Insight Into the Brain-Specific Alpha Isoform of the Scaffold Protein SH2B1 and Its Rare Obesity-Associated Variants
Total Page：16
File Type：pdf, Size：1020Kb
Insight into the Brain-Specific Alpha Isoform of the Scaffold Protein SH2B1 and its Rare Obesity-Associated Variants By Ray Morris Joe A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Cellular and Molecular Biology) in the University of Michigan 2017 Doctoral Committee: Professor Christin Carter-Su, Chair Professor Ken Inoki Professor Benjamin L. Margolis Professor Donna M. Martin Professor Martin G. Myers © Ray Morris Joe ORCID: 0000-0001-7716-2874 [email protected] All rights reserved 2017 Acknowledgements I’d like to thank my mentor, Christin Carter-Su, for providing me a second opportunity and taking a chance on me after my leave of absence without hesitation. It is an honor to have a mentor guide me through the many challenges I have faced as a graduate student. She has taught me to find passion in my profession, and to strive to push through obstacles and find ways to reach my goals. In addition, she has given me insight to find a balance between work and life by giving me freedom to independently perform my research as well as stories on family and child-rearing. Throughout the many countless days and nights writing grants, manuscripts, and analyzing/interpreting data, it was wonderful to share our past cultural identities with one another. I look forward to having Christy as a mentor, colleague, and friend for all my future endeavors I have after I leave her laboratory. I want to thank my thesis committee, Ken Inoki, Ben Margolis, Donna Martin, and Martin Myers for the numerous insights for my scientific training. Our exchange of ideas, constructive critiques, and positive motivation during our bi-annual committee meetings were an invaluable contribution that improved my experiments. I also want to thank Donna for providing me sound advice about graduate school and the mental approach I should consider accomplishing my thesis research. Furthermore, I want to thank you for being excellent role models during my graduate school studies. ii I want to extend my gratitude to the past Program in Biomedical Sciences Director, Lori Isom, and Director of Recruiting, Tiffany Porties, for their immediate inquiry to my initial departure from graduate school. They listened to the numerous issues I faced as a graduate student and provided positive feedback to motivate me to return to graduate school. In addition, I thank the past Cellular and Molecular Biology(CMB) Program Director, Jessica Schwartz, for engaging me to become a better graduate student and allowing me to be part of the CMB program without hesitation after my leave of absence. I’d like to thank numerous Carter-Su lab members and those involved in our joint laboratory meetings. Larry Argetsinger consistently provided excellent experimental advice, suggestions and numerous luncheon discussions on topics relating to science and/or life. Larry has always been enthusiastic to accompany me on many new experiments and/or provide help when needed through my tenure in the Carter-Su lab. I thank Jessica Schwartz for the helpful insights to improve my data, figures, and presentations. I thank Ram Menon and past laboratory members for helpful suggestions for improving my experimental quantitation and statistical analysis in our weekly lab meetings. I thank Joel Cline for providing helpful tips for my molecular biology experiments and humorous discussions on our pets and fantasy sport leagues. I thank past post-docs, Melissa Dobson, Hsaio-Wen Su, and Liliya Mancour for their friendship, mentorship, experimental suggestions, writing advice and navigational advice to survive graduate school. I thank past and current graduate students Michael Doche, Grace Lin, Brianne Docter, Anabel Flores, and Jessica Cote for our scientific and non-scientific conversations that have made life in the lab enjoyable and entertaining. I thank iii undergraduate work study students: Kenny Cha, Alex, Emily, Alvaro Malaga, Michelle Kim; who have made life in the lab easier. Special thanks to Erik Clutter for not only making my life easier but also bringing a friendly and positive attitude to the lab every day. Also, I thank the past and current undergraduate students I mentored (Kenny Cha, Michelle Sierant, Alvaro Malaga, Paul Vander); and Alex Bedard, Lauren DeSantis, Gowry Chandresakar for their positive and youthful energy they bring to the laboratory. I thank the administrative assistants: Barbara Hawkins, Deborah Tackett, Sarah Caine, Cathy Mitchell, Margarita Bekiares, Anne Many, Leah Peters, and Francine Bomar for maintaining organization and holding me to deadlines. I thank the past and current members of the Yin lab: Lei, Tony, Deqiang, Pei, and Nick; for helpful scientific feedback, experimental design, and critiques to improve my scientific communication. I want to thank my friends, Anthony Murphy, Conrad Valdez, John Shagonaby, Michaelina Martin, Corey Cunningham, Veronica Taylor, Allissa Waukau, Phillip Tapia, Bernadette Zwaans, Kiri Sailiata, Michael Gonzalez, Danielle Goodman, and Sara Wong for being outstanding and wonderful friends. I thank my mentors outside the University of Michigan, Margaret Werner-Washburne, A.D. Cropper, and Jacquelyn Bolman, for their guidance and foresight that I could accomplish my doctoral goals. Lastly, I want to thank my beautiful and wonderful wife, Shanda, for believing in me and standing beside me through these years and the many years to come. Also, I want to thank Shanda’s parents, Jim and Nancy Birkeland, for being my “Michigan parents” and providing help when needed. I thank my amazing sisters, Audrey and Andrea, for the many care-packages of green and red chile, pueblo bread and pies as iv well as their endless support. Finally, I want to thank my parents, Harrison and Margaret Joe, for their infinite love and support. v Table of Contents Acknowledgements………………………………………………………………..……….…ii List of Figures…………………………………………………………………………...……viii Abstract…...………………………………………………………………………………….…xi Chapter 1……………………………………………………………………………………......1 Introduction Chromosomal deletions and variants within SH2B1 promote obesity…………..….1 The scaffold protein SH2B1 is implicated in metabolic and neuronal cell signaling …………………………………………………………………...……8 Characterizing the cellular and molecular functions of SH2B1………………………............………………………………………...17 Alternative splicing of SH2B1 generates isoforms with unique C-terminal tails, enabling diversity in protein-protein interactions...…………………………25 PC12 cells as a model system for delineating cellular actions of SH2B1 in neurotrophic factor signaling………………………………………………29 Chapter 2………………………………………………………………………………………31 Functional Characterization of Obesity-Associated Variants Involving the α and β Isoforms of Human SH2B1 Abstract…………………………………………………………………………………31 Introduction………………………………………………………………………….....33 Materials and Methods………………………………………………………………..35 Results………………………………………………………………………………….39 Discussion……………………………………………………………………………...47 Acknowledgements……………………………………………………………….......51 Chapter 3………………………………………………………………………………………53 The C-Terminal Tail of the α Isoform of SH2B1 Inhibits the Ability of SH2B1 to Enhance NGF Functions and Alters SH2B1 Subcellular Localization, Both by a Phosphorylation-Dependent Mechanism vi Abstract…………………………………………………………………………………53 Introduction………………………………………………………………………….....55 Materials and Methods………………………………………………………………..59 Results…………………………………………………………………………............66 Discussion………………………………………………………………………..…….85 Chapter 4………………………………………………………………………………………95 Functional Roles of SH2B1 Obesity-Associated Variants in Response to Nerve Growth Factor Abstract…………………………………………………………………………......….95 Introduction………………………………………………………………………….....97 Materials and Methods……………..……………………………………….....…....100 Results…………………………………………………………………………….......105 Discussion……………………………………………………………………….……120 Chapter 5………………………………………………………………………………..……126 Summary and Future Directions…...……………………………………………….126 References…………………………………………………………………………………...140 vii List of Figures Figure 1.1. Schematic of the four human SH2B1 isoforms………..………………...........3 Figure 1.2. Schematic of SH2B1 isoforms and obesity-associated mutations identified in humans………………………………………………………………6 Figure 1.3. Model of illustrating some of the many roles of SH2B1β in response to NGF………………………………………………………………………….... 18 Figure 1.4. Schematic illustrating the alternative splicing of four SH2B1 isoforms........27 Figure 2.1. Identification of novel variants in SH2B1……………………………………...42 Figure 2.2. Comparison of SH2B1α and SH2B1β in vitro....………..……………………44 Figure 2.3. Characterization of novel human variants in SH2B1………………………...45 Figure 3.1. SH2B1α‘s C-terminal tail regulates SH2B1’s ability to enhance NGF-mediated neurite outgrowth and translocate to the nucleus……...….67 Figure 3.2. Mutating Tyr753 in SH2B1α’s C-terminal tail enables SH2B1α to enhance NGF-mediated neurite outgrowth…………………………..………70 Figure 3.3. Tyr753 in SH2B1α regulates the ability of SH2B1α to translocate to the nucleus……………………………………………………………...…….71 Figure 3.4. Loss of SH2B1α’s C-terminal tail or mutation of Tyr753 in the tail enables SH2B1α to enhance NGF-mediated gene transcription…………..73 viii Figure 3.5. SH2B1’s C-terminal tails modulate NGF-dependent phosphorylation of NGF signaling proteins………………………………………………...……..75 Figure 3.6. SH2B1’s C-terminal tails modulate NGF-mediated TrkA phosphorylation...........................................................................................78 Figure 3.7. TrkA phosphorylates