MOLECULAR MECHANISMS UNDERLYING INNATE IMMUNE KINASE TBK1-DRIVEN ONCOGENIC TRANSFORMATION APPROVED BY SUPERVISORY COMMITTEE Michael A White, Ph.D. Melanie H. Cobb, Ph.D. Lawrence Lum, Ph.D. John D. Minna, M.D. DEDICATION This work is dedicated to my mother and Arlene for their love and support. ACKNOWLEDGEMENTS I am very grateful to my mentor, Dr. Michael White, for his continuous support and guidance through the entire study. I really appreciate his inspiration, patience, and generosity. I would also like to thank my committee members, Dr. Cobb, Dr. Lum, and Dr. Minna, for their invaluable advice and discussion. I thank all the White lab members and my friends for their help, suggestion, and discussion. Particularly, I would like to thank Rosie, Michael, Brian, Tzuling, and Malia for their long-term support and collaboration. I would also like to thank my friends, Veleka, Pei-Ling, Jen-Chieh, Shu-Yi, Chih-Chiang, Jen-Shuan, Yi-Chun, and Yu-San for their friendship. I am grateful to Drs. Rolf Brekken, Zhijian James Chen, Xuetao Cao, Philip Tsichlis, Charles Yeaman, William Hahn, Keqiang Ye, and Shu-Chan Hsu, Bing Su, Dos Sarbassov, Mark Magnuson, David Sabatini, Thomas Tan, and Bert Vogelstein for many of the reagents used in these studies. Finally, and most importantly, I would like to thank my mother and Arlene for their unending support and encouragement. MOLECULAR MECHANISMS UNDERLYING INNATE IMMUNE KINASE TBK1-DRIVEN ONCOGENIC TRANSFORMATION by YI-HUNG OU DISSERTATION Presented to the Faculty of the Graduate School of Biomedical Sciences The University of Texas Southwestern Medical Center at Dallas In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY The University of Texas Southwestern Medical Center at Dallas Dallas, Texas April, 2013 Copyright by YI-HUNG OU, 2013 All Rights Reserved MOLECULAR MECHANISMS UNDERLYING INNATE IMMUNE KINASE TBK1-DRIVEN ONCOGENIC TRANSFORMATION Publication No. Yi-Hung Ou, Ph.D. The University of Texas Southwestern Medical Center at Dallas, 2013 Michael A. White, Ph.D. An essential kinase in innate immune signaling, TBK1 couples pathogen surveillance to induction of host defense mechanisms. The pathological activation of TBK1 in cancer can overcome programmed cell death cues, enabling cells to survive oncogenic stress. The mechanistic basis of TBK1 prosurvival signaling, however, has been enigmatic. Here we show that TBK1 directly activates AKT by phosphorylation of the canonical activation loop and hydrophobic motif sites independently of PDK1 and mTORC2. A population of AKT is bound to components of the exocyst complex. Upon mitogen stimulation, triggering of the innate immune response, re-exposure to glucose, or oncogene activation, TBK1 is recruited vii to the exocyst, where it activates AKT. In cells lacking TBK1, insulin activates AKT normally, but AKT activation by these exocyst-dependent mechanisms is impaired. Discovery and characterization of a 6-aminopyrazolopyrimidine derivative, as a selective low nanomolar TBK1 inhibitor, indicates this regulatory arm can be pharmacologically perturbed independently of canonical PI3K/PDK1 signaling. Thus, AKT is a direct TBK1 substrate that connects TBK1 to prosurvival signaling. Additionally, biochemical and cell biological evidence indicates critical roles of TBK1 and its analog IKKε in the amino acid-dependent activation of mTORC1. TBK1 and IKKε are activated by amino acids and both proteins interact with mTORC1. In TBK1 and/or IKKε-deficient cells, mTORC1 activation by amino acids is impaired. Of note, we also discovered a set of TBK1 substrates and interacting proteins participating in amino acid-dependent mTORC1 signaling. In conclusion, our results suggest that TBK1 not only supports physiological and oncogenic activation of AKT, but also plays a central role in the regulation of mTORC1 activation in response to amino acids. In addition, our studies reveal novel mTORC1 components and provide new insights into the regulation of the mTORC1 signaling network. TABLE OF CONTENTS ABSTRACT ........................................................................................................................ vii TABLE OF CONTENTS ..................................................................................................... ix PRIOR PUBLICATIONS .................................................................................................... xii LIST OF FIGURES ............................................................................................................. xiv LIST OF DEFINITIONS ..................................................................................................... xvi CHAPTER ONE: INTRODUCTION ..................................................................................... 1 HUMAN CANCER ......................................................................................................... 1 AKT SIGNALING PATHWAY ..................................................................................... 3 ACTIVATION OF AKT ........................................................................................... 4 REGULATION OF AKT BY E3 UBIQUITIN LIGASES ....................................... 5 SUPPRESSION OF AKT .......................................................................................... 7 DOWNSTREAM ACTIONS OF AKT ........................................................................... 8 CELL SURVIVAL AND CELL CYCLE ................................................................. 8 AKT AND mTORC1 SIGNALING .......................................................................... 8 AKT AND ANGIOGENESIS .................................................................................... 9 AKT AND METABOLISM ................................................................................... 10 AKT PATHWAY IN CANCER ............................................................................ 12 TBK1/IKKε SIGNALING NETWORK ....................................................................... 12 ROLES OF TBK1 AND IKKε IN INNATE IMMUNE RESPONSE ..................... 13 TBK1 IN CANCER .................................................................................................. 14 ix CHAPTER TWO: TBK1 DIRECTLY ENGAGES AKT/PKB SURVIVAL SIGNALING TO SUPPORT ONCOGENIC TRANSFORMATION .............................................................. 17 INTRODUCTION ......................................................................................................... 17 RESULTS ...................................................................................................................... 19 DISCUSSION ................................................................................................................ 48 MATERIAL AND METHODS .................................................................................... 52 CHAPTER THREE: AMINO ACID SENSING AND REGULATION OF mTORC1 ...... 65 INTRODUCTION ......................................................................................................... 65 mTORC1 SIGNALING ............................................................................................ 65 MOLECULAR COMPONENTS OF mTORC1 ............................................ 66 mTORC1 AND PROTEIN SYNTHESIS ...................................................... 66 mTORC1 AND AUTOPHAGY .................................................................... 67 mTORC1 AND METABOLISM ................................................................... 68 REGULATION OF mTORC1 ....................................................................... 69 mTORC1 AND AMINO ACID SENSING ................................................... 70 IKKε: AN INNATE IMMUNE RESPONSE AND CANCER GENE .................... 71 RESULTS ...................................................................................................................... 73 TBK1 CONTRIBUTES TO mTORC1ACTIVITY BY AMINO ACIDS ............... 73 IKKε CONTRIBUTES TO AMINO ACID-INDUCED mTORC1 SIGNALING. 78 IKKε INTERACTS WITH mTORC1 ............................................................ 78 AMINO ACIDS ELEVATE IKKε KINASE ACTIVITY ............................. 80 KNOCKDOWN OF IKKɛ SUPPRESSES mTORC1 SIGNALING ............. 80 CANDIDATE-BASED MINI SCREEN .................................................................. 84 THE MAP3K3-MAP2K5-ERK5 KINASE CASCADE SUPPORTS AMINO ACID- INDUCED MTORC1 ACTIVATION. .................................................................... 88 DISCUSSION ................................................................................................................ 91 CHAPTER FOUR: CONCLUDING REMARKS ............................................................... 95 BIBLIOGRAPHY ................................................................................................................ 96 PRIOR PUBLICATIONS Chang J, Meredith DM, Mayer PR, Borromeo MD, Lai HC, Ou YH, Johnson JE. Prdm13 mediates the balance of inhibitory and excitatory neurons in somatosensory circuits. Developmental Cell. 2013 (In Press) Herman M, Ciancanelli M*, Ou YH*, Lorenzo L, Guo Y, Sancho-Shimizu V, Pérez de Diego R, Pauwels E, Cardon A, Plancoulaine S, Klaudel-Dreszler M, Heropolitańska-Pliszka M, Rozenberg F, Lebon P, Tardieu M, White MA, Abel L, Zhang SY, and Casanova, JL. Dominant human TBK1 mutations impair TLR3 immunity and underlie herpes simplex encephalitis. The Journal of Experimental Medicine. 2012, 209(9):1567-82 Ou YH, Torres M, Ram RR, Formstecher E, Roland C,