Regulation of HIF-1α during Hypoxia by DAP5-Induced Translation of PHD2 by Jeffrey David Bryant Department of Molecular Genetics and Microbiology Duke University Date:_______________________ Approved: ___________________________ Matthias Gromeier, Supervisor ___________________________ Jack Keene ___________________________ Christopher Nicchitta ___________________________ Jen-Tsan Ashley Chi ___________________________ Christopher Counter 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 2018 ABSTRACT Regulation of HIF-1α during Hypoxia by DAP5-Induced Translation of PHD2 by Jeffrey David Bryant Department of Molecular Genetics and Microbiology Duke University Date:_______________________ Approved: ___________________________ Matthias Gromeier, Supervisor ___________________________ Jack Keene ___________________________ Christopher Nicchitta ___________________________ Jen-Tsan Ashley Chi ___________________________ Christopher Counter An abstract of a thesis 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 2018 Copyright by Jeffrey David Bryant 2018 Abstract Death associated protein 5 (DAP5) is an atypical isoform of the translation initiation scaffold eukaryotic initiation factors 4GI and II (eIF4GI/II), which recruit mRNAs to ribosomes in mammals. Unlike eIF4GI/II, DAP5 binds eIF2β, a subunit of the eIF2 complex that delivers methionyl-tRNA to ribosomes. Despite extensive work describing eIF4GI, an understanding of DAP5 activation is yet to be described. Here I describe our discovery that DAP5:eIF2β binding is regulated by DAP5 phosphorylation and can be stimulated by specific stimuli including protein kinase C PKC-Raf-ERK1/2 signals, mitosis and hypoxia, wherein DAP5:eIF2β binding determines DAP5’s influence on global and template-specific translation. However, DAP5 depletion causes an unanticipated surge of hypoxia-inducible factor 1α (HIF-1α), the transcription factor and master switch of the hypoxia response. The hypoxia response is tempered through HIF-1α hydroxylation by the oxygen-sensing prolyl hydroxylase-domain protein 2 (PHD2), and subsequent ubiquitination and degradation. Furthermore, we found that DAP5 regulates HIF-1α abundance and Akt signaling through DAP5:eIF2β- dependent translation of PHD2. DAP5:eIF2-induced PHD2 translation occurs during hypoxia-associated protein synthesis repression, indicating a role as a safeguard to reverse HIF-1α accumulation and curb the hypoxic response implying that DAP5:eIF2β binding may describe a conserved mechanism for selective stress induced translation. iv Dedication This is dedicated to the multitude of people who have supported me, offered me counsel, and/or put up with my frustrations over this long process. v Contents Abstract ......................................................................................................................................... iv List of Tables .................................................................................................................................. x List of Figures ............................................................................................................................... xi Acknowledgements .................................................................................................................. xiii 1. Introduction ............................................................................................................................... 1 1.1 Protein Synthesis in Eukaryotes ..................................................................................... 1 1.2 Initiation of Translation ................................................................................................... 2 1.2.1 Mechanisms of Translation Initiation ....................................................................... 2 1.2.1.1 Cap-dependent Translation ................................................................................ 3 1.2.1.2 Cap-independent Translation ............................................................................ 6 1.2.2 Function and Regulation of eIF4GI and its Homologues ...................................... 7 1.2.2.1 eIF4GI/II ................................................................................................................. 7 1.2.2.2 DAP5 ...................................................................................................................... 9 1.2.3 Function and Regulation of eIF2 ............................................................................. 11 1.2.3.1 Role of eIF2 during translation initiation ....................................................... 11 1.2.3.2 Regulatory eIF2 phosphorylation .................................................................... 14 1.3 Regulation of Translation by Cell Signaling and during Mitosis ............................ 15 1.3.1 MAPK Signaling ........................................................................................................ 16 1.3.1.1 ERK1/2 MAPKs .................................................................................................. 17 1.3.1.2 p38 and JNK MAPKs ......................................................................................... 19 vi 1.3.2 Regulation of Translation during Mitosis .............................................................. 20 1.3.2.1 CDK1 signaling .................................................................................................. 20 1.3.2.2 Protein synthesis during mitosis ..................................................................... 22 1.4 The Cellular Hypoxia Response ................................................................................... 23 1.4.1 The HIF-1 Transcription Factor ............................................................................... 23 1.4.1.1 HIF-1α activity ................................................................................................... 24 1.4.1.2 Regulation of HIF-1α ......................................................................................... 26 1.4.1.3 pVHL ................................................................................................................... 27 1.4.1.4 PHD2 .................................................................................................................... 27 1.4.2 Protein Synthesis in Hypoxia .................................................................................. 28 1.5 Introduction to the Dissertation Chapters .................................................................. 29 2. Materials and Methods........................................................................................................... 32 2.1 Cell lines, Inhibitors, and Stimulants ........................................................................... 32 2.2 Expression Plasmids and Stable Cell lines .................................................................. 33 2.3 Antibodies, Immunoprecipitation, Immunoblots, siRNA Transfection, and RT- qPCR ....................................................................................................................................... 34 2.4 In vitro Phosphorylation Assays and Phospho-proteomic Analysis ...................... 35 2.5 Polysome Profiling ......................................................................................................... 36 2.6 Hypoxia Treatment ........................................................................................................ 37 2.7 Statistics ........................................................................................................................... 38 3. Results ....................................................................................................................................... 39 3.1 PKC-Raf-ERK1/2 Signaling Induced DAP5:eIF2β Binding impacts Global Translation ............................................................................................................................. 39 vii 3.1.1 Introduction................................................................................................................ 39 3.1.2 Results ......................................................................................................................... 41 3.1.2.1 DAP5:eIF2β binding is induced by PKC-Raf-ERK1/2 signaling ................. 41 3.1.2.2 DAP5 depletion inhibits TPA-induced global translation ........................... 44 3.1.2.3 Loss of DAP5:eIF2β association results in decreased global translation ... 46 3.1.3 Discussion ................................................................................................................... 48 3.2 Phosphorylation of DAP5 regulates DAP5:eIF2β binding ....................................... 49 3.2.1 Introduction................................................................................................................ 49 3.2.2 Results ......................................................................................................................... 50 3.2.2.1 DAP5 is phosphorylated following PKC-Raf-ERK1/2 Signaling ................ 50 3.2.2.2 DAP5(S902) is phosphorylated by CK-2α ...................................................... 52 3.2.2.3 Phosphorylation