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UC San Diego Electronic Theses and Dissertations UC San Diego UC San Diego Electronic Theses and Dissertations Title Bcl3 and REG-gamma are the Regulators of NF-kappaB p50 and p52 Permalink https://escholarship.org/uc/item/2293k9sk Author Du, Qian Publication Date 2017 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERISTY OF CALIFORNIA, SAN DIEGO Bcl3 and REG-gamma are the Regulators of NF-kappaB p50 and p52 A thesis submitted in partial satisfaction of the requirements for the degree Master of Science in Chemistry by Qian Du Committee in Charge: Professor Gourisankar Ghosh, Chair Professor Simpson Joseph Professor Emmanuel Theodorakis Professor Wei Wang 2017 ii The Thesis of Qian Du is approved, and it is acceptable in quality and form for publication on microfilm and electronically: Chair University of California, San Diego 2017 iii DEDICATION This thesis is dedicated to my beloved parents, for their endless love, caring and under- standing throughout my life. This thesis is also dedicated to my beloved grandparents, for their kindness and devo- tion. Their selflessness will always be remembered. iv TABLE OF CONTENTS Signature Page……………………………………………………………………………ⅲ Dedication.......................................................................................................................…ⅳ Table of Contents…………………………………………………………………………ⅴ List of Figures…………………………………………………………………………….ⅵ Preface……………………………………………………….....………………………...ⅷ Acknowledgemnts………………………………………….……...……………………...ⅸ Abstract of the Thesis……………………………………...…………………...………....ⅺ CHAPTER Ⅰ: Introduction………………………………………………………………...1 CHAPTER Ⅱ: Material and Method……………………………………………………..10 CHAPTER Ⅲ: Bcl3 Phosphorylation Sites Results……………………………………..15 CHAPTER Ⅳ: p105 Processing Result…………………………………………………40 CHAPTER Ⅴ: Discussion………………………………………………………………..44 References………………………………………………………………………………..46 v LIST OF FIGURES Figure 1.1: NF-κB Protein Family………………………………...…………….……...……..3 Figure 1.2: IκB Protein Family (Oeckinghaus, A., & Ghosh, S. 2009)…………..….………..5 Figure 1.3: Bcl3 Structure (Jean Loup Huret., 2012)……………………………………..…..6 Figure 3.1: only p52:Bcl3 complex activate reporters’ activity…………………………...…16 Figure 3a.1: p52:Bcl3 single mutants affect reporters’ activity…………………..……..…...18 Figure 3a.2: p52:Bcl3 complex in control and AKT2 KD HeLa cells………...……..………19 Figure 3a.3: p52:Bcl3-Ser33 single mutants’ effect on reporter activity at control and AKT2 KD HeLa cells………………………………………...………………..……….………19 Figure 3b.1: p52:Bcl3 complex activity in control, IKK1 and IKK2 KD HeLa cells…….....21 Figure 3b. 2: p52:Bcl3-Ser446 phospho-mimetic mutant complex activity in control and IKK1 KD HeLa cells……………………………………………………………………22 Figure 3b. 3: p52:Bcl3-Ser446 phospho-mimetic mutant complex activity in control and IKK2 KD HeLa cells……………………………………………………………....……22 Figure 3b. 4: IKK XII inhibitor’s effect to basal level reporter activity ………………….....23 Figure 3b. 5: p52:Bcl3-Ser446 single mutants complex activity in control and IKK XII inhib- itor treated HeLa cells………………………………..………...……………..…..…….23 Figure 3c. 1: p52:Bcl3-Ser114 phospho-mimetic mutant complex activity in control and Erk2 KD HeLa cells…………………………………………….……………....………….....25 Figure 3c. 2: Erk II inhibitor’s effect to basal level repertory activity……….…................…26 Figure 3c. 3: p52:Bcl3-Ser114 single mutants complex activity in control and Erk II inhibitor treated HeLa cells…………………...…………………………...………………..…….26 Figure 3d. 1: Bcl3:p52:DNA ternary complex formation with Bcl3 from 293T cells…….....29 Figure 3d. 2: Bcl3:p52:DNA ternary complex disappearance by only phosphorylating Bcl3 Site 33 and 446 (Bcl3-EE)………………………………….………………..................30 vi Figure 3d. 3: Bcl3:p52:DNA ternary complex formation with Bcl3-S33ES114ES446E from E. coli cells…………………………………………………….……….…………...…..31 Figure 3d. 4: Bcl3:p52:DNA ternary complex formation with kinases inhibitor treatment in HeLa cells………......................................................…………………………………...32 Figure 3d. 5: Bcl3:p50:DNA ternary complex formation is different from p52…………..…33 Figure 3e. 1: p52:Bcl3-Ser366 single mutants complex affects reporter activity…...……….36 Figure 3e. 2: p52:Bcl3-Ser366 phospho-mimetic mutant complex affects reporter activity in control, AKT2 KD, Erk2 KD, IKK1 KD and IKK2 KD HeLa cells………..……….....37 Figure 3e. 3: p52:Bcl3 mutants complex affects reporter activity and its expression level check in Western blot…………………………………………...………...…..……..….38 Figure 3e. 4: Bcl3:p52:DNA ternary complex formation with Bcl3-S33ES114ES446E (EEE) and Bcl3-S33ES114ES366ES446E (EEEE) from E. coli……………………….…..….39 Figure 4a.1: p50 nuclear protein expression in Regα, Regγ and p50 KD HeLa cells and U2OS cells………………………………………………………………….……………….…41 Figure 4a.2: p50 nuclear protein expression in Regγ KD THP1 cells…………..………..….42 Figure 4b.1: gene expression level in Regα, Regγ and p50 KD HeLa cells…….…...………43 vii PREFACE This thesis is the final work my Masters study at the University of California, San Di- ego. It serves as documentation of my research during the study, which has been made from October 2015 until June 2017. It presents the results of a study towards uncovering the regu- latory features of Bcl3 and REG family proteins’ processing of p105. viii ACKNOWLEDGEMENTS I am using this opportunity to express my sincere gratitude to everyone who has sup- ported me throughout my study here in UCSD. Especially my advisor, Professor Gourisankar Ghosh for his continuous support of my study and related research, for his patience, motiva- tion and encouragement. His guidance helped me in all the time of research and writing this thesis; and my mentor Yidan Li who always patiently explain all the experiments, prepare knockdown cells for me and guide me to become an independent researcher. Without her ad- vices and our discussions on this project, it would not have been finished. Without their help, I couldn't have achieved this far. Besides my advisor, I would like to thank the rest of my thesis committee: professor Simpson Joseph, professor Emmanuel Theodorakis and professor Wei Wang for their support and almost immediate response to join the committee. Special thanks to professor Simpson Joseph for generously providing the plate reader for my luciferase assays, without his help, it is impossible for me to collect my results. I would also want to give thanks to all my fellow lab mates for the stimulating discus- sions, and for all the fun we have had in the last two years. Also, I want to thank my friends Boqing Gu, Samantha Natividad Cohen, Kyle Shumate, Rohit Ranjan, Si Hoon Lee and Miao Fan for helping me, supporting me and listening to me whenever I need. ix Finally, I would like to thank my family, especially my parents. For their understanding and support which make the difficulties become easy. x ABSTRACT OF THE THESIS Bcl3 and REG-gamma are the Regulators of NF-kappaB p50 and p52 by Qian Du Masters of Science in Chemistry University of California, San Diego, 2017 Professor Gourisankar Ghosh, Chair Apart from the canonical and non-canonical activation pathways of NF-κB family, the atypical IκB family member Bcl3, can regulate p50 and p52 homodimers’ activity. These dimers, known for their transcriptional repression, switch to activate transcription by associating with Bcl3. Several prior studies showed that transcriptional activity of Bcl3 requires extensive phosphorylation. In this study, I confirmed the significance of three phosphorylation sites of Bcl3, Ser33, 114 and 446 by using kinase knockdown cells and kinase-specific inhibitors; AKT, ERK2 and IKK kinases phosphorylate these sites, respectively. In addition, I also identified a new xi phosphorylation site of Bcl3, Ser366. My experiments suggest that both AKT and ERK2 are potential kinases targeting this site. I have also showed that p50 generation can be regulated by a proteasome regulator. This regulator maintains both a low level of free p50 in the nucleus and proper levels of gene expression at basal levels. Future experiments will determine if p52 generation is also regulated by these proteasome regulators. xii CHAPTER I: Introduction 1. NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), was discovered in 1980s by Sen and Baltimore. NF-κB is a family of structurally related transcription factors that function as mediators for cellular development and the immune system (Carmody, and Chen, 2007). The NF-κB family consists of five subunits: RelA/p65, RelB, c-Rel, p105/p50 and p100/p52. These proteins bind to form homo- and hetero-dimers through a shared highly homologous sequence near the N-terminus, referred to as the Rel Homology Region (RHR). This region contains the N-terminal domain (NTD), dimerization domain (DD) and nuclear localization signal (NLS) (Figure 1.1), which are essential for dimerization, DNA binding and nuclear localization (Baldwin, 1996; Ghosh et al., 1998). The consensus DNA binding target sequences of NF-κB dimers are known as κB sites or κB DNA. These κB DNA sites are located within the promoter/enhancer region of target genes (Baldwin, 1996; Ghosh et al., 1998). NF-κB dimers have overlapping but distinguishing binding specificities towards various κB sites (Hoffmann, Natoli, & Ghosh, 2006). The p50 and p52 subunits are generated from the precursor p105 and p100 respectively, through proteolytic processing and form one of the subclasses of NF-κB proteins. This subclass contains ankyrin repeats at their C-termini.
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