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Identification of Atat1 As a Major Alpha-Tubulin Acetyltransferase

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Identification of Atat1 As a Major Alpha-Tubulin Acetyltransferase Identification of Atat1 as a major alpha-tubulin acetyltransferase dispensable for mouse development Go Woon Kim Department of Anatomy and Cell Biology Faculty of Medicine McGill University Montreal, Quebec, Canada December 2012 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Master of Science © Go Woon Kim 2012 ABSTRACT Post-translational modification is one crucial cellular mechanism through which proteins are altered in structure and function. A variety of proteins different in function and cellular localization is affected by such modification. With a wide range of influence within a cell, lysine acetylation has emerged as one major post- translational modification that rivals to phosphorylation. Indeed, recent proteomic studies have revealed that lysine acetylation affects 5-10 % of mammalian and bacterial proteins. This modification is catalyzed by the opposing actions of lysine acetyltransferases and deacetylases. One of the first acetylated proteins identified in the mid-1980s is α-tubulin, a subunit of microtubules that comprise the cytoskeletal network. Although deacetylation of α-tubulin is well known to be catalyzed by HDAC6, the responsible acetyltransferase remained elusive until recently. The recent discovery of Atat1 (α-tubulin acetyltransferase 1) has yielded important insights into the role of acetylated α-tubulin in lower organisms such as C. elegans, but it remains unclear whether this is also the case in vivo in higher organisms such as mammals. Here, using mice devoid of Atat1, I demonstrate that Atat1 is a major acetyltransferase of α-tubulin dispensable for mouse development. I also show that Atat1 is highly expressed in testis, renal pelvis, gastrointestinal tract, and hippocampus, but development of these tissues appears normal in the absence of Atat1. Moreover, in a set of molecular and cellular studies, I have identified ATAT1 as a potential interaction partner of several proteins. Together, these findings provide direct support of Atat1 as an authentic II α-tubulin acetyltransferase, thereby setting up a solid foundation for additional studies of this enzyme and tubulin acetylation in mice and humans. III RÉ SUMÉ La modification post-traductionnelles des protéines est un mécanisme cellulaire essentiel par laquel la structure et la fonction des protéines peuvent-être affectées. Les modifications sont présentes dans la grande majorité de voie cellulaire et les protéines cibles possèdent une grande diversité de fonction et de localisation cellulaire. L’acétylation des lysines a émergé comme une modification de grande importance, ayant un impact majeur sur les cellules, à l’instar de la phosphorylation. Effet, des études récentes de protéomique ont révélées que de 5 à 10 % des protéines bactériennes et animales étaient affectées par l’acétylation des lysines. Cette mondification est catalysé par l’effet inverse de deux classes de protéines, les lysines acétyltransférase et les déactylases. L'une des premières protéines identifiées dans acétylés milieu des années 1980 est l’α- tubuline, une sous-unité des microtubules qui composent le réseau du cytosquelette. Malgré que l’enzyme responsable de la déacétylation de l’α- tubuline est connue sous le nom HDAC6, l’enzyme responsable de son acétylation est restée inconnue jusqu’à récemment. La découverte d’Atat1 (α- tubuline acétyltransférase1) a donné des informations importantes sur le rôle de l’ aétylation sur l’α-tubuline dans les organismses inférieurs comme C. elegans, mais il reste difficile de savoir si c’est aussi le cas in vivo dans les organismes supérieurs commes les mammifères. En utilisant des modèles murin de Atat1, je démontre que Atat1 est une acétyltransférase majeur de l’α-tubuline dispensable pour le développement normal. De plus, je démontre que le niveau d’expression d’Atat1 est particulièrement élevé dans les testicules, le bassinet du rein, le tractus IV gastro-intestinal, et l’hippocampe, pourtant, le développement de ces tissus semble normal en l'absence de Atat1. En outre, dans une série d'études moléculaires et cellulaires, j'ai identifié ATAT1 comme un partenaire potentiel d'interaction de plusieurs autres protéines. Ces résultats non seulement apporter un soutien supplémentaire de la Atat1 comme une acétyltransférase authentique de l’α-tubuline, mais aussi mettre en place une bonne base pour des études supplémentaires de cette protéine chez les souris et les humains. V ACKNOWLEDGEMENTS This thesis work presented herein has been completed with the help of many people. Firstly, I would like to thank my supervisor, Dr. Xiang-Jiao Yang for guiding me during the course of the studies and providing valuable suggestions and advice. His vast amount of experience and passion for science also helped me gain insight into the scientific research in depth. No matter whether it is short or long, science-related or not, discussion with him always made me learn and stimulated me to do better. Secondly, I would like to thank my colleagues, former and current, including Dr. H. Taniguchi, Dr. D. Walkinshaw, Dr. S. Tahmasebi, Dr. J. Nie, G. Gocevski, R. Weist, Y. Lu, C.J. Wu, K. Yan, L. You, H. Salem, M. Ghorbani, T. Sabri, H.I. Kim, L. Chen, H.Y. Ham, and many others for sharing scientific knowledge and laughs during the course of my research. I am particularly grateful to Dr. Taniguchi for teaching me very patiently about various techniques, especially those related to mice, in the first six months or so after I joined the lab. The experience that I have had with him and others in the lab is one memory so valuable that it can never be forgotten. Thirdly, special thanks should be given to M. Flamand in the neighbor lab for French translation of the abstract. I am also thankful to Dr. Craig Mandato, Dr. Stefano Stefani, and Dr. René St-Arnaud for serving as my mentor/committee members. Their kind and intellectual suggestions helped improve the research to be described herein. I would like to thank Dr. Jaeok Park for carefully reading and editing a close-to-final version of this thesis. VI Finally, I must thank all my friends and family, who supported me with never-failing love in every situation I have been. Their constant support equipped me with courage and strength to step forward to this achievement. The research presented here was supported by funding from CIHR, NSERC, and MDEIE (to Dr. X.J. Yang). VII PUBLICATIONS 1. Kim, G.W., et al., Dietary, metabolic, and potentially environmental modulation of the lysine acetylation machinery. Int J Cell Biol, 2010. 2010: p. 632739. 2. Kim, G.W. and X.J. Yang, Comprehensive lysine acetylomes emerging from bacteria to humans. Trends Biochem Sci, 2011. 36(4): p. 211-20. 3. Aka, J.A., G.W. Kim, and X.J. Yang, K-acetylation and its enzymes: overview and new developments. Handb Exp Pharmacol, 2011. 206: p. 1-12. VIII CONTRIBUTIONS OF AUTHORS CHAPTER II: Identification of Atat1 as a major α-tubulin acetyltransferase dispensable for mouse development I performed most of the experiments presented in this chapter, with several expression constructs given by others (see below). I also prepared all the figures and wrote the manuscript. In collaboration with Dr. Yang, I designed the experiments, edited, and finalized the manuscript. Dr. Jianyun Nie prepared the HA-AKAP8, HA-AKAP8L, HA-TUBB4, HA- TIF1β, and HA-MAGED2 expression constructs. Dr. Ed Seto and his colleagues at University of South Florida engineered the HA-RAC1 and FLAG-cortactin expression constructs. The FLAG-HSP90 plasmid construct was provided by Dr. Jason Young at McGill University. The mass spectrometry shown in Table 1 was carried out in collaboration with Dr. Jin Jing in Dr. Tony Pawson’s laboratory at University of Toronto. IX TABLE OF CONTENTS ABSTRACT................... ......................................................................................... II RÉSUMÉ .............................................................................................................. IV ACKNOWLEDGEMENTS.... .................................................................. ............VI PUBLICATIONS ................................................................................. ...............VIII CONTRIBUTIONS OF AUTHORS ..................................................... ...............IX TABLE OF CONTENTS ......................................................................... ...............X LIST OF ABBREVIATIONS...... ........................................................ ................XII CHAPTER I Literature Review 1. Post-translational modifications. ......................................................................2 1.1 Cellular function of post-translational modifications ..................................3 1.2 Lysine acetylation .........................................................................................3 2. Lysine acetyltransferases and deacetylases ..................................................4 2.1 Families of lysine acetyltransferases....... ....................................................5 2.2 Histone deacetylase superfamily ..................................................................5 2.3 Lysine acetylation in different cellular processes ......................................6 2.3.1 Lysine acetylation in chromatid cohesion complex ...............................6 2.3.2 Lysine acetylation in metabolism .............................................................7 2.3.3 Lysine acetylation in other cytoplasmic events ......................................8
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