Involvement of p120 catenin in myopodial assembly and nerve–muscle synapse formation

At developing neuromuscular junctions (NMJs), muscles initially contact motor axons by microprocesses, or myopodia, which are induced by nerves and nerve‐secreted agrin, but it is unclear how myopodia are assembled and how they influence synaptic differentiation at the NMJ. Here, we report that treatment of cultured muscle cells with agrin transiently depleted p120 catenin (p120ctn) from cadherin junctions in situ, and increased the tyrosine phosphorylation and decreased the cadherin‐association of p120ctn in cell extracts. Whereas ectopic expression of wild‐type p120ctn in muscle generated myopodia in the absence of agrin, expression of a specific dominant‐negative mutant form of p120ctn, which blocks filopodial assembly in nonmuscle cells, suppressed nerve‐ and agrin‐induction of myopodia. Significantly, approaching neurites triggered reduced acetylcholine receptor (AChR) clustering along the edges of muscle cells expressing mutant p120ctn than of control cells, although the ability of the mutant cells to cluster AChRs was itself normal. Our results indicate a novel role of p120ctn in agrin‐induced myopodial assembly and suggest that myopodia increase muscle–nerve contacts and muscle's access to neural agrin to promote NMJ formation. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

p120 catenin regulates dendritic spine and synapse development through Rho-family GTPases and cadherins

Both the cadherin-catenin complex and Rho-family GTPases have been shown to regulate dendrite development. We show here a role for p120 catenin (p120ctn) in regulating spine and synapse formation in the developing mouse brain. p120catenin gene deletion in hippocampal pyramidal neurons in vivo resulted in reduced spine and synapse densities along dendrites. In addition, p120 catenin loss resulted in reduced cadherin levels and misregulation of Rho-family GTPases, with decreased Rac1 and increased RhoA activity. Analyses in vitro indicate that the reduced spine density reflects aberrant Rho-family GTPase signaling, whereas the effects on spine maturation appear to result from reduced cadherin levels and possibly aberrant Rho-family GTPase signaling. Thus, p120ctn acts as a signal coordinator between cadherins and Rho-family GTPases to regulate cytoskeletal changes required during spine and synapse development.     

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.     

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.     

Identification of Src phosphorylation sites in the catenin p120ctn

p120-catenin (p120(ctn)) interacts with the cytoplasmic tail of cadherins and is thought to regulate cadherin clustering during formation of adherens junctions. Several observations suggest that p120 can both positively and negatively regulate cadherin adhesiveness depending on signals that so far remain unidentified. Although p120 tyrosine phosphorylation is a leading candidate, the role of this modification in normal and Src-transformed cells remains unknown. Here, as a first step toward pinpointing this role, we have employed two-dimensional tryptic mapping to directly identify the major sites of Src-induced p120 phosphorylation. Eight sites were identified by direct mutation of candidate tyrosines to phenylalanine and elimination of the accompanying spots on the two-dimensional maps. Identical sites were observed in vitro and in vivo, strongly suggesting that the physiologically important sites have been correctly identified. Changing all of these sites to phenylalanine resulted in a p120 mutant, p120-8F, that could not be efficiently phosphorylated by Src and failed to interact with SHP-1, a tyrosine phosphatase shown previously to interact selectively with tyrosine-phosphorylated p120 in cells stimulated with epidermal growth factor. Using selected tyrosine to phenylalanine p120 mutants as dominant negative reagents, it may now be possible to selectively block events postulated to be dependent on p120 tyrosine phosphorylation.     

The nerve-muscle synapse of the garter snake

Snake nerve-muscle preparations are well-suited for study of both motor innervation patterns at the systems level and NMJ function at the cellular level. Their small size (～100 myofibers) and thinness (one fiber) allows access to all NMJs in one muscle. Snake NMJs are of three types, two twitch subtypes and a single tonic type. Properties of the NMJs supplied by a particular motor neuron, and of the motor unit fibers they innervate, are precisely regulated by the motor neuron in a manner consistent with the Henneman Size Principle. Unlike its amphibian or mammalian cousins, the snake NMJ comprises ～50 (twitch) or ～20 (tonic) individual one-bouton synapses, similar to synapses found in the central nervous system. Each bouton releases a few quanta per stimulus. Larger fibers, which require more synaptic current to initiate contraction, receive nerve terminals that contain more boutons and express receptor patches with higher sensitivity to transmitter. Quantal analysis suggests that transmitter release sites in one bouton do not behave independently; rather, they may cooperate to reduce fluctuations and enhance reliability. After release, two mechanisms coexist for retrieval and reprocessing of spent vesicles–one involving clathrin-mediated endocytosis, the other macropinocytosis. Unanswered questions include how each mechanism is regulated in a use-dependent manner.     

p120 catenin is required for normal renal tubulogenesis and glomerulogenesis

Defects in the development or maintenance of tubule diameter correlate with polycystic kidney disease. Here, we report that absence of the cadherin regulator p120 catenin (p120ctn) from the renal mesenchyme prior to tubule formation leads to decreased cadherin levels with abnormal morphologies of early tubule structures and developing glomeruli. In addition, mutant mice develop cystic kidney disease, with markedly increased tubule diameter and cellular proliferation, and detached luminal cells only in proximal tubules. The p120ctn homolog Arvcf is specifically absent from embryonic proximal tubules, consistent with the specificity of the proximal tubular phenotype. p120ctn knockdown in renal epithelial cells in 3D culture results in a similar cystic phenotype with reduced levels of E-cadherin and active RhoA. We find that E-cadherin knockdown, but not RhoA inhibition, phenocopies p120ctn knockdown. Taken together, our data show that p120ctn is required for early tubule and glomerular morphogenesis, as well as control of luminal diameter, probably through regulation of cadherins.     

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.     

Monoallelic expression and asynchronous replication of p120 catenin in mouse and human cells

The number of autosomal mammalian genes subject to random monoallelic expression has been limited to genes highly specific to the function of chemosensory neurons or lymphocytes, making this phenomenon difficult to address systematically. Here we demonstrate that asynchronous DNA replication can be used as a marker for the identification of novel genes with monoallelic expression and identify p120 catenin, a gene involved in cell adhesion, as belonging to this class. p120 is widely expressed; its presence in available cell lines allowed us to address quantitative aspects of monoallelic expression. We show that the epigenetic choice of active allele is clonally stable and that biallelic clones express p120 at twice the level of monoallelic clones. Unlike previous reports about genes of this type, we found that expression of p120 can be monoallelic in one cell type and strictly biallelic in another. We show that in human lymphoblasts, the silencing of one allele is incomplete. These unexpected properties are likely to be wide-spread, as we show that the Tlr4 gene shares them. Identification of monoallelic expression of a nearly ubiquitous gene indicates that this type of gene regulation is more common than previously thought. This has important implications for carcinogenesis and definition of cell identity.     

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.     

Involvement of p120 carboxy-terminal domain in cadherin trafficking

P120 plays an essential role in cadherin turnover. The molecular mechanism involved, however, remains only partially understood. Here, using a gene trap targeting technique, we replaced the genomic sequence of p120 with HA-tagged p120 cDNA in mouse teratocarcinoma F9 cells. In the p120 knock-in (p120KI) cells, we found that the expression level of p120 was severely reduced and that the expression level of other components of the cadherin-catenin complex was also reduced. The stable expression of various p120 mutants in p120KI cells revealed that the armadillo repeat domain of p120 is sufficient to restore the expression level of E-cadherin. In p120KI cells, internalized E-cadherin was frequently detected as large aggregates. Transient expression of wild-type p120 and mutant p120 lacking the N-terminal region induced both relocalization of E-cadherin at the cell-cell boundaries and the disappearance of cytoplasmic E-cadherin aggregates. Transient expression of mutant p120 lacking the C-terminal region, however, only induced a small increase in E-cadherin signals at the cell-cell boundary. In these cells, the cytoplasmic E-cadherin signals became brighter and the expressed mutant p120 was incorporated in the E-cadherin aggregates. These results suggested the novel function of the p120 C-terminal region in regulating the trafficking of cytoplasmic E-cadherin.     

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.     

Interaction of nonreceptor tyrosine-kinase Fer and p120 catenin is involved in neuronal polarization

The neuronal cytoskeleton is essential for establishment of neuronal polarity, but mechanisms controlling generation of polarity in the cytoskeleton are poorly understood. The nonreceptor tyrosine kinase, Fer, has been shown to bind to microtubules and to interact with several actin-regulatory proteins. Furthermore, Fer binds p120 catenin and has been shown to regulate cadherin function by modulating cadherin-beta-catenin interaction. Here we show involvement of Fer in neuronal polarization and neurite development. Fer is concentrated in growth cones together with cadherin, beta-catenin, and cortactin in stage 2 hippocampal neurons. Inhibition of Fer-p120 catenin interaction with a cell-permeable inhibitory peptide (FerP) increases neurite branching. In addition, the peptide significantly delays conversion of one of several dendrites into an axon in early stage hippocampal neurons. FerP-treated growth cones also exhibit modified localization of the microtubule and actin cytoskeleton. Together, this indicates that the Fer-p120 interaction is required for normal neuronal polarization and neurite development.     

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.     

The function of p120 catenin in filopodial growth and synaptic vesicle clustering in neurons

At the developing neuromuscular junction (NMJ), physical contact between motor axons and muscle cells initiates presynaptic and postsynaptic differentiation. Using Xenopus nerve-muscle cocultures, we previously showed that innervating axons induced muscle filopodia (myopodia), which facilitated interactions between the synaptic partners and promoted NMJ formation. The myopodia were generated by nerve-released signals through muscle p120 catenin (p120ctn), a protein of the cadherin complex that modulates the activity of Rho GTPases. Because axons also extend filopodia that mediate early nerve-muscle interactions, here we test p120ctn's function in the assembly of these presynaptic processes. Overexpression of wild-type p120ctn in Xenopus spinal neurons leads to an increase in filopodial growth and synaptic vesicle (SV) clustering along axons, whereas the development of these specializations is inhibited following the expression of a p120ctn mutant lacking sequences important for regulating Rho GTPases. The p120ctn mutant also inhibits the induction of axonal filopodia and SV clusters by basic fibroblast growth factor, a muscle-derived molecule that triggers presynaptic differentiation. Of importance, introduction of the p120ctn mutant into neurons hinders NMJ formation, which is observed as a reduction in the accumulation of acetylcholine receptors at innervation sites in muscle. Our results suggest that p120ctn signaling in motor neurons promotes nerve-muscle interaction and NMJ assembly.     

p120 catenin is essential for mesenchymal cadherin-mediated regulation of cell motility and invasiveness

During epithelial tumor progression, the loss of E-cadherin expression and inappropriate expression of mesenchymal cadherins coincide with increased invasiveness. Reexpression experiments have established E-cadherin as an invasion suppressor. However, the mechanism by which E-cadherin suppresses invasiveness and the role of mesenchymal cadherins are poorly understood. We show that both p120 catenin and mesenchymal cadherins are required for the invasiveness of E-cadherin-deficient cells. p120 binding promotes the up-regulation of mesenchymal cadherins and the activation of Rac1, which are essential for cell migration and invasiveness. p120 also promotes invasiveness by inhibiting RhoA activity, independently of cadherin association. Furthermore, association of endogenous p120 with E-cadherin is required for E-cadherin-mediated suppression of invasiveness and is accompanied by a reduction in mesenchymal cadherin levels. The data indicate that p120 acts as a rheostat, promoting a sessile cellular phenotype when associated with E-cadherin or a motile phenotype when associated with mesenchymal cadherins.