The C-terminal domain of armadillo binds to hypophosphorylated teashirt to modulate wingless signalling in Drosophila

Wnt signalling is a key pathway for tissue patterning during animal development. In Drosophila, the Wnt protein Wingless acts to stabilize Armadillo inside cells where it binds to at least two DNA-binding factors which regulate specific target genes. One Armadillo-binding protein in Drosophila is the zinc finger protein Teashirt. Here we show that Wingless signalling promotes the phosphorylation and the nuclear accumulation of Teashirt. This process requires the binding of Teashirt to the C-terminal end of Armadillo. Finally, we present evidence that the serine/threonine kinase Shaggy is associated with Teashirt in a complex. We discuss these results with respect to current models of Armadillo/beta-catenin action for the transmission of the Wingless/Wnt pathway.     

A screen for mutations that suppress the phenotype of Drosophila armadillo, the beta-catenin homolog

During development signaling pathways coordinate cell fates and regulate the choice between cell survival or programmed cell death. The well-conserved Wingless/Wnt pathway is required for many developmental decisions in all animals. One transducer of the Wingless/Wnt signal is Armadillo/beta-catenin. Drosophila Armadillo not only transduces Wingless signal, but also acts in cell-cell adhesion via its role in the epithelial adherens junction. While many components of both the Wingless/Wnt signaling pathway and adherens junctions are known, both processes are complex, suggesting that unknown components influence signaling and junctions. We carried out a genetic modifier screen to identify some of these components by screening for mutations that can suppress the armadillo mutant phenotype. We identified 12 regions of the genome that have this property. From these regions and from additional candidate genes tested we identified four genes that suppress arm: dTCF, puckered, head involution defective (hid), and Dpresenilin. We further investigated the interaction with hid, a known regulator of programmed cell death. Our data suggest that Wg signaling modulates Hid activity and that Hid regulates programmed cell death in a dose-sensitive fashion.     

Activated armadillo/beta-catenin does not play a general role in cell migration and process extension in Drosophila.

Human beta-catenin and its fly homolog Armadillo are best known for their roles in cadherin-based cell-cell adhesion and in transduction of Wingless/Wnt signals. It has been hypothesized that beta-catenin may also regulate cell migration and cell shape changes, possibly by regulating the microtubule cytoskeleton via interactions with APC. This hypothesis was based on experiments in which a hyperstable mutant form of beta-catenin was expressed in MDCK cells, where it altered their migratory properties and their ability to send out long cellular processes. We tested the generality of this hypothesis in vivo in Drosophila. We utilized three model systems in which cell migration and/or process extension are known to play key roles during development: the migration of the border cells during oogenesis, the extension of axons in the nervous system, and the migration and cell process extension of tracheal cells. In all cases, cells expressing activated Armadillo were able to migrate and extend cell processes essentially normally. The one alteration from normal involved an apparent cell fate change in certain tracheal cells. These results suggest that only certain cells are affected by activation of Armadillo/beta-catenin, and that Armadillo/beta-catenin does not play a general role in inhibiting cell migration or process extension.     

beta-catenin is a target for the ubiquitin-proteasome pathway.

beta-catenin is a central component of the cadherin cell adhesion complex and plays an essential role in the Wingless/Wnt signaling pathway. In the current model of this pathway, the amount of beta-catenin (or its invertebrate homolog Armadillo) is tightly regulated and its steady-state level outside the cadherin-catenin complex is low in the absence of Wingless/Wnt signal. Here we show that the ubiquitin-dependent proteolysis system is involved in the regulation of beta-catenin turnover. beta-catenin, but not E-cadherin, p120(cas) or alpha-catenin, becomes stabilized when proteasome-mediated proteolysis is inhibited and this leads to the accumulation of multi-ubiquitinated forms of beta-catenin. Mutagenesis experiments demonstrate that substitution of the serine residues in the glycogen synthase kinase 3beta (GSK3beta) phosphorylation consensus motif of beta-catenin inhibits ubiquitination and results in stabilization of the protein. This motif in beta-catenin resembles a motif in IkappaB (inhibitor of NFkappaB) which is required for the phosphorylation-dependent degradation of IkappaB via the ubiquitin-proteasome pathway. We show that ubiquitination of beta-catenin is greatly reduced in Wnt-expressing cells, providing the first evidence that the ubiquitin-proteasome degradation pathway may act downstream of GSK3beta in the regulation of beta-catenin.     

Once upon a time there was beta-catenin in cadherin-mediated signalling

beta-Catenin was initially characterized as a protein interacting with the cadherin cytoplasmic tail and regulating cell-cell contacts and actin cytoskeleton interactions. Moreover, the gene coding for the Drosophila orthologue of beta-catenin, armadillo, was independently identified downstream of wingless in the segment-polarity signalling pathway. In fact, beta-catenin/Armadillo turned out to be key mediators of the Wnt/Wingless pathways in vertebrates and invertebrates. beta-Catenin participates in both adhesion and signalling functions in a mutually exclusive manner; bound to cadherins at the plasma membrane or 'unbound' in cytosolic or nuclear complexes. This model had placed beta-catenin at the crossroads between cadherin and Wnt signalling, leading to the dogma of inhibition of beta-catenin signalling by cadherins.     

Drosophila Twins regulates Armadillo levels in response to Wg/Wnt signal

Protein Phosphatase 2A (PP2A) has a heterotrimeric-subunit structure, consisting of a core dimer of approximately 36 kDa catalytic and approximately 65 kDa scaffold subunits complexed to a third variable regulatory subunit. Several studies have implicated PP2A in Wg/Wnt signaling. However, reports on the precise nature of PP2A role in Wg/Wnt pathway in different organisms are conflicting. We show that twins (tws), which codes for the B/PR55 regulatory subunit of PP2A in Drosophila, is a positive regulator of Wg/Wnt signaling. In tws(-) wing discs both short- and long-range targets of Wingless morphogen are downregulated. Analyses of tws(-) mitotic clones suggest that requirement of Tws in Wingless pathway is cell-autonomous. Epistatic genetic studies indicate that Tws functions downstream of Dishevelled and upstream of Sgg and Armadillo. Our results suggest that Tws is required for the stabilization of Armadillo/beta-catenin in response to Wg/Wnt signaling. Interestingly, overexpression of, otherwise normal, Tws protein induce dominant-negative phenotypes. The conflicting reports on the role of PP2A in Wg/Wnt signaling could be due to the dominant-negative effect caused by the overexpression of one of the subunits.     

Cell adhesion and signal transduction: the Armadillo connection

The products of the Drosophila segment polarity gene armadillo and its vertebrate homologue beta-catenin are components of the signal transduction pathway for Wingless/Wnt-1; this signal regulates cell-fate choices in embryos of the fruit fly Drosophila and vertebrates. Armadillo/beta-catenin is also a component of cell-cell adherens junctions in epithelia. How can these two seemingly distinct roles be reconciled? Evidence suggests that Armadillo has distinct functions: one in the adherens junction and one or more in the cytoplasm. The biochemical role of Armadillo may be to serve as a scaffold upon which different multiprotein complexes are assembled.