School of Biomedical Sciences The role of monoubiquitylation in the regulation of the transcription factor Elk-1 by Leo Kam Yuen Chow, B.Sc. (Hons) Thesis submitted to the University of Nottingham for the degree of Doctor of Philosophy September 2010 Contents Contents ............................................................................................................................... I Abstract ............................................................................................................................. VI Acknowledgements .......................................................................................................... VII Abbreviations .................................................................................................................. VIII 1. Introduction ................................................................................................................. 1 1.1. The ternary complex factor Elk-1 ............................................................................. 1 1.1.1. Overview ........................................................................................................... 1 1.1.2. Historical perspective ....................................................................................... 2 1.1.3. Domain structure of Elk-1 ................................................................................. 3 1.1.4. Ternary complex formation .............................................................................. 4 1.1.4.1. Serum response element .......................................................................... 4 1.1.4.2. ETS/DNA binding ....................................................................................... 5 1.1.4.3. TCF/SRF interaction .................................................................................. 8 1.1.5. Regulation of Elk-1 by post-translational modifications................................... 9 1.1.5.1. Elk-1 phosphorylation by MAPKs .............................................................. 9 1.1.5.2. The role of Elk-1 phosphorylation .......................................................... 10 1.1.6. Elk-1 target genes and biological function ..................................................... 12 1.2. Ubiquitylation ......................................................................................................... 14 1.2.1. Overview and historical perspective .............................................................. 14 1.2.2. Mechanism of ubiquitylation .......................................................................... 14 1.2.3. Ubiquitin enzymes .......................................................................................... 15 1.2.4. Ubiquitin E3 ligases ......................................................................................... 15 1.2.4.1. RING-domain E3 ligases .......................................................................... 16 1.2.4.2. CRLs regulation by neddylation .............................................................. 18 1.2.4.3. HECT-domain E3s .................................................................................... 19 1.2.5. E2 conjugating enzymes ................................................................................. 20 1.2.6. E1 activating enzymes..................................................................................... 21 1.2.7. Ubiquitylation linkage ..................................................................................... 21 1.2.7.1. Multiple mono- and mono-ubiquitylation .............................................. 22 I 1.2.7.2. Polyubiquitylation ................................................................................... 23 1.2.7.2.1. Lysine 48-linked polyubiquitylation .................................................... 24 1.2.7.2.2. Lysine 63-linked polyubiquitylation .................................................... 24 1.2.7.2.3. Polyubiquitylation via other linkages .................................................. 26 1.2.8. 26S proteasome-mediated proteolysis .......................................................... 27 1.2.9. Deubiquitylation ............................................................................................. 28 1.3. Regulation of ubiquitylation ................................................................................... 30 1.3.1. Regulation of ubiquitylation by phosphorylation ........................................... 30 1.3.1.1. Phosphodegron ....................................................................................... 30 1.3.1.2. Regulation of ubiquitin enzymes by phosphorylation ............................ 32 1.3.2. Regulation of ubiquitylation by subcellular location ...................................... 33 1.3.3. Regulation of ubiquitylation by complex formation ....................................... 34 1.4. Roles of ubiquitylation in transcription .................................................................. 35 1.4.1. Proteolysis-coupled transcription ................................................................... 35 1.4.2. The roles of 26S proteasome in transcription ................................................ 36 1.4.3. Hypothesized mode of proteolysis coupled transcription initiation .............. 38 1.5. Research aims and objectives ................................................................................. 40 2. Materials .................................................................................................................... 41 2.1. Antibodies ............................................................................................................... 41 2.2. Chemicals and commercial kits .............................................................................. 42 2.3. Bacterial Strains ...................................................................................................... 43 2.4. Plasmid Constructs ................................................................................................. 44 2.5. Oligonucleotides ..................................................................................................... 47 3. Methods ..................................................................................................................... 48 3.1. Nucleic Acid Techniques ......................................................................................... 48 3.1.1. DNA extraction ................................................................................................ 48 3.1.1.1. Small scale DNA preparation .................................................................. 48 3.1.1.2. Large scale DNA preparation .................................................................. 48 3.1.2. Restriction digestion of DNA ........................................................................... 49 3.1.3. Alkaline phosphatase treatment of DNA ........................................................ 49 3.1.4. Ligation of DNA ............................................................................................... 49 3.1.5. Oligonucleotide 32P [γ] ATP labelling ............................................................... 50 II 3.1.6. Production of [32P]-labelled double stranded oligonucleotides .................... 50 3.1.7. Site-Directed Mutagenesis.............................................................................. 51 3.2. Bacterial Techniques ............................................................................................... 51 3.2.1. Preparation of competent cells ...................................................................... 51 3.2.2. Transformation of competent cells with plasmid DNA .................................. 52 3.3. Gel Electrophoresis ................................................................................................. 52 3.3.1. Agarose gel electrophoresis ........................................................................... 52 3.3.1.1. DNA agarose gel ...................................................................................... 52 3.3.1.2. Extraction of DNA from agarose gels ...................................................... 53 3.3.2. Polyacrylamide gels ........................................................................................ 53 3.3.2.1. Non-denaturing polyacrylamide gel electrophoresis ............................. 53 3.3.2.2. SDS polyacrylamide gel electrophoresis ................................................. 54 3.4. Protein Techniques ................................................................................................. 55 3.4.1. Protein extraction ........................................................................................... 55 3.4.1.1. Preparation of whole cell extracts .......................................................... 55 3.4.1.2. Preparation of HeLa cytosolic extracts from suspension culture ........... 55 3.4.1.3. Preparation of nuclear extracts .............................................................. 56 3.4.2. Transfer of proteins to Polyvinylidene Fluoride membrane........................... 56 3.4.3. Western blotting ............................................................................................. 57 3.4.4. Electrophoretic Mobility Shift Assay (EMSA) .................................................. 57 3.4.5. Fast Performance Liquid Chromatography (FPLC) .......................................... 58 3.4.6. Dialysis ...........................................................................................................