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Duke University Dissertation Template Regulation of Eukaryotic Translation Initiation by Signal Transduction by Mayya Shveygert Department of Molecular Genetics and Microbiology Duke University Date:_______________________ Approved: ___________________________ Matthias Gromeier, Supervisor ___________________________ Christopher Nicchitta ___________________________ Jeffrey Rathmell ___________________________ Micah Luftig Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Molecular Genetics and Microbiology in the Graduate School of Duke University 2012 i v ABSTRACT Regulation of Eukaryotic Translation Initiation by Signal Transduction by Mayya Shveygert Department of Molecular Genetics and Microbiology Duke University Date:_______________________ Approved: ___________________________ Matthias Gromeier, Supervisor ___________________________ Christopher Nicchitta ___________________________ Jeffrey Rathmell ___________________________ Micah Luftig An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Molecular Genetics and Microbiology in the Graduate School of Duke University 2012 Copyright by Mayya Shveygert 2012 Abstract Eukaryotic translation initiation is a rate-limiting step of protein synthesis and is controlled by signal transduction in response to various extracellular cues and stresses. This thesis is focused on the eukaryotic initiation factor 4G (eIF4G), which participates in multiple steps of initiation: (i) eIF4G interaction with polyA-binding protein (PABP) links the 5’ pre-initiation complex to the 3’ poly(A) tail of mRNAs; (ii) eIF4G recruits 40S ribosomal subunit to mRNAs through interactions with the m7-G cap binding protein eIF4E; (iii) eIF4G binds to the eIF4E kinase, mitogen activated protein kinase (MAPK) interacting kinase 1 (Mnk1), modulating eIF4E phosphorylation. I studied how eIF4G function is affected by viral infection, mitogenic stimulation and during mitosis. First, I reported that herpes simplex virus 1 (HSV-1) infection leads to re- localization of PABP to the nucleus, dissociating it from translation initiation machinery. Next, I showed that MAPK-mediated phosphorylation of Mnk1 leads to Mnk1 conformational changes, enhancing its binding to eIF4G and, hence, increasing phosphorylation of its substrate, eIF4E. Finally, I demonstrated that Cdk1/cyclin B1 directly phosphorylates eIF4G in mitosis and speculated on the role of this phosphorylation event in mitotic translation. In summary, my work demonstrated that eIF4G plays key roles in the regulation of the translational response to viral infection, growth signaling and cell cycle progression. iv Dedication Dedicated to my parents. v Contents Abstract .................................................................................................................................... iv List of Tables............................................................................................................................ xi List of Figures ......................................................................................................................... xii List of abbreviations.............................................................................................................. xiv Acknowledgements ........................................................................................................... xviii 1. Introduction .......................................................................................................................... 1 1.1 Overview of translational regulation ......................................................................... 1 1.1.1 Post-transcriptional control of gene expression .................................................... 1 1.1.2 Regulation of protein synthesis.............................................................................. 3 1.1.3 Stages of translation ................................................................................................ 5 1.1.4 Translation initiation ............................................................................................... 5 1.2 Growth signaling pathways control translation initiation........................................ 9 1.2.1 Overview of the receptor-induced signaling pathways ....................................... 9 1.2.1 mTOR ..................................................................................................................... 11 1.2.1.1 Upstream activators of mTOR ...................................................................... 12 1.2.1.2 mTORC1 targets translational machinery .................................................... 15 1.2.2 Ras/MAPK pathway ............................................................................................. 17 1.3 eIF4G is a central protein for translation initiation ................................................. 21 1.3.1 Structure of eIF4G ................................................................................................. 21 1.3.2 eIF4G isoforms....................................................................................................... 25 vi 1.3.3 Initiation factors that interact with eIF4G ........................................................... 27 1.3.3.1 Cap-binding protein eIF4E ............................................................................ 27 1.3.3.2 RNA helicase eIF4A ....................................................................................... 28 1.3.3.3 Multi-subunit complex eIF3 .......................................................................... 29 1.3.3.4 Poly A-binding protein PABP ....................................................................... 30 1.3.4.4 eIF4G binds translational repressors ............................................................ 32 1.4 Role of eIF4G in biology and disease........................................................................ 33 1.4.1 Depletion of eIF4G leads to growth arrest phenotypes...................................... 33 1.4.2 The role of eIF4G in gametogenesis and in neurons .......................................... 37 1.4.3 Over-expression of eIF4G promotes tumorigenesis ........................................... 38 1.4.4 Signal transduction to eIF4G ................................................................................ 39 1.5 Translation in mitosis ................................................................................................ 42 1.5.1 Global down-regulation of translation in mitosis ............................................... 43 1.5.2 Selective upregulation of certain mRNAs in mitosis.......................................... 45 1.6 Regulation of translation during viral infection ...................................................... 48 1.6.1 Herpes simplex virus 1 ......................................................................................... 49 1.6.2 HSV-1 infection induces host protein synthesis shut-off ................................... 51 1.6.3 Host defense response and viral countermeasures ............................................ 52 2. Materials and Methods ...................................................................................................... 57 2.1 Cell culture and stable cell lines ................................................................................ 57 2.2 Expression constructs ................................................................................................ 58 2.2.1 Cloning for Chapter 3.1 ........................................................................................ 58 vii 2.2.2 Cloning for Chapter 3.2 ........................................................................................ 58 2.2.3 Cloning for Chapter 3.3 ........................................................................................ 60 2.3 Recombinant proteins ................................................................................................ 63 2.4 Immunofluorescence ................................................................................................. 63 2.5 DNA transfections ..................................................................................................... 64 2.6 Kinase inhibitors and activators ............................................................................... 65 2.7 m7G cap pull down, glutathione pull down, immunoprecipitation...................... 65 2.7.1 Chapter 3.1 ............................................................................................................. 65 2.7.2 Chapter 3.2 ............................................................................................................. 66 2.7.3 Chapter 3.3 ............................................................................................................. 67 2.8 Immunoblotting and antibodies ............................................................................... 68 2.8.1 Antibodies for Chapter 3.1 ................................................................................... 68 2.8.2 Antibodies for Chapter 3.2 ................................................................................... 69 2.8.3 Antibodies for Chapter 3.3 ................................................................................... 69 2.9 In vitro kinase assay ................................................................................................... 70 2.9.1.
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