University of Nebraska Medical Center DigitalCommons@UNMC Theses & Dissertations Graduate Studies Spring 5-4-2019 The Role of Zyxin and LIMD1 in Mitosis and Cancer Jiuli Zhou University of Nebraska Medical Center Follow this and additional works at: https://digitalcommons.unmc.edu/etd Part of the Cancer Biology Commons, and the Cell Biology Commons Recommended Citation Zhou, Jiuli, "The Role of Zyxin and LIMD1 in Mitosis and Cancer" (2019). Theses & Dissertations. 353. https://digitalcommons.unmc.edu/etd/353 This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@UNMC. It has been accepted for inclusion in Theses & Dissertations by an authorized administrator of DigitalCommons@UNMC. For more information, please contact [email protected]. THE ROLE OF ZYXIN AND LIMD1 IN MITOSIS AND CANCER by Jiuli Zhou A DISSERTATION Presented to the Faculty of the University of Nebraska Graduate College in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Pathology & Microbiology Graduate Program Under the Supervision of Professor Jixin Dong University of Nebraska Medical Center Omaha, Nebraska May, 2019 Supervisory Committee: Jennifer D. Black, Ph.D. Robert E. Lewis, Ph.D. Kaihong Su, Ph.D. i ACKNOWLEDGEMENTS Funding support: The China Scholarship Council; University of Nebraska Medical Center (UNMC) Graduate Studies Office Fellowship; National Cancer Institute/National Institutes of Health (NCI/NIH); Department of Defense; the COBRE Grant from the Nebraska Center for Cell Signaling/National Institute of General Medical Sciences (NIGMS/NIH). More important than the funding support, I would like to acknowledge the support and encouragement I received during my doctoral research. First and foremost, I would like to express my deepest appreciation to my advisor Dr. Jixin Dong. The completion of my dissertation would not have been possible without the support and nurturing of him. He taught me that it takes a long journey to dig deep into this field, which is not only a challenge of intelligence, but also a challenge of perseverance. Under his guidance, I am now equipped with a solid foundation for both molecular biology techniques and cancer research insights. Besides, I have strengthened my ability to design experiments and write papers and proposals independently. By pursuing various projects, I know how to deal with obstacles properly and turn disappointments into lessons, discoveries, alternatives and new plans set in motion. Thanks to his encouragement and patience for years, I have prepared myself to be a skillful and thoughtful researcher and will achieve my dream step by step. Additionally, Dr. Dong has led our lab to be a happy family, where I feel love and warmness. I would also like to extend my deepest gratitude to my supervisor committee, Dr. Jennifer Black, Dr. Kaihong Su, and Dr. Robert Lewis. They have set up themselves as excellent models for my research career. I really appreciate the time they have taken out of their busy schedules to help me out. Their insightful suggestion and critical comments ii have given me valuable clues for my projects and greatly improved my scientific knowledge. Besides, I am also grateful that they provided me with encouragement throughout the duration of my project. I am deeply indebted to my previous and present lab members: Shuping Yang, Lin Zhang, Xingcheng Chen, Seth Stauffer, Yu Ou, Zhan Wang, Yuanhong Chen, Yongji Zeng, Renya Zeng, and Ling Yin. I want to thank them for providing technique solutions as well as scientific suggestions on my research projects, and generous support and help when I was struggling. Special thanks to Yuanhong not only for helping us in research needs and laboratory management, but also for her countless supports and cares in my daily life like my family. Many thanks to my program director, Dr. Rakesh Singh and my program coordinator, Tuire Cechin for their help with my study and supports when I faced problems. I wish to thank UNMC core facilities, including Comparative Medicine, Advanced Microscope Core Facility, Tissue Sciences Facilities, Flow Cytometry Research Facility and DNA Sequencing Core for their great amount of assistance with my projects. My heartfelt appreciation also goes to my families and friends near and far. I especially thank my parents who nurtured me with endless love, and encouraged me to be a brave, honest, confident, and hard-working person. I also wish to express my thanks to my best friend, soul-mate, and husband Yijiu Ren. I married the best person, who never let me down and always stood by my side. Four years ago, I built a dream. Now, the dream builds me. I am so grateful for those who support me along the way of my Ph.D. training. They are a reason to my greatness in life and I will never walk alone. iii ABSTRACT: THE ROLE OF ZYXIN AND LIMD1 IN MITOSIS AND CANCER Jiuli Zhou, Ph.D. University of Nebraska, 2019 Supervisor: Jixin Dong, Ph.D. The Hippo signaling pathway, originally discovered in Drosophila, consists of a core kinase cascade and has been subsequently demonstrated to control tissue growth and tumorigenesis. The core of this pathway contains MST1/2 (Mammalian sterile 20-like kinase 1/2), LATS1/2 (large tumor suppressor 1/2) and downstream effector named Yes- associated protein (YAP) and PDZ-binding motif (TAZ). MST1/2 transduce their kinase activity mainly through directly phosphorylating LATS1/2. Once phosphorylated and activated, LATS1/2 subsequently phosphorylate and inhibit YAP/TAZ from translocating to nucleus, thereby suppressing the expression of downstream pro-growth and survival genes. While recent studies provide important insight into the tumor suppressor properties of this pathway, the underlying molecular mechanisms through which the Hippo components exert their oncogenic/suppressing function are poorly understood. Zyxin and LIM domains containing 1 (LIMD1) are adaptor/scaffold proteins with three LIM domains, which are involved in protein-protein interactions during cellular signaling transduction. Several recent studies showed that Zyxin/LIMD1 affect Hippo-YAP signaling. Our studies found that Zyxin and LIMD1 are phosphorylated in mitosis via novel sites. We further characterized the phospho-regulation of Zyxin and LIMD1 in mitosis and examined the functional significance of the phosphorylation in colon cancer and lung cancer, respectively. Moreover, we demonstrated that Zyxin regulates YAP activity through colon cancer oncogene Cyclin-Dependent Kinase 8 (CDK8). Mechanistically, we identified that iv CDK8 directly phosphorylates YAP and promotes its activation. Together, our findings revealed novel layers of regulation for Zyxin/LIMD1 in mitosis and their roles in tumorigenesis. v TABLE OF CONTENT ACKNOWLEDGEMENTS ................................................................................................. i ABSTRACT: THE ROLE OF ZYXIN AND LIMD1 IN MITOSIS AND CANCER ............... iii LIST OF FIGURES ....................................................................................................... viii LIST OF ABBREVIATION ............................................................................................... x Chapter 1: Zyxin promotes colon cancer tumorigenesis in a mitotic phosphorylation- dependent manner and through CDK8-mediated YAP activation* ………………………..1 Abstract ................................................................................................................. 2 1.1. Introduction .................................................................................................. 3 1.2. Materials and Methods ................................................................................ 5 1.2.1. Expression constructs .................................................................................. 5 1.2.2. Cell culture and transfection ........................................................................ 5 1.2.3. Cell cycle synchronization ............................................................................ 6 1.2.4. Establishment of cell lines ............................................................................ 6 1.2.5. CRISPR-double nikase-mediated knockout ................................................. 6 1.2.6. Kinase inhibitors .......................................................................................... 7 1.2.7. Recombinant protein purification and in vitro kinase assay .......................... 7 1.2.8. Antibodies .................................................................................................... 8 1.2.9. Phos-tag and western blot assay ................................................................. 8 1.2.10. Immunofluorescence staining and cofocal mircoscopy ............................... 8 1.2.11. Cell proliferation, migration (wound healing) and colony formation assays . 9 1.2.12. Quantitative real time PCR ......................................................................... 9 1.2.13. Animal studies ............................................................................................ 9 1.2.14. Immunohistochemistry (IHC) staining and scoring ....................................10 vi 1.2.15. Statistical analysis .....................................................................................11 1.3. Results .......................................................................................................12 1.3.1. Zyxin is phosphorylated by CDK1 in vitro during mitosis .............................12