Publications
64 results found
Delegkou GNN, Birkou M, Fragkaki N, et al., 2023, E2 Partner Tunes the Ubiquitylation Specificity of Arkadia E3 Ubiquitin Ligase, CANCERS, Vol: 15
Birkou M, Delegkou GN, Marousis KD, et al., 2022, Unveiling the Essential Role of Arkadia's Non-RING Elements in the Ubiquitination Process, INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, Vol: 23
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- Citations: 2
Birkou M, Raptis V, Marousis KD, et al., 2022, Impact of a single nucleotide polymorphism on the 3D protein structure and ubiquitination activity of E3 ubiquitin ligase arkadia, Frontiers in Molecular Biosciences, Vol: 9, ISSN: 2296-889X
Single nucleotide polymorphisms (SNPs) are genetic variations which can play a vital role in the study of human health. SNP studies are often used to identify point mutations that are associated with diseases. Arkadia (RNF111) is an E3 ubiquitin ligase that enhances transforming growth factor-beta (TGF-β) signaling by targeting negative regulators for degradation. Dysregulation of the TGF-β pathway is implicated in cancer because it exhibits tumor suppressive activity in normal cells while in tumor cells it promotes invasiveness and metastasis. Τhe SNP CGT > TGT generated an amino-acid (aa) substitution of Arginine 957 to Cysteine on the enzymatic RING domain of Arkadia. This was more prevalent in a tumor than in a normal tissue sample of a patient with colorectal cancer. This prompted us to investigate the effect of this mutation in the structure and activity of Arkadia RING. We used nuclear magnetic resonance (NMR) to analyze at an atomic-level the structural and dynamic properties of the R957C Arkadia RING domain, while ubiquitination and luciferase assays provided information about its enzymatic functionality. Our study showed that the R957C mutation changed the electrostatic properties of the RING domain however, without significant effects on the structure of its core region. However, the functional studies revealed that the R957C Arkadia exhibits significantly increased enzymatic activity supporting literature data that Arkadia within tumor cells promotes aggressive and metastatic behavior.
Xu H, Wu L, Nguyen HH, et al., 2021, Arkadia-SKI/SnoN signaling differentially regulates TGF-β-induced iTreg and Th17 cell differentiation, Journal of Experimental Medicine, Vol: 218, ISSN: 0022-1007
TGF-β signaling is fundamental for both Th17 and regulatory T (Treg) cell differentiation. However, these cells differ in requirements for downstream signaling components, such as SMAD effectors. To further characterize mechanisms that distinguish TGF-β signaling requirements for Th17 and Treg cell differentiation, we investigated the role of Arkadia (RNF111), an E3 ubiquitin ligase that mediates TGF-β signaling during development. Inactivation of Arkadia in CD4+ T cells resulted in impaired Treg cell differentiation in vitro and loss of RORγt+FOXP3+ iTreg cells in the intestinal lamina propria, which increased susceptibility to microbiota-induced mucosal inflammation. In contrast, Arkadia was dispensable for Th17 cell responses. Furthermore, genetic ablation of two Arkadia substrates, the transcriptional corepressors SKI and SnoN, rescued Arkadia-deficient iTreg cell differentiation both in vitro and in vivo. These results reveal distinct TGF-β signaling modules governing Th17 and iTreg cell differentiation programs that could be targeted to selectively modulate T cell functions.
Carthy JM, Ioannou M, Episkopou V, 2018, Arkadia via SNON enables NODAL-SMAD2/3 signaling effectors to transcribe different genes depending on their levels
<jats:title>Abstract</jats:title><jats:p>How cells assess levels of signaling and select to transcribe different target genes depending on the levels of activated effectors remains elusive. High NODAL-signalling levels specify anterior/head, lower specify posterior, and complete loss abolishes anterior-posterior patterning in the mammalian embryo. Here we show that cells assess NODAL-activated SMAD2 and SMAD3 (SMAD2/3) effector-levels by complex formation and pairing each effector with the co-repressor SNON, which is present in the cell before signaling. These complexes enable the E3-ubiquitin ligase Arkadia (RNF111) to degrade SNON. High SMAD2/3 levels can saturate and remove SNON, leading to derepression and activation of a subset of targets (high targets) that are highly susceptible to SNON repression. However, low SMAD2/3 levels can only reduce SNON preventing derepression/activation of high targets. Arkadia degrades SNON transiently only upon signaling exposure, leading to dynamic signaling-responses, which most likely initiate level-specific cell-fate decisions. Arkadia-null mouse embryos and Embryonic Stem Cells (ESC) cannot develop anterior tissues and head. However, SnoN/Arkadia, double-null embryos and ESCs are rescued confirming that Arkadia removes SNON, to achieve level-dependent cell-fates</jats:p><jats:sec><jats:title>One Sentence Summary</jats:title><jats:p>Signaling intensity induces equivalent degradation of a transcriptional repressor leading to level-dependent responses.</jats:p></jats:sec>
Birkou M, Chasapis CT, Marousis KD, et al., 2017, A Residue Specific Insight into the Arkadia E3 Ubiquitin Ligase Activity and Conformational Plasticity, JOURNAL OF MOLECULAR BIOLOGY, Vol: 429, Pages: 2373-2386, ISSN: 0022-2836
Arkadia (Rnf111) is an E3 ubiquitin ligase that plays a central role in the amplification of transforming growth factor beta (TGF-β) signaling responses by targeting for degradation the negative regulators of the pathway, Smad6 and Smad7, and the nuclear co-repressors Ski and Skil (SnoN). Arkadia's function in vivo depends on the really interesting new gene (RING)–H2 interaction with the E2 enzyme UbcH5b in order to ligate ubiquitin chains on its substrates. A conserved tryptophan (W972) in the C-terminal α-helix is widely accepted as essential for E2 recruitment and interaction and thus also for E3 enzymatic activity.The present NMR-driven study provides an atomic-level investigation of the structural and dynamical properties of two W972 Arkadia RING mutants, attempting to illuminate for the first time the differences between a functional and a nonfunctional mutant W972A and W972R, respectively. A TGF-β-responsive promoter driving luciferase was used to assay for Arkadia function in vivo. These experiments showed that the Arkadia W972A mutant has the same activity as wild-type (WT) Arkadia in enhancing TGF-β signaling responses, while W972R does not. Only minor structural differences exist between the W972A RING domain and WT-RING. In contrast, the W972R mutant hardly interacts with E2. The loss of function correlates with structural changes in the C-terminal α-helix and an increase in the distance between the Zn(II) ions. Our data show that the position occupied by W972 within WT Arkadia is critical for the function of RING and that it depends on the nature of the residue at this position.
Kicheva A, Bollenbach T, Ribeiro A, et al., 2014, Coordination of progenitor specification and growth in mouse and chick spinal cord, Science, Vol: 345, Pages: 1-11, ISSN: 0036-8075
Development requires tissue growth as well as cell diversification. To address how these processes are coordinated, we analyzed the development of molecularly distinct domains of neural progenitors in the mouse and chick neural tube. We show that during development, these domains undergo changes in size that do not scale with changes in overall tissue size. Our data show that domain proportions are first established by opposing morphogen gradients and subsequently controlled by domain-specific regulation of differentiation rate but not differences in proliferation rate. Regulation of differentiation rate is key to maintaining domain proportions while accommodating both intra- and interspecies variations in size. Thus, the sequential control of progenitor specification and differentiation elaborates pattern without requiring that signaling gradients grow as tissues expand.
Birkou M, Tsapardoni M, Marousis K, et al., 2013, CONFORMATIONAL DYNAMICS AND DRUG DESIGN APPROACHES THROUGH NMR, EUROPEAN JOURNAL OF PHARMACEUTICAL SCIENCES, Vol: 50, Pages: E37-E37, ISSN: 0928-0987
Martin N, Popov N, Aguilo F, et al., 2013, Interplay between Homeobox proteins and Polycomb repressive complexes in p16<SUP>INK4a</SUP> regulation, EMBO JOURNAL, Vol: 32, Pages: 982-995, ISSN: 0261-4189
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- Citations: 32
Kelly CE, Thymiakou E, Dixon JE, et al., 2013, Rnf165/Ark2C enhances BMP-smad signaling to mediate motor axon extension, PLoS Biology, Vol: 11, ISSN: 1544-9173
Little is known about extrinsic signals required for the advancement of motor neuron (MN) axons, which extend over long distances in the periphery to form precise connections with target muscles. Here we present that Rnf165 (Arkadia-like; Arkadia2; Ark2C) is expressed specifically in the nervous system and that its loss in mice causes motor innervation defects that originate during development and lead to wasting and death before weaning. The defects range from severe reduction of motor axon extension as observed in the dorsal forelimb to shortening of presynaptic branches of the phrenic nerve, as observed in the diaphragm. Molecular functional analysis showed that in the context of the spinal cord Ark2C enhances transcriptional responses of the Smad1/5/8 effectors, which are activated (phosphorylated) downstream of Bone Morphogenetic Protein (BMP) signals. Consistent with Ark2C-modulated BMP signaling influencing motor axons, motor pools in the spinal cord were found to harbor phosphorylated Smad1/5/8 (pSmad) and treatment of primary MN with BMP inhibitor diminished axon length. In addition, genetic reduction of BMP-Smad signaling in Ark2C+/− mice caused the emergence of Ark2C−/−-like dorsal forelimb innervation deficits confirming that enhancement of BMP-Smad responses by Ark2C mediates efficient innervation. Together the above data reveal an involvement of BMP-Smad signaling in motor axon advancement.
Redshaw N, Camps C, Sharma V, et al., 2013, TGF-beta/Smad2/3 signaling directly regulates several miRNAs in mouse ES cells and early embryos, PLoS One, Vol: 8, ISSN: 1932-6203
The Transforming Growth Factor-β (TGF-β) signaling pathway is one of the major pathways essential for normal embryonic development and tissue homeostasis, with anti-tumor but also pro-metastatic properties in cancer. This pathway directly regulates several target genes that mediate its downstream functions, however very few microRNAs (miRNAs) have been identified as targets. miRNAs are modulators of gene expression with essential roles in development and a clear association with diseases including cancer. Little is known about the transcriptional regulation of the primary transcripts (pri-miRNA, pri-miR) from which several mature miRNAs are often derived. Here we present the identification of miRNAs regulated by TGF-β signaling in mouse embryonic stem (ES) cells and early embryos. We used an inducible ES cell system to maintain high levels of the TGF-β activated/phosphorylated Smad2/3 effectors, which are the transcription factors of the pathway, and a specific inhibitor that blocks their activation. By performing short RNA deep-sequencing after 12 hours Smad2/3 activation and after 16 hours inhibition, we generated a database of responsive miRNAs. Promoter/enhancer analysis of a subset of these miRNAs revealed that the transcription of pri-miR-181c/d and the pri-miR-341∼3072 cluster were found to depend on activated Smad2/3. Several of these miRNAs are expressed in early mouse embryos, when the pathway is known to play an essential role. Treatment of embryos with TGF-β inhibitor caused a reduction of their levels confirming that they are targets of this pathway in vivo. Furthermore, we showed that pri-miR-341∼3072 transcription also depends on FoxH1, a known Smad2/3 transcription partner during early development. Together, our data show that miRNAs are regulated directly by the TGF-β/Smad2/3 pathway in ES cells and early embryos. As somatic abnormalities in functions known to be regulated by the TGF-β/Smad2/3 pathway under
Chasapis CT, Kandias NG, Episkopou V, et al., 2012, NMR-based insights into the conformational and interaction properties of Arkadia RING-H2 E3 Ub ligase, PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS, Vol: 80, Pages: 1484-1489, ISSN: 0887-3585
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- Citations: 26
Sharma V, Antonacopoulou AG, Tanaka S, et al., 2011, Enhancement of TGF-β Signaling Responses by the E3 Ubiquitin Ligase Arkadia Provides Tumor Suppression in Colorectal Cancer, CANCER RESEARCH, Vol: 71, Pages: 6438-6449, ISSN: 0008-5472
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- Citations: 29
Thymiakou E, Kelly CE, Episkopou V, 2011, BMP SIGNALING ENHANCEMENT BY ARKADIA2C IS REQUIRED FOR MOTOR NEURON AXON ELONGATION, Publisher: WILEY, Pages: 195-195, ISSN: 0022-3042
Bami M, Episkopou V, Gavalas A, et al., 2011, Directed neural differentiation of mouse embryonic stem cells is a sensitive system for the identification of novel hox gene effectors, PLoS One, Vol: 6, ISSN: 1932-6203
The evolutionarily conserved Hox family of homeodomain transcription factors plays fundamental roles in regulating cell specification along the anterior posterior axis during development of all bilaterian animals by controlling cell fate choices in a highly localized, extracellular signal and cell context dependent manner. Some studies have established downstream target genes in specific systems but their identification is insufficient to explain either the ability of Hox genes to direct homeotic transformations or the breadth of their patterning potential. To begin delineating Hox gene function in neural development we used a mouse ES cell based system that combines efficient neural differentiation with inducible Hoxb1 expression. Gene expression profiling suggested that Hoxb1 acted as both activator and repressor in the short term but predominantly as a repressor in the long run. Activated and repressed genes segregated in distinct processes suggesting that, in the context examined, Hoxb1 blocked differentiation while activating genes related to early developmental processes, wnt and cell surface receptor linked signal transduction and cell-to-cell communication. To further elucidate aspects of Hoxb1 function we used loss and gain of function approaches in the mouse and chick embryos. We show that Hoxb1 acts as an activator to establish the full expression domain of CRABPI and II in rhombomere 4 and as a repressor to restrict expression of Lhx5 and Lhx9. Thus the Hoxb1 patterning activity includes the regulation of the cellular response to retinoic acid and the delay of the expression of genes that commit cells to neural differentiation. The results of this study show that ES neural differentiation and inducible Hox gene expression can be used as a sensitive model system to systematically identify Hox novel target genes, delineate their interactions with signaling pathways in dictating cell fate and define the extent of functional overlap among different Hox genes
Messller S, Kropp S, Episkopou V, et al., 2011, The TGF-β signaling modulators TRAP1/TGFBRAP1 and VPS39/Vam6/TLP are essential for early embryonic development, IMMUNOBIOLOGY, Vol: 216, Pages: 343-350, ISSN: 0171-2985
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- Citations: 24
Thymiakou E, Episkopou V, 2011, Detection of Signaling Effector-Complexes Downstream of BMP4 Using <i>in situ</i> PLA, a Proximity Ligation Assay, JOVE-JOURNAL OF VISUALIZED EXPERIMENTS, ISSN: 1940-087X
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- Citations: 18
Guzman-Ayala M, Lee KL, Mavrakis KJ, et al., 2009, Graded Smad2/3 activation is converted directly into levels of target gene expression in embryonic stem cells, PLoS One, Vol: 4, Pages: 1-20, ISSN: 1932-6203
The Transforming Growth Factor (TGF) β signalling family includes morphogens, such as Nodal and Activin, with important functions in vertebrate development. The concentration of the morphogen is critical for fate decisions in the responding cells. Smad2 and Smad3 are effectors of the Nodal/Activin branch of TGFβ signalling: they are activated by receptors, enter the nucleus and directly transcribe target genes. However, there have been no studies correlating levels of Smad2/3 activation with expression patterns of endogenous target genes in a developmental context over time. We used mouse Embryonic Stem (ES) cells to create a system whereby levels of activated Smad2/3 can be manipulated by an inducible constitutively active receptor (Alk4*) and an inhibitor (SB-431542) that blocks specifically Smad2/3 activation. The transcriptional responses were analysed by microarrays at different time points during activation and repression. We identified several genes that follow faithfully and reproducibly the Smad2/3 activation profile. Twenty-seven of these were novel and expressed in the early embryo downstream of Smad2/3 signalling. As they responded to Smad2/3 activation in the absence of protein synthesis, they were considered direct. These immediate responsive genes included negative intracellular feedback factors, like SnoN and I-Smad7, which inhibit the transcriptional activity of Smad2/3. However, their activation did not lead to subsequent repression of target genes over time, suggesting that this type of feedback is inefficient in ES cells or it is counteracted by mechanisms such as ubiquitin-mediated degradation by Arkadia. Here we present an ES cell system along with a database containing the expression profile of thousands of genes downstream of Smad2/3 activation patterns, in the presence or absence of protein synthesis. Furthermore, we identify primary target genes that follow proportionately and with high sensitivity changes in Smad2/3 levels over 1
Kandias NG, Chasapis CT, Bentrop D, et al., 2009, High yield expression and NMR characterization of Arkadia E3 ubiquitin ligase RING-H2 finger domain, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Vol: 378, Pages: 498-502, ISSN: 0006-291X
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- Citations: 23
Bravou V, Antonacopoulou A, Papadaki H, et al., 2009, TGF-beta repressors SnoN and Ski are implicated in human colorectal carcinogenesis, Cellular Oncology, Vol: 31, Pages: 41-51, ISSN: 0884-6812
Background: The TGF-β signaling repressors SnoN and Ski have been critically implicated in human cancer.Methods: To explore the role of SnoN and Ski in the development and progression of colorectal cancer we examined their protein expression profile by immunohistochemistry in a series of human colorectal adenomas, carcinomas and lymph node metastases. The mRNA expression of SnoN was also quantified by Real-Time RT-PCR.Results: SnoN and Ski were overexpressed both in adenomas with severe dysplasia and colorectal carcinomas. Protein expression was cytoplasmic and nuclear with predominant cytoplasmic localization. The subcellular localization was related differently to pathologic variables of colorectal carcinomas. Although there was no significant association of protein levels with tumor invasion and metastasis, a significant correlation of nuclear SnoN and Ski with β-catenin pathway was observed. Moreover, SnoN mRNA did not differ in carcinomas as compared to normal control and there was no correlation between SnoN protein and mRNA levels.Conclusion: Our findings suggest that SnoN and Ski exert oncogenic effects in human colorectal carcinogenesis and their overexpression is implicated in early stage disease.
Serafimidis I, Rakatzi I, Episkopou V, et al., 2008, Novel effectors of directed and Ngn3-mediated differentiation of mouse embryonic stem cells into endocrine pancreas progenitors, STEM CELLS, Vol: 26, Pages: 3-16, ISSN: 1066-5099
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- Citations: 50
Kan L, Jalali A, Zhao L-R, et al., 2007, Dual function of <i>Sox1</i> in telencephalic progenitor cells, DEVELOPMENTAL BIOLOGY, Vol: 310, Pages: 85-98, ISSN: 0012-1606
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- Citations: 32
Levy L, Howell M, Das D, et al., 2007, Arkadia activates Smad3/Smad4-Dependent transcription by triggering signal-induced SnoN degradation, MOLECULAR AND CELLULAR BIOLOGY, Vol: 27, Pages: 6068-6083, ISSN: 0270-7306
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- Citations: 134
Nagano Y, Mavrakis KJ, Lee KL, et al., 2007, Arkadia induces degradation of SnoN and c-Ski to enhance transforming growth factor-β signaling, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 282, Pages: 20492-20501
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- Citations: 132
Donner AL, Ko F, Episkopou V, et al., 2007, Pax6 is misexpressed in <i>Sox1</i> null lens fiber cells, GENE EXPRESSION PATTERNS, Vol: 7, Pages: 606-613, ISSN: 1567-133X
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- Citations: 13
Donner AL, Episkopou V, Maas RL, 2007, <i>Sox2</i> and <i>Pou2f1</i> interact to control lens and olfactory placode development, DEVELOPMENTAL BIOLOGY, Vol: 303, Pages: 784-799, ISSN: 0012-1606
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- Citations: 91
Mavrakis KJ, Andrew RL, Lee KL, et al., 2007, Arkadia enhances nodal/TGF-β signaling by coupling phospho-Smad2/3 activity and turnover, PLOS BIOLOGY, Vol: 5, Pages: 586-603, ISSN: 1545-7885
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- Citations: 79
Wilson ME, Yang KY, Kalousova A, et al., 2005, The MHG box transcription factor Sox4 contributes to the development of the endocrine pancreas, DIABETES, Vol: 54, Pages: 3402-3409, ISSN: 0012-1797
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- Citations: 88
Ekonomou A, Kazanis I, Malas S, et al., 2005, Neuronal migration and ventral subtype identity in the telencephalon depend on SOX1., PLoS biology., Vol: 3
Little is known about the molecular mechanisms and intrinsic factors that are responsible for the emergence of neuronal subtype identity. Several transcription factors that are expressed mainly in precursors of the ventral telencephalon have been shown to control neuronal specification, but it has been unclear whether subtype identity is also specified in these precursors, or if this happens in postmitotic neurons, and whether it involves the same or different factors. SOX1, an HMG box transcription factor, is expressed widely in neural precursors along with the two other SOXB1 subfamily members, SOX2 and SOX3, and all three have been implicated in neurogenesis. SOX1 is also uniquely expressed at a high level in the majority of telencephalic neurons that constitute the ventral striatum (VS). These neurons are missing in Sox1-null mutant mice. In the present study, we have addressed the requirement for SOX1 at a cellular level, revealing both the nature and timing of the defect. By generating a novel Sox1-null allele expressing beta-galactosidase, we found that the VS precursors and their early neuronal differentiation are unaffected in the absence of SOX1, but the prospective neurons fail to migrate to their appropriate position. Furthermore, the migration of non-Sox1-expressing VS neurons (such as those expressing Pax6) was also affected in the absence of SOX1, suggesting that Sox1-expressing neurons play a role in structuring the area of the VS. To test whether SOX1 is required in postmitotic cells for the emergence of VS neuronal identity, we generated mice in which Sox1 expression was directed to all ventral telencephalic precursors, but to only a very few VS neurons. These mice again lacked most of the VS, indicating that SOX1 expression in precursors is not sufficient for VS development. Conversely, the few neurons in which Sox1 expression was maintained were able to migrate to the VS. In conclusion, Sox1 expression in precursors is not sufficient for VS neuro
Ekonomou A, Kazanis I, Malas S, et al., 2005, Neuronal migration and ventral subtype identity in the telencephalon depend on SOX1, PLoS Biology, Vol: 3, Pages: 1111-1122, ISSN: 1544-9173
Little is known about the molecular mechanisms and intrinsic factors that are responsible for the emergence of neuronal subtype identity. Several transcription factors that are expressed mainly in precursors of the ventral telencephalon have been shown to control neuronal specification, but it has been unclear whether subtype identity is also specified in these precursors, or if this happens in postmitotic neurons, and whether it involves the same or different factors. SOX1, an HMG box transcription factor, is expressed widely in neural precursors along with the two other SOXB1 subfamily members, SOX2 and SOX3, and all three have been implicated in neurogenesis. SOX1 is also uniquely expressed at a high level in the majority of telencephalic neurons that constitute the ventral striatum (VS). These neurons are missing in Sox1-null mutant mice. In the present study, we have addressed the requirement for SOX1 at a cellular level, revealing both the nature and timing of the defect. By generating a novel Sox1-null allele expressing β-galactosidase, we found that the VS precursors and their early neuronal differentiation are unaffected in the absence of SOX1, but the prospective neurons fail to migrate to their appropriate position. Furthermore, the migration of non-Sox1-expressing VS neurons (such as those expressing Pax6) was also affected in the absence of SOX1, suggesting that Sox1-expressing neurons play a role in structuring the area of the VS. To test whether SOX1 is required in postmitotic cells for the emergence of VS neuronal identity, we generated mice in which Sox1 expression was directed to all ventral telencephalic precursors, but to only a very few VS neurons. These mice again lacked most of the VS, indicating that SOX1 expression in precursors is not sufficient for VS development. Conversely, the few neurons in which Sox1 expression was maintained were able to migrate to the VS. In conclusion, Sox1 expression in precursors is not sufficient for VS neu
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