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Identifying and Characterizing Yeast PAS Kinase 1 Substrates Reveals Regulation of Mitochondrial and Cell Growth Pathways

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Identifying and Characterizing Yeast PAS Kinase 1 Substrates Reveals Regulation of Mitochondrial and Cell Growth Pathways Brigham Young University BYU ScholarsArchive Theses and Dissertations 2015-06-01 Identifying and Characterizing Yeast PAS Kinase 1 Substrates Reveals Regulation of Mitochondrial and Cell Growth Pathways Desiree DeMille Brigham Young University - Provo Follow this and additional works at: https://scholarsarchive.byu.edu/etd Part of the Microbiology Commons BYU ScholarsArchive Citation DeMille, Desiree, "Identifying and Characterizing Yeast PAS Kinase 1 Substrates Reveals Regulation of Mitochondrial and Cell Growth Pathways" (2015). Theses and Dissertations. 5930. https://scholarsarchive.byu.edu/etd/5930 This Dissertation is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected], [email protected]. Identifying and Characterizing Yeast PAS Kinase 1 Substrates Reveals Regulation of Mitochondrial and Cell Growth Pathways Desiree DeMille A dissertation submitted to the faculty of Brigham Young University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Julianne H. Grose, Chair William R. McCleary Laura C. Bridgewater David M. Thomson Steven M. Johnson Department of Microbiology & Molecular Biology Brigham Young University June 2015 Copyright © 2015 Desiree DeMille All Rights Reserved ABSTRACT Identifying and Characterizing Yeast PAS Kinase 1 Substrates Reveals Regulation of Mitochondrial and Cell Growth Pathways Desiree DeMille Department of Microbiology & Molecular Biology, BYU Doctor of Philosophy Glucose allocation is an important cellular process that is misregulated in the interrelated diseases obesity, diabetes and cancer. Cells have evolved critical mechanisms for regulating glucose allocation, one of which is sensory protein kinases. PAS kinase is a key sensory protein kinase that regulates glucose allocation in yeast, mice and man; and is a novel therapeutic target for the treatment of metabolic diseases such as obesity, diabetes and cancer. Despite its importance, the molecular mechanisms of PAS kinase function are largely unknown. Through large-scale protein-interaction studies, we have identified 93 novel binding partners for PAS kinase which help to expand its role in glucose allocation as well as suggest novel roles for PAS kinase including mitochondrial metabolism, cell growth/division, protein modification, stress tolerance, and gene/protein expression. From a subset of these binding partners, we identified 5 in vitro substrates of PAS kinase namely Mot3, Utr1, Zds1, Cbf1 and Pbp1. Additionally, we have further characterized Pbp1 and Cbf1 as PAS kinase substrates through both in vitro and in vivo evidence as well as phenotypic analysis. Evidence is provided for the PAS kinase- dependent phosphorylation and activation of Pbp1, which in turn inhibits cell proliferation through the sequestration of TORC1. In contract, PAS kinase-dependent phosphorylation of Cbf1 inhibits its activity, decreasing cellular respiration. This work elucidates novel molecular mechanisms behind PAS kinase function in both mitochondrial and cell growth pathways in eukaryotic cells, increasing our understanding of the regulation of central metabolism. Keywords: PAS kinase, Psk1, Cbf1, Pbp1, respiration, glucose allocation, diabetes, metabolism ACKNOWLEDGMENTS I would like to express appreciation for my mentor Dr. Julianne Grose especially for her enthusiasm and dedication to our research. I am grateful for her time and effort spent in helping me to succeed. I would like to give a big thank you to all of my graduate committee members, as well as other faculty whom I have had the opportunity to collaborate with, for their ideas and advice with troubleshooting. Finally, I would like to dedicate this dissertation to my loving, supportive and patient family, Lee, Kai and Mya. TABLE OF CONTENTS TITLE PAGE ................................................................................................................................... i ABSTRACT .................................................................................................................................... ii ACKNOWLEDGMENTS ............................................................................................................. iii TABLE OF CONTENTS ............................................................................................................... iv LIST OF TABLES .......................................................................................................................... x LIST OF FIGURES ....................................................................................................................... xi CHAPTER 1: PAS kinase: A nutrient sensing regulator of glucose homeostasis .......................... 1 ABSTRACT ................................................................................................................................ 2 INTRODUCTION ....................................................................................................................... 2 Conservation and functional domains of PAS kinase .............................................................. 3 Activation and regulation of PAS kinase ................................................................................. 7 PAS kinase phenotypes and substrates..................................................................................... 9 CONCLUSIONS ....................................................................................................................... 16 REFERENCES .......................................................................................................................... 18 CHAPTER 2: A comprehensive protein-protein interactome for yeast PAS kinase 1 reveals direct inhibition of respiration through the phosphorylation of Cbf1 ........................................... 22 ABSTRACT ........................................................................................................................... 23 iv INTRODUCTION ..................................................................................................................... 23 RESULTS .................................................................................................................................. 25 Identification of ΔN692Psk1, a construct with increased protein-binding proficiency ............... 25 PAS kinase binding partners identified through Y2H and copurification ........................ 27 Validation of the Psk1 interactome through in vitro kinase assays ........................................ 33 Bioinformatic analysis of the Psk1 interactome revealed both known and novel functions ................................................................................................................................ 34 Mapping and in vitro verification of Cbf1 phosphosites ........................................................ 38 Evidence for Psk1-dependent respiratory inhibition through the phosphorylation and inactivation of Cbf1 ................................................................................................................ 38 DISCUSSION ............................................................................................................................ 42 MATERIALS AND METHODS .............................................................................................. 46 Yeast cells, plasmids, and culture media ................................................................................ 46 Growth assays ........................................................................................................................ 48 Y2H library generation ........................................................................................................... 49 Y2H screens ........................................................................................................................... 49 HIS, Myc, and FLAG epitope protein purification ................................................................ 50 Quantitative mass spectrometry ............................................................................................. 52 v Bioinformatic analysis............................................................................................................ 53 In vitro kinase assays.............................................................................................................. 54 Mitochondrial respiration assays ............................................................................................ 54 ACKNOWLEDGMENTS ......................................................................................................... 55 REFERENCES .......................................................................................................................... 58 CHAPTER 3: PAS kinase is activated by direct SNF1-dependent phosphorylation and mediates inhibition of TORC1 through the phosphorylation and activation of Pbp1 .................................. 64 ABSTRACT .............................................................................................................................. 65 INTRODUCTION ..................................................................................................................... 65 RESULTS .................................................................................................................................
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