Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München Biochemical and cell biological characterisation of the SUMO E1 activating enzyme Aos1/Uba2 Katarzyna Chmielarska aus Kielce, Polen 2005 Erklärung Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung vom 29. Januar 1998 von Prof. Dr. Frauke Melchior and Prof. Dr. Patrick Cramer betreut. Ehrenwörtliche Versicherung Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet. München, 6.10.2005 Katarzyna Chmielarska Dissertation eingereicht am 6.10.2005 1. Gutachter: Prof. Dr. Patrick Cramer 2. Gutachter: Prof. Dr. Alexander Pfeifer Mündliche Prüfung am 20.12.2005 TABLE OF CONTENTS LIST OF ABBREVIATIONS 1 1 SUMMARY 4 2 INTRODUCTION 4 2.1 Overview 4 2.2 Molecular mechanism of Sumoylation 9 2.2.1 Enzymes mediating the SUMO pathway 9 2.2.2 SUMO-Activating Enzyme (E1) 10 2.2.3 SUMO-Conjugating Enzyme (E2) 13 2.2.4 E3 ligases 14 2.2.5 SUMO processing and deconjugation 18 2.3 Function of SUMO modifications 19 2.3.1 Molecular consequences of sumoylation 20 2.3.2 Pathways in which SUMO was found 21 3 MATERIALS AND METHODS 24 3.1 Materials 24 3.1.1 Chemicals and materials 24 3.1.2 Reagents and reaction kits 25 3.1.3 Buffers and solutions 26 3.1.4 Cell culture 28 3.1.5 Antibodies 29 3.1.6 Recombinant proteins 31 3.1.7 Plasmids 31 3.1.8 Important oligonucleotides 32 3.1.9 Bacterial strains 33 3.2 Molecular biology methods 34 3.2.1 Plasmid DNA purification (miniprep) 34 3.2.2 Plasmid DNA (midi, maxiprep) 34 3.2.3 Preparation of chemically competent cells 34 3.2.4 Estimation of DNA concentration 35 3.2.5 Digestion of plasmid DNA with restriction endonucleases 35 3.2.6 DNA purification 36 3.2.7 Ligation of DNA insert into vector DNA 36 3.2.8 Transformation into DH5 α 36 3.2.9 Polymerase Chain Reaction (PCR) 37 3.2.10 Mutagenesis 37 3.2.11 Sequencing 40 3.2.12 Organ extracts 40 3.2.13 RNA isolation 40 3.2.14 RT-PCR 41 3.3 Cell biology methods 41 3.3.1 Cell lines 41 3.3.2. Transient transfection 42 3.3.3 Fluorescence microscopy 43 3.3.4 Reporter luciferase assay 43 3.4 Biochemistry methods 44 3.4.1 SDS-PAGE 44 3.4.2 Coomassie staining 44 3.4.3 Silver staining 44 3.4.4 Immunoblotting 45 3.4.5 Expression and purification of recombinant proteins 46 3.4.6 Measurement of protein concentration 48 3.4.7 In vitro SUMOylation 48 3.4.8 Fluorescence Resonance Energy Transfer (FRET) 48 3.4.9 Generation and purification of rabbit or goat polyclonal antibodies 50 3.4.10 Immunoprecipitation 50 3.4.11 His-SUMO pull down 52 3.4.12 Mass Spectrometry 52 4 RESULTS 4.1 PART 1: Characterisation of an Uba2 splice variant 54 4.1.1 Identification of Uba2 splice variant 54 4.1.2 Generation and characterisation of Uba2 polyclonal antibody in goat 56 4.1.3 In vivo and in vitro analysis of Uba2 splice variant 59 4.1.4 Finding new binding partners for Uba2 full length and Uba2 splice variant 64 4.2 PART 2: Identification of a novel SUMO target 66 4.2.1 Affinity purification and characterisation of Aos1 polyclonal antibody 66 4.2.2 α Aos1 antibodies decorate the nucleus and Golgi structures 70 4.2.3 A small fraction of Aos1 and Uba2 are present in unsoluble pellet fractions 70 4.2.4 Aos1/Uba2 is present in Golgi fraction 73 4.2.5 Identification of a new binding partner (ELKS) for Aos1 73 4.2.6 Published functions of ELKS 76 4.2.7 Generation of recombinant ELKS 78 4.2.8 Generation and characterisation of ELKS polyclonal antibodies 81 4.2.9 Conformation of ELKS-Aos1 interaction 83 4.2.10 Subcellular localisation of ELKS 85 4.2.11 Recombinant of ELKS and Aos1 do not bind 88 4.2.12 Rab6 is not the binding factor between ELKS and Aos1 88 4.2.13 ELKS does not interact directly with SUMO in vitro 90 4.2.14 Identification of binding partner candidates for Aos1 and ELKS 91 4.2.15 ELKS interacts with RanBP2 in interphase cell extract 93 4.2.16 ELKS does not interact directly with BP2 ∆FG in vitro 94 4.2.17 ELKS does not inhibit sumoylation 95 4.2.18 ELKS is SUMO modified on 3 distinct lysine residues 98 4.2.19 ELKS sumoylation decreases its NF κΒ stimulating activity 106 5 APPENDIX 110 6 DISCUSSION 112 6.1 Characterisation of variant Uba2 112 6.2 Identification of a novel SUMO target 113 5.2.1 Identification of a new Aos1 binding partner 113 5.2.2 ELKS is a protein implicated in NF κB signaling and vesicle transport 114 6.3 Identification of additional ELKS and Aos1 binding partners suggest possible functions in vesicle transport and NF κκκB signaling 117 6.4 ELKS is a new SUMO substrate 118 6.5 SUMO does not change ELKS localisation and Rab6 interaction 119 6.6 ELKS negatively regulates the NF κκκB signaling pathway 120 6.7 RanBP2-an E3 ligase for ELKS? 122 7 REFERENCES 124 8 ACKNOWLEGMENTS 139 9 PUBLICATIONS 140 10 CURRICULUM VITAE 141 Abbreviations 1 ABBREVIATIONS aa amino acid Ac acetat AP aprotinin ATP adenosine-5‘-triphosphate bp base pair BSA bovine serum albumin cDNA complementary DNA Cys cysteine Da dalton dNTP 2‘-Didesoxynucleosid-5‘-triphospate ddH20 double-destilled water DEPC diethyl pyrocarbonate DMEM Dulbecco‘s modified Eagle‘s medium DMSO dimethyl sulfoxide DTT dithiothreitol E.coli Escherichia coli ECL enhanced chemoluminescence EDTA ethylenediaminetetraacetic EGTA ethyleneglycoltetra acetic acid FBS fetal bovine serum FCS fetal calf serum FITC fluorescein isothiocyanate FPLC fast protein liquid chromatography g centrifugation force g gramm GFP green fluorescent protein GTP guanosine-5‘-triphosphate h hour(s) HA Hemagglutinin Abbreviations 2 7EPES N-(2-Hydroxyethyl)piperazine-N‘-(2-ethanesulfonic acid) His Histidine HPLC high-performance liquid chromatography IF immunofluorescence IgG immunoglobulin G IP immunoprecipitation IPTG isopropylthiogalactoside kb kilobase(s) l liter L/P leupeptin/pepstatin LB Luria-Bertani-Medium m mili M molar (mol/l) min minute(s) mRNA messenger ribonucleic acid MW molecular weight µ micro n nano nc nitrocellulose NCS newborn calf serum NEM N-ethylmaleimide NF κκκB nuclear factor kappa B NPC nuclear pore complex OD optical density units ORF open reading frame p pico PAGE polyacrylamide gel electrophoresis pH potential hydrogen PBS phosphate buffered saline PCR polymerase chain reaction Abbreviations 3 2T-PCR reverse transcription-polymerase chain reaction pfu Pyrococcus furiosus (DNA polymerase) PMSF phenylmethanesulfonyl fluoride RIPA radio-immunoprecipitation assay RNase ribonuclease rpm revolutions per minute RT room temperature RT-PCR reverse transcription followed by PCR S. cerevisiae Saccharomyces cerevisiae SDS sodium dodecyl sulfate s second(s) TB transport buffer TEMED N, N, N‘,N‘-Tetramethylethylenediamine TNF ααα Gumor necrosis factor alpha Tris Tris-”(hydroxymethyl)-aminomethane Triton X-100 t-octylphenoxypolyethoxyethanol Tween 20 polyoxyethylenesorbitan monolaurate ub ubiquitin UV ultraviolet light v/v volume per volume w/v weight per volume V volt Vol volume WB Western Blot Summary 4 1 SUMMARY Small ubiquitin-related modifier (SUMO) is a protein that is attached to lysine residues in a variety of target proteins. Sumoylation of proteins can alter their intracellular localisation, stability, activity and interaction with other proteins. The pathway of sumoylation is analogous to that of ubiquitination. The reaction is ATP dependent and requires the E1-activating enzyme (Aos1/Uba2), the E2-conjugation enzyme (Ubc9) and for most target proteins SUMO E3 ligases. The aim of this study was to characterise the SUMO E1 enzyme, a heterodimer consisting of the subunits Aos1 and Uba2. On one hand I characterised an Uba2 splice variant, which lacks one exon encoding 50 amino acids. Using RT-PCR I could determine the tissue specific distribution of the Uba2 splice variant. I furthermore showed that Uba2 variant protein is still able to form an active E1 enzyme complex with Aos1. I could demonstrate that variant Aos1/Uba2 complex is fully active in RanGAP1 sumoylation with SUMO1 or SUMO2. This finding was surprising in light of the missing amino acids, and will have implications for the understanding of E1 function. A large part of my work was dedicated to the identification and characterization of a novel SUMO substrate called ELKS. According to literature, ELKS proteins have been linked to intracellular membrane traffic and NF κB signaling pathways. I identified ELKS in membrane fractions as a binding partner for the Aos1 subunit of the SUMO E1 enzyme and confirmed in vivo interaction with ELKS antibodies that I generated. Because recombinant proteins did not interact directly, I searched for potential bridging factors. Neither SUMO nor Ubc9 or Rab6 (one ELKS partner) mediated interaction between ELKS and Aos1. Performing a large scale immunoprecipitation and analysis by mass spectrometry, I could find several candidates, including nucleoporin RanBP2, a SUMO E3 ligase. This suggested that ELKS may be a target for sumoylation. Indeed, I could show that ELKS was SUMO-modified in vivo and in vitro . Moreover, RanBP2 enhanced ELKS sumoylation. By mass spectrometry I identified two SUMO acceptor sites in ELKS. Mutation of these two residues had no effect on ELKS localisation, but strongly inhibited ELKS induced NF κB activation. In conclusion, work described in this thesis implicates sumoylation as an important mechanism for ELKS function in NF κB signaling. Introduction 0 2 INTRODUCTION 2.1 Overview Posttranslational protein modifications Posttranslational modification of proteins is a fundamental mechanism of modulating their function, activity or localisation after their synthesis has been completed.