A Dissertation entitled Regulation of the Mitotic Checkpoint by Wenbin Ji Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biology _________________________________________ Dr. Song-Tao Liu, Committee Chair _________________________________________ Dr. Tomer Avidor-Reiss, Committee Member _________________________________________ Dr. Rafael Garcia-Mata, Committee Member _________________________________________ Dr. Donald Ronning, Committee Member _________________________________________ Dr. William Taylor, Committee Member _________________________________________ Dr. Kam Yeung, Committee Member _________________________________________ Dr. Amanda Bryant-Friedrich, Dean College of Graduate Studies The University of Toledo May 2017 Copyright 2017, Wenbin Ji This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Regulation of the Mitotic Checkpoint by Wenbin Ji Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biology The University of Toledo May 2017 The mitotic checkpoint is an evolutionarily conserved mechanism that preserves genomic integrity. The mitotic checkpoint can be viewed as a special signal transduction pathway. The signal amplification step for the mitotic checkpoint signaling involves the mitotic checkpoint protein MAD2 converting from open (O-MAD2) to closed (C-MAD2) conformation (MAD2 O-C conversion). In current models, the MAD2 O-C conversion is catalyzed by an unusual catalyst, the complex formed by MAD1 and C-MAD2 (the MAD1:C-MAD2 complex). Previously it was known that MAD1(485-584) (a MAD1 fragment spanning 485-584 residues that contains MAD2 Interacting Motif, or MIM) is responsible for forming the complex with C-MAD2. MAD1(597-718) (MAD1 C-terminal domain, CTD) also seemed essential for the mitotic checkpoint. Despite these findings, how MAD2 O-C conversion is catalyzed and regulated remains largely unclear. We found that deleting MAD1(1-485) (MAD1 N-terminal domain, NTD) or MAD1(597-718) (MAD1-CTD) resulted in a defective mitotic checkpoint, suggesting that both MAD1-NTD and MAD1-CTD are required for efficient catalysis of MAD2 O-C conversion. We further demonstrated that MAD1-NTD and MAD1-CTD bind to both O- iii MAD2 and C-MAD2, as expected for an enzyme to associate with its substrates and products. Moreover, our data showed that MAD1-NTD directly interacts with MAD1- CTD, suggesting possible coordination between different MAD1 domains. Additionally, we discovered that MPS1 kinase, which promotes MAD2 O-C conversion during mitosis, regulates the MAD1:C-MAD2 complex activity through directly phosphorylating both MAD1-NTD and MAD1-CTD. Our data revealed that MPS1 kinase reduced the interaction between MAD1-NTD and MAD1-CTD. Phosphorylation of MAD1-CTD by MPS1 seems required for a functional mitotic checkpoint. The newly characterized protein-protein interactions and their modulation by MPS1 kinase may indicate a delicately controlled regulatory mechanism for the catalytic activity of the MAD1:C-MAD2 complex. Our work has led to an updated mechanistic model to understand how the mitotic checkpoint signal is amplified by the unusual catalyst---the MAD1:C-MAD2 complex. In addition to MPS1 kinase, we also characterized other kinases that might regulate the mitotic checkpoint including Aurora B, BUB1, MELK and Abl. The results provided clues for better understanding the regulation of the mitotic checkpoint signal transduction pathway. We also investigated how a pseudokinase BUBR1 contributes to the mitotic checkpoint. We found that BUBR1(487-700) directly interacts with C-MAD2 and p31comet, which is a MAD2-binding protein and negative regulator of the mitotic checkpoint. Our study on BUBR1 will fill the knowledge gap on how the mitotic checkpoint complex (MCC), the effector of the mitotic checkpoint, assembles and functions. iv In summary, our work has demonstrated the functions of currently little appreciated MAD1 domains in the mitotic checkpoint and advanced the mechanistic understanding about how different MAD1 domains coordinate to catalyze MAD2 O-C conversion. In addition, our work provided insights into how mitotic kinases regulate the MAD2 O-C conversion and other aspects of the mitotic checkpoint in order to maintain the high fidelity of chromosome segregation. v Acknowledgements I would like to express my sincere appreciation and gratitude to my advisor, Dr. Song-Tao Liu, for giving me the opportunity to start this Ph.D. journey in his lab and for his patient guidance and mentorship through every step of acquiring this degree. I also gratefully appreciate my committee members for providing valuable advice and insight into my project. Gratitude also goes to my friends and lab mates, especially Dr. Michael Bekier and Dr. Kexi Wang for teaching me many lab skills and discussing the research, Brianne Sturt-Gillespie for her technical support and input, Dr. Yibo Luo and Dr. Ejaz Ahmad for the collaboration and support for the projects, Dr. Michael Moenk, Christopher Arnst and Hang Zhang for their help and companion in the lab. I would like to thank my family and friends, especially my parents and my wife for their constant love and understanding. Without their unconditional support and encouragement, this dissertation would not have been possible. vi Table of Contents Abstract .............................................................................................................................. iii Acknowledgements ............................................................................................................ vi Table of Contents .............................................................................................................. vii List of Tables ................................................................................................................... xii List of Figures .................................................................................................................. xiii List of Abbreviations ....................................................................................................... xvi List of Symbols ................................................................................................................ xix 1 Introduction .................................................................................................................. 1 1.1 Mitosis is one important stage of the cell cycle .................................................. 2 1.2 Regulations of the cell cycle ............................................................................... 3 1.3 The mitotic checkpoint ....................................................................................... 5 1.4 Molecular components of the mitotic checkpoint ............................................... 7 1.5 The effector of the mitotic checkpoint-the mitotic checkpoint complex (MCC) 9 1.6 The anaphase promoting complex/cyclosome (APC/C) ................................... 15 1.7 The mitotic checkpoint signal transduction pathway--signal initiation ............ 16 1.8 Mitotic checkpoint signal transduction pathway--signal amplification ............ 18 1.8.1 The MAD1:C-MAD2 complex catalyzes MAD2 O-C conversion .............. 19 1.8.2 The regulation of the MAD1:C-MAD2 complex ......................................... 21 vii 1.9 The mitotic checkpoint signal transduction pathway--effector formation ........ 24 1.10 The mitotic checkpoint signal transduction pathway--silencing ...................... 25 1.11 Questions to be addressed ................................................................................. 28 2 Hypothesis ................................................................................................................. 29 3 Materials and Methods............................................................................................... 31 3.1 Cell culture, synchronization and drug treatment ............................................. 31 3.2 DNA Constructs and Transfection .................................................................... 32 3.3 Recombinant protein expression ....................................................................... 33 3.4 Cell lysates, immunoblotting, immunoprecipitation and GST pulldown ......... 34 3.5 In vitro kinase assays ........................................................................................ 35 3.6 In vitro binding assays ...................................................................................... 35 3.7 Immunofluorescence and Live Cell Imaging .................................................... 36 3.8 APC/C activity assay using concentrated mitotic extracts ............................... 36 3.9 Statistics Analysis ............................................................................................. 37 4 Results ........................................................................................................................ 38 4.1 MAD1-NTD, MIM and CTD coordinate to catalyze MAD2 O-C conversion. 38 4.1.1 Both MAD1-NTD and CTD are required for maintaining a functional mitotic checkpoint ................................................................................................................. 38 4.1.2 MAD1-NTD and CTD can interact with both O-MAD2 and C-MAD2 ...... 44 4.1.3 The integrity of MAD1-NTD and