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A Dissertation entitled Regulation and Post-translational modifications of Borealin by Dipali A. Date Submitted to the Graduate Faculty as partial fulfillment of the requirements for The Doctor of Philosophy Degree in Biology ______________________________________________ Dr. William R. Taylor, Committee Chair ______________________________________________ Dr. Patricia Komuniecki, Dean College of Graduate Studies The University of Toledo August 2010 ii An Abstract of Regulation and Post-translational modifications of Borealin by Dipali A. Date As partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biology The University of Toledo August 2010 Cancer occurs when normal regulation of the cell division cycle is disrupted by genetic or environmental factors. Hence, understanding the molecular mechanisms that regulate cell division is essential for the development of anti-cancer therapeutics. Borealin/Dasra B/CDCA8 (Cell Division Cycle Associated 8) is a member of the chromosomal passenger complex (CPC) also composed of Aurora B, INCENP and Survivin. The CPC exhibits a dynamic pattern of localization during mitosis and plays important roles in chromosome segregation, spindle assembly checkpoint (SAC) and cytokinesis. We identified Borealin to be an E2F/Rb target; several genes repressed by Rb dependent pathways are highly expressed in various cancers. We observed that Borealin expression was elevated in lymphomas, brain, and colon cancers and down regulated in response to DNA damage in a p53 dependent manner. Borealin is regulated in a cell cycle dependent manner and we show that it is degraded by the 26S proteasome during the cell cycle. We identified a putative stability region between amino acids 141-168 that protects Borealin from proteolytic degradation. Further, over expression of CDH1, an activator of the Anaphase promoting complex (APC/C) caused a minimal decrease in Borealin levels. iii Therefore Borealin may be targeted by an E3 ligase other than APC/C. Post-translational modifications of the passenger proteins are essential in the regulation of the CPC. We observed that Borealin is phosphorylated in vivo, in response to increased expression of constitutively active Cdk1/Cyclin B1. In addition, we observed a reduced level of slow migrating phosphorylated Borealin species upon treatment with a combination of purvalanol (Cdk1 inhibitor) and ZM447439 (Aurora B inhibitor). However, Borealin was inefficiently phosphorylated by CDK1 in vitro. We further investigated candidate phosphatases belonging to the Cdc14 family. Immunofluorescence analysis revealed that Cdc14B and Borealin co-localize to the nucleolus of interphase cells. Conversely, overexpression of Cdc14B only caused a subtle decrease in mobility shift characteristic of Borealin phosphorylation. Also, Borealin was still dephosphorylated in cells lacking Cdc14B. However, shRNA mediated depletion of Cdc14A did induce phosphorylation of endogenous Borealin. Hence, Borealin maybe dephosphorylated by Cdc14A, while CDK1 and Aurora B may exert a combinatorial effect to induce phosphorylation of mitotic Borealin. iv Acknowledgments I am perpetually grateful to my advisor Dr. William R. Taylor for guiding me through every step of acquiring this degree. I am thankful to my committee members for their guidance towards the completion of my degree. I am especially thankful to Dr. Deborah Chadee and Dr.Song-Tao Liu for helpful discussions about my project. I would like to express my sincere gratitude towards my friends and lab mates Megan Drier and Michael Beiker for helping me conduct the experiments and survive through the ones that failed. In addition, I would like to acknowledge the past lab members Cara Jacobs and Harpreet Kaur for their work on the projects that I later continued. I would also like to acknowledge all the undergraduate students for making the laboratory work a fun experience. Finally, this dissertation would not have been possible without the love and support of my parents Mr. and Mrs. Ashok and Vaishali Date. A special thanks to my fiancé Preshit Gawade for being my support and motivation through this journey. v Table of Contents Abstract iii Acknowledgement v Table of contents vi List of Figures ix List of Abbreviations xi I. Introduction 1. Cell division and Cell Cycle Checkpoints 1 2. Chromosomal Passenger Complex 5 3. Regulation of Borealin 12 4. Post translational modifications of Borealin 16 II. Hypothesis 25 III. Materials and Methods 1.Cell lines and culture conditions 26 2.Drug treatments 26 3. Antibodies 27 4. Western Blotting 28 4. Transient Transfections 28 5. Immunofluoresence 29 vi 6. Invitro kinase Assay 30 7. Generation of recombinant adenoviruses 31 8. Immunoprecipitation 31 9. Generation of Cdc14 depleted cell lines 32 10. Analysis of DNA synthesis 33 IV. Results 1. Regulation of Borealin 1.1 Borealin is downregulated upon DNA damage 34 1.2 Borealin is downregulated in a p53 dependent manner 37 1.3 Borealin is downregulated in a Rb dependent manner 39 1.4 Effect of proteasome inhibition on the levels of endogenous 41 Borealin 1.5 Association of Borealin with Ubiquitin 46 2. Phosphorylation of Borealin 2.1 Borealin is phosphorylated in response to CDK1 51 overexpression in vivo 2.2 Borealin is not an optimal substrate for CDK1 in vitro 55 2.3 Effect of kinase inhibitors on Borealin phosphorylation 59 2.4 Effect of PLK1 overexpression on Borealin phosphorylation 62 3. Dephosphorylation of Borealin 3.1 Kinetics of Borealin dephosphorylation 64 3.2 Borealin and Cdc14B co-localize to the nucleolus of 66 interphase cells vii 3.3 Effect of Cdc14 overexpression on Borealin 68 3.4 Cdc14B does not mediate proteasome mediated degradation of 71 Borealin 3.5 Analysis of Cdc14 depletion on the status of Borealin 74 phosphorylation V. Discussion 76 VI. Conclusion 87 VII. References 89 VIII. Appendix 1. Analysis of Borealin phosphorylation with a phospho specific 96 antibody 2. Analysis of Borealin phosphorylation in cells over expressing 96 Borealin viii List of Figures 1. Stages of Cell Cycle 4 2. Structure of the CPC 7 3. Localization of the CPC 11 4. Potential degradation sites in Borealin 15 5. Phosphorylation sites mapped in Borealin 19 6. The effects of DNA damage on the levels of p53 and Borealin 35 7. The effect of DNA damage on Borealin levels in HT1080 cells 36 8. p53 and p21/waf1 are required for Borealin down-regulation in response 38 to DNA damage 9. Borealin is down-regulated in a Rb-dependent manner 40 10. Effect of proteasome inhibition on the levels of Borealin 42 11. Effect of proteasome inhibition on the levels of Borealin 44 12. Association of Borealin with ubiquitin 47 13. Effect of APC co-activators on Borealin 49 14. Characterization of recombinant adenoviruses 53 15. The effect of CDK1 on phosphorylation of Borealin in vivo 54 16. Borealin is not an optimal CDK1 substrate in vitro 56 17. CDK1 does not phosphorylate a Borealin peptide encompassing S219 58 ix 18. Effect of kinase inhibitors on Borealin phosphorylation during S-phase 60 19. Effect of kinase inhibitors on the phosphorylation of Borealin during 61 mitosis 20. Effect of PLK1 over expression on Borealin phosphorylation 63 21. Kinetics of Borealin phosphorylation 65 22. Borealin and Cdc14B co-localize to the nucleolus of interphase cells 67 23. Effect of Cdc14B overexpression on Borealin phosphorylation 69 24. Cdc14B does not mediate proteasome- mediated degradation of 73 Borealin 25. Effect of Cdc14 knock down on Borealin phosphorylation 75 26. Borealin down -regulation in response to DNA damage by an indirect 81 multi-component system 27. Analysis of Borealin phosphorylation with a phospho-specific antibody 98 28. Analysis of Borealin phosphorylation in cells overexpressing Borealin 100 x List of Abbreviations APC/C Anaphase Promoting Complex/Cyclosome ATM Ataxia-telangiectasia ATR Ataxia -Rad3-related protein ATP Adenosine triphosphate BIR domain baculovirus IAP repeat domain BPB Bromo phenol blue BrDu Bromodeoxyuridine BSA Bovine serum albumin BUB1/ 3 budding uninhibited by benzimidazole 1/3 BUBR1 budding uninhibited by benzimidazole related 1 CBF CCAAT-binding Factor CDK Cyclin dependent kinase CDK1-AF Cyclin dependent kinase T14A Y15F CDCA8 Cell Division Cycle Associated 8 CDC Cell Division Cycle CHK1/2 Csk homologous kinase 1 and 2 Clp1 Cdc14-related protein phosphatase 1 CMV Cytomegalovirus CPC Chromosomal Passenger Complex DAPI 4’, 6- Diamidino-2-phenylindole D box Destruction box DNA Deoxyribonucleic acid DMEM Dulbecco’s modified Eagle’s medium DMSO Dimethyl Sulfoxide DTT Dithiothreitol ECT2 Epithelial cell transforming gene 2 EDTA Ethylenediaminetetraacetic acid ERK1/ 2 Extracellular signal related kinases 1 and 2 E2F E2F promoter binding factor FBS fetal bovine serum GFP Green Fluorescent Protein GST Glutathione S transferase HA Hemagglutinin hDM2 Human double minute 2 HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HP-1 Heterochromatin protein-1 xi HRP Horse radish peroxidase HU Hydroxyurea IAP inhibitor of apoptosis INCENP Inner Centromeric Protein JNK c-Jun N terminal kinase MAD 1/2 mitotic arrest deficient 1 and 2 MAPK Mitogen activated protein kinase MCAK Mitotic centromere-associated kinesin MEK 1/2 MAPK/Erk kinase 1 and 2 MEF Mouse embryonic fibroblasts MgcRacGAP Male germ cell Rac-GTPase-activating protein MgCl2 Magnesium chloride MKLP1/ 2 Mitotic Kinesin-Like Protein 1 and 2 MOI Multiplicity of infection MPS1 Multipolar Spindle 1 Myt1 Membrane-associated inhibitory kinase NaCl Sodium Chloride NaF Sodium fluoride
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  • Discovery of BPR1K871, a Quinazoline Based

    Discovery of BPR1K871, a Quinazoline Based

    www.impactjournals.com/oncotarget/ Oncotarget, 2016, Vol. 7, (No. 52), pp: 86239-86256 Research Paper Discovery of BPR1K871, a quinazoline based, multi-kinase inhibitor for the treatment of AML and solid tumors: Rational design, synthesis, in vitro and in vivo evaluation Yung Chang Hsu1,*, Mohane Selvaraj Coumar2,*, Wen-Chieh Wang1,*, Hui-Yi Shiao1,*, Yi-Yu Ke1, Wen-Hsing Lin1, Ching-Chuan Kuo1, Chun-Wei Chang1, Fu-Ming Kuo1, Pei-Yi Chen1, Sing-Yi Wang1, An-Siou Li1, Chun-Hwa Chen1, Po-Chu Kuo1, Ching- Ping Chen1, Ming-Hsine Wu1, Chen-Lung Huang1, Kuei-Jung Yen1, Yun-I Chang1, John T.-A. Hsu1, Chiung-Tong Chen1, Teng-Kuang Yeh1, Jen-Shin Song1, Chuan Shih1, Hsing-Pang Hsieh1,3 1Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Zhunan, Taiwan, ROC 2Centre for Bioinformatics, School of Life Sciences, Pondicherry University, Kalapet, Puducherry, India 3Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan, ROC *These authors contributed equally to this work Correspondence to: Hsing-Pang Hsieh, email: [email protected] Keywords: acute myeloid leukemia, aurora kinase, FLT3, quinazoline, multi-kinase inhibitor Received: July 25, 2016 Accepted: November 07, 2016 Published: November 15, 2016 ABSTRACT The design and synthesis of a quinazoline-based, multi-kinase inhibitor for the treatment of acute myeloid leukemia (AML) and other malignancies is reported. Based on the previously reported furanopyrimidine 3, quinazoline core containing lead 4 was synthesized and found to impart dual FLT3/AURKA inhibition (IC50 = 127/5 nM), as well as improved physicochemical properties. A detailed structure-activity relationship study of the lead 4 allowed FLT3 and AURKA inhibition to be finely tuned, resulting in AURKA selective (5 and 7; 100-fold selective over FLT3), FLT3 selective (13; 30-fold selective over AURKA) and dual FLT3/AURKA selective (BPR1K871; IC50 = 19/22 nM) agents.