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Functional Characterization of Human Adck3 and Adck4, Mitochondrial Atypical Kinases

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Functional Characterization of Human Adck3 and Adck4, Mitochondrial Atypical Kinases FUNCTIONAL CHARACTERIZATION OF HUMAN ADCK3 AND ADCK4, MITOCHONDRIAL ATYPICAL KINASES by Brody Wheeler A thesis submitted to the Department of Biomedical and Molecular Sciences In conformity with the requirements for the degree of Master of Science Queen’s University Kingston, Ontario, Canada November, 2015 Copyright © Brody Wheeler, 2015 Abstract AarF domain containing kinases 3 and 4 (ADCK3 and ADCK4, respectively) are paralogous human mitochondrial proteins, co-orthologs of the yeast protein Coq8 and bacterial protein UbiB. These proteins are required in the biosynthesis of coenzyme Q, a lipid-soluble electron carrier well known for its role in energy metabolism, more specifically in the electron transport chain. Patients with mutations in ADCK3 experience an onset of neurological disorders from childhood, including cerebellar ataxia and exercise intolerance. Mutations in ADCK4 cause steroid-resistant nephrotic syndrome, and are also associated with coenzyme Q deficiency. Aside from this minimal characterization at the biochemical level, the precise biological functions of both ADCK3 and ADCK4 remain poorly understood. After extensive screening for soluble recombinant protein expression, N-terminal fusions with maltose-binding protein were found to facilitate the overexpression of human ADCK3 and ADCK4 truncations in Escherichia coli as soluble and biologically active entities. For the first time, our work revealed Mg2+-dependent ATPase activity of ADCK3, providing strong support for the theoretical prediction of these proteins being functional atypical kinases. Subsequent experimentation confirmed protein kinase activity for both ADCK3 and ADCK4. This observed kinase activity was inhibited by the presence of an atypical N-terminal extension – containing an invariant KxGQ motif – found within the kinase domains of these proteins, suggesting an autoinhibitory role for this motif. Additionally, ADCK3 was shown, through radiometric kinase assays, to phosphorylate a peptide corresponding to a potential phosphorylation site in ATP synthase F0 subunit 8, providing insight into a potential role of this atypical kinase family. i Acknowledgements I would like to express great appreciation and thanks to several people whom I have worked with during my time at Queen’s University. Without the support of the following people, much of my work would have not been possible. First, I would like to thank my supervisor Dr. Zongchao Jia. Dr. Jia has helped me both on an academic and on a personal level countless times over the past two years. Due to his acceptance and guidance, I have developed several practical skills in the field of biochemistry that I intend to use in my future endeavours. His continuous positive outlook on my project and support of my efforts have been crucial to any success I have achieved. I will be forever grateful for the experience he has provided me. I would like to thank the other members of the Jia lab, all of which have positively impacted both my project and experience at Queen’s University. A special thanks goes out to Natalie Roy for her technical assistance and friendship, both of which are extremely valuable. Thanks to Mona Rahman, Laura van Staalduinen, and Chelsy Prince for using their personal lab experiences to provide advice and suggestions that helped me greatly in several aspects of my project. Chelsy deserves additional thanks for constructing modified vectors for our lab that were extremely useful for my project. I would like to thank Gregory Hicks, Yichen “Jerry” Zhang and Michael Lee for their valuable conversations, and overall positive contributions to my time spent in the lab. I would also like to thank Alexander Andrew, who has become a great friend of mine, for all of his advice and support throughout my project. A special thanks goes out to the members of my supervisory committee, Dr. Robert Campbell and Dr. Graham Côté, for the guidance they provided and the valuable discussions we have had. I would like to also thank Dr. Côté for use of both his laboratory and valuable reagents ii to conduct several of my functional assays. A very special thank you goes to Yidai Yang, who sacrificed much of her time to help teach me both how to perform ATPase and kinase assays, as well as how to interpret the resultant data. Without her help, my project would not have been possible. I would also like to thank Kim Munro from the Protein Function Discovery facility at Queen’s University. Not only was he extremely helpful with conducting circular dichroism and isothermal calorimetry experiments, he was great at teaching how the instrumentation functioned. We have also had many great conversations over the past two years, and I would like to thank him for being yet another positive contributor to my experience here. I would like to express my thanks to Dr. Steven Pelech, Catherine Sutter and the rest of the technical staff at Kinexus Bioinformatics Corporation. Without their peptide synthesis and kinase expertise, peptide substitution analysis experiments would not have been possible. Finally, I would like to thank my family. I am forever grateful for the endless love and support I continue to receive from my family. I would not be here if it weren’t for them. From the bottom of my heart, thank you for everything. Thanks everyone. iii Table of Contents Abstract ............................................................................................................................................ i Acknowledgements ......................................................................................................................... ii List of Figures ................................................................................................................................ vi List of Tables ................................................................................................................................ vii List of Abbreviations ................................................................................................................... viii Chapter 1: Introduction ................................................................................................................... 1 1.1 Protein Kinases ...................................................................................................................... 1 1.1.1 Overview ........................................................................................................................ 1 1.1.2 Defining Characteristics ................................................................................................. 4 1.1.3 Atypical Kinases ............................................................................................................. 7 1.2 ADCK Proteins ..................................................................................................................... 8 1.2.1 ADCK Proteins ............................................................................................................... 8 1.2.2 Classification of ADCK proteins as atypical kinases ..................................................... 9 1.2.3 Structure of ADCK3 ..................................................................................................... 12 1.2.4 Activity of ADCK3 mutants ......................................................................................... 15 1.3 Project overview .................................................................................................................. 16 Chapter 2: Methods ....................................................................................................................... 19 2.1 Construction of ADCK3 and ADCK4 expression vectors .................................................. 19 2.2 Site-directed mutagenesis .................................................................................................... 20 2.3 Protein Expression ............................................................................................................... 21 2.4 Protein Purification ............................................................................................................. 22 2.5 Analyzing protein folding of ADCK3 via circular dichroism ............................................. 23 2.6 Crystallization Screening .................................................................................................... 24 2.7 Assaying ATPase activity ................................................................................................... 24 2.8 Assaying protein kinase activity ......................................................................................... 26 2.9 Peptide substitution analysis ............................................................................................... 27 Chapter 3: Results ......................................................................................................................... 29 3.1 Expression and purification of ADCK3 250-523 ................................................................ 29 3.2 Analyzing secondary structure of ADCK3 250-523 ........................................................... 32 iv 3.3 ATPase activity assays ........................................................................................................ 34 3.4 Crystallization of MBP-ADCK3 250-523 ........................................................................... 37 3.5 Expression and purification of ADCK3 258-644 and ADCK4 137-524 ............................ 38
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