DIPA-family coiled-coils bind conserved isoform-specific head domain of p120-catenin family: potential roles in hydrocephalus and heterotopia

p120-catenin (p120) modulates adherens junction (AJ) dynamics by controlling the stability of classical cadherins. Among all p120 isoforms, p120-3A and p120-1A are the most prevalent. Both stabilize cadherins, but p120-3A is preferred in epithelia, whereas p120-1A takes precedence in neurons, fibroblasts, and macrophages. During epithelial-to-mesenchymal transition, E- to N-cadherin switching coincides with p120-3A to -1A alternative splicing. These isoforms differ by a 101–amino acid “head domain” comprising the p120-1A N-terminus. Although its exact role is unknown, the head domain likely mediates developmental and cancer-associated events linked to p120-1A expression (e.g., motility, invasion, metastasis). Here we identified delta-interacting protein A (DIPA) as the first head domain–specific binding partner and candidate mediator of isoform 1A activity. DIPA colocalizes with AJs in a p120-1A- but not 3A-dependent manner. Moreover, all DIPA family members (Ccdc85a, Ccdc85b/DIPA, and Ccdc85c) interact reciprocally with p120 family members (p120, δ-catenin, p0071, and ARVCF), suggesting significant functional overlap. During zebrafish neural tube development, both knockdown and overexpression of DIPA phenocopy N-cadherin mutations, an effect bearing functional ties to a reported mouse hydrocephalus phenotype associated with Ccdc85c. These studies identify a novel, highly conserved interaction between two protein families that may participate either individually or collectively in N-cadherin–mediated development.     

Interactions of Plakoglobin and ��-Catenin with Desmosomal Cadherins: BASIS OF SELECTIVE EXCLUSION OF ��- AND ��-CATENIN FROM DESMOSOMES*

Plakoglobin and β-catenin are homologous armadillo repeat proteins found in adherens junctions, where they interact with the cytoplasmic domain of classical cadherins and with α-catenin. Plakoglobin, but normally not β-catenin, is also a structural constituent of desmosomes, where it binds to the cytoplasmic domains of the desmosomal cadherins, desmogleins and desmocollins. Here, we report structural, biophysical, and biochemical studies aimed at understanding the molecular basis of selective exclusion of β-catenin and α-catenin from desmosomes. The crystal structure of the plakoglobin armadillo domain bound to phosphorylated E-cadherin shows virtually identical interactions to those observed between β-catenin and E-cadherin. Trypsin sensitivity experiments indicate that the plakoglobin arm domain by itself is more flexible than that of β-catenin. Binding of plakoglobin and β-catenin to the intracellular regions of E-cadherin, desmoglein1, and desmocollin1 was measured by isothermal titration calorimetry. Plakoglobin and β-catenin bind strongly and with similar thermodynamic parameters to E-cadherin. In contrast, β-catenin binds to desmoglein-1 more weakly than does plakoglobin. β-Catenin and plakoglobin bind with similar weak affinities to desmocollin-1. Full affinity binding of desmoglein-1 requires sequences C-terminal to the region homologous to the catenin-binding domain of classical cadherins. Although pulldown assays suggest that the presence of N- and C-terminal β-catenin “tails” that flank the armadillo repeat region reduces the affinity for desmosomal cadherins, calorimetric measurements show no significant effects of the tails on binding to the cadherins. Using purified proteins, we show that desmosomal cadherins and α-catenin compete directly for binding to plakoglobin, consistent with the absence of α-catenin in desmosomes.     

The p120 family of cell adhesion molecules

p120 is the prototypic member of the p120 subfamily of armadillo-related proteins that includes p0071, delta-catenin/NPRAP, ARVCF and the more distantly related plakophilins 1-3. Like armadillo, beta-catenin and plakoglobin these proteins are involved in mediating cell-cell adhesion. Besides their junctional localization they also reveal a cytoplasmic and nuclear localization. Non-cadherin-associated, cytoplasmic p120 functions in Rho signaling and regulation of cytoskeletal organization and actin dynamics. The nuclear function remains largely unsolved. Some characteristics seem to be shared by the various members of the family but it seems unlikely that p120-related proteins have solely redundant functions and compete for interactions with identical binding partners. Stabilization of cadherins at the membrane seems a common function of p120, p0071, delta-catenin and ARVCF but it is not yet known if and how these proteins confer distinct properties to cellular junctions. Moreover, p0071, NPRAP and ARVCF have a C-terminal PDZ-binding motif that is lacking in p120 pointing to distinct roles of these proteins. PDZ domains are found in a series of proteins involved in establishing cell polarity in epithelial cells. Thus, p120 proteins may not only be master regulators of cadherin abundance and activity but play additional roles in regulating cell polarity. This review focuses on the putative roles of p120 proteins in cell polarity.     

Structure and Biochemistry of Cadherins and Catenins

Classical cadherins mediate specific adhesion at intercellular adherens junctions. Interactions between cadherin ectodomains from apposed cells mediate cell–cell contact, whereas the intracellular region functionally links cadherins to the underlying cytoskeleton. Structural, biophysical, and biochemical studies have provided important insights into the mechanism and specificity of cell–cell adhesion by classical cadherins and their interplay with the cytoskeleton. Adhesive binding arises through exchange of β strands between the first extracellular cadherin domains (EC1) of partner cadherins from adjacent cells. This “strand-swap” binding mode is common to classical and desmosomal cadherins, but sequence alignments suggest that other cadherins will bind differently. The intracellular region of classical cadherins binds to p120 and β-catenin, and β-catenin binds to the F-actin binding protein α-catenin. Rather than stably bridging β-catenin to actin, it appears that α-catenin actively regulates the actin cytoskeleton at cadherin-based cell–cell contacts.Cadherins constitute a large family of cell surface proteins, many of which participate in Ca²⁺-dependent cell adhesion that plays a fundamental role in the formation of solid tissues (Takeichi 1995; Tepass 1999; Gumbiner 2005). Many events in the development of multicellular assemblies are associated with changes in cadherin expression (Takeichi 1995; Honjo et al. 2000; Price et al. 2002). Expression of particular cadherins often correlates with formation of discrete tissue structures, and in mature tissues discrete cell layers or other cell assemblies are often demarcated by particular cadherins (Gumbiner 1996). Conversely, down-regulation or loss of cadherins correlates with an increased metastatic potential of the affected cells that arises from the loss of their adhesive properties (Hajra and Fearon 2002; Gumbiner 2005).The cadherins of vertebrates, and some of their invertebrate homologs, are the most highly characterized. “Classical” cadherins, associated with the adherens junction, and the closely related desmosomal cadherins feature an amino-terminal extracellular region or ectodomain that is followed by a transmembrane anchor and a carboxy-terminal intracellular region. Interactions between ectodomains on apposed cells mediate specific cell–cell contacts, whereas the intracellular region functionally links cadherins to the underlying cytoskeleton. This article focuses on structural, biophysical, and biochemical studies that have provided important mechanistic insights into the specificity of cell–cell adhesion and its interplay with the cytoskeleton (see also Meng and Takeichi 2009; Cavey and Lecuit 2009).     

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.     

Kaiso/p120-catenin and TCF/beta-catenin complexes coordinately regulate canonical Wnt gene targets

Beta-catenin-dependent or canonical Wnt signals are fundamental in animal development and tumor progression. Using Xenopus laevis, we report that the BTB/POZ zinc finger family member Kaiso directly represses canonical Wnt gene targets (Siamois, c-Fos, Cyclin-D1, and c-Myc) in conjunction with TCF/LEF (TCF). Analogous to beta-catenin relief of TCF repressive activity, we show that p120-catenin relieves Kaiso-mediated repression of Siamois. Furthermore, Kaiso and TCF coassociate, and combined Kaiso and TCF derepression results in pronounced Siamois expression and increased beta-catenin coprecipitation with the Siamois promoter. The functional interdependency is underlined by Kaiso suppression of beta-catenin-induced axis duplication and by TCF-3 rescue of Kaiso depletion phenotypes. These studies point to convergence of parallel p120-catenin/Kaiso and beta-catenin/TCF signaling pathways to regulate gene expression in vertebrate development and possibly carcinogenesis.     

GSK-3 Phosphorylates ��-Catenin and Negatively Regulates Its Stability via Ubiquitination/Proteosome-mediated Proteolysis

δ-Catenin was first identified because of its interaction with presenilin-1, and its aberrant expression has been reported in various human tumors and in patients with Cri-du-Chat syndrome, a form of mental retardation. However, the mechanism whereby δ-catenin is regulated in cells has not been fully elucidated. We investigated the possibility that glycogen-synthase kinase-3 (GSK-3) phosphorylates δ-catenin and thus affects its stability. Initially, we found that the level of δ-catenin was greater and the half-life of δ-catenin was longer in GSK-3β^−/− fibroblasts than those in GSK-3β^+/+ fibroblasts. Furthermore, four different approaches designed to specifically inhibit GSK-3 activity, i.e. GSK-3-specific chemical inhibitors, Wnt-3a conditioned media, small interfering RNAs, and GSK-3α and -3β kinase dead constructs, consistently showed that the levels of endogenous δ-catenin in CWR22Rv-1 prostate carcinoma cells and primary cortical neurons were increased by inhibiting GSK-3 activity. In addition, it was found that both GSK-3α and -3β interact with and phosphorylate δ-catenin. The phosphorylation of ΔC207-δ-catenin (lacking 207 C-terminal residues) and T1078A δ-catenin by GSK-3 was noticeably reduced compared with that of wild type δ-catenin, and the data from liquid chromatography-tandem mass spectrometry analyses suggest that the Thr¹⁰⁷⁸ residue of δ-catenin is one of the GSK-3 phosphorylation sites. Treatment with MG132 or ALLN, specific inhibitors of proteosome-dependent proteolysis, increased δ-catenin levels and caused an accumulation of ubiquitinated δ-catenin. It was also found that GSK-3 triggers the ubiquitination of δ-catenin. These results suggest that GSK-3 interacts with and phosphorylates δ-catenin and thereby negatively affects its stability by enabling its ubiquitination/proteosome-mediated proteolysis.δ-Catenin was first identified as a molecule that interacts with presenilin-1 (PS-1)² by yeast two-hybrid assay (1) and was found to belong to the p120-catenin subfamily of armadillo proteins, which characteristically contain 10 Arm repeats (2). In addition to its interaction with PS-1 and its abundant expression in brain (3, 4), several lines of evidence indicate that δ-catenin may play a pivotal role in cognitive function. First, the hemizygous loss of δ-catenin is known to be closely correlated with Cri-du-Chat syndrome, a severe form of mental retardation in humans (5). Second, severe learning deficits and abnormal synaptic plasticity were found in δ-catenin-deficient mice (6). Moreover, in δ-catenin^−/− mice, paired pulse facilitation (a form of short term plasticity) was found to be reduced, and long term potentiation, which is related to the forming and storage mechanisms of memory, was deficient (7, 8). Third, δ-catenin interacting molecules, such as PSs (1, 9), cadherins (10), S-SCAM (2), and PSD-95 (11), have been shown to play important roles in modulating synaptic plasticity. However, even though the maintenance of an adequate δ-catenin level is known to be critical for normal brain function, few studies have been undertaken to identify the factors that regulate δ-catenin stability in cells. We have previously demonstrated that PS-1 inhibits δ-catenin-induced cellular branching and promotes δ-catenin processing and turnover (12).Because of structural similarities among β-catenin, p120-catenin, and δ-catenin and to their shared binding partners (i.e. PS-1 (1, 9) and cadherins (10)), glycogen-synthase kinase-3 (GSK-3) drew our attention as a potential candidate effector of δ-catenin stability in cells. GSK-3 is a serine/threonine kinase and has two highly homologous forms, GSK-3α and GSK-3β, in mammals (13). Although GSK-3α and GSK-3β have similar structures, they differ in mass (GSK-3α (51 kDa) and GSK-3β (47 kDa) (13)) and to some extent in function (14). GSK-3 is a well established inhibitor of Wnt signaling. Moreover, it is known to phosphorylate β-catenin, which results in its degradation via ubiquitination/proteosome-dependent proteolysis (15). GSK-3 is ubiquitously distributed in the human body, but it is particularly abundant in brain (13), and it is interesting that δ-catenin is also abundant in the nervous system (4) and that GSK-3 participates in the progression of Alzheimer disease (16). The majority of GSK-3 substrates have the consensus sequence (Ser/Thr)-Xaa-Xaa-Xaa-(Ser/Thr) (17). Interestingly, we found that δ-catenin has several putative phosphorylation sites targeted by GSK-3, which suggests that δ-catenin can be regulated by GSK-3 in the same way as β-catenin.In this report, we demonstrate that both GSK-3α and -3β interact with and phosphorylate δ-catenin and that this leads to its subsequent ubiquitination and degradation via proteosome-dependent proteolysis. Our results strongly suggest that GSK-3 is a key regulator of δ-catenin stability in cells.     

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.     

A core function for p120-catenin in cadherin turnover

p120-catenin stabilizes epithelial cadherin (E-cadherin) in SW48 cells, but the mechanism has not been established. Here, we show that p120 acts at the cell surface to control cadherin turnover, thereby regulating cadherin levels. p120 knockdown by siRNA expression resulted in dose-dependent elimination of epithelial, placental, neuronal, and vascular endothelial cadherins, and complete loss of cell-cell adhesion. ARVCF and delta-catenin were functionally redundant, suggesting that proper cadherin-dependent adhesion requires the presence of at least one p120 family member. The data reveal a core function of p120 in cadherin complexes, and strongly predict a dose-dependent loss of E-cadherin in tumors that partially or completely down-regulate p120.     

Functional Wnt signaling is increased in idiopathic pulmonary fibrosis

Background

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease, characterized by distorted lung architecture and loss of respiratory function. Alveolar epithelial cell injury and hyperplasia, enhanced extracellular matrix deposition, and (myo)fibroblast activation are features of IPF. Wnt/β-catenin signaling has been shown to determine epithelial cell fate during development. As aberrant reactivation of developmental signaling pathways has been suggested to contribute to IPF pathogenesis, we hypothesized that Wnt/β-catenin signaling is activated in epithelial cells in IPF. Thus, we quantified and localized the expression and activity of the Wnt/β-catenin pathway in IPF.

Methodology/Principal Findings

The expression of Wnt1, 3a, 7b, and 10b, the Wnt receptors Fzd1-4, Lrp5-6, as well as the intracellular signal transducers Gsk-3β, β-catenin, Tcf1, 3, 4, and Lef1 was analyzed in IPF and transplant donor lungs by quantitative real-time (q)RT-PCR. Wnt1, 7b and 10b, Fzd2 and 3, β-catenin, and Lef1 expression was significantly increased in IPF. Immunohistochemical analysis localized Wnt1, Wnt3a, β-catenin, and Gsk-3β expression largely to alveolar and bronchial epithelium. This was confirmed by qRT-PCR of primary alveolar epithelial type II (ATII) cells, demonstrating a significant increase of Wnt signaling in ATII cells derived from IPF patients. In addition, Western blot analysis of phospho-Gsk-3β, phospho-Lrp6, and β-catenin, and qRT-PCR of the Wnt target genes cyclin D1, Mmp 7, or Fibronectin 1 demonstrated increased functional Wnt/β-catenin signaling in IPF compared with controls. Functional in vitro studies further revealed that Wnt ligands induced lung epithelial cell proliferation and (myo)fibroblast activation and collagen synthesis.

Conclusions/Significance

Our study demonstrates that the Wnt/β-catenin pathway is expressed and operative in adult lung epithelium. Increased Wnt/β-catenin signaling may be involved in epithelial cell injury and hyperplasia, as well as impaired epithelial-mesenchymal cross-talk in IPF. Thus, modification of Wnt signaling may represent a therapeutic option in IPF.     

Linkage of N-cadherin to multiple cytoskeletal elements revealed by a proteomic approach in hippocampal neurons

The CNS synapse is an adhesive junction differentiated for chemical neurotransmission and is equipped with presynaptic vesicles and postsynaptic neurotransmitter receptors. Cell adhesion molecule cadherins not only maintain connections between pre- and postsynaptic membranes but also modulate the efficacy of synaptic transmission. Although the components of the cadherin-mediated adhesive apparatus have been studied extensively in various cell systems, the complete picture of these components, particularly at the synaptic junction, remains elusive. Here, we describe the proteomic assortment of the N-cadherin-mediated synaptic adhesion apparatus in cultured hippocampal neurons. N-cadherin immunoprecipitated from Triton X-100-solubilized neuronal extract contained equal amounts of β- and α-catenins, as well as F-actin-related membrane anchor proteins such as integrins bridged with α-actinin-4, and Na(+)/K(+)-ATPase bridged with spectrins. A close relative of β-catenin, plakoglobin, and its binding partner, desmoplakin, were also found, suggesting that a subset of the N-cadherin-mediated adhesive apparatus also anchors intermediate filaments. Moreover, dynein heavy chain and LEK1/CENPF/mitosin were found. This suggests that internalized pools of N-cadherin in trafficking vesicles are conveyed by dynein motors on microtubules. In addition, ARVCF and NPRAP/neurojungin/δ2-catenin, but not p120ctn/δ1-catenin or plakophilins-1, -2, -3, -4 (p0071), were found, suggesting other possible bridges to microtubules. Finally, synaptic stimulation by membrane depolarization resulted in an increased 93-kDa band, which corresponded to proteolytically truncated β-catenin. The integration of three different classes of cytoskeletal systems found in the synaptic N-cadherin complex may imply a dynamic switching of adhesive scaffolds in response to synaptic activity.     

Guilt by association: What p120-catenin has to hide

Members of the p120-catenin family associate with cadherins and regulate their stability at the plasma membrane. How p120-catenin limits cadherin endocytosis has long remained a mystery. In this issue, Nanes et al. (2012. J. Cell Biol. doi:10.1083/jcb.201205029) identify a conserved acidic motif within cadherins that acts as a physical platform for p120-catenin binding. However, in the absence of p120-catenin, the motif acts as an endocytic signal. These results provide new insight into p120-catenin’s role as guardian of intercellular junction dynamics.Adhesion receptors of the classical cadherin family have a major role in establishing tissue organization and maintaining tissue homeostasis (Gumbiner, 1996). Classical cadherins are transmembrane glycoproteins that use their extracellular domains to establish calcium-dependent trans homophilic interactions with cadherins in neighboring cells. To enhance adhesive strength, cadherin ectodomains oligomerize through lateral (cis) interactions, whereas their cytoplasmic domains anchor to the actomyosin cytoskeleton. The cytoplasmic domain of cadherins is highly conserved and binds to proteins called catenins. p120-catenin (p120) associates with the transmembrane adjacent domain (juxtamembrane; JMD) of the cadherin cytoplasmic tail, whereas β-catenin interacts with the more distal portion of cadherin’s cytoplasmic domain. β-Catenin in turn, binds α-catenin, which, through multiple interactions, both indirect and direct, can associate with the actin cytoskeleton (Perez-Moreno and Fuchs, 2006).Cellular rearrangements are orchestrated by dynamic assembly/disassembly of cadherin complexes. The process is fueled by endocytosis of cadherin complexes (Le et al., 1999; de Beco et al., 2009). Endocytosis can be stimulated by proteins that associate with cadherin–catenin complexes, including proteases that shed the cadherin ectodomains, and the ubiquitin ligase Hakai (Fujita et al., 2002). Cadherin internalization can be regulated by different pathways depending on the cellular context, involving clathrin-dependent and clathrin-independent mechanisms. These endocytic processes must be carefully regulated, as an untimely destabilization of cadherin-mediated adhesion can lead to alterations in tissue architecture and growth, features of several diseases, including cancers (Mosesson et al., 2008).In the past decade, p120 catenins (p120, ARVCF, δ-catenin, and p0071) have emerged as critical regulators of cadherin-mediated adhesion (Reynolds, 2007). p120, the founding family member, is a component of cadherin complexes (Reynolds et al., 1994), and its association with the cadherin JMD is important for retaining cadherins at the membrane (Ireton et al., 2002). Moreover, p120 loss causes rapid internalization of cadherins, followed by proteasomal and/or lysosomal-mediated degradation (Davis et al., 2003; Xiao et al., 2003a,b, 2005; Miyashita and Ozawa, 2007).Although these studies expose p120 as a master regulator of cadherin levels at the membrane, exactly how p120 governs cadherin endocytosis rates has remained unclear. Based upon experiments in which endocytic machinery components (clathrin, dynamin, and AP2) have been impaired (Chiasson et al., 2009) or cadherin endocytic motifs have been mutated (Hong et al., 2010; Troyanovsky et al., 2007), researchers have posited that p120 binding to cadherins may in some way prevent junctional complex endocytosis. In this issue, Nanes et al. add new molecular insights into the mechanism. The authors show that the VE-cadherin JMD functions as a bimodal platform for either p120 binding or endocytic signaling. Moreover, they identify a key conserved amino acid residue within the JMD, which, when mutated, blocks endocytosis without the need for p120.Recently, the cocrystallization of p120 bound to E-cadherin’s JMD has yielded insights into the essential residues of this binding interface (Ishiyama et al., 2010). Previous studies had attributed the core function of p120-cadherin to its ability to bind and mask a dileucine endocytic motif present in the JMD (Miyashita and Ozawa, 2007; Hong et al., 2010). The crystal structure showed that interactions between p120 and the JMD domain might be sufficient to sterically prevent accessibility of the dileucine cadherin endocytic motif to endocytic adaptors such as the AP2-clathrin adaptor, thereby placing this motif at the crux of the bimodal switch controlling the mutually exclusive binding of either p120 or the endocytic machinery.The affinity of p120 and AP2 for the JMD dileucine motif is similar, pointing toward the existence of a balanced regulation of cadherin endocytic rates and cadherin retention at the membrane. However, evaluating this balance in cellular contexts has not been possible because of the inability to uncouple p120 binding to the JMD and endocytosis. Nanes et al. (2012) have now overcome this hurdle. They first used a simulated model of the p120–E-cadherin crystal structure, which highlighted a conserved p120-binding region that is present in the JMD of both VE- and E-cadherin. However, the VE-cadherin JMD lacked endocytic dileucine and tyrosine residues present in E-cadherin, which are involved in clathrin internalization and Hakai-dependent ubiquitination, respectively.Because both types of adherens junctions undergo dynamic endocytic-based remodeling, the authors astutely realized that they might be able to exploit VE- and E-cadherin differences to unearth novel endocytic signals within the sequence that might be conserved among cadherins. To this end, the author first used mutant VE-cadherin chimeric proteins, consisting of the cytoplasmic domain of VE-cadherin fused to the extracellular domain of the IL-2 receptor, and internalization assays. They discovered that the core p120-binding region on its own was endocytosed, in a fashion similar to the full VE-cadherin cytoplasmic tail. This occurred in a clathrin-dependent manner, as previously observed in Kowalzcyk’s laboratory (Chiasson et al., 2009). Point mutagenesis identified some mutants no longer able to bind p120, which is consistent with previous findings (Thoreson et al., 2000). But the authors made an interesting finding: mutations in a conserved acidic motif (DEE) within the p120-core binding region of the JMD displayed loss of p120 binding and also blocked cadherin internalization (Fig. 1). Moreover, DEE mutant VE-cadherins localized stably at the membrane even in the absence of p120, although with an increased diffusion within the membrane. This increase in mobility suggests a reduction in cadherin lateral clustering, a process modulated by the binding of p120 to the JMD (Yap et al., 1998). Interestingly, in crystal structures, the E-cadherin JMD binding to p120 induced oligomerization of the complex (Ishiyama et al., 2010).Open in a separate windowFigure 1.Model of VE-cadherin stabilization at the cell membrane. (A) VE-cadherin binds to p120 and β-catenin. p120 associates with the juxtamembrane (JMD) domain of the cadherin cytoplasmic tail, whereas β-catenin binds to the more distal portion (catenin binding domain, CBD). Cadherin internalization is triggered by p120 dissociation, exposing a conserved endocytic factor recognition motif (DEE; 646–648) within the JMD. (B) When this motif is mutated in VE-cadherin, adherens junctions are resistant to endocytosis independent of p120 binding.These new tools now allow uncoupling of p120 binding from cadherin endocytosis, which will be instrumental in unraveling new p120 cadherin roles in cell adhesion. The VE-cadherin mutant that fails to bind to p-120 still coimmunoprecipitates with β-catenin. These findings are intriguing, given that overexpression of p120 can rescue the otherwise poor adhesive properties of cadherins mutant for β-catenin binding (Ohkubo and Ozawa, 1999). In addition, interactions between p120 and α-catenin at adherens junctions seem to contribute in preventing cadherin endocytosis (Troyanovsky et al., 2011). Given these collective results, it will be interesting in the future to measure the binding affinities of endocytosis-uncoupled VE-cadherin mutants for its binding partners.Overall, these data provide strong evidence that the JMD landing pad provides the nuts and bolts of the decision of whether an adherens junction remains at the cell surface or whether it is internalized. But who makes the decision? Recent results from Gumbiner’s group provide a possible clue. They show that cadherin activation stimulates the dephosphorylation of specific Ser/Thr residues within the N-terminal domain of p120, and this in turn stabilizes intercellular adhesion (Petrova et al., 2012).The new tools developed by Kowalczyk’s group (Nanes et al., 2012) will pave the way for researchers to dig further into the mechanism. In the current study, the authors use their newfound tools to analyze the consequences to cell migration when p120-JMD binding is uncoupled from endocytosis. In scratched monolayers of endothelial cells, cell migration was decreased. Importantly, when they examined the VE-cadherin mutant in which p120 binding was blocked but cadherin internalization could proceed normally, cell migration was largely normal. These findings indicate that the migration defects seen in the cells expressing the E-cadherin mutant are rooted in inhibition of endocytosis, rather than lack of p120 recruitment to junctions. They further suggest that endocytic trafficking of cadherins is necessary to transiently destabilize cell–cell contacts that otherwise impede migration. This notion is particularly intriguing given that when E-cadherins are stabilized at intercellular junctions, they can sequester proteins that are required for integrin-based migration (Livshits et al., 2012). Kowalczyk’s findings (Nanes et al., 2012) now suggest a means by which dynamic changes in intercellular adhesion can be achieved to trigger such downstream events.Although less well characterized, there are other regulatory circuits that might also be affected by transiently liberating p120 from intercellular junctions. Thus, for example, p120 enhances cadherin stability through its ability to interact with afadin and Rap1, thereby bridging connections with nectin intercellular junctions (Hoshino et al., 2005). Other direct and indirect p120 associates that might affect cadherin internalization include the endocytic adaptor Numb (Sato et al., 2011) and the signaling enzyme γ-secretase (Kiss et al., 2008). Additionally, p120 can also regulate Rac1 activity, which influences cadherin endocytosis in a clathrin-independent way (Akhtar and Hotchin, 2001). Thus, removing p120 or devising additional mutations to uncouple these interactions may be needed to fully unravel all the mysteries underlying p120’s power in governing intercellular adhesion in tissue development and maintenance (Davis and Reynolds, 2006; Elia et al., 2006; Perez-Moreno et al., 2006; Smalley-Freed et al., 2010; Marciano et al., 2011; Stairs et al., 2011; Chacon-Heszele et al., 2012; Kurley et al., 2012). That said, by dissecting p120’s web at the crossroads between intercellular junction stabilization and endocytosis, Kowalczyk and coworkers (Nanes et al., 2012) now illustrate the power of their approach and provide new insights into how similar strategies might ultimately enable this molecular crossword puzzle to be solved.