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Characterization of NUMB As a Regulator of Anaplastic Lymphoma Kinase

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Characterization of NUMB As a Regulator of Anaplastic Lymphoma Kinase Western University Scholarship@Western Electronic Thesis and Dissertation Repository 6-28-2016 12:00 AM Characterization of NUMB as a Regulator of Anaplastic Lymphoma Kinase Ran Wei The University of Western Ontario Supervisor Dr. Shawn Li The University of Western Ontario Graduate Program in Biochemistry A thesis submitted in partial fulfillment of the equirr ements for the degree in Doctor of Philosophy © Ran Wei 2016 Follow this and additional works at: https://ir.lib.uwo.ca/etd Part of the Biochemistry Commons, Cancer Biology Commons, Cell Biology Commons, Molecular Biology Commons, and the Systems Biology Commons Recommended Citation Wei, Ran, "Characterization of NUMB as a Regulator of Anaplastic Lymphoma Kinase" (2016). Electronic Thesis and Dissertation Repository. 3804. https://ir.lib.uwo.ca/etd/3804 This Dissertation/Thesis is brought to you for free and open access by Scholarship@Western. It has been accepted for inclusion in Electronic Thesis and Dissertation Repository by an authorized administrator of Scholarship@Western. For more information, please contact [email protected]. Abstract Cellular events rely on protein-protein interactions that are often mediated by modular domains which recognize particular sequence motifs in binding partners. The NUMB protein is the first described cell fate determinant and multifaceted adaptor that is involved in a wide variety of cellular events. NUMB mainly mediates protein interactions via its modular PTB domain. Here we present a systematic investigation of the NUMB-PTB interactome by employing an integrative strategy combining both protein and peptide arrays. We profiled NUMB-PTB binding specificity and interacting proteins genome-wide. The receptor tyrosine kinases (RTKs) are found highly enriched in the interactome, raising the possibility that NUMB may act as a universal binding partner of RTKs. To further validate this hypothesis, we focused on the interaction between NUMB and Anaplastic Lymphoma Kinase (ALK), which promotes oncogenesis in a number of cancer types. Consistent with the prediction based on our proteomic study, NUMB-PTB directly binds to two motifs in ALK in vitro and in vivo. Intriguingly, functional analysis reveals that NUMB-ALK interaction regulates ALK activity antagonistically in an isoform dependent manner, by directing ALK to distinct post-endocytic trafficking destinations. Our study provides mechanistic insight into ALK regulation, explains the controversial behaviors of NUMB in tumorigenesis at the molecular level, and further reveals a biomarker of potential clinical value. Keywords NUMB, PTB, interactome, binding motifs, isoform, receptor tyrosine kinase, ALK, endocytosis, post-endocytic trafficking, recycling, degradation, MAPK, proliferation, cancer ii Co-Authorship Statement All chapters of this thesis were written by Ran Wei and edited by Dr. Shawn Li and Ms. Courtney Voss. Most experimental results and analysis presented in this thesis were completed by Ran Wei. The data presented in Chapter 2 is in preparation for publication. The chip-based NUMB interactome screening was assisted by Dr. David Schibli, Dr. Chengang Wu. The bioinformatics data analysis was assisted by Dr. Lei Li and Dr. Huadong Liu. The structural analysis was assisted by Dr. Tomo Kaneko. The data presented in Chapter 3 is in preparation for publication. All confocal microscopy, image processing and quantification were assisted by Xuguang Liu. iii Acknowledgments First and foremost, I would like to express my sincere gratitude to my supervisor Dr. Shawn Li for providing me with this great opportunity to study at Western and work on fabulous research projects. In the past years, Dr. Li has provided me continuous support and helped me a lot in and outside the laboratory. Dr. Li’s expertise assisted me in establishing a firm foundation in research. I could not wish for a better supervisor for my graduate studies. I would like to thank my committee Dr. David Litchfield, Dr. Eric Ball and Dr. Lina Dagnino for their invaluable suggestions for developing my project. Many thanks go to Dr. Marc Vigny for his insightful comments and his generous permission to share a number of critical experimental materials. I also would like to thank Ms. Barb Green for her continuous administrative service. Thank you all to Li lab members. I would like to give my special thanks and appreciation to Ms. Courtney Voss, Dr. Huadong Liu, Dr. Tomonori Kaneko, Dr. Shelley Sandiford and Dr. Gurpreet Dhami. My thanks also go to Dr. Lei Li, Dr. Mei Huang, Dr. Rakesh Joshi, Dr. Kyle Biggar, Dr. Lyugao Qin, Dr. Xing Li, Xuguang Liu, Qi Fang, Eric Liu and Wayne Wu. Because of you, Li lab is such an adorable and enjoyable place to work and study. Finally, I would like to thank my parents Guoqiang Wei and Xiaoqin Zhou for their understanding and encouragement. iv Table of Contents Abstract ................................................................................................................................ i Keywords ............................................................................................................................ ii Co-Authorship Statement................................................................................................... iii Acknowledgments.............................................................................................................. iv Table of Contents ................................................................................................................ v List of Figures and Tables.................................................................................................. ix List of Abbreviations ......................................................................................................... xi Chapter 1 ............................................................................................................................. 1 1 General introduction ................................................................................................... 1 1.1 The multifaceted adaptor protein NUMB ........................................................... 1 1.1.1 NUMB structure and isoforms ........................................................................ 2 1.1.2 NUMB in cancer as a tumor suppressor ......................................................... 5 1.1.3 The controversial functions of NUMB in cancer .......................................... 11 1.2 Receptor tyrosine kinases in cancer .................................................................. 13 1.2.1 Tyrosine phosphorylation ............................................................................. 13 1.2.2 Tyrosine phosphorylation mediated cell signaling ....................................... 15 1.2.3 Endocytosis regulates RTK signaling ........................................................... 20 1.2.4 ALK-positive cancers and ALK target therapeutics ..................................... 23 1.3 Scope of thesis project ...................................................................................... 26 1.4 Reference .......................................................................................................... 28 Chapter 2 ........................................................................................................................... 39 2 Investigation of NUMB-PTB interactome ................................................................ 39 2.1 Abstract ............................................................................................................. 39 2.2 Introduction ....................................................................................................... 40 2.3 Material and methods ........................................................................................ 44 v 2.3.1 Cell culture .................................................................................................... 44 2.3.2 GST-NUMB-PTB expression, purification and biotinylation ...................... 44 2.3.3 ProtoArray hybridization .............................................................................. 45 2.3.4 Transient transfection.................................................................................... 45 2.3.5 Immunoprecipitation, pull down and western blotting ................................. 45 2.3.6 Free peptide and peptide array synthesis ...................................................... 46 2.3.7 Far western assay for peptide membrane blotting ........................................ 46 2.3.8 Determination of dissociation constant ......................................................... 47 2.3.9 Consensus binding motif and SMALI matrix ............................................... 47 2.4 Results ............................................................................................................... 49 2.4.1 Screening NUMB PTB domain binding proteins using protein array .......... 49 2.4.2 Characterizing NUMB-PTB domain binding sites in the interactome ......... 51 2.4.3 Defining NUMB-PTB domain binding specificity ....................................... 56 2.4.4 Structural explanation of NUMB-PTB binding specificity .......................... 57 2.4.5 Verification of NUMB RTK interactors in vivo ........................................... 60 2.4.6 Predicting a genome-wide NUMB interactome by SMALI ......................... 61 2.5 Discussion ......................................................................................................... 63 2.5.1
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