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

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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

Chemistry

Qian Du

Committee in Charge:

Professor Gourisankar Ghosh, Chair Professor Simpson Joseph Professor Emmanuel Theodorakis Professor Wei Wang

2017

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 understanding throughout my life.

This thesis is also dedicated to my beloved grandparents, for their kindness and devo- tion. Their selflessness will always be remembered.

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

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 inhibitor 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

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 regulatory 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 discussions, 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.

Finally, I would like to thank my family, especially my parents. For their understanding and support which make the difficulties become easy.

ABSTRACT OF THE THESIS

Bcl3 and REG-gamma are the Regulators of NF-kappaB p50 and p52

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

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. When present as homodimers, these

precursors may suppress expression of certain genes by competing with other transactivating

NF-κB dimers at κB binding site (Collins et al., 2015); The other subclass includes the remaining Rel-related proteins—RelA/p65, RelB and c-Rel. Different from p50 and p52, this subclass of NF-κB has an activation domain called transactivation domain (TAD) (Bours,

1993). Dimers that form with a Rel protein can activate gene expression.

In unstimulated cells, NF-κB proteins are sequestered in the cytoplasm by inhibitor proteins—IκBs (Inhibitor of NF-κB). These inhibitors bind to the RHR of NF-kB dimers with high affinity and largely prevent nuclear localization NF-κB. However, upon stimulation, the NF-κB pathway is activated by the phosphorylation and proteasomal degradation of IκBs, thus releasing NF-κB dimer complexes. The NF-κB dimers can then easily move into the nucleus and activate gene expression by binding to the κB sites (Jacobs

& Harrison, 1998). The critical kinase that is activated by a variety of stimuli is known as

IKK (IκB Kinase).

Figure 1.1: NF-κB Protein Family

2. IκB Proteins

IκB proteins function primarily as inhibitors of NF-κB transcription factors, however, they can also regulate NF-κB activities through different mechanisms. These proteins can be classified into three classes - classical IκB (IκBα, β and ε), precursor IκB (p105/IκBγ and p100/IκBδ) and atypical IκB (Bcl3, IκBζ) (Wang, 2011). IκB proteins are similar in structure harboring multiple ankyrin repeats in the central region that folds into a single domain referred to as the ankyrin repeat domain (ARD) (Baldwin, 1996) (Figure 1.2). The ARDs are known to interact with specific members of the NF-κB dimers (Fujita, Nolan, Liou, Scott &

Baltimore, 1993). The classical IκB proteins (IκBα, IκBβ and IκBε) bind primarily to RelA and c-Rel containing NF-κB dimers. IκBα shows highest preference for NF-κB p50:Rel

heterodimer whereas IκBβ and IκBε prefer RelA and cRel homodimers (Wang, 2011).

Classical IκBs inhibits NF-κB by forming stable 1:1 complexes with NF-κB dimers. The N- terminal unstructured region contains the signal receiving region (SRR) where the two conserved serines are targeted by IKK for phosphorylation and two lysines can be ubiquitinylated by ubiquitin ligases. The C-terminal unstructured region contains the PEST

(proline, glutamic acid, serine and threonine) region.

The precursor IκB proteins (p105/IκBγ and p100/IκBδ) inhibit NF-κB by forming a large multi-protein assembly, known as the κBsome that contains multiple NF-κB and IκB proteins (Savinova et al., 2009). It appears that within a single κBsome complex, multiple

NF-κB dimers can be inhibited, although the precise mode of inhibition by these inhibitors is still under investigation. Signal induced complete degradation of these inhibitors releases

NF-κB. Unlike classical IκBs, the signal inducible phosphorylation sites are located within the C-terminal region of these inhibitors.

Atypical IκBs (Bcl3, IκBζ) function distinctly. They do not function as classical inhibitors like the two other classes of inhibitors. Indeed, their most defined function is at the level of transcription. These factors are known to activate genes by acting as co-activators of p50 and p52 homodimers. The ARD of these inhibitors contains seven AR and like other

IκBs they use the ARD to bind NF-κB (Wang, 2011).

Figure 1.2: IκB Protein Family (Oeckinghaus, A., & Ghosh, S. 2009)

3. Bcl3

B-cell Lymphoma 3-encoded protein (Bcl3) is a member of the atypical IκB protein family (Figure 1.3). It was discovered in B cell chronic lymphocytic leukemia (Zhang, Wang,

Claudio, Brown & Siebenlist, 2017) where chromosomal translocation transfers the Bcl3 gene behind a strong constitutive promoter resulting in 3-4-fold higher levels of Bcl3 protein.

Bcl3 is also overexpressed in many other non-lymphoid solid tumors. However, in these cases no chromosomal translocation is observed, and therefore, overexpression of Bcl3 occurs due to an unknown mechanism. It was shown that Bcl3 stabilizes p50 homodimer-

DNA complexes through inhibition of p50 ubiquitination. It is shown that the direct interaction between p50 and Bcl3 is required for Bcl3-mediated inhibition of pro- inflammatory gene expression, and the regions of Bcl3 that mediate interaction with p50

homodimers is at ankyrin repeats (ANK) 1, 6 and 7 and the N-terminal region of Bcl3

(Collins et al., 2015).

Figure 1.3: Bcl3 Structure (Jean Loup Huret., 2012)

4. Bcl3 Functions as Coactivator and Corepressor for Transcription

Bcl3 was identified in late 1980s, however, its functions could not be determined with great certainty. It was determined to be a constitutively expressed nuclear protein; this conclusion was made based on its nuclear localization in cancer cell models. Subsequently, it was also found that in most non-cancer cells Bcl3 is present in the cytoplasm (Oeckinghaus

& Ghosh, 2009). Several studies confirmed the role of Bcl3 in cell migration and metastasis when overexpressed. Lack of Bcl3 is responsible for immune deficiency and lack of expression of inflammatory genes (Chen, et al., 2016, Jeong, Kwon, & Shin, 2014).

Earlier transfection-based experiments showed that Bcl3 interacts with both p50 and p52 homodimers and can activate the expression of reporter genes through the protein- protein interactions between the ARD of Bcl3 and the DD of NF-κB dimers to form

Bcl3:p50/p52:DNA ternary complex (Schuster, Annemann, Plaza-Sirvent & Schmitz, 2013).

Interestingly, when Bcl3 is un-phosphorylated, such as being expressed in E. coli or baculovirus, the ternary complex is failed to form in vitro. Not only so, the un-

phosphorylated Bcl3 will function as classical IκBs to dissociate the NF-κB:DNA binary complexes. These and many other experiments showed that Bcl3 is heavily phosphorylated in vivo to activate transcriptional activity by forming the ternary complex with NF-κB and

DNA (Fujita, Nolan, Liou, Scott & Baltimore, 1993). So far only two phosphorylation sites have been identified. Both are located at the C-terminus. These sites (Ser394 and Ser398) are phosphorylated by GSK3β and are responsible for Bcl3 continuous basal degradation through proteasomal pathway using the E3 ligase TBLR1 (Viatour et al., 2004b).

Previous work in our lab showed that the Bcl3:p52 complex can interact with two different types of κB sites in vivo with two distinct transcriptional outcomes; The Bcl3:p52 complex upon binding to G/C-centric κB sites (GGGANG/CNTCCC) activates transcription.

However, upon binding to A/T-centric κB sites (GGGANA/TNTCCC) Bcl3 represses transcription. That is, as few a single base pair variation can drastically differentiate the transcriptional program (Wang, et al., 2017).

5. Constitutive Processing of p105 into p50

As indicated earlier, one of the Bcl3 binding partners is p50 homodimer. The sole source of p50 is its precursor protein p105. It is known that the proteasome is responsible for the generation of NF-κB subunits, p50 (and p52) which selectively degrades the C-terminus of p105 (Moorthy, et al., 2006). Processing is limited since only a pool of these precursors remains unprocessed. The unprocessed precursors act as inhibitors of NF-κB including the

processed products, p50 and p52, by forming larger assemblies called kappaBsomes

(Savinova et al., 2009). The processed product, p50 and p52, associate with themselves and other NF-κB subunits forming homo- and heterodimers. p105 and p100 can also undergo complete degradation which is required to release bound NF-κB subunits (Basak et al., 2007;

Lang et al., 2003). Processing and complete degradation of these precursors are under intense investigation since their discovery in 1990s since both forms are responsible for carrying out specific but opposite activities. Signal inducible complete degradation and processing are in general better understood with both processes requiring ubiquitination and 26S proteasome.

The mechanism of constitutive processing of p105 has remained a subject of intense investigation, however. Our laboratory previously reported that constitutive p105 processing does not require ATP or ubiquitination and that the free 20S proteasome is able to process p105 in vitro (Moorthy et al., 2006). However, many other mechanisms of constitutive p105 processing have also been reported including co-translational (Lin et al., 1998, 2000), ATP- dependent (Fan and Maniatis, 1991), poly and mono-ubiquitination-dependent (Kravtsova-

Ivantsiv et al., 2009; Orian et al., 2000; Orian et al., 1999; Palombella et al., 1994). Thus, the subject of constitutive p105 processing has remained unclear. Several recent reports have shown that many proteins that are degraded upon ubiquitination and in an ATP independent manner are indeed degraded by the 20S-bound to a different class of regulators known as

REG proteins (also known as PMSE, 11S, PA28). In addition, it is also unknown if the free

20S proteasome exists in vivo. Thus, it is critical to test if the 20S bound to REG is the true

processing enzyme for p105.

Thesis Focus:

As mentioned earlier, three sites of Bcl3 phosphorylation sites were determined, Ser33,

Ser114 and Ser446. Therefore, it is important to identify the function of these phosphorylation sites towards Bcl3’s biochemical and biological activities. Through experiments and analysis, a new phosphorylation site was also studied, which appears to play an important role in transcriptional regulation by functioning in conjunction with other sites.

Another focus of the study is to determine how REGγ, as one of the REG family members, can affect p105 processing. Previous member Yidan Li showed that REGγ does not facilitate processing but appears to completely degrade p105 and its processed product, p50. Based on my result, p50 levels are higher in cells devoid of REGγ. The higher levels of p50 repress expression of specific genes by competing with RelA or activates specific genes by synergizing with Bcl3/IκBζ.

Both these projects are part of larger ongoing projects in the laboratory. My emphasis was to learn different techniques to address these broad biological problems related to NF-

κB signaling to enhance our knowledge in this area.

CHAPTER II: Material and Method

1. Antibodies and Reagents

Antibody: Bcl3NR (gift from NIH, Bethesda, MD), p-Bcl3-S33 (BioBharati LifeScience Dvt.

Ltd.), IKK (sc-7184, Santa Cruz), Akt (sc-8312, Santa Cruz Biotechnology), Erk

(BioBharati LifeScience Dvt. Ltd.), p52 (1495)

Reagents: anti-Flag M2 (F1804), Flag-peptide, Akt VIII inhibitor (124018, Calbiochem), Erk

II inhibitor (FR180204), IKK inhibitor XII (401491)

PCR Reagents: RT-PCR (reagent, Super reverse transcriptase MuLV Kit BB-E0043,

BioBharati LifeScience Dvt. Ltd.)

EMSA Probe: Adenosine-5’-triphosphate γ-32P (PerkinElmer, 250uCi)

2. Mammalian Cell Culture and Transfection

HEK 293T, HeLa and U2OS cells were used for transient mammalian cell transfection.

Cells were plated the day before to reach around 60% confluency at the day of experiment and grew in Dulbecco’s modification of Eagle’s medium (DMEM) with 4.5g/L Glucose, L- glutamine and sodium pyruvate, supplemented with 10% fetal bovine serum and 1% penicillin- streptomycin-L-glutamate (PSG). pEYFP vector, Flag-p52 and Flag-tagged-Bcl3-WT and various mutants were transfected into cells using polyethyleneimine (PEI). Cell lysates were prepared 48 hours after transfection.

Knockdown cell line was performed by Yidan Li and prepared in five days following the procedure outline as follows. On day 1, cells at 70% confluency were infected with lentiviral shRNA vector and packaging vectors with DMEM without serum and PSG. 2× PEI was added, and the mixture was incubate for 15 minutes at room temperature then added to plate dropwise.

On day 3, the supernatant was collected and filtered through a 0.45μm syringe filter. The following day, media containing virus was removed and replenished with new media containing virus plus 10μg/mL polybrene and incubated overnight. On day 5, the virus containing mead was aspirated and replaced with fresh DMEM with 10% FMS and 1× PSG.

3. Protein Purifications

Mammalian cell proteins were purified through transient transfection in HeLa, HEK 293T and U2OS cells as described above. After 48 hours of incubation, cells were lysed by RIPA buffer (20mM Tris pH 8.0, 200mM NaCl, 1% Triton-X100, 2mM DTT, 5mM 4-nitriphenyl

phosphate di-Tris)Salt, 2mM Na2VO4 and 1mM PMSF) supplemented with protease inhibitor cocktail. To ensure efficient lysis, cell lysates were also sonicated for at least three times at 15 seconds burst intervals. After centrifugation, the clear cell lysates were incubated with 50μL

Anti-Flag M2 Affinity resin (Sigma A2220) overnight at 4℃. At the following day, the Flag resin was washed for three times with ice-cold 1×TBS buffer (25mM Tris pH 7.5, 140mM

NaCl) and eluted with 30μL of FLAG peptide (Sigma F3290) at 100μg/mL.

Proteins expressed and purified in vitro were first transformed into Rosetta cells and

grown in 1L LB/Kanamycin broth to O.D.600 if 0.6 at 37℃. Induction of protein expression was performed with 0.2mM IPTG overnight at room temperature. Cells were harvested and pellets were re-suspended in lysis buffer (20mM Tris-HCl pH 7.5, 150mM NaCl, 0.1mM PMSF,

10% Glycerol), supplemented with proteasome inhibitor (Sigma/Alrich). The resuspension mixture was then lysed through sonication and centrifuged to separate the insoluble fraction.

Ni resin was equilibrated with lysis buffer and used for protein binding at 4℃. The column was then washed with lysis buffer supplemented with 5mM imidazole (pH 7.5) and eluted with

(150mM NaCl, 25mM Tris pH7.5, 250 Imidazole pH7.5).

4. Luciferase Reporter Assays

HeLa cells were transfected first as described above with Flag-p52 (1-415), pEYFP-

Flag-Bcl3 WT, various mutants or with inhibitors together with luciferase reporter DNA with specific κB DNA promoters (5’-TCGACGGAAGGGGGTGACCCCTTGG-3’). After 48 hours of incubation, cells were lysed using 100μL lysis buffer (Dual-Luciferase Reporter

Assay System, Promega) and incubated in 4℃ for 30 minutes. After pelleting down the cell debris, 20μL whole cell lysates were mixed with 20μL of substrate (Dual-Luciferase Reporter

Assay System, Promega) in Costar plate and the absorbance was taken by microplate reader.

Bradford Assay (BioRad) was performed at the same day for estimation of protein concentration to complete the figure.

5. Electrophoretic Mobility Shift Assays (EMSA)

The DNA probe was 5’ end radiolabeled with 32P using T4-PNK (Promega) and [γ-32P]

ATP and annealed to the complementary strands. This oligonucleotide contains κB sites from different genes. Each reaction had a total volume of 6μL that contained 3μL binding buffer

(10mM Tris-HCl pH 7.5, 50mM NaCl, 10% (v/v) glycerol, 1% (v/v) NF-40, 1mM EDTA, 0.1 mg/mL poly(dI-dC)), 1μL ~10000cpm of 32P-labeled DNA probe, 2μL of proteins. The reactions were incubated in room temperature for about 20 minutes and loaded onto a 5% (w/v) non-denatured polyacrylamide gel at 200V for 1 hour in 25mM Tris base, 190mM glycine and

1mM EDTA. Before exposing to a phosphor imager overnight, the gel was dried for an hour and imaged the following day (Typhoon scanner 9400, Amershan Bioscience).

P-selectin (A/T): 5’-TCGACGGAAGGGGGTAACCCCTTGGG-3’

MHC: 5’-TCGACGGGCTGGGGATTCCCCATCTCG-3’

HIV: 5’-GGGGACTTTCCGCTGGGGACT-3’

6. RT-PCR

RNA isolation was done by using TRIzol reagent and followed the protocol as directed in their website. The reverse transcription of the isolated RNA was done by using Super reverse transcriptase MuLV Kit (BB-E0043, BioBharati LifeScience Dvt. Ltd.) under condition: 42℃

50min, 70℃ 15min and 4℃ forever. The following PCR reaction for the cDNA products were done by using Pfu, under the condition 95℃ 5min, 95℃ 30sec, 55℃ 30sec, 72℃ 30sec, go to

step 2 for 25 cycles and 72℃ 10min, 4℃ hold. Samples were run on 2% Agarose gel and quantified by using GAPDH primer.

CHAPTER III: Bcl3 Phosphorylation Sites Results

Bcl3 Is Phosphorylated at Three Sites

One of the research key focus in the laboratory is to identify functionally important phosphorylation sites of the oncoprotein Bcl3 and the kinases responsible to target these sites.

By carrying out mass-spectrometry (MS) experiments of 293T mammalian cells over- expressing Bcl3, we found 27 sites that may undergo phosphorylation. At least 19 of those sites are at high confidence level. Of these sites two were reproducible in all MS experiments (total of five independent experiments). These sites are Ser33 and Ser446.

An Assay to Test Bcl3-Dependent Gene Expression

To perform the gene expression experiments, a well-established luciferase reporter system was used. In the reporter, a known G/C-centric p52/Bcl3 DNA response element

(GGGGAGTCCCC) was inserted behind the transcription start site which is fused to the coding sequence of firefly gene, luciferase (Vopalensky, et al., 2008). The reporter plasmid, p52 and

Bcl3 plasmids were co-transfected into the cells and incubated for 48 hours. As shown in figure

3.1, Bcl3 or p52 or p52 + Bcl3 were co-transfected with P-Selectin (G/C) reporter to compare their luciferase activity. The result shows that p52 and Bcl3 co-transfection greatly enhanced the activity. p52 or Bcl3 alone showed no activity.

P-Selectin

20 (GGGGTGACCCC) 18 16 14 12 10 8 6 4 2 0 vector Bcl3-WT p52 p52+Bcl3-WT HeLa 1 0.8 1.3 18

Figure 3.1: only p52:Bcl3 complex activate reporters’ activity

A. AKT Phosphorylates Ser33 of Bcl3

It is previously determined using mass-spectrometry that Ser33 of Bcl3 is phosphorylated.

Efforts were made to identify the kinase targeting Ser33. The first clue came from the sequence of the region surrounding Ser33. This site is located within the near consensus of protein kinase,

AKT. In vitro phosphorylation confirmed that AKT phosphorylates Ser33. It was further shown that endogenous Bcl3 failed to localize into the nucleus of HeLa cells treated with the AKT inhibitor in response to LPS (lipopolysaccharide). Moreover, when serine 33 is mutated to alanine (phospho-inhibitory mutant), the mutant (S33A) undergoes constitutive degradation by the proteasome in the cytoplasm. Collectively these results confirmed that Ser33 17

phosphorylation is essential for its activity in vivo. It also suggested that AKT might be the kinase that phosphorylates Ser33 (Wang, et al., 2017). However, further proof was necessary to confirm this hypothesis.

Luciferase activity of phospho-resistant and phospho-mimetic mutants of Ser33 (S33A and S33E) were compared with Bcl3-WT (Figure 3a.1). S33A mutant showed greatly reduced the reporter activity and S33E activity was enhanced. These results indicate that the phosphorylation of Ser33 is essential. Similarly, I also tested phsopho-resistant and phospho- mimetic mutants at positions 114 and 446. As expected, luciferase activity was reduced for alanine mutant and enhanced for glutamic acid mutant.

One way to test AKT’s activity toward Bcl3 is to examine the Bcl3-dependent gene expression in cells with reduced levels of AKT. In this experiment, AKT kinase was knocked down (KD) in HeLa cells (done by others), detailed procedure was described above. The reporter activity in AKT2 KD cells is lower than in the control cell (Figure 3a.2). These results suggest the effect of AKT kinase depletion on Bcl3. The phospho-resistant (S33A) and phospho-mimetic (S33E) mutants were also tested in the AKT2 KD HeLa cells (Figure 3a.3).

The reporter activity of S33A mutant remained the same in both cell lines, however, the reporter activity was decreased in AKT2 KD cell with S33E mutant. The precise reason for this decrease is not clear. It is possible that AKT also phosphorylates other sites of Bcl3 that are important for transcriptional activity. These results further confirmed the previous conclusion that AKT is responsible for Bcl3-Ser33 phosphorylation. 18

P-Selectin (GGGGTGACCCC) 60

0 p52+Bcl3 p52+Bcl3 p52+Bcl3 p52+Bcl3 pEYFP p52+Bcl3 WT S33A S33E S446A S446E HeLa 1 5.8 0.5 43.5 2.9 49.1

P-Selectin (GGGGTGACCCC) 40

0 pEYFP p52+Bcl3-WT p52+Bcl3-S114A p52+Bcl3-S114E HeLa 1 14.5 9.8 38.3

Figure 3a.1: p52:Bcl3 single mutants affect reporters’ activity

P-Selectin (GGGGTGACCCC) 40

0 vector p52+Bcl3 WT pLKO 1 35.4 Akt2 KD 1 24.9

Figure 3a.2: p52:Bcl3 complex in control and AKT2 KD HeLa cells

P-Selectin (GGGGTGACCCC) 50

0 pEYFP p52+Bcl3 WT p52+Bcl3 S33A p52+Bcl3 S33E pLKO 1 5.8 0.5 43.5 Akt2 KD 1 1.8 0.5 10.3

Figure 3a.3: p52:Bcl3-Ser33 single mutants’ effect on reporter activity at control and AKT2

KD HeLa cells 20

B. IKK1/2 Phosphorylates Ser446

Based on previous experiments, Ser446 is also an important residue for Bcl3 function, which Ser446A mutant reduced reporter activity and Ser446E showed enhanced activity

(Figure 3a.1). Previous MS experiments of Bcl3 isolated from 293T cells treated or untreated with IKK inhibitor showed that Ser446 might be phosphorylated by IKK, further test was necessary to validate this conclusion.

The first approach to test this hypothesis is by using IKK1 and IKK2 knockdown (KD) cells (prepared by others). Reporter assay showed that both IKK1 and IKK2 could be the

Ser446 kinase since the activity was reduced in both KD cell lines (Figure 3b.1). Bcl3 phospho- mimetic mutant (Bcl3-S446E) with p52 were transfected in both types of IKK KD cell lines

(Figure 3b. 2, 3). The result shows that reporter activity of the mutant remained relatively similar in both KD cell lines, suggesting the possibility for both IKK1 and IKK2 to phosphorylate Ser446. It is known that these two kinases predominantly form a complex (Israel,

2009). Therefore, it cannot be concluded which kinase plays more important role towards the other.

To further verify the specificity of IKK kinase for Bcl3-Ser446 phosphorylation, IKK inhibitor XII was used to treat HeLa cell for luciferase assay. This inhibitor inhibits both IKK1 and IKK2 to similar extents. Figure 3b. 4 showed the inhibitor XII does not affect basal level of reporter activity. The experiment was done by transfecting Bcl3-WT and Ser446 single mutants with p52 to the inhibitor treated cell. As expected, the inhibitor greatly reduced the 21

reporter activity in all samples (Figure 3b. 5), which it remained at similar level for S446A samples either treated or un-treated with inhibitor XII; and Bcl3-S446E mutant showed reduced activity in inhibitor-treated cells (Figure 3b. 5). Collectively, these results suggest that

IKKs are responsible for Bcl3-Ser446 phosphorylation.

P-Selectin (GGGGTGACCCC)

0 vector p52+Bcl3 WT pLKO 1 35.4 IKK1 KD 1 3.4 IKK2 KD 1 19.7

Figure 3b.1: p52:Bcl3 complex activity in control, IKK1 and IKK2 KD HeLa cells 22

P-Selectin (GGGGTGACCCC) 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 pEYFP p52+ Bcl3-S446E pLKO 1 4.3 IKK1 KD 1 3.3

Figure 3b. 2: p52:Bcl3-Ser446 phospho-mimetic mutant complex activity in control and IKK1

KD HeLa cells

P-Selectin (GGGGTGACCCC) 30

0 pEYFP p52+Bcl3-S446E pLKO 1 26.1 IKK2 KD 1 22.7

Figure 3b. 3: p52:Bcl3-Ser446 phospho-mimetic mutant complex activity in control and IKK2

KD HeLa cells 23

P-Selectin (GGGGTGACCCC) 1.2

0.8

0.6

0.4

0.2

0 DMSO IKK XII 20uM HeLa 1 0.87

Figure 3b. 4: IKK XII inhibitor’s effect to basal level reporter activity

P-Selectin (GGGGTGACCCC)

16 14 12 10 8 6 4 2 0 pEYFP p52+Bcl3 WT p52+Bcl3-S446A p52+Bcl3-S446E DMSO 1 11.9 3.2 15.7 IKK XII 20uM 1 5.8 2.6 5.4 IKK XII 25uM 1 6.1 1.9 2.0

Figure 3b. 5: p52:Bcl3-Ser446 single mutants complex activity in control and IKK XII inhibitor treated HeLa cells 24

C. Erk2 Phosphorylates Ser114

Different from site 33 and 446, Ser114 was not shown to be a potential phosphorylation site by MS analysis. However, through EMSA experiments, Bcl3-S33E/S446E was not able to activate the Bcl3:p52:DNA ternary complex formation. Therefore, some other sites must exist that is assisting with this function. Because of the proline and serine rich feature of Bcl3 at N- and C-terminus, a bold assumption was made that MAP kinases (Erk1,2; Jun; p38), which have high tendency to phosphorylate sequences rich in proline/serine may contain kinase(s) responsible for Bcl3 phosphorylation. By treating HeLa cells with the kinases inhibitors during luciferase assay for Bcl3 transcription activity, Erk showed the least activation. In this case, our focus was drawn onto Erk kinase. In vitro kinase assay with Erk inhibitor was tested with five selected sites. Among those, Ser114 showed the most effect due to this inhibitor. Based on this observation, Erk was hypothesized to be the kinase for Bcl3-Ser114 phosphorylation

(Wang, et al., 2017).

To verify this point, luciferase assays were performed in Erk2 knockdown (KD) cells

(prepared by others). The reporter activity decreased in Erk2 KD cells compare to control cell line, which confirmed that Erk kinase is responsible for Bcl3 phosphorylation (Figure 3c. 1).

By mutating Ser114 to phospho-mimetic mutant (Bcl3-S114E), the activity was decreased comparing to control cell. This proves the effect of Erk towards site 114 of Bcl3 (Figure 3c. 1).

To further test the specificity of Erk for Bcl3-Ser114 phosphorylation, HeLa cells were treated with Erk II inhibitor. Figure 3c. 2 showed that Erk II inhibitor has no effect on basal 25

level activity of the luciferase reporter P-Selectin (G/C). The experiment continued by transfecting Bcl3-Ser114 phospho-resistant (Bcl3-S114A) and phospho-mimetic (Bcl3-S114E) mutants together with p52 to check the reporter activity (Figure 3c. 3). The result shows that

Bcl3-S114A transcription activity remains to be at similar level for both treated and un-treated cell; however, when tested with Bcl3-S114E, the reporter activity level decreased in Erk II inhibitor treated cell. Collectively, these data strongly suggests that Erk is responsible for Bcl3-

Ser114 phosphorylation.

P-Selectin (GGGGTGACCCC) 10 9 8 7 6 5 4 3 2 1 0 pEYFP p52+Bcl3-WT p52+ Bcl3 S114E pLKO 1 6.8 8.8 Erk2 KD 1 3.4 7.1

Figure 3c. 1: p52:Bcl3-Ser114 phospho-mimetic mutant complex activity in control and Erk2

KD HeLa cells 26

P-Selectin (GGGGTGACCCC) 1.4

1.2

0.8

0.6

0.4

0.2

0 DMSO Erk II 10uM HeLa 1 0.9

Figure 3c. 2: Erk II inhibitor’s effect to basal level repertory activity

P-Selectin (GGGGTGACCCC) 45 40 35 30 25 20 15 10 5 0 p52+Bcl3- p52+Bcl3- p52+Bcl3- pEYFP WT S114A S114E DMSO 1 14.5 9.8 38.3 Erk II 10uM 1 8.9 7.7 24.5

Figure 3c. 3: p52:Bcl3-Ser114 single mutants complex activity in control and Erk II inhibitor treated HeLa cells 27

D. Bcl3:p52:DNA Ternary Complex

To further investigate the significance of Bcl3 phosphorylation, the advantage was taken for its ability to form the ternary complex only when it is phosphorylated. As described in the introduction, Bcl3 isolated from 293T cells form a ternary complex with the p52:DNA binary complex. In contrast, Bcl3 derived from E. coli cells dissociates the p52:DNA binary complex.

If this is true and AKT, IKK and Erk2 phosphorylation of Bcl3 is essential for its transcriptional activity, then Bcl3 isolated from cells treated with inhibitors specific to these kinases will not allow formation of the ternary complex.

To test the condition for Bcl3:p52:DNA ternary complex formation, a previous

Electrophoretic Mobility Shift Assay (EMSA) was repeated using Bcl3-WT proteins purified from E. coli and 293T cells (Figure 3d. 1). In this experiment, it showed that p52 forms binary complex with P-Selectin (A/T) DNA (Figure 3d. 1, lane 2 and 3); un-phosphorylated Bcl3 purified from E. coli cells not only could not form ternary complex with p52 and reporter DNA as with Bcl3 purified from 293T mammalian cell, it removed p52 from the DNA and dissociated the binary complex (Figure 3d. 1, lane 4 and 5). Since E. coli purified un- phosphorylated Bcl3 has this feature, it is later used to test how phosphorylation of residues,

Ser33, Ser114 and Ser446 solely or together affect the ternary complex formation.

To test this, Bcl3-S33ES446E mutant was purified from E. coli cells. However, this mutant failed to form ternary complex with p52 and DNA (Figure 3d. 2, lane 6, 7 and 8), at the same time it also partially functioned as Bcl3-WT from E. coli, which removed some binary 28

complex binding of p52 and DNA (compare to lane 2 and 3 in Figure 3d. 2). Mutant Bcl3-

S33ES114ES446E (Bcl3-EEE) triple mutant was isolated from E. coli and Bcl3-

S33ES1114AS446A (Bcl3-EAA) isolated from 293T cells (figure 4c). Addition of phospho- mimetic S114E to the EE double mutant enabled Bcl3 to form the ternary complex (Figure 3d.

3, lane 6 and 7). Interestingly, when Ser114 and Ser446 were mutated to alanine (phospho- resistant) along with S33E phospho-mimetic mutant (Bcl3-EAA) isolated from 293T cells was tested, this mutant too could form the ternary complex. The ternary complex however shifted faster in EMSA gel (Figure 3d. 3, lane 8 and 9). This observation shows the importance of phosphorylation at site 114 and 446, at the same time, it implies the complexity of ternary complex formation by phosphorylated Bcl3.

Realizing the importance of phosphorylation of Ser33, Ser114 and Ser446, it is further tested how the putative kinases that phosphorylate these sites affect ternary complex formation.

Bcl3 proteins were purified from 293T cells treated with or without the inhibitors specific to these kinases discussed above. These proteins were used for EMSA (Figure 3d. 4). The result shows that Bcl3 isolated from AKT VIII and Erk II inhibitor treated cells, ternary complex could still form (Figure 3d. 4, lane 5 and 6) control (lane 4). However, in Bcl3 derived from

IKK inhibitor XII treated 293 cells the ternary complex mostly disassembled (lane 7). This observation indicates that IKK kinase is responsible for phosphorylation of sites on Bcl3 that facilitate ternary complex formation.

As one of the atypical IκB proteins, Bcl3 can not only form ternary complex with p52, it 29

can also activate another NF-κB member—p50 forming ternary complex. One may expect similar ternary complex formation between p50, Bcl3 and DNA as described for p52. However, the EMSA experiment shows that Bcl3-EEE (E. coli) forms a weaker ternary complex with p50 (Figure 3d. 5, lane 3 and 4) compare to p52 (Figure 3d. 3, lane 6 and 7). Moreover, the complex formation is even weaker with Bcl3-WT purified from 293T mammalian cells (Figure

3d. 5, lane 5 and 6). This result indicates the dissimilar preference between p52 and p50 towards ternary complex formation with Bcl3 proteins.

Figure 3d. 1: Bcl3:p52:DNA ternary complex formation with Bcl3 from 293T cells 30

Figure 3d. 2: Bcl3:p52:DNA ternary complex disappearance by only phosphorylating Bcl3 Site

33 and 446 (Bcl3-EE) 31

Figure 3d. 3: Bcl3:p52:DNA ternary complex formation with Bcl3-S33ES114ES446E from E. coli cells

Figure 3d. 4: Bcl3:p52:DNA ternary complex formation with kinases inhibitor treatment in HeLa cells

Figure 3d. 5: Bcl3:p50:DNA ternary complex formation is different from p52

E. Ser366 Is Another Possible Site for Phosphorylation

In addition to the Bcl3 phosphorylation sites discussed above, another site was also identified on Bcl3—Ser366, which is found to be a functionally important site for phosphorylation as well. It is shown previously that Bcl3-S33ES114AS446A (EAA) mutant can form ternary complex with p52 and DNA (Figure 3d. 3). Therefore, it is hypothesized that other phosphorylation sites might be present that compensate for the defect in Ser114 and

Ser446 phosphorylation. By using an online proteomics database which assembled all discovered Bcl3 phosphopeptides (PhosphoSitePlus-Bcl3 (human)), phospho-peptide containing Ser366 has been identified from several studies. This suggests that Ser366 is a highly probable site for phosphorylation.

Using luciferase assay system, the reporter activity was tested of the two phospho-resistant and phospho-mimetic mutants of Ser366 (S366A and S366E) (Figure 3e. 1). S366A mutant showed reduced activity and S366E showed enhanced activity as compare to Bcl3-WT. This result confirmed that Ser366 is an important residue for Bcl3 function. By verifying the effect of Ser366 at Bcl3 transcriptional activity, we wondered if any of the previously identified kinases be responsible for its phosphorylation. Therefore, luciferase assays were performed again in knockdown (KD) cell lines (prepared by others) (figure 5b). comparing to Bcl3-WT activity level, phospho-mimetic mutant (S366E) shows no reduction when IKK1 or IKK2 kinases were knocked down. This excludes the possibility for this site to be IKK phosphorylated (Figure 3e. 2); however, when AKT2 and Erk 2 kinases were knocked down, 35

the reporter level was decreased comparing to control cell type in S366E mutant (Figure 3e. 2). therefore, it can be deducted that AKT and Erk might be the possible kinases responsible for phosphorylating Ser366.

To further test Ser3666 function, two new mutants were made by including this site, Bcl3-

S33ES114ES366ES446E (Bcl3-EEEE) and Bcl3-S33ES114AS366AS446A (Bcl3-EAAA). To our surprise, even though Bcl3-EAAA showed decreased activity, the Bcl3-EEEE mutant did not induce enhanced reporter activity compare to WT as expected (Figure 3e. 3). Therefore, the Bcl3 protein expression was checked in all three cases (Figure 3e. 3) by blotting with Bcl3 antibody and GAPDH to ensure protein loading amount. The western blot indicates that the expression level between Bcl3-WT and EEEE mutant are similar. In this case, it can be concluded that Ser366, though important for activate Bcl3 function, may not interact together with the other three phosphorylation sites (Ser33, 114 and 446).

It is shown previously that Bcl3-EEE (E. coli) could form ternary complex with p52 and

DNA (Figure 3d. 3). Therefore, by introducing another phospho-mimetic mutation, we want to test if this new mutant Bcl3-EEEE purified from E. coli can also assist ternary complex formation. In this experiment, three different amounts of Bcl3-EEE and Bcl3-EEEE were added to the reaction, as expected, the ternary complex shows increasing binding stability as the Bcl3 protein amount increases (Figure 3e. 4, lane 11, 12 and 13); interestingly, the opposite case applies to Bcl3-EEE (E. coli), as the Bcl3 protein amount increases, this protein almost functions as Bcl3-WT (E. coli) (Figure 3e. 4, lane 8), as the protein amount decreases, ternary 36

complex shows with increasing binding stability (Figure 3e. 4, lane 9 and 10). The exact reason is not known yet, however, it is shown by another lab that constitutive expression of Bcl3 in certain cell types does not necessarily result in increase in the κB binding capacity of p52

(Caamaño, Perez, Lira & Bravo, 1996).

P-Selectin (GGGGTGACCCC) 12

0 p52+Bcl3- p52+Bcl3- pEYFP p52+Bcl3-WT S366A S366E HeLa 1 6.8 5.5 11.4

Figure 3e. 1: p52:Bcl3-Ser366 single mutants complex affects reporter activity 37

P-Selectin (GGGGTGACCCC) 16

0 pEYFP p52+Bcl3 WT p52+Bcl3-S366E pLKO 1 5.8 3.9 Akt2 KD 1 1.1 2.1 Erk2 KD 1 0.9 2.4 IKK1 KD 1 3.9 9.3 IKK2 KD 1 5.8 14.5

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

Figure 3e. 3: p52:Bcl3 mutants complex affects reporter activity and its expression level check in Western blot 39

Figure 3e. 4: Bcl3:p52:DNA ternary complex formation with Bcl3-S33ES114ES446E (EEE) and Bcl3-S33ES114ES366ES446E (EEEE) from E. coli

CHAPTER Ⅳ: p105 Processing Result

p50 is constitutively generated from a precursor protein, p105. It is known that the proteasome is involved in the p50 processing from p105. However, modulation of p50 level, if occurs, is not known. Our laboratory previously showed that that the core 20S proteasome generates p50 in a ubiquitination and ATP-independent mechanism. Since the regulatory REG subunits are involved degradation of many protein without ATP and ubiquitination, we wanted to test if these REG subunits are also involved p50 processing. Yidan Li prepared REGα, β and

γ knockdown cells in HeLa and 293T. She also tested by Western blotting that p50 levels is lower in REGγ knockdown cells. Therefore, my focus for this project is by using EMSA experiments to test the level of p50 present in the nucleus of these cells with or without treatment with LPS (lipopolysaccharide).

A. Depletion of REGγ Induces Nuclear p50 Levels

It is previously described that when REGγ was deficient in cells, p50 protein level would recover. To further prove this point, nuclear extracts were isolated from REGα and REGγ knockdown (KD) HeLa cells and tested in EMSA. Upon stimulation with TNF-α, both REGα and REGγ KD cells showed enhanced p50 binding ability to HIV probe (figure 4a.1, compare lane 3 to lane 4 and 5). The super-shift of p50 band in REGα and REGγ KD cells further confirmed this observation through incubation with p50 antibody (figure 4a.1, vilane 8 and 9).

However, in U2OS cells, p50 level was not expressed as high in HeLa cells. The results were not shown clearly (figure 4a.1, lane 10-17).

Similar experiment was repeated by using THP1 cell line by using MHC probe for better binding ability of p50. Upon stimulation by LPS, even though the p50 level seems to be higher at un-stimulated cell (figure 4a.2, lane 1 and 2, lane 6 and 7); however, when REGγ was knocked down, p50 level was significantly increased in LPS stimulated cell (figure 4a.2, lane

4 and 5, lane 8 and 9). These experiments prove the hypothesis that REGγ plays a role towards nuclear p50 level in cells.

Figure 4a.1: p50 nuclear protein expression in Regα, Regγ and p50 KD HeLa cells and U2OS cells 42

Figure 4a.2: p50 nuclear protein expression in Regγ KD THP1 cells

B. REGγ Depletion Alters Gene Expression Program by NF-κB Dimers

To study different gene expression level in REG knockdown (KD) cells, REGα, REGγ and p50 were knocked down in HeLa cells and extracted their RNA. By using their reverse transcription product, target gene primers were used during RT-PCR and results were run on a

2% Agarose gel to check the expression level (figure 4b.1). With GAPDH as control, gene IL-

6 and p105 seem to be strongly regulated by REGγ, when REGγ level goes down, the gene expression level increases. Different from other, gene IFN-γ, IL-1β, IFN-β, TNF-α and IP-10 are not affected by the KD condition of the cells.

Figure 4b.1: gene expression level in Regα, Regγ and p50 KD HeLa cells

CHAPTER Ⅴ: Discussion

It is known in most cells that NF-κB is present as an inactive complex bound by IκB proteins in cytoplasm. By receiving different stimuli, either the canonical or non-canonical pathway will be activated, which result in releasing and translocation of NF-κB dimers to cytoplasm. However, the debatable third NF-κB activation pathway was proposed some time ago. In the well-studied canonical and non-canonical pathways, IκB proteins will be degraded by IKK kinase upon stimulation; however, Bcl3—the atypical IκB protein has not been shown to be degraded by IKK, rather, it goes through phosphorylation and translocates into nucleus to facilitate p50 or p52 homodimers regulation towards transcription (Gilmore, 2006). It is not known the precise role for the activation of this pathway, however, a previous lab member

Vivien Wang has determined that this pathway is activated when the NF-κB dimers bind to

G/C centric κB DNAs, and repressed when bind to A/T centric κB DNAs (Wang, et al., 2017).

In my thesis, the hypothesis was verified that kinases AKT, Erk and IKK are responsible for phosphorylate Bcl3 at the MS determined sites Ser33, Ser114 and Ser446 respectively. A new site Ser366 was also identified, which locates at the C-terminus of Bcl3 protein that is important for Bcl3 function. Based on the prior knowledge, Bcl3 regulates p50 or p52 homodimers through formation of a ternary complex. Therefore, to further study these phosphorylation sites, new combinational mutants were created and tested of their complex formation with p52 and P-Selectin κB DNA. The result shows that when Bcl3 is

phosphorylated at site 33 and 446 (Bcl3-EE), this modification is not sufficient for ternary complex formation but this mutant loses the strong inhibitory potential of fully un- phosphorylated Bcl3. Phosphorylating one more residue, Ser114 (Bcl3-EEE) succeeded in forming the ternary complex (Figure 3d. 2, 3). Further experiments were done with Bcl3-

S33ES114AS446A (EAA) from 293T cells, and it is hypothesized that if the ternary complex formation exists, the binding will be significantly weaker; however, Bcl3-EAA could form ternary complex with p52 homodimers (Figure 3d. 3). This observation encouraged us to look for other possible site for phosphorylation. In this case, mutant Bcl3-S33ES114ES366ES446E

(EEEE) was created and tested in EMSA with p52 and P-Selectin κB DNA. As expected, Bcl3-

EEEE could form ternary complex with p52 and DNA. It is found that Bcl3-EEEE is more effective in the ternary complex formation that Bcl3-EEE (Figure 3e. 4). Collectively, these observations suggest that Bcl3 is not only a highly phosphorylated protein but its differential phosphorylation is essential for its diverse function including its ability to function with p50 or p52 homodimers.

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