The Role of Subcellular Distribution upon Signalling by Polyomavirus Middle Tumour-antigen Yunsi Zhou Imperial College London, Division of Surgery and Cancer Thesis submitted to Imperial College London for the degree of Doctor of Philosophy 1 ABSTRACT The middle T-antigen (MT) encoded by polyomavirus induces tumours by mimicking an activated cell surface receptor. A hydrophobic region close to the C-terminus locates MT to the plasma membrane where it initiates oncogenic signalling. HA or FLAG tags added to the C-terminus of MT could be detected on the outside of cells, demonstrating that MT is a transmembrane protein. Addition of a KDEL sequence retained MT in the endoplasmic reticulum, where it failed to transform cells as a consequence of its lack of binding to ShcA, PI3K, and PLC-γ1 despite the phosphorylation of the appropriate binding sites. Additional studies show that MT binding to PP2A is probably required in addition to the TMD for MT to exit the ER and migrate to the plasma membrane. MT on the cell surface is present in discrete clusters that contain phosphorylated ShcA and so represent active signalling complexes. New mutations in the hydrophobic region prevented MT clustering, and also inhibited cell transformation without altering the association of MT with its known binding proteins. Overall, these data, together with previous publications, illustrate that MT associates with signalling proteins at discrete subcellular membrane sites in its maturation pathway. MT binds to PP2A in the cytoplasm, to c-Src at the ER, and to ShcA, PI3K, and PLC-γ1 at subsequent locations en route to the plasma membrane. Formation of a large macromolecular complex in the plasma membrane is probably required for MT signalling. Similar cell surface complexes are observed with activated growth factor receptors, so it is possible that normal and oncogenic signalling from receptors is also dependent upon the assembly of large macromolecular complexes at the cell surface membrane. There was no obvious association of MT clusters with lipid rafts but there was colocalisation with the actin cytoskeleton. 2 DECLARATION OF ORIGINALITY Unless otherwise stated, all experiments were performed by me, Yunsi Zhou, the author of this thesis. COPYRIGHT DECLARATION The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work. 3 ACKNOWLEDGEMENTS I would like to thank my supervisors Professor Steve Dilworth and Dr Nick Dibb for their guidance throughout my project and their advice on my thesis. Many thanks to Dr Natalia Ichaso, who generated many of the plasmids that forms the basis of my project; our collaborators Dr Jitka Forstova and Dr Vojtech Zila at Charles University in Prague, who generated IEM images for our paper; Dr Nicola Brownlow at London Research Institute for her advice on experiment designs; Dr Rupa Sarkar for her help with RNA extraction and all the colleagues at the IRDB for their support. I would like to thank my father, Mingxuan Zhou, for moral support. With loving memories, I would like to give thanks to my mother, Yanfen Lu, for being a great role model in my life. I would also like to thank my grandma and the rest of my family as well as friends far and near for their continuous encouragement. Finally, I would like to give special thanks to Mark Refermat for all his patience, encouragement and support throughout my study, especially during my final year. 4 PUBLICATIONS Zhou AY, Ichaso N, Adamarek A, Zila V, Forstova J, Dibb NJ, Dilworth SM. Polyomavirus middle T-antigen is a transmembrane protein that binds signalling proteins in discrete subcellular membrane sites. J Virol. 2011 Apr;85(7):3046-54. Epub 2011 Jan 12. Zhou AY, Ichaso N, Shah K, Zila V, Forstova J, Dibb NJ, Gullick WJ, Dilworth SM. Oncogenic signalling by polyoma virus MT occurs in discrete membrane complexes. Manual script completed. 5 Table of Contents FIGURE AND TABLE LIST ........................................................................................................................ 10 ABBREVIATIONS .................................................................................................................................... 15 LIST OF MT MUTANTS ........................................................................................................................... 18 CHAPTER 1 – INTRODUCTION ....................................................................................................... 20 1.1 Overview ..................................................................................................................................... 20 1.2 Small DNA Tumour Viruses ......................................................................................................... 20 1.3 MPyV Genome ............................................................................................................................ 22 1.4 T-antigens .................................................................................................................................... 23 1.4.1 Large T-antigen .................................................................................................................... 25 1.4.2 Middle T-antigen .................................................................................................................. 25 1.4.3 Small T-antigen .................................................................................................................... 29 1.5 Late Replication Cycle Proteins ................................................................................................... 29 1.6 Cooperation of MPyV T-antigens induces tumour formation .................................................... 30 1.7 Transcription Factors in MT-induced Transformation ................................................................ 31 1.8 MT in Other Polyomaviruses ....................................................................................................... 32 1.8.1 Hamster Polyomavirus ......................................................................................................... 32 1.8.2 Human Merkel Cell Polyomavirus ........................................................................................ 32 1.9 MT Oncogenic Mouse Models .................................................................................................... 33 1.10 MT Binding Proteins .................................................................................................................. 34 1.10.1 PP2A ................................................................................................................................... 34 1.10.2 Src Family Tyrosine Kinase ................................................................................................. 36 1.10.3 PI3 Kinase ........................................................................................................................... 38 1.10.4 Shc ...................................................................................................................................... 40 1.10.5 PLC-γ1 ................................................................................................................................. 42 1.10.6 Regulatory Protein 14-3-3 .................................................................................................. 43 1.11 Proline –rich Region .................................................................................................................. 44 1.12 Additional Tyrosine Phosphorylation ........................................................................................ 44 1.13 Subcellular Localisation of MT .................................................................................................. 45 1.14 Cytoskeletons and Focal Adhesion Complexes ......................................................................... 47 1.14.1 Focal Adhesion Kinase ........................................................................................................ 48 1.14.2 Vinculin............................................................................................................................... 50 1.14.3 Integrin ............................................................................................................................... 51 1.15 Lipid Rafts .................................................................................................................................. 51 6 1.16 Membrane Localisation Region ................................................................................................ 53 1.17 Aims: ......................................................................................................................................... 57 CHAPTER 2 - MATERIAL AND METHODS ............................................................................................... 58 2.1 Cell Culture .................................................................................................................................. 58 2.2 Plasmids .....................................................................................................................................