Afadin: a Novel Actin Filament–Binding Protein with One PDZ

Total Page:16

File Type:pdf, Size:1020Kb

Afadin: a Novel Actin Filament–Binding Protein with One PDZ Afadin: A Novel Actin Filament–binding Protein with One PDZ Domain Localized at Cadherin-based Cell-to-Cell Adherens Junction Kenji Mandai,* Hiroyuki Nakanishi,* Ayako Satoh,* Hiroshi Obaishi,* Manabu Wada,* Hideo Nishioka,* Masahiko Itoh,§ Akira Mizoguchi,i Takeo Aoki,¶ Toyoshi Fujimoto,¶ Yoichi Matsuda,** Shoichiro Tsukita,§ and Yoshimi Takai*‡ *Takai Biotimer Project, ERATO, Japan Science and Technology Corporation, c/o JCR Pharmaceuticals Co., Ltd., Kobe 651-22, Japan; ‡Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita 565, Japan; §Department of Cell Biology, iDepartment of Anatomy and Neurobiology, Faculty of Medicine, Kyoto University, Kyoto 606-01, Japan; ¶Department of Anatomy and Cell Biology, Gunma University School of Medicine, Maebashi 371, Japan; and **Laboratory of Animal Genetics, School of Agricultural Sciences, Nagoya University, Nagoya 464-01, Japan Abstract. A novel actin filament (F-actin)–binding pro- p190 showed 90% identity over the entire sequence tein with a molecular mass of z205 kD (p205), which with the product of the AF-6 gene, which was found to was concentrated at cadherin-based cell-to-cell adherens be fused to the ALL-1 gene, known to be involved in junction (AJ), was isolated and characterized. p205 was acute leukemia. p190 is likely to be a rat counterpart of purified from rat brain and its cDNA was cloned from a human AF-6 protein. p205 bound along the sides of rat brain cDNA library. p205 was a protein of 1,829 F-actin but hardly showed the F-actin–cross-linking ac- amino acids (aa) with a calculated molecular mass of tivity. Northern and Western blot analyses showed that 207,667 kD. p205 had one F-actin–binding domain at p205 was ubiquitously expressed in all the rat tissues 1,631–1,829 aa residues and one PDZ domain at 1,016– examined, whereas p190 was specifically expressed in 1,100 aa residues, a domain known to interact with trans- brain. Immunofluorescence and immunoelectron mi- membrane proteins. p205 was copurified from rat brain croscopic studies revealed that p205 was concentrated with another protein with a molecular mass of 190 kD at cadherin-based cell-to-cell AJ of various tissues. We (p190). p190 was a protein of 1,663 aa with a calculated named p205 l-afadin (a large splicing variant of AF-6 molecular mass of 188,971 kD. p190 was a splicing vari- protein localized at adherens junction) and p190 s-afa- ant of p205 having one PDZ domain at 1,009–1,093 aa din (a small splicing variant of l-afadin). These results residues but lacking the F-actin–binding domain. Ho- suggest that l-afadin serves as a linker of the actin cy- mology search analysis revealed that the aa sequence of toskeleton to the plasma membrane at cell-to-cell AJ. n various cellular events, such as cell adhesion, cell mo- linkage between the actin cytoskeleton and the plasma mem- tility, and cell shape determination, specialized mem- brane is not fully understood. I brane domains are formed with transmembrane pro- To understand this molecular linkage, the cell adhesion teins, such as cell adhesion molecules, receptors, and sites have most extensively been studied (Geiger, 1983, channels, and these domains are often associated with the 1989; Geiger and Ginsberg, 1991; Turner and Burridge, actin cytoskeleton (Geiger, 1983, 1989; Geiger and Ginsberg, 1991; Luna and Hitt, 1992; Tsukita et al., 1992, 1997; 1991; Turner and Burridge, 1991; Luna and Hitt, 1992; Bretscher, 1993). The actin filament (F-actin)1–associated Tsukita et al., 1992, 1997; Bretscher, 1993). The linkage cell adhesion sites are subclassed into two types: cell-to-cell between the actin cytoskeleton and the plasma membrane and cell-to-matrix adherens junctions (AJ). Many linker plays crucial roles in these cellular events, and proteins link- proteins have been identified at cell-to-cell AJ where cad- ing the actin cytoskeleton to the transmembrane proteins herins interact with each other at the extracellular surface have been identified. However, the molecular basis of the (Takeichi, 1988; Geiger and Ginsberg, 1991; Takeichi, 1. Abbreviations used in this paper: aa, amino acid; AJ, adherens junction; Address all correspondence to Y. Takai, Department of Molecular Biol- F-actin, actin filament; FISH, fluorescence in situ hybridization; G-actin, ogy and Biochemistry, Osaka University Medical School, Suita 565, Osaka, actin monomer; GST, glutathione S-transferase; His6, six histidine resi- Japan. Tel.: 81-6-879-3410. Fax: 81-6-879-3419. E-mail: ytakai@molbio. dues; l-afadin, a large splicing variant of AF-6 protein localized at adherens med.osaka-u.ac.jp junction; S1, subfragment 1; s-afadin, a small splicing variant of l-afadin. The Rockefeller University Press, 0021-9525/97/10/517/12 $2.00 The Journal of Cell Biology, Volume 139, Number 2, October 20, 1997 517–528 http://www.jcb.org 517 1991; Tsukita et al., 1992). The cytoplasmic domain of cad- (Prasad et al., 1993), known to be involved in acute leuke- herin is associated with cytoplasmic proteins such as a-, b-, mia (Cimino et al., 1991). We named p205 l-afadin (a large and g-catenins (Ozawa et al., 1989; Nagafuchi et al., 1991; splicing variant of AF-6 protein localized at adherens junc- Takeichi, 1991; Tsukita et al., 1992). a-Catenin directly in- tion) and p190 s-afadin (a small splicing variant of l-afadin). teracts with F-actin (Rimm et al., 1995). a-Catenin also in- We describe here isolation and characterization of l-afadin. teracts indirectly with F-actin through a-actinin and/or ZO-1 (Knudsen et al., 1995; Itoh et al., 1997). Vinculin, an- other F-actin–binding protein, is concentrated at cell-to- Materials and Methods cell AJ, although its interacting molecule at cell-to-cell AJ 125 has not yet been identified (Geiger and Ginsberg, 1991; I-labeled F-Actin Blot Overlay Tsukita et al., 1992). At cell-to-matrix AJ where integrin 125I-labeled F-actin blot overlay was done as described (Chia et al., 1991; interacts with matrix proteins at the extracellular surface, Pestonjamasp et al., 1995). Briefly, purified actin monomer (G-actin) was 125 125 the cytoplasmic domain interacts directly or indirectly labeled with I-Bolton Hunter reagent. I-labeled G-actin (1 mg/ml, av- erage specific activity = 63.3 mCi/mg) was polymerized with 18 mg/ml of with F-actin–binding proteins, including a-actinin, vincu- gelsolin (molar ratio = 100:1; Sigma Chemical Co., St. Louis, MO) by incu- lin, and talin (Jockusch et al., 1995). Thus, many F-actin– bation for 10 min at 48C in a solution containing 20 mM Pipes, pH 7.0, 50 binding proteins appear to serve as linkers of the actin cy- mM KCl, and 2 mM MgCl2. Phalloidin was then added to give a final con- toskeleton to the plasma membrane cadherin and integrin. centration of 40 mM. The mixture was then incubated for another 15 min 125 The linkage between the actin cytoskeleton and the at room temperature and stored at 48C as I-labeled F-actin. The sample to be tested was subjected to SDS-PAGE and transferred to a nitrocellu- plasma membrane is also important for neuron-specific lose membrane. The membrane was blocked in TBS containing 5% defat- events, such as growth cone pathfinding and the subse- ted powder milk. The membrane was then incubated for 1 h at room tem- quent formation and maintenance of synaptic junction perature with 10 mg/ml of 125I-labeled F-actin in TBS containing 5% (Mitchison and Kirschner, 1988; Smith, 1988; Bentley and defatted powder milk and 4 mM phalloidin. After the incubation, the membrane was washed with TBS containing 0.5% Tween 20, followed by O’Connor, 1994; Lin et al., 1994; Tanaka and Sabry, 1995). autoradiography using an image analyzer (Fujix BAS-2000II; Fuji Photo However, it remains to be clarified which molecules link Film Co., Tokyo, Japan). the actin cytoskeleton to the plasma membrane in these For competition experiments, 125I-labeled F-actin was prepared as de- neuron-specific events. To address this issue, we attempted scribed above, except that the concentration of gelsolin was reduced to 7.2 125 to isolate novel F-actin–binding proteins from the rat brain mg/ml (molar ratio = 250:1). 20 mg/ml of I-labeled F-actin was incubated 125 for 30 min at room temperature with 0.42 mg/ml of myosin subfragment 1 by using a blot overlay method with I-labeled F-actin. (S1; Sigma Chemical Co.) in a solution containing 20 mM Pipes, pH 7.0, 58 We had isolated a neural tissue-specific F-actin–binding mM KCl, 2 mM MgCl2, 130 mM CaCl2, and 0.8 mM phalloidin. Where in- protein that is concentrated at synapse and named it dicated, 4 mM MgATP was added to the mixture. After the incubation, neurabin (Nakanishi et al., 1997). Neurabin has one F-actin– the mixture was diluted with an equal volume of TBS containing 10% de- fatted powder milk, and it was added to the blot membrane, followed by binding domain and one PDZ domain. The PDZ domain incubation for 1 h at room temperature. has been found in a variety of proteins, some of which are localized at cell-to-cell junctions, such as PSD-95/SAP90 at Purification of l-Afadin synaptic junction (Cho et al., 1992; Kistner et al., 1993), Dlg at septate junction (Woods and Bryant, 1991), and All the purification procedures were carried out at 0–48C. Fetal brains from 60 mother rats (17-d gestation) were homogenized with a solution ZO-1 and ZO-2 at tight junction (Stevenson et al., 1986; containing 10 mM Tris/Cl, pH 8.0, 2 mM EDTA, 5 mM EGTA, and a pro- Gumbiner, 1991; Itoh et al., 1993; Tsukita et al., 1993; Wil- tease inhibitor cocktail (1 mM PMSF, 20 mg/ml of leupeptin, and 1 mg/ml lott et al., 1993; Jesaitis and Goodenough, 1994).
Recommended publications
  • Gene Section Short Communication
    Atlas of Genetics and Cytogenetics in Oncology and Haematology OPEN ACCESS JOURNAL INIST -CNRS Gene Section Short Communication SSX2IP (synovial sarcoma, X breakpoint 2 interacting protein) Ghazala Khan, Barbara Guinn University of Bedfordshire, Division of Science, Park Square, Luton, Bedfordshire, UK (GK), University of Bedfordshire, Division of Science, Park Square, Luton, Bedfordshire, UK; Cancer Sciences Unit, University of Southampton, Southampton, UK; Department of Haematological Medicine, Kings College, London, UK (BG) Published in Atlas Database: March 2012 Online updated version : http://AtlasGeneticsOncology.org/Genes/SSX2IPID42407ch1p22.html DOI: 10.4267/2042/47489 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2012 Atlas of Genetics and Cytogenetics in Oncology and Haematology exons however the first one is not translated (de Bruijn Identity et al., 2002). Other names: ADIP Transcription HGNC (Hugo): SSX2IP The gene contains 33 introns. 18 different mRNAs are Location: 1p22.3 produced; 17 spliced and 1 un-spliced form (Thierry- Note Mieg and Thierry-Mieg, 2006). SSX2IP gene encodes the protein SSX2IP which Pseudogene interacts with the cancer-testis antigen SSX2. It is A pseudogene of this gene is found on chromosome 3 thought that SSX2IP regulates the function of SSX2 in (provided by RefSeq, Oct 2009 from Entrez Gene). the testes and malignant cells. The rodent equivalent is known as afadin DIL domain-interacting protein (ADIP) and the chicken orthologue is called clock- Protein controlled gene (LCG) (Breslin et al., 2007). Note SSX2IP was discovered due to its interaction with DNA/RNA SSX2 in a yeast two-hybrid system and believed to regulate the function of SSX2 in the testes and Note malignant cells (de Bruijn et al., 2002).
    [Show full text]
  • Epha2 Proteomics in Human Keratinocytes Reveals a Novel Association with Afadin and Epidermal Tight Junctions Bethany E
    © 2017. Published by The Company of Biologists Ltd | Journal of Cell Science (2017) 130, 111-118 doi:10.1242/jcs.188169 SPECIAL ISSUE: 3D CELL BIOLOGY SHORT REPORT EphA2 proteomics in human keratinocytes reveals a novel association with afadin and epidermal tight junctions Bethany E. Perez White1, Rosa Ventrella1, Nihal Kaplan1, Calvin J. Cable1, Paul M. Thomas2,3 and Spiro Getsios1,4,*,‡ ABSTRACT EphA2 increased susceptibility to chemically-induced skin EphA2 is a receptor tyrosine kinase that helps to maintain epidermal carcinogenesis (Guo et al., 2006), whereas ephrin-targeting of tissue homeostasis. A proximity-dependent biotin identification (BioID) EphA2 enhanced keratinocyte adhesion and differentiation (Lin approach was used to identify proteins in close proximity to EphA2 et al., 2010; Walsh and Blumenberg, 2011). EphA2 can positively within primary human keratinocytes and three-dimensional (3D) or negatively regulate intercellular junctions, including tight reconstituted human epidermis (RHE) cultures to map a putative junctions (Zhou et al., 2011; Tanaka et al., 2005; Larson et al., protein interaction network for this membrane receptor that exhibits a 2008; Miao et al., 2014; Miura et al., 2009) that contribute to skin polarized distribution in stratified epithelia. Although a subset of known barrier function (Niessen, 2007). Importantly, EphA2 is expressed EphA2 interactors were identified in the BioID screen, >97% were in a differentiation-dependent, polarized manner within human uniquely detected in keratinocytes with over 50% of these vicinal epidermis (Fig. 1A). proteins only present in 3D human epidermal culture. Afadin (AFDN), a We adapted the method of unbiased, proximity-dependent biotin cytoskeletal and junction-associated protein, was present in 2D and 3D identification (BioID) (Roux et al., 2012) to identify near neighbors keratinocyte cultures, and validated as a so-far-unknown EphA2- of EphA2 in normal human epidermal keratinocytes (NHEKs) 2+ interacting protein.
    [Show full text]
  • Afadin, a Ras/Rap Effector That Controls Cadherin Function, Promotes Spine and Excitatory Synapse Density in the Hippocampus
    The Journal of Neuroscience, January 4, 2012 • 32(1):99–110 • 99 Development/Plasticity/Repair Afadin, A Ras/Rap Effector That Controls Cadherin Function, Promotes Spine and Excitatory Synapse Density in the Hippocampus Gerard M. J. Beaudoin III,1 Claude M. Schofield,2* Tulip Nuwal,3* Keling Zang,1 Erik M. Ullian,1,2† Bo Huang,3† and Louis F. Reichardt1,4 Departments of 1Physiology, 2Opthamology, 3Pharmaceutical Chemistry, and 4Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California 94158 Many molecules regulate synaptogenesis, but intracellular signaling pathways required for their functions are poorly understood. Afadin is a Rap-regulated, actin-binding protein that promotes cadherin complex assembly as well as binding many other cell adhesion mole- cules and receptors. To examine its role in mediating synaptogenesis, we deleted afadin (mllt1), using a conditional allele, in postmitotic hippocampal neurons. Consistent with its role in promoting cadherin recruitment, afadin deletion resulted in 70% fewer and less intense N-cadherin puncta with similar reductions of ␤-catenin and ␣N-catenin puncta densities and 35% reduction in EphB2 puncta density. Its absence also resulted in 40% decreases in spine and excitatory synapse densities in the stratum radiatum of CA1, as determined by morphology, apposition of presynaptic and postsynaptic markers, and synaptic transmission. The remaining synapses appeared to function normally. Thus, afadin is a key intracellular signaling molecule for cadherin recruitment and is necessary for spine and synapse formation in vivo. Introduction 2007). In Drosophila, absence of N-cadherin prevents normal Synapses are formed at contacts between neurons as a result of synapse formation between photoreceptor axons and their tar- intercellular interactions that result in stepwise assembly of syn- gets (Clandinin and Feldheim, 2009).
    [Show full text]
  • Rap1 Acts Via Multiple Mechanisms to Position Canoe/Afadin and Adherens Junctions and Mediate Apical-Basal Polarity Establishment
    bioRxiv preprint doi: https://doi.org/10.1101/170977; this version posted July 31, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Rap1 acts via multiple mechanisms to position Canoe/Afadin and adherens junctions and mediate apical-basal polarity establishment Teresa T. Bonello1, Kia Z. Perez-Vale2, Kaelyn D. Sumigray3, and Mark Peifer1,2,3* 1 Department of Biology, University of North Carolina at Chapel Hill, CB#3280, Chapel Hill, NC 27599-3280, USA 2 Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA 3 Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA Running Title Active Rap1 positions Canoe and AJs 6950 words * To whom correspondence should be addressed Email: [email protected] Phone: (919) 962-2272 Abbreviations used: α-cat, alpha-catenin; β-cat, beta-catenin; AJ, adherens junction; Arm, Armadillo; Baz, BazooKa; CA, constitutively active; Cno, Canoe; DE-cad, Drosophila E-cadherin; Dzy, Dizzy; GAP, GTPase activating protein; GDP, guanosine diphosphate; GEF, guanine nucleotide exchange factor; GFP, green fluorescent protein; GTP, guanosine triphosphate; IF, immunofluorescence; MIP, maximum intensity projection; RA, Ras-associated; RFP, red fluorescent protein; SAJ, spot adherens junction; shRNA, short hairpin RNA; TCJ, tricellular junction; WT, wildtype 1 bioRxiv preprint doi: https://doi.org/10.1101/170977; this version posted July 31, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved.
    [Show full text]
  • Vinexin Family (SORBS) Proteins Play Different Roles in Stiffness- Sensing and Contractile Force Generation Takafumi Ichikawa1,2, Masahiro Kita1, Tsubasa S
    © 2017. Published by The Company of Biologists Ltd | Journal of Cell Science (2017) 130, 3517-3531 doi:10.1242/jcs.200691 RESEARCH ARTICLE Vinexin family (SORBS) proteins play different roles in stiffness- sensing and contractile force generation Takafumi Ichikawa1,2, Masahiro Kita1, Tsubasa S. Matsui3,4, Ayaka Ichikawa Nagasato1, Tomohiko Araki3, Shian-Huey Chiang5, Takuhito Sezaki1, Yasuhisa Kimura1, Kazumitsu Ueda1,2, Shinji Deguchi3,4, Alan R. Saltiel5,* and Noriyuki Kioka1,2,‡ ABSTRACT generating actin stress fibers (SFs) (Geiger et al., 2001). This Vinexin, c-Cbl associated protein (CAP) and Arg-binding protein 2 ‘ ’ (ArgBP2) constitute an adaptor protein family called the vinexin mechanical linkage acts as a molecular clutch to transmit the force (SORBS) family that is targeted to focal adhesions (FAs). Although derived from non-muscle myosin-II-dependent contraction to the numerous studies have focused on each of the SORBS proteins and ECM. Cells on more rigid substrates exert greater contractile forces partially elucidated their involvement in mechanotransduction, a than those on soft substrates (Hoffman et al., 2011; Roca-Cusachs comparative analysis of their function has not been well addressed. et al., 2012; LaCroix et al., 2015). These alterations can lead to Here, we established mouse embryonic fibroblasts that individually stiffness-dependent biochemical signals. Among the numerous FA scaffolding proteins, vinculin is one of expressed SORBS proteins and analysed their functions in an ‘ ’ identical cell context. Both vinexin-α and CAP co-localized with the main clutch molecules that can regulate force transmission. vinculin at FAs and promoted the appearance of vinculin-rich FAs, Vinculin consists of an N-terminal head region and a C-terminal tail α region separated by a flexible proline-rich linker region (Bakolitsa whereas ArgBP2 co-localized with -actinin at the proximal end of – FAs and punctate structures on actin stress fibers (SFs), and induced et al., 2004; Borgon et al., 2004).
    [Show full text]
  • Kobe University Repository : Thesis
    Kobe University Repository : Thesis Afadin Regulates Puncta Adherentia Junction Formation and 学位論文題目 Presynaptic Differentiation in Hippocampal Neurons(アファディンは海 Title 馬ニューロンにおいてアドへレンスジャンクションの形成と前シナプ スの分化を調節している) 氏名 Toyoshima, Daisaku Author 専攻分野 博士(医学) Degree 学位授与の日付 2014-03-25 Date of Degree 公開日 2015-03-01 Date of Publication 資源タイプ Thesis or Dissertation / 学位論文 Resource Type 報告番号 甲第6188号 Report Number 権利 Rights JaLCDOI URL http://www.lib.kobe-u.ac.jp/handle_kernel/D1006188 ※当コンテンツは神戸大学の学術成果です。無断複製・不正使用等を禁じます。著作権法で認められている範囲内で、適切にご利用ください。 PDF issue: 2021-09-26 Afadin Regulates Puncta Adherentia Junction Formation and Presynaptic Differentiation in Hippocampal Neurons アファディンは海馬ニューロンにおいてアドへレンスジャンクション の形成と前シナプスの分化を調節している 豊嶋大作, 萬代研二, 丸尾知彦、Irwan Supriyanto、 富樫英、井上貴仁、森正弘、高井義美 神戸大学大学院医学研究科医科学専攻 小児科学 (指導教員:飯島一誠 教授) 豊嶋大作 Key words: Afadin, puncta adherentia junction, presynapse, hippocampal neuron Manuscript Click here to download Manuscript: Toyoshima_text_revised_2nd_final.pdf 1 Afadin Regulates Puncta Adherentia Junction Formation and 2 Presynaptic Differentiation in Hippocampal Neurons 3 4 Daisaku Toyoshima1,3,4, Kenji Mandai1,3, Tomohiko Maruo1,3, Irwan Supriyanto2,3, 5 Hideru Togashi1,3, Takahito Inoue1,3, Masahiro Mori2,3 & Yoshimi Takai1,3 6 7 1. Department of Biochemistry and Molecular Biology, Kobe University Graduate 8 School of Medicine, Kobe, Hyogo 650-0047, Japan. 9 2. Faculty of Health Sciences, Kobe University Graduate School of Health Sciences, 10 Kobe, Hyogo 654-0142, Japan. 11 3. CREST, Japan Science and Technology Agency, Kobe, Hyogo 650-0047, Japan. 12 4. Present address: Department of Pediatrics, Kobe University Graduate School of 13 Medicine, Kobe, Hyogo 650-0017, Japan. 14 15 Correspondence should be addressed to Y.T. ([email protected]), K.M. 16 ([email protected]).
    [Show full text]
  • The Adherens Junction Protein Afadin Is an AKT Substrate That Regulates Breast Cancer Cell Migration
    Published OnlineFirst November 22, 2013; DOI: 10.1158/1541-7786.MCR-13-0398 Molecular Cancer Signal Transduction Research The Adherens Junction Protein Afadin Is an AKT Substrate that Regulates Breast Cancer Cell Migration Sivan Elloul, Dmitriy Kedrin, Nicholas W. Knoblauch, Andrew H. Beck, and Alex Toker Abstract The PI3K–AKT signaling pathway regulates all phenotypes that contribute to progression of human cancers, including breast cancer. AKT mediates signal relay by phosphorylating numerous substrates, which are causally implicated in biologic responses such as cell growth, survival, metabolic reprogramming, migration, and invasion. Here a new AKT substrate is identified, the adherens junction protein Afadin, which is phosphorylated by AKT at Ser1718. Importantly, under conditions of physiologic IGF-1 signaling and oncogenic PI3K and AKT, Afadin is phosphorylated by all AKT isoforms, and this phosphorylation elicits a relocalization of Afadin from adherens junctions to the nucleus. Also, phosphorylation of Afadin increased breast cancer cell migration that was dependent on Ser1718 phosphorylation. Finally, nuclear localization of Afadin was observed in clinical breast cancer specimens, indicating that regulation of Afadin by the PI3K–AKT pathway has pathophysiologic significance. Implications: Phosphorylation of the adhesion protein Afadin by AKT downstream of the PI3K pathway, leads to redistribution of Afadin and controls cancer cell migration. Mol Cancer Res; 12(3); 464–76. Ó2013 AACR. Introduction of the proteins that function to transduce PI3K and Akt The phosphoinositide 3-kinase (PI3K) and Akt signaling signaling are enzymes with catalytic pockets, this pathway is pathway orchestrates virtually all aspects of epithelial and highly druggable and numerous phase I and II clinical trials tumor cell behavior, from initial transformation to dysplasia are underway with small-molecule inhibitors targeting PI3K and ultimately the dissemination of cancer cells to distant or Akt isoforms for single agent or combination therapy, metastatic sites (1).
    [Show full text]
  • Elucidating the Role of EPAC-Rap1 Signaling Pathway in the Blood
    Elucidating the Role of EPAC-Rap1 Signaling Pathway in the Blood-Retinal Barrier By Carla Jhoana Ramos A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Cellular and Molecular Biology) in The University of Michigan 2017 Doctoral Committee: Professor David A. Antonetti, Chair Professor Christin Carter-Su Associate Professor Philip J. Gage Professor Ben Margolis Assistant Professor Ann L. Miller Image of exchange factor directly activated by cAMP (EPAC), Ras-related protein (Rap1) and Zonula occludens-1 (ZO-1) immunofluorescent staining in bovine retinal endothelial cells, obtained by confocal microscopy. Carla Jhoana Ramos [email protected] ORCID iD: 0000-0001-5041-6758 © Carla Jhoana Ramos 2017 DEDICATION This dissertation is dedicated to my mother Victoria de Jesus Larios de Ramos, my father Jose Carlos Ramos, and my younger brother Hilmar Ramos. My parents migrated from El Salvador to the USA in search of better opportunities and dedicated their lives to providing us with a good education. They have instilled in me determination, perseverance and strength. Finally, my experience observing my mother cope with more than 26 years of diabetes and 10 years of mild diabetic retinopathy led me into diabetes research at the University of Michigan. ii ACKNOWLEDGEMENTS The completion of this dissertation has been made possible by the support from my community of mentors, friends and family. I would like to first thank my mentor David Antonetti, who gave me the opportunity to train in his laboratory under his guidance during these last 6 years. Additionally, I would like to thank my thesis committee: Ben Margolis, Christin Carter-Su, Philip Gage, and Ann Miller whose suggestions, questions, and scientific discussions helped improve the quality of this thesis and for their constant support and encouragement.
    [Show full text]
  • AFDN (Afadin, Adherens Junction Formation Factor)
    Atlas of Genetics and Cytogenetics in Oncology and Haematology OPEN ACCESS JOURNAL INIST-CNRS Gene Section Review AFDN (afadin, adherens junction formation factor) Jean Loup Huret, Philippe Dessen [email protected] (JLH); UMR 1170 INSERM, Gustave Roussy, 114 rue Edouard Vaillant, 94805 Villejuif 94805, France (PHD) Published in Atlas Database: December 2018 Online updated version : http://AtlasGeneticsOncology.org/Genes/AF6ID6.html Printable original version : http://documents.irevues.inist.fr/bitstream/handle/2042/70479/12-2018-AF6ID6.pdf DOI: 10.4267/2042/70479 This article is an update of : Huret JL. MLLT4 (myeloid/lymphoid or mixed-lineage leukemia (trithorax). Atlas Genet Cytogenet Oncol Haematol 1997;1(2) This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2019 Atlas of Genetics and Cytogenetics in Oncology and Haematology Abstract Afadin, the protein coded by AFDN (6q27), also known as AF6 or MLLT4, is a cytoskeletal and junction-associated protein that links nectins, transmembrane proteins, to the F-actin (actin cytoskeleton) in a type of cell-cell junctions: the adherens junctions (AJs). Afadin plays an important role in AJs integrity and apical-basal polarity. There is growing evidence of it's role in carcinogenesis. Keywords Afadin; AFDN; AF6; MLLT4; Cytoskeleton; Cell- cell junctions; Adherens junctions; Apical-basal polarity; Epithelial-mesenchymal transition; Tight junctions; Mitotic spindle orientation; Migration; Nectin; Actin; Acute myeloid leukemia; Breast Figure 1 Localization of AFDN (also called AF6 or MLLT4) cancer; Colon cancer; Pancreatic cancer; - Courtesy Mariano Rocchi. Endometrial cancer; Gastric cancer; Osteosarcoma; Neurone synapse. DNA/RNA Identity Description HGNC (Hugo): AFDN The AFDN gene, located on 6q27, has a genomic size of 145,030 bases and encodes for several Location: 6q27 transcripts on the position chr6:167826991- Other names: MLLT4, AF6 167972020 in the + strand.
    [Show full text]
  • A Novel Interface Consisting of Homologous Immunoglobulin Superfamily Members with Multiple Functions
    Cellular & Molecular Immunology (2010) 7, 11–19 ß 2010 CSI and USTC. All rights reserved 1672-7681/10 $32.00 www.nature.com/cmi REVIEW A novel interface consisting of homologous immunoglobulin superfamily members with multiple functions Zhuwei Xu and Boquan Jin Immunoglobulin superfamily (IgSF) members account for a large proportion of cell adhesion molecules that perform important immunological functions, including recognizing a variety of counterpart molecules on the cell surface or extracellular matrix. The findings that CD155/poliovirus receptor (PVR) and CD112/nectin-2 are the ligands for CD226/platelet and T-cell activation antigen 1 (PTA1)/DNAX accessory molecular-1 (DNAM-1), CD96/tactile and Washington University cell adhesion molecule (WUCAM) and that CD226 is physically and functionally associated with lymphocyte function-associated antigen-1 (LFA-1) on natural killer (NK) and activated T cells have largely expanded our knowledge about the functions of CD226, CD96, WUCAM and LFA-1 and their respective ligands, CD155, CD112, intercellular adhesion molecule (ICAM)-1 and junctional adhesion molecule (JAM)-1. The interactions of these receptors and their ligands are involved in many key functions of immune cells including naive T cells, cytotoxic T cells, NK cells, NK T cells, monocytes, dendritic cells, mast cells and platelets/megakaryocytes. Cellular & Molecular Immunology (2010) 7, 11–19; doi:10.1038/cmi.2009.108 Keywords: CD112; CD155; CD226; IgSF; LFA-1 INTRODUCTION LIGANDS FOR CD226 AND CD96 The concept of the immunoglobulin superfamily (IgSF) was proposed Nectin-2/CD112 has been identified as a ligand for CD226, whereas in the 1980s.1 IgSF molecules are generally transmembrane glycopro- PVR/CD155 serves as a ligand for both CD226 and CD96 receptors.
    [Show full text]
  • Loss of AF6/Afadin, a Marker of Poor Outcome in Breast Cancer, Induces Cell Migration, Invasiveness and Tumor Growth
    Oncogene (2011) 30, 3862–3874 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE Loss of AF6/afadin, a marker of poor outcome in breast cancer, induces cell migration, invasiveness and tumor growth G Fournier1,4,5, O Cabaud1,4,5, E Josselin1,4,5, A Chaix2,4,5, J Ade´laı¨de4,5, D Isnardon4,5, A Restouin3,4,5, R Castellano3,4,5, P Dubreuil2,4,5, M Chaffanet1,4,5, D Birnbaum1 and M Lopez1,4,5 1Inserm, UMR 891, Laboratoire d’oncologie mole´culaire, CRCM, Marseille, France; 2Inserm, UMR 891, Laboratoire d’he´matopoı¨e`se fonctionnelle et mole´culaire, CRCM, Marseille, France; 3Inserm, UMR 891, Plateforme TrGET essais pre´-cliniques, CRCM, Marseille, France; 4Institut Paoli-Calmettes, Marseille, France and 5Univ. Me´diterrane´e, Marseille, France Afadin/AF6, an F-actin-binding protein, is ubiquitously leading to improvement of classification and prognosis expressed in epithelia and has a key role during develop- (Sorlie et al., 2001). Despite this, adverse evolution can ment, through its regulatory role in cell–cell junction still be observed for patients with apparent good organization. Afadin loss of expression in 15% of breast prognosis at diagnosis and vice versa. New molecular carcinoma is associated with adverse prognosis and markers that help define prognosis are needed. We increased risk of metastatic relapse. To determine the previously showed that afadin expression is lost in 15% role of afadin in breast cancer, we studied the functional of breast cancer irrespective of the molecular subtype consequences of afadin protein extinction using in vitro (Letessier et al., 2007).
    [Show full text]
  • Afadin Orients Cell Division to Position the Tubule Lumen in Developing Renal Tubules Lei Gao1, Zhufeng Yang1, Chitkale Hiremath1, Susan E
    © 2017. Published by The Company of Biologists Ltd | Development (2017) 144, 3511-3520 doi:10.1242/dev.148908 RESEARCH ARTICLE Afadin orients cell division to position the tubule lumen in developing renal tubules Lei Gao1, Zhufeng Yang1, Chitkale Hiremath1, Susan E. Zimmerman1, Blake Long1, Paul R. Brakeman2, Keith E. Mostov3, David M. Bryant4, Katherine Luby-Phelps5 and Denise K. Marciano1,* ABSTRACT can polarize along its apical-basal axis, which orients the cell In many types of tubules, continuity of the lumen is paramount to relative to the underlying extracellular matrix. The apical surface tubular function, yet how tubules generate lumen continuity in vivo is faces the luminal space, the lateral surface contacts adjacent cells, not known. We recently found that the F-actin-binding protein afadin is and the basal surface contacts the basal lamina. Additionally, the required for lumen continuity in developing renal tubules, though its individually polarized cells must coordinate their polarity with mechanism of action remains unknown. Here, we demonstrate that surrounding cells along the long axis of the tubule, in a process afadin is required for lumen continuity by orienting the mitotic spindle referred to as planar cell polarity. during cell division. Using an in vitro 3D cyst model, we find that afadin In the developing kidney, epithelial tubules are derived from localizes to the cell cortex adjacent to the spindle poles and orients progenitor cells from two embryonic sources: the cap mesenchyme the mitotic spindle. In tubules, cell division may be oriented relative to and the ureteric epithelium (Saxen, 1987). The cap mesenchyme two axes: longitudinal and apical-basal.
    [Show full text]