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Investigating the Nedd4-Mediated

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Investigating the Nedd4-Mediated INVESTIGATING THE NEDD4-MEDIATED UBIQUITINATION OF PMEPA1, AND ITS POTENTIAL ROLE IN THE REGULATION OF THE ANDROGEN RECEPTOR AS PART OF THE STEROID RESPONSE PATHWAY IN PROSTATIC CANCER HELEN MARGARET MARKS School of Biological & Chemical Sciences, Queen Mary, University of London THESIS SUBMITTED TO THE UNIVERSITY OF LONDON FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Page 1 of 229 Declaration DECLARATION BY CANDIDATE I declare that the work presented in this thesis is my own and that the thesis presented is the one upon which I expect to be examined. Any quotation or paraphrase from the published or unpublished work of another person has been duly acknowledged in the work which I present for examination. Signed (Candidate) .................................................. Date: .................. Page 2 of 229 Abstract ABSTRACT Ubiquitination is an extremely important post-translational modification, regulating a wide variety of cellular processes including proteasomal degradation, subcellular targeting, endocytosis and DNA repair. The HECT class of E3 ligases catalyse the final step of ubiquitin conjugation to the substrate; the Nedd4 family make up 9 members of this class in humans, and are implicated in pathologies ranging from congenital ion channel misregulation to cancer, via the TGF-ß signalling pathway. The Nedd4-like proteins contain WW substrate recognition domains, which recognise and bind proline–rich PY motifs. This work focuses on the interaction between Nedd4 and PMEPA1, a membrane protein showing altered expression in prostate cancer and a known Nedd4 substrate. PMEPA1 is recognised as important in several cancers, although its detailed function is not yet known; it is upregulated in prostate cancer and has been postulated to decrease cellular androgen receptor (AR) via a negative feedback loop involving Nedd4 in a ubiquitin-proteasome dependent process. Misregulation of the AR response to testosterone is associated with a more advanced form of prostatic cancer and poor patient prognosis, for which there are currently few treatment options available. PMEPA1 contains two well-documented canonical (PPxY) PY motifs both required for interaction with Nedd4. This work details the identification and characterisation of a third motif with a variant PY (vPY) sequence, QPTY. Our findings indicate that the loss of this vPY motif leads to a significant reduction in Nedd4-dependent ubiquitination in vitro. In addition, the nature of the amino acid residues surrounding the vPY motif also appears to play a role in the functionality of the site. Alongside these experiments, immunodetection of protein levels in HeLa and LNCaP cell lysates was used in conjunction with confocal microscopy to shed light on the interactions between PMEPA1, Nedd4 and AR in vitro and in vivo. A possible non-proteasomal role for ubiquitination in the PMEPA1-AR interaction, as opposed to the simple ubiquitin-proteasome mediated degradation previously proposed, is discussed in the light of this new data. This work has expanded previous knowledge of the specificity of the PY-WW interaction, as well as providing a basis for further investigation, and possibly clinical targeting, of the role of PMEPA1 and AR in prostate cancer. Page 3 of 229 Acknowledgements ACKNOWLEDGEMENTS Thankyou to the Biotechnology & Biological Sciences Research Council for the funding to complete this project, and to Queen Mary, University of London for being home for the last four years. I am hugely grateful to Dr. Jim Sullivan for giving me the opportunity to take on this project, and for his patience, support and expertise throughout. He has always made time for my questions, even the stupid ones, and his insight and depth of knowledge are inspiring. I hope one day to be able to put away that much red wine at lunchtime and still go back to work! Dr. Tatiana Novoselova was an absolutely invaluable source of help and information and is much missed as a member of the group, not least for her incredible ability to keep even Jim’s lab bench clean. Thankyou especially to Dr. Ruth Rose, who has become a great friend as well as colleague over the last few years. Her ability to know exactly when to offer tea, advice and puppy cuddles, as well as her enormous internal database of protein biochemistry knowledge, has played a large role in getting me to this stage. I am grateful to Dr. James Wilkinson of Nanotemper, Germany for his help in obtaining the microscale thermophoresis data, even allowing me to take over his kitchen at home to repeat some experiments, and to Dr. Robert Janes and Prof. Richard Pickersgill, who have both provided invaluable time, advice and equipment to assist my attempts at structural biology. Mr. Mahesh Pancholi, Ms. Jen Armstrong-McKay, Ms. Sarah Tirrell and Ms. Alison Penfold have all given me the luxury of always having a London base, as well Page 4 of 229 Acknowledgements as lots of laughs, advice, KFC, Nando’s and wine. Thanks so much guys, I promise to start paying my way more now. Mr. Jay Paul, Mr. Steve Pestaille, Dr. Raj Joseph and Dr. Michaela Egertova have all gone above & beyond the call of duty in helping with technical & administrative problems, lending out supplies as well as hands when I have been desperate. My Mum and Dad have been incredibly trusting and patient throughout, despite my appalling failure to ever explain what it is I have actually been doing all these years instead of getting a job. I hope this goes some way towards a thankyou, and I hope to make you proud. Finally, I couldn’t have produced this labour of love without the financial & emotional support of Mr. Caspar Berry, who has been by my side and seen me at my highest and lowest, without flinching or letting me throw the laptop out of the window. Thankyou, now and every day. Page 5 of 229 Contents CONTENTS Title Page 1. Signed Declaration 2. Abstract 3. Acknowledgements 4. Contents 6. Figures list 12. 1. Introduction 15. 1.1 Ubiquitin and ubiquitination 15. 1.1.1 Ubiquitination in the context of post-translational modifications 15. 1.1.2 Ubiquitin discovery 17. 1.1.3 Ubiquitin structure 18. 1.1.4 Types of ubiquitination and their function 19. 1.1.5 Enzymes involved in the ubiquitination reaction 25. 1.2 HECT type ligases and the Nedd4 family 28. 1.2.1 Types of ubiquitin ligase enzymes 28. 1.2.2 Structure of the Nedd4 family ligases 31. 1.2.3 Members of the Nedd4 family and their functions 33. 1.2.3.1 In the genus Saccharomyces 33. 1.2.3.2 In the genus Drosophila 36. 1.2.3.3 In mammals 36. 1.3 PY motifs and PMEPA1 39. 1.3.1 PY motifs as WW domain-interacting motifs 39. 1.3.2 Different permutations of PY motifs & the roles of multiple PY motifs 41. 1.3.3 Identification of PMEPA1 as a Nedd4 substrate, and the PY motifs in PMEPA1 43. 1.4 Androgen receptor function, regulation and interplay with PMEPA1 45. 1.4.1 Function and structure of the androgen receptor 45. 1.4.2 Role in prostate cancer 48. 1.4.3 Regulatory mechanisms 54. 1.4.4 A published feedback loop between AR and PMEPA1 59. Page 6 of 229 Contents 1.5 Summary and outstanding questions 62. 1.6 Hypothesis 63. 2 Materials & Methods 64. 2.1 Cloning 64. 2.1.1 Plasmids 64. 2.1.2 Primer design 66. 2.1.3 Polymerase chain reaction 66. 2.1.3.1 Kod Hot Start Master Mix 68. 2.1.3.2 Phusion Hot Start Flex Master Mix 68. 2.1.3.3 DNA digestion and cleanup for SDM 69. 2.1.3.4 Agarose gel electrophoresis 69. 2.1.3.5 Gel slice extraction 70. 2.1.4 Restriction digestion and ligation 71. 2.1.5 Bacterial transformation 71. 2.1.6 Plasmid DNA preparation 74. 2.1.6.1 Small (mini) scale extraction via kit 74. 2.1.6.2 Alkaline lysis 76. 2.1.6.3 Larger (midi) scale extraction via kit 76. 2.2 Recombinant protein production 77. 2.2.1 Small scale 77. 2.2.2 Large scale 78. 2.2.3 Buffer exchange and FPLC 79. 2.3 Protein analysis 80. 2.3.1 Protein separation and visualisation 80. 2.3.1.1 Polyacrylamide gel electrophoresis (PAGE) 80. 2.3.1.2 Coomassie Brilliant Blue staining 82. 2.3.2 Western blotting 82. 2.3.3 Odyssey detection 84. 2.3.4 Enhanced chemiluminescence (ECL) detection 84. 2.4 Mammalian cell culture and transfections 85. 2.4.1 Cell culture 85. 2.4.1.1 Maintenance and passage 85. 2.4.1.2 Freezing and thawing cell cultures 85. 2.4.2 Transfection of mammalian cells 87. 2.4.2.1 FuGene 87. 2.4.2.2 JetPRIME 88. Page 7 of 229 Contents 2.4.2.3 Calcium chloride 88. 2.4.3 Harvesting mammalian cells for western blotting 89. 2.4.3.1 TCA precipitation 89. 2.4.3.2 Detergent lysis 90. 2.5 Immunoprecipitation 90. 2.5.1 IgG sepharose 90. 2.5.2 Protein A sepharose 92. 2.5.3 HisSelect resin 93. 2.6 Recombinant protein analyses 94. 2.6.1 In vitro assays 94. 2.6.1.1 Ubiquitination assays 94. 2.6.1.2 SUMOylation assays 95. 2.6.2 Microscale thermophoresis 95. 2.7 Cell imaging 96. 2.7.1 Fixing and mounting 96. 2.7.2 Immunostaining 97. 2.7.3 Confocal microscopy 97. 2.8 Statistical & in silico analysis 98. 2.8.1 Relative ubiquitination 98. 2.8.2 Localisation of cellular proteins 98. 2.8.3 In silico prediction of PMEPA1 localisations 100. 2.8.4 In silico production of 3D molecular images 100.
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