Targeting the Nck/PERK Interaction to Improve Β Cell Function and Survival

Targeting the Nck/PERK interaction to improve β cell function and survival

George Kefalas

Division of Experimental Medicine

Department of Medicine

McGill University

Montreal, Quebec, Canada

December 2017

A thesis submitted to McGill University in partial fulfillment of

the requirements of the degree of Master of Science

Abstract

The failure of pancreatic β cells to produce insulin is a central problem in both Type 1 and

Type 2 diabetes, however the molecular mechanisms leading to β cell failure remain unclear.

Consequently, investigating mechanisms that promote β cell function and survival is paramount in the development of therapies to prevent and treat both types of diabetes.

PKR-like endoplasmic reticulum kinase (PERK) is an ER transmembrane protein involved in maintaining β cell homeostasis. Previously, our group identified the adaptor protein Nck1 as a negative regulator of PERK. Indeed, we demonstrated that Nck1, by directly binding PERK autophosphorylated on Tyr561, limits PERK activation and signaling. In accordance, we found that

Nck1 depletion in β cells promotes PERK activity and signaling, increases insulin biosynthesis, and promotes cell viability in response to diabetes-related stresses.

Herein, we explore the therapeutic potential of abrogating the interaction between Nck and

PERK in order to improve β cell function and survival. To do so, we used a peptide containing the minimal sequence of PERK that is involved in binding Nck1 conjugated to the cell permeable protein transduction domain from the HIV protein TAT. In the current study, we confirm that the synthetic phosphopeptide TAT-pY561 sequesters Nck and prevents its interaction with PERK.

Moreover, we demonstrate that TAT-pY561 penetrates the β cell membrane and promotes basal

PERK activity. Furthermore, we report that pretreatment of β cells with TAT-pY561 inhibits apoptosis induced by glucolipotoxicity. Finally, we further characterize TAT-pY561 at the biochemical level by comparing its affinity for Nck with unphosphorylated and mutant peptide variants. Taken together, our data support the potential of sequestering Nck using a synthetic peptide to enhance basal PERK activity and create more resilient β cells.

Résumé

L’incapacité des cellules β du pancréas endocrine à produire suffisamment d'insuline est un problème majeur chez les diabétiques. Cependant, les mécanismes moléculaires menant à la défaillance des cellules β restent flous. Par conséquent, l’identification de mécanismes contrôlant la fonction des cellules β pancréatiques est d’importance majeure pour le design de thérapies efficaces visant à prévenir et traiter le diabète.

Plusieurs études ont révélé le rôle essentiel de la protéine kinase du réticulum endoplasmique PERK dans le maintien fonctionnel des cellules β pancréatiques. Dans ce contexte, notre groupe a découvert que l’adaptateur Nck1 limite l’activation de PERK. En effet, nous avons démontré que la liaison directe de Nck1 au résidu Tyr561 phosphorylé de PERK réduit l’activité basale de PERK. Nous avons par ailleurs révélé que la déplétion de Nck1 dans les cellules β augmente l'activité basale de PERK et la biosynthèse de l'insuline, et améliore la viabilité des cellules β en réponse aux stress liés au diabète.

Dans cette thèse, nous avons exploré le potentiel thérapeutique de prévenir l'interaction entre Nck et PERK afin d'améliorer la fonction et la survie des cellules β. Dans cette optique, nous avons utilisé le peptide TAT-pY561 composé de la séquence minimale de PERK impliquée dans la liaison avec Nck1 conjuguée au domaine de transduction protéique de la protéine TAT du VIH.

Dans cette étude, nous confirmons que le phosphopeptide synthétique TAT-pY561 séquestre Nck et empêche son interaction avec PERK. En outre, nous démontrons que TAT-pY561 est capable de pénétrer la membrane des cellules β et d’augmenter l’activité basale de PERK. De plus, nous rapportons que le prétraitement des cellules β avec TAT-pY561 inhibe l'apoptose induite par la glucolipotoxicité. Finalement, nous avons plus amplement caractérisé le peptide TAT-pY561 en comparant son affinité pour Nck avec une variante non phosphorylée ou mutée du peptide

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TAT-pY561. Dans leur ensemble, ces données supportent le potentiel de séquestration de Nck à travers l’usage d’un peptide synthétique afin d’améliorer l'activité basale de PERK et de créer des cellules β plus robustes.

Acknowledgments

The authorship of this thesis will be attributed to only one person, however multiple people are responsible for its creation and for all of the efforts behind it.

First and foremost, I’d like to express my gratitude to my supervisor, Dr. Louise Larose, for taking a chance on me and providing me with the opportunity to pursue my graduate studies in her laboratory. Thank you for always challenging me and for mentoring me in such a way that helped me grow as both a scientist and a person. The lessons I have learned from you will stick with me throughout my entire life as a scientist. Thanks also to all members of the Larose lab from the past few years, especially Dr. Lama Yamani, Dr. Hui Li, Dr. Julie Dusseault, Dr. Bing Li, Nida

Haider, and Cindy Baldwin. I would not have enjoyed my time as a graduate student nearly as much if it were not for the pleasant environment you all helped to create.

Throughout my graduate studies I had the privilege of being supported by many brilliant and caring researchers. Thanks to the members of my research advisory committee, Dr. Geoffrey

Hendy, Dr. Stéphane Laporte, and Dr. Simon Wing, for the stimulating discussions that were held concerning my project. Thanks to our collaborators, Dr. Jennifer Estall and Dr. Nathalie Jouvet, for the passion they demonstrated towards this project. Finally, thanks to the Canadian Institutes of Health Research, the Fonds de recherche du Québec – Santé, and the McGill University

Division of Experimental Medicine for providing me with the financial support to pursue these graduate studies.

Next, I would like to thank the faculty, staff, and students of the Polypeptide Laboratories and the MeDiC program for all that they have done to make it so enjoyable to come to work every day. I never once dreaded coming to the lab simply because I knew the laughs we’d share would brighten up my day. Over the past few years, the MUHC-RI has served as more than just an

v academic setting for me; it’s truly been a home away from home. While many people can claim to have met and worked with amazing colleagues here, I am honored to say that it is here that I built amazing friendships, ones that I am certain will last a lifetime.

Finally, I’d like to acknowledge my family and friends, especially those who undertook this journey of graduate school with me over the past two years: Alexandra Lewis, Andrew Dixon,

Joseph Szymborski, Arielle Leone, and Vivian Stavrakos. Through all the highs and lows of this era of our lives, it’s been a blast having you all by my side. Lastly, I’d like to thank my parents,

Anna and Peter, and my late grandfather, George, for all that they have done for me over the past

25 years to make this entire experience a possibility for me.

Preface & Author Contributions

This thesis was written in accordance with the guidelines outlined by the Faculty of Graduate and

Postdoctoral Studies of McGill University. The author wrote the entire thesis, accompanied by editorial comments by Dr. Louise Larose. Experimental design, laboratory work, and data analysis contributing to the final results shown in this thesis were performed by the author under the supervision and guidance of Dr. Louise Larose. Cindy Baldwin (research technician) assisted with several experimental protocols.

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Table of Contents Abstract ...... ii Résumé ...... iii Acknowledgments...... v Preface & Author Contributions ...... vii Table of Contents ...... viii List of Figures ...... xi List of Abbreviations ...... xii Chapter 1: Introduction ...... 1 1.1 Glucose Homeostasis & β Cell Physiology ...... 2 1.1.1 Anatomy of the Pancreas ...... 3 1.1.2 Insulin Biogenesis ...... 4 1.1.3 Insulin Secretion ...... 6 1.1.4 Insulin Signaling ...... 8 1.1.5 Clinical Significance ...... 10 1.1.5.1 Type 1 Diabetes ...... 10 1.1.5.2 Type 2 Diabetes ...... 11 1.1.5.3 Monogenic Diabetes ...... 12 1.2 PERK ...... 13 1.2.1 Unfolded Protein Response...... 13 1.2.1.1 PERK Signaling ...... 14 1.2.1.2 IRE1α Signaling ...... 16 1.2.1.3 ATF6α Signaling ...... 17 1.2.2 PERK & β Cell Homeostasis ...... 19 1.2.2.1 β Cell Development and Survival ...... 19 1.2.2.2 β Cell Proliferation ...... 20 1.2.2.3 Insulin Processing & Secretion ...... 21 1.2.2.4 PERK-Dependent Signaling Network ...... 22 1.2.2.5 Pathogenesis of Diabetes ...... 25 1.3 Nck Adaptor Proteins ...... 25 1.3.1 Discovery ...... 26 1.3.2 Evolution ...... 27 1.3.3 Domains & Protein Interactions...... 27

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1.3.4 Regulation ...... 28 1.3.5 Biological Processes ...... 29 1.3.5.1 Cytoskeletal Remodeling ...... 30 1.3.5.2 mRNA Translation ...... 31 1.3.5.3 Unfolded Protein Response ...... 32 1.3.6 Physiology...... 35 1.3.6.1 Embryonic Development ...... 35 1.3.6.2 Immunology & Pathogen Entry ...... 35 1.3.6.3 Renal Physiology ...... 37 1.3.6.4 Insulin Signaling ...... 39 1.3.6.5 Adipogenesis ...... 39 1.3.6.6 β Cell Physiology ...... 40 1.4 Rationale and Objectives ...... 42 Chapter 2: Materials & Methods ...... 43 2.1 Cell Culture & Treatments ...... 44 2.1.1 Palmitate Preparation ...... 44 2.1.2 Synthetic Peptides ...... 44 2.2 Cell Lysis...... 45 2.3 Western Blotting & Antibodies ...... 45 2.4 Recombinant Protein Preparation...... 46 2.5 In vitro Pull-Down Assays ...... 46 2.5.1 GST Protein Pull-Down Assays...... 47 2.5.2 Streptavidin Pull-Down Assays ...... 47 2.6 Confocal Microscopy ...... 47 2.7 Flow Cytometry...... 48 2.8 Statistical Analysis ...... 48 Chapter 3: Results ...... 49 3.1 TAT-pY561 Binds Nck ...... 50 3.1.1 Relative Specificity of TAT-pY561 Towards Nck ...... 54 3.2 TAT-pY561 Abrogates the Nck1/PERK Interaction ...... 56 3.2.1 Unphosphorylated and Mutant Peptides ...... 57 3.3 FITC-TAT-pY561 Enters MIN6 Cells ...... 58 3.4 TAT-pY561 Promotes Basal PERK Activation ...... 59 3.5 TAT-pY561 Protects Against Palmitate-Induced Apoptosis ...... 60

Chapter 4: Discussion ...... 64 4.1 Summary of Findings ...... 65 4.1.1 Biochemical Characterization of TAT-pY561 ...... 65 4.1.2 TAT-pY561 Promotes PERK Activation ...... 66 4.1.2.1 Role of Nck2 ...... 68 4.1.3 TAT-pY561 Treatment Confers Resistance Against Glucolipotoxicity ...... 68 4.1.3.1 PERK-Independent Effects of Nck Sequestration ...... 69 4.2 Translational Implications ...... 69 4.3 Future Perspectives ...... 70 4.3.1 Targeting the Nck/PERK Interaction Using CRISPR/Cas9 ...... 72 4.3.2 Studying the Mechanistic Basis of PERK Regulation by Nck1 ...... 72 4.4 Concluding Remarks ...... 73 References ...... 74 Appendix: Permissions to Reproduce Material ...... 86

List of Figures

Figure 1.1: Glucose Homeostasis ...... 2 Figure 1.2: Posttranslational Modifications of Insulin ...... 5 Figure 1.3: Glucose-Stimulated Insulin Secretion ...... 7 Figure 1.4: Progression of Type 2 Diabetes ...... 11 Figure 1.5: Unfolded Protein Response ...... 18 Figure 1.6: PERK & β Cell Homeostasis ...... 24 Figure 1.7: Nck Proteins ...... 26 Figure 1.8: Regulation of PERK Activity by Nck1 ...... 33 Figure 1.9: Role of Nck in Nephrin Signaling in Podocytes ...... 38 Figure 1.10: PERK Signaling in β Cells ...... 41 Figure 1.11: TAT-pY561 Amino Acid Sequence ...... 42 Figure 2.1: TAT Peptides ...... 45 Figure 3.1: TAT-pY561 binds Nck ...... 51 Figure 3.2: Y/F peptides compete for Nck binding with lower affinity than TAT-pY561 ...... 52 Figure 3.3: TAT-F does not bind Nck ...... 53 Figure 3.4: TAT-pY561 specifically binds the Nck1 SH2 domain...... 55 Figure 3.5: TAT-pY561 prevents the Nck1/PERK interaction ...... 56 Figure 3.6: Y/F peptides compete with PERK for Nck binding with lower affinity than TAT-pY561 ...... 57 Figure 3.7: FITC-TAT-pY561 enters MIN6 cells ...... 58 Figure 3.8: TAT-pY561 promotes basal PERK activation ...... 59 Figure 3.9: TAT-pY561 protects MIN6 cells against palmitate-induced caspase-3 cleavage ...... 61 Figure 3.10: TAT-pY561 protects INS-1 832/13 cells against palmitate/glucose-induced caspase-3 cleavage ... 62 Figure 3.11: TAT-pY561 protects INS-1 832/13 cells against palmitate/glucose-induced apoptosis ...... 63 Figure 4.1: Modulation of PERK Activation and the UPR ...... 67

List of Abbreviations

7-AAD 7-aminoactinomycin D ADP adenosine diphosphate AMP adenosine monophosphate AMPK AMP-activated protein kinase ANOVA analysis of variance Arp2/3 actin-related protein 2/3 ATF4 activating transcription factor 4 ATF6α activating transcription factor 6α ATP adenosine triphosphate BCAP B cell adaptor for PI3K BCR B cell receptor BiP binding immunoglobulin protein BMI body mass index BSA bovine serum albumin C/EBP CCAAT/enhancer-binding protein cAMP cyclic adenosine monophosphate Casp3 caspase-3 CD3 cluster of differentiation 3 Cdc42 cell division control protein 42 cDNA complementary deoxyribonucleic acid CHOP C/EBP-homologous protein Clv cleaved CRISPR clustered regularly interspaced short palindromic repeats DAPI 4',6-diamidino-2-phenylindole DMEM Dulbecco’s Modified Eagle Medium DNA deoxyribonucleic acid DTT dithiothreitol EDTA ethylenediaminetetraacetic acid EGF epidermal growth factor EGFR epidermal growth factor receptor eIF eukaryotic initiation factor EPAC exchange protein directly activated by cAMP EPEC enteropathogenic Escherichia coli ER endoplasmic reticulum ERAD ER-associated degradation ERK extracellular signal-regulated kinase FBS fetal bovine serum FFA free fatty acid FITC fluorescein isothiocyanate FL full-length GAP GTPase activating protein GCN2 general control non-depressible-2 GDP guanosine diphosphate

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GLP-1 glucagon-like peptide-1 GLUT2 glucose transporter 2 GLUT4 glucose transporter 4 GPCR G protein-coupled receptor GRB growth factor receptor-bound protein GST glutathione S-transferase GTP guanosine triphosphate HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HIV human immunodeficiency virus HRI heme-regulated inhibitor HRP horseradish peroxidase IFN interferon IFNAR1 type I interferon receptor α chain INS-1 rat insulinoma pancreatic β cells IP3 inositol 1,4,5-trisphosphate IR insulin receptor IRE1 inositol-requiring enzyme-1 IRS insulin receptor substrate JNK c-Jun N-terminal kinase KATP ATP-sensitive potassium channel Keap1 Kelch-like ECH-associated protein 1 MEF mouse embryonic fibroblast MIN6 mouse insulinoma pancreatic β cells mRNA messenger ribonucleic acid mTORC mammalian/mechanistic target of rapamycin complex Nck non-catalytic region of tyrosine kinase NF-E2 nuclear factor erythroid-derived 2 Nr2e1 Nuclear receptor subfamily 2 group E member 1 Nrf2 NF-E2-related factor 2 PA palmitate PAK p21-activated kinase PBS phosphate-buffered saline PC prohormone convertase PDGF platelet-derived growth factor PDGFR platelet-derived growth factor receptor PDI protein disulfide isomerase PECAM-1 platelet endothelial cell adhesion molecule-1 PERK PKR-like ER kinase PI3K phosphatidylinositol-3-kinase PIP2 phosphatidylinositol 4,5-bisphosphate PIP3 phosphatidylinositol 3,4,5-trisphosphate PKA protein kinase A PKR protein kinase RNA-dependent PLC phospholipase C PMSF phenylmethylsulfonyl fluoride PP1 protein phosphatase 1

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PPARγ peroxisome proliferator-activated receptor-γ PPI protein-protein interaction PTD protein transduction domain PTP1B protein tyrosine phosphatase 1B PVDF polyvinylidene difluoride RIDD regulated IRE1-dependent decay RNA ribonucleic acid RPMI Roswell Park Memorial Institute 1640 Medium RRP readily releasable pool SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis SEM standard error of the mean Ser serine SERCA sarcoplasmic/ER Ca2+-ATPase SH2 Src homology 2 SH3 Src homology 3 shRNA short hairpin ribonucleic acid SP site protease T1D type 1 diabetes T2D type 2 diabetes Tat transactivator of transcription TCR T cell receptor Thr threonine Tir translocated intimin receptor tRNA transfer ribonucleic acid Tyr tyrosine uORF upstream open reading frame UPR unfolded protein response VDCC voltage-gated calcium channel WASP Wiskott-Aldrich syndrome protein WB Western blot WIP WASP-interacting protein WRS Wolcott-Rallison syndrome XBP1 X-box-binding protein 1

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Chapter 1: Introduction

1.1 Glucose Homeostasis & β Cell Physiology

In order to sustain normal bodily function, organisms utilize various homeostatic mechanisms to control physiological parameters such as blood composition. In one such mechanism, the pancreas secretes insulin, a hormone responsible for maintaining blood glucose levels within the physiological range of 4-6 mM [1]. When blood glucose levels increase, the pancreas releases insulin into the blood stream, allowing it to act in an endocrine manner on peripheral tissues including adipose, muscle, and liver (Figure 1.1). In these tissues, insulin signaling leads to increased glucose uptake or storage, thus restoring blood glucose homeostasis.

In adipose tissue, glucose is stored in the form of triglycerides, whereas in muscle and liver, glucose is stored in the form of glycogen.

↑ Plasma Glucose

Adipose

Insulin

Muscle

Pancreas Islet of Langerhans (Endocrine)

Acinar Cell (Exocrine) Liver

Figure 1.1: Glucose Homeostasis Pancreatic β cells, located within the islets of Langerhans, respond to rising levels of plasma glucose by secreting insulin, which acts on peripheral tissues to restore blood glucose homeostasis.

The action of insulin is opposed by glucagon, another pancreatic hormone. When blood glucose levels are low, glucagon is released into the blood stream and acts to increase blood glucose levels by inducing glycogenolysis and gluconeogenesis in the liver and lipolysis in adipose tissue. The balance between these two counteracting hormones controls the tight regulation of glucose homeostasis in the body.

1.1.1 Anatomy of the Pancreas

The pancreas is a glandular organ that consists of both endocrine and exocrine components.

The function of both these components is to secrete proteins; consequently, the pancreas has the greatest rate of protein synthesis of any mammalian organ [2]. The exocrine pancreas, consisting mainly of acinar cells, comprises 75-90% of the mass of the entire organ [3]. Acinar cells are responsible for the synthesis and secretion of enzymes into the duodenum, where they are involved in the digestion of proteins, lipids, carbohydrates, and nucleic acids.

The endocrine pancreas, consisting of dispersed clusters of cells called islets of

Langerhans, accounts for only 1-2% of the organ mass [1]. Five different endocrine cell types are found within these islets: glucagon-producing α (alpha) cells, insulin-producing β (beta) cells, pancreatic polypeptide-producing γ (gamma) cells, somatostatin-producing δ (delta) cells, and ghrelin-producing ε (epsilon) cells [1]. Across mammalian species, β cells are the most numerous of all islet endocrine cells, comprising 75% of mouse islets and 54% of human islets [4]. However, mouse and human islets display markedly different endocrine cell spatial organization patterns; in mouse islets, β cells compose a central core surrounded by α and δ cells, while in human islets, α,

β, and δ cells appear to be dispersed throughout the islet [4].

1.1.2 Insulin Biogenesis

Most animals, including humans, have a single copy of the insulin gene, but mice and rats have two copies, Ins1 and Ins2 [5, 6]. These genes, only differing in their intronic regions and chromosomal location, both produce the same 110-amino acid precursor protein known as preproinsulin [7]. As with other secreted proteins, preproinsulin contains an N-terminal signal peptide which facilitates nascent preproinsulin translocation into the lumen of the endoplasmic reticulum (ER). In addition to the signal peptide, preproinsulin consists of three segments: the B- chain, the C-peptide, and the A-chain. During co-translational insertion into the ER lumen, the signal peptide undergoes proteolytic cleavage, creating the prohormone proinsulin (Figure 1.2A)

[8]. Proinsulin then undergoes folding and posttranslational modifications, including the formation of three intramolecular disulfide bonds, including two between the A- and B-chains (A7-B7, A20-

B19) and one within the A-chain (A7-A11) [9, 10]. Deletion of any single insulin disulfide bond results in loss of ordered secondary structure and increased susceptibility to proteolysis [11]. Once this tertiary structure has been established, the folded proinsulin is trafficked to the Golgi apparatus where it is packaged into immature secretory granules. Subsequent granule maturation involves acidification of the granule lumen [12], providing the optimal conditions in which prohormone convertases (PC) 1/3 and 2 can cleave proinsulin [13]. The coordinated action of PC1/3, PC2, and carboxypeptidase E result in the excision of the C-peptide, producing the mature form of insulin, a 51-amino acid protein consisting of a B-chain and an A-chain (Figure 1.2B) [14].

The ability of β cells to respond to changes in glucose levels by increasing insulin biogenesis is largely due to transcriptional regulation [10]. Exposure to elevated glucose for as little as 15 minutes is sufficient to induce a 2- to 5-fold increase in insulin mRNA levels within 60 to 90 minutes [15]. Several studies have identified regulatory elements within the promoter region

4 of the insulin gene that respond to elevated glucose [16-18]. Moreover, exposure to elevated glucose increases the stability of insulin mRNA in β cells, thereby augmenting the effects of glucose on insulin transcription [19]. Finally, glucose controls insulin biosynthesis by stimulating its translational initiation and elongation [20-23].

Figure 1.2: Posttranslational Modifications of Insulin (A) During translation, preproinsulin is translocated to the ER where its signal peptide is cleaved, forming proinsulin. (B) Once packaged into secretory granules, excision of the C-peptide produces the mature form of insulin. Figure adapted from the Beta Cell Biology Consortium and the Public Library of Science.

1.1.3 Insulin Secretion

A single mouse β cell contains roughly 13,000 insulin granules, each of which contains approximately 200,000 insulin molecules [10, 24]. Less than 1% of these granules comprise the readily releasable pool (RRP), a population of granules that are pre-docked at the cell membrane, while the remainder of granules are contained in a reserve pool [25]. The first phase of insulin secretion is characterized by the rapid exocytosis of the RRP, occurring within five minutes of glucose stimulation, while the second phase requires mobilization of the reserve pool of granules, a process which occurs within 30 minutes of initial glucose stimulation.

Despite not expressing glucose receptors, β cells can indirectly sense circulating levels of glucose, the main stimulus for insulin secretion. Glucose enters β cells via glucose transporter 2

(GLUT2) by facilitated diffusion. Glucose is then phosphorylated by glucokinase, an enzyme expressed almost exclusively in β cells, allowing it to enter the glycolytic pathway. Pyruvate, a product of glycolysis, is further oxidized through the tricarboxylic acid cycle, generating ATP and resulting in an increased ATP/ADP ratio. This increased ratio induces the closing of ATP-sensitive potassium (KATP) channels, resulting in the depolarization of the cell membrane [26].

Consequently, voltage-dependent calcium channels (VDCCs) are activated, resulting in the influx of extracellular calcium into the cytoplasm which can subsequently trigger insulin granule exocytosis [27]. In summary, β cells sense increases in extracellular glucose levels using ATP as a proxy and consequently activate the first phase of insulin granule secretion via a KATP-dependent mechanism (Figure 1.3).

In addition to the canonical glucose-Ca2+ pathway, other signals activated by glucose can potentiate insulin secretion. For example, glucagon-like peptide-1 (GLP-1), a hormone released by intestinal enteroendocrine cells following ingestion of a carbohydrate-rich meal [28], binds a G

6 protein-coupled receptor (GPCR) on β cells that activates adenylate cyclase. In turn, elevated cyclic AMP (cAMP) levels activate protein kinase A (PKA) as well as a nucleotide exchange factor referred to as exchange protein directly activated by cAMP (EPAC) [1]. Both PKA and

EPAC potentiate insulin secretion by increasing intracellular Ca2+ levels; PKA phosphorylates

VDCCs, thus increasing their activity [29], while EPAC mobilizes calcium from internal stores

[30]. Moreover, EPAC further potentiates insulin secretion by increasing the density of insulin granules near the plasma membrane [31]. Thus, incretin hormones such as GLP-1 promote insulin secretion by multiple cAMP-mediated pathways (Figure 1.3).

Figure 1.3: Glucose-Stimulated Insulin Secretion Following its entry into pancreatic β cells through GLUT2, glucose is metabolized to produce 2+ ATP. KATP channel closure results in VDCC-mediated Ca influx into the cytoplasm, triggering insulin release. GLP-1 promotes insulin secretion by multiple cAMP-mediated pathways. Figure reproduced with permission from [14].

In addition to glucose, β cells can respond to other nutrients in the blood, including amino acids and free fatty acids. Despite the fact that individual amino acids are poor insulin secretagogues, certain combinations of amino acids can promote insulin secretion. For example, glutamine alone does not stimulate insulin secretion; however, leucine can activate glutamate dehydrogenase, which allows glutamine entry into the tricarboxylic acid cycle, thus producing

ATP and enhancing insulin secretion [32]. Free fatty acids (FFAs) also play a role in stimulating the secretion of insulin from β cells [33, 34]. Indeed, long-chain FFAs bind free fatty acid receptor

1, a GPCR expressed in β cells, leading to the activation of phospholipase C and the subsequent hydrolysis of PIP2 to IP3 and diacylglycerol. The secondary messenger IP3 travels to the ER, binds to the IP3 receptor, and stimulates the release of calcium into the cytoplasm, further amplifying insulin secretion [35, 36]. Finally, amino acids and FFAs also stimulate insulin secretion via the incretin effect, by inducing release of GLP-1 at the level of the intestine [37, 38].

1.1.4 Insulin Signaling

Upon its release into the blood stream, insulin acts in an endocrine manner on multiple cell types including adipocytes, hepatocytes, and myocytes. Insulin initiates signaling by binding to the insulin receptor (IR), a tetrameric protein composed of two extracellular α subunits and two transmembrane β subunits. Upon insulin binding to the α subunits, IR undergoes a conformational change leading to the activation of the tyrosine kinase activity harbored in the β subunits. This results in transautophosphorylation among β subunits, leading to the recruitment and phosphorylation of insulin receptor substrate (IRS) proteins. Specifically, IRS-1 contains over ten potential tyrosine phosphorylation sites [39], suggesting that it could act as a multisite docking protein for effector proteins containing phosphotyrosine-binding domains or Src homology 2

(SH2) domains, both known to bind phosphotyrosine residues [40, 41]. Indeed, IRS-1 has been shown to bind two SH2 domain-containing proteins, phosphatidylinositol-3-kinase (PI3K) [39] and growth factor receptor-bound protein 2 (Grb2) [42], in a non-competitive manner. These interactions play a crucial role in propagating signals from IR to downstream effectors.

Following its recruitment to the plasma membrane via IRS-1, PI3K phosphorylates PIP2 to generate PIP3. Next, the protein kinase Akt is recruited to the cell membrane via a high affinity

308 interaction with PIP3. Once correctly positioned, Akt is phosphorylated on Thr by phosphoinositide-dependent kinase 1 [43] and on Ser473 by the mammalian target of rapamycin complex 2 (mTORC2) [44, 45]. Of three isoforms, Akt2 is most abundant in insulin-sensitive tissues and appears to play a predominant role in mediating insulin action [46]. In liver, active Akt phosphorylates and inhibits glycogen synthase kinase 3, a negative regulator of glycogen synthase, an enzyme involved in glycogen synthesis [47]. Thus, Akt activation promotes glucose storage as glycogen. Akt also inhibits gluconeogenesis and glycogenolysis, thus preventing the production and release of glucose by the liver [48, 49]. In muscle and adipose tissue, insulin-mediated Akt activation leads to translocation of the glucose transporter GLUT4 to the membrane, thus stimulating glucose uptake [50]. Moreover, in liver and adipose tissue, insulin acts to increase lipogenesis, another process by which extracellular glucose levels are decreased.

In addition to peripheral tissues, insulin has been shown to induce signaling within the islet of Langerhans. Upon glucose stimulation, insulin is secreted and acts in an autocrine manner to induce IR signaling in β cells, as well as downstream IRS-1 and PI3K signaling [51]. Although historically, insulin signaling in β cells has been suggested to provide negative feedback by negatively regulating β cell function, more recent studies have identified insulin as a positive regulator of transcription, translation, proliferation, and β cell survival [52]. Finally, insulin also

9 acts in a paracrine manner within the islet of Langerhans, specifically on α cells. Indeed, α cell- specific knockout of IR results in enhanced glucagon secretion, supporting a role for insulin in the negative regulation of α cell secretory function [53]. In summary, insulin appears to act in an autocrine manner on β cells as a form of positive feedback, while opposing the action of α cells in a paracrine manner.

1.1.5 Clinical Significance

Diabetes mellitus is a disease characterized by the failure of pancreatic β cells to produce enough insulin, resulting in uncontrolled blood glucose levels. In 2015, 415 million people suffered from diabetes worldwide, a figure expected to rise by over 50% by 2040 [54]. Diabetes disorders are generally classified into two main categories: type 1 diabetes and type 2 diabetes.

1.1.5.1 Type 1 Diabetes

Type 1 diabetes (T1D) is caused by an autoimmune reaction against pancreatic β cells.

More specifically, this disease is characterized by an immunological response against self-islet proteins, including proinsulin [55] and mature insulin [56], resulting in β cell destruction.

Susceptibility to T1D is controlled by multiple factors, the most important one being variability in the major histocompatibility complex genes [57]. Moreover, genetic variations in genes unrelated to immune responses may increase susceptibility to T1D, including genes involved in β cell senescence and apoptosis [58]. Given the nature of the disease, it is also referred to as insulin- dependent diabetes mellitus, and it can be managed by treatment with exogenous insulin.

1.1.5.2 Type 2 Diabetes

Type 2 diabetes (T2D) is the most common type of diabetes, accounting for over 90% of all cases. The development of diabetes is preceded by the development of insulin resistance in peripheral tissues, a pathological condition leading to hyperglycemia [59]. In order to restore glucose homeostasis in this condition, β cells attempt to compensate by increasing insulin secretion. Mechanistically, this compensation occurs via an increase of β cell proliferation and enhanced glucose sensitivity [60-62]. However, over time, β cells fail to compensate for insulin resistance, resulting in the progressive decline of β cell function, associated with an increase in β cell apoptosis (Figure 1.4) [63, 64].

Figure 1.4: Progression of Type 2 Diabetes Early in the development of T2D, β cells compensate for insulin resistance by increasing insulin synthesis. However, over time, β cell exhaustion inevitably results in β cell dysfunction and death. Figure reproduced with permission from [64].

Genome-wide association studies have identified genetic variations that predispose people to developing T2D. Indeed, genes with important roles in β cell physiology have been shown to affect susceptibility to T2D, including KCNJ11, encoding a KATP channel controlling insulin secretion [65], and WFS1, a membrane protein that maintains calcium homeostasis in the ER [66].

Due to the chronic state of insulin resistance, T2D is considered a non-insulin dependent form of the disease; thus, exogenous insulin is insufficient to manage hyperglycemia. Common therapies used to manage T2D include biguanides and thiazolidinediones, which sensitize peripheral tissues to insulin, and sulfonylureas, which act on β cells to enhance insulin secretion. The most widely used antidiabetic drug is Metformin, a member of the biguanide class of drugs that reduces glucose levels by diminishing hepatic gluconeogenesis and enhancing glucose uptake by skeletal muscle

[67, 68].

1.1.5.3 Monogenic Diabetes

The genetic component of T2D is considered to be polygenic, which means that multiple genes contribute to the diabetic phenotype. On the other hand, several rare forms of diabetes are caused by an abnormality in a single gene. One such form of monogenic diabetes arises due to mutations in the human INS gene. Indeed, multiple missense mutations in proinsulin and insulin have been identified, affecting insulin biosynthesis and structure and ultimately resulting in diabetes [69]. This finding has been corroborated through the characterization of the Akita mouse, a model of diabetes caused by a mutation in the Ins2 gene that results in proinsulin misfolding in the ER [70, 71]. Another form of monogenic diabetes, Wolcott-Rallison syndrome, is caused by mutations in the EIF2AK3 gene [72]. This gene encodes the ER transmembrane protein PERK, a protein that will be described in depth in the following section.

1.2 PERK

Through a signaling network known as the integrated stress response, eukaryotic cells respond to physiological disturbances by attenuating general translation via phosphorylation of the

α subunit of eukaryotic initiation factor 2 (eIF2α) on Ser51. In mammals, this process is mediated by four protein kinases, each responding to a distinct type of stress: heme-regulated inhibitor

(HRI), induced by heme deficiency [73]; protein kinase RNA-dependent (PKR), induced by viral infection and the presence of dsRNA [73]; general control non-depressible-2 (GCN2), induced by amino acid depravation [74]; and PKR-like endoplasmic reticulum kinase (PERK), induced by perturbations in protein folding [75, 76]. PERK is a type-I transmembrane protein that consists of an ER luminal domain, a transmembrane domain, and a cytoplasmic domain harboring enzymatic activity. PERK was originally defined as a serine/threonine kinase given its ability to phosphorylate its substrates, or be autophosphorylated, exclusively on serine and threonine residues. However, studies later revealed that PERK is autophosphorylated on Tyr561 and Tyr615, suggesting that PERK also possesses tyrosine kinase activity [77, 78]. PERK signaling pathways form a major branch of the unfolded protein response (UPR), a signaling network described in the section below.

1.2.1 Unfolded Protein Response

The endoplasmic reticulum is an organelle involved in the synthesis of secretory and transmembrane proteins. Upon delivery to the ER lumen, nascent polypeptides undergo posttranslational modifications and are folded into their native structure. This process involves various ER chaperone proteins, including protein disulfide isomerase (PDI), which catalyzes the formation of disulfide bonds between cysteine residues within proteins, and binding

13 immunoglobulin protein (BiP), which binds nascent proteins and assists in their folding, translocation, and if necessary, their degradation. When the protein folding needs of the cell exceed the folding capacity of the ER, misfolded or unfolded proteins accumulate in the ER lumen, creating a condition referred to as ER stress. In response to this stress, the cell employs various signaling pathways, collectively known as the unfolded protein response, to restore ER homeostasis. Three branches of the UPR have been characterized, all of which are mediated by

ER transmembrane proteins: PERK, inositol-requiring enzyme-1α (IRE1α), and activating transcription factor 6α (ATF6α) (Figure 1.5).

1.2.1.1 PERK Signaling

Under basal conditions, PERK is maintained in an inactive state via the interaction of its luminal domain with BiP, an ER resident protein [79]. In response to ER stress, BiP dissociation results in PERK dimerization [80] and hyperphosphorylation of its cytoplasmic domain [75, 79].

This state of hyperphosphorylation is not induced in a mutant form of PERK lacking kinase activity

(K618A), suggesting that this phenomenon is a result of autophosphorylation [79]. Such PERK phosphorylation, notably on Thr980 in mouse and Thr974 in rat, induces a major conformational change in the PERK kinase domain, enhancing both its activity and its affinity for the eIF2 complex [81, 82], thus resulting in eIF2α phosphorylation on Ser51 (Figure 1.5) [83].

In one of the first steps of translation initiation, eIF2, composed of α, β, and γ subunits, forms a ternary complex with methionine-tRNA and GTP. This ternary complex, along with other eIFs, binds to the 40S ribosomal subunit, forming a complex which is ready to bind mRNA and migrate to the initiation codon. At the end of initiation, the GTP molecule bound to eIF2γ is hydrolysed to GDP, resulting in the dissociation of eIFs from the ribosomal subunit [84]. To enable

14 the formation of a new ternary complex, GDP must be replaced by GTP, a process performed by the guanine exchange factor eIF2B. When phosphorylated, however, eIF2α has a greater affinity for eIF2B, which paradoxically results in the reduction of its guanine exchange function [85].

Thus, PERK-mediated phosphorylation of eIF2α prevents the formation of a new ternary complex, resulting in the attenuation of general translation, providing the cell with optimal conditions to alleviate ER stress.

Paradoxically, this condition allows preferential translation of selective mRNAs, including that encoding activating transcription factor 4 (ATF4). Initiation of ATF4 translation involves ribosomal association at one of two upstream open reading frames (uORFs), uORF1 and uORF2, located in the 5’ untranslated region of ATF4 mRNA. uORF2 overlaps with the start codon of the

ATF4 coding sequence, thus acting as an inhibitory element. During physiological conditions, eIF2-GTP is abundant, and ribosomes initiated at uORF1 can reinitiate at uORF2, thus blocking

ATF4 expression [86]. During ER stress conditions, eIF2α phosphorylation results in decreased levels of eIF2-GTP; thus, ribosomes are slower to reinitiate downstream of uORF1, bypassing the inhibitory uORF2 and instead reinitiating at the ATF4 coding region, resulting in increased ATF4 expression [86]. ATF4 upregulates genes involved in amino acid metabolism and reduction- oxidation reactions, both of which contribute to reducing ER stress [87]. On the other hand, ATF4 also induces expression of C/EBP-homologous protein (CHOP), a transcription factor that induces expression of several pro-apoptotic genes [88]. CHOP also induces expression of growth and arrest

DNA damage-inducible 34, a protein that participates in a negative feedback mechanism that leads to dephosphorylation of eIF2α [89]. Thus, the PERK-ATF4-CHOP signalling axis mediates a major part of the UPR-dependent transcriptional and translational network regulating cell fate according to the intensity and duration of ER stress.

Moreover, PERK also induces activation of the transcription factor NF-E2-related factor 2

(Nrf2). In unstressed cells, Nrf2 remains sequestered in the cytoplasm due to its interaction with

Kelch-like ECH-associated protein 1 (Keap1), a cytoskeletal anchor protein [90]. PERK- dependent phosphorylation of Nrf2 on an unidentified site results in dissociation of the Keap1/Nrf2 complex, permitting Nrf2 translocation into the nucleus where it acts to increase expression of antioxidant genes [91, 92]. Independent of PERK, Nrf2 has also been shown to induce expression of antioxidant genes, including NAPDH quinone oxidoreductase 1, glutathione S-transferase, and heme oxygenase 1 [93, 94]. These data suggest that in addition to eIF2α-mediated attenuation of general translation, the Nrf2 pathway also contributes to PERK-mediated cell survival, a notion supported by the fact that in vitro deletion of Nrf2 reduces cell survival following ER stress [91].

1.2.1.2 IRE1α Signaling

The first UPR sensor to be discovered was IRE1, a transmembrane protein encoded by a gene identified in S. cerevisiae that appeared to be required for cell viability under stress conditions

[95]. This protein is evolutionary conserved from yeast to mammals, where two isoforms of IRE1 exist: IRE1α, the ubiquitously expressed form, and IRE1β, which exhibits tissue-specific expression [96-98]. IRE1α consists of an ER luminal domain, responsible for monitoring the status of protein folding, and a cytoplasmic region, consisting of a kinase domain and an endonuclease domain [95, 98]. PERK and IRE1α have functionally interchangeable ER luminal domains, both of which are bound by BiP to maintain a state of inactivity [79]. In response to ER stress conditions,

BiP dissociates from IRE1α, resulting in dimerization and autophosphorylation of IRE1α on Ser724

[99]. These events lead to the activation of the IRE1α endoribonuclease domain that specifically cleaves the mRNA encoding X-box-binding protein 1 (XBP1) into its spliced form (XBP1s), a highly active transcription factor (Figure 1.5) [100]. Upon entering the nucleus, XBP1s regulates 16 the transcription of various genes involved in protein folding, ER biogenesis, and ER-associated degradation (ERAD), with the goal of restoring ER homeostasis. In order to reduce the load of protein folding, the endoribonuclease activity of IRE1α also degrades mRNAs encoding proteins that are targeted to the ER for modifications, a process referred to as regulated IRE1-dependent decay (RIDD) [101]. For example, when β cells are exposed to ER stress, the IRE1 pathway is activated and Ins1 and Ins2 transcripts are rapidly degraded, thereby reducing the protein folding load required by the ER during insulin synthesis [102].

1.2.1.3 ATF6α Signaling

Like IRE1α and PERK, ATF6α is an ER transmembrane protein consisting of a luminal domain bound by BiP under physiological conditions. However, unlike the other two UPR sensors,

ER stress-induced BiP dissociation does not result in phosphorylation of the ATF6α cytoplasmic domain. Instead, ER stress induces ATF6α translocation from the ER membrane to the Golgi, where it undergoes proteolytic cleavage in its luminal domain by SP1 and SP2 (Figure 1.5) [103,

104]. Cleaved ATF6α is a transcription factor that upregulates genes involved in restoring ER homeostasis such as PDI-like enzymes, chaperones, and ERAD-associated proteins [104-106].

Interestingly, cleaved ATF6α also induces expression of XBP1 [100], demonstrating the crosstalk that exists between different signaling arms of the UPR.

Figure 1.5: Unfolded Protein Response ER stress triggers the activation of three ER transmembrane receptors: PERK, IRE1α, and ATF6α. PERK phosphorylation results in eIF2α-mediated general translation inhibition and preferential ATF4 translation. IRE1α phosphorylation results in XBP1 mRNA splicing, producing a protein- encoding transcript, and RIDD. Activated ATF6α translocates to the Golgi where it is cleaved. ATF4, XBP1, and cleaved ATF6α are transcription factors that drive coordinated responses to restore ER homeostasis. Figure reproduced from [107].

1.2.2 PERK & β Cell Homeostasis

PERK was first identified as an eIF2α kinase in rat pancreatic islets and was consequently named pancreatic eIF2α kinase [76]. In pancreatic β cells, PERK is highly expressed and partially activated under physiological conditions [108]. Over the past two decades, multiple studies have described various roles for PERK in β cell development and insulin biogenesis both under physiological conditions and in the pathogenesis of diabetes (Figure 1.6).

1.2.2.1 β Cell Development and Survival

Shortly following the discovery of PERK, loss of function of PERK was linked with

Wolcott-Rallison syndrome (WRS), a genetic disease characterized by neonatal diabetes associated with non-autoimmune-mediated β cell destruction in humans [72]. Indeed, WRS families displayed distinct mutations in the Perk gene producing truncated or inactive versions of

PERK [72]. Since then, multiple mutations in Perk have been reported in WRS patients, all of which are located within or in the vicinity of the catalytic domain [109], further supporting the notion that PERK activity is crucial for proper β cell physiology. In agreement, mice lacking PERK

(Perk-/-) reproduce several phenotypes of WRS patients, including pancreatic defects [108, 110].

Indeed, although the endocrine pancreas appears functional and morphologically normal early after birth, with time β cells are destroyed, as indicated by decreased β cell mass and insulin immunostaining, potentially due to an increase in apoptosis [108, 110]. On the other hand, expression of the insulin genes, Ins1 and Ins2, as well as Mafa, encoding a transcription factor regulating Ins1 and Ins2 transcription, is repressed in Perk-/- β cells during embryonic development, suggesting that impaired β cell differentiation during the neonatal period is part of the mechanism by which PERK deficiency leads to β cell failure [111]. However, postnatal

19 conditional PERK deletion results in β cell death through apoptosis, revealing that PERK also regulates β cell homeostasis in adult mice [112]. Moreover, in vivo treatment with a PERK kinase inhibitor throughout postnatal life results in reduced pancreas weight accompanied by lower serum insulin levels, confirming the role of PERK activity in postnatal β cell development or survival

[113, 114].

1.2.2.2 β Cell Proliferation

Total β cell mass is controlled by a balance between proliferation and apoptosis. The concept that decreased β cell mass resulting from PERK deficiency is due to increased ER stress and β cell apoptosis was challenged by Zhang et al., who reported that although pancreas-specific deletion in mice leads to abnormally lower neonatal β cell mass, increased apoptosis was not detected [111]. In contrast, β cell proliferation was reduced in both late embryonic and early neonatal stages in Perk-/- mice, impeding postnatal gain of pancreatic β cell mass [111]. In agreement, analysis of genes differentially expressed in Perk-/- islets mainly identified genes encoding cell cycle or proliferation factors [111]. Further supporting a direct link between PERK and β cell proliferation, expression of a dominant negative PERK mutant in rat β cells reduces proliferation [115]. Therefore, the role of PERK in β cells during development is to promote proliferation rather than to prevent cell death. In contrast, although conditional deletion of PERK in young and adult mice also results in the loss of β cell mass, β cell proliferation is dramatically increased [112], highlighting differential PERK-dependent regulation of β cell homeostasis during embryonic development and adulthood.

1.2.2.3 Insulin Processing & Secretion

Consistent with an important role of PERK in regulating pancreatic  cell function, several studies have revealed that PERK is essential for insulin synthesis and processing. Global and pancreas-specific PERK knockout mice exhibit a highly distended ER phenotype in β cells, suggesting abnormal retention of proinsulin [111]. Accordingly, PERK-deficient mice display impaired glucose-stimulated insulin secretion and develop diabetes [111]. In a similar vein, ablation of PERK activity in vitro via transduction of a PERK dominant negative mutant causes abnormal accumulation of proinsulin in the ER, resulting in reduced insulin content and glucose- induced insulin secretion [115]. Specifically, proinsulin accumulation was associated with a defect in anterograde trafficking between the ER and the Golgi [116]. Consistently, in vitro treatment with a PERK kinase inhibitor results in the delayed maturation of insulin [117]. Finally, PERK control of insulin biogenesis may arise from its ability to regulate  cell calcium dynamics. Indeed, insulin synthesis and glucose-stimulated insulin secretion are largely dependent on intracellular

Ca2+ levels regulated by the sarcoplasmic/ER Ca2+-ATPase (SERCA), and in vitro treatment with a PERK kinase inhibitor reduces Ca2+ uptake by inhibiting the activity of SERCA through a calcineurin-dependent pathway [118]. Therefore, PERK deletion, by impairing calcium homeostasis, may indirectly impede insulin synthesis and secretion. Furthermore, β cell death associated with PERK inhibition may also be attributed to Ca2+ depletion resulting from reduced

SERCA activity [119].

1.2.2.4 PERK-Dependent Signaling Network

Given that PERK responds to ER stress by attenuating translation through the phosphorylation of eIF2α on Ser51, it was postulated that reduced phosphorylation of eIF2α may contribute to β cell dysfunction in models of PERK deficiency. Indeed, mice harboring a homozygous Eif2αS51A mutation, abolishing phosphorylation on that residue, display pancreatic β cell deficiency associated with decreased levels of both pancreatic and serum insulin, in addition to dying shortly after birth [120]. Concomitantly, heterozygous Eif2αS51A mice, which unlike their homozygous counterparts are postnatally viable, display defective proinsulin trafficking and reduced insulin secretion when fed a high-fat diet [121]. Furthermore, mice harboring a β cell- specific homozygous Eif2αS51A mutation displayed increased β cell death and reduced insulin production, defects that were attenuated by feeding mice an antioxidant-rich diet [122], suggesting that PERK signaling through eIF2α phosphorylation preserves β cell function, in part, by preventing oxidative stress. Interestingly, the endocrine pancreata of both Atf4-/-- [122-124] and

Chop-/- mice [124] appear morphologically and functionally normal, indicating that the requirement of a functional PERK-eIF2α signaling axis under physiological conditions in β cells involves attenuation of general translation, rather than induction of ATF4 and CHOP. On the other hand, CHOP-mediated apoptosis seems to be involved in β cell loss in various diabetic mouse models. Indeed, in Akita mice, which express a mutant form of insulin (Ins2C96Y) that misfolds and accumulates in the ER, creating ER stress [70, 71], prevention of full PERK activation is protective

[116] and CHOP knockout is sufficient to protect islet cells from apoptosis, delaying the onset of diabetes [125]. Concomitantly, in leptin receptor mutant (db/db) mice, a common genetic model of diabetes [126, 127], CHOP knockout protects β cells against oxidative stress and apoptosis by promoting expression of antioxidant genes and preventing expression of proapoptotic genes [128].

In addition to phosphorylation of eIF2α leading to general attenuation of protein synthesis, the effects of PERK in β cells also appear to be mediated by Nrf2, which plays a crucial role in inducing antioxidant gene expression in pancreatic islets [129]. Indeed, ex vivo pharmacological activation of Nrf2 in human islets, which results in increased antioxidant gene expression, improves β cell survival in response to oxidative stress [130]. In addition, in vivo β cell-specific

Nrf2 depletion results in a reduction in islet size [129], while suppressing expression of Nrf2 by silencing Nr2e1 in mouse insulinoma MIN6 cells augments palmitate-induced oxidative stress resulting in decreased proliferation and higher rates of apoptosis [131]. Finally, knockdown of

Keap1, a negative regulator of Nrf2 [90], preserves β cell mass and prevents the onset of diabetes in db/db mice via induction of Nrf2 signaling [132].

In the past few years, increasing evidence highlights the concept that PERK regulation of

β cell homeostasis proceeds through alternative mechanisms. Indeed, PERK antagonizes interferon signaling, thus protecting against pancreatic tissue injury, by mediating phosphorylation, ubiquitination, and degradation of the type I interferon (IFN) receptor subunit IFNAR1 [133, 134].

PERK depletion in pancreatic islets results in elevated expression of IFN ligands as well as increased IFNAR1 levels and signaling, which could contribute to loss of islet mass and the development of diabetes [114]. Moreover, knockout of IFNAR1 or administration of an IFNAR1 neutralizing antibody protects β cells against the damaging effects of PERK kinase inhibition or genetically-induced PERK depletion [114]. These findings support a model in which PERK regulates IFNAR1 expression and signaling in pancreatic β cells to protect against IFN-induced injury, providing a novel and alternative mechanism by which PERK functions to maintain β cell survival.

IFNAR1 IFNAR2

P Ub Degradation

SERCA

eIF2 Translation P P α β P γ PERK ATF4

ER Lumen P Nrf2

Cytoplasm

Nucleus

Antioxidant genes Chop P Nrf2 ATF4

Apoptosis

Figure 1.6: PERK & β Cell Homeostasis The ER transmembrane protein PERK directly regulates eIF2α phosphorylation, Nrf2 activity, and IFNAR1 degradation, and indirectly impacts SERCA activity in physiological conditions to control β cell homeostasis (solid arrows). In pathological conditions, aberrant PERK activity leads to ATF4-mediated CHOP induction linked to β cell apoptosis (dashed arrows).

1.2.2.5 Pathogenesis of Diabetes

Increasing evidence supports a strong correlation between aberrant PERK activation and signaling and the pathogenesis of β cell dysfunction in diabetes in both mice and humans.

Pancreatic islets of db/db mice display increased levels of eIF2α phosphorylation, Atf4 and Chop, and reduced expression of genes regulating β-cell function [135, 136]. Similarly, β cells of Akita mice display increased ER stress [71], and expression of Ins2C96Y in rat insulinoma INS-1 cells induces eIF2α phosphorylation and Chop expression, leading to apoptosis [137]. In humans, expression of CHOP is elevated in the islets of T1D patients [138] and in pancreatic sections of

T2D patients [135]. Furthermore, both cytosolic and nuclear CHOP levels are greater in obese

T2D pancreata compared to obese nondiabetic counterparts [139]. Taken together, these studies identify a strong correlation between aberrations in PERK signaling and the pathogenesis of diabetes in both mice and humans.

1.3 Nck Adaptor Proteins

The Nck (non-catalytic region of tyrosine kinase) family of adaptor proteins consists of two members in humans and mice: Nck1 and Nck2. Both are composed exclusively of Src homology 2 (SH2) and Src homology 3 (SH3) domains, and lack any other functional or enzymatic motifs [140]. SH2 domain-containing proteins bind phosphotyrosine residues on activated receptors and cytoplasmic phosphoproteins, while SH3 domain-containing proteins bind proline- rich amino acid sequences (PxxP) as well as atypical motifs such as RxxK [41, 141]. Through these domains, Nck1 and Nck2 couple cell surface receptors to downstream effector proteins, thus playing a crucial role in signal transduction in various biological processes [142].

1.3.1 Discovery

Nck1 (then named Nck) was first identified in 1990, when its gene was isolated from a human melanoma cDNA library [143]. Two years later, a partial cDNA fragment of a mouse Nck homolog was reported as a binding partner of epidermal growth factor receptor (EGFR) [144]. In this screen, five SH2 domain-containing proteins were identified and named GRBs (growth factor receptor-bound proteins), with Grb4 showing an overall 64% amino acid identity with the previously identified human Nck [144]. Based on the rationale that this identity was too low for human NCK and mouse Grb4 to be considered a pair of orthologs, Chen et al. aimed to identify a mouse gene that has a higher homology to human NCK, and a human gene that has a higher homology to mouse Grb4 [140]. They identified paralogous genes in both humans and mice, renaming NCK to Nck1/Nckα, and Grb4 to Nck2/Nckβ [140]. In humans and mice, these paralogs share 68% amino acid identity, while across species, both Nck1 and Nck2 show 96% identity to each other (Figure 1.7) [140]. In humans, these genes reside on different chromosomes – NCK1 on chromosome 3, located at 3q21, and NCK2 on chromosome 2, located at 2q12 [140, 145].

Figure 1.7: Nck Proteins In humans and mice, Nck1 and Nck2, both composed of three N-terminal SH3 domains and a single C-terminal SH2 domain, share 68% amino acid identity. Individual SH2 or SH3 domains show a high degree of homology, while differences are mainly located within linker regions. Figure reproduced from [146].

1.3.2 Evolution

Orthologs of Nck have been detected in various species, including Caenorhabditis elegans

(nematode), Drosophila melanogaster (fruit fly), Xenopus laevis (frog), Danio rerio (zebrafish), and Gallus gallus (chicken) [6]. The family of Nck proteins in C. elegans consists of Nck-1A and

Nck-1B, both of which are encoded by the nck-1 gene [147]. Mutations in this gene are associated with defects in neuronal guidance and neuronal cell position [147]. Mutations in dreadlocks, the

D. melanogaster ortholog of Nck, disrupt photoreceptor cell axon guidance and targeting in the eye [148]. Moreover, in X. laevis, Nck has been implicated in mesoderm patterning during early development [149, 150].

While the Nck orthologs in the three aforementioned species are all encoded by single genes, other species have two or more Nck-encoding genes, suggesting the occurrence of a duplication event throughout evolution [6]. Notably, zebrafish have four Nck-encoding genes, while mice, rats, and humans have two each [6].

1.3.3 Domains & Protein Interactions

Both Nck1 and Nck2 are composed of three N-terminal SH3 domains (SH3.1, SH3.2,

SH3.3) and a single C-terminal SH2 domain (Figure 1.7) [143]. All three SH3 domains consist of a WW motif as well as a distal Y residue, forming a structure of pockets that allows for the binding of proline-rich peptides [151]. Via their SH3 domains, Nck proteins bind various intracellular proteins involved in signal transduction, including IRS-1 [152], the CD3ε subunit of the T cell receptor [153], Wiskott-Aldrich syndrome protein [154], the netrin-1 receptor DCC [155], and the

β subunit of eIF2 [156].

Using a phosphopeptide library, Sonyang et al. studied the sequence selectivity of the Nck1

SH2 domain and identified the preferred sequence to be pYDE(P/D/V) [157]. Frese et al. identified a phosphopeptide derived from the Escherichia coli translocated intimin receptor (Tir) as the strongest natural ligand of both Nck1 and Nck2, and performed sequential mutation on this sequence to identify the optimal profile for binding their SH2 domains [158]. To maintain high affinity binding, the phosphotyrosine residue (position 0), the aspartic acid residue (position +1), and the valine residue (position +3) were determined to be optimal [158]. In fact, all positions -2 to +6 (except for +5) participate in creating a stable interaction between the phosphopeptide and the Nck SH2 domain [158]. Via their SH2 domains, Nck proteins have been demonstrated to interact with a variety of proteins, including EGFR [159], platelet-derived growth factor receptor

(PDGFR) [159], nephrin [160, 161], platelet endothelial cell adhesion molecule-1 (PECAM-1)

[162], and PERK [78]. While some proteins bind Nck1 and Nck2 through a common site, others experience differential binding; for example, Nck1 binds human PDGFRβ phosphorylated on Y751 while Nck2 interacts with human PDGFRβ on Y1009 [163, 164]. Interestingly, the SH2 domains of

Nck1 and Nck2 have virtually identical binding profiles, suggesting that the distinguishing characteristics of Nck1 and Nck2 involve regions outside their SH2 domains [158].

1.3.4 Regulation

Both Nck1 and Nck2 are known to be phosphorylated in response to various stimuli, including EGF, PDGF, and angiotensin II [159, 165-167]. In order to explain the differential binding profiles of these two similar proteins, it is plausible that differential regulation of Nck1 and Nck2 by posttranslational modifications such as phosphorylation could be a contributing factor. Indeed, computational analyses have indicated that the SH2 domains of the two Nck

28 proteins bear unique phosphorylation sites [158], a phenomenon that could potentially explain the differences in the interactomes between the two proteins despite their similarities in amino acid identity and binding profiles.

In addition to regulation by phosphorylation, various studies have also demonstrated that

Nck expression is regulated under different conditions. A role for Nck in insulin resistance was suggested by Bonini et al., who demonstrated that Nck levels were significantly elevated in the livers and adipose tissue of KK-Ay mice, a model of obesity, insulin resistance, and non-insulin dependent diabetes mellitus [168]. On the other hand, Nck levels were found to be decreased in the livers of streptozotocin-treated rats, a model of insulin-dependent diabetes mellitus [169].

While these studies did not differentiate between Nck1 and Nck2, the regulation of these two proteins is not necessarily linked; interestingly, Dusseault et al. observed a decrease in Nck2, but not Nck1 expression, in the omental white adipose tissue of severely obese human subjects compared to their moderately obese counterparts [170].

In addition to its role in the pathogenesis of metabolic disease, the regulation of Nck expression has also been linked to cancer. More specifically, our group has identified a role for

Nck in melanoma progression [171]. Particularly, Labelle-Côté et al. observed increased levels of

Nck2 mRNA and protein in human metastatic melanoma cell lines, while no such pattern was seen for Nck1 [171].

1.3.5 Biological Processes

SH2/SH3-domain containing adaptor proteins, including Nck1 and Nck2, play important roles in recruiting proline-rich effector proteins to tyrosine-phosphorylated kinases or their substrates. As such, these proteins are involved in signal transduction pathways that are crucial for

29 various biological processes, including cytoskeletal remodeling, protein translation, and regulation of the unfolded protein response.

1.3.5.1 Cytoskeletal Remodeling

One of the earliest identified biological functions of Nck was its role in cytoskeletal remodeling. Via an SH3 domain-dependent interaction, Nck binds various proteins involved in this process, including WASP [172], WIP [173], and PAK [174, 175].

The Wiskott-Aldrich syndrome protein (WASP) is involved in the transduction of signals to the actin cytoskeleton. Upon a conformational change induced by cooperative binding of the

Rho GTPase Cdc42 and the membrane phospholipid PIP2, WASP binds and activates the actin- related protein 2/3 (Arp2/3) complex, resulting in the nucleation of actin polymerization [176,

177]. The role of Nck in this process has been studied using the vaccinia virus, which spreads between cells by inducing host cell actin polymerization. The viral protein A36R directly interacts with Nck through its phosphorylated Tyr112 residue, resulting in the recruitment of WASP to the site of actin assembly and thus allowing for the dissemination of the virus [178]. Nck also recruits

WASP-interacting protein (WIP) to this complex, which contributes to actin polymerization by stabilizing WASP levels and promoting the formation and stability of new actin filaments [173,

179, 180].

Based on this finding, it was suggested that the vaccinia virus achieves actin-based motility by mimicking the endogenous signaling pathway that induces actin polymerization, likely downstream of receptor tyrosine kinases [178]. Indeed, Rivera et al. demonstrated that PDGF- induced cytoskeletal remodeling, including actin bundle disassembly and membrane protrusion formation, failed to occur in Nck-deficient mouse embryonic fibroblasts [181]. In fact, PDGFR

30 activation strengthens the interaction between Nck and p21-activated kinase (PAK), bringing forth a plausible mechanistic link between receptor tyrosine kinase activation and cytoskeletal remodeling [175]. Finally, Chen et al. demonstrated that blocking Nck2 by microinjection of an anti-Nck2 antibody in vitro inhibits PDGF-stimulated actin polymerization, providing further evidence that Nck plays a crucial role in transducing signals from growth factor receptors to the actin cytoskeleton [164]. Taken together, these results support the notion that Nck acts as an important adaptor protein in the regulation of cytoskeletal remodeling by forming complexes between tyrosine-phosphorylated receptor proteins and downstream effectors.

1.3.5.2 mRNA Translation

Initiation of translation is a complex process organized by the coordinated action of eukaryotic initiation factors (eIFs). Our group first suggested a role for Nck in this process, demonstrating that Nck and eIF2β interact and colocalize in ribosomal-enriched cellular fractions and that Nck1 overexpression enhances both cap-dependent and cap-independent translation [156].

Phosphorylation of eIF2α on Ser51 is a regulatory event associated with the inhibition of translation [182]. Our group has identified Nck as an inhibitor of eIF2α phosphorylation under specific stress conditions. Indeed, we demonstrated that Nck negatively regulates eIF2α phosphorylation mediated by eIF2α kinases PERK [78, 183-185], PKR [184, 186, 187], and HRI

[184], but not by the eIF2α kinase GCN2 [184]. In another study, we demonstrated that Nck exists in a complex with protein phosphatase 1 (PP1), an enzyme that dephosphorylates eIF2α, providing an alternative mechanism by which Nck maintains eIF2α in a hypophosphorylated state [188].

The mammalian/mechanistic target of rapamycin complex 1 (mTORC1) controls mRNA translation in response to various metabolic stimuli [189]. Recently, it has been demonstrated that

31 mTORC1 promotes the phosphorylation of eIF2β on Ser2, leading to the recruitment of Nck1 to the eIF2 complex [190]. This event is correlated with eIF2α dephosphorylation, likely mediated by PP1, and results in the activation of general translation [188, 190].

Eukaryotic mRNA is characterized by a 7-methylguanosine cap found at the 5’ end of the transcript, a structure involved in mRNA splicing, transport, stability, and translation [191]. The formation of this cap structure is coordinated by a complex consisting of a capping enzyme and a kinase that renders the mRNA into a capping substrate [192]. Interestingly, Mukherjee et al. observed that Nck1 is crucial for the assembly of this complex, identifying binding sites for both the capping enzyme and the kinase on adjacent SH3 domains of Nck1 [193]. Accordingly, they demonstrated that the capping complex dissociates upon Nck1 depletion in vitro [193]. Thus, Nck1 promotes translation not only by attenuating eIF2α phosphorylation, but also by promoting formation of the 5’ mRNA cap. In summary, studies by our group and others have compiled convincing evidence identifying Nck as a positive regulator of mRNA translation.

1.3.5.3 Unfolded Protein Response

The unfolded protein response (UPR; described in 1.2.1) is an adaptive signaling network responsible for maintaining ER homeostasis. Our group first identified Nck as a negative regulator of the UPR by demonstrating that Nck1 or Nck2 overexpression diminishes ER stress-induced

PERK activation, eIF2α phosphorylation, and ATF4 and CHOP expression [185, 188]. On the other hand, Nck1-/-/Nck2-/- mouse embryonic fibroblasts (MEFs) display increased PERK activation and signaling [78, 185, 188], both basally and in response to ER stress, further supporting the notion that Nck1 and Nck2 negatively regulate PERK activation. Concomitantly,

MIN6 cells depleted of Nck1 display increased basal PERK phosphorylation, eIF2α Ser51

32 phosphorylation, and Nrf2 nuclear localization [78, 183]. The mechanistic basis for this regulation was partially uncovered by Yamani et al., who demonstrated that Nck1 and Nck2 bind PERK in an SH2 domain-dependent interaction [78]. Moreover, PERK phosphorylation at Tyr561 appears to be crucial for this interaction and regulation, as binding fails when this residue is replaced with a non-phosphorylatable phenylalanine (Y561F) [78]. Consequently, Perk-/- MEFs transiently expressing PERK Y561F display increased PERK activity compared to those expressing wild type

PERK [78]. Taken together, these data strongly demonstrate that Nck1 binds and negatively regulates PERK activation and signaling (Figure 1.8).

Nck1 Present Nck1 Absent

T980 P P P Nck1 S51 eIF2 PERK 2 3 3 3 P PERK α β γ

ER ER Cytoplasm Cytoplasm

Figure 1.8: Regulation of PERK Activity by Nck1 Nck1, through its SH2 domain, binds PERK autophosphorylated on Tyr561 and maintains it in an inactive state. In Nck1-deficient cells, PERK dimerizes and is transautophosphorylated on Thr980, resulting in increased PERK activation and phosphorylation of eIF2α.

The mechanism by which Nck1 limits PERK activation is not fully understood. By binding

PERK on Tyr561, Nck1 may act as a canonical adaptor protein, recruiting a phosphatase which dephosphorylates an activation site of PERK. Moreover, Nck1 may physically block PERK dimerization or phosphorylation of an activation site. Finally, Nck1 binding may induce a conformational change in PERK, resulting in its increased affinity for BiP. On the other hand, it is entirely possible that phosphorylation of PERK on Tyr561 may be the direct cause of PERK inhibition, and the role of Nck1 is simply to protect this site from dephosphorylation.

In addition to regulating PERK signaling, Nck has also been implicated in the regulation of the IRE1α branch of the UPR. Nck1 knockdown in hepatocytes in vitro impairs ER stress- induced IRE1α signaling, as indicated by reduced phosphorylation of its substrate c-Jun N- terminal kinase (JNK) and decreased XBP1 splicing [194]. Consistently, Nck1-/- mice display attenuated hepatic IRE1α activation compared to wild type mice in response to ER stress associated with diet-induced obesity [194]. Taken together, these results suggest that Nck1 is required for full activation of IRE1α in response to various conditions inducing ER stress.

The molecular basis of the interaction between Nck and IRE1α is not well understood.

Studies using recombinant Nck1 have demonstrated that Nck1 directly interacts with IRE1α through its SH3.1 domain, and that this interaction is abolished upon ER stress-induced IRE1α activation [195]. However, recent studies using cell lysates have disputed this finding, arguing that

Nck1 associates with IRE1α mainly through its SH2 domain [196].

1.3.6 Physiology

Through its numerous functions in various biological processes, Nck has proven to be a crucial player in various physiological systems, including in embryonic development, the immune system, renal physiology, insulin signaling, adipogenesis, and β cell homeostasis.

1.3.6.1 Embryonic Development

The generation of Nck-deficient mouse strains has contributed greatly to our understanding of the roles of Nck in physiology, notably in the embryonic development stage. For example, concomitant loss of both Nck1 and Nck2 results in multiple morphological abnormalities and embryonic lethality at E9.5 [197]. Moreover, MEFs derived from these double knockout mice display impaired motility [197]. In wild type mice, Nck1 and Nck2 are both prominently expressed in the developing nervous system, suggesting that they may play a role in cytoskeletal organization in the development of mesodermal structures, and that in their absence, the nervous system fails to develop. These results have been corroborated in Xenopus laevis, where the single Nck isoform expressed has been implicated in mesoderm patterning during early development [149, 150].

Interestingly, mice deficient for either Nck1 or Nck2 are both viable, suggesting that they may be functionally redundant in the context of embryonic development [197].

1.3.6.2 Immunology & Pathogen Entry

B cells are lymphocytes that function in the adaptive immune system by recognizing antigens, presenting antigens, and secreting antibodies, all of which are dependent on signaling initiated by the B cell receptor (BCR), a protein composed of a membrane-bound immunoglobulin molecule and an intracellular signaling component. Following antigen recognition, multiple

35 signaling pathways downstream of BCR are utilized in order to stimulate B cell development and activation, including the PI3K/Akt pathway [198]. Recruitment of PI3K to BCR requires the B cell adaptor for PI3K (BCAP), a substrate of tyrosine phosphorylation that acts as a dock for PI3K binding [199]. Castello et al. identified Nck a crucial player in this pathway, demonstrating that

Nck directly binds BCR via its SH2 domain, while its SH3 domains bind BCAP [200]. Thus, Nck is required for proper formation of the BCR-BCAP-PI3K complex. Consistently, B cells derived from Nck-deficient mice display impaired Akt activation and antibody production in response to

BCR-activating stimuli [200].

T cells, another type of lymphocyte, are activated through the T cell receptor (TCR)-CD3 complex, consisting of a TCRα/TCRβ heterodimer, a CD3δ/CD3ε heterodimer, a CD3γ/CD3ε heterodimer, and a CD3ζ/CD3ζ homodimer. TCR activation involves a conformational change induced by ligand binding, resulting in Nck1 binding to a proline-rich sequence in the cytoplasmic tail of CD3ε via its SH3.1 domain [153, 201]. This interaction is supported by the subsequent binding of the Nck1 SH2 domain to a phosphotyrosine residue on the CD3ε tail, resulting in full

TCR activation [201]. Consistently, preventing the Nck1-CD3ε interaction by expressing Nck1 mutated in its SH3.1 domain impairs T cell activation, as indicated by decreased Akt and extracellular signal-regulated kinase (ERK) phosphorylation [201]. Moreover, depletion of Nck1, but not Nck2, in human T cells reduces TCR-induced ERK phosphorylation and interleukin expression, demonstrating a non-redundant function of the two Nck isoforms [202].

Several regulatory mechanisms exist to prevent immune responses against self-antigens.

Autoimmunity occurs following the failure of such mechanisms, and is generally treated through the use of immunosuppressants, some of which aim to attenuate TCR activation and signaling.

Therapies targeting the Nck-CD3ε have shown potential in their ability to decrease T cell

36 activation in the context of autoimmune disease. Treatment with an antibody that binds the proline- rich sequence of CD3ε and blocks Nck1 binding results in diminished T cell proliferation in response to TCR activation [153]. Moreover, treatment of T cells with AX-024, a compound that binds the CD3ε motifs responsible for binding the Nck1 SH3.1 domain, inhibits canonical TCR signaling and T cell proliferation [203]. Furthermore, oral administration of AX-024 protects mice against autoimmune diseases such as psoriasis and multiple sclerosis while not inducing general immunosuppression in response to pathogen infection [203]. Of note, this study demonstrates the pharmacological potential of targeting the Nck interactome to counteract pathological conditions.

Ironically, Nck has also been implicated in the infection processes of pathogens such as enteropathogenic Escherichia coli (EPEC) and the vaccinia virus. EPEC adheres to intestinal enterocyte membranes, allowing for insertion of Tir and subsequent clustering [154]. Tir clustering induces tyrosine phosphorylation on its cytosolic tail and recruitment of Nck1 or Nck2, triggering localized actin assembly via recruitment of WASP and Arp2/3 [204, 205]. Similarly, the vaccinia virus membrane protein A36R is phosphorylated following intracellular replication, leading to the recruitment of Nck, WASP, and Arp2/3, and resulting in the formation of motile plasma membrane projections [178].

1.3.6.3 Renal Physiology

The nephron is the basic functional unit of the kidney involved in blood filtration. At the beginning of the nephron, a network of capillaries known as the glomerulus makes contact with the Bowman’s capsule, forming the glomerular filtration barrier. The Bowman’s capsule contains cells called podocytes, which extend actin-based foot processes that wrap around the glomerular capillaries, leaving junctions between them known as slit diaphragms. The primary molecular

37 component of the slit diaphragm is nephrin, a transmembrane protein that serves both as a structural component and as part of a signaling complex regulating cytoskeletal dynamics [206].

During slit diaphragm formation, engagement of the nephrin ectodomain activates the Src family kinase Fyn, resulting in tyrosine phosphorylation of the cytoplasmic domain of nephrin [161, 207].

Nck binding through its SH2 domain to nephrin at three phosphotyrosine residues has been reported to lead to actin polymerization [160, 161]. Moreover, when recruited to nephrin, the

SH3.1 and SH3.3 domains of Nck interact with Fyn, resulting in increased phosphorylation of nephrin on tyrosine residues [208]. Thus, the nephrin-Nck complex acts as a link between slit diaphragm dynamics and cytoskeletal reorganization in podocytes (Figure 1.9). Consistently, podocyte-specific deletion of Nck leads to decreased nephrin phosphorylation, abnormal foot process morphology, and proteinuria [209].

Figure 1.9: Role of Nck in Nephrin Signaling in Podocytes At the podocyte slit diaphragm, Fyn phosphorylation of nephrin leads to Nck recruitment through its SH2 domain. The SH3 domains of Nck transduce signals to the actin cytoskeleton while also recruiting Fyn, creating a positive feedback loop. Figure reproduced from [208].

1.3.6.4 Insulin Signaling

Insulin receptor signaling is regulated, in part, by IR dephosphorylation by protein tyrosine phosphatase 1B (PTP1B) [210]. Nck was first implicated in insulin signaling when it was demonstrated that upon insulin stimulation, the SH2 domain of Nck directly binds IRS-1 phosphorylated on Y147 [152]. Through its SH3 domains, Nck interacts with PTP1B, recruiting it to the IR/IRS-1 complex [211]. Thus, Nck attenuates insulin signaling by recruiting PTP1B to its substrate, IR, resulting in its dephosphorylation and decreased signaling. Consistently, Nck1 depletion in hepatocytes in vitro enhances PI3K-dependent Akt activation [212].

Nck also appears to negatively regulate IR signaling through a unique second mechanism.

Hyperactivation of IRE1α has been shown to impair insulin signaling through phosphorylation of

IRS-1 by JNK on Ser307 [213]. Given that Nck1 positively regulates IRE1α-mediated JNK phosphorylation [194], it was postulated that Nck1 depletion could ameliorate insulin signaling by reducing IRS-1 phosphorylation on Ser307. Indeed, livers from obese Nck1-/- mice display reduced

IRS-1 phosphorylation on Ser307 compared to their wild type counterparts, and consequently display enhanced Akt activation when placed on a high fat diet [194]. Consistently, obese Nck1-/- mice display enhanced insulin sensitivity compared to their wild type littermates and clear glucose more efficiently [194].

1.3.6.5 Adipogenesis

Adipocytes are the main cellular constituents of adipose tissue and are involved in important metabolic processes including energy storage in the form of triglycerides and hormone synthesis and secretion. Our group has identified Nck2 as a negative regulator of adipogenesis, the process by which adipocytes mature [170, 214]. Indeed, Nck2 depletion promotes the expression

39 of peroxisome proliferator-activated receptor-γ (PPARγ), a major transcription factor involved in this process, while cells overexpressing Nck2 display impaired PPARγ nuclear translocation [170,

214]. Consistently, Nck2-/- mice exhibit larger epidydimal and subcutaneous white adipose tissue depots, as well as greater circulating levels of adipokines than their wild type counterparts [170].

Interestingly, an inverse correlation between BMI and Nck2 levels in white adipose tissue was detected in obese human subjects, supporting the notion that Nck2 negatively regulates adipogenesis in vivo in humans.

1.3.6.6 β Cell Physiology

Given that PERK activity is crucial for β cell physiology (1.2.2) and that Nck1 is a negative regulator of PERK activity (1.3.5.3), it is plausible that Nck1 deficiency may improve β cell homeostasis. Indeed, our group has demonstrated that Nck1 deficiency improves insulin biogenesis and is protective against diabetes-relevant stresses [78, 183].

Major findings related to the role of Nck1 in β cells were elucidated through the study of

MIN6 cells stably expressing shRNA targeting Nck1 [78]. Compared to control cells, shNck1 cells express more proinsulin and display more insulin secretory granules [78, 183]. Consistently, pancreatic islets from Nck1-/- mice contain more insulin than islets from wild type mice [78]. In addition to promoting insulin biogenesis, Nck1 depletion also appears to ameliorate β cell survival in response to various diabetogenic stresses. Specifically, shNck1 cells display lower levels of cleaved caspase-3 than control cells in response to palmitate treatment, indicative of reduced apoptosis [183]. Moreover, shNck1 cells exhibit increased viability compared to control cells when treated with cytotoxic levels of palmitate, glucose, or H2O2 [183], supporting the notion that Nck1 depletion is beneficial for β cells.

Mechanistically, multiple pathways downstream of PERK are involved in ameliorating β cell function in shNck1 cells. Nck1 depletion correlates with increased levels of ATF4 and Nrf2 in the nucleus, as well as increased expression of antioxidant target genes [183]. Moreover, Nck1 depletion results in increased levels of sestrin2, which acts through AMPK and mTORC1 to enhance autophagy and consequently promote cell survival (Figure 1.10) [183].

ER stress

Silencing Nck1 PERK

P Nck1 P P P P P  Sestrin2

P  pAMPK P P eIF2 eIF2 Nrf2   Translation ~Translation  mTORC1  PI3K/Akt

ATF4  Autophagy P Translation and ATF4  P transcription Nrf2  Translation and transcription ATF4 Nrf2  Survival

Nucleus

GST Sestrin2 GST CHOP Antioxidant Genes Others Antioxidant Genes Others

Adaptive Response Cell Death/Apoptosis

Figure 1.10: PERK Signaling in β Cells In the absence of Nck1 or under acute ER stress conditions, slight PERK hyperactivity initiates an adaptive response that contributes to improved β cell function and survival, including enhanced autophagy and increased expression of antioxidant genes. Under chronic ER stress conditions, PERK is highly activated, resulting in β cell death. Figure reproduced with permission from [183].

1.4 Rationale and Objectives

Recent evidence by our group has demonstrated that Nck1 depletion in β cells promotes slight basal activation of the PERK arm of the UPR, which in turn enhances insulin biogenesis and confers resistance to diabetes-related stresses [78, 183]. Building off these findings, we hypothesize that preventing the interaction between Nck1 and PERK will mimic the effects of

Nck1 depletion. To achieve this, we sought to synthesize a cell-permeable peptide that has the ability to bind and sequester Nck. To satisfy the requirement of cell permeability, we will use the protein transduction domain (PTD) from the HIV protein transactivator of transcription (TAT)

[215, 216]. The amino acid sequence of the PTD, YGRKKRRQRRR, contains a high ratio of positively charged residues, allowing it to interact favorably with the exterior cell membrane, resulting in endocytosis [217]. To create the Nck-sequestering component of the peptide, we will use the amino acid sequence derived from mouse PERK that is involved in binding Nck1, including the phosphorylated Tyr561 residue and flanking amino acids. Our final synthetic peptide, named

TAT-pY561, consists of these two peptides connected together through a flexible GG linker (Figure

1.11). The objectives of the current study are to confirm the ability of TAT-pY561 to bind Nck and prevent the Nck/PERK interaction in vitro and to determine the therapeutic potential of this peptide by studying its ability to promote PERK activation in β cells, enhance insulin biosynthesis, and confer resistance to diabetes-related stresses.

PERK P 2 P Nck1 Y G R K K R R Q R R R G G T E S K Y D S V S A D TAT Protein Nck-Binding Sequence Transduction Domain (mPERK 557-567)

Figure 1.11: TAT-pY561 Amino Acid Sequence The synthetic phosphopeptide TAT-pY561 consists of the HIV TAT protein transduction domain fused to the amino acid sequence derived from mouse PERK that is involved in binding Nck1.

Chapter 2: Materials & Methods

2.1 Cell Culture & Treatments

Mouse insulinoma (MIN6) cells were cultured in high glucose Dulbecco’s Modified Eagle

Medium (DMEM; Gibco), supplemented with 15% fetal bovine serum (FBS; Gibco), 71.5 μM β- mercaptoethanol (Sigma), and antibiotic-antimycotic (Gibco). Rat insulinoma (INS-1 832/13) cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium (Gibco), supplemented with 10% FBS, 71.5 μM β-mercaptoethanol, 10mM HEPES (BioShop), 1mM sodium pyruvate (Sigma), and antibiotic-antimycotic. All cells were maintained at 37°C in a 5%

CO2 environment.

2.1.1 Palmitate Preparation

Sodium palmitate (Sigma) was dissolved in warm 50% ethanol to prepare a 150 mM stock solution, which was then conjugated to free fatty acid (FFA)-free bovine serum albumin (BSA) under overnight agitation at 37°C, achieving a final concentration of 15 mM and a final FFA:BSA molar ratio of 5:1. The FFA-free BSA solution was prepared in Krebs-Ringer Buffer (135 mM NaCl,

5 mM KCl, 1 mM MgSO4, 0.4 mM K2HPO4, 20 mM HEPES pH 7.4, 1 mM CaCl2).

2.1.2 Synthetic Peptides

The Nck-sequestering peptide TAT-pY561 was designed and synthesized based on the minimal sequence of PERK that is involved in binding Nck1, conjugated to the PTD of the cell-penetrating

TAT peptide. Similar peptides, unphosphorylated (TAT-Y) or harboring a Y561F mutation (TAT-

F), were used as negative controls. For differing experimental purposes, a peptide tagged with the fluorophore fluorescein isothiocyanate (FITC-TAT-pY561) and biotinylated peptides (Biotin-TAT- pY561 and Biotin-TAT-F) were also synthesized (Figure 2.1). All peptides were synthesized by

Bio Basic Canada Inc. Stock solutions (5mM) of each peptide were prepared in phosphate-buffered saline (PBS), and were stored short-term at 4°C and long-term at -80°C.

T ENK YDS V SGE Human (561-571) T E S K YDS V S AD Mouse (557-567) PERK T E S K YDS V S AD Rat (553-563)

P TAT 561 PTD T E S K YDS V S AD TAT-pY

TAT PTD T E S K YDS V S AD TAT-Y

TAT PTD T E S K F DS V S AD TAT-F Synthetic P TAT 561 F PTD T E S K YDS V S AD FITC-TAT-pY Peptides P TAT 561 B PTD T E S K YDS V S AD Biotin-TAT-pY

TAT B PTD T E S K F DS V S AD Biotin-TAT-F

Figure 2.1: TAT Peptides Schematic representation of the amino acid sequences of (upper) the PERK juxtamembrane domain, centered on the tyrosine residue involved in binding Nck, and (lower) synthetic peptides containing the conserved sequence of PERK conjugated to the PTD of TAT.

2.2 Cell Lysis

Unless otherwise indicated, cells were lysed in PLC lysis buffer (50 mM HEPES, 150 mM NaCl,

10% glycerol, 1% Triton® X-100 (Sigma), 1 mM EGTA, 1.5 mM MgCl2, 10 mM Na2P4O7,

100 mM NaF), supplemented before use with 10 μg/μl aprotinin, 10 μg/μl leupeptin, 1 mM PMSF, and 100 μM Na3VO4.

2.3 Western Blotting & Antibodies

Proteins were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-

PAGE) and transferred onto polyvinylidene difluoride (PVDF) membrane. Membranes were then blocked in 5% milk or BSA in tris-buffered saline containing 0.01% Tween® 20 and subsequently

45 immunoblotted with indicated antibodies. Low molecular weight synthetic peptides were fixed by incubating PVDF membranes in 1% KOH immediately following transfer [218]. Polyclonal Nck antibody, recognizing both protein isoforms, was generated as previously described [219].

Polyclonal phosphospecific PERK Y561 antibody, which also recognizes TAT-pY561, was generated as previously described [78]. HRP-conjugated streptavidin, which recognizes Biotin-

TAT-pY561, was purchased from Thermo Fisher Scientific. Caspase-3 antibody, which recognizes both full-length and cleaved forms of the protein, was purchased from Cell Signaling Technology.

Total PERK (C33E10) and phosphospecific PERK T980 (16F8) antibodies were purchased from

Cell Signaling Technology. GST (B-14) antibody was purchased from Santa Cruz Biotechnology.

Tubulin antibody was purchased from Sigma-Aldrich.

2.4 Recombinant Protein Preparation

Recombinant glutathione S-transferase (GST) fusion proteins were purified from bacteria and immobilized on glutathione beads. Recombinant Nck1 was obtained by thrombin cleavage of

GST-Nck1. Following separation by SDS-PAGE, the expression of all recombinant proteins was verified by Coomassie Brilliant Blue staining.

2.5 In vitro Pull-Down Assays

Pull-downs were performed in freshly prepared binding buffer (10% glycerol, 0.5% Triton® X-

100, 100 mM NaCl, 20 mM Tris pH 7.5, 0.5 mM EDTA, 1 mM DTT, 10 μg/μl aprotinin, 10 μg/μl leupeptin, 1 mM PMSF, and 100 μM Na3VO4). In all cases, beads were washed in binding buffer, and proteins were recovered in Laemmli buffer and analyzed by SDS-PAGE.

2.5.1 GST Protein Pull-Down Assays

For TAT-pY561 pull-downs, GST proteins (5 μg) were incubated with TAT-pY561 (100 nM –

10 μM) for 3 hours at 4°C. For pull-downs of recombinant Nck1, GST-PERK (50 ng) was incubated with recombinant Nck1 (20 ng) for 3 hours at 4°C in the presence or absence of synthetic peptide (TAT-pY561, TAT-F, TAT-Y). For pull-downs of cell lysate, GST-PERK (5 μg) was incubated with MIN6 cell lysate (100 μg) prepared in binding buffer for 3 hours at 4°C in the presence or absence of synthetic peptide (TAT-pY561, TAT-F, TAT-Y).

2.5.2 Streptavidin Pull-Down Assays

Biotin-TAT-pY561 (1 μM) was preincubated with high capacity streptavidin agarose (Thermo

Scientific) or streptavidin magnetic beads (GenScript) for 1 hour at 4°C and then incubated with cell lysate (100 μg) for 2 hours at 4°C in the presence or absence of synthetic peptide (TAT-pY561,

TAT-F, TAT-Y).

2.6 Confocal Microscopy

Cells were seeded on glass-bottom plates and treated with several concentrations of FITC-TAT- pY561. The next day, cells were fixed in 4% formaldehyde solution in PBS for 15 minutes at room temperature. Cells were then washed and stained with 4',6-diamidino-2-phenylindole (DAPI), a fluorescent stain that binds strongly to DNA, for 3 minutes at room temperature. Image acquisition was performed using the confocal laser scanning microscope LSM-510 Meta (Zeiss).

2.7 Flow Cytometry

Following treatments, cell culture media containing detached cells was collected in flow cytometry tubes, and adherent cells were trypsinized, harvested, and combined with their respective culture media. Cells were stained with FITC-annexin V and 7-aminoactinomycin D (7-AAD; BioLegend) as per the manufacturer’s instructions and subsequently analyzed by flow cytometry analysis using the FACSCanto™ II system (BD Biosciences).

2.8 Statistical Analysis

Data are presented as the mean ± standard error of the mean (SEM). Statistical significance was determined as indicated in each figure (GraphPad Prism).

Chapter 3: Results

3.1 TAT-pY561 Binds Nck

To determine if TAT-pY561 binds Nck, we performed in vitro pull-down assays using GST fusion proteins. We tested the ability of GST alone, GST-Nck1, and GST-Nck2 to bind TAT-pY561. GST alone proved unable to bind, whereas both GST-Nck1 and GST-Nck2 successfully bound the peptide (Figure 3.1A). In order to better understand the molecular basis of this interaction, we performed pull-down assays using constructs of GST-Nck1 and GST-Nck2 containing point mutations in their respective SH2 domains known to abrogate their binding activity [220]. These mutants proved unable to bind TAT-pY561 (Figure 3.1A), suggesting that both isoforms of Nck interact with the peptide through their SH2 domains. To confirm this interaction using an alternative experimental approach, Biotin-TAT-pY561 was bound to streptavidin-coupled beads, and the peptide’s ability to bind lysate-derived Nck was evaluated using pull-down assays and immunoblot. Nck derived from both mouse and rat cell lysates was successfully pulled down by

Biotin-TAT-pY561 (Figure 3.1B), while the unbiotinylated TAT-pY561 successfully competed with its biotinylated counterpart for Nck binding (Figure 3.2A). Of note, the same peptide containing a phenylalanine residue rather than a phosphotyrosine residue (Biotin-TAT-F) was unable to pull down detectable levels of Nck, demonstrating the importance of the phosphotyrosine residue in the interaction between TAT-pY561 and Nck (Figure 3.3). Consistently, the unphosphorylated

TAT-Y and mutant TAT-F peptides competed with Biotin-TAT-pY561 for Nck binding with a much lower affinity than TAT-pY561 (Figure 3.2B-D).

Nck2 Nck2 Nck1 Nck2 Nck1

- - - -

Pull-Down

GST GST GST GST GST SH2M SH2M TAT-pY561 (10 μM) + + + + +

WB: TAT-pY561

70 WB: GST

25 B

MIN6 Lysate - + - INS-1 832/13 Lysate - - + Biotin-TAT-pY561 (1 μM) + + + WB: Nck Streptavidin WB: Streptavidin Pull-Down

WB: Nck Lysate

Figure 3.1: TAT-pY561 binds Nck (A) Recombinant GST proteins were incubated with TAT-pY561 in vitro. Bound proteins were resolved by SDS-PAGE and analyzed by immunoblot. Binding of TAT-pY561 to GST proteins was determined using an antibody against PERK pY561 (SH2M: SH2 domain mutant; Nck1 R308K; Nck2 R312K). Amount of GST protein used in each pull-down was revealed by immunoblot using a GST antibody. Expected molecular weight: GST: 26kDa; GST-Nck: 66kDa. (B) MIN6 or INS- 1 832/13 cell lysates were incubated with Biotin-TAT-pY561 immobilized on streptavidin beads. Bead-bound proteins and total cell lysate proteins were resolved by SDS-PAGE and analyzed by immunoblot.

A Streptavidin Pull-Down MIN6 Lysate + - + + + + + Biotin-TAT-pY561 (1 μM) - + + + + + + TAT-pY561 (M) - - - 10-7 10-6 10-5 10-4 WB: Nck

WB: Streptavidin

B D Streptavidin Pull-Down 2 .0 Y MIN6 Lysate + + + + + F

561 k

Biotin-TAT-pY (1 μM) + + + + + c 1 .5 p Y

N /

-7 -6 -5 -4 1

TAT-Y (M) - 10 10 10 10 6

i p

WB: Nck -

d 1 .0

- n

WB: Streptavidin i

t o

i 0 .5 B C Streptavidin Pull-Down 0 .0 MIN6 Lysate + + + + + 0 -7 -6 -5 -4 -3 L o g [P e p tid e ] (M ) Biotin-TAT-pY561 (1 μM) + + + + + TAT-F (M) - 10-7 10-6 10-5 10-4 WB: Nck

WB: Streptavidin

Figure 3.2: Y/F peptides compete for Nck binding with lower affinity than TAT-pY561 MIN6 cell lysates were incubated with Biotin-TAT-pY561 immobilized on streptavidin beads in the presence of increasing concentrations of (A) TAT-pY561, (B) TAT-Y, or (C) TAT-F. Bound proteins were resolved by SDS-PAGE and analyzed by immunoblot. (D) Quantification of Nck bound to Biotin-TAT-pY561 (n = 3-4). Data represent the mean ± SEM.

Unbound Streptavidin Lysate Pull-Down

WB: Nck

WB: Streptavidin

Figure 3.3: TAT-F does not bind Nck MIN6 cell lysates were incubated with Biotin-TAT-pY561 or Biotin-TAT-F immobilized on streptavidin beads. Bead-bound and unbound proteins were analyzed by immunoblot.

3.1.1 Relative Specificity of TAT-pY561 Towards Nck

The SH2 domain is a structurally conserved domain found within many proteins involved in signal transduction [41], through which they bind other proteins containing phosphotyrosine residues such as the one found in the TAT-pY561 peptide. Thus, we wanted to confirm that the peptide shows specificity towards the SH2 domain of Nck1 compared to that of any other SH2 domain- containing protein. To test this, we performed pull down assays, incubating various GST fusion proteins with TAT-pY561. These proteins included full-length SH2-domain containing proteins

(Nck1, Grb2), as well as a sample of lone SH2 domains (Nck1, Shc, PI3K, RasGAP, PLCγ). We confirmed that TAT-pY561 binds Nck1 while it fails to bind Grb2, and that the peptide demonstrates specificity towards the Nck1 SH2 domain compared to any of the other SH2 domains tested

(Figure 3.4A). Despite identifying low levels of binding between TAT-pY561 and both Shc and

RasGAP SH2 domains in this assay, these proteins were not detected in Biotin-TAT-pY561 pull- downs of MIN6 lysate (Figure 3.4B). Taken together, these results suggest that TAT-pY561 exhibits a relatively high specificity for binding the SH2 domain of Nck1.

A Ras Ras Pull-down Nck1 Shc PI3K GAP GAP PLCγ PLCγ (GST Fusion Protein) Nck1 Grb2 SH2 SH2 SH2N SH2N SH2C SH2N SH2C TAT-pY561 (10-7 M) + + + + + + + + +

WB: TAT-pY561

55 WB: GST

B Streptavidin Pull-Down 0 0.1 0.1 0.1 1.0 1.0 1.0 Biotin-TAT-pY561 (μg)

1.0 0.1 0.5 1.0 0.1 0.5 1.0 MIN6 Lysate (mg) Lysate WB: Nck

WB: Streptavidin

WB: RasGAP

WB: Shc

Figure 3.4: TAT-pY561 specifically binds the Nck1 SH2 domain (A) Recombinant GST proteins (full-length or lone SH2 domains) were incubated with TAT-pY561 in vitro. Bound proteins were resolved by SDS-PAGE and analyzed by immunoblot. Binding of TAT-pY561 to GST proteins was determined using an antibody against PERK pY561. Amount of GST protein used in each pull-down was revealed by immunoblot using a GST antibody. (B) MIN6 cell lysates were incubated with Biotin-TAT-pY561 immobilized on streptavidin beads. Bound proteins were resolved by SDS-PAGE and analyzed by immunoblot with indicated antibodies.

3.2 TAT-pY561 Abrogates the Nck1/PERK Interaction

Given that TAT-pY561 binds the SH2 domain of Nck1 in a similar manner to PERK, we next wanted to determine if it could prevent Nck1 from interacting with PERK. To do so, we performed multiple pull down assays, incubating GST-PERK with MIN6 lysate and increasing concentrations of TAT-pY561, essentially allowing GST-PERK and TAT-pY561 to compete for lysate-derived Nck binding. Consistent with our hypothesis, we found that with an increase in concentration of TAT- pY561, there was a decrease in Nck binding to GST-PERK in a dose-dependent manner (Figure

3.5A-B). This result was also observed when GST-PERK was incubated with purified recombinant

Nck1, where the interaction was also abolished with increasing concentrations of TAT-pY561

(Figure 3.5C-D). These results support our hypothesis that in addition to binding Nck1, the peptide competes with PERK for Nck1 binding.

A B 1 .5

GST-PERK Pull-Down

i d

-8 -7 -6 -5 -4 561 n 1 .0 Lysate

0 10 10 10 10 10  TAT-pY (M) i

K R

WB: Nck E

P 0 .5

c N WB: GST (PERK) 0 .0 0 -7 -6 -5 -4 -3

5 6 1 L o g [T A T -p Y ] (M )

C D 1 .5

GST-PERK Pull-Down

n i

0 5·10-9 10-8 5·10-8 10-7 5·10-7 10-6  TAT-pY561 (M) d

n 1 .0

WB: Nck K

R E

P 0 .5

c N

0 .0 0 -8 -7 -6 -5

5 6 1 L o g [T A T -p Y ] (M )

Figure 3.5: TAT-pY561 prevents the Nck1/PERK interaction (A) MIN6 cell lysate or (C) purified Nck1 was incubated with GST-PERK in the presence of increasing concentrations of TAT-pY561. Bound proteins were resolved by SDS-PAGE and analyzed by immunoblot using an Nck antibody. Quantification of MIN6 lysate-derived Nck (B) or purified Nck1 (D) bound to GST-PERK. Data represent the mean ± SEM.

3.2.1 Unphosphorylated and Mutant Peptides

Next, we performed similar pull-down assays to determine the ability of control peptides to abrogate the GST-PERK/Nck interaction. Both TAT-Y and TAT-F, which we previously demonstrated bind Nck with a lower affinity than TAT-pY561, were able to compete with GST-

PERK for Nck binding, however they did so with lower efficiency than TAT-pY561 at the same concentration (Figure 3.6).

A GST-PERK Pull-Down GST-PERK + + + + MIN6 Lysate + + + + TAT Peptide (10-5 M) - pY Y F

WB: Nck

WB: GST (PERK)

B GST-PERK Pull-Down GST-PERK + + + + + Purified Nck1 - + + + + TAT Peptide (10-7 M) - - pY Y F WB: Nck

WB: GST (PERK)

Figure 3.6: Y/F peptides compete with PERK for Nck binding with lower affinity than TAT- pY561 (A) MIN6 cell lysate or (B) purified Nck1 were incubated with GST-PERK in the presence of identical concentrations of TAT-pY561, TAT-Y, or TAT-F. Bead-bound proteins were resolved by SDS-PAGE and analyzed by immunoblot using an Nck antibody. Presence of GST-PERK was confirmed using a GST antibody.

3.3 FITC-TAT-pY561 Enters MIN6 Cells

Prior to studying the biochemical characteristics of TAT-pY561 in cells, we sought to confirm that the peptide penetrates the β cell membrane. To test this, we treated MIN6 cells with increasing concentrations of the fluorescently labeled FITC-TAT-pY561 and observed increasing peptide entry by confocal microscopy (Figure 3.7). Furthermore, we stained the nuclei of these cells with

DAPI and noted that the peptide was exclusively localized to the cytoplasm. Nck1 has been demonstrated to bind PERK on the cytoplasmic side of the ER membrane; thus, the common localization of Nck1 and TAT-pY561 to the cytoplasm supports their potential to interact in vivo.

FITC-TAT-pY561 0 μM 10 μM 20 μM 50 μM

FITC-TAT-pY561

DNA

Merge

Figure 3.7: FITC-TAT-pY561 enters MIN6 cells MIN6 cells treated with increasing concentrations of FITC-TAT-pY561 for 24 hours were stained with DAPI and then visualized using confocal microscopy.

3.4 TAT-pY561 Promotes Basal PERK Activation

Previously, our group demonstrated that stable depletion of Nck1 in MIN6 cells promotes basal

PERK activation and signaling [78, 183]. Thus, we hypothesized that Nck sequestration upon treatment with TAT-pY561 would have a similar effect. Indeed, 4-day treatment of MIN6 cells with

10 μM TAT-pY561 resulted in significantly elevated basal PERK activation as indicated by increased phosphorylation of PERK on its activation site Thr980 (Figure 3.8A). Interestingly, increased basal PERK activity was also detected in rat INS-1 823/12 cells similarly treated with

TAT-pY561, as indicated by increased phosphorylation of PERK on Thr974 (Figure 3.8B).

A TAT-pY561 (10 μM) - - - + + + 1 .5 *

980 n i

pPERK (T ) l

u 1 .0

PERK R 0 .5

P p

Tubulin 0 .0 5 6 1 - T A T -p Y MIN6 cells B TAT-pY561 (10 μM) - - - + + + 2 .5 170 * pPERK (T974)

n 2 .0 i

130 l

u b

u 1 .5

/ PERK

K 1 .0

E P

p 0 .5 Tubulin 0 .0 5 6 1 - T A T -p Y INS-1 832/13 cells

Figure 3.8: TAT-pY561 promotes basal PERK activation Lysates from (A) MIN6 or (B) INS-1 832/13 cells cultured in the absence or presence of 10 μM TAT-pY561 for 4 days were subjected to immunoblotting with indicated antibodies. Bar charts represent the mean ± SEM of 3 independent experiments. Statistical significance was evaluated using the one sample t test (*p < 0.05).

3.5 TAT-pY561 Protects Against Palmitate-Induced Apoptosis

PERK activation associated with stable Nck1 depletion has been shown to protect MIN6 cells against palmitate-induced apoptosis [183], a process characterized by a caspase cascade which includes the cleavage of caspase-3. Nck sequestration via pre-treatment of MIN6 cells with TAT- pY561 prior to palmitate treatment mimicked the effect of Nck1 depletion, resulting in decreased levels of caspase-3 cleavage (Figure 3.9). This protective effect was also seen in INS-1 832/13 cells, where TAT-pY561 pretreatment protected against caspase-3 cleavage induced by cotreatment of palmitate and glucose (Figure 3.10). Moreover, analysis by flow cytometry revealed a lower proportion of late apoptotic and dead cells following cotreatment of palmitate and glucose in INS-1

832/13 cells pretreated with TAT-pY561 compared to untreated cells stressed under the same conditions (Figure 3.11).

TAT-pY561 (10 μM) - - - - + + + +

PA (1mM) - + + + - + + +

Casp3 (FL)

Casp3 (Clv)

Tubulin

TAT-pY561

MIN6 cells

1 .5 1 .5

)

u F

/ 1 .0 1 .0

)

C *

(

p 3

s 0 .5 0 .5

a C 0 .0 0 .0 5 6 1 5 6 1 - T A T -p Y - T A T -p Y

Figure 3.9: TAT-pY561 protects MIN6 cells against palmitate-induced caspase-3 cleavage MIN6 cells were cultured in the absence or presence of 10 μM TAT-pY561 for 3 days and treated for an additional 24 hours ± 1 mM palmitate. Lysates were subjected to immunoblotting with indicated antibodies (FL: full-length; Clv: cleaved). Bar charts represent the mean ± SEM of 3 independent experiments. Statistical significance was evaluated using the one sample t test (*p < 0.05).

TAT-pY561 (10 μM) - - - - + + +

PA (1 mM) + Glu (25 mM) - + + + + + +

Casp3 (FL)

Casp3 (Clv)

Tubulin

TAT-pY561

INS-1 832/13 cells

1 .5 1 .5

)

F u

/ 1 .0 1 .0

) C

( **

* v

(

p 3

s 0 .5 0 .5

a C 0 .0 0 .0 5 6 1 5 6 1 - T A T -p Y - T A T -p Y

Figure 3.10: TAT-pY561 protects INS-1 832/13 cells against palmitate/glucose-induced caspase-3 cleavage INS-1 832/13 cells were cultured in the absence or presence of 10 μM TAT-pY561 for 3 days and treated for an additional 24 hours ± 1 mM palmitate and 25 mM glucose. Lysates were subjected to immunoblotting with indicated antibodies (FL: full-length; Clv: cleaved). Bar charts represent the mean ± SEM of 4 independent experiments. Statistical significance was evaluated using the one sample t test (*p < 0.05, **p < 0.01).

A B 1 5 0 Control PA PA/Glucose C o n tro l 5 6 1

- T A T -p Y D

5 0,055% 0,94% 5 0,93% 13,0% 5 9,80% 46,6% A

10 10 10 A

- 1 0 0

4 4 4 n i 10 10 10 % ***

x 5 0

n A

3 3 3 0 10 10 10 - P A P A + G

0 0 0 C 6 0

+ ****

3 3 3 D

-10 98,4% 0,61% -10 80,2% 5,84% -10 17,9% 25,7%

A A

- 4 0

3 3 4 5 3 3 4 5 3 3 4 5 7

-10 0 10 10 10 -10 0 10 10 10 -10 0 10 10 10 /

n %

561 561 561 i AAD TAT-pY TAT-pY + PA TAT-pY + PA/Glucose

x 2 0

n 7 5 0,029% 0,79% 5 0,41% 11,2% 5 4,08% 33,5% n 10 10 10 A 0 - P A P A + G 4 4 4 10 10 10

D 1 5

+ D

3 3 3 A

10 10 10 A ****

- 1 0

c V

0 0 0

% i

x 5 3 3 3 e -10 98,1% 1,10% -10 82,0% 6,34% -10 41,2% 21,2% n

n * A 3 3 4 5 3 3 4 5 3 3 4 5 -10 0 10 10 10 -10 0 10 10 10 -10 0 10 10 10 0 - P A P A + G Annexin V

Figure 3.11: TAT-pY561 protects INS-1 832/13 cells against palmitate/glucose-induced apoptosis (A) Representative flow cytometry plots of INS-1 832/13 cells cultured in the absence or presence of 10 μM TAT-pY561 for 3 days and treated for an additional 24 hours ± 2 mM palmitate (PA) and 25 mM glucose (G). Apoptosis was analyzed by FITC-Annexin V and 7-AAD staining. The proportion of live (B) [Annexin V- / 7-AAD-], late apoptotic (C) [Annexin V+ / 7-AAD+], and dead (D) [Annexin V- / 7-AAD+] cells were quantified. Bar charts represent the mean ± SEM of triplicate measurements. Statistical significance was evaluated by two-way ANOVA with Bonferroni’s multiple comparisons test (*p < 0.05, ***p < 0.001, ****p < 0.0001). The effect of TAT-pY561 on early apoptotic cell proportion (Annexin V+ / 7-AAD-) was not statistically significant in either direction in any conditions.

Chapter 4: Discussion

4.1 Summary of Findings

Previously, our group identified Nck1 as a negative regulator of PERK signaling and demonstrated that Nck1 deficiency promotes PERK activation in various cell lines [78, 185, 188].

Given the importance of PERK in maintaining β cell homeostasis (1.2.2), we previously hypothesized that Nck1 deficiency may improve β cell function and survival. Indeed, Yamani et al. demonstrated that stable depletion of Nck1 in MIN6 cells leads to enhanced insulin biogenesis and increased resistance to diabetes-related stresses [78, 183]. Based on these findings, we proposed that Nck sequestration using the synthetic cell-permeable peptide TAT-pY561 could reproduce the beneficial effects of Nck1 depletion. The objective of the current study was therefore to characterize this peptide in its ability to bind Nck, prevent the Nck/PERK interaction, promote basal PERK activation in β cells, enhance insulin biosynthesis, and confer resistance to diabetes- related stresses.

4.1.1 Biochemical Characterization of TAT-pY561

We previously demonstrated that both Nck1 and Nck2 directly interact with PERK in an

SH2 domain-dependent manner [78]. Consistently, TAT-pY561, containing the minimal sequence of PERK involved in binding Nck, also directly binds both Nck1 and Nck2 (Figure 3.1A).

Moreover, we demonstrated that Biotin-TAT-pY561 binds Nck from both mouse and rat β cell lysates (Figure 3.1B). Interestingly, the unbiotinylated TAT-pY561 peptide competed with its biotinylated counterpart for Nck binding, while the unphosphorylated TAT-Y and mutant TAT-F peptides competed with a much lower affinity (Figure 3.2). These data suggest that the phosphorylated tyrosine residue is necessary for high affinity binding between TAT-pY561 and

Nck in vitro. The fact that TAT-Y and TAT-F, at high concentrations, compete for Nck binding

65 suggests that the amino acids flanking the tyrosine residue also contribute to the stability of the interaction. Moreover, these data provide insight into the molecular basis of the interaction between Nck and PERK, supporting our previous finding that the phosphorylation of the Tyr561 residue of PERK is crucial for its interaction with Nck1 [78], while also supporting the classical notion that the amino acids surrounding the phosphotyrosine residue contribute to SH2 domain binding [157, 158].

Based on the findings that TAT-pY561 interacts with Nck, we proposed that by binding to its SH2 domain, TAT-pY561 would neutralize Nck, thus preventing it from interacting with PERK.

Indeed, we found that TAT-pY561 competed with PERK for lysate-derived Nck binding in a dose- dependent manner (Figure 3.5), while TAT-Y and TAT-F did so with a lower efficiency at the same concentration (Figure 3.6). These observations further support our aforementioned statement that amino acids other than the phosphotyrosine (pY561) residue contribute to the efficacy of the peptide. Based on this conclusion, it would be interesting to examine an array of peptides of varying lengths to determine the optimal and necessary sequence of PERK required to sequester

Nck. Furthermore, peptides containing single mutations in amino acids surrounding Tyr561 could also be of interest to determine which of these residues are relevant for Nck-specific interactions.

4.1.2 TAT-pY561 Promotes PERK Activation

Consistent with our original hypothesis, Nck sequestration by TAT-pY561 enhances basal

PERK activation in β cells (Figure 3.8). It is well established that PERK activity is crucial for proper β cell physiology (1.2.2). Indeed, we have previously demonstrated that slight basal PERK activation induced by Nck1 depletion correlates with enhanced β cell function and survival [78,

183]. In a similar vein, others have demonstrated that β cells with a more active UPR are more

66 likely to proliferate [221]. On the other hand, it is well established that hyperactivation of the UPR, such as in the case of diabetes, is detrimental for β cell function and survival (1.2.2.5). It is therefore important to note that the increased basal PERK activity seen in Nck1-depleted cells and cells treated with TAT-pY561 is subtle in comparison to the intensity of activation associated with the proapoptotic UPR. Accordingly, we propose a model wherein increased basal PERK activity initiates a signaling network that extends the window in which PERK activation contributes to the adaptive UPR rather than the apoptotic UPR, thus rendering cells more resistant to ER stress

(Figure 4.1).

A Mild UPR Activation  Chronic UPR Activation  Cytoprotective Mechanisms Cell Death Response to ER stress in β cells

Unfolded Protein Response

B Mild UPR Activation  Chronic UPR Activation  Extended adaptive Cytoprotective Mechanisms Cell Death response in β cells depleted of Nck1 or treated with TAT-pY561 Unfolded Protein Response

Figure 4.1: Modulation of PERK Activation and the UPR (A) In pancreatic β cells, mild or transient activation of the UPR induces cytoprotective mechanisms; in contrast, stress resulting in intense or chronic UPR activation results in apoptosis. (B) Cells depleted of Nck1 or treated with TAT-pY561 have an extended window in which PERK activation induces an adaptive response rather than an apoptotic response, thus rendering these cells more proficient in dealing with physiological and pathological stress.

4.1.2.1 Role of Nck2

Our knowledge on the role of Nck in β cells comes from studies involving shNck1 MIN6 cells or Nck1-/- mice [78, 183]. Interestingly, we previously reported that Nck2 binds PERK and negatively regulates PERK activation in the context of adipocyte differentiation [78, 170].

However, the role of Nck2 in the regulation of PERK in β cells has not yet been addressed.

Although Nck1 and Nck2 share a high degree of identity and interacting partners, these adaptor proteins also display exclusive binding partners and expression profiles. Therefore, it is possible that in β cells, they could act non-redundantly in limiting PERK activation and signaling. It remains to be determined whether TAT-pY561, which binds both Nck1 and Nck2, is acting exclusively by alleviating the negative regulation exerted on PERK by Nck1.

4.1.3 TAT-pY561 Treatment Confers Resistance Against Glucolipotoxicity

The most physiologically relevant finding of the current study is the ability of TAT-pY561 to protect against apoptosis. Consistent with our previous findings in shNck1 MIN6 cells, we found that pretreating MIN6 cells with TAT-pY561 protected them against palmitate-induced caspase-3 cleavage, a marker of apoptosis (Figure 3.9). In a similar vein, we reported that pretreatment of

INS-1 832/13 cells with TAT-pY561 conferred protection against caspase-3 cleavage induced by glucolipotoxicity (Figure 3.10). Finally, a lower proportion of cells stressed with palmitate and glucose were found to be late apoptotic or dead following treatment with TAT-pY561 (Figure

3.11). While our approach aims to protect β cells against ER stress-induced apoptosis, others have previously used TAT-conjugated peptides to protect against apoptosis induced by oxidative stress in different systems [222, 223]. Given that we previously reported that Nck1 depletion improves cell viability in response to H2O2 treatment [183], it would be interesting to determine if TAT-

68 pY561 pretreatment would have the same effect as depleting Nck1 on β cell survival in response to oxidative stress. Moreover, it could be interesting to design and test a combination therapy of the aforementioned peptides along with TAT-pY561 with the goal of concurrently protecting pancreatic

β cells against multiple types of diabetes-relevant stresses.

4.1.3.1 PERK-Independent Effects of Nck Sequestration

We previously demonstrated that increased basal PERK activity induced by stable Nck1 depletion correlates with ameliorated β cell physiology [78, 183]. In the current study, we support these findings by demonstrating that TAT-pY561 treatment induces a similar increase in basal

PERK activity that correlates with an increased resistance against glucolipotoxicity. In both studies, we attributed effects of Nck depletion or sequestration to an increase in basal PERK activity; however, in doing so we exclude any other cellular processes involving Nck-mediated signaling. Apart from the aforementioned studies, no other role of Nck in β cells has been reported.

However, it is possible that by neutralizing Nck using TAT-pY561, we may confer protection against apoptosis in a manner independent of PERK activity. In order to address this issue, we could characterize β cells engineered to express a PERK mutant that fails to interact with Nck, an approach that will be discussed in an upcoming section.

4.2 Translational Implications

Protein-protein interactions (PPIs) are involved in nearly all cellular processes and as such are considered prime targets for therapeutics. Specifically, targeting PPIs involving Nck has shown promise in multiple disease models. In several pathological conditions, oxidative stress stimulates proinflammatory gene expression through a signaling mechanism involving Nck coupling the

69 tyrosine phosphorylation of PECAM-1 to the activation of PAK [162]. Consistently, neutralizing

Nck using a synthetic peptide containing the Nck-binding sequence of PAK suppresses oxidative stress-induced proinflammatory gene expression in vitro, while treatment of mice subjected to ischemia/reperfusion injury with this peptide leads to reduced leukocyte-endothelial interactions and blunted vascular permeability [162]. In another disease model, targeting the interaction between Nck1 and CD3ε results in diminished TCR signaling and protects mice against autoimmune diseases (1.3.6.2) [153, 203]. In accordance with our current work, these studies support the notion that targeting PPIs involving Nck is a valuable approach in the treatment of diseases.

In our current study, we provide evidence that sequestering Nck using TAT-pY561 confers protection to β cells against ER stress, a relevant condition in multiple pathological contexts.

Human islets from both T1D and T2D patients are characterized by aberrant activation of the UPR, resulting in β cell apoptosis (1.2.2.5) [135, 138, 139]. In this context, it is plausible that treatment with TAT-pY561 could protect β cells against ER stress-induced apoptosis, thus delaying the onset of diabetes. Moreover, ER stress-induced apoptosis is prevalent in pancreatic islets following islet transplantation, a key limitation to the success of the therapy meant to treat T1D [224]. We hypothesize that incubation of pancreatic islets with TAT-pY561 will confer protection against ER stress-induced apoptosis, thus increasing the yield of surviving islets following transplantation.

4.3 Future Perspectives

Further investigation is required to determine whether treatment with TAT-pY561 could enhance insulin biosynthesis and glucose-stimulated insulin secretion in β cells. To test this, we will treat insulin-secreting β cells with TAT-pY561 for four days and measure insulin levels in

70 culture media following glucose stimulation as well as insulin content within the cells. It would also be of great interest to treat isolated human islets from healthy, T1D, and T2D donors ex vivo with TAT-pY561 to compare the effectiveness of the peptide on improving β cell function under different pathological conditions and upon transplantation.

Others have investigated the potential of delivering cell permeable peptides to pancreatic islets in vivo. Delivery through the pancreatic duct results in successful transduction of membrane permeable and biologically active peptides into islet cells, including those located in the inner core region [225]. This technique could be used to administer TAT-pY561 to mouse islets to monitor the ability of this peptide to improve insulin biosynthesis. Moreover, it would be interesting to assess the ability of the peptide to delay the onset of diabetes in Akita mice, a model wherein β cell- specific ER stress results in apoptosis [70, 71].

In general, the therapeutic use of peptide compounds is limited by their low metabolic stability in vivo [226]. Therefore, it would be of interest to develop a small molecule compound that has a greater half-life than TAT-pY561 and could also bind the SH2 domain of Nck with an equal or greater affinity. To do so, we could develop peptidomimetics by making alterations to the

TAT-pY561 peptide sequence; for example, we could substitute proteinogenic amino acids with nonproteinogenic ones to ameliorate the proteolytic stability of the compound [226]. Moreover, with a greater understanding of the structural basis of the interaction between TAT-pY561 and Nck, we could synthesize a conformationally restricted analog of TAT-pY561 that maintains the secondary structure required for binding Nck, thus increasing the affinity of the interaction [227].

4.3.1 Targeting the Nck/PERK Interaction Using CRISPR/Cas9

In line with the considerations discussed earlier (4.1.3.1), studying the effects of TAT- pY561 does not definitively address the function of the interaction between Nck and PERK, as

TAT-pY561 also blocks the SH2 domain of Nck from binding partners other than PERK. To specifically abrogate the Nck/PERK interaction, we will edit the endogenous Perk gene in mouse

β cells using CRISPR/Cas9, creating a PERK Y561F mutant through homologous repair using a donor template identical to the Perk gene except for a single nucleotide substitution that encodes a phenylalanine residue rather than tyrosine. In this context, PERK and Nck will be unable to interact, while all other PERK- or Nck-mediated interactions will be preserved. Analysis of PERK activity, insulin biosynthesis, and survival to stress in these cells will provide further evidence that targeting the interaction between Nck and PERK is a therapeutically viable strategy to improve β cell physiology.

4.3.2 Studying the Mechanistic Basis of PERK Regulation by Nck1

In this study, we targeted the Nck/PERK interaction using TAT-pY561 with the goal of alleviating PERK of its negative regulation by Nck1. However, the mechanism by which Nck1 limits PERK activation is not understood. Given that Perk-/- MEFs expressing PERK Y561F, a mutant unable to bind Nck1, display increased basal and ER stress-induced PERK activity, we postulated that Nck1 protects the phosphorylation of this inhibitory site in wild type cells, thus maintaining PERK in a less active state [78]. However, it is not known whether the negative charge imparted by phosphorylation of the Tyr561 residue and/or the recruitment of Nck1 is the mechanism by which Nck1 limits PERK activation. In order to address this issue, the CRISPR/Cas9 genome editing system could be used to create cells in which endogenous PERK is replaced by a Y561E

72 mutant. This variant will be unable to bind Nck but will maintain a negative charge at position 561 due to the presence of a glutamic acid residue, thus acting as a phosphomimetic mutant. If PERK

Y561E behaves like wild type PERK, then we could deduce that the charge imparted on Y561 by phosphorylation limits PERK activation, and that the only role of Nck in this context is to protect the inhibitory phosphosite. On the other hand, if PERK Y561E exhibits increased basal activity compared to wild type PERK, as is hypothesized for PERK Y561F, then we could deduce that the absence of Nck binding is the cause of this alleviated negative regulation. If this is the case, the next logical step would be to determine the mechanism by which Nck binding results in reduced

PERK activity. This may be attributed to a conformational change in PERK induced by Nck binding, or by Nck physically preventing PERK dimerization. On the other hand, in this context,

Nck may be acting as a canonical adaptor protein, recruiting a kinase or phosphatase through its

SH3 domains to contribute to PERK regulation. Overall, understanding the mechanistic basis of the interaction between Nck and PERK will allow us to better develop strategies to target this interaction with the goal of enhancing basal PERK activity and improving β cell function.

4.4 Concluding Remarks

In this thesis, we characterized the synthetic peptide TAT-pY561, demonstrating its ability to sequester Nck and phenocopy Nck1 depletion in pancreatic β cells using a pharmacologically- relevant strategy. Overall, studies reported in this thesis demonstrate the therapeutic relevance of targeting the interaction between Nck and PERK to improve β cell function and survival.

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