armadillo: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

Gene name - armadillo

Synonyms -

Cytological map position - 2B15

Function - cytoskeletonal element

Keywords - segment polarity gene, junctional protein, tumor suppressor

Symbol - arm

FlyBase ID:FBgn0000117

Genetic map position - 1-[0.4]

Classification - plakoglobin homolog

Cellular location - sub-surface

NCBI link: Entrez Gene
arm orthologs: Biolitmine

Recent literature
Balmer, S., Dussert, A., Collu, G. M., Benitez, E., Iomini, C. and Mlodzik, M. (2015). Components of intraflagellar transport Complex A function independently of the cilium to regulate canonical Wnt signaling in Drosophila. Dev Cell 34: 705-718. PubMed ID: 26364750
The development of multicellular organisms requires the precisely coordinated regulation of an evolutionarily conserved group of signaling pathways. Temporal and spatial control of these signaling cascades is achieved through networks of regulatory proteins, segregation of pathway components in specific subcellular compartments, or both. In vertebrates, dysregulation of primary cilia function has been strongly linked to developmental signaling defects, yet it remains unclear whether cilia sequester pathway components to regulate their activation or cilia-associated proteins directly modulate developmental signaling events. To elucidate this question, an RNAi-based screen was conducted in Drosophila non-ciliated cells to test for cilium-independent loss-of-function phenotypes of ciliary proteins in developmental signaling pathways. The results show no effect on Hedgehog signaling. In contrast, the screen identified several cilia-associated proteins as functioning in canonical Wnt signaling. Further characterization of specific components of Intraflagellar Transport complex A uncovered a cilia-independent function in potentiating Wnt signals by promoting β-catenin/Armadillo activity.

Hall, E. T. and Verheyen, E. M. (2015). Ras-activated Dsor1 promotes Wnt signaling in Drosophila development. J Cell Sci. [Epub ahead of print]. PubMed ID: 26542023
Wnt/Wingless (Wg) and Ras/MAPK signaling both play fundamental roles in growth, cell-fate determination, and when dysregulated, can lead to tumorigenesis. Several conflicting modes of interaction between Ras/MAPK and Wnt signaling have been identified in specific cellular contexts, causing synergistic or antagonistic effects on target genes. This study found novel evidence that the dual specificity kinase Downstream of Raf1 (Dsor1), also known as MEK. is required for Wnt signaling. Knockdown of Dsor1 results in loss of Wingless target gene expression, as well as reductions in stabilized Armadillo (Arm; Drosophila beta-catenin). A close physical interaction was found between Dsor1 and Arm; catalytically inactive Dsor1 causes a reduction inactive Arm. These results suggest that Dsor1 normally counteracts the Axin-mediated destruction of Arm. Ras-Dsor1 activity is independent of upstream activation by EGFR, rather it appears to be activated by the insulin-like growth factor receptor to promote Wg signaling. Together our results suggest novel crosstalk between Insulin and Wg signaling via Dsor1.
Franz, A., Shlyueva, D., Brunner, E., Stark, A. and Basler, K. (2017). Probing the canonicity of the Wnt/Wingless signaling pathway. PLoS Genet 13(4): e1006700. PubMed ID: 28369070
The hallmark of canonical Wnt signaling is the transcriptional induction of Wnt target genes by the β-catenin/TCF complex. Several studies have proposed alternative interaction partners for β-catenin or TCF, but the relevance of potential bifurcations in the distal Wnt pathway remains unclear. This study examined, on a genome-wide scale, the requirement for Armadillo (Arm, Drosophila β-catenin) and Pangolin (Pan, Drosophila TCF) in the Wnt/Wingless(Wg)-induced transcriptional response of Drosophila Kc cells. Using somatic genetics, it was demonstrated that both Arm and Pan are absolutely required for mediating activation and repression of target genes. Furthermore, by means of STARR-sequencing Wnt/Wg-responsive enhancer elements were identified and it was found that all responsive enhancers depend on Pan. Together, these results confirm the dogma of canonical Wnt/Wg signaling and argue against the existence of distal pathway branches in this system.
Zhang, T., Hsu, F. N., Xie, X. J., Li, X., Liu, M., Gao, X., Pei, X., Liao, Y., Du, W. and Ji, J. Y. (2017). Reversal of hyperactive Wnt signaling-dependent adipocyte defects by peptide boronic acids. Proc Natl Acad Sci U S A 114(36): E7469-e7478. PubMed ID: 28827348
Deregulated Wnt signaling and altered lipid metabolism have been linked to obesity, diabetes, and various cancers, highlighting the importance of identifying inhibitors that can modulate Wnt signaling and aberrant lipid metabolism. This study has established a Drosophila model with hyperactivated Wnt signaling caused by partial loss of axin, a key component of the Wnt cascade. The Axin mutant larvae are transparent and have severe adipocyte defects caused by up-regulation of beta-catenin transcriptional activities. This study demonstrates pharmacologic mitigation of these phenotypes in Axin mutants by identifying bortezomib and additional peptide boronic acids. The suppressive effect of peptide boronic acids on hyperactive Wnt signaling is dependent on alpha-catenin; the rescue effect is completely abolished with the depletion of alpha-catenin in adipocytes. These results indicate that rather than targeting the canonical Wnt signaling pathway directly, pharmacologic modulation of beta-catenin activity through alpha-catenin is a potentially attractive approach to attenuating Wnt signaling in vivo.
Kaur, P., Saunders, T. E. and Tolwinski, N. S. (2017). Coupling optogenetics and light-sheet microscopy, a method to study Wnt signaling during embryogenesis. Sci Rep 7(1): 16636. PubMed ID: 29192250
Optogenetics allows precise, fast and reversible intervention in biological processes. Light-sheet microscopy allows observation of the full course of Drosophila embryonic development from egg to larva. Bringing the two approaches together allows unparalleled precision into the temporal regulation of signaling pathways and cellular processes in vivo. To develop this method, this study investigated the regulation of canonical Wnt signaling during anterior-posterior patterning of the Drosophila embryonic epidermis. Cryptochrome 2 (CRY2) from Arabidopsis Thaliana was fused to mCherry fluorescent protein and Drosophila beta-catenin to form an easy to visualize optogenetic switch. Blue light illumination caused oligomerization of the fusion protein and inhibited downstream Wnt signaling in vitro and in vivo. Temporal inactivation of beta-catenin confirmed that Wnt signaling is required not only for Drosophila pattern formation, but also for maintenance later in development. We anticipate that this method will be easily extendable to other developmental signaling pathways and many other experimental systems.
Roper, J. C., Mitrossilis, D., Stirnemann, G., Waharte, F., Brito, I., Fernandez-Sanchez, M. E., Baaden, M., Salamero, J. and Farge, E. (2018). The major beta-catenin/E-cadherin junctional binding site is a primary molecular mechano-transductor of differentiation in vivo. Elife 7. PubMed ID: 30024850
In vivo, the primary molecular mechanotransductive events mechanically initiating cell differentiation remain unknown. This study finds the molecular stretching of the highly conserved Y654-beta-catenin-D665-E-cadherin binding site as mechanically induced by tissue strain. It triggers the increase of accessibility of the Y654 site, target of the Src42A kinase phosphorylation leading to irreversible unbinding. Molecular dynamics simulations of the beta-catenin/E-cadherin complex under a force mimicking a 6 pN physiological mechanical strain predict a local 45% stretching between the two alpha-helices linked by the site and a 15% increase in accessibility of the phosphorylation site. Both are quantitatively observed using FRET lifetime imaging and non-phospho Y654 specific antibody labelling, in response to the mechanical strains developed by endogenous and magnetically mimicked early mesoderm invagination of gastrulating Drosophila embryos. This is followed by the predicted release of 16% of beta-catenin from junctions, observed in FRAP, which initiates the mechanical activation of the beta-catenin pathway process.
Zhang, N., Parr, C., Birch, A. M., Goldfinger, M. H. and Sastre, M. (2018). The Amyloid Precursor Protein binds to beta-catenin and modulates its cellular distribution. Neurosci Lett. PubMed ID: 30176342
Accumulating evidence has shown that the processing of the amyloid precursor protein (APP) and the formation of amyloid-beta are associated with the canonical Wnt/ beta-catenin signalling pathway. It was recently published that the Drosophila homologue of APP (Appl) is a conserved modulator of Wnt PCP signalling, suggesting a potential regulation of this pathway by APP. The aim of this study was to investigate the potential interaction of APP with the canonical Wnt pathway. APP overexpression in N2a cells led to alterations in the subcellular distribution of beta-catenin by physically binding to it, preventing its translocation to the nucleus and precluding the transcription of Wnt target genes. In addition, studies in APP transgenic mice and human Alzheimer's disease (AD) brain tissue showed the cellular co-localization of APP and beta-catenin and binding of both proteins, suggesting the formation physical complexes of APP and beta-catenin, yet not present in healthy controls. Furthermore, a reduction in the levels of nuclear beta-catenin was detected in AD brains compared to controls as well as a decrease in the expression of the inactive phosphorylated Glycogen synthase kinase 3 (GSK3) isoform. Therefore, these findings indicate a reciprocal regulation of Wnt/beta-catenin signalling pathway and APP processing involving a physical interaction between APP and beta-catenin.
White, K. A., Grillo-Hill, B. K., Esquivel, M., Peralta, J., Bui, V. N., Chire, I. and Barber, D. L. (2018). beta-Catenin is a pH sensor with decreased stability at higher intracellular pH. J Cell Biol. PubMed ID: 30315137
beta-Catenin functions as an adherens junction protein for cell-cell adhesion and as a signaling protein. beta-catenin function is dependent on its stability, which is regulated by protein-protein interactions that stabilize beta-catenin or target it for proteasome-mediated degradation. This study shows that beta-catenin stability is regulated by intracellular pH (pHi) dynamics, with decreased stability at higher pHi in both mammalian cells and Drosophila melanogaster. beta-Catenin degradation requires phosphorylation of N-terminal residues for recognition by the E3 ligase beta-TrCP. While beta-catenin phosphorylation was pH independent, higher pHi induced increased beta-TrCP binding and decreased beta-catenin stability. An evolutionarily conserved histidine in beta-catenin (found in the beta-TrCP DSGIHS destruction motif) is required for pH-dependent binding to beta-TrCP. Expressing a cancer-associated H36R-beta-catenin mutant in the Drosophila eye was sufficient to induce Wnt signaling and produced pronounced tumors not seen with other oncogenic beta-catenin alleles. This study identified pHi dynamics as a previously unrecognized regulator of beta-catenin stability, functioning in coincidence with phosphorylation.
Hall, E. T., Hoesing, E., Sinkovics, E. and Verheyen, E. M. (2018). Actomyosin contractility modulates Wnt signaling through adherens junction stability. Mol Biol Cell: mbcE18060345. PubMed ID: 30540525
Actomyosin contractility can influence the canonical Wnt signaling pathway in processes like mesoderm differentiation and tissue stiffness during tumorigenesis. This study found that increased non-muscle myosin II activation and cellular contraction inhibited Wnt target gene transcription in developing Drosophila imaginal discs. This effect is due to myosin-induced accumulation of cortical F-actin resulting in clustering and accumulation of E-cadherin to the adherens junctions. This results in E-cadherin titrating any available beta-catenin, the Wnt pathway transcriptional co-activator, to the adherens junctions in order to maintain cell-cell adhesion under contraction. Decreased levels of cytoplasmic beta-catenin result in insufficient nuclear translocation for full Wnt target gene transcription. This study shows the consequences of modulating myosin phosphatase. This work elucidates a mechanism in which the dynamic promotion of actomyosin contractility refines patterning of Wnt transcription during development and maintenance of epithelial tissue in organisms.
Tsai, C. R. and Galko, M. J. (2019). Casein kinase 1alpha decreases beta-catenin levels at adherens junctions to facilitate wound closure in Drosophila larvae. Development. PubMed ID: 31511254
Skin wound repair is essential to restore barrier function and prevent infection after tissue damage. Wound-edge epidermal cells migrate as a sheet to close the wound. However, it is still unclear how cell-cell junctions are regulated during wound closure (WC). To study this, adherens junctions were examined during WC in Drosophila larvae. beta-catenin is reduced at the lateral cell-cell junctions of wound-edge epidermal cells in the early healing stages. Destruction complex components, including Ck1alpha, GSK3beta and beta-TrCP suppress beta-catenin levels in the larval epidermis. Tissue-specific RNAi targeting these genes also caused severe WC defects. The Ck1alpha (RNAi) -induced WC defect is related to adherens junctions because loss of either beta-catenin or E-cadherin significantly rescued this WC defect. In contrast, TCF (RNAi) does not rescue the Ck1alpha (RNAi) -induced WC defect, suggesting that Wnt signaling is not related to this defect. Direct overexpression of beta-catenin recapitulates most of the features of Ck1alpha reduction during wounding. Finally, loss of Ck1alpha also blocked junctional E-cadherin reduction around the wound. These results suggest that Ck1alpha and the destruction complex locally regulate cell adhesion to facilitate efficient wound repair.
Vishal, K., Lovato, T. L., Bragg, C., Chechenova, M. B. and Cripps, R. M. (2020). FGF signaling promotes myoblast proliferation through activation of wingless signaling. Dev Biol. PubMed ID: 32445643
Indirect flight muscles (IFMs) are the largest muscles in Drosophila and are made up of hundreds of myonuclei. The generation of these giant muscles requires a large pool of wing disc associated adult muscle precursors (AMPs), however the factors that control proliferation to form this myoblast pool are incompletely known. This study examined the role of fibroblast growth factor (FGF) signaling in the proliferation of wing disc associated myoblasts. The components of FGF signaling are expressed in myoblasts and surrounding epithelial cells of the wing disc. Next, this study showed that attenuation of FGF signaling results in a diminished myoblast pool. This reduction in the pool size is due to decreased myoblast proliferation. By contrast, activating the FGF signaling pathway increases the myoblast pool size and restores the proliferative capacity of FGF knockdown flies. Finally, the results demonstrate that the FGF receptor Heartless acts through up-regulating β-catenin/Armadillo signaling to promote myoblast proliferation. These studies identify a novel role for FGF signaling during IFM formation and uncover the mechanism through which FGF coordinates with Wingless signaling to promote myoblast proliferation.
Vuong, L. T., Won, J. H., Nguyen, M. B. and Choi, K. W. (2020). Role of Armadillo repeat 2 and kinesin-II motor subunit Klp64D for wingless signaling in Drosophila. Sci Rep 10(1): 13864. PubMed ID: 32807823
Armadillo (Arm) is crucial for transducing Wingless (Wg) signaling. Previous work has shown that Klp64D, a motor subunit of Drosophila kinesin-II, interacts with Arm for Wg signaling. Molecular basis for this interaction has remained unknown. This study has identified a critical Arm repeat (AR) required for binding Klp64D and Wg signaling. Arm/β-catenin family proteins contain a conserved domain of 12 Arm repeats (ARs). Five of these ARs can interact with Klp64D, but only the second AR (AR2) binds to the cargo/tail domain of Klp64D. Overexpression of AR2 in wing imaginal disc is sufficient to cause notched wing margin. This phenotype by AR2 is enhanced or suppressed by reducing or increasing Klp64D expression, respectively. AR2 overexpression inhibits Wg signaling activity in TopFlash assay, consistent with its dominant-negative effects on Klp64D-dependent Wg signaling. Overexpression of the Klp64D cargo domain also results in dominant-negative wing notching. Genetic rescue data indicate that both AR2 and Klp64D cargo regions are required for the function of Arm and Klp64D, respectively. AR2 overexpression leads to an accumulation of Arm with GM130 Golgi marker in Klp64D knockdown. This study suggests that Wg signaling for wing development is regulated by specific interaction between AR2 and the cargo domain of Klp64D.
Kinsey, S. D., Vinluan, J. P., Shipman, G. A. and Verheyen, E. M. (2021). Expression of human HIPKs in Drosophila demonstrates their shared and unique functions in a developmental model. G3 (Bethesda) 11(12). PubMed ID: 34849772
Homeodomain-interacting protein kinases (HIPKs) are a family of four conserved proteins essential for vertebrate development, as demonstrated by defects in the eye, brain, and skeleton that culminate in embryonic lethality when multiple HIPKs are lost in mice. While HIPKs are essential for development, functional redundancy between the four vertebrate HIPK paralogues has made it difficult to compare their respective functions. Because understanding the unique and shared functions of these essential proteins could directly benefit the fields of biology and medicine, this study addressed the gap in knowledge of the four vertebrate HIPK paralogues by studying them in the fruit fly Drosophila melanogaster, where reduced genetic redundancy simplifies functional assessment. The single hipk present in the fly allowed the performance of rescue experiments with human HIPK genes that provide new insight into their individual functions not easily assessed in vertebrate models. Furthermore, the abundance of genetic tools and established methods for monitoring specific developmental pathways and gross morphological changes in the fly allowed for functional comparisons in endogenous contexts. Rescue experiments were performed to demonstrate the extent to which each of the human HIPKs can functionally replace Drosophila Hipk for survival and morphological development. The ability of each human HIPK to modulate Armadillo/β-catenin levels, JAK/STAT activity, proliferation, growth, and death, each of which have previously been described for Hipks, but never all together in comparable tissue contexts. Finally, novel developmental phenotypes induced by human HIPKs were characterized to gain insight to their unique functions. Together, these experiments provide the first direct comparison of all four vertebrate HIPKs to determine their roles in a developmental context.
Hale, C., Moulton, J. K., Otis, Y. and Ganter, G. (2022). ARMADILLO REGULATES NOCICEPTIVE SENSITIVITY IN THE ABSENCE OF INJURY Mol Pain: 17448069221111155. PubMed ID: 35712882
Abnormal pain has recently been estimated to affect ~50 million adults each year within the United States. With many treatment options for abnormal pain, such as opioid analgesics, carrying numerous deleterious side effects, research into safer and more effective treatment options is crucial. To help elucidate the mechanisms controlling nociceptive sensitivity, the Drosophila melanogaster larval nociception model has been used to characterize well-conserved pathways through the use of genetic modification and/or injury to alter the sensitivity of experimental animals. Mammalian models have provided evidence of β-catenin signaling involvement in neuropathic pain development. By capitalizing on the conserved nature of β-catenin functions in the fruit fly, this study describes a role for Armadillo, the fly homolog to mammalian β-catenin, in regulating baseline sensitivity in the primary nociceptor of the fly, in the absence of injury, using under- and over-expression of Armadillo in a cell-specific manner. Underexpression of Armadillo resulted in hyposensitivity, while overexpression of wild-type Armadillo or expression of a degradation-resistant Armadillo resulted in hypersensitivity. Neither underexpression nor overexpression of Armadillo resulted in dendritic morphological changes that could contribute to behavioral phenotypes observed. These results showed that focused manipulation of Armadillo expression within the nociceptors is sufficient to modulate baseline response in the nociceptors to a noxious stimulus and that these changes are not shown to be associated with a morphogenetic effect.
Kassel, S., Hanson, A. J., Benchabane, H., Saito-Diaz, K., Cabel, C. R., Goldsmith, L., Taha, M., Kanuganti, A., Ng, V. H., Xu, G., Ye, F., Picker, J., Port, F., Boutros, M., Weiss, V. L., Robbins, D. J., Thorne, C. A., Ahmed, Y. and Lee, E. (2023). USP47 deubiquitylates Groucho/TLE to promote Wnt-β-catenin signaling. Sci Signal 16(771): eabn8372. PubMed ID: 36749823
The Wnt-β-catenin signal transduction pathway is essential for embryonic development and adult tissue homeostasis. Wnt signaling converts TCF from a transcriptional repressor to an activator in a process facilitated by the E3 ligase XIAP. XIAP-mediated monoubiquitylation of the transcriptional corepressor Groucho (also known as TLE) decreases its affinity for TCF, thereby allowing the transcriptional coactivator β-catenin to displace it on TCF. Through a genome-scale screen in cultured Drosophila melanogaster cells, this study identified the deubiquitylase USP47 as a positive regulator of Wnt signaling. USP47 was found to be required for Wnt signaling during Drosophila and Xenopus laevis development, as well as in human cells, indicating evolutionary conservation. In human cells, knockdown of USP47 inhibited Wnt reporter activity, and USP47 acted downstream of the β-catenin destruction complex. USP47 interacted with TLE3 and XIAP but did not alter their amounts; however, knockdown of USP47 enhanced XIAP-mediated ubiquitylation of TLE3. USP47 inhibited ubiquitylation of TLE3 by XIAP in vitro in a dose-dependent manner, suggesting that USP47 is the deubiquitylase that counteracts the E3 ligase activity of XIAP on TLE. These data suggest a mechanism by which regulated ubiquitylation and deubiquitylation of TLE enhance the ability of β-catenin to cycle on and off TCF, thereby helping to ensure that the expression of Wnt target genes continues only as long as the upstream signal is present.
DeSantis, D. F., Neal, S. J., Zhou, Q. and Pignoni, F. (2023). Peripodial adherens junctions regulate Ajuba-Yorkie signaling to preserve fly eye morphology. Biol Open 12(3). PubMed ID: 36912729
The Drosophila eye develops from the larval eye disc, a flattened vesicle comprised of continuous retinal and peripodial epithelia (PE). The PE is an epithelium that plays a supporting role in retinal neurogenesis, but gives rise to cuticle in the adult. This study reports that the PE is also necessary to preserve the morphology of the retinal epithelium. Depletion of the adherens junction (AJ) components β-Catenin (β-Cat), DE-Cadherin or α-Catenin from the PE leads to altered disc morphology, characterized by retinal displacement (RDis); so too does loss of the Ajuba protein Jub, an AJ-associated regulator of the transcriptional coactivator Yorkie (Yki). Restoring AJs or overexpressing Yki in β-Cat deficient PE results in suppression of RDis. Additional suppressors of AJ-dependent RDis include knockdown of Rho kinase (Rok) and Dystrophin (Dys). Furthermore, knockdown of βPS integrin (Mys) from the PE results in RDis, while overexpression of Mys can suppress RDis induced by the loss of β-Cat. It is thus proposed that AJ-Jub-Yki signaling in PE cells regulates PE cell contractile properties and/or attachment to the extracellular matrix to promote normal eye disc morphology.
Gignac, S. J., MacCharles, K. R., Fu, K., Bonaparte, K., Akarsu, G., Barrett, T. W., Verheyen, E. M. and Richman, J. M. (2023). Mechanistic studies in Drosophila and chicken give new insights into functions of DVL1 in dominant Robinow syndrome. Dis Model Mech 16(4). PubMed ID: 36916233
The study of rare genetic diseases provides valuable insights into human gene function. The autosomal dominant or autosomal recessive forms of Robinow syndrome are genetically heterogeneous, and the common theme is that all the mutations lie in genes in Wnt signaling pathways. Cases diagnosed with Robinow syndrome do survive to adulthood with distinct skeletal phenotypes, including limb shortening and craniofacial abnormalities. This study focused on mutations in dishevelled 1 (DVL1), an intracellular adaptor protein that is required for both canonical (β-catenin-dependent) or non-canonical (requiring small GTPases and JNK) Wnt signaling. We expressed human wild-type DVL1 or DVL1 variants alongside the endogenous genome of chicken and Drosophila. This design is strategically suited to test for functional differences between mutant and wild-type human proteins in relevant developmental contexts. The expression of variant forms of DVL1 produced a major disorganization of cartilage and Drosophila wing morphology compared to expression of wild-type DVL1. Moreover, the variants caused a loss of canonical and gain of non-canonical Wnt signaling in several assays. These data point to future therapies that might correct the levels of Wnt signaling, thus improving skeletal growth.
Hamada-Kawaguchi, N., Nore, B. F., Zain, R., Engstrom, Y., Smith, C. I. E. and Yamamoto, D. (2023). Exposure to Therapeutic BTK Inhibitors Induces Phenocopying of Btk29A Mutants in the Fruit Fly Drosophila melanogaster. Front Biosci (Landmark Ed) 28(6): 124. PubMed ID: 37395037
Bruton's tyrosine kinase (BTK) is a non-receptor type tyrosine kinase originally identified as the genetic signature responsible for X-linked agammaglobulinemia (XLA) when mutated. Its functional form is required for B lymphocyte maturation in both humans and mice, whereas loss-of-function causes a different form of developmental defect in the fruit fly, Drosophila melanogaster. Ibrutinib and other therapeutic inhibitors of BTK have been extensively used to successfully treat various leukemias and lymphomas. Btk29A type 2 is the ortholog of BTK in the fruit fly. Feeding wild-type flies an ibrutinib-containing diet induces phenocopying of Btk29A mutants, i.e., failure in the fusion of left and right halves of the dorsal cuticles, partial loss of wing tissues and dysregulation of germ cell production. It has been previously reported that Btk29A phosphorylates Drosophila Arm (β-catenin), and ibrutinib reduces phosphorylation at Tyrosine142 of endogenously expressed β-catenin in Cos7 cells transfected with Btk29A type 2 cDNA. Thus, Drosophila is suitable for screens of novel BTK inhibitor candidates and offers a unique in vivo system in which the mode of action of BTK inhibitors can be examined at the molecular, cellular, and organismal levels.
Vuong, L. T. and Mlodzik, M. (2023). Wg/Wnt-signaling induced nuclear translocation of β-catenin is attenuated by a β-catenin peptide through its interaction with IFT-A in development and cancer cells. bioRxiv. PubMed ID: 37398005
Wnt/Wingless (Wg) signaling is critical for many developmental patterning processes and linked to diseases, including cancer. Canonical Wnt-signaling is mediated by β-catenin, Armadillo/Arm in Drosophila transducing signal activation to a nuclear response. The IFT-A/Kinesin-2 complex is required to promote the nuclear translocation of β-catenin/Arm. This study defined a small conserved N-terminal Arm/β-catenin (Arm (34-87)) peptide, which binds IFT140, as a dominant interference tool to attenuate Wg/Wnt-signaling in vivo. Expression of Arm (34-87) is sufficient to antagonize endogenous Wnt/Wg-signaling activation resulting in marked reduction of Wg-signaling target gene expression. This effect is modulated by endogenous levels of Arm and IFT140, with the Arm (34-87) effect being enhanced or suppressed, respectively. Arm (34-87) thus inhibits Wg/Wnt-signaling by interfering with the nuclear translocation of endogenous Arm/β-catenin. Importantly, this mechanism is conserved in mammals with the equivalent β-catenin (34-87) peptide blocking nuclear translocation and pathway activation, including in cancer cells. This work indicates that Wnt-signaling can be regulated by a defined N-terminal peptide of Arm/β-catenin, and thus this might serve as an entry point for potential therapeutic applications to attenuate Wnt/β-catenin signaling.

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).

Tissue remodeling during maturation of the Drosophila wing

The final step in morphogenesis of the adult fly is wing maturation, a process not well understood at the cellular level due to the impermeable and refractive nature of cuticle synthesized some 30 h prior to eclosion from the pupal case. Advances in GFP technology now make it possible to visualize cells using fluorescence after cuticle synthesis is complete. Between eclosion and wing expansion, the epithelia within the folded wing begin to delaminate from the cuticle and that delamination is complete when the wing has fully expanded. After expansion, epithelial cells lose contact with each other, adherens junctions are disrupted, and nuclei become pycnotic. The cells then change shape, elongate, and migrate from the wing into the thorax. During wing maturation, the Timp gene product, tissue inhibitor of metalloproteinases, and probably other components of an extracellular matrix are expressed that bond the dorsal and ventral cuticular surfaces of the wing following migration of the cells. These steps are dissected using the batone and Timp genes and ectopic expression of αPS integrin, inhibitors of Armadillo/β-catenin nuclear activity and baculovirus caspase inhibitor p35. It is concluded that an epithelial-mesenchymal transition is responsible for epithelial delamination and dissolution (Kiger, 2007; full text of article).

The following outline is proposed of that program based upon cell behavior: delamination and severing contacts; changing cell shape; and migration and ECM synthesis.

Stage 1, delamination and severing contacts

A signaling role for integrins during the prepupal apposition has been proposed that prepares cells for integrin-based adhesion of the epithelia at the pupal apposition. The observation that wing epithelial cells persist in the blistered regions produced by ectopic αPS integrin expression suggests that the integrin interaction also prepares cells to respond to the later signal that induces epithelial delamination and dissolution. This signal is also blocked in the mutant batone, which prevents wing expansion. Some cells begin to delaminate from the cuticle before wing expansion has begun, and all have delaminated by the time expansion is complete. Delamination must involve severing of ECM contacts. The precision of the cellular array in a newly open wing must derive from cell–cell contacts between stretched cells that are maintained following delamination. Each cell then compacts and becomes round (as judged by the increase in fluorescence intensity). The round cells have evidently severed their junctions with adjacent cells because the precise array of cells begins to break up and Arm-GFP moves from the cell membrane to the cytoplasm (Kiger, 2007).

It would appear that disturbing the normal state of Arm/β-catenin signaling activity in epithelial cells blocks delamination. Delamination is blocked by ectopic expression of Pygo in the epithelial cells, which blocks expression of Arm target genes in a variety of tissues, and by ectopic expression of Shaggy, which blocks expression of Arm target genes by phosphorylating cytoplasmic Arm, promoting its degradation and depleting nuclear Arm. Ectopic expression of stabilized forms of Arm not subject to Shaggy phosphorylation evidently has a dominant-negative effect on Arm signaling activity in the maturing wing, blocking delamination of epithelial cells. This interpretation is supported by the following observations. First, no effect is produced by ectopic expression of wild-type Arm using the same Gal4-30A driver, consistent with other reports, very likely indicating the efficiency with which wild-type Arm is eliminated by phosphorylation and degradation through the proteasome. Second, a very low level of nuclear Arm is sufficient for target gene expression. The Arm-GFP fusion protein used here is fully active and completely covers homozygosity for a null arm allele, yet nuclear Arm-GFP cannot be detected in cells receiving a Wingless signal. Thus, it is reasonable that non-physiologically high levels of stable forms of Arm could have a dominant-negative effect, not unlike the inhibitory effect of over-expression of Pygo on Arm-directed transcription. (Kiger, 2007).

Arguing against an interpretation that the effects of ectopic gene expression might be non-specific, note that Gal4-30A-driven expression of p35 does not block delamination. Nor does Gal4-30A-driven expression of either αPS integrin or wild-type transcription factor Pangolin/dTCF/LEF-1, or a dominant-negative form of CREB have any effect on wing maturation (Kiger, 2007).

Stage 2, changing cell shape

The round cells then begin to change shape, extending thin cytoplasmic filaments, and elongate into spindles that associate with similarly shaped cells forming streams. The fact that p35 expression interrupts developmental progression at the round cell stage clearly separates Stage 1 from the changes in cell shape, cell migration, and ECM synthesis events that follow. In some cellular contexts caspase inhibition prevents cell migration independently of blocking apoptosis. It has been shown that the nuclei of wing cells cease to retain nuclear-targeted GFP and begin to fragment their DNA at what appears to be the round cell stage, consistent with the observation of pycnotic nuclei at this stage (Kiger, 2007).

Stage 3, migration and ECM synthesis

The cells migrate toward the hinge and into the body of the fly, leaving behind components, perhaps including tissue inhibitor of metalloproteinases, of an ECM that will bond dorsal and ventral cuticular surfaces. It is noteworthy that Timp deficiency does not interfere with cell migration. ECM assembly must be the final step in the developmental program. The nonautonomous action of Timp in bonding cuticle secreted by mutant Timp clones suggests that Timp is present in abundance and diffuses over large distances in the wing to participate in ECM formation (Kiger, 2007).

Precisely how ectopic expression of the various UAS transgenes studied in this paper produces wing blisters or collapsed wings is not wholly clear. It seems doubtful that cells that fail to delaminate during early phases of tissue remodeling would secrete ECM components normally. Yet a variable number of cells in these wings do delaminate and leave the wing, presumably because of variation in the level of Gal4-30A expression. These cells might be expected to secrete the necessary ECM components, although the level of critical component(s) may be insufficient for normal bonding to occur in some cases. Blister formation might also be caused by the presence of numbers of undelaminated cells physically preventing ECM from bonding the underlying cuticle. Note that when ectopic p35 expression is limited, a moderate number of round, delaminated cells can become bound in the wing without producing blisters (Kiger, 2007).

The presence of true hemocytes in the wing raises the question of whether these cells play a role in wing maturation. If Gal4-30A was to be expressed in these cells, as well as in epithelial cells, interpretation of ectopic expression studies would be complicated. No cells were detected expressing DsRedGFP fluorescence that did not express ywing-GFP fluorescence, suggesting that Gal4-30A is not expressed in true hemocytes. The observations that Hemese-Gal4-driven expression of Shaggy or of Pygo has no effect on wing maturation strongly suggest that the effects of Shaggy and Pygo on wing maturation are not mediated by true hemocytes exclusively, if at all. While the possibility that Timp and/or other ECM components are supplied by true hemocytes cannot be ruled out, the bulk of the evidence supports an active role for epithelial cells in bonding the wing surfaces. Precocious death of epithelial cells induced by Gal4-30A-driven expression of Ricin A in late pupal epithelial cells prevents bonding of dorsal and ventral cuticle after eclosion. Because the wing cuticle is fully formed, the induced cell death must have occurred after cuticle deposition but before eclosion. UV irradiation after eclosion blocks both epithelial cell delamination and bonding of the wing surfaces. In addition, it is clear that mitotic clones of defective epithelial cells affect bonding of the wing surfaces. Mitotic clones mutant for an integrin gene produce blisters in the wing cuticle as do mitotic clones ectopically expressing PKAc (Kiger, 2007).

These studies describe for the first time the developmental program that completes morphogenesis of the adult fly. The requirement for a normal state of Arm/β-catenin signaling activity suggests that an epithelial–mesenchymal transition (EMT) transforms epithelial cells into mobile fibroblasts in the wing (Kiger, 2007).

The best known example of an EMT in Drosophila is neuroblast delamination. In embryonic central nervous system formation, Wingless signaling has been shown to induce nonautonomously the delamination of specific neuroectoderm cells to form S2 neuroblasts. In peripheral nervous system formation, Wingless signaling is required for bristle formation at the wing margin, and ectopic expression of Wingless induces ectopic bristles in the wing blade. The ability of Wingless to induce neuroectoderm cells to form neuroblasts is tightly regulated by Notch in both the central and peripheral nervous systems. Evidence supports the idea that Notch modulates Wingless signaling by associating directly with Arm/β-catenin to regulate its transcriptional activity (Kiger, 2007).

Arm/β-catenin signaling appears to be characteristic of EMTs. Translocation of Arm/β-catenin into the nucleus precedes gastrulation in Drosophila, the sea urchin, and zebrafish. EMTs occur in the vertebrate neural crest when cells delaminate from the neural epithelium and migrate throughout the embryo. In the avian neural crest, dominant-negative forms of β-catenin and LEF/TCF inhibit delamination of cells from the epithelium, G1/S transition, and transcription of target genes. β-Catenin and LEF/TCF proteins are observed to translocate to the nuclei of avian neural crest cells only during delamination and to be absent during advanced stages of migration. EMTs are also a characteristic of cancer formation and can be initiated in some cancers by aberrant β-catenin activity (Kiger, 2007).

Multiple ways of activating Arm/β-catenin signaling exist. There are two independently regulated pathways that can target Arm/β-catenin to the proteasome, the Shaggy/Glycogen synthase kinase 3 degradation complex and the Seven in Absentia Homologue/ubiquitin ligase degradation complex. Multiple G-protein-coupled receptors target the Shaggy/Glycogen synthase kinase 3 degradation complex for inhibition. Further studies are necessary to identify the hormone(s), receptor(s) and signal transduction mechanisms acting in the wing maturation program and to relate this work to the extensive studies of the hormonal signals controlling wing expansion and cuticle tanning (Kiger, 2007).

Wnt pathway activation by ADP-ribosylation

Wnt/β-catenin signal transduction directs metazoan development and is deregulated in numerous human congenital disorders and cancers. In the absence of Wnt stimulation, a multi-protein 'destruction complex', assembled by the scaffold protein Axin, targets the key transcriptional activator β-catenin for proteolysis. Axin is maintained at very low levels that limit destruction complex activity, a property that is currently being exploited in the development of novel therapeutics for Wnt-driven cancers. This study used an in vivo approach in Drosophila to determine how tightly basal Axin levels must be controlled for Wnt/Wingless pathway activation, and how Axin stability is regulated. For nearly all Wingless-driven developmental processes, a three- to four-fold increase in Axin was found to be insufficient to inhibit signaling, setting a lower-limit for the threshold level of Axin in the majority of in vivo contexts. Further, both the tumor suppressor Adenomatous polyposis coli (APC) and the ADP-ribose polymerase Tankyrase (Tnks) were found to have evolutionarily conserved roles in maintaining basal Axin levels below this in vivo threshold, and separable domains were defined in Axin that are important for APC- or Tnks-dependent destabilization. Together, these findings reveal that both APC and Tnks maintain basal Axin levels below a critical in vivo threshold to promote robust pathway activation following Wnt stimulation (Yang, 2016).

The Wnt/β-catenin signal transduction pathway directs fundamental processes during metazoan development and tissue homeostasis, whereas deregulation of Wnt signalling underlies numerous congenital disorders and carcinomas. Two multimeric protein complexes with opposing functions -- the cytoplasmic destruction complex and the plasma membrane-associated signalosome -- control the stability of the transcriptional co-factor β-catenin to coordinate the state of Wnt pathway activation. In the absence of Wnt stimulation, β-catenin is targeted for proteasomal degradation by the destruction complex, which includes the two tumour suppressors: Axin and Adenomatous polyposis coli (APC), and two kinases: casein kinase α (CK1α) and glycogen synthase kinase 3 (GSK3). Engagement of Wnt with its transmembrane receptors, Frizzled and low-density lipoprotein receptor-related protein 5/6 (herein LRP6), induces rapid LRP6 phosphorylation, recruitment of Axin to phospho-LRP6, and assembly of the signalosome, which includes two other Axin-associated components, GSK3 and Dishevelled (Dvl). Signalosome assembly results in the inhibition of β-catenin proteolysis; consequently stabilized β-catenin promotes the transcriptional regulation of Wnt pathway target genes (Yang, 2016).

As a key component in both the destruction complex and the signalosome, Axin is tightly regulated. Under basal conditions, Axin is maintained at very low levels, and serves as the concentration-limiting scaffold for assembly of the destruction complex. Following Wnt exposure, the rapid association of phospho-Axin with phospho-LRP6 triggers Axin dephosphorylation, inducing a conformational change that inhibits Axin's interaction with both the destruction and signalosome complexes. Axin is subsequently degraded; however, Axin proteolysis occurs several hours after Wnt exposure, and thus does not regulate Axin's essential role during the initial activation of the Wnt pathway (Yang, 2016).

The mechanisms that rapidly reprogram Axin from inhibitory to stimulatory roles following Wnt exposure remain uncertain. In current models, Wnt stimulation induces Axin's dissociation from the destruction complex, thereby promoting its interaction with the signalosome. As Wnt stimulation induces Axin dephosphorylation, decreased phosphorylation was postulated to facilitate the dissociation of Axin from the destruction complex; however, recent work revealed that the interaction of Axin with LRP6 precedes Axin dephosphorylation, and that dephosphorylation serves to inhibit, rather than enhance this interaction (Kim, 2013) Furthermore, some findings have challenged prevailing models, providing evidence that Axin's interaction with the destruction complex is not diminished upon Wnt stimulation. Thus, whereas the rapid switch in Axin function following Wnt stimulation is essential for the activation of signalling, the underlying mechanisms remain uncertain (Yang, 2016).

During investigation of this critical process, an unanticipated role was discovered for the ADP-ribose polymerase Tankyrase (Tnks) in the reprogramming of Axin activity following Wnt exposure. As Tnks-mediated ADP-ribosylation is known to target Axin for proteolysis, small molecule Tnks inhibitors have become lead candidates for development in the therapeutic targeting of Wnt-driven cancers. This study identified a novel mechanism through which Tnks regulates Axin: by promoting Axin's central role in rapid Wnt pathway activation. Wnt stimulation was found to modulate Axin levels biphasically in both Drosophila and human cells. Unexpectedly, Axin is rapidly stabilized following Wnt stimulation, before its ultimate proteolysis hours later. In an evolutionarily conserved process, the ADP-ribosylated pool of Axin is preferentially increased immediately following Wnt exposure. ADP-ribosylation enhances Axin's association with phospho-LRP6, providing a mechanistic basis for the rapid switch in Axin function following Wnt stimulation. These results thus indicate that Tnks inhibition not only increases basal Axin levels, but also impedes the Wnt-dependent interaction between Axin and LRP6, suggesting a basis for the potency of Tnks inhibitors in Wnt-driven cancers. Thus, Tnks not only targets Axin for proteolysis independently of Wnt stimulation, but also promotes Axin's central role in Wnt pathway activation, which may be relevant to the context-dependent activation of Wnt signalling and the treatment of Wnt-driven cancers with Tnks inhibitors (Yang, 2016).

Wnt exposure induces biphasic regulation in the level of Axin, and a large increase in the level of ADP-ribosylated Axin immediately after stimulation. ADP-ribosylation enhances the interaction of Axin with phospho-LRP6, and promotes the activation of Wnt signalling. These findings lead to three major revisions of the current model for the role of Tnks in the activation of the Wnt pathway. First, Tnks serves bifunctional roles under basal conditions and after stimulation, revealing a remarkable economy and coordination of pathway components. Second, the results provide a mechanistic basis for the rapid reprogramming of Axin function in response to Wnt stimulation, and thereby reveal an unanticipated role for Tnks in this process. These findings suggest that Wnt exposure either rapidly increases the ADP-ribosylation of Axin or inhibits the targeting of ADP-ribosylated Axin for proteasomal degradation, through mechanisms yet to be elucidated. Finally, pharmacologic inactivation of Tnks was shown to diminish the interaction of Axin with LRP6, revealing a previously unknown mechanism through which small molecule Tnks inhibitors disrupt Wnt signalling, distinct from their known role in stabilizing the destruction complex inhibitors (Yang, 2016).

In the absence of Wnt stimulation, the concentration-limiting levels of Axin regulate its scaffold function in the destruction complex. As components of the destruction complex participate in other signalling pathways, the low levels of Axin were proposed to maintain modularity of the Wnt pathway. The new findings indicate that Axin levels are not only regulated in the absence of Wnt, but also regulated biphasically following Wnt stimulation. This sequential modulation of Axin divides activation of the pathway into an early, fast phase and a delayed long-term phase. During embryogenesis, the earliest expression of Wg triggers the rapid appearance of Axin in segmental stripes, which is a novel hallmark for the initial activation of the pathway. The findings reveal that Wnt exposure induces a rapid increase in the total level of Axin, and importantly, a preferential increase in the level of the ADP-ribosylated Axin. The early Axin stripes are absent in Tnks null mutant embryos and are also absent when the Tnks binding domain in Axin is deleted. Therefore, it is proposed that Axin ADP-ribosylation contributes to Axin stabilization and to the rapid response to Wg stimulation (Yang, 2016).

It is postulated that the initial increase in levels of ADP-ribosylated Axin jump-starts the response to Wnt stimulation by enhancing the Axin-LRP6 interaction, whereas the subsequent decrease in Axin levels prolongs the duration of signalling by reducing destruction complex assembly. Thus, Wnt stimulation induces rapid increases in the levels of not only cytoplasmic β-catenin, but also ADP-ribosylated Axin. Previous work that coupled mathematical modelling with experimental analysis revealed that several Wnt signalling systems were responsive to the relative change in β-catenin levels, rather than their absolute value. This dependence was proposed to impart robustness and resistance to noise and cellular variation. The current data raise the possibility that a similar principle applies to changes in Axin levels on the Axin-LRP6 interaction, as the marked increase in ADP-ribosylated Axin levels following Wnt stimulation is evolutionarily conserved. Thus, the relative change in levels of ADP-ribosylated Axin may promote signalling following Wnt exposure by facilitating the fold change in β-catenin levels (Yang, 2016).

The current findings have relevance for the context-specific in vivo roles of Tnks in Wnt signalling suggested in previous studies. Tnks inhibition disrupts Wnt signalling in a number of cultured cell lines, but in vivo studies in several model organisms suggested that the requirement for Tnks in promoting Wnt signalling is restricted to specific cell types or developmental stages. In mice, functional redundancy exists between the two Tnks homologues, such that Tnks single mutants are viable and fertile, whereas double mutants display embryonic lethality without overt Wnt-related phenotypes. However, a missense mutation in the TBD of Axin2 that is predicted to disrupt ADP-ribosylation resulted in either activating or inhibiting effects on Wnt signalling that were dependent on developmental stage. Tnks inhibitors resulted in the same paradoxical effects, suggesting complex roles in mouse embryonic development. Analogously, treatment of fish with Tnks inhibitors resulted in no observed defects in Wnt-mediated processes during development; however, the regeneration of injured fins in adults, a process that requires Wnt signalling, was disrupted (Yang, 2016).

Similarly, the finding that Drosophila Tnks null mutants are viable (Wang, 2016a; Wang, 2016b; Feng, 2014) was unexpected, as Tnks is highly evolutionarily conserved, and no other Tnks homologues exist in fly genomes. Nonetheless, the current studies reveal that a less than twofold increase in Axin levels uncovers the importance of Tnks in promoting Wg signalling during embryogenesis. Therefore, it is postulated that Tnks loss can be compensated during development unless Axin levels are increased, but that the inhibition of Wg signalling resulting from Tnks inactivation cannot be attributed solely to increased Axin levels. Furthermore, Drosophila Tnks is essential for Wg target gene activation in the adult intestine, and exclusively within regions of the gradient where Wg is present at relatively low concentration. Thus, the context-specific roles of Tnks observed in different model organisms may reflect the mechanisms described in this study, which reveal that the Wnt-induced association of Axin with LRP6 occurs even in the absence of Axin ADP-ribosylation, but is markedly enhanced in its presence. It is postulated that by enhancing this interaction, Tnks-dependent ADP-ribosylation of Axin serves to amplify the initial response to Wnt stimulation, and thus is essential in a subset of in vivo contexts (Yang, 2016).

The recent discovery that Tnks enhances signalling in Wnt-driven cancers has raised the possibility that Tnks inhibitors will offer a promising new therapeutic option. Indeed, preclinical studies have supported this possibility. Tnks inhibitors were thought previously to disrupt Wnt signalling solely by increasing the basal levels of Axin, and thus by increasing destruction complex activity. However, the current findings indicate that the degree to which the basal level of Axin increases following Tnks inactivation is not sufficient to disrupt Wnt signalling in some in vivo contexts. Instead, the results reveal that Tnks inhibition simultaneously disrupts signalling at two critical and functionally distinct steps: by promoting activity of the destruction complex and by diminishing an important step in signalosome assembly: the Wnt-induced interaction between LRP6 and Axin. On the basis of these findings, it is proposed that the efficacy of Tnks inhibitors results from their combined action at both of these steps, providing a rationale for their use in the treatment of a broad range of Wnt-driven cancers. Therefore, these results suggest that in contrast with the current focus on tumours in which attenuation of the destruction complex aberrantly activates Wnt signalling (such as those lacking APC), the preclinical testing of Tnks inhibitors could be expanded to include cancers that are dependent on pathway activation by Wnt stimulation. These include the colorectal, gastric, ovarian and pancreatic cancers that harbour inactivating mutations in RNF43, a negative Wnt feedback regulator that promotes degradation of the Wnt co-receptors Frizzled and LRP6 (Yang, 2016).

Reduced SERCA function preferentially affects Wnt signaling by retaining E-Cadherin in the endoplasmic reticulum

Calcium homeostasis in the lumen of the endoplasmic reticulum is required for correct processing and trafficking of transmembrane proteins, and defects in protein trafficking can impinge on cell signaling pathways. This study shows that mutations in the endoplasmic reticulum calcium pump SERCA disrupt Wingless signaling by sequestering Armadillo/beta-catenin away from the signaling pool. Armadillo remains bound to E-cadherin, which is retained in the endoplasmic reticulum when calcium levels there are reduced. Using hypomorphic and null SERCA alleles in combination with the loss of the plasma membrane calcium channel Orai allowed definition of three distinct thresholds of endoplasmic reticulum calcium. Wingless signaling is sensitive to even a small reduction, while Notch and Hippo signaling are disrupted at intermediate levels, and elimination of SERCA function results in apoptosis. These differential and opposing effects on three oncogenic signaling pathways may complicate the use of SERCA inhibitors as cancer therapeutics (Suisse, 2019).

Transmembrane proteins must pass through the secretory pathway to reach the cell surface, where they can interact with other cells and respond to signaling cues. Disrupting the environment in the first secretory compartment, the endoplasmic reticulum (ER), causes misfolding of transmembrane and secreted proteins and elicits a stress response that can either restore proteostasis or trigger apoptosis. The ER acts as a store of intracellular calcium (Ca2+) that can be rapidly released into the cytoplasm to trigger a variety of cellular responses. The sarcoplasmic-ER ATPase (SERCA) actively pumps Ca2+ into the ER, increasing its concentration to 1,000-fold higher than in the cytosol. Depletion of Ca2+ from the ER is sensed by Stromal interaction molecule (Stim), which encodes an endoplasmic reticulum-membrane protein that is an essential component of the store-operated calcium entry mechanism, which in neurons regulates flight. Stim, which accumulates at ER-plasma membrane junctions and activates Orai, a Ca2+ channel in the plasma membrane that mediates store-operated calcium entry (SOCE). SERCA colocalizes with Stim-Orai complexes, allowing entering Ca2+ to be pumped directly into the ER. SOCE maintains Ca2+ homeostasis in the ER so that Ca2+-binding proteins can fold correctly. In the absence of SERCA, the cell-surface receptor Notch, which has extracellular EGF and Lin-12/Notch repeats that interact with Ca2+, fails to mature (Suisse, 2019).

Wnt signaling relies on the bifunctional β-catenin protein, which acts as an essential linker between E-cadherin (E-Cad) and α-catenin at adherens junctions (AJs), but also enters the nucleus and regulates target gene expression in cells that receive a Wnt signal. In the absence of Wnt, cytoplasmic β-catenin is phosphorylated within a destruction complex, leading to its ubiquitination and degradation. Junctional β-catenin is distinct from the pool available for Wnt signaling, and excess E-Cad can remove β-catenin from the signaling pool. The extracellular domain of E-Cad binds Ca2+ ions at the junctions between cadherin domains, giving it a rigid structure. The cadherin family also includes the large protocadherins Fat and Dachsous, which restrict growth by activating the Hippo signaling pathway and regulate planar cell polarity. The precise conformation of these molecules depends on Ca2+ binding by only a subset of their cadherin domain linkers (Suisse, 2019).

There has been significant interest in using SERCA inhibitors such as thapsigargin as cancer therapeutics due to their ability to induce ER stress and apoptosis. Their general toxicity means that they would need to be targeted to specific cancer cell types. However, activating mutations in Notch that are found in certain types of leukemia may make this receptor especially sensitive to reduced SERCA function. This study, shows that a hypomorphic mutation in Drosophila SERCA preferentially affects signaling by the Wnt Wingless (Wg), because E-Cad is retained in the ER and sequesters bound Armadillo (Arm)/β-catenin. Complete loss of SERCA function leads to apoptosis, but an intermediate reduction in ER Ca2+ induced by mutating orai in the hypomorphic SERCA background disrupts Hippo signaling, leading to overgrowth and Notch signaling. These results imply that Wnt-driven cancers may be the most sensitive to SERCA inhibition but highlight the risk that inhibitors may activate cell proliferation through the Hippo pathway (Suisse, 2019).

Transmembrane proteins must pass through the secretory pathway to reach the cell surface, where they can interact with other cells and respond to signaling cues. Disrupting the environment in the first secretory compartment, the endoplasmic reticulum (ER), causes misfolding of transmembrane and secreted proteins and elicits a stress response that can either restore proteostasis or trigger apoptosis. The ER acts as a store of intracellular calcium (Ca2+) that can be rapidly released into the cytoplasm to trigger a variety of cellular responses. The sarcoplasmic-ER ATPase (SERCA) actively pumps Ca2+ into the ER, increasing its concentration to 1,000-fold higher than in the cytosol. Depletion of Ca2+ from the ER is sensed by Stim, which accumulates at ER-plasma membrane junctions and activates Orai, a Ca2+ channel in the plasma membrane that mediates store-operated calcium entry (SOCE). SERCA colocalizes with Stim-Orai complexes, allowing entering Ca2+ to be pumped directly into the ER (Alonso, 2012). SOCE maintains Ca2+ homeostasis in the ER so that Ca2+-binding proteins can fold correctly. In the absence of SERCA, the cell-surface receptor Notch, which has extracellular EGF and Lin-12/Notch repeats that interact with Ca2+, fails to mature (Suisse, 2019 and references therein).

Wnt signaling relies on the bifunctional β-catenin protein, which acts as an essential linker between E-cadherin (E-Cad) and α-catenin at adherens junctions (AJs), but also enters the nucleus and regulates target gene expression in cells that receive a Wnt signal. In the absence of Wnt, cytoplasmic β-catenin is phosphorylated within a destruction complex, leading to its ubiquitination and degradation. Junctional β-catenin is distinct from the pool available for Wnt signaling, and excess E-Cad can remove β-catenin from the signaling pool. The extracellular domain of E-Cad binds Ca2+ ions at the junctions between cadherin domains, giving it a rigid structure. The cadherin family also includes the large protocadherins Fat and Dachsous, which restrict growth by activating the Hippo signaling pathway and regulate planar cell polarity. The precise conformation of these molecules depends on Ca2+ binding by only a subset of their cadherin domain linkers (Suisse, 2019).

There has been significant interest in using SERCA inhibitors such as thapsigargin as cancer therapeutics due to their ability to induce ER stress and apoptosis. Their general toxicity means that they would need to be targeted to specific cancer cell types. However, activating mutations in Notch that are found in certain types of leukemia may make this receptor especially sensitive to reduced SERCA function (Roti, 2013). This study shows that a hypomorphic mutation in Drosophila SERCA preferentially affects signaling by the Wnt Wingless (Wg), because E-Cad is retained in the ER and sequesters bound Armadillo (Arm)/β-catenin. Complete loss of SERCA function leads to apoptosis, but an intermediate reduction in ER Ca2+ induced by mutating orai in the hypomorphic SERCA background disrupts Hippo signaling, leading to overgrowth and Notch signaling. These results imply that Wnt-driven cancers may be the most sensitive to SERCA inhibition but highlight the risk that inhibitors may activate cell proliferation through the Hippo pathway (Suisse, 2019).

Characterization of a hypomorphic SERCA mutant allele revealed that E-Cad trafficking is especially sensitive to reduced ER Ca2+ levels and that retention of E-Cad in the ER under these mild stress conditions sequesters Arm away from the pool available for Wg signaling. A similar ER retention of E-Cad and desmosomal cadherins, leading to the loss of cell adhesion, has been demonstrated in human keratinocytes in Darier disease, which results from a mutation in SERCA2. In addition, ER stress promotes the differentiation of mouse intestinal stem cells, suggesting that this may be a physiological mechanism to reduce the Wnt signaling that is required for stem cell maintenance. Ca2+ is essential for the homophilic binding of cadherin extracellular domains that mediates cell adhesion. Cadherin monomers contain multiple cadherin domains separated by hinge regions that can each bind three Ca2+ ions, stabilizing the molecule to form a rod-like structure that is resistant to protease cleavage. In larger cadherins, some of the linker regions are Ca2+ free and remain flexible. Cadherin folding into the correct conformation may thus be very sensitive to Ca2+ levels in the ER. In mammalian cells, Tg-induced ER stress leads to O-GlcNAc glycosylation of the E-Cad cytoplasmic domain, blocking its exit from the ER. However, this modification depends on caspase induction by ER stress-induced apoptosis, which does not occur in SERCAdsm mutant clones. It is also possible that E-Cad is not affected by ER Ca2+ levels directly, but is especially sensitive to the general reduction in secretion caused by the loss of SERCA (Suisse, 2019).

Arm that is bound to E-Cad at the ER membrane appears to be unavailable for Wg signaling. In mammalian cells, β-catenin forms a complex with E-Cad during co-translation in the ER and helps to transport E-Cad from the ER to the Golgi. Depleting ER Ca2+ levels may enhance the binding of Arm to E-Cad at the ER, as low extracellular Ca2+ induces rapid Arm recruitment to E-Cad at the plasma membrane. Because E-Cad competes with adenomatous polyposis coli and Axin to bind to the Arm domains, a stronger Arm-E-Cad interaction could both protect Arm from degradation and prevent it from translocating into the nuclei of Wg-receiving cells. The mechanism by which β-catenin enters the nucleus is poorly understood, and it is possible that mislocalization at the ER membrane would exclude it from docking with the partner proteins required for nuclear import (Suisse, 2019).

Using two SERCA alleles and a SERCA orai mutant combination, this study produced three distinct levels of ER Ca2+ that revealed the differential sensitivities of three oncogenic pathways. Wg signaling is the most sensitive, as it is disturbed by the weak allele SERCAdsm; while Notch trafficking is also abnormal in this mutant background, Notch target genes can still be activated. A further reduction in ER Ca2+ produced by disrupting SOCE prevents Notch and Hippo signaling, probably through effects on the trafficking of Notch and the large protocadherin Fat, but only complete loss of SERCA induces apoptosis. These findings have important implications for the use of SERCA inhibitors such as Tg as cancer therapeutics, even when targeted to specific cell types. Although it may be possible to selectively block Wnt-driven cancers with low doses of such inhibitors, the level of inhibition needed to prevent Notch signaling is likely to actually enhance tumor invasiveness by downregulating FAT family members and thus disrupting Hippo signaling (Suisse, 2019).


Amino Acids - 843

Structural Domains

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).

armadillo continued: Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

date revised: 18 February 2024 

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