Identification of Proteins that Regulate c-Myc Stability and Function Dissertation by: Julienne Rebeca Escamilla-Powers Presented to the Department of Medical and Molecular Genetics and the Oregon Health and Sciences School of Medicine in partial fulfillment of the requirements for the degree of Doctor of Philosophy August 2008 Table of Contents Table of Contents Table of Contents ............................................................................................................ i List of Tables and Figures.............................................................................................. iv List of Abbreviations ..................................................................................................... vi Acknowledgements ....................................................................................................... ix Abstract ........................................................................................................................ xii Chapter One: Introduction .............................................................................................1 c-Myc ..........................................................................................................................2 HBP1 ......................................................................................................................... 17 Chapter Two: A Conserved Pathway that Controls c-Myc Protein Stability Through Opposing Phosphorylation Events Occurs in Yeast ........................................................ 30 Abstract: .................................................................................................................... 31 Introduction: .............................................................................................................. 32 Results: ...................................................................................................................... 35 Discussion: ................................................................................................................ 57 Chapter Three: Use of a Yeast Two-Hybrid Assay to Identify c-Myc-interacting Proteins ...................................................................................................................................... 65 Abstract: .................................................................................................................... 66 Introduction: .............................................................................................................. 67 Results: ...................................................................................................................... 72 Discussion ................................................................................................................. 82 i Table of Contents Chapter Four: The Tumor Suppressor Protein HBP1 Negatively Regulates c-Myc Activity ......................................................................................................................... 87 Abstract: .................................................................................................................... 88 Introduction: .............................................................................................................. 89 Results: ...................................................................................................................... 93 Discussion: .............................................................................................................. 117 Chapter Five: Summary and Discussion ...................................................................... 122 Use of yeast as a model system to study c-Myc ........................................................ 123 HBP1 negatively regulates c-Myc activity ............................................................... 130 Chapter Six: Materials and Methods ........................................................................... 138 Plasmids, shRNA and siRNA................................................................................... 139 Cell lines and Transfections ..................................................................................... 143 Yeast strains, media and reagents ............................................................................. 144 CPRG Assay ............................................................................................................ 144 DNA extraction from Yeast ..................................................................................... 145 Antibodies ............................................................................................................... 146 Galactose Induction Assay ....................................................................................... 147 In vitro Kinase Assay ............................................................................................... 148 Western Blotting and Quantitation ........................................................................... 148 Co-Immunoprecipitations ........................................................................................ 149 Luciferase Assay ..................................................................................................... 150 Chromatin Immunoprecipitation (ChIP) Assays ....................................................... 150 RNA isolation and qRT-PCR ................................................................................... 151 ii Table of Contents Appendix ..................................................................................................................... 152 Contributions to Projects and Figures ....................................................................... 152 Funding ................................................................................................................... 154 References ................................................................................................................... 155 iii List of Figures and Tables List of Tables and Figures Figure 1.1: Transcriptional regulation by the Myc/Max/Mad network of proteins ............6 Figure 1.2: Schematic of the c-Myc protein. ....................................................................9 Figure 1.3: Summary of the pathways controlling c-Myc phosphorylation and ubiquitin- mediated degradation. ............................................................................................ 14 Figure 1.4: HBP1 functional domains and binding partners............................................ 20 Figure 1.5: Potential modes of HBP1 activity. ............................................................... 26 Figure 2.1: Interdependent phosphorylation of c-Myc at T58 and S62 in yeast. .............. 39 Table 2.1: Mammalian proteins known to regulate c-Myc protein stability and their S. cerevisiae orthologs ............................................................................................... 40 Figure 2.2: The yeast kinases Kss1 and Rim11 phosphorylate c-Myc and thereby control c-Myc protein stability. .......................................................................................... 45 Figure 2.3: Mutation of the yeast peptidyl prolyl isomerase, Ess1, does not significantly affect c-Myc phosphorylation or overall stability. .................................................. 49 Figure 2.4: Loss of yeast PP2A activity increases c-Myc S62 phosphorylation and c-Myc half-life. ................................................................................................................. 53 Figure 2.5: Mutation of the Fbw7 structural homolog, Cdc4, does not result in c-Myc stabilization. .......................................................................................................... 56 Figure 2.6: A conserved pathway to control protein degradation. ................................... 59 Figure 3.1: Screening for c-Myc interacting proteins in a yeast two-hybrid assay with three reporter genes. ............................................................................................... 71 Figure 3.2: Schematic of mating strategy used to rescreen potential c-Myc interacting proteins .................................................................................................................. 77 iv List of Figures and Tables Table 3.1: c-DNA isolated in yeast two-hybrid screen as those expressing c-Myc interacting proteins ................................................................................................ 79 Figure 3.3: c-Myc interacts with the ribosomal proteins L12 and L3 .............................. 81 Figure 4.1: HBP1 is a c-Myc interacting protein ............................................................ 96 Figure 4.2: HBP1 inhibits c-Myc induced transcription. ............................................... 102 Figure 4.3: Knockdown of HBP1 results in increased c-Myc transcriptional activity. .. 104 Figure 4.4: HBP1 inhibits transactivation by c-Myc MBI point mutants....................... 106 Figure 4.5: HBP1 inhibits expression of endogenous c-Myc target genes. .................... 108 Figure 4.6: HBP1 inhibits binding of c-Myc to its target gene promoters. .................... 111 Figure 4.7: c-Myc interacts with the C-terminus of HBP1 ............................................ 113 Figure 4.8: Multiple c-Myc domains are required for binding to HBP1 ........................ 116 Figure 5.1: Schematic of a role for isomerization in regulation of c-Myc