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Regulation of MDM2 and the P53 Family by the NEDD8 Pathway

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Regulation of MDM2 and the P53 Family by the NEDD8 Pathway Regulation of MDM2 and the p53 family by the NEDD8 pathway by Ian Robert Watson A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Laboratory Medicine and Pathobiology University of Toronto © Copyright by Ian Robert Watson 2010 Regulation of MDM2 and the p53 family by the NEDD8 pathway Ian Robert Watson Doctor of Philosophy Laboratory Medicine and Pathobiology University of Toronto 2010 Abstract NEDD8 is an ubiquitin-like protein sharing approximately 60% amino acid identity with ubiquitin and has biological roles in cell cycle progression, viability and development. Recently, a number of oncoproteins and tumor suppressors have been identified as NEDD8 substrates, including MDM2 and p53. MDM2 is an oncogenic E3 ligase that promotes NEDD8 modification and ubiquitin-mediated degradation of the tumor suppressor transcription factor, p53. Cellular stresses such as DNA damage lead to p53 activation due, in part, to MDM2 destabilization by mechanisms that are not completely understood. Studies in mice demonstrate the biological role of MDM2 is to negatively regulate p53 function, however, when overexpressed or amplified, MDM2 has p53-independent oncogenic functions presumably due to the regulation of additional substrates. One such substrate may be the p53 family member, p73. p73 exists as multiple isoforms and accumulating evidence suggests that the N-terminal isoforms dictate its role in tumorigenesis. The full-length pro-apoptotic TAp73 isoforms are induced by chemotherapies and are able to transactivate p53-target genes to initiate cell cycle arrest and apoptosis. Conversely, the N-terminally truncated ΔNp73 isoforms lack the transactivation ii domain (TAD) and consequently act as dominant-negative inhibitors for all TA isoforms of the p53 family, and are overexpressed in human tumors. Here, we report that TAp73, but not ΔNp73, is covalently modified by NEDD8 in an MDM2-dependent manner, attenuating its transactivation function and promoting cytoplasmic localization of neddylated TAp73. These results provide the first evidence of a covalent post-translational modification exclusively targeting the TA isoforms of p73, and identify the MDM2-TAp73 interaction as a promising therapeutic target. We also demonstrate that the stability of MDM2 is regulated by the NEDD8 pathway and identify NEDP1 as a chemotherapy-induced isopeptidase that deneddylates MDM2, resulting in MDM2 destabilization, concomitant with p53 activation. This study identifies a novel p53 activating mechanism in response to chemotherapy. In conclusion, the work presented herein has helped characterize the function of NEDD8 modification of MDM2 and the p53 family, and identify mechanisms by which MDM2 and the NEDD8 pathway may be targeted in the development of anti-cancer therapeutics. iii Acknowledgments First, I would like to thank my supervisors Dr. Michael Ohh and Dr. Meredith Irwin. Throughout the duration of my PhD they have never wavered in their support, fostered my research potential and given me confidence in my research abilities. I could not have asked for a better supervisory team. I would also like to thank my advisory committee members, Dr. Dwayne Barber and Dr. Samuel Benchimol, who have provided exceptional guidance and have always offered their time graciously. I owe a great deal of gratitude to my undergraduate supervisor, Dr. Sandy Der, for first providing me the opportunity to study in the department of Laboratory Medicine and Pathobiology and Dr. Irene Hwang, the PhD student whose training has stayed with me to this day. Throughout the duration of my study, I have had the great opportunity to work alongside a number of talented trainees in the labs of Dr. Ohh and Dr. Irwin who have provided at one time or another, assistance, direction and advice. In particular, I would like to thank: Dr. Alvaro Blanch, who was a key contributor in Chapter 2, and has always taken the time to provide advice in matters related to the lab, career, and life in general—I will sadly miss his guidance when I am gone; Dr. Loretta Lau, who was always there for me to provide help and direction during her time as the senior lab member; Bryan Li, who was a key contributor to Chapter 3—I appreciate all his hard work and efforts; our lab manager, Lynn Cheng, for always being there for lab matters and anything beyond; Dr. Fiona Robinson, for her careful reading of this thesis; and Joanne Lau, my fellow PhD student, who shared all the same experiences with me as we started our graduate studies together. Personally, I would like to thank my parents for their support throughout my education and research studies. Most importantly, I would like to thank my wife, Christine DeSantis, for her patience, encouragement and understanding. She never ceases to amaze me with her thoughts, consideration, and awareness. I am truly appreciative of her support; without her I would not be where I am today. iv Table of Contents Abstract ii Acknowledgments iv Table of Contents v List of Figures viii Abbreviations x Chapter 1: Introduction to ubiquitin-like proteins, the p53 family and MDM2 1 1 INTRODUCTION 1 1.1 Ubiquitin-proteasome pathway 1 1.1.2 NEDD8: Ubiquitin-like protein (UBL) 4 1.1.3 The role of NEDD8 in cancer 6 1.1.4 p53: The guardian of the genome 7 1.1.5 Structure and function of the p53 family 10 1.1.6 p63 and p73: Dual tumor suppressor and oncogenic functions 13 1.1.7 p63 and p73: Roles in development 14 1.1.8 p63 and p73: Mouse models of cancer 15 1.1.9 p63 and p73: Alterations in human cancer and role in chemotherapy response 16 1.1.10 Mechanisms of p53 activation in response to DNA damage 17 1.1.11 MDM2 regulation of p53 18 1.1.12 MDM2 studies in mice 21 1.1.13 MDM2: p53-independent oncogenic functions 22 1.1.14 MDM2 destabilization: Mechanism of p53 activation in response to DNA damage 23 1.1.15 Regulation of the p53 family by ubiquitin and ubiquitin-like modifications 24 1.2 SIGNIFICANCE 26 CHAPTER 2: MDM2-mediated NEDD8 modification of TAp73 regulates its transactivation function 28 2.1 HYPOTHESIS AND RATIONALE 28 v 2.2 RESULTS AND DISCUSSION 30 2.2.1 TAp73 is modified by NEDD8 via MDM2 30 2.2.2 NEDP1 deneddylates modified TAp73β 34 2.2.3 Np73 does not undergo MDM2-mediated neddylation 36 2.2.4 Neddylation of p73 occurs under physiological conditions 38 2.2.5 The neddylation pathway attenuates TAp73 transcriptional activity 40 2.2.6 NEDD8 modification of TAp73 promotes cytoplasmic localization 44 2.3 DISCUSSION 48 2.4 FUTURE DIRECTIONS 50 2.5 MATERIALS AND METHODS 52 2.5.1 Cells 52 2.5.2 Antibodies 52 2.5.3 Plasmids 53 2.5.4 Immunoprecipitation and immunoblotting 53 2.5.5 Subcellular fractionation 54 2.5.6 Dual-luciferase reporter assay 55 CHAPTER 3: Chemotherapy induces NEDP1-mediated destabilization of MDM2 56 3.1 HYPOTHESIS AND RATIONALE 56 3.2 RESULTS AND DISCUSSION 58 3.2.1 Neddylation stabilizes MDM2 58 3.2.2 NEDP1-mediated deneddylation promotes MDM2 destabilization 62 3.2.3 Chemotherapy increases NEDP1 levels 64 3.2.4 NEDP1 modulates p53-apoptotic response to chemotherapy 68 3.3 DISCUSSION 71 3.4 FUTURE DIRECTIONS 73 3.5 MATERIAL AND METHODS 75 3.5.1 Cells 75 vi 3.5.2 Antibodies and reagents 75 3.5.3 Plasmids 76 3.5.4 Immunoprecipitation and immunoblotting 77 3.5.5 Protein turnover assays 77 3.5.6 RNAi 78 3.5.7 TUNEL assays 78 Chapter 4: CONCLUSIONS AND FUTURE DIRECTIONS 80 APPENDIX 87 REFERENCES 98 vii List of Figures Page Chapter 1 Figure 1.1.1 General overview of the ubiquitin and ubiquitin-like protein 4 conjugation pathways Figure 1.1.2 Structure of the p73 isoforms 11 Figure 1.1.3 Schematic representation of the gene structure of the p53 family 12-13 Figure 1.1.4 Structure of MDM2 protein 20 Figure 1.1.5 MDM2 promotion of p53 ubiquitylation 21 Chapter 2 Figure 2.1 TAp73 is modified by NEDD8 32-33 Figure 2.2 NEDP1, a NEDD8 specific cysteine protease, deneddylates modified 35 TAp73 Figure 2.3 The Np73 isoform lacking the MDM2-binding site is not conjugated 37 by NEDD8 Figure 2.4 Endogenous p73 is modified by NEDD8 39 Figure 2.5 NEDD8 pathway inhibits TAp73-mediated transactivation 42-43 Figure 2.6 Neddylated TAp73 species are found preferentially in the cytoplasm 46-47 Chapter 3 Figure 3.1 Neddylation of MDM2 increases its protein stability 60-61 Figure 3.2 NEDP1-mediated deneddylation decreases MDM2 stability 63 Figure 3.3 Chemotherapy increases NEDP1-mediated p53 activation 66 viii Figure 3.4 NEDP1 levels increase in response to chemotherapy independent of 67 ATM and p53 status Figure 3.5 siRNA-mediated downregulation of NEDP1 enhances chemoresistance 69-70 Figure 3.6 A model of NEDP1-mediated activation of p53 apoptotic response 70 Chapter 4 Figure 4.1 MLN4924 inhibits NEDD8 conjugation by targeting the NEDD8- 84 activating enzyme (NAE) Appendix Figure A.1 TAp73 is modified by NEDD8, but not ubiquitin, in the presence of 87 overexpressed NEDD8 Figure A.2 Endogenous p73 is modified by NEDD8 88 Figure A.3 TAp73 1-344 truncation mutant is not modified by NEDD8 89-90 Figure A.4 MDM2(C464A) RING finger mutant does not promote cytoplasmic 91 neddylated TAp73 species localization Figure A.5 Ectopic NEDD8 expression prolongs MDM2 half-life 92 Figure A.6 Chemotherapy induces the expression of NEDP1 93 Figure A.7 Ectopic expression of p53 has negligible effect on NEDP1 expression 94 Figure A.8 siRNA-mediated NEDP1 knockdown decreases p53 activation in 95-96 response to doxorubicin Figure A.9 DNA-PK status determines induction of NEDP1 in glioma cell lines 97 ix Abbreviations AMP: adenosine 5’-monophosphate APP-BP1: amyloid
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