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.     

The Nonreceptor Protein Tyrosine Phosphatase PTP1B Binds to the Cytoplasmic Domain of N-Cadherin and Regulates the Cadherin–Actin Linkage

Cadherin-mediated adhesion depends on the association of its cytoplasmic domain with the actin-containing cytoskeleton. This interaction is mediated by a group of cytoplasmic proteins: α-and β- or γ- catenin. Phosphorylation of β-catenin on tyrosine residues plays a role in controlling this association and, therefore, cadherin function. Previous work from our laboratory suggested that a nonreceptor protein tyrosine phosphatase, bound to the cytoplasmic domain of N-cadherin, is responsible for removing tyrosine-bound phosphate residues from β-catenin, thus maintaining the cadherin–actin connection (Balsamo et al., 1996). Here we report the molecular cloning of the cadherin-associated tyrosine phosphatase and identify it as PTP1B. To definitively establish a causal relationship between the function of cadherin-bound PTP1B and cadherin-mediated adhesion, we tested the effect of expressing a catalytically inactive form of PTP1B in L cells constitutively expressing N-cadherin. We find that expression of the catalytically inactive PTP1B results in reduced cadherin-mediated adhesion. Furthermore, cadherin is uncoupled from its association with actin, and β-catenin shows increased phosphorylation on tyrosine residues when compared with parental cells or cells transfected with the wild-type PTP1B. Both the transfected wild-type and the mutant PTP1B are found associated with N-cadherin, and recombinant mutant PTP1B binds to N-cadherin in vitro, indicating that the catalytically inactive form acts as a dominant negative, displacing endogenous PTP1B, and rendering cadherin nonfunctional. Our results demonstrate a role for PTP1B in regulating cadherin-mediated cell adhesion.     

p120-catenin is a key component of the cadherin-gamma-secretase supercomplex

In this work, we show several previously unknown features of p120-catenin in a cadherin–catenin complex that are critical for our understanding of cadherin-based adhesion and signaling. We show that in human epithelial A-431 cells, nearly all p120 molecules engage in high-affinity interaction with E-cadherin–catenin complexes located at the cellular surface. p120 is positioned in proximity to α-catenin in the complex with cadherin. These findings suggest a functional cooperation between p120 and α-catenin in cadherin-based adhesion. A low level of cadherin-free p120 molecules, in contrast, could facilitate p120-dependent signaling. Finally, we present compelling evidence that p120 is a key linker cementing the E-cadherin–catenin complex with the transmembrane protease γ-secretase. The cell–cell contact location of this supercomplex makes it an important candidate for conducting different signals that rely on γ-secretase proteolytic activity.     

E-cadherin phosphorylation occurs during its biosynthesis to promote its cell surface stability and adhesion

E-cadherin is highly phosphorylated within its β-catenin–binding region, and this phosphorylation increases its affinity for β-catenin in vitro. However, the identification of key serines responsible for most cadherin phosphorylation and the adhesive consequences of modification at such serines have remained unknown. In this study, we show that as few as three serines in the β-catenin–binding domain of E-cadherin are responsible for most radioactive phosphate incorporation. These serines are required for binding to β-catenin and the mutual stability of both E-cadherin and β-catenin. Cells expressing a phosphodeficient (3S>A) E-cadherin exhibit minimal cell–cell adhesion due to enhanced endocytosis and degradation through a lysosomal compartment. Conversely, negative charge substitution at these serines (3S>D) antagonizes cadherin endocytosis and restores wild-type levels of adhesion. The cadherin kinase is membrane proximal and modifies the cadherin before it reaches the cell surface. Together these data suggest that E-cadherin phosphorylation is largely constitutive and integral to cadherin–catenin complex formation, surface stability, and function.     

p120-catenin and β-catenin differentially regulate cadherin adhesive function

Vascular endothelial (VE)-cadherin, the major adherens junction adhesion molecule in endothelial cells, interacts with p120-catenin and β-catenin through its cytoplasmic tail. However, the specific functional contributions of the catenins to the establishment of strong adhesion are not fully understood. Here we use bioengineering approaches to identify the roles of cadherin–catenin interactions in promoting strong cellular adhesion and the ability of the cells to spread on an adhesive surface. Our results demonstrate that the domain of VE-cadherin that binds to β-catenin is required for the establishment of strong steady-state adhesion strength. Surprisingly, p120 binding to the cadherin tail had no effect on the strength of adhesion when the available adhesive area was limited. Instead, the binding of VE-cadherin to p120 regulates adhesive contact area in a Rac1-dependent manner. These findings reveal that p120 and β-catenin have distinct but complementary roles in strengthening cadherin-mediated adhesion.     

Loss of ��-Catenin Decreases the Strength of Single E-cadherin Bonds between Human Cancer Cells

The progression of several human cancers correlates with the loss of cytoplasmic protein α-catenin from E-cadherin-rich intercellular junctions and loss of adhesion. However, the potential role of α-catenin in directly modulating the adhesive function of individual E-cadherin molecules in human cancer is unknown. Here we use single-molecule force spectroscopy to probe the tensile strength, unstressed bond lifetime, and interaction energy between E-cadherins expressed on the surface of live human parental breast cancer cells lacking α-catenin and these cells where α-catenin is re-expressed. We find that the tensile strength and the lifetime of single E-cadherin/E-cadherin bonds between parental cells are significantly lower over a wide range of loading rates. Statistical analysis of the force displacement spectra reveals that single cadherin bonds between cancer cells feature an exceedingly low energy barrier against tensile forces and low molecular stiffness. Disassembly of filamentous actin using latrunculin B has no significant effect on the strength of single intercellular E-cadherin bonds. The absence of α-catenin causes a dominant negative effect on both global cell-cell adhesion and single E-cadherin bond strength. These results suggest that the loss of α-catenin alone drastically reduces the adhesive force between individual cadherin pairs on adjoining cells, explain the global loss of cell adhesion in human breast cancer cells, and show that the forced expression of α-catenin in cancer cells can restore both higher intercellular avidity and intercellular E-cadherin bond strength.The reduction of intercellular adhesion in a solid tumor is a critical step in the progression of tumor cells to metastasis (1). How normal cells lose their ability to form strong adhesions within a tissue is not well understood (2, 3). The loss of adhesion between adjoining epithelial cells and the ensuing onset of metastasis occur through an epithelial-to-mesenchymal transition that often correlates with the loss of cytoplasmic protein α-catenin and a poor prognosis in a wide range of cancers, including breast (4), esophageal (5), gastric (6, 7), cervical (8), and colorectal cancer (9). In normal epithelial tissues, α-catenin localizes to junctions that organize at the interface between adjacent epithelial cells through clustering of cell surface adhesion transmembrane molecule cadherin and its association to the cytoskeleton (10, 11). On the extracellular side, structural studies suggest that cadherin molecules form molecular pairs that interact with cadherin pairs on an adjacent cell through their distal Ca²⁺-binding domains (12). On the intracellular side, cadherin pairs are connected to the cytoskeleton network through specific linker proteins. Until recently it was believed that one critical linker protein between the cytoplasmic domain of cadherin and the actin cytoskeleton was α-catenin, because it can both bind filamentous actin (F-actin) and E-cadherin through β-catenin (13, 14). However, a recent study indicates that α-catenin can either bind the E-cadherin-β-catenin complex as monomer or cross-link actin filaments as homodimer but cannot bind both E-cadherin-β-catenin and F-actin simultaneously (15). Therefore, whether the loss of α-catenin plays a direct role in the loss of adhesion in human cancer cells is unclear.Our recent data using engineered Chinese hamster ovarian cells suggest that α-catenin mediates the rapid strengthening of individual intercellular E-cadherin/E-cadherin bonds following initial molecular recognition between cells bearing E-cadherin molecules (16). Furthermore, α-catenin mediates the formation of additional E-cadherin/E-cadherin bonds once a first bond is formed between adjoining cells to form a nascent intercellular junction (16). Here we hypothesize that the loss of cytoplasmic protein α-catenin in human cancer cells greatly affects the ability of E-cadherin molecules on the surface of these cells to form firm adhesion by reducing the strength of individual intercellular E-cadherin/E-cadherin bonds.Our strategy is to compare parental breast cancer cells that lack α-catenin (MDA-MB-468 cells; denoted here MDA468) with these cells when α-catenin is introduced and exploit high resolution live cell single-molecule force spectroscopy (17) to probe the strength of individual E-cadherin/E-cadherin bonds between adjacent cells (18). The cells are juxtaposed for a controlled time of contact, the probability of successful interactions is subsequently measured, and the mechanical properties (tensile strength, molecular stiffness, and reactive compliance) and biochemical properties (interaction energy, dissociation rate, and bond lifetime) of single intercellular E-cadherin/E-cadherin bonds are analyzed.Our main hypothesis cannot be readily tested using purified proteins. Our ability to measure molecular interactions between live cells (17) rather than recombinant proteins ensures that the proper orientation of cadherin on the cell surfaces and its post-translational modifications are physiological. Moreover, using living cells ensures that the cytoplasmic domain of transmembrane receptors (here human E-cadherin) can interact with cytoplasmic proteins (in particular β-catenin and α-catenin), thereby allowing cell signaling pathways that can influence cell adhesion to function normally.     

In Vitro Degradation of Endothelial Catenins by a Neutrophil Protease

It has been recently proposed that adhesion of polymorphonuclear cells (PMNs) to human umbilical vein endothelial cells leads to the disorganization of the vascular endothelial cadherin–dependent endothelial adherens junctions. Combined immunofluorescence and biochemical data suggested that after adhesion of PMNs to the endothelial cell surface, β-catenin, as well as plakoglobin was lost from the cadherin/catenin complex and from total cell lysates. In this study we present data that strongly suggest that the adhesion-dependent disappearance of endothelial catenins is not mediated by a leukocyte to endothelium signaling event, but is due to the activity of a neutrophil protease that is released upon detergent lysis of the cells.     

Absolute β-catenin concentrations in Wnt pathway-stimulated and non-stimulated cells

The intracellular level of the proto-oncoprotein β-catenin is a parameter for the activity of the Wnt pathway, which has been linked to carcinogenesis. The paper introduces a novel sandwich-based ELISA for the determination of the β-catenin concentration in lysates from cells or tissues. The advantages of the method were proven by determining β-catenin levels in cell lines and in cells after activation of the Wnt pathway. Analysis revealed high β-catenin concentrations in the cell lines HeLa, KB, HT1080, MCF-7, U-87 and U-373, which had not been described before. β-Catenin concentrations were compared in HEK293 and C57MG cells after activation of the Wnt pathway. The β-catenin concentrations increased by different factors depending on whether the Wnt pathway was activated by incubation with LiCl or with Wnt-3a-conditioned medium. This finding indicated that the β-catenin level depends on the way and level of Wnt pathway activation. The quantitative analysis of β-catenin in colorectal tumours revealed high β-catenin levels in tumours with truncating mutations in the APC gene.     

The Intracellular Domain of Cadherin-11 is not Required for the Induction of Cell Aggregation, Adhesion or Gap-Junction Formation

The cadherin family of cell adhesion molecules demonstrates calcium-dependent hemophilic binding, leading to cellular recognition and adhesion. The adhesion mediated by the classical type 1 cadherins is strengthened through catenin-mediated coupling of the cytoplasmic domain to the cytoskeleton. This cytoskeletal interaction may not be essential for the adhesion promoted by all cadherins, several of which lack cytosolic catenin-binding sequences. Cadherin-11, a classical cadherin, possesses a cytoplasmic domain that interacts with catenins, but may also occur as a variant form expressing a truncated cytoplasmic domain. To study the role of the cytoplasmic sequence in cadherin-11 mediated adhesion we have constructed and expressed a truncated cadherin-11 protein lacking the cytoplasmic domain and unable to bind β-catenin. Expression of the truncated cadherin-11 in MDA-MB-435S human mammary carcinoma cells reduced their motility and promoted calcium-dependent cell aggregation, frequent cell contacts, and functional gap-junctions. We conclude that the intracellular catenin-binding domain of cadherin-11, and by inference cytoskeletal interaction, is not required for the initiation and formation of cell adhesion.     

A functional analysis of Wnt inducible signalling pathway protein ?1 (WISP-1/CCN4)

Wnt-1 inducible signalling pathway protein 1 (WISP-1/CCN4) is an extracellular matrix protein that belongs to the Cyr61 (cysteine-rich protein 61), CTGF (connective tissue growth factor) and NOV (CCN) family and plays a role in multiple cellular processes. No specific WISP-1 receptors have been identified but emerging evidence suggests WISP-1 mediates its downstream effects by binding to integrins. Here we describe a functional analysis of integrin receptor usage by WISP-1. Truncated WISP-1 proteins were produced using a baculovirus expression system. Full length WISP-1 and truncated proteins were evaluated for their ability to induce adhesion in A549 epithelial cells and β-catenin activation and CXCL3 secretion in fibroblasts (NRK49-F cells). Subsequent inhibition of these responses by neutralising integrin antibodies was evaluated. A549 cells demonstrated adhesion to full-length WISP-1 whilst truncated proteins containing VWC, TSP or CT domains also induced adhesion, with highest activity observed with proteins containing the C-terminal TSP and CT domains. Likewise the ability to induce β-catenin activation and CXCL3 secretion was retained in truncations containing C-terminal domains. Pre-treatment of A549s with either integrin αVβ5, αVβ3 or β1 neutralising antibodies partially inhibited full length WISP-1 induced adhesion whilst combining integrin αVβ5 and β1 antibodies increased the potency of this effect. Incubation of NRK49-F cells with integrin neutralising antibodies failed to effect β-catenin translocation or CXCL3 secretion. Analysis of natural WISP-1 derived from human lung tissue showed the native protein is a high order oligomer. Our data suggest that WISP-1 mediated adhesion of A549 cells is an integrin-driven event regulated by the C-terminal domains of the protein. Activation of β-catenin signalling and CXCL3 secretion also resides within the C-terminal domains of WISP-1 but are not regulated by integrins. The oligomeric nature of native WISP-1 may drive a high avidity interaction with these receptors in vivo.     

Independent interactions of phosphorylated β-catenin with E-cadherin at cell-cell contacts and APC at cell protrusions

Background

The APC tumour suppressor functions in several cellular processes including the regulation of β-catenin in Wnt signalling and in cell adhesion and migration.

Findings

In this study, we establish that in epithelial cells N-terminally phosphorylated β-catenin specifically localises to several subcellular sites including cell-cell contacts and the ends of cell protrusions. N-terminally phosphorylated β-catenin associates with E-cadherin at adherens junctions and with APC in cell protrusions. We isolated APC-rich protrusions from stimulated cells and detected β-catenin, GSK3β and CK1α, but not axin. The APC/phospho-β-catenin complex in cell protrusions appears to be distinct from the APC/axin/β-catenin destruction complex. GSK3β phosphorylates the APC-associated population of β-catenin, but not the cell junction population. β-catenin associated with APC is rapidly phosphorylated and dephosphorylated. HGF and wound-induced cell migration promote the localised accumulation of APC and phosphorylated β-catenin at the leading edge of migrating cells. APC siRNA and analysis of colon cancer cell lines show that functional APC is required for localised phospho-β-catenin accumulation in cell protrusions.

Conclusions

We conclude that N-terminal phosphorylation of β-catenin does not necessarily lead to its degradation but instead marks distinct functions, such as cell migration and/or adhesion processes. Localised regulation of APC-phospho-β-catenin complexes may contribute to the tumour suppressor activity of APC.     

Involvement of ZO-1 in Cadherin-based Cell Adhesion through Its Direct Binding to α Catenin and Actin Filaments

ZO-1, a 220-kD peripheral membrane protein consisting of an amino-terminal half discs large (dlg)-like domain and a carboxyl-terminal half domain, is concentrated at the cadherin-based cell adhesion sites in non-epithelial cells. We introduced cDNAs encoding the full-length ZO-1, its amino-terminal half (N-ZO-1), and carboxyl-terminal half (C-ZO-1) into mouse L fibroblasts expressing exogenous E-cadherin (EL cells). The full-length ZO-1 as well as N-ZO-1 were concentrated at cadherin-based cell–cell adhesion sites. In good agreement with these observations, N-ZO-1 was specifically coimmunoprecipitated from EL transfectants expressing N-ZO-1 (NZ-EL cells) with the E-cadherin/α, β catenin complex. In contrast, C-ZO-1 was localized along actin stress fibers. To examine the molecular basis of the behavior of these truncated ZO-1 molecules, N-ZO-1 and C-ZO-1 were produced in insect Sf9 cells by recombinant baculovirus infection, and their direct binding ability to the cadherin/catenin complex and the actin-based cytoskeleton, respectively, were examined in vitro. Recombinant N-ZO-1 bound directly to the glutathione-S-transferase fusion protein with α catenin, but not to that with β catenin or the cytoplasmic domain of E-cadherin. The dissociation constant between N-ZO-1 and α catenin was ~0.5 nM. On the other hand, recombinant C-ZO-1 was specifically cosedimented with actin filaments in vitro with a dissociation constant of ~10 nM. Finally, we compared the cadherin-based cell adhesion activity of NZ-EL cells with that of parent EL cells. Cell aggregation assay revealed no significant differences among these cells, but the cadherin-dependent intercellular motility, i.e., the cell movement in a confluent monolayer, was significantly suppressed in NZ-EL cells. We conclude that in nonepithelial cells, ZO-1 works as a cross-linker between cadherin/catenin complex and the actin-based cytoskeleton through direct interaction with α catenin and actin filaments at its amino- and carboxyl-terminal halves, respectively, and that ZO-1 is a functional component in the cadherin-based cell adhesion system.     

α—catenin在肿瘤中的多种角色

α-连环蛋白（α-catenin）可将钙粘素／B连环蛋白（cadherin／β-catenin）复合体连接于肌动蛋白丝（actinfilament,F-actin）,为细胞表面黏附分子与细胞骨架提供了稳固的机械连接。近年,人们对α—catenin的研究发现,其作用不仅仅是稳固cadherin／catenin的结构、参与细胞黏附,还在肿瘤细胞侵袭、迁移中发挥着重要作用。此外,α-catenin还可能参与cadherin／catenin与Ras／MAPK,NF—KB和Wnt等通路之间的串话,涉及了细胞的膜动态、增殖和凋亡的调节。     

Smad3 Prevents ��-Catenin Degradation and Facilitates ��-Catenin Nuclear Translocation in Chondrocytes

Our previous study demonstrated that transforming growth factor (TGF)-β activates β-catenin signaling through Smad3 interaction with β-catenin in chondrocytes. In the present studies, we further investigated the detailed molecular mechanism of the cross-talk between TGF-β/Smad3 and Wnt/β-catenin signaling pathways. We found that C-terminal Smad3 interacted with both the N-terminal region and the middle region of β-catenin protein in a TGF-β-dependent manner. Both Smad3 and Smad4 were required for the interaction with β-catenin and protected β-catenin from an ubiquitin-proteasome-dependent degradation. In addition, the formation of the Smad3-Smad4-β-catenin protein complex also mediated β-catenin nuclear translocation. This Smad3-mediated regulatory mechanism of β-catenin protein stability enhanced the activity of β-catenin to activate downstream target genes during chondrogenesis. Our findings demonstrate a novel mechanism between TGF-β and Wnt/β-catenin signaling pathways during chondrocyte development.     

Retained Cell�CCell Adhesion in Serrated Neoplastic Pathway as Opposed to Conventional Colorectal Adenomas

The molecular features of serrated polyps of colorectum remain to be elucidated. The expression pattern of adhesive molecules (E-cadherin, α-catenin, and β-catenin) has not been examined in serrated neoplastic pathway. The expression of E-cadherin, α-catenin, and β-catenin were analyzed by immunohistochemistry in 32 hyperplastic polyps (HPs), 28 sessile serrated adenomas (SSAs), 37 traditional serrated adenomas (TSAs), 51 traditional adenomas (TAs), and 10 normal colonic tissues (NCs). Retained membranous expression for E-cadherin, α-catenin, and β-catenin was more frequent in HPs, SSAs, and TSAs than that in TAs (p < 0.001). Nuclear labeling of β-catenin was detected in 19.6% of TAs, but in none of HPs, SSAs, and TSAs (p < 0.001). Cytoplasmic accumulation of β-catenin was found in 3.1% of HPs, 3.6% of SSAs, and 21.6% of TSAs, significantly lower than that in TAs (60.8%, p < 0.001). The membranous co-expression of E-cadherin, α-catenin, and β-catenin was more frequent in HPs (68.8%), SSAs (60.7%), and TSAs (37.8%) than that in TAs (7.8%, p < 0.001). Cell adhesion function is retained in serrated neoplastic pathway. Wnt signaling pathway plays a less active role in the development of colorectal serrated polys than in TAs.