Receptor protein-tyrosine phosphatase RPTPmu binds to and dephosphorylates the catenin p120(ctn)

RPTPmu is a prototypic receptor-like protein-tyrosine phosphatase (RPTP) that mediates homotypic cell-cell interactions. Intracellularly, RPTPmu consists of a relatively large juxtamembrane region and two phosphatase domains, but little is still known about its substrate(s). Here we show that RPTPmu associates with the catenin p120(ctn), a tyrosine kinase substrate and an interacting partner of cadherins. No interaction is detectable between RPTPmu and beta-catenin. Furthermore, we show that tyrosine-phosphorylated p120(ctn) is dephosphorylated by RPTPmu both in vitro and in intact cells. Complex formation between RPTPmu and p120(ctn) does not require tyrosine phosphorylation of p120(ctn). Mutational analysis reveals that both the juxtamembrane region and the second phosphatase domain of RPTPmu are involved in p120(ctn) binding. The RPTPmu-interacting domain of p120(ctn) maps to its unique N terminus, a region distinct from the cadherin-interacting domain. A mutant form of p120(ctn) that fails to bind cadherins can still associate with RPTPmu. Our findings indicate that RPTPmu interacts with p120(ctn) independently of cadherins, and they suggest that this interaction may serve to control the tyrosine phosphorylation state of p120(ctn) at sites of cell-cell contact.     

Diverse functions of p120ctn in tumors

p120ctn is a member of the Armadillo protein family. It stabilizes the cadherin-catenin adhesion complex at the plasma membrane, but also has additional roles in the cytoplasm and nucleus. Extensive alternative mRNA splicing and multiple phosphorylation sites generate additional complexity. Evidence is emerging that complete loss, downregulation or mislocalization of p120ctn correlates with progression of different types of human tumors. It remains to be determined whether a causal relationship exists between specific isoform expression, subcellular localization or selective phosphorylation of p120ctn on the one hand and tumor prognosis on the other.     

p120 catenin and phosphorylation: Mechanisms and traits of an unresolved issue

p120 catenin is a scaffold protein that interacts with cadherin cytoplasmic domain and acts as a crucial component of the signalling that regulates the cycle of adherens junction formation and disassembly. Here, we review the nature of stimuli that modulate p120ctn function and are translated as serine/threonine and tyrosine phosphorylation events at this multisite substrate for a variety of protein kinases. We also highlight recent findings that tentatively link phosphorylation of p120ctn to its role as a signal integrator capable to influence the state of the cadherin adhesive bond, the cytoskeleton and cell motility.     

The Human DF3/MUC1 carcinoma-associated antigen signals nuclear localization of the catenin p120(ctn)

The human DF3/MUC1 glycoprotein is aberrantly overexpressed by carcinoma cells. The present studies show that MUC1 associates with the Armadillo protein, p120(ctn). The cytoplasmic domain of MUC1 binds directly to p120. The functional significance of the MUC1-p120 association is supported by the demonstration that MUC1 induces nuclear localization of p120. These findings demonstrate that MUC1 confers cell membrane to nuclear signaling by interactions with p120.     

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.     

Inhibition of RhoA by p120 catenin

RhoA organizes actin stress fibres and is necessary for cell transformation by oncogenes such as src and ras. Moreover, RhoA is implicated in cadherin clustering during the formation of adherens junctions. The catenin p120 has also been implicated in cadherin clustering through an unknown mechanism. Here we show that p120 selectively inhibits RhoA activity in vitro and in vivo. RhoA inhibition and the interaction of p120 with cadherins are mutually exclusive, suggesting a mechanism for regulating the recruitment and exchange of RhoA at nascent cell-cell contacts. By affecting RhoA activation, p120 could modulate cadherin functions, including suppression of invasion, neurite extension and junction formation.     

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.     

The regulatory or phosphorylation domain of p120 catenin controls E-cadherin dynamics at the plasma membrane

In contrast to growth factor-stimulated tyrosine phosphorylation of p120, its relatively constitutive serine/threonine phosphorylation is not well understood. Here we examined the role of serine/threonine phosphorylation of p120 in cadherin function. Expression of cadherins in cadherin-null cells converted them to an epithelial phenotype, induced p120 phosphorylation and localized it to sites of cell contact. Detergent solubility and immunofluorescence confirmed that phosphorylated p120 was at the plasma membrane. E-cadherin constructs incapable of traveling to the plasma membrane did not induce serine/threonine phosphorylation of p120, nor did cadherins constructs incapable of binding p120. However, an E-cadherin cytoplasmic domain construct artificially targeted to the plasma membrane did induce serine/threonine phosphorylation of p120, suggesting phosphorylation occurs independently of signals from cadherin dimerization and trafficking through the ER/Golgi. Solubility assays following calcium switch showed that p120 isoform 3A was more effective at stabilizing E-cadherin at the plasma membrane relative to isoform 4A. Since the major phosphorylation domain of p120 is included in isoform 3A but not 4A, we tested p120 mutated in the known phosphorylation sites in this domain and found that it was even less effective at stabilizing E-cadherin. These data suggest that serine/threonine phosphorylation of p120 influences the dynamics of E-cadherin in junctions.     

Rho1 regulates Drosophila adherens junctions independently of p120ctn

During animal development, adherens junctions (AJs) maintain epithelial cell adhesion and coordinate changes in cell shape by linking the actin cytoskeletons of adjacent cells. Identifying AJ regulators and their mechanisms of action are key to understanding the cellular basis of morphogenesis. Previous studies linked both p120catenin and the small GTPase Rho to AJ regulation and revealed that p120 may negatively regulate Rho. Here we examine the roles of these candidate AJ regulators during Drosophila development. We found that although p120 is not essential for development, it contributes to morphogenesis efficiency, clarifying its role as a redundant AJ regulator. Rho has a dynamic localization pattern throughout ovarian and embryonic development. It preferentially accumulates basally or basolaterally in several tissues, but does not preferentially accumulate in AJs. Further, Rho1 localization is not obviously altered by loss of p120 or by reduction of core AJ proteins. Genetic and cell biological tests suggest that p120 is not a major dose-sensitive regulator of Rho1. However, Rho1 itself appears to be a regulator of AJs. Loss of Rho1 results in ectopic accumulation of cytoplasmic DE-cadherin, but ectopic cadherin does not accumulate with its partner Armadillo. These data suggest Rho1 regulates AJs during morphogenesis, but this regulation is p120 independent.     

p120, a p120-related protein (p100), and the cadherin/catenin complex

Cadherins and catenins play an important role in cell-cell adhesion. Two of the catenins, beta and gamma, are members of a group of proteins that contains a repeating amino acid motif originally described for the Drosophila segment polarity gene armadillo. Another member of this group is a 120-kD protein termed p120, originally identified as a substrate of the tyrosine kinase pp60src. In this paper, we show that endothelial and epithelial cells express p120 and p100, a 100-kD, p120- related protein. Peptide sequencing of p100 establishes it as highly related to p120. p120 and p100 both appear associated with the cadherin/catenin complex, but independent p120/catenin and p100/catenin complexes can be isolated. This association is shown by coimmunoprecipitation of cadherins and catenins with an anti-p120/p100 antibody, and of p120/p100 with cadherin or catenin antibodies. Immunocytochemical analysis with a p120-specific antibody reveals junctional colocalization of p120 and beta-catenin in epithelial cells. Catenins and p120/p100 also colocalize in endothelial and epithelial cells in culture and in tissue sections. The cellular content of p120/p100 and beta-catenin is similar in MDCK cells, but only approximately 20% of the p120/p100 pool associates with the cadherin/catenin complex. Our data provide further evidence for interactions among the different arm proteins and suggest that p120/p100 may participate in regulating the function of cadherins and, thereby, other processes influenced by cell-cell adhesion.     

A novel role for p120 catenin in E-cadherin function

Indirect evidence suggests that p120-catenin (p120) can both positively and negatively affect cadherin adhesiveness. Here we show that the p120 gene is mutated in SW48 cells, and that the cadherin adhesion system is impaired as a direct consequence of p120 insufficiency. Restoring normal levels of p120 caused a striking reversion from poorly differentiated to cobblestone-like epithelial morphology, indicating a crucial role for p120 in reactivation of E-cadherin function. The rescue efficiency was enhanced by increased levels of p120, and reduced by the presence of the phosphorylation domain, a region previously postulated to confer negative regulation. Surprisingly, the rescue was associated with substantially increased levels of E-cadherin. E-cadherin mRNA levels were unaffected by p120 expression, but E-cadherin half-life was more than doubled. Direct p120-E-cadherin interaction was crucial, as p120 deletion analysis revealed a perfect correlation between E-cadherin binding and rescue of epithelial morphology. Interestingly, the epithelial morphology could also be rescued by forced expression of either WT E-cadherin or a p120-uncoupled mutant. Thus, the effects of uncoupling p120 from E-cadherin can be at least partially overcome by artificially maintaining high levels of cadherin expression. These data reveal a cooperative interaction between p120 and E-cadherin and a novel role for p120 that is likely indispensable in normal cells.     

p120-Catenin prevents neutrophil transmigration independently of RhoA inhibition by impairing Src dependent VE-cadherin phosphorylation

Leukocyte transendothelial migration (TEM) is regulated by several signaling pathways including Src family kinases (SFK) and the small RhoGTPases. Previous studies have shown that vascular endothelial-cadherin (VE-cad) forms a complex with β-,γ-, and p120-catenins and this complex disassociates to form a transient gap during leukocyte TEM. Additionally, p120-catenin (p120-1A) overexpression in human umbilical vein endothelial cells (HUVEC) stabilizes VE-cad surface expression, prevents tyrosine phosphorylation of VE-cad, and inhibits leukocyte TEM. Based on reports showing that p120 overexpression in fibroblasts or epithelial cells inhibits RhoA and activates Rac and Cdc42 GTPases, and on other reports showing that RhoA activation in endothelial cells is necessary for leukocyte TEM, we reasoned that p120 overexpression inhibited TEM through inhibition of RhoA. To test this idea, we overexpressed a mutant p120 isoform, p120-4A, which does not interact with RhoA. p120-4A colocalized with VE-cad in HUVEC junctions and enhanced VE-cad surface expression, similar to overexpression of p120-1A. Interestingly, overexpression of either p120-4A or p120-1A dramatically blocked TEM, and overexpression of p120-1A in HUVEC did not affect RhoA basal activity or activation of RhoA and Rac induced by thrombin or ICAM-1 crosslinking. In contrast, biochemical studies revealed that overexpression of p120-1A reduced activated pY416-Src association with VE-cad. In summary, p120 overexpression inhibits neutrophil TEM independently of an effect on RhoA or Rac and instead blocks TEM by preventing VE-cad tyrosine phosphorylation and association of active Src with the VE-cad complex.     

Tissue distribution and cell type-specific expression of p120ctn isoforms.

Cadherin-based molecular complexes play a major role in cell-cell adhesion. At the adherens junctions the intracellular domain of cadherins specifically interacts with beta-catenin and p120ctn, members of the Armadillo repeat protein family. Differential splicing and utilization of the alternative translation initiation codons lead to many p120ctn isoforms. Two major p120ctn isoforms are expressed in mouse tissues. In this study we used indirect immunofluorescence to demonstrate significant tissue specificity in expression of the p120ctn isoforms. The short isoform is abundant at cell-cell adhesion junctions in epidermis, palatal, and tongue epithelia, in the ducts of excretory glands, bronchiolar epithelium, and in mucosal epithelia of esophagus, forestomach, and small intestine. In contrast, the long isoform, containing an amino terminus highly conserved within the p120ctn subfamily, is expressed at vascular-endothelial cell junctions in blood vessels, at cell-cell junctions in the serosal epithelium lining the internal organs, in choroid plexus of brain, in the pigment epithelium of retina, and in structures such as the outer limiting membrane of retina and intercalated discs of cardiomyocytes. The tissue- and cell type-specific expression of p120ctn isoforms suggests a role for the long p120ctn isoform in cell structures responsible for stable tissue integrity, compared to the role of the short isoform in cell-cell adhesion in the external epithelia with rapid turnover.     

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.     

Identification of a new catenin: the tyrosine kinase substrate p120cas associates with E-cadherin complexes.

p120cas is a tyrosine kinase substrate implicated in ligand-induced receptor signaling through the epidermal growth factor, platelet-derived growth factor, and colony-stimulating factor receptors and in cell transformation by Src. Here we report that p120 associates with a complex containing E-cadherin, alpha-catenin, beta-catenin, and plakoglobin. Furthermore, p120 precisely colocalizes with E-cadherin and catenins in vivo in both normal and Src-transformed MDCK cells. Unlike beta-catenin and plakoglobin, p120 has at least four isoforms which are differentially expressed in a variety of cell types, suggesting novel means of modulating cadherin activities in cells. In Src-transformed MDCK cells, p120, beta-catenin, and plakoglobin were heavily phosphorylated on tyrosine, but the physical associations between these proteins were not disrupted. Association of p120 with the cadherin machinery indicates that both Src and receptor tyrosine kinases cross talk with proteins important for cadherin-mediated cell adhesion. These results also strongly suggest a role for p120 in cell adhesion.     

Numb controls E-cadherin endocytosis through p120 catenin with aPKC

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

Conformational epitopes at cadherin calcium-binding sites and p120-catenin phosphorylation regulate cell adhesion

We investigated changes in cadherin structure at the cell surface that regulate its adhesive activity. Colo 205 cells are nonadhesive cells with a full but inactive complement of E-cadherin-catenin complexes at the cell surface, but they can be triggered to adhere and form monolayers. We were able to distinguish the inactive and active states of E-cadherin at the cell surface by using a special set of monoclonal antibodies (mAbs). Another set of mAbs binds E-cadherin and strongly activates adhesion. In other epithelial cell types these activating mAbs inhibit growth factor-induced down-regulation of adhesion and epithelial morphogenesis, indicating that these phenomena are also controlled by E-cadherin activity at the cell surface. Both types of mAbs recognize conformational epitopes at different interfaces between extracellular cadherin repeat domains (ECs), especially near calcium-binding sites. Activation also induces p120-catenin dephosphorylation, as well as changes in the cadherin cytoplasmic domain. Moreover, phospho-site mutations indicate that dephosphorylation of specific Ser/Thr residues in the N-terminal domain of p120-catenin mediate adhesion activation. Thus physiological regulation of the adhesive state of E-cadherin involves physical and/or conformational changes in the EC interface regions of the ectodomain at the cell surface that are mediated by catenin-associated changes across the membrane.