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Characterization of Human Cdc14b's Function in Centrosome Cycle Control

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Characterization of Human Cdc14b's Function in Centrosome Cycle Control University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 12-2008 Characterization of Human Cdc14B's function in centrosome cycle control Jun Wu University of Tennessee - Knoxville, [email protected] Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Recommended Citation Wu, Jun, "Characterization of Human Cdc14B's function in centrosome cycle control. " PhD diss., University of Tennessee, 2008. https://trace.tennessee.edu/utk_graddiss/2789 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Jun Wu entitled "Characterization of Human Cdc14B's function in centrosome cycle control." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, with a major in Life Sciences. Yisong Wang, Major Professor We have read this dissertation and recommend its acceptance: Brynn Voy, Hayes McDonald, Bruce Mckee, Sundar Venkatachalam Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) To the Graduate Council: I am submitting herewith a dissertation written by Jun Wu entitled “Characterization of Human Cdc14B's function in centrosome cycle control.” I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, with a major in Life Sciences. Yisong Wang Major Professor We have read this dissertation and recommend its acceptance: Brynn Voy Hayes McDonald Bruce Mckee Sundar Venkatachalam Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official student records.) Characterization of Human Cdc14B's function in centrosome cycle control A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville Jun Wu December 2008 DEDICATION I dedicate this dissertation to my parents Hongfen Xu and Lusheng Wu, my wife Di Xu and daughter Olivia Wu, for their love and support. ii ACKNOWLEDGMENTS Firstly, I would like to thank my advisor, Dr. Yisong Wang, for his encouragement and guidance throughout my research, especially during difficult times in the lab due to lack of funding. I would also like to thank my committee members, Dr. Brynn Voy, Dr. W. Hayes McDonald, Dr. Bruce Mckee and Dr. Sundar Venkatachalam, who have advised me not only in my committee meetings but also in our numerous discussions along the course of my research. I thank all members of our lab for constructive advice. I am especially grateful to Dr. Yie Liu and Dr. Hyekyung P. Cho whom I have learned great amount of scientific knowledge and experimental skills. I wish to thank Dr. John Dunlap for his invaluable assistance with electronmicroscopic work. Finally, I would like to thank my friends also labmates Dr. Richard Giannone and David B. Rhee for their encouragement, support and helpful discussions. iii ABSTRACT Centrosomes are the only non-membranous organelles in most vertebrate cells and their major function is to nucleate microtubules, hence often recognized as the microtubule-organizing center (MTOC). Much like chromosome centrosome duplicates only once during the S phase of each cell cycle. The fidelity and timing of this duplication event will ensure equal division of duplicated chromosomes into the daughter cells. As a consequence, numerical and/or structural centrosome abnormalities will cause chromosome missegregation and lead to the generation of multiple mitosis and ultimately chromosomal instability, which typify many cancers. The molecular mechanism of centrosome duplication remains unclear. Previous studies found that a fraction of human proline-directed phosphatase Cdc14B associates with centrosomes. However, Cdc14B’s involvement in centrosome cycle control has never been explored. In this study, we identify Cdc14B as a negative regulator in centrosome cycle control: depletion of Cdc14B by RNA interference leads to centriole amplification in both HeLa and normal human fibroblast BJ and MRC-5 cells; ectopic expression of Cdc14B leads to stepwise loss of centrioles and attenuates centriole amplification in HU/APH arrested S phase cells and cells treated with proteasome inhibitor Z-L3VS. This inhibitory function requires centriole-associated Cdc14B catalytic activity. In addition, our data suggests counterbalancing effects between Cdc14B phosphatase and kinases such as Plk4, Cdk2/Cyclin-E/A in centrosome duplication control potentially through modulating phosphorylation status of their common downstream effectors, HsSas-6 and B23 respectively. Taken together, these results iv suggest a potential function for Cdc14B phosphatase in maintaining the fidelity of centrosome duplication cycle. v TABLE OF CONTENTS Chapter 1: Introduction to centrosome biology….…………………….……...…..…..…1 Chapter 2: Cdc14B, a primer……………………...………………………..…………...27 Chapter 3: Cdc14B associates with centriole in a cell cycle dependent manner……….50 Chapter 4: Cdc14B phosphatase is a negative regulator for centrosome duplication......83 Chapter 5: Potential counterbalance effect between Cdc14B phophatase and Plk4 kinase in regulating centrosome duplication…………………………………………………...126 Chapter 6: Role of Cdc14B phosphatase in counterbalancing Cdk2/Cyclin-E/A in centrosome cycle control through regulation of its substrates………………………….154 References……………………………………………………………………...............190 Vita………………….……………………………………………………………….....220 vi LIST OF FIGURES Figure 1.1: Centrosome structure.………………………………………………….…..…5 Figure 1.2: Schematic view of the centrosome cycle.……...................………….…….....8 Figure 1.3: Procentriole assembly in C. elegans...………………………………..…..…11 Figure 1.4: General mechanisms of cytokinesis in animal cells…...……….....................19 Figure 2.1: Substrates and functions of Cdc14 during mitosis....…...................…….…..29 Figure 2.2: The FEAR and MEN networks.......................................................................32 Figure 2.3: The SIN pathway in S. pombe....……………………………...................…..38 Figure 2.4: The cell cycle and G2 DNA damage response in human cells ………….….48 Figure 3.1: A fraction of Cdc14BWT-EGFP associates with centrioles ……………...….56 Figure 3.2: Colocalization of Cdc14BWT-EGFP and C-Nap1..…………………..……..58 Figure 3.3: Centrin-GFP vs C-Nap1DsRed during cell cycle...………….………………59 Figure 3.4: A fraction of Cdc14B associates with centrioles..……………………...……62 Figure 3.5: Colocalization of endogenous Cdc14B and C-Nap1...........................………63 Figure 3.6: Isolated centrosomes contain Cdc14B...………………….…………………65 Figure 3.7: Cdc14B centrosomal retention requires Cdc14B phosphatase activity...……68 Figure 3.8: Mapping of Cdc14B to centrioles…………………………………………...73 Figure 4.1: Centrosome duplication cycle...…………………………………………….88 Figure 4.2: Depletion of Cdc14B leads to centriole amplification in HeLa cells....….…97 Figure 4.3: Specificity of Chicken anti-Cdc14B antibody and centrosome amplification in Cdc14B knockdown cells using γ-tubulin as a centrosome marker....………………..99 vii Figure 4.4: Depletion of Cdc14B causes centriole amplification in normal human fibroblast cells…………………………………………………...……………………...103 Figure 4.5: Stepwise loss of centrioles in cells with overexpressed Cdc14BWT-EGFP...105 Figure 4.6: Cdc14B phosphatase activity is required to prevent HU-induced centriole amplification...…………………………………………………………………..……...109 Figure 4.7: Cdc14B catalytic activity is required to prevent HU-induced centrosome overduplication...………………………………………………………………..……...111 Figure 4.8: Cdc14B phosphatase activity is required to prevent Z-L3-VS–induced centriole overduplication... ...…….. ...……………………..………………...………...114 Figure 4.9: Polyploidy in Cdc14B knockdown cells ………………………………..…117 Figure 5.1: Cdc14B phosphatase suppresses centriole amplification triggered by deregulated Plk4 expression ……………...……………………………………………133 Figure 5.2: Counterbalance effect between Cdc14B and Plk4...…………...……..……136 Figure 5.3: HsSas-6 as a potential downstream effector of Cdc14B in centriole duplication control...………………………....……………….......………………..…...139 Figure 5.4: Downregulation of Cdc14B expression in G1 stabilizes HsSas-6........……141 Figure 5.5: Cdc14B antagonizes Plk4 kinase activity in centriole duplication control...148 Figure 6.1: Cdc14B phosphatase activity is required to prevent DXR-induced centriole overduplication...….........................…………...………………....…………………….164 Figure 6.2: Cdc14A interacts with B23...…......…………….......……..………....…….166 Figure 6.3: Cdc14B interacts with B23 and dephosphorylates phospho-Thr-199 of B23 in vitro..…................…………...……………....……………….......….…………..……...168 Figure 6.4: B23 as a potential substrate of Cdc14B.…………....….………..…………171 viii Figure 6.5: Cdc14B-mediated inhibition of centrosome overduplication does not depend on B23...……………………………………………………………………….....…..…174 Figure 6.6: B23 is a downstream mediator of Cdc14B in suppressing
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