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DISSECTING the EXTRACELLULAR MECHANISMS of NODAL and ACTIVIN SIGNALING REGULATION by Senem Aykul a DISSERTATION Submitted To

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DISSECTING the EXTRACELLULAR MECHANISMS of NODAL and ACTIVIN SIGNALING REGULATION by Senem Aykul a DISSERTATION Submitted To DISSECTING THE EXTRACELLULAR MECHANISMS OF NODAL AND ACTIVIN SIGNALING REGULATION By Senem Aykul A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Biochemistry and Molecular Biology – Doctor of Philosophy 2017 ABSTRACT DISSECTING THE EXTRACELLULAR MECHANISMS OF NODAL AND ACTIVIN SIGNALING REGULATION By Senem Aykul Transforming Growth Factor-β (TGF-β) family ligands are key regulators of multiple cellular processes including cell proliferation, differentiation, and death. Dysregulation of TGF-β family signaling thus contributes to many human diseases, including cancers, fibrosis, and musculoskeletal disorders. Because of its roles in human diseases, understanding the mechanism and regulation of TGF-β family signal transduction is essential and will help the development of new therapeutic agents that could be used to target the family for treating a number of devastating diseases. The basic mechanisms of TGF-β family action are well established. A dimeric ligand binds two ‘type I’ and two ‘type II’ receptors to form a hexameric complex. Assembly of the ligand-receptor complex in the extracellular space leads to phosphorylation of SMAD transcription factors in the intracellular space, their translocation to the nucleus, and expression of target genes. However, beyond ligands and receptors, many additional factors contribute critically to physiological TGF-β family signaling, including membrane-bound co- receptors, secreted inhibitors, and other ligands that are present on or near the surface of a cell. Elucidating the molecular interplay of all the components that form the TGF-β signal transduction system of a particular cell type or tissue is essential for understanding TGF-β signaling in a cellular context. The goal of my thesis was to investigate the extracellular regulation mechanisms of the TGF-β family ligands, Nodal and Activin using biophysical approaches and to define the physiological consequences using cell-based approaches. In the first chapter of my dissertation, I introduced the TGF-β family and described how I expressed and purified TGF-β family ligands, receptors, and extracellular regulators. I also introduced a powerful technique called Surface Plasmon Resonance (SPR) that I used to determine the binding modes between TGF-β family members. In the second chapter of my dissertation, I elucidated the molecular function of TGF-β family signaling co-receptor Cripto-1 and its homolog. I demonstrated that soluble Cripto-1 inhibits ligand induced signaling, but the membrane-anchored form potentiates the signaling, suggesting an extracellular capture and endocytic trafficking mechanism. In the third chapter of my dissertation, I characterized the function of the TGF-β family ligand Nodal and its natural inhibitor Cerberus in breast cancer cell lines. I found that Cerberus significantly suppresses migration, invasion, and colony formation of Nodal expressing breast cancer cells. In the fourth chapter of my dissertation, I defined the function of human Cerberus in TGF-β family signaling. I showed that full-length and short forms of human Cerberus share TGF-β family ligand binding and inhibition mechanism, but only full-length Cerberus suppresses MDA- MB-231 breast cancer cell migration. In the fifth chapter of my dissertation, I evaluated how the population of different ligands affects signaling outcomes. I demonstrated that high affinity TGF-β family ligands compete with low affinity ligands for receptor binding and antagonize low affinity ligand signaling. In the sixth chapter of my dissertation, I discussed the possible future directions for this project. Collectively, the results of these experiments provide new information about TGF-β family signaling and regulation, and potentially lead to evaluation of new therapeutics targeting family members in different diseases. ACKNOWLEDGEMENTS First and foremost, I would like to thank my supervisor, Dr. Erik Martinez Hackert for his guidance, encouragement, and patience throughout the six years of my PhD studies. He guided me with his great ideas and expertise, but he also taught me how to approach a scientific problem, interpret and criticize the results. His working ethics, open-mindedness, intelligence, scientific vision and passion for science have always motivated me. I am very fortunate to have an amazing advisor like him. I also want to thank all previous and current members of the Dr. Martinez lab. Especially, I would like to thank Dr. Washington Mutatu. He taught me all the basic techniques when I first joined the lab, but he also has been a really good friend supporting me all the time. Special thanks to all undergraduate assistants worked in the lab, specifically Wendi Ni, Wongsirikul Will, Jake Reske, Elisa Chaparro, Sabeth Dalbo, Alice Chu and Melissa Meschkewitz. I would like to thank my committee members, Dr. Leslie Kuhn, Dr. Min Hao Kuo, Dr. David Weliky and Dr. Shelagh Ferguson-Miller. Each of them was available to share their expertise and made me think about my research in a new and deeper way. Finally, my most heartfelt appreciation goes to my mom and dad, and the rest of my family. Their presence, support, love, and encouragement are invaluably important for me. I owe them everything I have. I also would like to thank Asim Addemir and Dilek Addemir for considering me as a member of their family and supporting me all the time since I came to the USA. iv TABLE OF CONTENTS LIST OF TABLES …………………………………………………………………………. viii LIST OF FIGURES …………………………………………………………………………. ix KEY TO ABBREVIATIONS …………………………………………………………… xi CHAPTER 1 - INTRODUCTION ……………………………………………………….. 1 Discovery of the TGF-β family ……………………………………………………….......2 TGF-β family ligands and receptors ……………………………………………………... 3 Extracellular regulation of TGF-β family signaling …………………………................... 7 Why is it important to study TGF-β family signaling? ………………………………….. 11 A general approach for expression of TGF-β family ligands and receptors …………….. 12 A general approach to examine the interactions between TGF-β family signaling components …………………………………………………………………… 16 REFERENCES ……………………………………………………………………………... 20 CHAPTER 2 – BIOCHEMICAL AND CELLLULAR ANALYSIS REVEAL LIGAND BINDING SPECIFICITIES, A MOLECULAR BASIS FOR LIGAND RECOGNITION, AND MEMBRANE ASSOCIATION DEPENDENT ACTIVITIES OF CRIPTO-1 AND CRYPTIC ……………………………………………...31 Abstract ……………………………………………………………………………….. 32 Introduction …………………………………………………………………………. 32 Materials and Methods ………………………………………………………………. 34 TGF-β family ligands ……………………………………………………………….. 34 Expression plasmids ………………………………………………………………… 35 Protein purification …………………………………………………………………. 35 Cell lines ……………………………………………………………………………..35 Surface plasmon resonance …………………………………………………………. 36 Cross-linking ………………………………………………………………………... 37 Reporter assays ……………………………………………………………………... 37 Immunoblotting …………………………………………………………………….. 39 Statistics …………………………………………………………………………….. 39 Results ……………………………………………………………………………………………… 39 Production of soluble Cripto-1 and Cryptic ………………………………………… 39 Cripto-1 and Cryptic bind distinct ligands ………………………………………….. 42 All Cripto-1 domains are required for ligand binding …………………………….... 45 Cripto-1 glycosylation is necessary for ligand binding ……………………………...46 Soluble Cripto-1 does not bind type I receptors strongly …………………………... 47 Cripto-1 and Cryptic block ligand association with type II and some type I receptors ……………………………………………………………….. 51 Soluble Cripto-1 and Cryptic inhibit signaling ……………………………………... 55 Membrane associated Cripto-1 potentiates BMP-4 signaling ………………………. 57 v Discussion ………………………………………………………………………………..60 REFERENCES ……………………………………………………………………………… 65 CHAPTER 3 –HUMAN CERBERUS PREVENTS NODAL-RECEPTOR BINDING, INHIBITS NODAL SIGNALING, AND SUPPRESSES NODAL-MEDIATED BREAST CANCER PHENOTYPES ……………………………… 73 Abstract ……………………………………………………………………………….. 74 Introduction …………………………………………………………………………. 74 Materials and Methods ………………………………………………………………. 78 Materials …………………………………………………………………………….. 78 Construction of expression plasmids …………………………………………………… 78 Protein purification ………………………………………………………………….. 78 Cell lines ……………………………………………………………………………..79 Immunoblotting ……………………………………………………………………... 79 Surface plasmon resonance …………………………………………………………. 81 Migration assay ……………………………………………………………………... 82 Invasion assay ………………………………………………………………………. 82 Soft agar colony formation assay ………………………………………………….... 83 Reporter assay …………………………………………………………………………… 83 Statistics …………………………………………………………………………………. 83 Results ………………………………………………………………………………….... 84 Nodal binds ALK4, BMPRII and Cripto-1 …………………………………………. 84 Human Cerberus binds Nodal with high affinity …………………………………… 87 Human Cerberus prevents Nodal interactions and inhibits Nodal signaling ……….. 90 Nodal is expressed in invasive breast cancer cell lines and induces invasion ……… 93 Cerberus suppresses aggressive phenotypes of Nodal expressing breast cancer cells …………………………………………………………………... 95 Discussion ……………………………………………………………………………… 100 REFERENCES ………………………………………………………………………………. 105 CHAPTER 4 – NEW LIGAND BINDING FUNCTION OF HUMAN CERBERUS AND ROLE OF PROTEOLYTIC PROCESSING IN REGULATING LIGAND RECEPTOR INTERACTIONS AND ANATGONISTIC ACTIVITY …………112 Abstract ……………………………………………………………………………… 113 Introduction ………………………………………………………………………... 114 Materials and Methods ……………………………………………………………...116 Materials …………………………………………………………………………… 116 Construction of expression plasmids ………………………………………………….. 116 Protein purification
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