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Role of the Protein Kinase TBK1 in Insulin-Stimulated Glucose Transport

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Role of the Protein Kinase TBK1 in Insulin-Stimulated Glucose Transport Role of the Protein Kinase TBK1 in Insulin-Stimulated Glucose Transport by Maeran Uhm A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Molecular and Integrative Physiology) in the University of Michigan 2015 Doctoral Committee: Professor Alan R. Saltiel, Chair Associate Professor Ken Inoki Associate Professor Jiandie Lin Professor Jessica Schwartz Professor John A. Williams ACKNOWLEDGEMENTS I would like to gratefully thank my advisor, Dr. Alan Saltiel, for his support, guidance, understanding, patience and encouragement in science and life. I feel extremely fortunate to have an advisor who affords his graduate students the freedom to explore their own ideas and pursue various projects without objection. He has taught me how to question thoughts, express my ideas, and communicate with people. This allowed me to grow as a better scientist. He is one of the smartest people I know and a great scientific leader. I hope that I could be as enthusiastic and energetic as Alan and become a good scientist like him in the future. I would also like to thank my committee members, Dr. John Williams, Jessica Schwartz, Jiandie Lin, and Ken Inoki for serving as my committee members and their helpful discussions and guidance over the course of my graduate career. Everyone has given me always brilliant comments and suggestions. I would like to thank all of my past and current colleagues in the Saltiel lab, including Louise Chang, Shian-Huey Chiang, Stuart Decker, Dave Bridges, Nicole Maher, Christina Sherry, Binbin Lu, Sheela Karunanithi, Alan Cheng, David Buchner, Irit Hochberg, Xiao-Wei Chen, Jon Mowers, Dara Leto, Tingting Xiong, Carey Lumeng, Melissa McGill, Shannon Reilly, Yuliya Skorobogatko, Prasanth Puthanveetil, Peng Zhao, Tae Hyeon Koo, BreAnne Poirier, Brandon Pang, Jerry Yan, Joshua Castle, Kai Yuan, Silvia Novakova, and Jamie Yost Brandon. Thank ii you all for your friendship, help, and advice during my graduate training. I also would like to thank my other colleagues, including Siming Li, Grace Wang, and Di Ma for their friendship and collaboration. My special thanks also go to Dr. Ken Inoki, Ormond MacDougald, Bishr Omary, and Michele Boggs for giving me an opportunity to be trained at the University of Michigan even before the matriculation. You truly allowed me to become a PhD student. Of course, I could not overcome many of the obstacles in my life without the endless support and love of my family. Last but not the least, I would like to give my special thanks to my lovely husband, Juil Yum, for his support, patience, and understanding throughout my graduate training. Many thanks to my family, including my parents (Dongseob Uhm and Jinsoon Park), my parents-in-law (Taewoon Yum and Soonyi Jung), my brother Hyunsik Uhm, and my baby Jay Yum for their support and prayers. None of this would have been possible without the love and patience and you made me who I am. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS ................................................................................... ii LIST OF FIGURES ............................................................................................... vi LIST OF ABBREVIATIONS ............................................................................. viii ABSTRACT ..............................................................................................................x Chapter 1 Introduction ............................................................................................1 I. Insulin-stimulated glucose uptake .......................................................................2 II. Insulin signaling pathways in GLUT4 translocation .......................................14 III. Small G proteins in insulin-stimulated glucose uptake ..................................23 IV. Roles of IKK-related kinases ..........................................................................34 V. Summary ..........................................................................................................38 References .............................................................................................................40 Chapter 2 Novel function of the protein kinase TBK1 in insulin-stimulated glucose transport ....................................................................................................59 Introduction ...........................................................................................................59 Results and Discussion .........................................................................................60 Materials and Methods ..........................................................................................75 iv References .............................................................................................................80 CHAPTER 3 Exo84, a bona fide direct substrate of TBK1 ...............................82 Introduction ...........................................................................................................82 Results and Discussion .........................................................................................83 Materials and Methods ..........................................................................................99 References ...........................................................................................................105 CHAPTER 4 Regulation of engagement and disengagement of GLUT4 vesicles from the exocyst by TBK1 .....................................................................107 Introduction .........................................................................................................107 Results and Discussion .......................................................................................108 Materials and Methods ........................................................................................144 References ...........................................................................................................154 Chapter 5 Conclusions and future directions ...................................................156 References ...........................................................................................................161 v LIST OF FIGURES Figure 1.1 Schematic diagram of GLUT4 trafficking itinerary. ..................................................... 6 Figure 1.2 The exocyst in GLUT4 exocytosis. ............................................................................. 12 Figure 1.3 Insulin signaling regulates GLUT4 trafficking. .......................................................... 22 Figure 1.4 The regulation of G protein cycle by GEFs, GAPs, and GDIs.................................... 25 Figure 1.5 Small G proteins in GLUT4 trafficking. ..................................................................... 27 Figure 1.6 Structural comparison of the classical and noncanonical IKKs. ................................. 35 Figure 2.1 Differentiation of 3T3-L1 fibroblasts under different conditions. .............................. 61 Figure 2.2 SiRNA-mediated knockdown of TBK1 inhibits insulin-stimulated glucose uptake. .. 63 Figure 2.3 Ectopic overexpression of a kinase-inactive mutant of TBK1 reduces insulin- stimulated glucose uptake. ............................................................................................................ 64 Figure 2.4 TBK1 activity is required for insulin-stimulated glucose uptake. ............................... 66 Figure 2.5 SiRNA-mediated knockdown of TBK1 inhibits insulin-stimulated GLUT4 translocation. ................................................................................................................................. 68 Figure 2.6 TBK1 regulates insulin-stimulated glucose uptake in parallel with Akt. .................... 70 Figure 2.7 TBK1 and Akt parallel pathways are required for insulin-stimulated glucose uptake.72 Figure 2.8 TBK1 regulates insulin-stimulated GLUT4 translocation in parallel with Akt. ......... 73 Figure 2.9 Hypothetical model of the role of TBK1 in insulin-stimulated GLUT4 translocation. ....................................................................................................................................................... 74 Figure 3.1 TBK1 phosphorylates Exo84 in vitro. ......................................................................... 84 Figure 3.2 TBK1 phosphorylates Exo84 in vitro in a dose-dependent manner. ........................... 85 Figure 3.3 TBK1 but not other kinases phosphorylates Exo84 in vitro........................................ 87 Figure 3.4 TBK1 phosphorylates Exo84 in vivo. .......................................................................... 88 Figure 3.5 The interaction between Exo84 and TBK1 is specific. ............................................... 90 Figure 3.6 Sec5 is a substrate of TBK1. ....................................................................................... 92 Figure 3.7 The helical domain of Exo84 interacts with TBK1. .................................................... 94 Figure 3.8 TBK1 directly interacts with Exo84 via the helical domain of Exo84 in vitro. .......... 96 Figure 3.9 TBK1 directly interacts with Exo84 via the coiled-coil domain of TBK1 in vitro. .... 97 Figure 4.1 TBK1 does not directly interact with RalA. .............................................................. 109 Figure 4.2 The interaction of Exo84 with RalA is impaired by WT TBK1 overexpression. ..... 110 Figure 4.3
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