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Characterization of Novel Post-Translational Modifications of Microtubule Associated Proteins and Immunologically Related Modifications of Dna Topoisomerase Ii

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Characterization of Novel Post-Translational Modifications of Microtubule Associated Proteins and Immunologically Related Modifications of Dna Topoisomerase Ii CHARACTERIZATION OF NOVEL POST-TRANSLATIONAL MODIFICATIONS OF MICROTUBULE ASSOCIATED PROTEINS AND IMMUNOLOGICALLY RELATED MODIFICATIONS OF DNA TOPOISOMERASE II DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Min Ding, M.S. The Ohio State University 1996 Dissertation Committee: Approved by Dale D. Vandre, Ph.D., Adviser Robert M. DePhilip, Ph.D. Adviser Amanda A. Simcox, Ph.D. Molecular, Cellular and Developmental Biology Kenneth H. Jones, Ph.D. Graduate Program UMI Number: 9639227 UMI Microform 9639227 Copyright 1996, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 ABSTRACT As a component of the cytoskeleton, microtubules (MTs) play indispensable roles during the cell cycle and neuronal development. MT dynamics and functions are regulated by a group of proteins called microtubule associated proteins (MAPs), which in turn are regulated by post-translational modifications. It has been established that MAPs are phosphorylated in a cell cycle dependent and/or developmentally regulated fashion. One phosphorylation site, recognized by the monoclonal antibody MPM-2, is found on both MAP4 in mitotic cells and MAP1B in differentiating PC12 cells. MPM-2 antibody also recognizes mitotic DNA topoisomerase II, an enzyme required during mitosis. This study documents work on post-translational modification of MAPs as well as the phosphorylation site shared by MAPs and topoisomerase II. It is shown here that MAPI, MAP2, and MAP4 are glycosylated. In particular, MAP2 and MAP4 are modified by a single O- linked N-Acetylglucosamine (O-GlcNAc), adding MAPs to the family of intracellular O-GlcNAc modified proteins. At least 10% of brain MAP2 is modified by O-GlcNAc. Multiple O-GlcNAc modification sites exist on MAP2, and these are all located in the MAP2 projection domain. The phosphorylation of MAPs was examined using a series of synthetic phosphopeptides based upon the MPM-2 phosphoepitope shared between certain MAPs and topoisomerase II. The most essential element ii of the MPM-2 epitope identified here consists of an aromatic amino acid -2 residues N-terminal to the phosphorylated threonine. An aromatic amino acid at the +2 position is also important for the antibody recognition. This sequence is indeed the in vivo MPM-2 epitope as substantiated by a rabbit polyclonal antibody generated against the topoisomerase II synthetic phosphopeptide. This affinity purified antibody, PTE1, recognized a band that co-migrates with DNA topoisomerase II on immunoblots of mitotic cells. Preincubation with the phosphopeptide, but not the dephosphopeptide, abolished PTETs ability to recognize this band. PTE1 also recognizes a distinctive group of proteins in mitotic HeLa cell lysates. Immunocytochemical localization has shown that PTE1 specifically stains mitotic chromosomes, spindle poles and midbodies. These results indicate that proteins recognized by the PTE1 antibody share phosphoepitopes recognized by the MPM-2 antibody, including those present on several MAPs. To My Parents Boqi Ding and Mindi Shen, Whose Unconditional Support Is the Foundation of All My Achievements and To My Wife Hong Lu, Whose Boundless Love Makes My Life Complete ACKNOWLEDGMENTS Looking back on the past twenty-nine years, joining Dr. Dale D. Vandre’s lab in the summer of 1993 was one of the wisest decisions I have ever made. Under his guidance, I have matured from a rookie in scientific research to a semi­ independent investigator. I am grateful for his confidence in me, and his willingness to let me explore new ideas and experiment with new approaches. I admire his style of scientific investigation and his philosophy of graduate training. What I have learned from him is invaluable now, and will be invaluable throughout my career. Dr. Vandre has been both a mentor and a friend, sharing with me his experience and insights not only in biological research but also in career development and personal life. His advice and support have helped make some of my personal aspirations come true. I also want to thank him for his patience and painstaking effort in correcting the linguistic errors in my manuscripts. Without his support, this dissertation would not be possible. I wish to thank Professors Robert M. DePhilip, Kenneth H. Jones, John M. Robinson, and Amanda A. Simcox for their constant interest in my research and valuable suggestions. I want to especially thank Professor Amanda A. Simcox, who had acted kindly as my temporary adviser during my first year at OSU. I wish to thank past and current graduate students in Dr. Vandre's lab, Dr. Yunhi Choi, Mrs. Yang Feng, Mrs. Christine Kondratick, and Mr. Colin Lowry, for their companionship during my endeavor here. Especially I want to thank Yunhi for the MAP4 samples she generously provided, and Yang for allowing me to steal her reagents from time to time. I enjoyed Christine’s sincere friendship, and my conversations with Colin that made some monotonous intervals between experiments interesting. I also want to thank my friends who helped to make Columbus our home away from home, Dr. Timothy Cain, Mr. Chong Fu, Mr. Yiping Jia, Mr. Qiang Tong, and Mr. Guoshan Tsen. Among the many memorable moments we have shared together are our passionate discussions of science and our five-thousand-mile cross-country trip, and everything in between. I am deeply indebted to my parents, Boqi Ding and Mindi Shen, for their unconditional love and tremendous sacrifice. They have always put their sons' needs before theirs, and have done everything in their power to make sure we grow up physically, intellectually, and morally sound. They have exemplified to us the meaning of love without reservation, pride in endeavors disregarding results, and perseverance before the most daunting obstacles. I am forever grateful for the solid foundation they molded inside me, upon which, I build everything I have and will have. I am also grateful for my grandfathers. One started the family tradition of love, self-sacrification, and respect for knowledge. The story of the other, whom I have never met, from an illiterate blacksmith’s son to a self-made factory manager vi more than half century ago, has always inspired me for the best. Finally, I want to thank my wife, Hong Lu, for your unwavering love and support. I thank you for taking care of most of our mundane chores in your busy schedule to allow me more time for my aspirations. You help me to unwind when the going gets tough, and to focus when my imagination deviated too far from the reality. I am particularly grateful for your understanding and/or tolerance of my many unconventional thoughts, hobbies, and aspirations. I am lucky to have you as my wife, and I am looking forward to share life’s ups and downs with you for the many years to come. VITA March 13, 1967 ......................... Born - Shanghai, China 1989 ........................................... Bachelor of Science, Genetics and Genetic Engineering, Fudan University, Shanghai, China 1989-1991 .................................. Assistant Engineer (Biotechnology), Jiangxi National Pharmaceutical Company, Nanchang, PRC 1991 -1992 ................................. Graduate Teaching Assistant, Department of Biology, Northeast Louisiana University, Monroe, Louisiana 1995........................................... Master of Science, Program in Molecular, Cellular and Developmental Biology, The Ohio State University Columbus, Ohio 1992-present .......................... Graduate Teaching and Research Associate Program in Molecular, Cellular and Developmental Biology, The Ohio State University Columbus, Ohio PUBLICATIONS 1. Ding, M., Robinson, J.M., Burry, R.B., and Vandre, D.D. Human Phagocytic Leukocytes Have an Extremely Dynamic Microtubule Array. (1993) Molecular Biology of the Cell 4, 451 a. [abstract] viii 2. Ding, M., and Vandre, D.D. Evidence For the Glycoprotein Nature of High Molecular Weight MAPs. (1994) Molecular Biology of the Cell 5,167a. [abstract] 3. Ding, M., Vandre, D.D., Behrens, B.C., and Robinson, J.M. The Microtubule Cytoskeleton in Human Leukocytes is a Highly Dynamic Structure. (1995) European Journal of Cell Biology 66, 234-245. 4. Ding, M. and Vandre, D.D. High Molecular Weight Microtubule Associated Proteins Contain O-linked N-Acetylglucosamine. (1996) Journal of Biological Chemistry 271(21), 12555-12561. FIELDS OF STUDY Major Field: Molecular, Cellular and Developmental Biology TABLE OF CONTENTS Page ABSTRACT............................................................................................................ ii DEDICATION........................................................................................................ iv ACKNOWLEDGMENTS..................................................................................... v VITA..................................................................................................................... viii LIST OF DIAGRAM.............................................................................................. xii LIST OF TABLES.............................................................................................. xiii LIST OF FIGURES............................................................................................. xiv LIST OF ABBREVIATIONS................................................................................ xvi
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