The Role of Pirh2 E3 Ligases in Ubiquitination and p73 Regulation by Rami Mahmoud Abou Zeinab A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Medical Sciences - Laboratory Medicine and Pathology University of Alberta © Rami Mahmoud Abou Zeinab, 2015 Abstract Cancer and tumor suppressors have been highly associated with many cancer researches (1). The main function of these proteins is to detect any error in DNA that might eventually leads to abnormal cell division and repair these errors; hence eliminating the risk of cancer development (2,3). Many researches focused on the p53 family protein consisting of three proteins: p53, p63, and p73 (4). p53 was the first to be discovered, and many scientists refer to this protein as the “master switch”, or the “guardian of genome” because of its critical role in coordinating cellular processes (4,5). p53 acts as a checkpoint in the cell cycle monitoring all the cellular responses. At this level, should any stress signal occur affecting the cellular mechanism, p53 will be highly expressed and will trigger a variety of programs including a series of repressing and activating responses (6). Unfortunately, 50-60% of human tumors show mutations in the p53 proteins and the remainder, despite bearing wild type (WT) proteins, shows a dysfunctional system (6, 7). More attention later on also included p73 proteins after revealing their role in cell cycle arrest and apoptosis (8). The exact mechanism for the tight regulation of these proteins is not fully understood; however ubiquitination has been shown to be the master regulatory process (9, 10). Several ubiquitin E3 ligases such as MDM2, Pirh2, AIP4, UBE4B, etc.; are shown to act as regulators (11). Interestingly among all E3 ligases, Pirh2 is the only one over-expressed in a wide range of human tumors: 41, 64, 61, 70 and 82% in primary breast cancer, hepatocellular carcinoma, head and neck, prostate, and lung cancer respectively (12). Knowing that Pirh2 is overexpressed, the first objective of this thesis was focused on examining the self-regulatory process of Pirh2 including the optimal conditions and the role of E2 enzymes. Also we mapped the domains of Pirh2 to reveal that in addition to the RING domain, commonly known for the catalytic activity, portions of the C terminal domain are also essential for Pirh2 self-ubiquitination mechanism. Besides, lysine chains (K48, K63, ii and KO), known for their role in determining the substrate’s fate, were analyzed showing lack of significance on the contrary to p53 ubiquitination, where K48 lysine chains are utilized. The second objective of this thesis was to analyze Pirh2 and p73 correlation knowing that p73 share up to 80% homology with p53 at the DNA binding sequence (13). Interestingly, Pirh2 has been shown to bind, ubiquitinate and down-regulate p73 tumor suppressor function without altering p73 protein level. Lack of degradation was explained with the use of lysine chains 63 that do not trigger proteosomal degradation post ubiquitination. The third objective of this thesis focused on Pirh2-p73 regulatory pathway especially when studies have shown that the same substrate, p73 in our case, has been shown to be regulated by other ligase: AIP4 (14). In this study, we reveal for the first time a novel correlation between two ligases within the same signalling pathway where Pirh2 physically interacts with AIP4 and significantly down-regulates its expression. This down-regulation is shown to involve the ubiquitination of AIP4 by Pirh2. At the p73 level, we showed that Pirh2 ceased the AIP4-p73 negative regulatory pathway. In-vivo and in-vitro ubiquitination analyses regarding p73 isoforms (α & β) confirmed the decrease of p73-AIP4 induced ubiquitination when Pirh2 is introduced. At the translation level and regarding p73 cell cycle arrest function in specific, Pirh2 secured p73 G1 arrest role despite overexpressing AIP4. Overall, our findings added valuable knowledge over the E3 ligase self-regulation and also the substrate regulation. Also we introduced a novel concept correlating two independent signalling pathways. This opens a gateway to explain how E3 ligases differentiate between regulating multiple substrates that may belong to the same family of proteins as it is the case for p53 and p73 proteins; hence, providing a new framework for the development of novel anti-cancer targeting strategies. iii Preface This thesis represents collaborative work, led by Dr. Roger Leng at the University of Alberta. Chapter 2 of this thesis has been published as: Rami Abou Zeinab, Hong Wu, Consolato Sergi, Roger Leng. 2013. Residues 240-250 in the C-terminal of Pirh2 protein complement the function of the RING domain in self ubiquitinating Pirh2 protein. PlOS ONE, 8 (12): e82803, 1-8. I was first author of this manuscript, and contributed to the practical work, manuscript writing, and data analysis as outlined in chapter 2. Chapter 3 of this thesis has been published as: Hong Wu, Rami Abou Zeinab, Elsa R. Flores, Roger Leng. 2011. Pirh2, a Ubiquitin E3 ligase, inhibits p73 Transcriptional Activity by promoting its Ubiquitination. Molecular Cancer Research, 9 (12): 1780-1790. I was second author of this submitted manuscript and did the in vivo and in-vitro ubiquitination analysis as outlined in chapter 3. Chapter 4 of this thesis is to be submitted as: Rami Abou Zeinab, Hong Wu, Consolato Sergi, Roger Leng. 2014. Pirh2 E3 ligase inhibits the AIP4-p73 negative regulatory pathway by mediating AIP4 expression and ubiquitination. In progress to be submitted by December 2014. I am the first author of this manuscript, and contributed to all practical work, manuscript writing, and data analysis as outlined in chapter 4. iv Acknowledgments I would like to record my deep acknowledgments to all those who encouraged me in forging a success out of this work. Let me first start with my supervisor, Dr. Roger Leng, whose support, motivation, and guidance were truly beyond description. Thank you for believing in me and in my potentials. I doubt that I will ever be able to convey my full appreciation and I will always remember that I was extremely lucky to be part of your laboratory. I would like to thank my committee members, Dr. Fiona Bamforth, Dr. Gordon Chan, and Dr. Consolato Sergi, for all their contributions to my work. Also I was delighted to interact with remarkable professors during my candidacy and thesis examination; thank you: Dr. Manijeh Pasdar and Dr. Sujata Persad for all your kind words. Also thank you Dr. Arnim Pause for being my external examiner on my PhD thesis defence. My salutes go to many faculty and staff members including: Dr. Richard Lehner, Dr. Monika Keelan, Dr. Jonathan Martin, Dr. Judith Hugh, Mrs. Jennifer McPhee, and Ms. Cheryl Titus. I will never forget the nice moments that we spent together. I have learned a lot from you. My sincere gratitude to all the funding agencies that sponsored me including: Alberta Cancer Foundation, Women and Children’s Health Research Institute, Hair Massacure, and LMP department for the several awards I was granted. Without your support, my accomplishments would not have been the same. All the lab work done in the Leng’s laboratory would not have been possible without the support of the research team starting with our Research Associate: Dr. Hong Wu; whose assistance and cooperative friendly personality made the working environment extremely smooth and joyful. Of course our postdocs and graduate students, salutes to all of you and best of luck in your careers. I would like to thank all my friends and colleagues; the time we spent together was valuable and unforgettable. Forgive me if I will not talk about each one of you alone since I am lacking the words able to describe what each one of you had offered me during the past 4 years. Thanks to: Ariel, Jihong, Antoinette, Nour, Hadeel, Luciana, Nickolas, Reem, Redha, Suzan, Russ, Randy, Jelske. Exceptional thanks to my family for their endless love, care, and support. You are my inspiration in life to keep going on. Without your continuous encouragement it would be v impossible for me to accomplish what I have achieved till this moment. Your sacrifices made me discover the real miracle of self-realization. My last thank you goes to my love, the person who accepted me as I am and was by my side till this day. vi Table of Contents Title Page ............................................................................................................... i Abstract ................................................................................................................ ii Preface ................................................................................................................ iv Acknowledgments .................................................................................................. v Table of Contents ................................................................................................. vii List of Abbreviations .............................................................................................. xi Authorizations ..................................................................................................... xiv List of Figures ..................................................................................................... xxii CHAPTER 1- General Introduction ............................................................................. 1 1.1 Cancer and Tumor Suppressors ........................................................................ 2 1.2 The p53 Network ............................................................................................ 2 1.3 p53 overview ................................................................................................. 5 1.3.1 Discovery of p53 ...................................................................................... 5 1.3.2 Role of p53 as tumor suppressor ................................................................ 6 1.3.3 Regulation of p53 ................................................................................... 11 1.4 The ubiquitin system ..................................................................................... 12 1.4.1 Ubiquitination enzymes ........................................................................... 15 1.4.2 Ubiquitination signalling and function ....................................................... 18 1.5 E3 ligases and p53 ....................................................................................... 21 1.5.1 RING-H2 ligases .................................................................................... 21 1.5.1.1 MDM2 ...................................................................................... 21 1.5.1.2 Pirh2 ....................................................................................... 27 1.5.1.3 COP1 ....................................................................................... 33 1.5.2 AIP4 HECT ligases .................................................................................. 34 1.5.3 Additional groups E3 ligases (UBE4B and CHIP) ......................................... 38 1.6 p73 overview ............................................................................................... 39 vii 1.6.1 Role of p73 in cancer .............................................................................. 42 1.6.2 p73 regulation ....................................................................................... 44 1.6.3 p73 and E3 ligases ................................................................................. 45 1.7 Aim of thesis ................................................................................................ 48 1.8 References ................................................................................................... 51 CHAPTER 2- Residues 240-250 in the c-terminus of pirh2 are essential for Pirh2 self- ubiquitination ...................................................................................................... 71 2.1 Introduction ................................................................................................. 72 2.2 Material and Methods .................................................................................... 73 2.2.1 Plasmids and antibodies .......................................................................... 73 2.2.2 Cell Culture and DNA transfection ............................................................ 74 2.2.3 Expression and recombinant protein preparation ........................................ 74 2.2.4 In vitro self-ubiquitination assay .............................................................. 75 2.2.5 In vivo self-ubiquitination assay ............................................................... 75 2.2.6 Ubiquitination integrated density ............................................................. 76 2.3 Results ........................................................................................................ 76 2.3.1 Pirh2 in vitro self-ubiquitination optimal conditions ..................................... 76 2.3.2 Mapping Pirh2 domains for self-ubiquitination in vitro ................................. 80 2.3.3 Residues 240-250 in the C-terminal domain are essential for Pirh2 self- ubiquitination ................................................................................................ 84 2.3.4 Pirh2 preferentially utilizes Lys-48 of ub to mediate p53 ubiquitination in vitro .................................................................................................................... 87 2.4 Discussion ................................................................................................... 92 2.5 References .................................................................................................. 94 CHAPTER 3- Pirh2 represses p73 transcriptional activity by promoting its ubiquitination 98 3.1 Introduction ................................................................................................. 99 3.2 Material and Methods .................................................................................. 101 3.2.1 Plasmids and antibodies ........................................................................ 101 viii 3.2.2 Cell Culture and DNA transfection ......................................................... 102 3.2.3 Expression and recombinant protein preparation ...................................... 102 3.2.4 Immunoprecipitation ............................................................................ 102 3.2.5 In vitro self-ubiquitination assay ............................................................ 103 3.2.6 In vivo self-ubiquitination assay ............................................................ 103 3.3 Results ...................................................................................................... 104 3.3.1 Pirh2 promotes the ubiquitination in vivo ................................................ 104 3.3.2 Pirh2 promotes the ubiquitination in vitro using k63 lysine chains .............. 107 3.4 Discussion ................................................................................................. 111 3.5 References ................................................................................................ 113 CHAPTER 4- Pirh2 E3 ligase inhibits the AIP4-p73 negative regulatory pathway by mediating AIP4 expression and ubiquitination ........................................................ 117 4.1 Introduction ............................................................................................... 118 4.2 Material and Methods .................................................................................. 119 4.2.1 Plasmids and antibodies ........................................................................ 119 4.2.2 Cell Culture and DNA transfection ......................................................... 120 4.2.3 siRNA experiments ............................................................................... 120 4.2.4 Expression and recombinant protein preparation ...................................... 121 4.2.5 Immunoprecipitation ............................................................................ 121 4.2.6 In vitro ubiquitination assay .................................................................. 121 4.2.7 In vivo ubiquitination assay ................................................................... 122 4.2.8 Cell cycle analysis ................................................................................ 123 4.3 Results ...................................................................................................... 123 4.3.1 In vivo and in vitro binding of Pirh2 and AIP4 utilizing the N-terminal domain of Pirh2 and HECT domain of AIP4 ..................................................................... 123 4.3.2 Pirh2 down-regulates AIP4 protein expression whereas AIP4 up-regulates Pirh2 expression ................................................................................................... 130 ix 4.3.3 p73 expression in response to AIP4 and/or Pirh2 overexpression/ knockdown at the endogenous and exogenous level .............................................................. 133 4.3.4 Pirh2 induces AIP4 ubiquitination and ceases AIP4-p73 ubiquitination in vivo and in vitro.................................................................................................. 138 4.3.5 Pirh2 restores p73 cell cycle arrest function ............................................. 148 4.4 Discussion ................................................................................................. 151 4.5 References ................................................................................................ 154 CHAPTER 5- General Discussion ........................................................................... 158 5.1 Thesis Overview ......................................................................................... 159 5.2 Pirh2 self ubiquitination and p53 family association ......................................... 162 5.3 Pirh2-AIP4-p73 story ................................................................................... 168 5.4 Limitation and future directions .................................................................... 171 5.5 Closing remarks ......................................................................................... 173 5.6 References ................................................................................................ 174 x