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NPM/B23: the Effector of CDK2 in the Control of Centrosome Duplication and Mrna Processing

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NPM/B23: the Effector of CDK2 in the Control of Centrosome Duplication and Mrna Processing UNIVERSITY OF CINCINNATI Date: October 7th, 2004 I, ________________Yukari Tokuyama___________________________, hereby submit this work as part of the requirements for the degree of: Doctor of Philosophy in: Cell and Molecular Biology It is entitled: NPM/B23: The Effector of CDK2 in the Control of Centrosome Duplication and mRNA Processing This work and its defense approved by: Chair: Kenji Fukasawa, Ph.D. Robert W. Brackenbury, Ph.D. Wallace S. Ip, Ph.D. Erik S. Knudsen, Ph.D. Yolanda Sanchez, Ph.D. NPM/B23: THE EFFECTOR OF CDK2 IN THE CONTROL OF CENTROSOME DUPLICATION AND mRNA PROCESSING 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 Cell Biology, Neurobiology, and Anatomy of the College of Medicine 2004 by Yukari Tokuyama B.S. Texas A&M University, 1998 Committee Chair: Kenji Fukasawa, Ph.D. Robert W. Brackenbury, Ph.D. Wallace S. Ip, Ph.D. Erik S. Knudsen, Ph.D. Yolanda Sanchez, Ph.D. Abstract Nucleophosmin (NPM/B23) is a phosphoprotein predominantly localized in the nucleolus, and is believed to function in ribosome assembly, cytonuclear shuttling, and molecular chaperoning. Here we report novel functions of NPM/B23 in initiation of centrosome duplication and mRNA processing. The centrosome, a major microtubule organizing center of cells, directs the formation of bipolar mitotic spindles, which is essential for proper chromosome segregation to daughter cells. Cancer cells frequently contain abnormal numbers of centrosomes suggesting that centrosome amplification is the major contributing factor for chromosome instability. It has been shown that cyclin- dependent kinase 2 (CDK2) activity is required for initiation of centrosome duplication. NPM/B23 is a substrate of CDK2/Cyclin E in initiation of centrosome duplication. Here we identified that threonine 199 (Thr199) of NPM/B23 is the major phosphorylation site of CDK2/Cyclin E. NPM/B23 associates with single centrosomes and upon phosphorylation by CDK2/Cyclin E, NPM/B23 is lost from the centrosomes resulting in initiation of centrosome duplication. A non-phosphorylatable mutant of NPM/B23 (NPM/T199A) acts as a dominant negative, resulting in inhibition of centrosome duplication. Thus, NPM/B23 is a key regulator in initiation of centrosome duplication. We further examined the role of CDK2/Cyclin E phosphorylation of NPM/B23 on Thr199 (phospho-Thr199 NPM). We found that phospho-Thr199 NPM specifically localizes to nuclear structures known as nuclear speckles, which consist of pre-mRNA splicing factors. We found that phosphorylation on Thr199 of NPM/B23 enhances NPM/B23’s RNA binding activity. Also, non-phosphorylated NPM/B23 binds with specific splicing factors, whereas phosphorylation of NPM/B23 decreases these interactions. Moreover, we found that phosphorylation of NPM/B23 on Thr199 represses pre-mRNA splicing. These findings suggest an interesting connection between cell cycle progression and mRNA processing. We also found NPM/B23 to be ubiquitinated. NPM/B23 is maximally ubiquitinated at the G1/S transition of the cell cycle, when CDK2/Cyclin E is also maximally active. Furthermore, phosphorylation on Thr199 targets NPM/B23 for ubiquitination. Studies have shown that ubiquitin ligases such as SCF and BRCA1-BARD1, localize to centrosomes, and ubiquitin-mediated proteolysis plays an important role in centrosome duplication. Thus, it is possible that ubiquitin modification of NPM/B23 is a key event in the centrosome duplication cycle. Acknowledgments This work would not have been possible without the help, support, and guidance of so many people. Especially, I am grateful to my advisor, Dr. Kenji Fukasawa, for his guidance and patience throughout these years. He has been a great advisor, strengthening and shaping me to become a scientist. I am thankful to my committee members for all their advise and keeping me on the right path. The past and present members of the Fukasawa lab have made my graduate career enjoyable. I would especially like to thank Dr. Masaru Okuda who has taught me the basics when I first joined the lab. Also, I would like to thank Dr. Pheruza Tarapore and Dr. Henning Horn for their countless help and support…thank you for putting up with me for all these years! I would also like to thank all my friends who have supported and encouraged me throughout graduate school. I am most grateful to my friends Rick Flannery and Robin Kuns for their friendship, support and encouragement. They have always been there for me in the toughest of times, without them I am not sure if I would have made it. This dissertation is dedicated to my family (mom, dad and Mayumi). Thank you for your understanding, endless support and encouragement…words can’t really describe how grateful I am to have a wonderful family! TABLE OF CONTENTS Page List of Tables and Figures…………………………………………………………….2 Chapter I: Introduction………………………………………………………………..4 A. NPM/B23 as a multi-functional protein………………………………5 B. NPM/B23 functional domains………………………………………...7 C. NPM/B23 function is mediated by phosphorylation events…………..9 D. Centrosomes and the cell cycle……………………………………….10 E. Involvement of CDK2/Cyclin E in regulation of centrosome duplication……………………………………………..14 F. Nuclear Speckles……………………………………………………...15 G. Pre-mRNA splicing…………………………………………………...17 H. Ubiquitination……………………………………………...…………20 I. References………………………………………………………...…..22 Chapter II-IV: Results Chapter II: Specific Phosphorylation of Nucleophosmin on Thr199 by Cyclin-dependent Kinase 2-Cyclin E and Its Role in Centrosome Duplication……………………………………………….....29 A. Abstract……………………………………………………………….30 B. Introduction…………………………………………………………...32 C. Materials and Methods………………………………………………..34 D. Results ………………………………………………………………...39 E. Discussion…………………………………………………………….47 F. References…………………………………………………………….52 G. Figures and Legends……….….………...…………………………….56 Chapter III: CDK2/CyclinE-Mediated Phosphorylation on Threonine 199 of Nucleophosmin/B23 Localizes to Nuclear Speckles and Represses Pre-mRNA Splicing……………………………………...……………...70 A. Abstract……………………………………………………………….71 B. Introduction…………………………………………………………...72 C. Materials and Methods………………………………………………..74 D. Results ………………………………………………………………...79 E. Discussion…………………………………………………………….88 F. References…………………………………………………………….92 G. Figures and Legends………….……………………………………….97 Chapter IV: Ubiquitination of Nucleosphomin/B23………………….……………....107 A. Abstract……………………………………………...………………108 B. Introduction………………………………………………………….109 C. Materials and Methods………………………………………………112 1 D. Results……………………………………………………………….114 E. Discussion…………………………………………………………...117 F. References…………………………………………………………...119 G. Figures and Legends……………………….……………...………....122 Chapter V: Summary and Conclusions……………………………………………….128 A. Summary………………………………………………....…………..129 B. NPM/B23 and centrosome duplication………………………………129 C. NPM/B23 at the nuclear speckles……………………………………133 D. References……………………………………………………………137 Chapter VI: Future Directions………………………………………………………...140 LIST OF TABLES AND FIGURES Chapter I: Introduction Figure 1. The structure of NPM/B23………………………………………………….8 Figure 2. Schematic diagram of the centrosome……………………………………...11 Figure 3. The centrosome (centriole) duplication cycle………………………………13 Figure 4. Schematic diagram of nuclear speckles…………………………………….16 Figure 5. Spliceosome complex formation along splicing reaction…………………..19 Chapter II: Specific Phosphorylation of Nucleophosmin on Thr199 by Cyclin- dependent Kinase 2-Cyclin E and Its Role in Centrosome Duplication Figure 1. CDK2/cyclin E phosphorylates NPM/B23 in vitro on threonine residue(s)…………………………………………………………………...63 Figure 2. NPM/B23 is phosphorylated by CDK2/cyclin E on threonine 199 (Thr199) in vitro……………………………………………………………64 Figure 3. The site of NPM/B23 phosphorylated in vitro by CDK2/cyclin E is phosphorylated in vivo……………………………………………………...65 Figure 4. Initiation of centrosome duplication is blocked by NPM/T199A expression…………………………………….…………………………….66 Figure 5. Expression of NPM/T199A mutant specifically inhibits centrosome duplication…………………………………………….……………………67 Figure 6. Expression of NPM/T199A mutant results in a high frequency of monopolar mitosis………………………………………………………….68 Figure 7. NPM/T199A physically associates with centrosomes……………………...69 Chapter III: CDK2/CyclinE-Mediated Phosphorylation on Threonine 199 of Nucleophosmin/B23 Localizes to Nuclear Speckles and Represses Pre-mRNA Splicing Figure 1. Phospho-Thr199 NPM antibody specifically recognizes NPM/B23 phosphorylated on Thr199 by CDK2/Cyclin E..…………………………..101 2 Figure 2. Cell-cycle dependent changes in the level of phospho-Thr199 NPM……….102 Figure 3. Phospho-Thr199 NPM co-localizes with splicing factors and re-distributes upon transcription inhibition………………………………...103 Figure 4. Phosphorylation on Thr199 enhances the RNA-binding activity of NPM/B23…………………………………………………………………..104 Figure 5. Phosphorylation on Thr 199 influences association of NPM/B23 with splicing factors……………………………………………………………..105 Figure 6. Phosphorylation on Thr 199 represses splicing in vitro……...…………….106 Chapter IV: CDK2/CyclinE Phosphorylation Dependent Ubiquitination of Nucleosphomin/B23 Figure 1. Proteasome inhibitor blocks centrosome duplication………………………124 Figure 2. Proteasome inhibition prevents loss of centrosomal NPM/B23……………125 Figure 3. NPM/B23 is ubiquitinated during G1 phase in vivo………………………..126 Figure 4. In vitro ubiquitination
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