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.     

Rapid suppression of activated Rac1 by cadherins and nectins during de novo cell-cell adhesion

Cell-cell adhesion in simple epithelia involves the engagement of E-cadherin and nectins, and the reorganization of the actin cytoskeleton and membrane dynamics by Rho GTPases, particularly Rac1. However, it remains unclear whether E-cadherin and nectins up-regulate, maintain or suppress Rac1 activity during cell-cell adhesion. Roles for Rho GTPases are complicated by cell spreading and integrin-based adhesions to the extracellular matrix that occur concurrently with cell-cell adhesion, and which also require Rho GTPases. Here, we designed a simple approach to examine Rac1 activity upon cell-cell adhesion by MDCK epithelial cells, without cell spreading or integrin-based adhesion. Upon initiation of cell-cell contact in 3-D cell aggregates, we observed an initial peak of Rac1 activity that rapidly decreased by ～66% within 5 minutes, and further decreased to a low baseline level after 30 minutes. Inhibition of E-cadherin engagement with DECMA-1 Fab fragments or competitive binding of soluble E-cadherin, or nectin2alpha extracellular domain completely inhibited Rac1 activity. These results indicate that cadherins and nectins cooperate to induce and then rapidly suppress Rac1 activity during initial cell-cell adhesion, which may be important in inhibiting the migratory cell phenotype and allowing the establishment of initially weak cell-cell adhesions.     

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.     

Transmembrane control of cadherin-mediated cell adhesion: a 94 kDa protein functionally associated with a specific region of the cytoplasmic domain of E-cadherin.

Cadherins are a family of transmembrane glycoproteins which play a key role in Ca(2+)-dependent cell-cell adhesion. Cytoplasmic domains of these molecules are anchored to the cell cytoskeleton and are required for cadherin function. To elucidate how the function of cadherins is controlled through their cytoplasmic domains, we deleted five different regions in the cytoplasmic domain of E-cadherin. After transfecting L cells with cDNA encoding the mutant polypeptides, we assayed aggregating activity of these transfectants; all these mutant proteins were shown to have an extracellular domain with normal Ca(2+)-sensitivity and molecular weight. Two mutant polypeptides with deletions in the carboxy half of the cytoplasmic domain, however, did not promote cell-cell adhesion and had also lost the ability to bind to the cytoskeleton, whereas the mutant molecules with deletions of other regions retained the ability to promote cell adhesion and to anchor to the cytoskeleton. Thus, the cytoplasmic domain contains a subdomain which was involved in the cell adhesion and cytoskeleton-binding functions. When E-cadherin in F9 cells or in L cells transfected with wild-type or functional mutant cadherin polypeptides was solubilized with nonionic detergents and immunoprecipitated, two additional 94 and 102 kDa components were coprecipitated. The 94 kDa component, however, was not detected in the immunoprecipitates from cells expressing the mutant cadherins which had lost the adhesive function. These results suggest that the interaction of the carboxy half of the cytoplasmic domain with the 94 kDa component regulates the cell binding function of the extracellular domain of E-cadherin.     

Cell binding function of E-cadherin is regulated by the cytoplasmic domain.

Cadherins are a family of transmembrane glycoproteins responsible for Ca2+-dependent cell-cell adhesion. Their amino acid sequences are highly conserved in the cytoplasmic domain. To study the role of the cytoplasmic domain in the function of cadherins, we constructed expression vectors with cDNAs encoding the deletion mutants of E-cadherin polypeptides, in which the carboxy terminus was truncated at various lengths. These vectors were introduced into L cells by transfection, and cell lines expressing the mutant E-cadherin molecules were isolated. In all transfectants obtained, the extracellular domain of the mutant E-cadherins was exposed on the cell surface, and had normal Ca2+-sensitivity and molecular size. However, these cells did not show any Ca2+-dependent aggregation, indicating that the mutant molecules cannot mediate cell-cell binding. The mutant E-cadherin molecules could be released from cells by nonionic detergents, whereas a fraction of normal E-cadherin molecules could not be extracted with the detergent and appeared to be anchored to the cytoskeleton at cell-cell junctions. These results suggest that the cytoplasmic domain regulates the cell-cell binding function of the extracellular domain of E-cadherin, possibly through interaction with some cytoskeletal components.     

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.     

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.     

Two cell adhesion molecules, nectin and cadherin, interact through their cytoplasmic domain-associated proteins

We have found a new cell-cell adhesion system at cadherin-based cell-cell adherens junctions (AJs) consisting of at least nectin and l-afadin. Nectin is a Ca(2+)-independent homophilic immunoglobulin-like adhesion molecule, and l-afadin is an actin filament-binding protein that connects the cytoplasmic region of nectin to the actin cytoskeleton. Both the trans-interaction of nectin and the interaction of nectin with l-afadin are necessary for their colocalization with E-cadherin and catenins at AJs. Here, we examined the mechanism of interaction between these two cell-cell adhesion systems at AJs by the use of alpha-catenin-deficient F9 cell lines and cadherin-deficient L cell lines stably expressing their various components. We showed here that nectin and E-cadherin were colocalized through l-afadin and the COOH-terminal half of alpha-catenin at AJs. Nectin trans-interacted independently of E-cadherin, and the complex of E-cadherin and alpha- and beta-catenins was recruited to nectin-based cell-cell adhesion sites through l-afadin without the trans-interaction of E-cadherin. Our results indicate that nectin and cadherin interact through their cytoplasmic domain-associated proteins and suggest that these two cell-cell adhesion systems cooperatively organize cell-cell AJs.     

Cadherin-directed actin assembly: E-cadherin physically associates with the Arp2/3 complex to direct actin assembly in nascent adhesive contacts

Cadherin cell adhesion molecules are major determinants of tissue patterning which function in cooperation with the actin cytoskeleton. In the context of stable adhesion, cadherin/catenin complexes are often envisaged to passively scaffold onto cortical actin filaments. However, cadherins also form dynamic adhesive contacts during wound healing and morphogenesis. Here actin polymerization has been proposed to drive cell surfaces together, although F-actin reorganization also occurs as cell contacts mature. The interaction between cadherins and actin is therefore likely to depend on the functional state of adhesion. We sought to analyze the relationship between cadherin homophilic binding and cytoskeletal activity during early cadherin adhesive contacts. Dissecting the specific effect of cadherin ligation alone on actin regulation is difficult in native cell-cell contacts, due to the range of juxtacrine signals that can arise when two cell surfaces adhere. We therefore activated homophilic ligation using a specific functional recombinant protein. We report the first evidence that E-cadherin associates with the Arp2/3 complex actin nucleator and demonstrate that cadherin binding can exert an active, instructive influence on cells to mark sites for actin assembly at the cell surface.     

CD148 Tyrosine Phosphatase Promotes Cadherin Cell Adhesion

CD148 is a transmembrane tyrosine phosphatase that is expressed at cell junctions. Recent studies have shown that CD148 associates with the cadherin/catenin complex and p120 catenin (p120) may serve as a substrate. However, the role of CD148 in cadherin cell-cell adhesion remains unknown. Therefore, here we addressed this issue using a series of stable cells and cell-based assays. Wild-type (WT) and catalytically inactive (CS) CD148 were introduced to A431D (lacking classical cadherins), A431D/E-cadherin WT (expressing wild-type E-cadherin), and A431D/E-cadherin 764AAA (expressing p120-uncoupled E-cadherin mutant) cells. The effects of CD148 in cadherin adhesion were assessed by Ca²⁺ switch and cell aggregation assays. Phosphorylation of E-cadherin/catenin complex and Rho family GTPase activities were also examined. Although CD148 introduction did not alter the expression levels and complex formation of E-cadherin, p120, and β-catenin, CD148 WT, but not CS, promoted cadherin contacts and strengthened cell-cell adhesion in A431D/E-cadherin WT cells. This effect was accompanied by an increase in Rac1, but not RhoA and Cdc42, activity and largely diminished by Rac1 inhibition. Further, we demonstrate that CD148 reduces the tyrosine phosphorylation of p120 and β-catenin; causes the dephosphorylation of Y529 suppressive tyrosine residue in Src, a well-known CD148 site, increasing Src activity and enhancing the phosphorylation of Y228 (a Src kinase site) in p120, in E-cadherin contacts. Consistent with these findings, CD148 dephosphorylated both p120 and β-catenin in vitro. The shRNA-mediated CD148 knockdown in A431 cells showed opposite effects. CD148 showed no effects in A431D and A431D/E-cadherin 764AAA cells. In aggregate, these findings provide the first evidence that CD148 promotes E-cadherin adhesion by regulating Rac1 activity concomitant with modulation of p120, β-catenin, and Src tyrosine phosphorylation. This effect requires E-cadherin and p120 association.     

IQGAP1 and calmodulin modulate E-cadherin function

Ca(2+)-dependent cell-cell adhesion is mediated by the cadherin family of transmembrane proteins. Adhesion is achieved by homophilic interaction of the extracellular domains of cadherins on adjacent cells, with the cytoplasmic regions serving to couple the complex to the cytoskeleton. IQGAP1, a novel RasGAP-related protein that interacts with the cytoskeleton, binds to actin, members of the Rho family, and E-cadherin. Calmodulin binds to IQGAP1 and regulates its association with Cdc42 and actin. Here we demonstrate competition between calmodulin and E-cadherin for binding to IQGAP1 both in vitro and in a normal cellular milieu. Immunocytochemical analysis in MCF-7 (E-cadherin positive) and MDA-MB-231 (E-cadherin negative) epithelial cells revealed that E-cadherin is required for accumulation of IQGAP1 at cell-cell junctions. The cell-permeable calmodulin antagonist CGS9343B significantly increased IQGAP1 at areas of MCF-7 cell-cell contact, with a concomitant decrease in the amount of E-cadherin at cell-cell junctions. Analysis of E-cadherin function revealed that CGS9343B significantly decreased homophilic E-cadherin adhesion. On the basis of these data, we propose that disruption of the binding of calmodulin to IQGAP1 enhances the association of IQGAP1 with components of the cadherin-catenin complex at cell-cell junctions, resulting in impaired E-cadherin function.     

Disruption of epithelial cell-cell adhesion by exogenous expression of a mutated nonfunctional N-cadherin.

Cadherins, a family of transmembrane cell-cell adhesion receptors, require interactions with the cytoskeleton for normal function. To assess the mechanisms of these interactions, we studied the effect of exogenous expression of a mutant N-cadherin, cN390 delta; on epithelial cell-cell adhesion. The intracellular domain of cN390 delta was intact but its extracellular domain was largely deleted so that this molecule was not functional for cell adhesion. cDNA of cN390 delta was attached to the metallothionein promoter, and introduced into the keratinocyte line PAM212 expressing endogenous E- and P-cadherin. When the expression of cN390 delta was induced by Zn2+, cadherin-dependent adhesion of the transfected cells was inhibited, resulting in the dispersion of cell colonies, although their contacts were maintained under high cell density conditions. In these cultures, cN390 delta was expressed not only on the free surfaces of the cells but also at cell-cell junctions. The endogenous cadherins were concentrated at cell-cell junctions under normal conditions. As a result of cN390 delta expression, however, the endogenous cadherins localizing at the cell-cell junctions were largely diminished, suggesting that these molecules were replaced by the mutant molecules at these sites. As a control, we transfected the same cell line with cDNA of a truncated form of N-cadherin cadherin whose intracellular C terminus had been deleted leaving the extracellular domain intact. This molecule had no effect on cell-cell adhesion, nor did it localize to cell-cell contact sites. We also found that the association of the endogenous cadherins with alpha- and beta-catenins and plakoglobin was not affected by the expression of cN390 delta, which also formed a complex with these molecules, suggesting that no competition occurred between the endogenous and exogenous cadherins for these cytoplasmic proteins. These and other additional results suggest that the nonfunctional cadherins whose intracellular domain is intact occupy the sites where the endogenous cadherins should localize, through interactions with the cytoskeleton, and inhibit the cadherin adhesion system.     

Myosin VI and vinculin cooperate during the morphogenesis of cadherin cell cell contacts in mammalian epithelial cells

Cooperation between cadherins and the actin cytoskeleton controls many aspects of epithelial biogenesis. We report here that myosin VI critically regulates the morphogenesis of epithelial cell-cell contacts. As epithelial monolayers mature in culture, discontinuous cell-cell contacts are initially replaced by continuous (cohesive) contacts. Myosin VI is recruited to cell contacts as they become linear and cohesive, where it forms a biochemical complex with epithelial cadherin (E-cadherin). Myosin VI is necessary for strong cadherin adhesion, for cells to form cohesive linear contacts, and for the integrity of the apical junctional complex. We find that vinculin mediates this effect of myosin VI. Myosin VI is necessary for vinculin and E-cadherin to interact. A combination of gain and loss of function approaches identifies vinculin as a downstream effector of myosin VI that is necessary for the integrity of intercellular contacts. We propose that myosin VI and vinculin form a molecular apparatus that generates cohesive cell-cell contacts in cultured mammalian epithelia.     

The Juxtamembrane Region of the Cadherin Cytoplasmic Tail Supports Lateral Clustering, Adhesive Strengthening, and Interaction with p120ctn

Cadherin cell–cell adhesion molecules form membrane-spanning molecular complexes that couple homophilic binding by the cadherin ectodomain to the actin cytoskeleton. A fundamental issue in cadherin biology is how this complex converts the weak intrinsic binding activity of the ectodomain into strong adhesion. Recently we demonstrated that cellular cadherins cluster in a ligand-dependent fashion when cells attached to substrata coated with the adhesive ectodomain of Xenopus C-cadherin (CEC1-5). Moreover, forced clustering of the ectodomain alone significantly strengthened adhesiveness (Yap, A.S., W.M. Brieher, M. Pruschy, and B.M. Gumbiner. Curr. Biol. 7:308–315). In this study we sought to identify the determinants of the cadherin cytoplasmic tail responsible for clustering activity. A deletion mutant of C-cadherin (CT669) that retained the juxtamembrane 94–amino acid region of the cytoplasmic tail, but not the β-catenin–binding domain, clustered upon attachment to substrata coated with CEC1-5. Like wild-type C-cadherin, this clustering was ligand dependent. In contrast, mutant molecules lacking either the complete cytoplasmic tail or just the juxtamembrane region did not cluster. The juxtamembrane region was itself sufficient to induce clustering when fused to a heterologous membrane-anchored protein, albeit in a ligand-independent fashion. The CT669 cadherin mutant also displayed significant adhesive activity when tested in laminar flow detachment assays and aggregation assays. Purification of proteins binding to the juxtamembrane region revealed that the major associated protein is p120^ctn. These findings identify the juxtamembrane region of the cadherin cytoplasmic tail as a functionally active region supporting cadherin clustering and adhesive strength and raise the possibility that p120^ctn is involved in clustering and cell adhesion.     

Intracellular domain of desmoglein 3 (pemphigus vulgaris antigen) confers adhesive function on the extracellular domain of E-cadherin without binding catenins

For the extracellular (EC) domain of E-cadherin to function in homophilic adhesion it is thought that its intracytoplasmic (IC) domain must bind alpha- and beta-catenins, which link it to the actin cytoskeleton. However, the IC domain of pemphigus vulgaris antigen (PVA or Dsg3), which is in the desmoglein subfamily of the cadherin gene superfamily, does not bind alpha- or beta-catenins. Because desmogleins have also been predicted to function in the cell adhesion of desmosomes, we speculated that the PVA IC domain might be able to act in a novel way in conferring adhesive function on the EC domain of cadherins. To test this hypothesis we studied aggregation of mouse fibroblast L cell clones that expressed chimeric cDNAs encoding the EC domain of E-cadherin with various IC domains. We show here that the full IC domain of PVA as well as an IC subdomain containing only 40 amino acids of the PVA intracellular anchor (IA) region confer adhesive function on the E-cadherin EC domain without catenin-like associations with cytoplasmic molecules or fractionation with the cell cytoskeleton. This IA region subdomain is evolutionarily conserved in desmogleins, but not classical cadherins. These findings suggest an important cell biologic function for the IA region of desmogleins and demonstrate that strong cytoplasmic interactions are not absolutely necessary for E- cadherin-mediated adhesion.     

beta-Catenin associates with the actin-bundling protein fascin in a noncadherin complex

Catenins were first characterized as linking the cytoplasmic domains of cadherin cell-cell adhesion molecules to the cortical actin cytoskeleton. In addition to their essential role in modulating cadherin adhesivity, catenins have more recently been indicated to participate in cell and developmental signaling pathways. beta-Catenin, for example, associates directly with at least two receptor tyrosine kinases and transduces developmental signals within the Wnt pathway. Catenins also complex with the tumor suppressor protein adenomatous polyposis coli (APC), which appears to have a role in regulating cell proliferation. We have used the yeast two-hybrid method to reveal that fascin, a bundler of actin filaments, binds to beta-catenin's central Armadillo repeat domain. Western blotting of immunoprecipitates from cell line and mouse and rat brain extracts indicate that this interaction exists in vivo. Fascin and beta-catenin's association was further substantiated in vitro using purified proteins isolated from recombinant bacterial and baculoviral sources. Immunoprecipitation analysis indicates that fascin additionally binds to plakoglobin, which is highly homologous to beta-catenin but not to p120cas, a newly described catenin which contains a more divergent Armadillo-repeat domain. Immunoprecipitation, in vitro competition, and domain-mapping experiments demonstrate that fascin and E-cadherin utilize a similar binding site within beta-catenin, such that they form mutually exclusive complexes with beta-catenin. Immunofluorescence microscopy reveals that fascin and beta-catenin colocalize at cell-cell borders and dynamic cell leading edges of epithelial and endothelial cells. In addition to cell-cell borders, cadherins were unexpectedly observed to colocalize with fascin and beta-catenin at cell leading edges. It is conceivable that beta-catenin participates in modulating cytoskeletal dynamics in association with the microfilament-bundling protein fascin, perhaps in a coordinate manner with its functions in cadherin and APC complexes.     

Cadherin engagement regulates Rho family GTPases.

The formation of cell-cell adherens junctions is a cadherin-mediated process associated with reorganization of the actin cytoskeleton. Because Rho family GTPases regulate actin dynamics, we investigated whether cadherin-mediated adhesion regulates the activity of RhoA, Rac1, and Cdc42. Confluent epithelial cells were found to have elevated Rac1 and Cdc42 activity but decreased RhoA activity when compared with low density cultures. Using a calcium switch method to manipulate junction assembly, we found that induction of cell-cell junctions increased Rac1 activity, and this was inhibited by E-cadherin function-blocking antibodies. Using the same calcium switch procedure, we found little effect on RhoA activity during the first hour of junction assembly. However, over several hours, RhoA activity significantly decreased. To determine whether these effects are mediated directly through cadherins or indirectly through engagement of other surface proteins downstream from junction assembly, we used a model system in which cadherin engagement is induced without cell-cell contact. For these experiments, Chinese hamster ovary cells expressing C-cadherin were plated on the extracellular domain of C-cadherin immobilized on tissue culture plates. Whereas direct cadherin engagement did not stimulate Cdc42 activity, it strongly inhibited RhoA activity but increased Rac1 activity. Deletion of the C-cadherin cytoplasmic domain abolished these effects.     

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.     

Arp2/3 activity is necessary for efficient formation of E-cadherin adhesive contacts

Classical cadherin adhesion molecules are fundamental determinants of cell-cell recognition that function in cooperation with the actin cytoskeleton. Productive cadherin-based cell recognition is characterized by a distinct morphological process of contact zone extension, where limited initial points of adhesion are progressively expanded into broad zones of contact. We recently demonstrated that E-cadherin ligation recruits the Arp2/3 actin nucleator complex to the plasma membrane in regions where cell contacts are undergoing protrusion and extension. This suggested that Arp2/3 might generate the protrusive forces necessary for cell surfaces to extend upon one another during contact assembly. We tested this hypothesis in mammalian cells by exogenously expressing the CA region of N-WASP. This fragment, which potently inhibits Arp2/3-mediated actin assembly in vitro, also effectively reduced actin assembly at cadherin adhesive contacts. Blocking Arp2/3 activity by this strategy profoundly reduced the ability of cells to extend cadherin adhesive contacts but did not affect cell adhesiveness. These findings demonstrate that Arp2/3 activity is necessary for cells to efficiently extend and assemble cadherin-based adhesive contacts.     

Receptor protein tyrosine phosphatase PTPmu associates with cadherins and catenins in vivo

The extracellular segment of the receptor-type type protein tyrosine phosphatase PTPmu, possesses an MAM domain, an immunoglobulin domain, and four fibronectin type-III repeats. It binds homophilically, i.e., PTPmu on the surface of one cell binds to PTPmu on an apposing cell, and the binding site lies within the immunoglobulin domain. The intracellular segment of PTPmu has two PTP domains and a juxtamembrane segment that is homologous to the conserved intracellular domain of the cadherins. In cadherins, this segment interacts with proteins termed catenins to mediate association with the actin cytoskeleton. In this article, we demonstrate that PTPmu associates with a complex containing cadherins, alpha- and beta-catenin in mink lung (MvLu) cells, and in rat heart, lung, and brain tissues. Greater than 80% of the cadherin in the cell is cleared from Triton X-100 lysates of MvLu cells after immunoprecipitation with antibodies to PTPmu; however, the complex is dissociated when lysates are prepared in more stringent, SDS-containing RIPA buffer. In vitro binding studies demonstrated that the intracellular segment of PTPmu binds directly to the intracellular domain of E-cadherin, but not to alpha- or beta-catenin. Consistent with their ability to interact in vivo, PTPmu, cadherins, and catenins all localized to points of cell-cell contact in MvLu cells, as assessed by immunocytochemical staining. After pervanadate treatment of MvLu cells, which inhibits cellular tyrosine phosphatase activity including PTPmu, the cadherins associated with PTPmu are now found in a tyrosine- phosphorylated form, indicating that the cadherins may be an endogenous substrate for PTPmu. These data suggest that PTPmu may be one of the enzymes that regulates the dynamic tyrosine phosphorylation, and thus function, of the cadherin/catenin complex in vivo.