The catenin, p120ctn, is a common membrane-associated protein in various epithelial and non-epithelial cells and tissues

The cadherin-binding catenin p120ctn was originally identified as an Src-tyrosine kinase substrate. More recently, p120ctn has been shown in some cell types to be associated with catenin/cadherin complexes of adherens junctions. To address the question whether p120ctn is restricted to certain cell types or whether it is a general cellular component we investigated tissue distribution of p120ctn by immunohistochemistry and immunoblotting in the rat. We found p120ctn to be widely distributed in several tissues where it is mainly restricted to the plasma membrane. In various epithelia p120ctn was found in association with different adherens junctions such as the zonula adherens and puncta adherentia. In addition, p120ctn was localized along infoldings of the basal cell membrane, most prominently in renal proximal and distal tubules. pl20ctn was not restricted to epithelia. It was also found at intercalated discs of cardiomyocytes. In the nervous system, immunostaining was particularly prominent in areas rich in synapses suggesting that pl20ctn is a component of synaptic adherens junctions as well. By immunoblotting, four different isoforms of pl20ctn could be detected displaying similar electrophoretic mobilities as the isoforms 1A, 1B, 2A, and 2B reported from mice. Whereas all epithelia assayed contained at least two isoforms, testis, heart, brain, and retina contained a single 110-kDa band that corresponds to isoform 1B in mice.     

Early enterocytic differentiation of HT-29 cells: biochemical changes and strength increases of adherens junctions

We have characterized the modulation of cell-cell adhesion and the structure of adherens junctions in the human colon adenocarcinoma HT-29 cell line that differentiates into enterocytes after glucose substitution for galactose in the medium. We demonstrate that differentiated cells (HT-29 Gal) rapidly established E-cadherin-mediated interactions in aggregation assays. This effect is not due to an increase in E-cadherin expression during this early stage of cell differentiation, but rather results from the maturation of preexisting adherens junctions. These junctions are characterized by the redistribution of E-cadherin to the basolateral membrane and its co-localization with the actin cytoskeleton. Subcellular fractionation studies indicate that actin-associated E-cadherins bind beta-catenin and p120ctn. Furthermore, the p120ctn/E-cadherin association is upregulated. These data reveal a cooperative interaction between p120ctn and E-cadherin that corresponds to mature functional adherens junctions able to initiate tight cell-cell adhesion required for epithelium architecture and further affirm the gatekeeper role of p120ctn.     

P120-catenin isoforms 1A and 3A differently affect invasion and proliferation of lung cancer cells

Different isoforms of p120-catenin (p120ctn), a member of the Armadillo gene family, are variably expressed in different tissues as a result of alternative splicing and the use of multiple translation initiation codons. When expressed in cancer cells, these isoforms may confer different properties with respect to cell adhesion and invasion. We have previously reported that the p120ctn isoforms 1 and 3 were the most highly expressed isoforms in normal lung tissues, and their expression level was reduced in lung tumor cells. To precisely define their biological roles, we transfected p120ctn isoforms 1A and 3A into the lung cancer cell lines A549 and NCI-H460. Enhanced expression of p120ctn isoform 1A not only upregulated E-cadherin and β-catenin, but also downregulated the Rac1 activity, and as a result, inhibited the ability of cells to invade. In contrast, overexpression of p120ctn isoform 3A led to the inactivation of Cdc42 and the activation of RhoA, and had a smaller influence on invasion. However, we found that isoform 3A had a greater ability than isoform 1A in both inhibiting the cell cycle and reducing tumor cell proliferation. The present study revealed that p120ctn isoforms 1A and 3A differently regulated the adhesive, proliferative, and invasive properties of lung cancer cells through distinct mechanisms.     

Differential effects of substrate-analogue inhibitors on nitric oxide synthase dimerization

Cigarette smoking has been linked to almost all major types of cancer. Emerging evidence suggests that smoking initiates transformed cell growth and migration by disrupting cell-cell interactions in the polarized mucosal epithelium. Together with other adherens junction proteins, p120-catenin (p120ctn) maintains cell-cell adhesion through its direct interaction with E-cadherin (E-cad). Mislocalization and/or loss of p120ctn have been reported in all lung cancer subtypes and are related to poor prognosis. Here, we showed that p120ctn modulates smoke-induced cell migration via the EGFR/Src-P pathway. Chemical blockade of EGFR/Src signaling inhibited smoke-induced activation of cofilin (an actin severing protein) and promoted cell migration in the presence of p120ctn but had little effect on blocking migration in the absence of p120ctn. These data suggested that smoke-induced cell migration was mediated via an EGFR/Src-dependent signaling pathway in cells that expressed p120ctn, but upon loss of p120ctn, migration continued to occur via an alternative, EGFR/Src-independent pathway. Thus, gradual loss of membrane p120ctn with lung cancer progression may contribute to reduced effectiveness of conventional chemotherapies, such as those directed against EGFR.     

A dileucine motif targets MCAM-l cell adhesion molecule to the basolateral membrane in MDCK cells

Melanoma cell adhesion molecule (MCAM), an adhesion molecule belonging to the Ig superfamily, is an endothelial marker and is expressed in different epithelia. MCAM is expressed as two isoforms differing by their cytoplasmic domain: MCAM-l and MCAM-s (long and short). In order to identify the respective role of each MCAM isoform, we analyzed MCAM isoform targeting in polarized epithelial Madin-Darby canine kidney (MDCK) cells using MCAM-GFP chimeras. Confocal microscopy revealed that MCAM-s and MCAM-l were addressed to the apical and basolateral membranes, respectively. Transfection of MCAM-l mutants established that a single dileucine motif (41-42) of the cytoplasmic domain was required for MCAM-l basolateral targeting in MDCK cells. Although double labelling experiments showed that MCAM-l is not a component of adherens junctions and focal adhesions, its expression on basolateral membranes suggests that MCAM-l is involved in epithelium insuring.     

p120ctn and P-Cadherin but Not E-Cadherin Regulate Cell Motility and Invasion of DU145 Prostate Cancer Cells

Background

Adherens junctions consist of transmembrane cadherins, which interact intracellularly with p120ctn, ß-catenin and α-catenin. p120ctn is known to regulate cell-cell adhesion by increasing cadherin stability, but the effects of other adherens junction components on cell-cell adhesion have not been compared with that of p120ctn.

Methodology/Principal Findings

We show that depletion of p120ctn by small interfering RNA (siRNA) in DU145 prostate cancer and MCF10A breast epithelial cells reduces the expression levels of the adherens junction proteins, E-cadherin, P-cadherin, ß-catenin and α-catenin, and induces loss of cell-cell adhesion. p120ctn-depleted cells also have increased migration speed and invasion, which correlates with increased Rap1 but not Rac1 or RhoA activity. Downregulation of P-cadherin, β-catenin and α-catenin but not E-cadherin induces a loss of cell-cell adhesion, increased migration and enhanced invasion similar to p120ctn depletion. However, only p120ctn depletion leads to a decrease in the levels of other adherens junction proteins.

Conclusions/Significance

Our data indicate that P-cadherin but not E-cadherin is important for maintaining adherens junctions in DU145 and MCF10A cells, and that depletion of any of the cadherin-associated proteins, p120ctn, ß-catenin or α-catenin, is sufficient to disrupt adherens junctions in DU145 cells and increase migration and cancer cell invasion.     

p120 catenin regulates the actin cytoskeleton via Rho family GTPases

Cadherins are calcium-dependent adhesion molecules responsible for the establishment of tight cell-cell contacts. p120 catenin (p120ctn) binds to the cytoplasmic domain of cadherins in the juxtamembrane region, which has been implicated in regulating cell motility. It has previously been shown that overexpression of p120ctn induces a dendritic morphology in fibroblasts (Reynolds, A.B. , J. Daniel, Y. Mo, J. Wu, and Z. Zhang. 1996. Exp. Cell Res. 225:328-337.). We show here that this phenotype is suppressed by coexpression of cadherin constructs that contain the juxtamembrane region, but not by constructs lacking this domain. Overexpression of p120ctn disrupts stress fibers and focal adhesions and results in a decrease in RhoA activity. The p120ctn-induced phenotype is blocked by dominant negative Cdc42 and Rac1 and by constitutively active Rho-kinase, but is enhanced by dominant negative RhoA. p120ctn overexpression increased the activity of endogenous Cdc42 and Rac1. Exploring how p120ctn may regulate Rho family GTPases, we find that p120ctn binds the Rho family exchange factor Vav2. The behavior of p120ctn suggests that it is a vehicle for cross-talk between cell-cell junctions and the motile machinery of cells. We propose a model in which p120ctn can shuttle between a cadherin-bound state and a cytoplasmic pool in which it can interact with regulators of Rho family GTPases. Factors that perturb cell-cell junctions, such that the cytoplasmic pool of p120ctn is increased, are predicted to decrease RhoA activity but to elevate active Rac1 and Cdc42, thereby promoting cell migration.     

N-terminal 1-54 amino acid sequence and Armadillo repeat domain are indispensable for P120-catenin isoform 1A in regulating E-cadherin

P120-catenin (p120ctn) exerts important roles in regulating E-cadherin and invasiveness in cancer cells. However, the mechanisms by which p120ctn isoforms 1 and 3 modulate E-cadherin expression are poorly understood. In the current study, HBE, H460, SPC and LTE cell lines were used to examine the effects of p120ctn isoforms 1A and 3A on E-cadherin expression and cell invasiveness. E-cadherin was localized on the cell membrane of HBE and H460 cells, while it was confined to the cytoplasm in SPC and LTE cells. Depletion of endogenous p120ctn resulted in reduced E-cadherin expression; however, p120ctn ablation showed opposite effects on invasiveness in the cell lines by decreasing invasiveness in SPC and LTE cells and increasing it in HBE and H460 cells. Restitution of 120ctn isoform 1A restored E-cadherin on the cell membrane and blocked cell invasiveness in H460 and HBE cells, while it restored cytoplasmic E-cadherin and enhanced cell invasiveness in SPC and LTE cells. P120ctn isoform 3A increased the invasiveness in all four cell lines despite the lack of effect on E-cadherin expression, suggesting a regulatory pathway independent of E-cadherin. Moreover, five p120ctn isoform 1A deletion mutants were constructed and expressed in H460 and SPC cells. The results showed that only the M4 mutant, which contains N-terminal 1-54 amino acids and the Armadillo repeat domain, was functional in regulating E-cadherin and cell invasiveness, as observed in p120ctn isoform 1A. In conclusion, the N-terminal 1-54 amino acid sequence and Armadillo repeat domain of p120ctn isoform 1A are indispensable for regulating E-cadherin protein. P120ctn isoform 1A exerts opposing effects on cell invasiveness, corresponding to the subcellular localization of E-cadherin.     

Regulation of E-cadherin endocytosis by nectin through afadin, Rap1, and p120ctn

Adherens junctions (AJs) are a major cell-cell adhesion structure in epithelial cells that are formed by two major cell-cell adhesion molecules, E-cadherin and nectin. We have previously shown that nectin first forms cell-cell adhesion and then recruits non-trans-interacting E-cadherin to the nectin-based cell-cell adhesion sites, which gradually trans-interact there, eventually forming AJs. We have examined here the effect of trans-interacting nectin on non-trans-interacting E-cadherin endocytosis. Trans-interacting nectin capable of associating with afadin, but not trans-interacting nectin mutant incapable of associating with afadin, inhibited non-trans-interacting E-cadherin endocytosis in intact cells. Afadin is a nectin- and actin filament-binding protein that connects nectin to the actin cytoskeleton. Studies on the mode of action of the nectin-afadin system using cell-free assay revealed that afadin associated with nectin bound Rap1 activated by trans-interacting nectin, interacted with p120ctn, and strengthened the binding of p120ctn to E-cadherin, eventually reducing non-trans-interacting E-cadherin endocytosis. Afadin, which did not bind Rap1, was inactive in this capacity. These results indicate that trans-interacting nectin inhibits non-trans-interacting E-cadherin endocytosis through afadin, Rap1, and p120ctn and thereby further accumulates non-trans-interacting E-cadherin to the nectin-based cell-cell adhesion sites for the formation of AJs.     

Desmoplakin controls microvilli length but not cell adhesion or keratin organization in the intestinal epithelium

Maintaining proper cell-cell adhesion in the intestine is essential for tissue homeostasis and barrier function. This adhesion is thought to be mediated by cell adhesion structures, including tight junctions, adherens junctions, and desmosomes, which concentrate in the apical junctional region. While clear roles for adherens and tight junctions have been established in simple epithelia, the function of desmosomes has not been addressed. In stratified epithelia, desmosomes impart mechanical strength to tissues by organizing and anchoring the keratin filament network. In this paper, we report that the desmosomal protein desmoplakin (DP) is not essential for cell adhesion in the intestinal epithelium. Surprisingly, when DP is lacking, keratin filament localization is also unperturbed, although keratin filaments no longer anchor at desmosomes. Unexpectedly, DP is important for proper microvillus structure. Our study highlights the tissue-specific functions of desmosomes and reveals that the canonical functions for these structures are not conserved in simple epithelium.     

Differential modulation of cadherin-mediated cell-cell adhesion by platelet endothelial cell adhesion molecule-1 isoforms through activation of extracellular regulated kinases

The role of platelet endothelial cell adhesion molecule-1 (PECAM-1) in endothelial cell-cell interactions and its contribution to cadherin-mediated cell adhesion are poorly understood. Such studies have been difficult because all known endothelial cells express PECAM-1. We have used Madin-Darby canine kidney (MDCK) cells as a model system in which to evaluate the role of PECAM-1 isoforms that differ in their cytoplasmic domains in cell-cell interactions. MDCK cells lack endogenous PECAM-1 but form cell-cell junctions similar to those of endothelial cells, in which PECAM-1 is concentrated. MDCK cells were transfected with two isoforms of murine PECAM-1, Delta15 and Delta14&15, the predominant isoforms expressed in vivo. Expression of the Delta15 isoform resulted in apparent dedifferentiation of MDCK cells concomitant with the loss of adherens junctions, down-regulation of E-cadherin, alpha- and beta-catenin expression, and sustained activation of extracellular regulated kinases. The Delta15 isoform was not concentrated at cell-cell contacts. In contrast, the Delta14&15 isoform localized to sites of cell-cell contact and had no effect on MDCK cell morphology, cadherin/catenin expression, or extracellular regulated kinase activity. Thus, the presence of exon 14 in the cytoplasmic domain of PECAM-1 has dramatic effects on the ability of cells to maintain adherens junctions and an epithelial phenotype. Therefore, changes in the expression of exon 14 containing PECAM-1 isoforms, which we have observed during development, may have profound functional consequences.     

An octapeptide in the juxtamembrane domain of VE-cadherin is important for p120ctn binding and cell proliferation

The cadherins are a family of adhesive proteins involved in cell-cell homophilic interactions. VE-cadherin, expressed in endothelial cells, is involved in morphogenesis, regulation of permeability, and cellular proliferation. The cytoplasmic tails of cadherins contain two major domains, the juxtamembrane domain that plays a role in the intercellular localization of the protein and also serves for binding of p120ctn, and a C-terminal domain that associates with beta- or gamma-catenin. A highly conserved region present in the juxtamembrane domain of the cadherins has been shown to be necessary for p120ctn binding in E-cadherin. Using a mutant VE-cadherin lacking a highly conserved octapeptide, we demonstrated that it is required for p120ctn binding to VE-cadherin as determined by immunoprecipitation and colocalization studies. By immunofluorescence, this mutant protein has a topographical distribution similar to that of the wild-type VE-cadherin and, therefore, we conclude that the topographical distribution of VE-cadherin is independent of this motif. In addition, although cell-cell association is present in cells expressing this mutant form of VE-cadherin, we found that the strength of adhesion is decreased. Finally, our results for the first time demonstrate that the interaction of VE-cadherin with p120 catenin plays an important role in cellular growth, suggesting that the binding of p120 catenin to cadherins may regulate cell proliferation.     

Regulation of the assembly and adhesion activity of E-cadherin by nectin and afadin for the formation of adherens junctions in Madin-Darby canine kidney cells

The Ca2+-independent immunoglobulin-like molecule nectin first forms cell-cell adhesion and then assembles cadherin at nectin-based cell-cell adhesion sites, resulting in the formation of adherens junctions (AJs). Afadin is a nectin- and actin filament-binding protein that connects nectin to the actin cytoskeleton. Here, we studied the roles and modes of action of nectin and afadin in the formation of AJs in cultured MDCK cells. The trans-interaction of nectin assembled E-cadherin, which associated with p120(ctn), beta-catenin, and alpha-catenin, at the nectin-based cell-cell adhesion sites in an afadin-independent manner. However, the assembled E-cadherin showed weak cell-cell adhesion activity and might be the non-trans-interacting form. This assembly was mediated by the IQGAP1-dependent actin cytoskeleton, which was organized by Cdc42 and Rac small G proteins that were activated by the action of trans-interacting nectin through c-Src and Rap1 small G protein in an afadin-independent manner. However, Rap1 bound to afadin, and this Rap1-afadin complex then interacted with p120(ctn) associated with non-trans-interacting E-cadherin, thereby causing the trans-interaction of E-cadherin. Thus, nectin regulates the assembly and cell-cell adhesion activity of E-cadherin through afadin, nectin signaling, and p120(ctn) for the formation of AJs in Madin-Darby canine kidney cells.     

Misexpression of the catenin p120(ctn)1A perturbs Xenopus gastrulation but does not elicit Wnt-directed axis specification

Modulators of cadherin function are of great interest given that the cadherin complex actively contributes to the morphogenesis of virtually all tissues. The catenin p120(ctn) (formerly p120cas) was first identified as a src- and receptor-protein tyrosine kinase substrate and later shown to interact directly with cadherins. In common with beta-catenin and plakoglobin (gamma-catenin), p120(ctn) contains a central Armadillo repeat region by which it binds cadherin cytoplasmic domains. However, little is known about the function of p120(ctn) within the cadherin complex. We examined the role of p120(ctn)1A in early vertebrate development via its exogenous expression in Xenopus. Ventral overexpression of p120(ctn)1A, in contrast to beta-catenin, did not induce the formation of duplicate axial structures resulting from the activation of the Wnt signaling pathway, nor did p120(ctn) affect mesoderm induction. Rather, dorsal misexpression of p120(ctn) specifically perturbed gastrulation. Lineage tracing of cells expressing exogenous p120(ctn) indicated that cell movements were disrupted, while in vitro studies suggested that this may have been a consequence of reduced adhesion between blastomeres. Thus, while cadherin-binding proteins beta-catenin, plakoglobin, and p120(ctn) are members of the Armadillo protein family, it is clear that these proteins have distinct biological functions in early vertebrate development. This work indicates that p120(ctn) has a role in cadherin function and that heightened expression of p120(ctn) interferes with appropriate cell-cell interactions necessary for morphogenesis.     

N-cadherin and Cx43alpha1 gap junctions modulates mouse neural crest cell motility via distinct pathways

Our previous studies showed an essential role for connexin 43 or alpha1 connexin (Cx43alpha1) gap junctions in the modulation of neural crest cell motility. Cx43alpha1 gap junctions and N-cadherin containing adherens junctions are expressed in migrating cardiac neural crest cells. Analysis of the N-cadherin knockout (KO) mouse model revealed that N-cadherin is essential for gap junction mediated dye coupling but not for expression of Cx43alpha1 gap junctions in neural crest cells. Time lapse videomicroscopy and motion analysis showed that the motility of N-cadherin KO neural crest cells were altered, but the motility changes differed compared to Cx43alpha1 KO neural crest cells. These observations suggest that the role of N-cadherin in cell motility is not simply mediated via the modulation of Cx43alpha1 mediated cell-cell communication. This was confirmed by a parallel analysis of wnt-1 deficient neural crest cells, which also showed a reduction in dye coupling, and yet no change in cell motility. Analysis of p120 catenin (p120ctn), an Amardillo family protein known to play a role in cell motility, showed that it is colocalized with N-cadherin and Cx43alpha1 in migrating neural crest cells. This subcellular distribution was altered in the N-cadherin and Cx43alpha1 KO neural crest cells. Given these results, we propose that N-cadherin and Cx43alpha1 may modulate neural crest cell motility by engaging in a dynamic cross-talk with the cell's locomotory apparatus through p120ctn signaling.     

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.     

Selective uncoupling of p120(ctn) from E-cadherin disrupts strong adhesion

p120(ctn) is a catenin whose direct binding to the juxtamembrane domain of classical cadherins suggests a role in regulating cell-cell adhesion. The juxtamembrane domain has been implicated in a variety of roles including cadherin clustering, cell motility, and neuronal outgrowth, raising the possibility that p120 mediates these activities. We have generated minimal mutations in this region that uncouple the E-cadherin-p120 interaction, but do not affect interactions with other catenins. By stable transfection into E-cadherin-deficient cell lines, we show that cadherins are both necessary and sufficient for recruitment of p120 to junctions. Detergent-free subcellular fractionation studies indicated that, in contrast to previous reports, the stoichiometry of the interaction is extremely high. Unlike alpha- and beta-catenins, p120 was metabolically stable in cadherin-deficient cells, and was present at high levels in the cytoplasm. Analysis of cells expressing E-cadherin mutant constructs indicated that p120 is required for the E-cadherin-mediated transition from weak to strong adhesion. In aggregation assays, cells expressing p120-uncoupled E-cadherin formed only weak cell aggregates, which immediately dispersed into single cells upon pipetting. As an apparent consequence, the actin cytoskeleton failed to insert properly into peripheral E-cadherin plaques, resulting in the inability to form a continuous circumferential ring around cell colonies. Our data suggest that p120 directly or indirectly regulates the E-cadherin-mediated transition to tight cell-cell adhesion, possibly blocking subsequent events necessary for reorganization of the actin cytoskeleton and compaction.