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UNIVERSITY OF CINCINNATI Date:___________________ I, _________________________________________________________, hereby submit this work as part of the requirements for the degree of: in: It is entitled: This work and its defense approved by: Chair: _______________________________ _______________________________ _______________________________ _______________________________ _______________________________ CHARACTERIZATION OF TANKYRASE STRUCTURE & FUNCTION; EVIDENCE FOR A ROLE AS A MASTER SCAFFOLDING PROTEIN A dissertation submitted to the Division of Research and Advanced Studies of the University of Cincinnati in partial fulfillment of the requirements for the degree of DOCTORATE OF PHILOSOPHY (Ph.D.) in the Department of Molecular Genetics, Biochemistry and Microbiology College of Medicine 2004 by Manu De Rycker B.S., University of Antwerp, 1995 M.S., University of Gent, 1998 Committee Chair: Carolyn Price, Ph.D. ABSTRACT Tankyrases are novel poly(ADP-ribose) polymerases that have SAM and ankyrin protein- interaction domains. They are found at telomeres, centrosomes, nuclear pores and the Golgi- apparatus, and participate in telomere length regulation and resolution of sister chromatid association. Their other function(s) are unknown and it has been difficult to envision a common role at such diverse cellular locations. We isolated the chicken tankyrase homologs and examined their interaction partners, subcellular location and domain functions to learn more about their mode of action. Cross-species sequence comparison indicated that tankyrase domain structure is highly conserved and supports division of the ankyrin domain into five subdomains, each separated by a highly conserved LLEAAR/K motif. GST-pull down experiments demonstrated that the ankyrin domains of both proteins interact with chicken TRF1. Analysis of total cellular and nuclear proteins showed that cells contain approximately twice as much tankyrase 1 as tankyrase 2. Although ≥90% of each protein is cytoplasmic, both tankyrase 1 and 2 were also nuclear. This nuclear location, together with its ability to interact with TRF1, point to a telomeric function for tankyrase 2. This work shows that tankyrases polymerize through their SAM domain to assemble large protein complexes. In vitro polymerization is reversible but still allows interaction with ankyrin-domain binding proteins. Polymerization also occurs in vivo, with SAM-dependent association of overexpressed tankyrase leading to the formation of large tankyrase-containing vesicles, disruption of Golgi structure and inhibition of apical secretion. Finally, tankyrase polymers are dissociated efficiently by poly(ADP-ribosy)lation. This disassembly is prevented by mutation of the PARP domain. Our findings indicate that tankyase 1 promotes both assembly and disassembly of large protein complexes. Thus, tankyrases appear to be master scaffolding proteins that regulate the formation of dynamic protein networks at different cellular locations. This implies a common scaffolding function for tankyrases at each location with specific tankyrase interaction partners conferring location-specific roles to each network, such as telomere compaction or regulation of vesicle trafficking. ACKNOWLEDGEMENTS First of all I would like to thank my advisor, Carolyn Price. She has not only given me the freedom to venture far away from the telomeres, but has also joined me in my excitement about the project. She has been a great guide in many ways, and I thank her for her advice, trust and support and for being a great friend. I would also like to thank my committee members, Yoli Sanchez, Joanna Groden, Jim Stringer and Jun Ma, for providing direction and thought-provoking questions during and outside our committee meetings. Thanks to all the members of the Price lab, past and present, for providing a great atmosphere to work in. Special thanks to Angela and Fred, for putting up with me (and my radio) and for being great friends. I thank the Albert J. Ryan foundation for their support. Many friends have made these five years fun and they will be dearly missed. Special thanks to Robyn and Tim for all the great times we had together. It is hard to express how much I am indebted to my parents, Paul and Mickie, and my sister, Kim. They have supported me in countless ways during these many years of study, and I can’t wait to spend more time with them the coming years. Finally I would like to thank the most important person in my life. Words are inadequate to express how much it means to me that Sandra stayed here with me for all these years. She moved almost 7000 kilometers from home to be with me, to support me and to enjoy life together. Knowing that I would see her every day has been my single greatest driving-force. TABLE OF CONTENTS 1. INTRODUCTION .................................................................................................................. 5 1.1. Tankyrase domain structure............................................................................................ 6 1.1.1. Ankyrin repeat domain ........................................................................................... 6 1.1.2. The Sterile-Alpha-Motif (SAM)............................................................................. 9 1.1.3. The poly-(ADP-ribose) polymerase (PARP) domain........................................... 11 1.1.4. Conclusions........................................................................................................... 14 1.2. Tankyrase functions...................................................................................................... 15 1.2.1. Function at telomeres............................................................................................ 15 1.2.2. Function in vesicle trafficking and insulin signaling............................................ 17 1.2.3. Other functions......................................................................................................20 2. GOAL OF THESIS............................................................................................................... 22 2.1. Hypothesis..................................................................................................................... 22 2.2. Overview....................................................................................................................... 22 3. MATERIALS AND METHODS.......................................................................................... 24 3.1. Cell lines....................................................................................................................... 24 3.2. Isolation of chicken tankyrase genes ............................................................................ 24 3.3. Genomic library screen and knock-out construct ......................................................... 24 3.4. Electroporations ............................................................................................................ 26 3.5. Analysis of DT40 genomic DNA by Southern blot...................................................... 27 3.6. Antibodies and protein detection .................................................................................. 28 3.7. Expression constructs....................................................................................................30 1 3.8. Protein expression and purification .............................................................................. 32 3.9. GST pull-downs............................................................................................................ 35 3.10. Radioligand binding assays .......................................................................................... 36 3.11. Yeast two-hybrid analysis of SAM self-association..................................................... 36 3.12. Gel filtration.................................................................................................................. 37 3.13. Electron microscopy ..................................................................................................... 38 3.14. Pelleting and dilution assays......................................................................................... 39 3.15. Poly-(ADP-ribose) binding assay ................................................................................. 40 3.16. Tankyrase activity assay ............................................................................................... 40 3.17. Co-immunoprecipitations ............................................................................................. 40 3.18. Immunofluorescence..................................................................................................... 41 3.19. Yeast two-hybrid screen ............................................................................................... 42 4. CHARACTERIZATION OF THE CHICKEN TANKYRASES. ........................................ 44 4.1. Summary....................................................................................................................... 44 4.2. Introduction................................................................................................................... 45 4.3. Results........................................................................................................................... 47 4.3.1. Identification of chicken tankyrase genes............................................................. 47 4.3.2. Conservation of tankyrase proteins......................................................................
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