Novel Regulation of Mtor Complex 1 Signaling by Site-Specific Mtor Phosphorylation
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Novel Regulation of mTOR Complex 1 Signaling by Site-Specific mTOR Phosphorylation by Bilgen Ekim Üstünel A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Cell and Developmental Biology) in The University of Michigan 2012 Doctoral Committee: Assistant Professor Diane C. Fingar, Chair Associate Professor Billy Tsai Associate Professor Anne B. Vojtek Assistant Professor Patrick J. Hu Assistant Professor Ken Inoki “Our true mentor in life is science.” (“Hayatta en hakiki mürşit ilimdir.”) Mustafa Kemal Atatürk, the founder of Turkish Republic © Bilgen Ekim Üstünel 2012 Acknowledgements This thesis would not have been possible without the enormous support and encouragement of my Ph.D. advisor Diane C. Fingar. I am sincerely thankful for her research insight and guidance during my Ph.D. training. I would like to express my great appreciation to Billy Tsai, Anne B. Vojtek, Ken Inoki, and Patrick J. Hu for serving on my thesis committee, whose advice and help have been valuable. I would like to thank all members of the Fingar, Tsai, and Verhey labs for the discussion in our group meetings. I also would like to thank the CDB administrative staff, especillay Kristen Hug, for their help. I thank Ed Feener for performing the liquid chromatography tandem mass spectrometry analysis to identify novel phosphorylation sites on mTOR and Steve Riddle for performing the in vitro kinome screen to identify candidate kinases for mTOR S2159 phosphorylation site. I thank Brian Magnuson, Hugo A. Acosta-Jaquez, and Jennifer A. Keller for contributing to my first-author paper published in Molecular and Cellular Biology Journal in 2011. I also thank Diane C. Fingar for generating data presented in Figure 2.2 Part D. I cannot finish without saying how grateful I am with my family; my father Halil İbrahim Ekim, my mother Meral Ekim, my sister Çiğdem Ekim, and most importantly my husband Eser Üstünel for their unconditional love and support. ii Table of Contents Acknowledgements .............................................................................................. ii List of Figures .......................................................................................................iv List of Abbreviations ............................................................................................. v Abstract ................................................................................................................ x Chapter .................................................................................................................. 1. Introduction ................................................................................................... 1 2. mTOR Kinase Domain Phosphorylation Promotes mTORC1 Signaling, Cell Growth, and Cell Cycle Progression .................................................... 38 3. Novel Regulation of mTORC1 by TBK1 and IKKε ...................................... 76 4. Conclusion ................................................................................................ 107 References ....................................................................................................... 113 iii List of Figures Figure 1.1 mTOR domain structure and phosphorylation sites 35 1.2 Intra- and extra-cellular signals regulate mTORC1 signaling 36 1.3 S6K1 isoforms, domain structure, and phosphorylation sites 37 1.4 4E-BP1 domain structure and phosphorylation sites 37 2.1 Identification of S2159 and T2164 as novel sites of mTOR phosphorylation 62 2.2 mTOR S2159/T2164 phosphorylation promotes mTORC1 signaling to S6K1 64 2.3 mTOR S2159/T2164 phosphorylation increases the sensitivity and duration of insulin-stimulated mTORC1 signaling 66 2.4 Phosphorylation of mTOR S2159/T2164 promotes mTORC1 signaling to 4E-BP1 68 2.5 mTOR S2159/T2164 phosphorylation weakens the mTOR-raptor and raptor-PRAS40 interactions and augments mTORC1 intrinsic kinase activity 69 2.6 mTOR S2159/T2164 phosphorylation promotes mTORC1-mediated cell growth and G1-phase cell cycle progression 71 2.7 mTOR-S2159D/T2164E (DE) but not S2159A/T2164A (AA) promotes cell growth to an increased cell size during a cell cycle block 73 2.8 Model 75 3.1 In vitro kinome screen to identify potential mTOR S2159 kinases 98 3.2 Recombinant TBK1 and IKKε phosphorylate mTOR S2159 in vitro 100 3.3 TBK1/IKKε and mTOR coimmunoprecipitate 102 3.4 TBK1-/- and IKKε-/- MEFs show reduced mTOR S2159 phosphorylation 103 3.5 Overexpression of TBK1 and IKKε increases mTOR S2159 phosphorylation and S2481 autophosphorylation in intact cells 104 3.6 Activation of TLR3 and TLR4 promotes mTOR signaling in RAW264.7 murine macrophages 106 iv List of Abbreviations 4E-BP1 Eukaryotic translation initiation factor 4E binding protein 5’TOP 5’track of polypyrimidines m7GTP 7-Methylguanosine 5’-triphosphate AGC Protein kinase A, Protein kinase G and Protein kinase C AMP Adenosine monophosphate AMPK Adenosine monophosphate activated kinase AP-1 Activator protein 1 ATG Autophagy-related protein ATM Ataxia telangiectasia mutated ATR Ataxia telangiectasia and Rad3-related protein CBP CREB binding protein CD Cluster of differentiation Cdc Cell division control CNS Central nervous system Deptor DEP domain-containing mTOR-interacting protein DNA-PK DNA dependent protein kinase dsRNA Double stranded RNA eEF2 Eukaryotic elongation factor 2 eEF2K Eukaryotic elongation factor 2 kinase EGF Epidermal growth factor EGFR Epidermal growth factor receptor eIF Eukaryotic translation initiation factor EMT Epithelial mesenchymal transition ERK Extracellular signal-regulated kinase v FAK Focal adhesion kinase FDA Food and Drug Administration FIP200 FAK family-interacting protein of 200 kDa FKBP12 FK506-binding protein 12-kDa FRAP The FKBP-12-rapamycin associated protein FRB FKBP12-rapamcyin binding GAP GTPase activating protein GEF GTP exchange factor Glut4 Glucose transporter type 4 GSK3β Glycogen synthase kinase 3 beta GTP Guanosine 5’ triphosphate HEAT Huntingtin, elongation factor 3, protein phosphatase 2A (PP2A), and the yeast PI3-kinase TOR1 HIF-1α Hypoxia-inducible factor 1-alpha HM Hydrophobic motif hVPS34 Mammalian vacuolar protein sorting 34 homologue IB Immunoblot IGF Insulin-like growth factor IκBα I-kappa-B-alpha IKK Inhibitor of nuclear factor-kappa B- kinase ILK Integrin-linked kinase IP Immunoprecipitate IRS Insulin receptor substrate IVK In vitro kinase JAK Janus kinase KOG1 Kontroller of growth protein 1 LKB1 Liver kinase B1 LPS Lipopolysaccharide LRL Leucine rich repeat MAP4K3 MAP kinase kinase kinase kinase 3 MAPK Mitogen-activated protein (MAP) kinase vi MAPKAPK-2 MAP kinase-activated protein kinase 2 MBH Mediobasal hypothalamus MEF Mouse embryonic fibroblast MEK MAPK/ERK kinase mLST8 mTOR associated lethal with Sec 13 8/G protein β subunit-like MP1 MEK-binding partner 1 mRNA Messenger RNA mSin1 Mammalian stress-activated protein kinase interacting protein mTOR Mammalian target of rapamycin mTORC mTOR complex MyD88 Myeloid differentiation primary response protein NDRG1 N-Myc downregulated gene 1 NEMO NF-kappa-B essential modulator NF-1 Neurofibromatosis-related protein NF-kB Nuclear factor-kappa B NLS Nuclear localization signal PA Phosphatidic acid PAK1 p21-activated kinase 1 PAMP Pathogen associated molecular patterns pDC Plasmacytoid dendritic cell PDCD4 Programmed cell death protein 4 PDCD4 PDGF Platelet derived growth factor PDK1 Phosphoinositide-dependent kinase-1 PGC1α Peroxisome-proliferator-activated receptor gamma coactivator-1 alpha PI3K Phosphatidylinositol 3 kinase PIKK Phosphoinositide kinase-related kinase PIP3 Phosphatidylinositol-3,4,5-trisphosphate PKCα Protein kinase c alpha isoform PLD Phospholipase D vii Pol DNA polymerase Poly(I:C) Polyinosinic:polycytidylic acid PPARγ Peroxisome proliferator-activated receptor gamma PRAS40 Proline rich Akt substrate of 40 KDa P-Rex1 Phosphatidylinositol-3,4,5-trisphosphate -dependent Rac exchange factor PRR5/Protor Proline-rich protein 5 PTEN Phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase Rac 1 Ras-related C3 botulinum toxin substrate 1 RAFT1 Rapamycin and FKBP12 target RAPT Rapamycin target Raptor Regulatory associated protein of mTOR Ras Rat sarcoma RCC Renal cell carcinoma rDNA Ribosomal DNA Redd1 Regulated in development and DNA damage response Rheb Ras homolog enriched in brain Rictor Rapamycin-insensitive companion of mTOR RNAi RNA interference ROS Reactive oxygen species RR Rapamycin resistant rRNA Ribosomal RNA S6K S6 kinase SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SEP Sirolimus effector protein SGK1 Serum/Glucocorticoid Regulated Kinase 1 SH3 SRC Homology 3 Domain SKAR S6K1 Aly/REF-like target SLC7A5/SLC3A2 Solute carrier family 7, member 5/solute carrier family 3, member 2 viii SREBP Sterol response element binding protein STAT3 Signal transducer and activator of transcription 3 Tssk3 Testis-specific serine/threonine-protein kinase 3 TBK1 TANK-binding kinase 1 TIF Transcription initiation factor TIR Toll/interleukin-1 receptor domain TM Turn motif TNFα Tumor necrosis factor alpha TOS TOR signaling TRAM TRIF-related adaptor molecule tRNA Transfer RNA TRIF Toll/IL-1 Receptor Domain-Containing Adaptor Inducing IFN-β TSC Tuberous sclerosis complex TTRAP Trafficking protein particle UBF Upstream-binding factor ULK Unc-51-like kinase UPR Unfolded protein response VEGF Vascular endothelial growth factor YY1 Yin-yang 1 ix Abstract Aberrant regulation of the kinase mTOR (mammalian target of rapamycin)