Insights into NEDD8 function and the regulation of its conjugation system Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) vorgelegt von Dana Pagliarini an der Mathematisch‐Naturwissenschaftliche Sektion Fachbereich Biologie Tag der mündlichen Prüfung: 07. Juni 2013 1. Referent: Prof. Dr. Martin Scheffner 2. Referent: Prof. Dr. Thomas U. Mayer Für meine Eltern Table of contents Abbreviations i Abstract ii Zusammenfassung iii 1. Introduction 1 1.1 Ubiquitin‐proteasome system 1 1.1.1 Ubiquitin‐conjugation cascade 2 1.1.2 Modes of ubiquitination 4 1.1.3 Ubiquitin recycling and the proteasome 6 1.2 Ubiquitin‐like proteins (UBLs) 6 1.2.1 SUMO 7 1.2.2 Other UBLs 8 1.3 NEDD8 9 1.3.1 Substrates and functions of NEDD8 10 1.3.1.1 Cullins 10 1.3.1.2 NEDD8 and transcriptional regulation 12 1.3.1.3 Further substrates and functions of NEDD8 13 1.3.2 NEDD8‐conjugation cascade 14 1.3.2.1 APPBP1/UBA3, the NEDD8‐activating enzyme 16 1.3.2.2 NEDD8‐conjugating enzymes 18 1.4 Aim of the studies 21 2. Material and Methods 22 2.1 Material 22 2.1.1 Solutions and media 22 2.1.2 Chemicals and Reagents 24 2.1.3 Bacterial strains 25 2.1.4 Mammalian cell lines 26 2.1.5 Antibodies 26 2.1.6 Primers 27 2.1.7 Plasmids constructed and used in this study 28 2.1.8 Other plasmids used in this study 29 2.1.9 DNA‐ and protein markers 30 2.2 Methods 30 2.2.1 PCR and cloning 30 2.2.1.1 Polymerase chain reaction (PCR) 30 2.2.1.2 Gene synthesis 30 2.2.1.3 Site directed mutagenesis 30 2.2.1.4 Restriction digest 31 2.2.1.5 Agarose gel electrophoresis 31 2.2.1.6 Purification of DNA from agarose gels 31 2.2.1.7 Ligation 31 2.2.1.8 Transformation of DNA into chemical competent E. coli 31 2.2.1.9 Preparation of DNA in low and high scale 31 2.2.1.10 Measurement of DNA and RNA concentrations 32 2.2.1.11 DNA sequencing 32 2.2.2 Maintenance of bacterial cultures and mammalian cell lines 32 2.2.2.1 Bacterial cultivation and preparation of glycerol stocks 32 2.2.2.2 Maintenance of mammalian cell lines 32 2.2.2.3 Freezing of cells in liquid nitrogen 32 2.2.3 Protein expression and ‐purification 32 2.2.3.1 Expression and purification of GST‐fusion proteins in E. coli 32 2.2.3.2 Expression and purification of His‐tagged proteins in E. coli 33 2.2.3.3 Expression and purification of NEDD8 and ubiquitin for click reaction 33 2.2.3.4 Expression and purification of PCNA for click reaction 33 2.2.3.5 In vitro translation 34 2.2.4 Protein analysis 34 2.2.4.1 Bradford assay 34 2.2.4.2 SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE) 35 2.2.4.3 Coomassie Blue and colloidal Coomassie staining 35 2.2.4.4 Fluorography 35 2.2.4.5 Western Blot 35 2.2.5 In vitro assays 36 2.2.5.1 Methanol‐Chloroform precipitation of proteins 36 2.2.5.2 GST‐pulldown assay 36 2.2.5.3 Affinity chromatography 36 2.2.5.4 In vitro NEDDylation assay 37 2.2.5.5 Thioester assay 37 2.2.5.6 Cu(I)‐catalyzed Huisgen azide‐alkyne cycloaddition 37 2.2.5.7 Transient transfection 37 2.2.5.8 TNN cell lysis and ß‐galactosidase assay 38 2.2.5.9 Immunoprecipitation 38 2.2.5.10 Cycloheximide chase 38 2.2.5.11 Cellular fractionation 39 2.2.5.12 Preparation of total RNA 39 2.2.5.13 Reverse transcription 39 2.2.5.14 Antibody purification 39 2.2.6 In cellulo assays 40 2.2.6.1 In cellulo ubiquitination and NEDDylation assays 40 2.2.6.2 Immunofluorescence 40 3. Results 41 3.1 AutoNEDDylation of NEDD8‐conjugating enzymes increases the affinity to their cognate E1 41 3.1.1 Ubc12 and Nce2 form a thioester bond with NEDD8 but not with ubiquitin 42 3.1.2 NEDD8‐conjugating enzymes are autoNEDDylated 43 3.1.3 HPNI mutants of the NEDD8‐conjugating enzymes are not impaired in thioester formation but in autoNEDDylation 45 3.1.4 AutoNEDDylation of the NEDD8 E2 enzymes predominantly occurs in their N terminus 46 3.1.5 AutoNEDDylation enhances the affinity of Ubc12 and Nce2 to the NEDD8 E1 enzyme APPBP1/UBA3 50 3.1.6 Fusion of NEDD8 to its E2s leads to an enhanced localization in the nucleus 51 3.2 PCNA as a new substrate for the NEDD8‐conjugation pathway 54 3.2.1 PCNA interacts with NEDD8 and ubiquitin 54 3.2.2 PCNA is NEDDylated in cells being dependent on K164 56 3.2.3 PCNA NEDDylation depends on the activity of APPBP1/UBA3 in cells 58 3.2.4 NEDDylation of PCNA is enhanced by the E3 ligase Rad18 60 3.2.5 NEDD8‐conjugating enzymes do not bind to PCNA in cells 60 3.2.6 PCNA Y211F, a phosphorylation deficient mutant, is NEDDylated in cells 61 3.2.7 Cu(I)‐catalyzed Huisgen azide‐alkyne cycloaddition as a tool to study functions of NEDDylated PCNA 62 3.3 A new isoform of the NEDD8‐conjugating enzyme Nce2 65 3.3.1 mRNA encoding Nce2 isoform 2 is expressed in HEK293T cells 67 3.3.2 Tertiary structure prediction for Nce2 isoform 2 reveals an unstructured, flexible C terminus 67 3.3.3 Nce2 isoform 2 forms a thioester bond with NEDD8 but not with ubiquitin and is NEDDylated in vitro 68 3.3.4 Nce2 isoform 2 is NEDDylated and ubiquitinated in cells 70 3.3.5 Nce2 isoform 2 has a shorter half‐life than isoform1 and is degraded by the proteasome 72 3.3.6 Nce2 isoform 1 and 2 differ in their subcellular localization 74 3.3.7 Development of an antibody specifically recognizing Nce2 isoform 2 75 4. Discussion 77 4.1 AutoNEDDylation as a regulatory mechanism of NEDD8‐conjugating enzymes 77 4.1.1 NEDD8‐conjugating enzymes are autoNEDDylated in their unique N terminus 77 4.1.1.1 Indications for endogenous autoNEDDylation of Ubc12 and Nce2 77 4.1.1.2 The HPNI‐motif of Ubc12 and Nce2 is important for isopeptide bond formation 78 4.1.1.3 The N termini of Ubc12 and Nce2 are crucial for an efficient autoNEDDylation 79 4.1.2 Possible functions of autoNEDDylation 81 4.1.2.1 AutoNEDDylation as regulator of the subcellular localization of Ubc12 and Nce2 82 4.1.2.2 Acetylation of NEDD8 E2 enzymes as a competitive modification to NEDDylation 82 4.1.2.3 AutoNEDDylation enhances the affinity of NEDD8 E2s to APPBP1/UBA3 83 4.1.2.4 Further possible functions and impacts of autoNEDDylation 85 4.2 Interplay between PCNA and the NEDD8 system 88 4.2.1 PCNA as an interaction partner of NEDD8 88 4.2.2 PCNA as a substrate for NEDD8 89 4.2.2.1 Evidence for NEDDylation of PCNA in vitro and in cellulo 89 4.2.2.2 Effects of MLN4924 on the NEDDylation of PCNA 91 4.2.2.3 Hints for possible functions of NEDDylated PCNA 92 4.2.2.4 Click reaction as tool to identify functions of monoNEDDylated PCNA 94 4.3 A second isoform of Nce2 with individual properties 96 4.3.1 Nce2 isoform 2 as a splice variant with an extended C terminus 96 4.3.2 mRNA of the second isoform of Nce2 is present in HEK293T cells 96 4.3.3 Nce2 isoform 2 is active in vitro and can be NEDDylated 97 4.3.4 The cellular localization distinguishes Nce2 isoform 2 from isoform 1 99 4.3.5 A C‐terminal unstructured region promotes insolubility in bacteria and rapid degradation in cells 100 4.3.6 Possible functions of Nce2 variants 101 5. References 105 Eidesstattliche Erklärung 124 Danksagung 125 Abbreviations aa amino acid(s) bp base pairs cDNA complementary DNA DMSO Dimethylsulfoxide dNTP Deoxynucleoside triphosphate DTT Dithiothreitol EDTA Ethylenediaminetetraacetic acid E1 UBL‐activating enzyme E2 UBL‐ conjugating enzyme E3 UBL ligase FCS Fetal calf serum GST Glutathione‐S‐transferase HA‐tag Hemagglutinin‐tag His‐tag 6x Histidin‐tag IP Immunoprecipitation IPTG Isopropyl‐β‐D‐thiogalactopyranoside kDa kilo Dalton mRNA messenger RNA Ni‐NTA Nickel‐nitrilotriacetic acid OD optical density ONPG Ortho‐nitrophenyl‐β‐galactoside PBS Phosphate buffered saline PCR polymerase chain reaction rpm revolutions per minute RT reverse transcription SDS Sodium dodecyl sulfate UBL ubiquitin‐like protein wt wild‐type i Abstract NEDD8 belongs to the family of ubiquitin‐like proteins which share a common basic structure. In an enzymatic cascade, NEDD8 can be attached to other proteins via its C terminus (“NEDDylation”). NEDDylation plays an important role in various cellular processes including cell cycle, transcription or the regulation of protein stability. Previous studies revealed NEDD8 to have many yet uncharacterized substrates. During this work, the sliding clamp PCNA, which functions in replication and DNA damage repair, was identified as a new substrate for NEDDylation. PCNA is modified with NEDD8 at the same lysine (K164) residue that can be targeted for SUMOylation and ubiquitination. Rad18, the E3 ligase for ubiquitination of PCNA upon DNA damage, enhances monoNEDDylation of PCNA.