes surface proteins internalin (Inl)A and InlB interact with the junctional protein E-cadherin and the hepatocyte growth factor (HGF) receptor Met, respectively, on the surface of epithelial cells to mediate bacterial entry. Here we show that InlA triggers two successive E-cadherin post-translational modifications, i.e. the Src-mediated tyrosine phosphorylation of E-cadherin followed by its ubiquitination by the ubiquitin-ligase Hakai. E-cadherin ubiquitination induces the recruitment of clathrin that is required for optimal bacterial internalization. We also show that the initial clustering of E-cadherin at the bacterial entry site requires caveolin, a protein normally involved in clathrin-independent endocytosis. Strikingly clathrin and caveolin are also recruited at the site of entry of E-cadherin-coated sepharose beads and functional experiments demonstrate that these two proteins are required for bead entry. Together these results not only document how the endocytosis machinery is recruited and involved in the internalization of a zippering bacterium, but also strongly suggest a functional link between E-cadherin endocytosis and the formation of adherens junctions in epithelial cells.     

Slit-Robo signaling induces malignant transformation through Hakai-mediated E-cadherin degradation during colorectal epithelial cell carcinogenesis

The Slit family of guidance cues binds to Roundabout (Robo) receptors and modulates cell migration. We report here that ectopic expression of Slit2 and Robo1 or recombinant Slit2 treatment of Robo1-expressing colorectal epithelial carcinoma cells recruited an ubiquitin ligase Hakai for E-cadherin (E-cad) ubiquitination and lysosomal degradation, epithelial-mesenchymal transition (EMT), and tumor growth and liver metastasis, which were rescued by knockdown of Hakai. In contrast, knockdown of endogenous Robo1 or specific blockade of Slit2 binding to Robo1 prevented E-cad degradation and reversed EMT, resulting in diminished tumor growth and liver metastasis. Ectopic expression of Robo1 also triggered a malignant transformation in Slit2-positive human embryonic kidney 293 cells. Importantly, the expression of Slit2 and Robo1 was significantly associated with an increased metastatic risk and poorer overall survival in colorectal carcinoma patients. We conclude that engagement of Robo1 by Slit2 induces malignant transformation through Hakai-mediated E-cad ubiquitination and lysosomal degradation during colorectal epithelial cell carcinogenesis.     

Reduced surface expression of epithelial E-cadherin evoked by interferon-gamma is Fyn kinase-dependent

Interferon gamma (IFNγ) is an important regulatory cytokine that can exert a pro-inflammatory effect in the gut, where it has been shown to increase epithelial permeability via disruption of the tight junctions. Here we investigated the potential for IFNγ to regulate the adherens junction protein E-cadherin, an important mediator of normal epithelial tissue function, using the model T84 human colonic epithelial cell line. IFNγ (10 ng/ml) stimulated increased internalization of E-cadherin as assessed by immunofluorescence microscopy; internalization was reversed when cells were treated with PP1 (125 nM), a Src kinase-selective inhibitor. Immunoprecipitation studies demonstrated loss of E-cadherin from membrane fractions following IFNγ treatment and a corresponding increase in cytosolic E-cadherin and its binding partners, p120-catenin and beta-catenin: effects that were Src-kinase dependent. E-cadherin and p120-catenin phosphorylation was increased by IFNγ treatment and siRNA studies showed this was dependent upon the Src-kinase isoform Fyn. E-cadherin ubiquitinylation and subsequent proteasomal degradation stimulated by IFNγ was found to be dependent upon Fyn and the E-cadherin-selective ubiquitin ligase, Hakai. Use of Fyn and Hakai siRNA inhibited the internalization of E-cadherin as shown by immunoblotting and confocal fluorescence microscopy. Finally, IFNγ treatment resulted in a more fragile T84 cell monolayer with increased cell detachment in response to physical stress, which was prevented by PP1 and siRNA targeting Fyn or Hakai. Collectively, these results demonstrate a Fyn kinase-dependent mechanism through which IFNγ regulates E-cadherin stability and suggest a novel mechanism of disruption of epithelial cell contact, which could contribute to perturbed epithelial barrier function.     

Minus end–directed motor KIFC3 suppresses E-cadherin degradation by recruiting USP47 to adherens junctions

The adherens junction (AJ) plays a crucial role in maintaining cell–cell adhesion in epithelial tissues. Previous studies show that KIFC3, a minus end–directed kinesin motor, moves into AJs via microtubules that grow from clusters of CAMSAP3 (also known as Nezha), a protein that binds microtubule minus ends. The function of junction-associated KIFC3, however, remains to be elucidated. Here we find that KIFC3 binds the ubiquitin-specific protease USP47, a protease that removes ubiquitin chains from substrates and hence inhibits proteasome-mediated proteolysis, and recruits it to AJs. Depletion of KIFC3 or USP47 promotes cleavage of E-cadherin at a juxtamembrane region of the cytoplasmic domain, resulting in the production of a 90-kDa fragment and the internalization of E-cadherin. This cleavage depends on the E3 ubiquitin protein ligase Hakai and is inhibited by proteasome inhibitors. E-cadherin ubiquitination consistently increases after depletion of KIFC3 or USP47. These findings suggest that KIFC3 suppresses the ubiquitination and resultant degradation of E-cadherin by recruiting USP47 to AJs, a process that may be involved in maintaining stable cell–cell adhesion in epithelial sheets.     

Overexpression of ADAM9 enhances growth factor-mediated recycling of E-cadherin in human colon cancer cell line HT29 cells

Growth factor-mediated stimulation of epithelial cells induces the disassembly of E-cadherin-mediated cell-cell adhesion. We found that overexpression of a disintegrin and metalloprotease 9 (ADAM9) enhanced growth factor-mediated induction of endocytosis and dynamic recycling of E-cadherin in HT29 human colon cancer cells. In addition, ubiquitination and degradation of E-cadherin were reduced in these cells. ADAM9 constitutively interacted with E-cadherin, and the two proteins co-localized at the plasma membrane of HT29 cells. Administration of a metalloprotease inhibitor or overexpression of an ADAM9 mutant lacking metalloprotease activity attenuated growth factor-dependent endocytosis and recycling of E-cadherin as well as scattering of HT29 cells. These results suggest that the metalloprotease activity of ADAM9 mediates growth factor-induced endocytosis and dynamic recycling of E-cadherin and prevents E-cadherin degradation.     

Recycling of E-cadherin: a potential mechanism for regulating cadherin dynamics.

E-Cadherin plays critical roles in many aspects of cell adhesion, epithelial development, and the establishment and maintenance of epithelial polarity. The fate of E-cadherin once it is delivered to the basolateral cell surface, and the mechanisms which govern its participation in adherens junctions, are not well understood. Using surface biotinylation and recycling assays, we observed that some of the cell surface E-cadherin is actively internalized and is then recycled back to the plasma membrane. The pool of E-cadherin undergoing endocytosis and recycling was markedly increased in cells without stable cell-cell contacts, i.e., in preconfluent cells and after cell contacts were disrupted by depletion of extracellular Ca2+, suggesting that endocytic trafficking of E-cadherin is regulated by cell-cell contact. The reformation of cell junctions after replacement of Ca2+ was then found to be inhibited when recycling of endocytosed E-cadherin was disrupted by bafilomycin treatment. The endocytosis and recycling of E-cadherin and of the transferrin receptor were similarly inhibited by potassium depletion and by bafilomycin treatment, and both proteins were accumulated in intracellular compartments by an 18 degrees C temperature block, suggesting that endocytosis may occur via a clathrin-mediated pathway. We conclude that a pool of surface E-cadherin is constantly trafficked through an endocytic, recycling pathway and that this may provide a mechanism for regulating the availability of E-cadherin for junction formation in development, tissue remodeling, and tumorigenesis.     

Clathrin dependent endocytosis of E-cadherin is regulated by the Arf6GAP isoform SMAP1

E-cadherin is a central component of the adherens junction in epithelial cells and continuously undergoes endocytosis via clathrin-coated vesicles and/or caveolae depending on the cell type. In this study, we examined the role of SMAP1, a clathrin-interacting GTPase-activating protein (GAP) for the ADP-ribosylation factor 6 (Arf6) GTPase, in E-cadherin endocytosis. Mardin-Darby canine kidney (MDCK) epithelial cells were used as a model, and SMAP1 localized in the cytoplasm and along the adherens junction where E-cadherin was present. Next, activity of SMAP1 was compared with that of other Arf6GAPs (and/or an effector of Arf6-GTP), namely GIT1 and AMAP2/DDEF2. Overexpression of SMAP1 but not GIT1 nor AMAP2/DDEF2 strongly inhibited basal, as well as phorbolester-induced, internalization of E-cadherin. Notably, AMAP2/DDEF2 rather enhanced the caveolae-mediated incorporation of a membrane protein other than E-cadherin. Thus, in MDCK cells, E-cadherin appeared to be endocytosed solely through SMAP1-regulated clathrin-coated vesicles. Furthermore, MDCK cells overexpressing SMAP1 showed a reduced degree of cell migration compared to untransfected cells, as assessed by wound healing and Transwell assays, and this reduction in migration appeared to be due to the accumulation of E-cadherin at the adherens junction in cells overexpressing SMAP1. Collectively, SMAP1 likely represents a key Arf6GAP in clathrin dependent endocytosis of E-cadherin in MDCK cells. This activity of SMAP1 in E-cadherin turnover may be involved in epithelial organization and/or epithelial-mesenchymal transition.     

Numb controls E-cadherin endocytosis through p120 catenin with aPKC

Cadherin trafficking controls tissue morphogenesis and cell polarity. The endocytic adaptor Numb participates in apicobasal polarity by acting on intercellular adhesions in epithelial cells. However, it remains largely unknown how Numb controls cadherin-based adhesion. Here, we found that Numb directly interacted with p120 catenin (p120), which is known to interact with E-cadherin and prevent its internalization. Numb accumulated at intercellular adhesion sites and the apical membrane in epithelial cells. Depletion of Numb impaired E-cadherin internalization, whereas depletion of p120 accelerated internalization. Expression of the Numb-binding fragment of p120 inhibited E-cadherin internalization in a dominant-negative fashion, indicating that Numb interacts with the E-cadherin/p120 complex and promotes E-cadherin endocytosis. Impairment of Numb induced mislocalization of E-cadherin from the lateral membrane to the apical membrane. Atypical protein kinase C (aPKC), a member of the PAR complex, phosphorylated Numb and inhibited its association with p120 and α-adaptin. Depletion or inhibition of aPKC accelerated E-cadherin internalization. Wild-type Numb restored E-cadherin internalization in the Numb-depleted cells, whereas a phosphomimetic mutant or a mutant with defective α-adaptin-binding ability did not restore the internalization. Thus, we propose that aPKC phosphorylates Numb to prevent its binding to p120 and α-adaptin, thereby attenuating E-cadherin endocytosis to maintain apicobasal polarity.     

Cell Regulation by Phosphotyrosine-Targeted Ubiquitin Ligases

Three classes of E3 ubiquitin ligases, members of the Cbl, Hakai, and SOCS-Cul5-RING ligase families, stimulate the ubiquitination of phosphotyrosine-containing proteins, including receptor and nonreceptor tyrosine kinases and their phosphorylated substrates. Because ubiquitination frequently routes proteins for degradation by the lysosome or proteasome, these E3 ligases are able to potently inhibit tyrosine kinase signaling. Their loss or mutational inactivation can contribute to cancer, autoimmunity, or endocrine disorders, such as diabetes. However, these ligases also have biological functions that are independent of their ubiquitination activity. Here we review relevant literature and then focus on more-recent developments in understanding the structures, substrates, and pathways through which the phosphotyrosine-specific ubiquitin ligases regulate diverse aspects of cell biology.     

The FAM deubiquitylating enzyme localizes to multiple points of protein trafficking in epithelia, where it associates with E-cadherin and beta-catenin

Ubiquitylation is a necessary step in the endocytosis and lysosomal trafficking of many plasma membrane proteins and can also influence protein trafficking in the biosynthetic pathway. Although a molecular understanding of ubiquitylation in these processes is beginning to emerge, very little is known about the role deubiquitylation may play. Fat Facets in mouse (FAM) is substrate-specific deubiquitylating enzyme highly expressed in epithelia where it interacts with its substrate, beta-catenin. Here we show, in the polarized intestinal epithelial cell line T84, FAM localized to multiple points of protein trafficking. FAM interacted with beta-catenin and E-cadherin in T84 cells but only in subconfluent cultures. FAM extensively colocalized with beta-catenin in cytoplasmic puncta but not at sites of cell-cell contact as well as immunoprecipitating with beta-catenin and E-cadherin from a higher molecular weight complex ( approximately 500 kDa). At confluence FAM neither colocalized with, nor immunoprecipitated, beta-catenin or E-cadherin, which were predominantly in a larger molecular weight complex ( approximately 2 MDa) at the cell surface. Overexpression of FAM in MCF-7 epithelial cells resulted in increased beta-catenin levels, which localized to the plasma membrane. Expression of E-cadherin in L-cell fibroblasts resulted in the relocalization of FAM from the Golgi to cytoplasmic puncta. These data strongly suggest that FAM associates with E-cadherin and beta-catenin during trafficking to the plasma membrane.     

Cdc42 regulates E-cadherin ubiquitination and degradation through an epidermal growth factor receptor to Src-mediated pathway

E-cadherins play an essential role in maintaining epithelial polarity by forming Ca2+-dependent adherens junctions between epithelial cells. Here, we report that Ca2+ depletion induces E-cadherin ubiquitination and lysosomal degradation and that Cdc42 plays an important role in regulating this process. We demonstrate that Ca2+ depletion induces activation of Cdc42. This in turn up-regulates epidermal growth factor receptor (EGFR) signaling to mediate Src activation, leading to E-cadherin ubiquitination and lysosomal degradation. Silencing Cdc42 blocks activation of EGFR and Src induced by Ca2+ depletion, resulting in a reduction in E-cadherin degradation. The role of Cdc42 in regulating E-cadherin ubiquitination and degradation is underscored by the fact that constitutively active Cdc42(F28L) increases the activity of EGFR and Src and significantly enhances E-cadherin ubiquitination and lysosomal degradation. Furthermore, we found that GTP-dependent binding of Cdc42 to E-cadherin is critical for Cdc42 to induce the dissolution of adherens junctions. Our data support a model that activation of Cdc42 contributes to mesenchyme-like phenotype by targeting of E-cadherin for lysosomal degradation.     

Spatial regulation of Dia and Myosin-II by RhoGEF2 controls initiation of E-cadherin endocytosis during epithelial morphogenesis

E-cadherin plays a pivotal role in epithelial morphogenesis. It controls the intercellular adhesion required for tissue cohesion and anchors the actomyosin-driven tension needed to change cell shape. In the early Drosophila embryo, Myosin-II (Myo-II) controls the planar polarized remodelling of cell junctions and tissue extension. The E-cadherin distribution is also planar polarized and complementary to the Myosin-II distribution. Here we show that E-cadherin polarity is controlled by the polarized regulation of clathrin- and dynamin-mediated endocytosis. Blocking E-cadherin endocytosis resulted in cell intercalation defects. We delineate a pathway that controls the initiation of E-cadherin endocytosis through the regulation of AP2 and clathrin coat recruitment by E-cadherin. This requires the concerted action of the formin Diaphanous (Dia) and Myosin-II. Their activity is controlled by the guanine exchange factor RhoGEF2, which is planar polarized and absent in non-intercalating regions. Finally, we provide evidence that Dia and Myo-II control the initiation of E-cadherin endocytosis by regulating the lateral clustering of E-cadherin.     

Inducible expression of p120Cas1B isoform corroborates the role for p120-catenin as a positive regulator of E-cadherin function in intestinal cancer cells

Over the past decade, the exact function of p120-catenin in regulation of E-cadherin/catenins complex has remained particularly controversial. We have previously reported that E-cadherin-mediated adhesion is tightly regulated by tyrosine phosphorylation of catenins. However, this effect is not observed in human colon carcinoma cell line Caco-2. Here, we have generated inducible Caco-2 clones that display p120Cas1B, a p120-catenin isoform poorly expressed by these cells. As a result, neither expression of the transgene nor tyrosine phosphorylation of catenins induces redistribution of E-cadherin to the cytosol and disassembly of adherens and tight junctions. In contrast, E-cadherin appears markedly increased reinforcing cell-cell adhesion. Interestingly, a substantial decrease in p120-catenin levels is found in MDCK cells expressing Snail, in which E-cadherin expression is strongly inhibited. Additionally, we show that the specific depletion of p120-catenin decreases cell-cell contacts, and increases cell motility and scattering of colonies established by HT-29 M6 cells. Together our results corroborate that p120-catenin plays an essential role in the maintenance of the required E-cadherin protein levels that prevent the loss of epithelial characteristics occurred during tumorigenesis.     

Regulation of endocytosis, nuclear translocation, and signaling of fibroblast growth factor receptor 1 by E-cadherin

Fibroblast growth factor (FGF) receptors (FGFRs) signal to modulate diverse cellular functions, including epithelial cell morphogenesis. In epithelial cells, E-cadherin plays a key role in cell-cell adhesion, and its function can be regulated through endocytic trafficking. In this study, we investigated the location, trafficking, and function of FGFR1 and E-cadherin and report a novel mechanism, based on endocytic trafficking, for the coregulation of E-cadherin and signaling from FGFR1. FGF induces the internalization of surface FGFR1 and surface E-cadherin, followed by nuclear translocation of FGFR1. The internalization of both proteins is regulated by common endocytic machinery, resulting in cointernalization of FGFR1 and E-cadherin into early endosomes. By blocking endocytosis, we show that this is a requisite, initial step for the nuclear translocation of FGFR1. Overexpression of E-cadherin blocks both the coendocytosis of E-cadherin and FGFR1, the nuclear translocation of FGFR1 and FGF-induced signaling to the mitogen-activated protein kinase pathway. Furthermore, stabilization of surface adhesive E-cadherin, by overexpressing p120ctn, also blocks internalization and nuclear translocation of FGFR1. These data reveal that conjoint endocytosis and trafficking is a novel mechanism for the coregulation of E-cadherin and FGFR1 during cell signaling and morphogenesis.     

Loss of density-dependent growth inhibition and dissociation of α-catenin from E-cadherin

Normal human breast epithelial (HBE) cells at early (9th) passage ceased growth and formed a monolayer when they reached confluence. Immunostaining and Western blotting revealed that α- and β-catenins colocalized and coprecipitated with E-cadherin, suggesting a complex formation of E-cadherin with α- and β-catenins in early passage cells. In contrast, HBE cells at late (12–13th) passage did not cease growth after confluence but stratified. The late passage cells exhibited enhanced colony forming ability in soft agar compared with early passage cells, however, they had a definite proliferating lifespan and were primarily diploid. In late passage cells grown as multilayers, α-catenin was expressed but did not colocalize or coprecipitate with E-cadherin, suggesting its dissociation from E-cadherin. Coimmunoprecipitation of α-actinin with α-catenin suggested an indirect link between the E-cadherin-β-catenin complex and α-actinin via α-catenin in early, but not in late passage cells. β-Catenin in late passage cells was tyrosine phosphorylated and was not dephosphorylated following the addition of inhibitors of tyrosine kinases. Inhibition of dephosphorylation of β-catenin in early passage cells by vanadate, an inhibitor of protein tyrosine phosphatases, caused overgrowth of cells beyond the saturation density and loss of α-catenin from the E-cadherin-β-catenin complex. The results suggest that E-cadherin requires its association with α-actinin-associated α-catenin to maintain epithelial monolayers and accomplish the density-dependent inhibition of growth. In addition, association between E-cadherin and α-catenin is suggested to be prevented by the presence of tyrosine phosphorylated β-catenin which associates with E-cadherin. J. Cell. Physiol. 173:54–63, 1997. © 1997 Wiley-Liss, Inc.     

Loss of epithelial differentiation and gain of invasiveness correlates with tyrosine phosphorylation of the E-cadherin/beta-catenin complex in cells transformed with a temperature-sensitive v-SRC gene

Loss of histotypic organization of epithelial cells is a common feature in normal development as well as in the invasion of carcinomas. Here we show that the v-src oncogene is a potent effector of epithelial differentiation and invasiveness. MDCK epithelial cells transformed with a temperature-sensitive mutant of v-src exhibit a strictly epithelial phenotype at the nonpermissive temperature for pp60v-src activity (40.5 degrees C) but rapidly loose cell-to-cell contacts and acquire a fibroblast-like morphology after culture at the permissive temperature (35 degrees C). Furthermore, the invasiveness of the cells into collagen gels or into chick heart fragments was increased at the permissive temperature. The profound effects of v-src on intercellular adhesion were not linked to changes in the levels of expression of the epithelial cell adhesion molecule E-cadherin. Rather, we observed an increase in tyrosine phosphorylation of E-cadherin and, in particular, of the associated protein beta-catenin. These results suggest a mechanism by which v-src counteracts junctional assembly and thereby promotes invasiveness and dedifferentiation of epithelial cells through phosphorylation of the E-cadherin/catenin complex.     

Phosphorylation and ubiquitination are necessary for Na,K-ATPase endocytosis during hypoxia

As a cellular adaptative response, hypoxia decreases Na,K-ATPase activity by triggering the endocytosis of its alpha(1) subunit in alveolar epithelial cells. Here, we present evidence that the ubiquitin conjugating system is important in the Na,K-ATPase endocytosis during hypoxia and that ubiquitination of Na,K-ATPase alpha(1) subunit occurs at the basolateral membrane. Endocytosis and ubiquitination were prevented when the Ser 18 in the PKC phosphorylation motif of the Na,K-ATPase alpha(1) subunit was mutated to an alanine, suggesting that phosphorylation at Ser-18 is required for ubiquitination. Mutation of the four lysines surrounding Ser 18 to arginine prevented Na,K-ATPase ubiquitination and endocytosis during hypoxia; however, only one of them was sufficient to restore hypoxia-induced endocytosis. We provide evidence that ubiquitination plays an important role in cellular adaptation to hypoxia by regulating Na,K-ATPase alpha(1)-subunit endocytosis.