DOCSLIB.ORG

Closely Related Rap1a and 1B Proteins Functions Suggesting Distinct Roles for the Rap1a Null Mice Have Altered Myeloid Cell

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Closely Related Rap1a and 1B Proteins Functions Suggesting Distinct Roles for the Rap1a Null Mice Have Altered Myeloid Cell Rap1a Null Mice Have Altered Myeloid Cell Functions Suggesting Distinct Roles for the Closely Related Rap1a and 1b Proteins This information is current as Yu Li, Jingliang Yan, Pradip De, Hua-Chen Chang, Akira of September 25, 2021. Yamauchi, Kent W. Christopherson II, Nivanka C. Paranavitana, Xiaodong Peng, Chaekyun Kim, Veerendra Munugulavadla, Reuben Kapur, Hanying Chen, Weinian Shou, James C. Stone, Mark H. Kaplan, Mary C. Dinauer, Donald L. Durden and Lawrence A. Quilliam Downloaded from J Immunol 2007; 179:8322-8331; ; doi: 10.4049/jimmunol.179.12.8322 http://www.jimmunol.org/content/179/12/8322 http://www.jimmunol.org/ References This article cites 79 articles, 49 of which you can access for free at: http://www.jimmunol.org/content/179/12/8322.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 25, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Rap1a Null Mice Have Altered Myeloid Cell Functions Suggesting Distinct Roles for the Closely Related Rap1a and 1b Proteins1 Yu Li,2*¶ Jingliang Yan,*¶ Pradip De,ʈ Hua-Chen Chang,†‡§ Akira Yamauchi,§ Kent W. Christopherson II,** Nivanka C. Paranavitana,3*¶ Xiaodong Peng,§ Chaekyun Kim,§# Veerendra Munugulavadla,§ Reuben Kapur,*‡§ Hanying Chen,§ Weinian Shou,*§ James C. Stone,†† Mark H. Kaplan,†‡§ Mary C. Dinauer,†‡§ Donald L. Durden,ʈ and Lawrence A. Quilliam4*¶ The Ras-related GTPases Rap1a and 1b have been implicated in multiple biological events including cell adhesion, free radical Downloaded from production, and cancer. To gain a better understanding of Rap1 function in mammalian physiology, we deleted the Rap1a gene. Although loss of Rap1a expression did not initially affect mouse size or viability, upon backcross into C57BL/6J mice some ,Rap1a؊/؊ embryos died in utero. T cell, B cell, or myeloid cell development was not disrupted in Rap1a ؊/؊ mice. However macrophages from Rap1a null mice exhibited increased haptotaxis on fibronectin and vitronectin matrices that correlated with decreased adhesion. Chemotaxis of lymphoid and myeloid cells in response to CXCL12 or CCL21 was significantly reduced. In contrast, an increase in FcR-mediated phagocytosis was observed. Because Rap1a was previously copurified with the human http://www.jimmunol.org/ neutrophil NADPH oxidase, we addressed whether GTPase loss affected superoxide production. Neutrophils from Rap1a؊/؊ mice had reduced fMLP-stimulated superoxide production as well as a weaker initial response to phorbol ester. These results suggest that, despite 95% amino acid sequence identity, similar intracellular distribution, and broad tissue distribution, Rap1a and 1b are not functionally redundant but rather differentially regulate certain cellular events. The Journal of Immunology, 2007, 179: 8322–8331. he small GTPase Ras is an important regulator of cell but encoded by a separate gene, also exists (7) in addition to three growth and differentiation (1). It operates as a molecular Rap2 proteins that share ϳ65% identity with Rap1 (2, 8, 9). Like switch, alternating between inactive GDP and active Ras, these GTPases are activated by guanine nucleotide exchange by guest on September 25, 2021 T 5 GTP-bound states and is the prototype for more than 130 Ras- factors (GEFs) and turned off by GTPase activating proteins related proteins that regulate a myriad of biological functions (1). (GAPs). The activation of Raps by a large number of diversely Rap1a/Krev-1/Smg p21 was originally identified because of ho- regulated GEFs suggests that they play ubiquitous roles in cell mology with Ras (2), by purification of GTP binding proteins (3, biology triggered by multiple extracellular stimuli and associated 4), by copurification with the neutrophil NADPH oxidase (5), and signaling pathways, reviewed in Ref. 10–12. Although Rap1 and by its ability to revert the phenotype of Ras-transformed cells (6). 2 are regulated by the same GEFs and GAPs, until very recently A closely related protein, Rap1B, sharing 95% amino acid identity little was known about their biological functions. Even less is known about the functional redundancy of Rap1a and 1b. It was originally reported that Rap1 operates as an antagonist of † *Department of Biochemistry and Molecular Biology, Department of Microbiology Ras, competing with it for downstream effectors such as c-Raf-1. and Immunology, ‡Department of Pediatrics, Medical and Molecular Genetics, and §Wells Center for Pediatric Research, Indiana University School of Medicine, Indi- Although this can occur in certain cell types (13), Rap1 has more anapolis, IN 46202; ¶Walther Cancer Institute, Indianapolis, IN 46208; ʈAflac Cancer recently been found to play a number of additional roles. These Center and Blood Disorders, Department of Pediatrics, Emory University, Atlanta, GA 30322; #Inha University College of Medicine, Incheon, Korea; **Rush University include activation of the ERK/MAPK pathway via B-Raf, promo- Medical Center, Chicago, IL 60612; and ††Department of Biochemistry, University of tion of cell-matrix adhesion through inside-out activation of inte- Alberta, Edmonton, Alberta, Canada grins via RAPL and RIAM, and regulation of cell-cell junctions Received for publication June 26, 2007. Accepted for publication October 12, 2007. (reviewed in Refs. 12, 14, 15). Overactivation of Rap1 has also The costs of publication of this article were defrayed in part by the payment of page been recently associated with hemopoietic malignancy (16, 17). charges. This article must therefore be hereby marked advertisement in accordance Loss of the only rap1 gene in Drosophila is embryonic lethal (18). with 18 U.S.C. Section 1734 solely to indicate this fact. Similarly, loss of C3G/RapGEF1, the most ubiquitously expressed 1 This work was supported by an Indiana University School of Medicine Biomedical Research Grant, the Indiana Genomics Initiative of Indiana University, supported in Rap exchange factor is lethal to mice (19, 20). However, because part by Lilly Endowment Incorporated, and by National Institutes of Health Grants C3G also acts on additional Ras family proteins, it was not clear CA108647 and HL69974 (all to L.A.Q.). Y.L. was supported by American Heart Association predoctoral fellowship 0110245Z. 2 Current address: Amgen, One Amgen Center Drive Thousand Oaks, CA 91302. 3 Current address: Applied Biosystems, 2130 Woodward Street, Austin, TX 78744. 5 Abbreviations used in this paper: GEF, guanine nucleotide exchange factor; GAP, 4 Address correspondence and reprint requests to Dr. Lawrence A. Quilliam, Depart- GTPase activating protein; ES cells, embryonic stem cells; KO, knockout; WT, wild ment of Biochemistry and Molecular Biology, 635 Barnhill Drive, MS-4053, Indiana type; MEF, mouse embryo fibroblast; BMD, bone marrow-derived; University School of Medicine, Indianapolis, IN 46202. E-mail address: [email protected] Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 www.jimmunol.org The Journal of Immunology 8323 whether this lethality was solely due to the loss of Rap1 activity fragment as template to confirm the long arm structure and correct inte- (21–24). gration 3Ј to the recombination site. PstI-digested ES cell genomic DNA To better understand the role of Rap1 in mammalian cell biol- was hybridized with a neo probe using a XhoI-SalI fragment from the neomycin resistance gene to confirm correct integration of 5Ј to the re- ogy and to address possible redundancy between the 1a and 1b combination site. Four ES cell clones were confirmed to contain the tar- Ϫ/Ϫ proteins, we generated a mouse lacking the rap1a gene. rap1A geted disruption and used to generate Rap1a KO mice by standard proce- mice initially exhibited no overt phenotype. However, upon back- dures (30) using C57BL/6J blastocysts and pseudopregnant females as crossing mice into the C57BL/6J strain, some embryonic lethality foster mothers. Chimeric mice were successfully generated from just one clone. These were screened by PCR and by Southern blotting for the pres- was observed. Because of the high abundance of Rap1a in human ence of the disrupted Rap1a allele and mated with C57BL/6J mice to platelets (25) and Rap1a in human neutrophils (26) where it asso- produce F1 heterozygous mice. Heterozygotes for the disrupted gene were ciates with the phagocyte NADPH oxidase (5, 27), we examined identified by PCR using digit samples along with three primers, the com- several properties of Rap1a-null white blood cells. Although there mon primer Rap1a 5Ј KO, the neo specific primer ADPR266, and the wild-type (WT) allele specific primer RapExon (5Ј-CCGTAAAATCTGT were no obvious defects in blood cell development, changes in Ј TCTCTC AAGTC-3 ). Confirmed heterozygous F1 mice were mated with macrophage adhesion, haptotaxis,
Load more

Recommended publications
  • Supplementary Materials
    Supplementary Materials COMPARATIVE ANALYSIS OF THE TRANSCRIPTOME, PROTEOME AND miRNA PROFILE OF KUPFFER CELLS AND MONOCYTES Andrey Elchaninov1,3*, Anastasiya Lokhonina1,3, Maria Nikitina2, Polina Vishnyakova1,3, Andrey Makarov1, Irina Arutyunyan1, Anastasiya Poltavets1, Evgeniya Kananykhina2, Sergey Kovalchuk4, Evgeny Karpulevich5,6, Galina Bolshakova2, Gennady Sukhikh1, Timur Fatkhudinov2,3 1 Laboratory of Regenerative Medicine, National Medical Research Center for Obstetrics, Gynecology and Perinatology Named after Academician V.I. Kulakov of Ministry of Healthcare of Russian Federation, Moscow, Russia 2 Laboratory of Growth and Development, Scientific Research Institute of Human Morphology, Moscow, Russia 3 Histology Department, Medical Institute, Peoples' Friendship University of Russia, Moscow, Russia 4 Laboratory of Bioinformatic methods for Combinatorial Chemistry and Biology, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Moscow, Russia 5 Information Systems Department, Ivannikov Institute for System Programming of the Russian Academy of Sciences, Moscow, Russia 6 Genome Engineering Laboratory, Moscow Institute of Physics and Technology, Dolgoprudny, Moscow Region, Russia Figure S1. Flow cytometry analysis of unsorted blood sample. Representative forward, side scattering and histogram are shown. The proportions of negative cells were determined in relation to the isotype controls. The percentages of positive cells are indicated. The blue curve corresponds to the isotype control. Figure S2. Flow cytometry analysis of unsorted liver stromal cells. Representative forward, side scattering and histogram are shown. The proportions of negative cells were determined in relation to the isotype controls. The percentages of positive cells are indicated. The blue curve corresponds to the isotype control. Figure S3. MiRNAs expression analysis in monocytes and Kupffer cells. Full-length of heatmaps are presented.
    [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]
  • DLL1- and DLL4-Mediated Notch Signaling Is Essential for Adult Pancreatic Islet
    Page 1 of 41 Diabetes DLL1- and DLL4-mediated Notch signaling is essential for adult pancreatic islet homeostasis (running title –Role of Delta ligands in adult pancreas) Marina Rubey1,2,6*, Nirav Florian Chhabra1,2*, Daniel Gradinger1,2,7, Adrián Sanz-Moreno1, Heiko Lickert2,4,5, Gerhard K. H. Przemeck1,2, Martin Hrabě de Angelis1,2,3** 1 Helmholtz Zentrum München, Institute of Experimental Genetics and German Mouse Clinic, Neuherberg, Germany 2 German Center for Diabetes Research (DZD), Neuherberg, Germany 3 Chair of Experimental Genetics, Centre of Life and Food Sciences, Weihenstephan, Technische Universität München, Freising, Germany 4 Helmholtz Zentrum München, Institute of Diabetes and Regeneration Research and Institute of Stem Cell Research, Neuherberg, Germany 5 Technische Universität München, Medical Faculty, Munich, Germany 6 Present address Marina Rubey: WMC Healthcare GmbH, Munich, Germany 7 Present address Daniel Gradinger: PSI CRO AG, Munich, Germany *These authors contributed equally **Corresponding author: Prof. Dr. Martin Hrabě de Angelis, Helmholtz Zentrum München, German Research Center for Environmental Health, Institute of Experimental Genetics, Ingolstädter Landstr.1, 85764 Neuherberg, Germany. Phone: +49-89-3187-3502. Fax: +49- 89-3187-3500. E-mail address: [email protected] Word count – 4088 / Figures – 7 Diabetes Publish Ahead of Print, published online February 6, 2020 Diabetes Page 2 of 41 Abstract Genes of the Notch signaling pathway are expressed in different cell types and organs at different time points during embryonic development and adulthood. The Notch ligand Delta- like 1 (DLL1) controls the decision between endocrine and exocrine fates of multipotent progenitors in the developing pancreas, and loss of Dll1 leads to premature endocrine differentiation.
    [Show full text]
  • Development of Novel Drugs Targeting Chaperones of Oncogenic K-Ras
    Farid Ahmad Siddiqui Farid Ahmad Siddiqui // Development of Novel Drugs Development of Novel Drugs Targeting Chaperones of Oncogenic K-Ras of Oncogenic Chaperones K-Ras Drugs Targeting of Novel Development Targeting Chaperones of Oncogenic K-Ras // 2021 9 789521 240317 ISBN 978-952-12-4031-7 Development of Novel Drugs Targeting Chaperones of Oncogenic K-Ras Farid Ahmad Siddiqui Cell Biology Faculty of Science and Engineering, Åbo Akademi University Turku Bioscience Centre University of Turku & Åbo Akademi University Turku, Finland, 2021 From the Turku Bioscience Centre, University of Turku and Åbo Akademi University, Faculty of Science and Engineering, Åbo Akademi University, Turku, Finland Supervised by Prof. Daniel Abankwa, PhD Department of Life Sciences and Medicine University of Luxembourg, Belval campus Luxembourg Reviewed by Prof. Olli Mikael Carpen, PhD Faculty of Medicine University of Helsinki Helsinki, Finland and Prof. Klaus Elenius, PhD Faculty of Medicine University of Turku Turku, Finland Opponent Prof. Krishnaraj Rajalingam, PhD Head, Cell Biology Unit, UMC-Mainz, Germany Author’s address Turku Bioscience Centre Åbo Akademi University Tykistökatu 6 20520 Turku Finland Email: [email protected] ISBN 978-952-12-4031-7 (printed) ISBN 978-952-12-4032-4 (digital) Painosalama Oy, Turku, Finland 2021 TABLE OF CONTENTS ABSTRACT .......................................................................................................... 6 ABSTRAKT (Swedish Abstract) .........................................................................
    [Show full text]
  • Gene Section Short Communication
    Atlas of Genetics and Cytogenetics in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS Gene Section Short Communication RAP1A (RAP1A, member of RAS oncogene family) Jean de Gunzburg Laboratoire de Signalisation Intracellulaire et Oncogenèse INSERM U-528 Institut Curie Section de Recherche 26, rue d'Ulm, 75248 Paris Cedex 05, France (Jd) Published in Atlas Database: May 2001 Online updated version : http://AtlasGeneticsOncology.org/Genes/RAP1AID272.html DOI: 10.4267/2042/37748 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2001 Atlas of Genetics and Cytogenetics in Oncology and Haematology two isoforms, Rap1A and Rap1B that share 95% Identity identity and are encoded by two different genes. Rap1 HGNC (Hugo): RAP1A proteins share 50% identity with Ras proteins, Location: 1p13.3 including the regions involved in GDP/GTP binding (hence Rap1A has very similar biochemical properties to Ras), C-terminal CAAX domain leading to prenylation (geranylgeranylation in the case of Rap1A), and effector region identical to that of Ras proteins causing Ras and Rap1 to share some potential effectors. Probe(s) - Courtesy Mariano Rocchi, Resources for Molecular Expression Cytogenetics. Ubiquitous ; higher in brain and hemopo•etic tissues. DNA/RNA Localisation Description Rap1 is bound to membranes. In many cell types, it is found in a perinuclear compartment overlapping the 6 coding exons covering 18095 bp on chromosome 1. Golgi. Rap1 proteins (A and B) are phosphorylated near the C-ter by cAMP-dependent protein kinase. This Protein results in translocation of part of the Rap1 pool to the Description cytosol.
    [Show full text]
  • Rap1 Regulates Hepatic Stellate Cell Migration Through the Modulation of Rhoa Activity in Response to TGF‑Β1
    INTERNATIONAL JOURNAL OF MOleCular meDICine 44: 491-502, 2019 Rap1 regulates hepatic stellate cell migration through the modulation of RhoA activity in response to TGF‑β1 MI-YOUNG MOON1, HEE-JUN KIM2, MO-JONG KIM2, SUNHO UHM1, JI‑WON PARK1, KI-TAE SUK3, JAE‑BONG PARK4, DONG-JUN KIM3 and SUNG-EUN KIM1 1Department of Internal Medicine, Hallym University Sacred Heart Hospital, College of Medicine, Hallym University, Anyang, Gyeonggi 14068; 2Ilsong Institute of Life Science, Hallym University, Anyang, Gyeonggi 14066; 3Department of Internal Medicine, Hallym University Chuncheon Sacred Heart Hospital, College of Medicine, Hallym University, Chuncheon, Gangwon 24253; 4Department of Biochemistry, College of Medicine, Hallym University, Chuncheon, Gangwon 24252, Republic of Korea Received November 1, 2018; Accepted May 28, 2019 DOI: 10.3892/ijmm.2019.4215 Abstract. Although the migration of hepatic stellate cells activation of RhoA in TGF‑β1-stimulated HSC‑T6 cells. These (HSCs) is important for hepatic fibrosis, the regulation of this findings suggest that TGF‑β1 regulates Rap1, resulting in the migration is poorly understood. Notably, transforming growth suppression of RhoA, activation of and formation of F‑actin factor (TGF)-β1 induces monocyte migration to sites of injury during the migration of HSCs. or inflammation during the early phase, but inhibits cell migra- tion during the late phase. In the present study, the role of Introduction transforming protein RhoA signaling in TGF-β1-induced HSC migration was investigated. TGF‑β1 was found to increase Hepatic fibrosis is characterized by the excessive deposition the protein and mRNA levels of smooth muscle actin and of extracellular matrix (ECM) mediated by activated hepatic collagen type I in HSC‑T6 cells.
    [Show full text]
  • RAP1-Mediated MEK/ERK Pathway Defects in Kabuki Syndrome
    The Journal of Clinical Investigation RESEARCH ARTICLE RAP1-mediated MEK/ERK pathway defects in Kabuki syndrome Nina Bögershausen,1,2,3 I-Chun Tsai,4 Esther Pohl,1,2,3 Pelin Özlem Simsek Kiper,5 Filippo Beleggia,1,2,3 E. Ferda Percin,6 Katharina Keupp,1,2,3 Angela Matchan,7 Esther Milz,1,2,3 Yasemin Alanay,5,8 Hülya Kayserili,9 Yicheng Liu,1,2,3 Siddharth Banka,10 Andrea Kranz,11 Martin Zenker,12 Dagmar Wieczorek,13 Nursel Elcioglu,14 Paolo Prontera,15 Stanislas Lyonnet,16 Thomas Meitinger,17 A. Francis Stewart,11 Dian Donnai,10 Tim M. Strom,17,18 Koray Boduroglu,5 Gökhan Yigit,1,2,3 Yun Li,1,2,3 Nicholas Katsanis,4 and Bernd Wollnik1,2,3 1Institute of Human Genetics, 2Center for Molecular Medicine Cologne (CMMC), and 3Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany. 4Center for Human Disease Modeling and Department of Cell Biology, Duke University, Durham, North Carolina, USA. 5Pediatric Genetics Unit, Department of Pediatrics, Hacettepe University Medical Faculty, Ankara, Turkey. 6Department of Medical Genetics, Gazi University Faculty of Medicine, Ankara, Turkey. 7Oxford Gene Technology (OGT), Begbroke Science Park, Begbroke, Oxfordshire, United Kingdom. 8Pediatric Genetics, Department of Pediatrics, Acibadem University, School of Medicine, Istanbul, Turkey. 9Medical Genetics Department, Koç University, School of Medicine, Istanbul, Turkey. 10Department of Genetic Medicine, St. Mary’s Hospital, Manchester Academic Health Sciences Centre (MAHSC), University of Manchester, Manchester, United Kingdom. 11Genomics, Bio-Innovationszentrum, Dresden University of Technology, Dresden, Germany. 12Institute of Human Genetics, University Hospital Magdeburg, Magdeburg, Germany.
    [Show full text]
  • Rap1a and Rap1b Ras-Family Proteins Are Prominently Expressed in the Nucleus of Squamous Carcinomas: Nuclear Translocation of GTP-Bound Active Form
    Oncogene (2003) 22, 6243–6256 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc Rap1A and rap1B ras-family proteins are prominently expressed in the nucleus of squamous carcinomas: nuclear translocation of GTP-bound active form Raj S Mitra1,4, Zhaocheng Zhang1,4, Bradley S Henson1, David M Kurnit2, Thomas ECarey 1,3, Nisha J D’Silva*,1 1Department of Oral Medicine, Pathology and Oncology, University of Michigan School of Dentistry, Ann Arbor, MI 48109-1078, USA; 2Departments of Pediatrics and Human Genetics, University of Michigan Medical School, Ann Arbor, MI 481091, USA; 3Department of Otolaryngology, Laboratory of Head and Neck Cancer Biology, The University of Michigan Medical School and the Comprehensive Cancer Center, Ann Arbor, MI 48109-0506, USA We recently showed that rap1 regulates growth and exclusively in eukaryotes (Takai et al., 2001). Those proliferation in normal keratinocytes, which provoked us SMGs that have been identified (currently 4100) have to investigate its expression and regulation in malignant homology to ras, and belong to one of the five cells. Rap1 is variably expressed in whole cell lysates of subfamilies namely ras, rho, rab, sar1/arf and ran squamous cell carcinoma (SCC) cell lines. Immunoblot (Takai et al., 2001). These ras-like proteins shuttle analysis of nuclear and cytosolic fractions and immuno- between inactive GDP- and active GTP-bound states. histochemistry revealed that in addition to cytoplasmic Ras subfamily proteins, including ras and rap1, are key expression, SCC cells also exhibit prominent punctate players in receptor-linked signaling pathways that rap1 expression in the nucleus.
    [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]
  • Distinct Actions of Rab3 and Rab27 Gtpases on Late Stages of Exocytosis of Insulin
    © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd doi:10.1111/tra.12182 Distinct Actions of Rab3 and Rab27 GTPases on Late Stages of Exocytosis of Insulin Victor A. Cazares1,2, Arasakumar Subramani1, Johnny J. Saldate1,2, Widmann Hoerauf1,2 and Edward L. Stuenkel1,2,∗ 1Department of Molecular & Integrative Physiology, University of Michigan, Ann Arbor, MI 48109, USA 2Neuroscience Graduate Program, University of Michigan, Ann Arbor, MI 48109, USA ∗Corresponding author: Edward L. Stuenkel, [email protected] Abstract Rab GTPases associated with insulin-containing secretory granules (SGs) readily releasable pool (RRP). By comparison, nucleotide cycling of Rab3 are key in targeting, docking and assembly of molecular complexes GTPases, but not of Rab27A, is essential for a kinetically rapid filling of governing pancreatic β-cell exocytosis. Four Rab3 isoforms along with the RRP with SGs. Aside from these distinct functions, Rab3 and Rab27A Rab27A are associated with insulin granules, yet elucidation of the GTPases demonstrate considerable functional overlap in building the distinct roles of these Rab families on exocytosis remains unclear. To readily releasable granule pool. Hence, while Rab3 and Rab27A coop- define specific actions of these Rab families we employ Rab3GAP erate to generate release-ready SGs in β-cells, they also direct unique and/or EPI64A GTPase-activating protein overexpression in β-cells from kinetic and functional properties of the exocytotic pathway. wild-type or Ashen mice to selectively transit the entire Rab3 fam- Keywords exocytosis, GTPase, insulin secretion, membrane fusion, Rab ily or Rab27A to a GDP-bound state. Ashen mice carry a sponta- proteins neous mutation that eliminates Rab27A expression.
    [Show full text]
  • Site-Specific Processing of Ras and Rap1 Switch I by a MARTX Toxin
    ARTICLE Received 8 Jan 2015 | Accepted 1 May 2015 | Published 8 Jun 2015 DOI: 10.1038/ncomms8396 OPEN Site-specific processing of Ras and Rap1 Switch I by a MARTX toxin effector domain Irena Antic1,*, Marco Biancucci1,*, Yueming Zhu2, David R. Gius2 & Karla J.F. Satchell1 Ras (Rat sarcoma) protein is a central regulator of cell growth and proliferation. Mutations in the RAS gene are known to occur in human cancers and have been shown to contribute to carcinogenesis. In this study, we show that the multifunctional-autoprocessing repeats-in- toxin (MARTX) toxin-effector domain DUF5Vv from Vibrio vulnificus to be a site-specific endopeptidase that cleaves within the Switch 1 region of Ras and Rap1. DUF5Vv processing of Ras, which occurs both biochemically and in mammalian cell culture, inactivates ERK1/2, thereby inhibiting cell proliferation. The ability to cleave Ras and Rap1 is shared by DUF5Vv homologues found in other bacteria. In addition, DUF5Vv can cleave all Ras isoforms and KRas with mutations commonly implicated in malignancies. Therefore, we speculate that this new family of Ras/Rap1-specific endopeptidases (RRSPs) has potential to inactivate both wild-type and mutant Ras proteins expressed in malignancies. 1 Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, 303 East Chicago Avenue, Ward 6-225, Chicago, Illinois 60611, USA. 2 Department of Radiation Oncology and Pharmacology, Feinberg School of Medicine, Northwestern University, 303 East Superior Avenue, Lurie 3-119, Chicago, Illinois 60611, USA. * These authors contributed equally to this work. Correspondence and requests for materials should be addressedto K.J.F.S.
    [Show full text]
  • Small Gtpases of the Ras and Rho Families Switch On/Off Signaling
    International Journal of Molecular Sciences Review Small GTPases of the Ras and Rho Families Switch on/off Signaling Pathways in Neurodegenerative Diseases Alazne Arrazola Sastre 1,2, Miriam Luque Montoro 1, Patricia Gálvez-Martín 3,4 , Hadriano M Lacerda 5, Alejandro Lucia 6,7, Francisco Llavero 1,6,* and José Luis Zugaza 1,2,8,* 1 Achucarro Basque Center for Neuroscience, Science Park of the Universidad del País Vasco/Euskal Herriko Unibertsitatea (UPV/EHU), 48940 Leioa, Spain; [email protected] (A.A.S.); [email protected] (M.L.M.) 2 Department of Genetics, Physical Anthropology, and Animal Physiology, Faculty of Science and Technology, UPV/EHU, 48940 Leioa, Spain 3 Department of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Granada, 180041 Granada, Spain; [email protected] 4 R&D Human Health, Bioibérica S.A.U., 08950 Barcelona, Spain 5 Three R Labs, Science Park of the UPV/EHU, 48940 Leioa, Spain; [email protected] 6 Faculty of Sport Science, European University of Madrid, 28670 Madrid, Spain; [email protected] 7 Research Institute of the Hospital 12 de Octubre (i+12), 28041 Madrid, Spain 8 IKERBASQUE, Basque Foundation for Science, 48013 Bilbao, Spain * Correspondence: [email protected] (F.L.); [email protected] (J.L.Z.) Received: 25 July 2020; Accepted: 29 August 2020; Published: 31 August 2020 Abstract: Small guanosine triphosphatases (GTPases) of the Ras superfamily are key regulators of many key cellular events such as proliferation, differentiation, cell cycle regulation, migration, or apoptosis. To control these biological responses, GTPases activity is regulated by guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs), and in some small GTPases also guanine nucleotide dissociation inhibitors (GDIs).
    [Show full text]