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Recommended Citation Abt, Kimberly. (2018). Characterizing Nab2 Phosphorylation and its Role in Regulating Egr Transcription Factors. In BSU Honors Program Theses and Projects. Item 303. Available at: http://vc.bridgew.edu/honors_proj/303 Copyright © 2018 Kimberly Abt

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Characterizing Nab2 Phosphorylation and its Role in Regulating Egr Transcription Factors

Kimberly Abt

Submitted in Partial Completion of the Requirements for Departmental Honors in Biology

Bridgewater State University

May 7, 2018

Dr. Kenneth Adams, Thesis Advisor Dr. Merideth Krevosky, Committee Member Dr. Heather Marella, Committee Member

Characterizing Nab2 Phosphorylation and its Role in Regulating Egr Transcription Factors

Kimberly Abt

Department of Biological Sciences, Bridgewater State University

Honors Thesis

Thesis Director:

Dr. Kenneth Adams

Thesis Committee Members:

Dr. Merideth Krevosky

Dr. Heather Marella

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TABLE OF CONTENTS

1. ABSTRACT ...... 2

2. INTRODUCTION ...... 4 2.1 Egr family members are transcription factors 4 2.2 Induction of Egr family members and their role in the nervous system 6 2.3 Post-translational regulation of Egr family members 8 2.4 NGFI-A binding (Nab1 and Nab2) are corepressors of Egr1-3 8 3. MATERIALS AND METHODS ...... 10 3.1 Mammalian cell culture 10 3.2 Generating Nab2 phosphorylation site mutants 10 3.3 Cell transfection 11 3.4 Cell harvest and Western blot analysis 12 3.5 Mammalian two-hybrid assay 13 4. RESULTS ...... 15 4.1 Endogenous Nab2 exhibits a transient mobility 15 shift following NGF treatment 4.2 Endogenous Nab2 mobility shift is due to ERK-mediated phosphorylation 17 4.3 Exogenous Nab2 is constitutively phosphorylated independent of ERK 20 4.4 Identification of nine putative phosphorylation sites on Nab2 22 4.5 Evaluation of the effect of Nab2 phosphorylation on Nab2/Egr1 interactions using mammalian two-hybrid assay 26 5. DISCUSSION ...... 30

6. ACKNOWLEDGEMENTS ...... 33

7. REFERENCES ...... 34

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1. Abstract The early growth response (Egr) family of transcription factors is composed of four members

Egr1-4, which play roles in various cell behaviors including proliferation, apoptosis, and differentiation in a cell type and stimulus specific manner. Roles for Egr1-3 are especially well documented in the nervous system: Egr 1 and 3 regulate expression of involved in synaptic plasticity and long term potentiation, Egr2 contributes to hindbrain development and peripheral nerve myelination, and Egr 1 and 2 also contribute to the transcriptional network that drives neuronal differentiation in PC12 cells. However, the mechanisms that regulate Egr activity are not fully understood. One mechanism of Egr regulation involves interactions with coregulators NGFI-A binding 1 and 2 (Nab1 and Nab2). Most studies demonstrate that Nab1 and 2 repress Egr transactivation when bound; however, one report indicates Nabs can enhance Egr transactivation in a target -specific manner. As such, the molecular mechanism controlling the effect of Nabs on the activity of Egr1-3 remain unclear.

Here, we provide evidence that Nab2 is rapidly phosphorylated in PC12 cells following treatment with nerve growth factor (NGF) based on an upward mobility shift in Western blot that is reversed by phosphatase treatment. The mobility shift was inhibited by the mitogen- activated protein kinase kinase (MEK) inhibitor (UO126), indicating Nab2 phosphorylation is mediated at least in part by extracellular-regulated kinase (ERK). Nine sites on Nab2 that match the consensus sequence for ERK phosphorylation were identified and site-directed mutagenesis was conducted to generate alanine mutants for each. Unexpectedly, exogenous

Nab2 was constitutively phosphorylated independent of NGF treatment; nevertheless, the

Nab2-S126A, -S129A, -S138A, T157A, and -T401A mutants exhibited downward mobility Abt, K 3 shifts, indicating they are likely phosphorylation sites. Mammalian two-hybrid assays were then conducted to detect Nab2-Egr1 interactions and to evaluate whether the mutation of each of the phosphorylation sites affect their interactions. Experiments thus far have tested the effect of three Nab2 phosphorylation site mutants (S126A, T157A, and S401A), none of which affect Nab2 interactions with Egr1.

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2. Introduction

2.1 Egr family members are transcription factors

The Egr family of transcription factors are immediate-early genes and consist of four members:

Egr1 (also known as NGFI-A, Krox-24, Zif-268, Znf225, AT225, G0S30, TIS8), Egr2 (also known as

Krox-20, AT591, CMT1D, and CMT4E), Egr3 (also known as PILOT), and Egr4 (also known as NGIF-

C, and pAT133). Egr family members contain a conserved DNA binding domain, which is composed of three tandem Cys2-His2-type zinc-finger motifs at the carboxyl terminus (figure 1)

(Christy and Nathans 1989; Pavletich and Pabo 1991). The zinc-finger domain is structured as an antiparallel beta pleated sheet and an alpha helix that are held together by a zinc atom and hydrophobic residues (figure 2) (Gashler and Sukhatme 1995). There is ninety-percent homology within the DNA binding domain of Egr family members, suggesting they bind similar DNA sequences and/or similar target genes. Egr family members bind to DNA sequences that are GC- rich and contain the nine consensus site: 5’-GCG(G/T)GGGCG-3’ (Christy and Nathans

1989; Nardelli et al. 1991; Patwardhan et al. 1991; Cao et al. 1993). Unlike the carboxyl-terminus, the amino terminus of Egr family members is variable. Despite this variability, all Egr family members contain an activation domain at the amino terminus through which they activate target genes, and the activation domain of Egr1 has been the best characterized (Thiel et al. 2000). Egr1-

3 also contain a conserved repression (R1 domain). NGFI-A binding proteins (Nab1 and Nab2) have been shown to bind to the R1 domain and negatively regulate the transcriptional activity of

Egr1-3 (Russo et al. 1995; Svaren et al. 1996; Thiel et al. 2000).

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2.2 Induction of Egr family members and their role in the nervous system

Egr family members are induced in response to several stimuli, including growth factors,

cytokines, neurotransmitters, as well as cell stressors such as hypoxia and oxidative stress

(O'Donovan et al. 1999; Pagel and Deindl 2012; Thiel et al. 2014). After induction, Egr proteins

contribute to several different cell behaviors including differentiation, proliferation, and

apoptosis in a cell-type and stimulus-type specific manner (Khachigian et al. 1997; Lee et al. 2004;

Pagel et al. 2012). Roles of Egr proteins are particularly well documented in the nervous system.

One of the original characterizations of Egr1 stemmed from studies on the neuronal differentiation of PC12 cells in response to NGF (Milbrandt 1987). PC12 cells are a frequently utilized model to investigate the molecular mechanism of neuronal differentiation. Following

NGF treatment, PC12 cells develop several features that are characteristic of neurons, such as cessation of cell proliferation, up-regulation of neuronal genes, neurite outgrowth, and development of electrical excitability (Greene and Tischler 1976). Egr1 is induced within 1 hour of NGF treatment and is required for subsequent differentiation, as expression of a dominant- negative Egr blocks differentiation in response to NGF (Levkovitz et al. 2001). Although not fully characterized, several Egr1 transcriptional targets that contribute to differentiation have been identified, including p35, Arc, Vgf, Sik1, Phlda1, Kctd11, kdm6b, Hbegf, and Ankrd34c (Harada et al. 2001; Adams et al. 2017).

Another role for Egr1 was discovered from the observation that induction of long term potentiation (LTP) in the dentate gyrus of the hippocampus is associated with the rapid induction and activation of Egr1 (Cole et al. 1989; Wisden et al. 1990). Egr1 was later shown to play a critical role in LTP when Egr1 mutant mice could not maintain LTP induction in the dentate gyrus for Abt, K 7

longer than 24 hours (Jones et al. 2001). More recently, overexpression of Egr1 in the forebrain

of transgenic mice was shown to enhance LTP in the dentate gyrus (Penke et al. 2014). Egr3 is

also essential for normal LTP and for hippocampal and amygdala-dependent learning and

memory, as Egr3-deficient mice have profound defects in hippocampal LTP as well as

hippocampal and amygdala dependent learning and memory (Li et al. 2007). Egr2 mRNA is also

upregulated in the hippocampus in response to LTP, but it is not associated with increased

protein levels, suggesting that Egr2 may not play a role in LTP or LTP-associated learning a

memory (Cheval et al. 2012).

Although the role of Egr2 in LTP is unclear, Egr2 is important in hindbrain development.

Egr2 is expressed in specific hindbrain nuclei in early development of the mouse brain (Wilkinson

et al. 1989). Egr2 is part of a transcriptional cascade that regulates Hox which is

necessary for proper segmentation of the hindbrain during development (Sham et al. 1993).

Moreover, Egr2 mutant mice die within two weeks after birth because of improper segmentation

in the hindbrain during development (Schneider-Maunoury et al. 1993).

Egr2 is also involved in peripheral nerve myelination. Peripheral myelinating cells

(Schwann cells) express Egr2 before the onset of myelination and transgenic mice with mutant

Egr2 have improperly differentiated Schwann cells and incomplete myelination of peripheral axons (Topilko et al. 1994). Egr2 expression is maintained in mature myelinating cells (Zorick et al. 1996), and the maintenance of peripheral nerve myelination requires continuous Egr2 expression, as Egr2 knockout mice have hypomyelinated nerves at 4 and 6 weeks of age (Decker et al. 2006). In humans, patients with congenital hypomyelinating neuropathy (CHN) contain one dominant or two recessive mutations in Egr2 (Warner et al. 1998). Further, the severity of patient Abt, K 8

symptoms in human myelinopathies is directly correlated with the inability of a mutant Egr2 to

bind DNA (Warner et al. 1999).

2.3 Post-translational regulation of Egr family members

Egr family members can be modified by a variety of post-translational modifications. Mechanisms

of post-translational modification have been best characterized in Egr1. Egr1 contains a number

of O- and N-linked glycosylation sites, but little is known about their role except that one N-linked

glycosylation within the DNA binding domain may regulate Egr1 binding (Muller et al. 1991). Egr1

is phosphorylated by casein kinase II, tyrosine kinases, and protein kinase C, all of which increase

the half-life of Egr1 and its DNA binding activity (Cao et al. 1992; Cao et al. 1993; Huang et al.

1998). In addition, Egr1 is phosphorylated by Akt at two sites (S350 and T309), which is necessary

for subsequent Egr1 sumoylyation by ARF/sumoylyation ligase (Yu et al. 2004; Yu et al. 2009).

One study showed that sumoylyation of Egr1 triggered its ubiquitination and consequent

degradation (Manente et al. 2011). Lastly, Egr1 contains an acetylation site within its DNA binding

domain that can be acetylated by the p300/CBP complex, which reduces transcriptional activity

of Egr1 (Yu et al. 2009).

2.4 NGFI-A Binding Proteins (Nab1 and Nab2) are corepressors of Egr1-3

In addition to post-translational modification, Egr1-3 are regulated through protein-protein

interactions with coregulators Nab1 and Nab2. Nab1 was first identified by its interaction with

Egr1 and Egr2 in a yeast two-hybrid screen (Russo et al. 1995). In this study, Nab1 repressed

transactivation of Egr1 but had no effect on Egr3 activity. Nab2 was later identified as a protein

with high homology to Nab1 in two domains (figure 3), termed Nab conserved domain 1 (NCD1) Abt, K 9 and 2 (NCD2) (Svaren et al 1996). Nab2 similarly acts as a transcriptional corepressor of Egr1-3.

Nab1 and Nab2 interact with Egr1-3 via the R1 domain (Svaren et al. 1998). Nab corepression of

Egr1-3 transactivation also involves the recruitment of other proteins. Nab2 interacts with chromatin helicase DNA-binding protein 4 (CHD4) through its CHD4-interacting domain (CID)

(Srinivasan et al. 2006). CHD4 is a subunit of the nucleosome remodeling and deaceteylase

(NuRD) complex, which represses transcription via histone decaceteylation and nucleosome mobilization.

Although there is much data suggesting that Nab corepressors play a critical role in regulation of Egr activity, whether there are molecular mechanisms that govern interactions between Nab1-2 and Egr1-3, as well as the effect of Nab on Egr activity upon binding, is largely unknown. This study extends our understanding of additional mechanisms that regulate Nab2 and potentially Egr function. Abt, K 10

3. Materials and Methods

3.1 Mammalian cell culture

Human brain neuroglioma (H4) cells, rat adrenal medulla pheochromocytoma (PC12) cells, and

human cervical cancer (HeLa) cells were grown and maintained for experimentation. H4 and HeLa

cells were maintained in growth medium consisting of Dulbecco’s modification of Eagle’s

Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C and 10% CO2. PC12

cells were maintained in growth medium consisting of DMEM supplemented with 10% FBS and

5 5% horse serum (HS) at 37°C and 10% CO2. For Western blot experiments, 5 x 10 PC12 or H4

cells were plated per 60 mm dish in 4 mL of growth medium and incubated for 24-48 hours until cell harvest. For luciferase assays, 106 PC12 or 5 x 105 HeLa cells were plated per well of a 6-well

cell culture dish in 2 mL of growth medium and incubated for 24-48 hours until cell harvest.

3.2 Generating Nab2 phosphorylation site mutants

An expression construct containing Nab2 cDNA (pJDM-Nab2) was received from Jeffrey

Milbrandt at Washington University School of Medicine in St. Louis. The Nab2 cDNA was subcloned into the expression vector pcDNA3 (pcDNA3-Nab2) by Kenneth Adams and Sergey

Kletsov. An oligonucleotide encoding a FLAG tag was also inserted at the 3’ end of the Nab2 cDNA,

resulting in a pcDNA3-Nab2-FLAG construct. To generate Nab2 phosphorylation site mutants,

site-directed mutagenesis (SDM) reactions were performed on pcDNA3-Nab2-FLAG to change

codons for serine/threonine that are situated immediately upstream of a proline residue to

alanine. SDM reactions were performed to generate nine single site mutants: S114A, S126A,

S129A, S138A, T157A, S172A, S176A, T401A, and S446A. Additional SDM reactions were

performed to generate three double mutants (S126A/S129A, S126A/T401A, and S129A/T401A) Abt, K 11

and one triple mutant (S126A/S129A/T401A). All SDM reactions were performed using Agilent

Technologies QuikChange II XL site-directed mutagenesis kit (Agilent Technologies, 200515-5)

according to manufacturer’s instructions.

Following SDM, XL-10 Gold Ultracompetent cells (Agilent Technologies, 200517-4) were

transformed with the products of each SDM reaction according to manufacturer’s instructions

and spread onto Luria-Bertani (LB) agar plates supplemented with ampicillin at 100 µg/mL.

Clones from each transformation were isolated and inoculated in 6 mLs of LB broth containing

Ampicillin at 100 µg/mL. The mutant Nab2 constructs were then purified from broth culture by

using the QIAprep Spin Miniprep Kit (Qiagen, 27106) according to manufacturers’ instructions.

To confirm mutagenesis of pcDNA3-Nab2-FLAG at each site, 200-300 ng/µL samples of each

construct were sent to Eurofins for sequencing using a SP6 or a T7 primer. After confirmation of

mutagenesis by sequencing, 200 mL of LB Broth containing Ampicillin at 100 ng/mL were

inoculated with 200 µl of cultures grown for miniprep, and the mutant Nab2 constructs were

then purified from broth culture by using the QIAprep Spin Maxiprep Kit (Qiagen, 12163)

according to manufacturers’ instructions.

3.3 Cell Transfection

For Western blot experiments, PC12 (106/60 mm plate), and H4 cells (5 x 105/60 mm plate) were plated 24 hours before transfection. Transfection mixtures were prepared containing 500 µl

DMEM, 10 μl of TransIT-LT1 reagent (Mirus, MIR 2300), and 2 μg of plasmid DNA. The mixtures were then incubated for 15-30 min before application to appropriate mammalian cell cultures.

For luciferase assay, PC12 cells (106/well) or HeLa cells (5 x 105/well) were plated 24 hours before Abt, K 12

transfection. Transfection mixtures were prepared containing 250 µl DMEM, 5 μl of TransIT-LT1

reagent (Mirus), and 1-2 μg of total plasmid DNA. The mixtures were then incubated for 15-30

min before application to each well of the appropriate mammalian cell line. After application of transfection mixtures, cultures were incubated at 37°C, 10% CO2 for 24-48 h before harvest.

3.4 Cell harvest and Western blot analysis

Cells were washed twice with 1X phosphate-buffered saline (PBS), lysed directly in 200-300 μl of

Laemmli buffer, and heated to 100°C for 5 min. Ten to twenty μl of each lysate were loaded and

electrophoresed through 10 % SDS-polyacrylamide gels, transferred to nitrocellulose

membranes, and Western blot conducted to detect Nab2-FLAG phosphorylation mutants with

mouse anti-FLAG (Sigma, F3165, 1:10,000) alongside β-actin with mouse anti-actin antibodies

(Sigma, A5441, 1:20,000 dilution) as a loading control. Membranes were submerged in blocking

buffer (5% nonfat dried milk in TBS + 0.2% Tween-20) for 30 min at room temperature, after

which they were incubated with primary antibody diluted in blocking buffer for 12–18 h at 4°C.

Membranes were then washed three times for 1 hour in TBS + 0.2% Tween-20 and incubated

with secondary antibody (goat anti-mouse HRP-conjugate, Bio-Rad, 170–6516, 1:3000 dilution)

diluted in blocking buffer for 1–2 h at room temperature. Membranes were washed three times

for 1 hour in TBS + 0.2% Tween-2. Bands were then detected by incubation in chemiluminescence

reagent followed by exposure to X-ray film (Western Lightning Plus-ECL, Enhanced

Chemiluminescence Substrate, Perkin Elmer, NEL105001).

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3.6 Mammalian two-hybrid assay

Reporter and expression constructs used for mammalian two-hybrid assays were received from

Dr. John Svaren at the University of Wisconsin-Madison and included (1) a luciferase reporter construct with four upstream Gal4 binding sites (Gal4 reporter), (2) an expression construct encoding the Egr1 R1 domain fused to the Gal4 DNA binding domain (R1-DBD), and (3) an expression construct encoding full length Nab2 fused to the Gal4 activation domain (Nab2-VP16).

HeLa cells were transfected with 1-2 μg of a total plasmid DNA that consisted of varying combinations of: pcDNA3, Gal4 reporter, R1-DBD, Nab2-VP16, and/or Nab2-VP16 phosphorylation site mutants (phosphorylation mutants were prepared as previously described in generating Nab2 phosphorylation site mutants and sequencing was performed as previously described but with a CMV forward primer). Cells were harvested 24-48 hours after transfection, rinsed once in ice cold 1X phosphate-buffered saline, and resuspended in 100 μL of lysis buffer

(0.1 M potassium phosphate, pH 7.8, 1% Triton X-100, 1 mM dithiothreitol, 2 mM EDTA). The samples were vortexed briefly, incubated on ice for 15 min, and centrifuged in a for 1 min at maximum speed to remove cellular debris. The supernatant was then collected and 30 μL of each sample was loaded in triplicate in a 96-well plate. Immediately prior to measurement of luminescence, 100 μL of assay buffer was added to each well (30 mM Tricine, 3 mM ATP, 15 mM

MgSO4, 10 mM dithiothreitol, 1 mM D-luciferin, pH 7.8). Luminescence was measured using an integration time of 10 seconds using a Synergy II Biotek plate reader with Gen5 software. The raw luminescence from two of the triplicate wells were selected and used to calculate the average luminescence of each sample. Raw values were converted into fold induction by dividing the raw luminescence of the sample (reporter/R1-DBD, reporter/ Nab2-VP16 or reporter/R1- Abt, K 14

DBD/Nab2-VP16) by the raw value of luminescence from reporter alone. Each experiment was repeated three times and the p-value (p < 0.001) was calculated using one-tailed students t-test.

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4. Results

4.1 Nab2 exhibits a transient mobility shift following NGF treatment

During a previous study investigating the role of Nab2 in neuronal differentiation of PC12 cells,

Western blot analysis showed an unexpected mobility shift of Nab2 following NGF treatment

(figure 4). In the experiment, PC12 cells were treated with NGF for 0-6 hours and subjected to

Western blot. The upward mobility shift was detected by 1 h, after which the shift gradually returned to baseline between 2-6 hours following NGF treatment, suggesting that NGF treatment induces a transient post-translational modification of Nab2 in PC12 cells.

Although the data presented in figure 4 suggests that Nab2 is post-translationally modified in response to NGF, another possibility is that the upper molecular weight bands detected after 1 hour of NGF treatment represent newly synthesized Nab2 protein that is larger than pre-existing Nab2 in untreated cells. While growth factors induce post-translational modification of numerous proteins, they also induce Nab2 transcription and up-regulate global protein synthesis rates. Thus it was important to discern whether the upper molecular weight

Nab2 bands resulted from post-translational modification of pre-existing Nab2 or synthesis of

Nab2 in a larger (perhaps precursor) form. To test this possibility, Western blot analysis was performed on PC12 cells treated with NGF in the presence of cylcoheximide (CHX), which inhibits global protein synthesis (figure 5). Nab2 exhibited an upward mobility shift both in the presence and absence of CHX, confirming that NGF induces post-translational modification of pre-existing

Nab2. However, Nab2 levels were higher in the absence of CHX, suggesting that induction of

Nab2 expression is occurring at the same time as post-translational modification.

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4.2 Endogenous Nab2 mobility shift is due to ERK-mediated phosphorylation

A variety of post-translational modifications could account for the Nab2 mobility shift (e.g. phosphorylation, ubiquitination, and sumoylyation). To evaluate if phosphorylation is the cause of the Nab2 mobility shift in response to NGF treatment, PC12 cells were treated with NGF for either 10 minutes or 1 hour, lysed, and the cell lysates were treated with lambda protein phosphatase prior to Western blot analysis (figure 6). The phosphatase induced a downward mobility shift of Nab2 in untreated PC12 cells, suggesting that Nab2 is basally phosphorylated in the absence of NGF. Treatment with NGF for 10 minutes or 1 hour induced similar upward mobility shifts that were reversed by phosphatase treatment, suggesting that the shifts are due to phosphorylation.

Previous research has demonstrated that NGF treatment activates two cell signaling pathways: (1) Ras/Raf/MEK/ERK and (2) PI-3 kinase/Akt (figure 7). To determine if ERK signaling and/or PI-3 kinase signaling are responsible for Nab2 phosphorylation in response to NGF, the effects of a MEK inhibitor (UO126) and a PI-3 kinase inhibitor (LY294002) on the Nab2 mobility shift in response to NGF treatment were examined. PC12 cells were pre-treated with UO126 or

LY294002 for 30 minutes before NGF treatment for an additional 10 minutes or 1 hour. In addition, treatments were done in the presence and absence of CHX to ensure that any observed mobility shift was not due to differences in Nab2 expression. Following treatments, the cells were harvested and subjected to Western blot analysis to evaluate Nab2 mobility (figure 8). UO126 treatment inhibited the Nab2 mobility shift induced by NGF treatment for 10 minutes or 1 hour both in the presence and absence of CHX. LY294002 did not inhibit the Nab2 mobility shift in either the presence or absence of CHX. This suggests that ERK signaling is necessary for Nab2 Abt, K 18 phosphorylation and that PI-3 kinase/Akt signaling does not mediate Nab2 phosphorylation in

PC12 cells treated with NGF.

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4.3 Exogenous Nab2 is constitutively phosphorylated independent of ERK

Having demonstrated that endogenous Nab2 is phosphorylated in PC12 cells, subsequent

experiments focused on identifying specific phosphorylation sites on Nab2 by performing

mutagenesis and transfection. However, before mutating Nab2, the mobility patterns of exogenous Nab2 prior to and after NGF treatment were examined alongside the effect of phosphatase treatment. An expression construct containing the Nab2 cDNA (pJDM-Nab2) was generously donated by Dr. Jeffrey Milbrandt at Washington University, St. Louis. PC12 cells were either untransfected or transfected with pJDM-Nab2 and then treated with NGF for 10 minutes

(figure 9). Cell lysates were harvested, treated with phosphatase, and Western blot was performed. As expected, NGF induced an upward mobility shift of endogenous Nab2 which was reversed by phosphatase treatment. However, NGF treatment does not induce an upward Abt, K 20

mobility shift of exogenous Nab2, indicating that either NGF does not does not induce

phosphorylation of exogenous Nab2 or NGF-induced phosphorylation does not cause a mobility

shift. Phosphatase treatment of transfected lysates with or without NGF treatment caused a

downward mobility shift, indicating that exogenous Nab2 is constitutively phosphorylated when

expressed in PC12 cells.

The basal phosphorylation state of exogenous Nab2 was unexpected. To determine if

serum starvation is necessary to reduce the phosphorylation state of exogenous Nab2, cells were

transfected with empty vector or pcDNA3-Nab2-FLAG and serum starved for 0-2 days. Cell lysates

were harvested and western blot analysis was performed using an anti-FLAG antibody (figure 10).

A quadruplet banding pattern of Nab2 was observed and the banding pattern was consistent

throughout 0-2 days of serum starvation. This data suggests that exogenous Nab2 is basally phosphorylated independent of growth factors.

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4.4 Identification of nine putative phosphorylation sites on Nab2

The mobility shift of endogenous Nab2 in response to NGF combined with the downward mobility

shift of endogenous and exogenous Nab2 induced by phosphatase treatment provide strong

evidence that Nab2 is phosphorylated under basal conditions and further phosphorylated in

response to NGF treatment. Data from figure 6 indicate that phosphorylation of endogenous

Nab2 is ERK mediated, but these results could not be replicated with exogenous Nab2 on account

of the observed constitutive phosphorylation of Nab2 after transfection (figure 10). Despite this

discrepancy, serine and threonine residues on Nab2 that matched consensus sites for ERK

phosphorylation were identified as candidate ERK phosphorylation sites. ERK is a proline-directed kinase that preferentially phosphorylates serine and threonine residues that lie directly upstream of a proline residue (i.e., S/TP sites), with a stronger preference for PXS/TP sites. Nab2 contains nine S/TP sites (S114, S126, S129, S138, T157, S172, S176, T401 and S446), two of which match Abt, K 22 the PXS/TP consensus sequence (S129 and S176). Site directed mutagenesis was performed on pcDNA3-Nab2-FLAG at the codon for each of the indicated residues to change the codon for serine/threonine to a codon for alanine (table 1). Samples of each mutant plasmid were subjected to Sanger sequencing to confirm successful mutation (figure 11).

After confirmation of mutagenesis by sequencing, each single site mutant of pcDNA3-

Nab2-FLAG (S114A, S126A, S129A, S138A, T157A, S172A, S176A, T401A, and S446A) was transfected into PC12 cells alongside wild-type. Cell lysates were harvested and Western blot analysis performed using an anti-FLAG antibody to evaluate the effect of the mutation(s) on the mobility patterns of exogenous Nab2 (figure 12). Several mutants exhibited an altered banding pattern compared to the quadruplet band pattern of wild-type Nab2-FLAG. Of the nine mutants, the S126A, S129A, and T401A exhibited the greatest change, with bands that migrated alongside the lower molecular weight bands of wild-type Nab2 FLAG. The S129A mutant also exhibited a similar band collapse but it did not appear to be as significant as that seen in S126A and T401A.

After observing the effect of S126A, S129A, and T401A on Nab2 mobility, double and triple mutants of pcDNA3-Nab2-FLAG at these sites were generated by site-directed mutagenesis, which were again confirmed by Sanger sequencing (data not shown). Each double and triple mutant (S126A/S129A, S16A/T401A, S129A/T401A, and S126A/S129A/T401A) was transfected into PC12 cells alongside wild-type pcDNA3-Nab2-FLAG, followed by harvest and

Western blot analysis with anti-FLAG antibody (figure 13). As in previous experiments, the single site mutants exhibited similarly collapsed banding patterns. The S126A/S129A double mutant did not yield a greater band collapse, while the double mutants with T401A both exhibited greater collapse. The triple mutant exhibited the greatest band collapse, yielding a single band that Abt, K 23 migrated similarly to the lowest molecular weight band detected in the wildtype Nab2-FLAG transfections. This data indicates Nab2 is likely phosphorylated at multiple sites, each of which contributes to altered mobility of Nab2 in Western blot.

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4.5 Evaluation of the effect of Nab2 phosphorylation on Nab2/Egr1 interactions using mammalian two-hybrid assay

After demonstrating that Nab2 is phosphorylated and identifying potential phosphorylation sites on Nab2, mammalian two-hybrid assays were used to determine whether Nab2 phosphorylation affects its ability to form protein-protein interactions with Egr1. The assay entails transfecting cells with three constructs: (1) a luciferase reporter with four upstream Gal4 binding sites

(reporter), (2) an expression construct encoding the Egr1 R1 domain fused to the Gal4 DNA binding domain (R1-DBD), and (3) an expression construct encoding full length Nab2 fused to the

Gal4 activation domain (Nab2-VP16). Following transfection, the R1-DBD fusion protein will bind the reporter; if Nab2-VP16 then binds the R1 domain, it will bring the VP16 domain to the reporter, resulting in luciferase expression, which can be detected and quantified by luciferase assay using a luminometer.

To first demonstrate Nab2/Egr1 interactions, HeLa cells were transfected with reporter alone, reporter + R1-DBD, reporter + Nab2-VP16, or reporter + R1-DBD + Nab2-VP16. Cells were then harvested and subjected to luciferase assay to quantify relative luciferase expression levels.

Luminescence levels from cells transfected with reporter + R1-DBD + Nab2-VP16 increased approximately 60-fold compared to reporter alone when 1 ug of total DNA was transfected and

135-fold when 2 ug of total DNA was transfected (Figure 14). This data suggests that the R1-DBD and Nab2-VP16 are interacting and activating transcription to the luciferase reporter, thus indicating that the mammalian two-hybrid is working successfully.

After ensuring that the mammalian two-hybrid could be used to measure interactions between Nab2 and the R1 domain of Egr1, mutagenesis was performed on the Nab2-VP16 Abt, K 27 construct in order to use the mammalian-two hybrid assay as a model for investigating the effect of Nab2 phosphorylation on its interaction with Egr1. Three single site phosphorylation mutants

(S126A, T157A, and S401A) of Nab2-VP16 construct were generated using site-directed mutagenesis and confirmed by Sanger sequencing as previously described (figure 15). HeLa cells were then transfected with reporter alone, reporter + R1-DBD, reporter + Nab2-VP16 wild-type, reporter + R1-DBD + Nab2-VP16 wild-type/S126A/T157A/S401A. Cell were then harvested and subjected to luciferase assay. Luminescence from cells transfected with reporter + R1-DBD + wild-type Nab2-VP16 increased approximately 300-fold compared to reporter alone.

Luminescence from cells transfected with reporter, R1-DBD, and single site Nab2-VP16 phosphorylation mutants (S126A/T157A/T401A) also increased approximately 250-300 fold compared to reporter alone, which did not significantly differ from wild-type Nab2. Altogether, this data confirms that protein-protein interactions between Nab2 and the Egr1 R1 domain can be detected by the mammalian two hybrid assay and indicates that the interaction is not affected by phosphorylation at S126, T157, and T401 on Nab2.

Abt, K 28

Abt, K 29

Abt, K 30

5. Discussion

This study demonstrates that the transcriptional coregulator Nab2 is transiently phosphorylated in PC12 cells in response to NGF in an ERK-dependent manner. NGF induced an upward mobility shift of Nab2 in Western blot within 10 minutes of treatment, which was reversed by incubating cell lysates with lambda protein phosphatase and was inhibited by pre-treatment with a MEK inhibitor U0126. The upward mobility shift of Nab2 in response to NGF was not reversed by the

PI3K inhibitor LY249002.

Given the effect of NGF treatment on endogenous Nab2, we expected to observe the same transient upward mobility shift of exogenous Nab2 following NGF treatment. However,

Western blot did not show an upward mobility shift of exogenous Nab2 in response to NGF; instead, exogenous Nab2 migrated as a quadruplet of bands ranging in size from the lowest molecular weight band of endogenous Nab2 before NGF treatment to the highest molecular weight band after NGF treatment. Nevertheless, lambda phosphatase treatment of lysates from transfectants caused a downward shift of exogenous Nab2 to a molecular weight similar to that of endogenous Nab2 before NGF treatment. These data suggest that exogenous Nab2 is basally phosphorylated. Serum starvation inhibited ERK activity based on levels of ERK phosphorylation, but had no effect on the exogenous Nab2 banding pattern, suggesting that phosphorylation of exogenous Nab2 may be ERK-independent.

Despite the discrepancy in endogenous versus exogenous Nab2 band shifts, potential ERK phosphorylation sites were investigated. ERK is a proline-directed kinase that phosphorylates serine or threonine residues that lie directly upstream of a proline residue. Nine potential ERK consensus sites were identified within the Nab2 sequence: S114, S126, S129, T157, S172, S176, Abt, K 31

T401, and S446. After site-directed mutagenesis of each serine/threonine residue to alanine and

transfection of mutant constructs into PC12 cells, Western blot showed a change in Nab2 banding

pattern in five phosphorylation mutants: S126A, S129A, S138A, T157A, and T401A. The greatest

change in banding pattern was seen in the S126A, S129A, and T401A mutants. Double and triple

phosphorylation mutants at these three sites (S126A/S129A, S126A/S401A, S129A/S401A,

S126A/S129A/S401A) depicted an additive effect on the banding pattern change of Nab2 in

Western blot. Of the five phosphorylation mutants that affected the Nab2 banding pattern, four

lie between the NCD1 and NCD2 domain (S126A, S129A, S138A, T157A) and one is in the CID

domain (T401A). The area between NCD1 and NCD2 does not have a characterized function, but

the CID domain modulates Nab2 interaction with CHD4 and the NuRD complex. The Nab2-T401A mutant may affect Nab2’s ability to interact with CHD4, which would be an interesting area for further study.

After identifying potential sites of ERK-mediated phosphorylation on Nab2, we examined

whether Nab2 phosphorylation at these sites would affect its ability to interact with Egr1 using a

mammalian two-hybrid assay. We first showed that mammalian two-hybrid assay is capable of

detecting interaction between wild-type Nab2 and the R1 domain of Egr1, and optimized

conditions for this protocol. We then used site-directed mutagenesis to generate four mutants

of the Nab2 construct (S126A, T157A, and T401A) and evaluated the effect that the mutation had

on Egr1 interactions. We found that the interaction between the R1 domain of Egr1 and the

phosphorylation mutants did not differ from wild-type Nab2. These findings need to be further

investigated because consistent clones were not able to be used for experimentation with the

phosphorylation mutants which could have caused the high variability in luciferase fold induction Abt, K 32 in the experiments which accounted for the high standard deviation. Nab2-S129A and -S138A mutants for mammalian two-hybrid analysis also needs to be generated and evaluated for any effect on interactions with the Egr1 R1 domain. Lastly, mammalian two-hybrid assays conducted thus far have not included co-transfection with a β-galactosidase expression construct to normalize luminescence data for transfection efficiency, which will be included in future experiments.

It is possible that phosphorylation of Nab2 affects its interactions with proteins other than

Egr1. For example, Nab2 interacts with the CHD4 domain of the NuRD complex, which modulates transcriptional repression. Nab2 phosphorylation could potentially alter Nab2 binding to CHD4, and this could effect transcriptional repression done by the NuRD complex. In addition, phosphorylation of Nab2 could affect Nab2’s interactions with other Nab proteins (Nab1 and

Nab2). Nab2 can multimerize with Nab1 or Nab2 through the NCD1 domain, but Nab2 phosphorylation could modify this interaction. Furthermore, Nab2 has been primarily characterized as a corepressor of Egr1, but one study has shown that Nab2 can enhance Egr1 transactivation. Nab2 phosphorylation could potentially play a role in determining whether or not Nab2 activates or represses Egr1 independent of Nab2 directly binding to Egr1. In conclusion, the data presented here provides strong evidence that Nab2 is phosphorylated both basally and in response to ERK signaling induced by NGF treatment of PC12 cells. This post-translational modification could have a number of effects on Nab2 and its interactions with other proteins in the cell. Learning more about the consequence of Nab2 phosphorylation in PC12 cells will be an interesting project for future members of the Adams lab.

Abt, K 33

6. Acknowledgements

I am grateful to the Department of Biological Sciences and the Office of Undergraduate Research at Bridgewater State University for providing funding and supplies for this project. I am also thankful for the generous donation of expression constructs by Dr. Jeffrey Milbrandt

(Washington University) and Dr. John Svaren (University of Wisconsin-Madison).

I am incredibly grateful to Dr. Ken Adams for mentoring me for the past two years. His

unwavering enthusiasm, consistent support, and genuine willingness to help me grow

throughout this process means so much to me. I would also like to thank Dr. Merideth Krevosky

and Dr. Heather Marella for their participation on my thesis committee and their supportive

feedback on my work. In addition, I would like to thank my fellow lab mates Dodger Adams,

Nicole Berry, Terrain Edwards-Grant, Sergey Kletsov, and Jacquelyn LaVallee for their comradery.

II would also like to thank Dr. Alexandra Adams for checking in with me throughout this process and keeping me sane.

Lastly, I would like to thank Dr. Jenny Shanahan and Kacey O’Donnell for their consistent support and encouragement over the past three years. Lastly, I would like to thank all of the faculty and staff (especially Debbie Fiore, Liz Chappuis, Maria Armour, and Tracey Fagan) in the biology department for their kindness and support during my time at Bridgewater.

Abt, K 34

7. References

Adams KW, Kletsov S, Lamm RJ, Elman JS, Mullenbrock S, Cooper GM. 2017. Role for Egr1 in the

Transcriptional Program Associated with Neuronal Differentiation of PC12 Cells. PLoS One

12: e0170076.

Cao X, Mahendran R, Guy GR, Tan YH. 1992. Protein phosphatase inhibitors induce the sustained

expression of the Egr-1 gene and the hyperphosphorylation of its gene product. The

Journal of biological chemistry 267: 12991-12997.

-. 1993. Detection and characterization of cellular EGR-1 binding to its recognition site. The

Journal of biological chemistry 268: 16949-16957.

Cheval H, Chagneau C, Levasseur G, Veyrac A, Faucon-Biguet N, Laroche S, Davis S. 2012.

Distinctive features of Egr transcription factor regulation and DNA binding activity in CA1

of the hippocampus in synaptic plasticity and consolidation and reconsolidation of fear

memory. Hippocampus 22: 631-642.

Christy B, Nathans D. 1989. DNA binding site of the growth factor-inducible protein Zif268.

Proceedings of the National Academy of Sciences of the United States of America 86: 8737-

8741.

Cole AJ, Saffen DW, Baraban JM, Worley PF. 1989. Rapid increase of an immediate early gene

messenger RNA in hippocampal neurons by synaptic NMDA receptor activation. Nature

340: 474-476.

Decker L, Desmarquet-Trin-Dinh C, Taillebourg E, Ghislain J, Vallat JM, Charnay P. 2006.

Peripheral myelin maintenance is a dynamic process requiring constant Krox20

expression. J Neurosci 26: 9771-9779. Abt, K 35

Gashler A, Sukhatme VP. 1995. Early growth response protein 1 (Egr-1): prototype of a zinc-finger

family of transcription factors. Prog Nucleic Acid Res Mol Biol 50: 191-224.

Greene LA, Tischler AS. 1976. Establishment of a noradrenergic clonal line of rat adrenal

pheochromocytoma cells which respond to nerve growth factor. Proceedings of the

National Academy of Sciences of the United States of America 73: 2424-2428.

Harada T, Morooka T, Ogawa S, Nishida E. 2001. ERK induces p35, a neuron-specific activator of

Cdk5, through induction of Egr1. Nat Cell Biol 3: 453-459.

Huang RP, Fan Y, deBelle I, Ni Z, Matheny W, Adamson ED. 1998. Egr-1 inhibits apoptosis during

the UV response: correlation of cell survival with Egr-1 phosphorylation. Cell Death Differ

5: 96-106.

Jones MW, Errington ML, French PJ, Fine A, Bliss TV, Garel S, Charnay P, Bozon B, Laroche S, Davis

S. 2001. A requirement for the immediate early gene Zif268 in the expression of late LTP

and long-term memories. Nat Neurosci 4: 289-296.

Khachigian LM, Anderson KR, Halnon NJ, Gimbrone MA, Jr., Resnick N, Collins T. 1997. Egr-1 is

activated in endothelial cells exposed to fluid shear stress and interacts with a novel

shear-stress-response element in the PDGF A-chain promoter. Arterioscler Thromb Vasc

Biol 17: 2280-2286.

Lee CG, Cho SJ, Kang MJ, Chapoval SP, Lee PJ, Noble PW, Yehualaeshet T, Lu B, Flavell RA,

Milbrandt J et al. 2004. Early growth response gene 1-mediated apoptosis is essential for

transforming growth factor beta1-induced pulmonary fibrosis. J Exp Med 200: 377-389. Abt, K 36

Levkovitz Y, O'Donovan KJ, Baraban JM. 2001. Blockade of NGF-induced neurite outgrowth by a

dominant-negative inhibitor of the egr family of transcription regulatory factors. J

Neurosci 21: 45-52.

Li L, Yun SH, Keblesh J, Trommer BL, Xiong H, Radulovic J, Tourtellotte WG. 2007. Egr3, a synaptic

activity regulated transcription factor that is essential for learning and memory. Mol Cell

Neurosci 35: 76-88.

Manente AG, Pinton G, Tavian D, Lopez-Rodas G, Brunelli E, Moro L. 2011. Coordinated

sumoylation and ubiquitination modulate EGF induced EGR1 expression and stability.

PLoS One 6: e25676.

Milbrandt J. 1987. A nerve growth factor-induced gene encodes a possible transcriptional

regulatory factor. Science 238: 797-799.

Muller HJ, Skerka C, Bialonski A, Zipfel PF. 1991. Clone pAT 133 identifies a gene that encodes

another human member of a class of growth factor-induced genes with almost identical

zinc-finger domains. Proceedings of the National Academy of Sciences of the United States

of America 88: 10079-10083.

Nardelli J, Gibson TJ, Vesque C, Charnay P. 1991. Base sequence discrimination by zinc-finger

DNA-binding domains. Nature 349: 175-178.

O'Donovan KJ, Tourtellotte WG, Millbrandt J, Baraban JM. 1999. The EGR family of transcription-

regulatory factors: progress at the interface of molecular and systems neuroscience.

Trends Neurosci 22: 167-173.

Pagel JI, Deindl E. 2012. Disease progression mediated by egr-1 associated signaling in response

to oxidative stress. Int J Mol Sci 13: 13104-13117. Abt, K 37

Pagel JI, Ziegelhoeffer T, Heil M, Fischer S, Fernandez B, Schaper W, Preissner KT, Deindl E. 2012.

Role of early growth response 1 in arteriogenesis: impact on vascular cell proliferation

and leukocyte recruitment in vivo. Thromb Haemost 107: 562-574.

Patwardhan S, Gashler A, Siegel MG, Chang LC, Joseph LJ, Shows TB, Le Beau MM, Sukhatme VP.

1991. EGR3, a novel member of the Egr family of genes encoding immediate-early

transcription factors. Oncogene 6: 917-928.

Pavletich NP, Pabo CO. 1991. Zinc finger-DNA recognition: crystal structure of a Zif268-DNA

complex at 2.1 A. Science 252: 809-817.

Penke Z, Morice E, Veyrac A, Gros A, Chagneau C, LeBlanc P, Samson N, Baumgartel K, Mansuy

IM, Davis S et al. 2014. Zif268/Egr1 gain of function facilitates hippocampal synaptic

plasticity and long-term spatial recognition memory. Philos Trans R Soc Lond B Biol Sci

369: 20130159.

Russo MW, Sevetson BR, Milbrandt J. 1995. Identification of NAB1, a repressor of NGFI-A- and

Krox20-mediated transcription. Proceedings of the National Academy of Sciences of the

United States of America 92: 6873-6877.

Schneider-Maunoury S, Topilko P, Seitandou T, Levi G, Cohen-Tannoudji M, Pournin S, Babinet C,

Charnay P. 1993. Disruption of Krox-20 results in alteration of rhombomeres 3 and 5 in

the developing hindbrain. Cell 75: 1199-1214.

Sham MH, Vesque C, Nonchev S, Marshall H, Frain M, Gupta RD, Whiting J, Wilkinson D, Charnay

P, Krumlauf R. 1993. The zinc finger gene Krox20 regulates HoxB2 (Hox2.8) during

hindbrain segmentation. Cell 72: 183-196. Abt, K 38

Srinivasan R, Mager GM, Ward RM, Mayer J, Svaren J. 2006. NAB2 represses transcription by

interacting with the CHD4 subunit of the nucleosome remodeling and deacetylase (NuRD)

complex. The Journal of biological chemistry 281: 15129-15137.

Svaren J, Sevetson BR, Apel ED, Zimonjic DB, Popescu NC, Milbrandt J. 1996. NAB2, a corepressor

of NGFI-A (Egr-1) and Krox20, is induced by proliferative and differentiative stimuli.

Molecular and cellular biology 16: 3545-3553.

Svaren J, Sevetson BR, Golda T, Stanton JJ, Swirnoff AH, Milbrandt J. 1998. Novel mutants of NAB

corepressors enhance activation by Egr transactivators. The EMBO journal 17: 6010-6019.

Thiel G, Kaufmann K, Magin A, Lietz M, Bach K, Cramer M. 2000. The human transcriptional

repressor protein NAB1: expression and biological activity. Biochim Biophys Acta 1493:

289-301.

Thiel G, Muller I, Rossler OG. 2014. Expression, signaling and function of Egr transcription factors

in pancreatic beta-cells and insulin-responsive tissues. Mol Cell Endocrinol 388: 10-19.

Topilko P, Schneider-Maunoury S, Levi G, Baron-Van Evercooren A, Chennoufi AB, Seitanidou T,

Babinet C, Charnay P. 1994. Krox-20 controls myelination in the peripheral nervous

system. Nature 371: 796-799.

Warner LE, Mancias P, Butler IJ, McDonald CM, Keppen L, Koob KG, Lupski JR. 1998. Mutations in

the early growth response 2 (EGR2) gene are associated with hereditary myelinopathies.

Nat Genet 18: 382-384.

Warner LE, Svaren J, Milbrandt J, Lupski JR. 1999. Functional consequences of mutations in the

early growth response 2 gene (EGR2) correlate with severity of human myelinopathies.

Hum Mol Genet 8: 1245-1251. Abt, K 39

Wilkinson DG, Bhatt S, Chavrier P, Bravo R, Charnay P. 1989. Segment-specific expression of a

zinc-finger gene in the developing nervous system of the mouse. Nature 337: 461-464.

Wisden W, Errington ML, Williams S, Dunnett SB, Waters C, Hitchcock D, Evan G, Bliss TV, Hunt

SP. 1990. Differential expression of immediate early genes in the hippocampus and spinal

cord. Neuron 4: 603-614.

Yu J, de Belle I, Liang H, Adamson ED. 2004. Coactivating factors p300 and CBP are

transcriptionally crossregulated by Egr1 in prostate cells, leading to divergent responses.

Mol Cell 15: 83-94.

Yu J, Zhang SS, Saito K, Williams S, Arimura Y, Ma Y, Ke Y, Baron V, Mercola D, Feng GS et al. 2009.

PTEN regulation by Akt-EGR1-ARF-PTEN axis. The EMBO journal 28: 21-33.

Zorick TS, Syroid DE, Arroyo E, Scherer SS, Lemke G. 1996. The transcription factors SCIP and Krox-

20 mark distinct stages and cell fates in Schwann cell differentiation. Mol Cell Neurosci 8:

129-145.