armadillo: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References
Gene name - armadillo
Cytological map position - 2B15
Function - cytoskeletonal element
Symbol - arm
Genetic map position - 1-[0.4]
Classification - plakoglobin homolog
Cellular location - sub-surface
Armadillo and alpha-Catenin are components of a multiprotein complex that both maintains and initiates formation of sheets of epithelial cells. These proteins are part of the adherens junction, a biochemical crossroad where cells are attached one to another, and signals that adhesion has taken place are communicated to the interior of cells. The proteins anchor the adherens junction to the actin cytoskeleton, thus connecting surface and interior, providing them with contractile function.
Armadillo can reversably associate with the adherens junction; that is, it can take up with it, or leave it alone. The cytoplasmic localization of ARM is regulated by phosphorylation, the attachment of a phosphate residue to a protein using kinase enzymes. This is the main mechanism of the cell for intermolecular communication. Zeste-white 3, a serine threonine kinase, lies upstream of ARM, and positively regulates the phosphorylation of ARM. Tyrosine phosphorylation modifies the adherens junction, increases the level of cytoplasmic rather than junctional ARM, but probably does not result in disassembly of the junction. Wingless signal negatively regulates ARM phosphorylation (Heifer, 1994). The role of Wingless in cell adhesion, cell boundary formation, cell mobility and gene expression is currently viewed via glimpses of incomplete yet tantalizing information (Peiper, 1994a and b).
How do wingless signals get to the nucleus? The pathway has now been worked out in Drosophila based on studies with vertebrates. A component of the wingless pathway has been identified in Xenopus. A maternally expressed Xenopus homolog of mammalian HMG box factors (XTcf-3) binds to ß-catenin, the vertebrate homolog of Armadillo. XTcf-3 (Drosophila homolog: Pangolin) is a transcription factor that mediates ß-Catenin-induced axis formation in Xenopus embryos. It is proposed that the ßcatenin-XTcf-3 complex is responsible for activation of targets genes in response to upstream Wnt signals that allow cytoplasmic ß-catenin to interact with XTcf-3 (Molenaar, 1996). These observations suggest that Armadillo, plus the associated transcription factor, Pangolin, are the nuclear affectors of Wingless.
An extensive screen has been carried out to identify genes interacting with Armadillo. Two viable fly stocks have been generated by altering the level of Armadillo available for signaling. Flies from one stock overexpress Armadillo (Armover) and, as a result, have increased vein material and bristles in their wings. Flies from the other stock have reduced cytoplasmic Armadillo following overexpression of the intracellular domain of Drosophila E-cadherin (Armunder). These flies display a wing-notching phenotype typical of wingless mutations. Both misexpression phenotypes can be dominantly modified by removing one copy of genes known to encode members of the wingless pathway. This study identifies and describes further mutations that dominantly modify the Armadillo misexpression phenotypes. These mutations are in genes encoding three different functions: establishment and maintenance of adherens junctions, cell cycle control, and Egfr signaling (Greaves, 1999).
Mutations in 17 genes (26 deficiencies) were characterized that interact with Armover and/or Armunder. Interaction strength varies from deficiency to point mutation, suggesting that several genes in the original deficiencies could have contributed to, or modified, the interaction. Only for 7 of the 17 genes have interactions been identical between the point mutation and the corresponding starting deficiency. The 17 genes were sorted into four groups described below.
The candidate gene approach does not always yield an interaction. For example, two obvious candidates uncovered by an interacting deficiency on chromosome II, Kelch and cadherin-N, do not modify either Arm misexpression phenotype (the deficiency modifies both). The lack of interaction with cadherin-N is particularly surprising considering the high degree of homology of its intracellular domain with that of E-cadherin . Ultimately, there remain 28 deficiencies without a mutant that could account for the interaction (this includes the Kelch/cadherin-N deficiency). These map to 10 overlapping chromosomal regions. Testing smaller deficiencies further refines the chromosomal location of each interactor. Once a small interacting region was identified, P-element-induced mutations were obtained and tested for an interaction with Armover and Armunder. This approach identified six P-element-induced mutations that modify the Arm wing phenotypes in a manner similar to the original deficiencies. All enhance Armover and suppress Armunder, both in the eye and the wing. These interactions are similar to those with sgg/zw3, which encodes a negative regulator of Armadillo levels. An interacting P insertion mutates fasciclin3 and yet another insertion disrupts twins. The remaining four are in genes not yet cloned (Greaves, 1999).
Among the interactors identified was naked (nkd), a mutant that has long been associated with excess Wg activity. The embryonic phenotype of nkd mutants is characterized by an excess of naked cuticle, just like that of sgg/zw3 mutants or embryos overexpressing Wg. In the case of sgg/zw3, this phenotype clearly follows from overactivation of the pathway, irrespective of whether endogenous wg is present or not. In contrast, wg/nkd double mutants resemble the wg single mutant, suggesting that nkd is upstream of wg. More precisely, since nkd mutants have enlarged stripes of Engrailed [and concomitant Hh] expression, nkd has been proposed to be a negative regulator of Engrailed expression. Broader hh expression in nkd embryos (as a result of widened engrailed expression) is thought to induce ectopic stripes of wg expression; this would cause the naked cuticle phenotype. However, in wing imaginal discs, wg expression is not controlled by engrailed or hh and therefore the finding that nkd modifies the Armover and Armunder phenotypes in the wing implies a more widespread role of nkd in Wg signaling. Maybe absence of nkd function renders cells more responsive to Wg. This would explain why endogenous Wg is required for the nkd phenotype to arise. It would also be consistent with the genetic interactions that are detected in the wing. Note that, so far, no function has been ascribed to nkd in disc development (Greaves, 1999).
The screen identified several genes involved in the assembly or maintenance of adherens junctions. shotgun (encoding DE-cadherin) itself is not very illuminating since it is expected that the phenotype caused by an excess of intracellular cadherin domain will be suppressed by decreasing endogenous cadherin levels. Still, this interaction shows that the level of overexpression afforded by the Gal4p system is within physiological levels. Interaction with fat and dachsous suggests that these two nonclassical cadherins interact (maybe directly) with Arm. Initial analysis of the intracellular domain of Fat and Dachsous fail to identify an Arm/ß-catenin binding site homologous to that found in E-cadherin. However, subsequent sequence examination suggests the existence of a bipartite site. Genetic interactions with fat and dachsous strongly suggest that this proposed site is functional, and thus removing one copy of the fat or dachsous gene would release additional Arm to the cytoplasm and make it available for use in Wg transduction. Interactions with fat and dachsous in the eye confirm the ability of these genes to modify cytoplasmic Arm levels. It also indicates that these genes are expressed in the eye and may be functional there (Greaves, 1999).
Cadherin-N (CadN) binds to Arm. Therefore the failure of CadN to interact in this screen suggests that CadN may not be expressed to significant levels in the posterior compartment of wing imaginal discs or in eye precursors. In contrast, crumbs (crb) and stardust (sdt) do interact. The proteins encoded by these genes are not thought to participate in junctional complexes per se. Rather, they control the biogenesis of the junctions. It is suggested that decreasing the activity of crb or sdt has a quantitative effect on the number or size of adherens junctions and this would lead to more Arm being released from the membrane and made available for Wg signaling (Greaves, 1999).
The interaction with discs-large (dlg) is somewhat surprising since DLG is presumed to act in septate junctions. DLG localizes at septate junctions once adherens junction contacts are established. However, ZO-1, the vertebrate homolog of Dlg, has been shown to interact with ß-catenin. The genetic interaction of dlg with Armover and Armunder implies either that Dlg binds Arm in vivo, or that altering Dlg levels affects septate junctions, which in turn are needed for the stability of adherens junctions. In support for the latter alternative the dlgM52 mutation leads to disruption not only of septate but also adherens junctions. In fact, in dlgM52 mutants, Arm no longer localizes to the membrane. This could lead to increased Arm in the cytoplasm (Greaves, 1999).
The interactions with genes encoding components of the Egf pathway were initially dismissed because argos and Egfr, which have opposite effects on the Egf pathway, interact similarly with Armunder and Armover. However, recent work has demonstrated an antagonism between the Wg and Egf pathways in the embryonic epidermis. This antagonism is probably not universal since another embryonic function of Wg, the maintenance of Engrailed expression, is not affected by Egf signaling. However, the interactions that were uncovered in the wing suggest that the Egf-Wg antagonism may not be limited to cuticle patterning. It is noteworthy that, in the wing, only an interaction with Armover (which involves ectopic bristles) was seen. It may thus be that Wg and Egf only compete at places where specialized cuticular structure are formed, although, while denticles are negatively regulated by Wg, bristles are made in response to Wg signaling. No explanation is available as to why argos, a negative regulator of Egf signaling, should interact in the same manner as Egfr (Greaves, 1999).
Genes encoding cell cycle components also interact with Arm misexpression lines; this was unexpected. However, recent work by others has established a link between Wg and the cell cycle. At the wing margin of the anterior compartment, wg suppresses string (stg) transcription via induction of the proneural genes achete (ac) and scute (sc). This is followed by G2 arrest and the formation of specialized sensory bristles. In the posterior compartment no such G2 arrest takes place, and therefore there is no simple explanation as to why stg should enhance the number of ectopic noninnervated bristles induced by excess wg signaling. This and the finding that stg suppresses the Armunder phenotype implies that somehow loss of stg activity potentiates Wg signaling (Greaves, 1999).
Work in both fruit flies and vertebrates has hinted at the central role played by Arm/ß-catenin in many cellular functions, most notably, Wg signaling, cell adhesion, and, more recently, Egf signaling and the cell cycle. It is expected that the characterization of the P-induced mutations identified during the screen described here will broaden the perspective on Arm function. It is encouraging that one such P has been inserted in a gene that has previously been implicated in cell adhesion (fasciclin3). The interaction with twins (tws), a regulatory subunit of protein phosphatase 2A (PP2A), is also promising. A vertebrate regulatory subunit of PP2A has recently been shown to regulate ß-catenin activity. It will be interesting, therefore, to determine if Arm, Apc, or Axin are substrates of PP2A. It is hoped that further experiments with tws and fas3, as well as the characterization of the remaining four mutants, will help an understanding of how diverse functions like cell adhesion and cell cycle and signaling might be integrated by the usage of one common component, Arm (Greaves, 1999).
ARM has an N-terminal acidic region that is 43% homologous to human Plakoglobin, 13 central copies of a 42 amino acid repeat that is 70% identical to Plakoglobin, and a C-terminal glycine proline rich region 36% homologous to Plakoglobin (Peiper, 1990).
Beta-catenin binds to cadherins, Tcf-family transcription factors, and the tumor suppressor gene product Adenomatous Polyposis Coli (APC). A core region of beta-catenin, composed of 12 copies of a 42 amino acid sequence motif known as an armadillo repeat, mediates these interactions. The three-dimensional structure of a protease-resistant fragment of beta-catenin containing the armadillo repeat region has been determined. The 12 repeats form a superhelix of helices that features a long, positively charged groove. Although unrelated in sequence, the beta-catenin binding regions of cadherins, Tcfs, and APC are acidic and are proposed to interact with this groove (A. H. Huber, 1997).
date revised: 2 January 2000
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