p120 catenin associates with microtubules: inverse relationship between microtubule binding and Rho GTPase regulation

p120 catenin (p120ctn), an armadillo protein and component of the cadherin adhesion complex, has been found recently to induce a dendritic morphology by regulating Rho family GTPases. We have identified specific serines within the Arm repeat domain that, when mutated to alanine, promote p120ctn association with interphase microtubules, leading to microtubule reorganization and stabilization. The mutant p120ctn also localized to the mitotic spindle and centrosomes. In contrast to wild-type p120ctn, the microtubule-associated p120ctn mutant did not activate Rac1 and did not induce a dendritic morphology. In addition, we show that a basic motif within the p120ctn Arm repeat domain known to be required for the inhibition of RhoA is also required for binding to microtubules. We therefore propose that binding of p120ctn to microtubules is inversely related to its ability to regulate Rho GTPases.     

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

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

Presenilins interact with armadillo proteins including neural-specific plakophilin-related protein and beta-catenin

Abstract : Missense substitutions in the presenilin 1 (PS1) and presenilin 2 (PS2) proteins are associated with early-onset familial Alzheimer's disease. We have used yeast-two-hybrid and coimmunoprecipitation methods to show that the large cytoplasmic loop domains of PS1 and PS2 interact specifically with three members of the armadillo protein family, including β-catenin, p0071, and a novel neuronal-specific armadillo protein—neural plakophilin-related armadillo protein (NPRAP). The PS1 : NPRAP interaction occurs between the arm repeats of NPRAP and residues 372-399 at the C-terminal end of the large cytoplasmic loop of PS1. The latter residues contain a single arm -like domain and are highly conserved in the presenilins, suggesting that they form a functional armadillo protein binding site for the presenilins.     

Designed armadillo repeat proteins as general peptide-binding scaffolds: consensus design and computational optimization of the hydrophobic core

Armadillo repeat proteins are abundant eukaryotic proteins involved in several cellular processes, including signaling, transport, and cytoskeletal regulation. They are characterized by an armadillo domain, composed of tandem armadillo repeats of approximately 42 amino acids, which mediates interactions with peptides or parts of proteins in extended conformation. The conserved binding mode of the peptide in extended form, observed for different targets, makes armadillo repeat proteins attractive candidates for the generation of modular peptide-binding scaffolds. Taking advantage of the large number of repeat sequences available, a consensus-based approach combined with a force field-based optimization of the hydrophobic core was used to derive soluble, highly expressed, stable, monomeric designed proteins with improved characteristics compared to natural armadillo proteins. These sequences constitute the starting point for the generation of designed armadillo repeat protein libraries for the selection of peptide binders, exploiting their modular structure and their conserved binding mode.     

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.     

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.     

Requirement of the juxtamembrane domain of the cadherin cytoplasmic tail for morphogenetic cell rearrangement during myotome development.

During development, the activity of cadherin cell adhesion molecules is assumed to be regulated to allow for cell rearrangement or translocation. Previous studies suggest that the juxtamembrane (JM) domain of the cadherin cytoplasmic tail, which contains the site for binding to p120ctn, has a regulatory function in this adhesion system. To study the possible role of JM domain-dependent cadherin regulation in embryonic cell rearrangement, we ectopically expressed a series of N-cadherin mutants in developing somites of chicken embryos. When a JM domain-deficient N-cadherin was expressed, the morphogenetic expansion of the myotome was strongly suppressed. However, a triple alanine substitution in the JM domain, which specifically inhibited the p120ctn binding, had no effect on myotome development. Furthermore, a dominant negative N-cadherin, which had a deletion at the extracellular domain but maintained the normal cytoplasmic tail, did not affect myotome expansion; although it disrupted intersomite boundaries. Overexpression of p120ctn also did not affect myotome expansion, but it did perturb myofiber orientation. These and other observations suggest that the JM domain of N-cadherin has a regulatory role in myotome cell rearrangement in which molecules other than p120ctn are involved. The p120ctn molecule itself seems to play a critical role in the arrangement of myofibers.     

Plakophilins: Multifunctional proteins or just regulators of desmosomal adhesion?

Plakophilins 1-3 are members of the p120(ctn) family of armadillo-related proteins. The plakophilins have been characterized as desmosomal proteins, whereas p120(ctn) and the closely related delta-catenin, ARVCF and p0071 associate with adherens junctions and play essential roles in stabilizing cadherin mediated adhesion. Recent evidence suggests that plakophilins are essential components of the desmosomal plaque where they interact with desmosomal cadherins as well as the cytoskeletal linker protein desmoplakin. Plakophilins stabilize desmosomal proteins at the plasma membrane and therefore may function in a manner similar to p120(ctn) in the adherens junctions. The three plakophilins reveal distinct expression patterns, and although partially redundant in their function, mediate distinct effects on desmosomal adhesion. Besides a structural role, a function in signaling has been postulated in analogy to other armadillo proteins such as beta-catenin. At least plakophilins 1 and 2 are also localized in the nucleus, and all three proteins occur in a cytoplasmic pool. This review aims to summarize the current knowledge of plakophilin function in the context of cell adhesion, signaling and their putative role in diseases.     

Chromosomal Mapping of HumanarmadilloGenes Belonging to the p120/Plakophilin Subfamily

Armadillo-like proteins are characterized by a series of armadillo repeats that are typically 42 to 45 amino acids in length. Three major subfamilies of Armadillo-like proteins can be distinguished on the basis of their number of repeats, their overall sequence similarity, and dispersion of the repeats throughout the protein. One of these is the p120^ctn/plakophilin subfamily, which contains at least six members. We mapped the corresponding human genes by PCR on a monochromosomal cell hybrid mapping panel and by fluorescencein situhybridization. The gene for plakophilin-1 (PKP1) was located at 1q32, the plakophilin-2 gene (PKP2) was located at 12p13, while the gene for p0071 was located at 2q23–q31. We confirmed the chromosomal localization of the p120^ctngene (CTNND1) at 11q11, the ARVCF gene at 22q11, and the δ-catenin/NPRAP gene (CTNND2) at 5p15. Although some of the Armadillo proteins are highly related to one another, the corresponding genes are dispersed throughout the human genome.     

The protein-tyrosine phosphatase SHP-1 binds to and dephosphorylates p120 catenin

A prominent tyrosine-phosphorylated protein of approximately 100 kDa (designated pp100) in epidermal growth factor (EGF)-stimulated A431 cells was found to be a main interaction partner of the protein-tyrosine phosphatase SHP-1 in pull-down experiments with a glutathione S-transferase-SHP-1 fusion protein. Binding was largely mediated by the N-terminal SH2 domain of SHP-1 and apparently direct and independent from the previously described association of SHP-1 with the activated EGF receptor. pp100 was partially purified and identified by mass spectrometric analysis of tryptic fragments, partial amino acid sequencing, and use of authentic antibodies as the 3A isoform of the Armadillo repeat protein superfamily member p120 catenin (p120(ctn)). Different p120(ctn) isoforms expressed in human embryonal kidney 293 cells, exhibited differential binding to SHP-1 that correlated partly with the extent of EGF-dependent p120(ctn) tyrosine phosphorylation. Despite strong phosphorylation, p120(ctn) isoforms 3B and 3AB bound, however, less readily to SHP-1. SHP-1 associated transiently with p120(ctn) in EGF-stimulated A431 cells stably transfected with a tetracycline-responsive SHP-1 expression construct, and p120(ctn) exhibited elevated phosphorylation upon a tetracycline-mediated decrease in the SHP-1 level. Functions of p120(ctn), which are regulated by tyrosine phosphorylation, may be modulated by the described SHP-1-p120(ctn) interaction.     

p120(ctn) acts as an inhibitory regulator of cadherin function in colon carcinoma cells

p120(ctn) binds to the cytoplasmic domain of cadherins but its role is poorly understood. Colo 205 cells grow as dispersed cells despite their normal expression of E-cadherin and catenins. However, in these cells we can induce typical E-cadherin-dependent aggregation by treatment with staurosporine or trypsin. These treatments concomitantly induce an electrophoretic mobility shift of p120(ctn) to a faster position. To investigate whether p120(ctn) plays a role in this cadherin reactivation process, we transfected Colo 205 cells with a series of p120(ctn) deletion constructs. Notably, expression of NH2-terminally deleted p120(ctn) induced aggregation. Similar effects were observed when these constructs were introduced into HT-29 cells. When a mutant N-cadherin lacking the p120(ctn)-binding site was introduced into Colo 205 cells, this molecule also induced cell aggregation, indicating that cadherins can function normally if they do not bind to p120(ctn). These findings suggest that in Colo 205 cells, a signaling mechanism exists to modify a biochemical state of p120(ctn) and the modified p120(ctn) blocks the cadherin system. The NH2 terminus-deleted p120(ctn) appears to compete with the endogenous p120(ctn) to abolish the adhesion-blocking action.     

p120-ctn: A nexus for contextual signaling via Rho GTPases

p120 catenin (p120) is the prototypic member of a subfamily of armadillo repeat domain proteins involved in intercellular adhesion. Recent evidence indicates that p120 associates with classical cadherins and regulates their stability. Ectopic p120 expression results in a variety of morphological effects, and promotes cell migration. There is now strong evidence that p120 acts, at least in part, through regulation of Rho GTPases. The data suggest that p120 may act as a signaling nexus, conveying messages from the cellular micro- and macro-environment to the cell's interior. By regulating Rho GTPases in a context-dependent manner p120 can exert profound effects on cellular responses from synaptic plasticity to vesicle trafficking, as well as regulate the motile vs. sessile, and possibly the proliferative vs. quiescent phenotype of epithelial cells. Here, we review the new evidence on the relationship of p120 to Rho GTPases, and discuss potential roles for the p120-Rho connection in normal and malignant cells.     

Autoinhibition by an internal nuclear localization signal revealed by the crystal structure of mammalian importin alpha

Importin alpha is the nuclear import receptor that recognizes classical monopartite and bipartite nuclear localization signals (NLSs). The structure of mouse importin alpha has been determined at 2.5 A resolution. The structure shows a large C-terminal domain containing armadillo repeats, and a less structured N-terminal importin beta-binding domain containing an internal NLS bound to the NLS-binding site. The structure explains the regulatory switch between the cytoplasmic, high-affinity form, and the nuclear, low-affinity form for NLS binding of the nuclear import receptor predicted by the current models of nuclear import. Importin beta conceivably converts the low- to high-affinity form by binding to a site overlapping the autoinhibitory sequence. The structure also has implications for understanding NLS recognition, and the structures of armadillo and HEAT repeats.     

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.