DOCSLIB.ORG

CHK2 Is Negatively Regulated by Protein Phosphatase 2A

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
CHK2 Is Negatively Regulated by Protein Phosphatase 2A University of South Florida Scholar Commons Graduate Theses and Dissertations Graduate School 5-31-2010 CHK2 is Negatively Regulated by Protein Phosphatase 2A Alyson Freeman University of South Florida Follow this and additional works at: https://scholarcommons.usf.edu/etd Part of the American Studies Commons, and the Biology Commons Scholar Commons Citation Freeman, Alyson, "CHK2 is Negatively Regulated by Protein Phosphatase 2A" (2010). Graduate Theses and Dissertations. https://scholarcommons.usf.edu/etd/3443 This Dissertation is brought to you for free and open access by the Graduate School at Scholar Commons. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. CHK2 is Negatively Regulated by Protein Phosphatase 2A by Alyson K. Freeman A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Cancer Biology College of Arts and Sciences University of South Florida Major Professor: Alvaro N. A. Monteiro, Ph.D. Gary W. Litman, Ph.D. Gary W. Reuther, Ph.D. Kenneth L. Wright, Ph.D. Date of Approval: January 14, 2010 Keywords: DNA damage response, phosphorylation, kinase, ionizing radiation, DNA double-strand break © Copyright 2010, Alyson K. Freeman DEDICATION To my husband Scott N. Freeman, my parents James P. and Carolyn C. Fay, and my sister Erin M. Fay. ACKNOWLEDGEMENTS I first and foremost would like to thank my mentor, Dr. Alvaro Monteiro. I could not have asked for a better experience in graduate school and I am grateful for all that I have learned from him. I would also like to thank my committee members Dr. Gary Litman, Dr. Gary Reuther, and Dr. Kenneth Wright for their guidance and expertise over the years. I thank Dr. Fred Bunz for taking the time to serve as the outside chairperson for my defense. Thank you to former lab members Marcelo Carvalho, Virna Dapic, Sylvia Marsillac, Jonathan Rios-Doria and current members Huey Nguyen, Melissa Price, Michael Sweet, Aneliya Velkova, and Nicholas Woods for their help, the great scientific discussions, and for making the lab a enjoyable place to be. I thank Cathy Gaffney for her support and all the work she does for the Cancer Biology Program. I want to extend a special thank you to my husband, Scott Freeman, who has given me endless support and advice as someone who went through this exact process two years before me. TABLE OF CONTENTS LIST OF FIGURES iv LIST OF ABBREVIATIONS vi ABSTRACT viii INTRODUCTION 1 DNA Damage Response Pathway 1 Sensors - Mre11-Rad50-Nbs1 and Rad9-Rad1-Hus1 Complexes 4 Signal Transducing Kinases - ATM and ATR 4 Effector Kinases - CHK1 and CHK2 5 Mediators - H2AX, BRCA1, MDC1, and 53BP1 7 Effector Proteins - Cdc25, E2F1, and p53 8 Checkpoint Kinase CHK2 10 CHK2 Structure and Activation 10 CHK2 in Cancer 13 CHK2 Phenotype 15 Protein Serine/Threonine Phosphatases 17 PP2A Structure and Regulation 17 PP2A Function 21 PP2A in Cancer 22 Protein Serine/Threonine Phosphatases in the DNA Damage Response 23 DNA Damage Response Proteins that are Negatively Regulated 23 ATM and ATR 25 RPA 26 CHK1 26 CHK2 28 H2AX 31 P53 32 DNA Damage Response Proteins Whose Activities are Enhanced 35 ATM 35 ATR 36 DNA-PK 36 BRCA1 37 P53 38 i Summary and Rationale 38 MATERIALS AND METHODS 40 Cloning 40 Yeast Two-Hybrid 40 Cell Culture, Transfection, and Reagents 41 Immunoprecipitations 41 Immunoblotting 42 Kinase Activity Assay 43 Subcellular Fractionation 44 Phosphatase Activity Assay 44 Protein Stability 45 RESULTS 46 CHK2 Binds to the B'α Subunit of PP2A 46 CHK2 Interacts with the B'α Subunit of PP2A in Yeast 46 CHK2 and B'α Bind in Mammalian Cells 51 Binding Between CHK2 and PP2A is Modulated by DNA Damage 54 B'α and CHK2 Dissociate upon IR-induced DNA Damage 54 CHK2 Binds to Many PP2A Subunits 58 B'α and CHK2 are Dissociated After DNA Damage Caused by IR and Doxorubicin, but not B'α and CHK1 61 Analysis of the IR-induced Dissociation of CHK2 and B'α 63 Dissociation is an Early Event and Occurs in a Dose-dependent Manner 63 Re-Association of CHK2 and B'α does Not Correlate With Resolution of DNA Damage 67 The Activity of ATM, but not PP2A, is Necessary for the Dissociation of CHK2 and B'α 71 Mutation of CHK2 Serines 33 and 35 Affects Binding to B'α 75 Analysis of the Effects of CHK2 on PP2A 77 CHK2 can Phosphorylate B'α in vitro 77 PP2A Localization is Not Influenced by IR 79 PP2A Activity is Unaffected by IR and is Not Influenced by CHK2 82 Analysis of the Effects of PP2A on CHK2 87 Changes in CHK2 Phosphorylation Due to IR 87 Changes in CHK2 Kinase Activity after IR 91 Mutation of CHK2 Serines 33 and 35 Affects Protein Stability 94 ii DISCUSSION 97 LIST OF REFERENCES 103 ABOUT THE AUTHOR END PAGE iii LIST OF FIGURES Figure 1. The DNA damage response pathway. 3 Figure 2. The structure of CHK2. 12 Figure 3. PP2A holoenzyme structure. 20 Figure 4. Protein serine/threonine phosphatases in the DNA damage response pathway. 24 Figure 5. B'α binds to CHK2 amino acids 1-107. 49 Figure 6. CHK2 binds to B'α amino acids 89-322. 50 Figure 7. CHK2 binds to FLAG-B'α in mammalian cells. 52 Figure 8. Endogenous CHK2 and endogenous B'α bind in mammalian cells. 53 Figure 9. Binding between CHK2 and B'α is dissociated after DNA damage. 56 Figure 10. Dissociation of CHK2 and B'α is verified using another CHK2 antibody. 57 Figure 11. CHK2 binds to many PP2A B' subunits and they are dissociated after IR. 59 Figure 12. Endogenous PP2A C and CHK2 can bind and they are dissociated after IR. 60 Figure 13. Dissociation of CHK2 and B'α is caused by DNA damage induced by IR and doxorubicin. 62 Figure 14. Dissociation of CHK2 and B'α occurs in a dose-dependent manner. 65 Figure 15. Dissociation of CHK2 and B'α is and early event after IR. 66 Figure 16. CHK2 and B'α are able to re-associate hours after 6 Gy IR. 69 Figure 17. CHK2 and B'α are able to re-associate hours after 50 Gy IR. 70 iv Figure 18. PP2A phosphatase activity is not required for CHK2 binding to or dissociating from B'α. 73 Figure 19. Inhibition of ATM and DNA-PK kinase activity prevents the IR- induced dissociation of B'α and CHK2. 74 Figure 20. Mutation of CHK2 serines 33 and 35 affects binding to B'α. 76 Figure 21. CHK2 can phosphorylate B'α in vitro. 78 Figure 22. B'α localization is not changed by IR. 80 Figure 23. Localization of endogenous PP2A proteins do not change after IR. 81 Figure 24. Total PP2A phosphatase activity does not change after IR. 84 Figure 25. Phosphatase activity of PP2A containing B'α does not change after IR. 85 Figure 26. Total PP2A activity does not differ in HCT116 wild-type and CHK2-/- cells. 86 Figure 27. Inhibition of PP2A activity increases CHK2 activity. 89 Figure 28. Mutation of CHK2 serines 33 and 35 affects CHK2 phosphorylation at other sites. 90 Figure 29. B'α overexpression negatively affects CHK2 kinase activity. 92 Figure 30. Mutation of CHK2 serines 33 and 35 affects CHK2 kinase activity. 93 Figure 31A. Mutation of CHK2 serines 33 and 35 affects CHK2 protein stability. 95 Figure 31B. Mutation of CHK2 serines 33 and 35 affects CHK2 protein stability. 96 Figure 32. Model of PP2A regulation of CHK2 through binding and dissociation. 102 v LIST OF ABBREVIATIONS 4-NQO 4-nitroquinoline-N-oxide 9-1-1 Rad9-Rad1-Hus1 AT Ataxia-telangiectasia ATLD AT-like disorder ATM Ataxia telangiectasia mutated ATR ATM-Rad3-related kinase ATRIP ATR interacting protein BRCA1 Breast cancer susceptibility 1 CHK2 Checkpoint kinase 2 CHK1 Checkpoint kinase 1 CPT Camptothecin DDR DNA damage response DNA-PK DNA-dependent protein kinase DOX Doxorubicin DSB DNA double-strand breaks ES Embryonic stem HR Homologous recombination HU Hydroxyurea IB Immunoblot IP Immunoprecipitation vi IR Ionizing radiation MEF Mouse embryonic fibroblast MMC Mitomycin C MMS Methylmethane sulfonate MRN Mre11-Rad50-Nbs1 NBS Nijmegen Breakage Syndrome NCS Neocarzinostatin NHEJ Non-homologous end joining NLS Nuclear localization signal OA Okadaic acid PCNA Proliferating cell nuclear antigen PIKK PI-3K like kinase PP2A Protein phosphatase 2A PPP Phosphoprotein phosphatase PPM Metal-dependent protein phosphatase PSP Protein serine/threonine phosphatase RDS Radioresistant DNA synthesis RFC Replication factor C RPA Replication protein A SSB Single-strand breaks VPR Viral protein R vii CHK2 IS NEGATIVELY REGULATED BY PROTEIN PHOSPHATSE 2A Alyson K. Freeman ABSTRACT Checkpoint kinase 2 (CHK2) is an effector kinase of the DNA damage response pathway and although its mechanism of activation has been well studied, the attenuation of its activity following DNA damage has not been explored. Here, we identify the B'α subunit of protein phosphatase 2A (PP2A), a major protein serine/threonine phosphatase of the cell, as a CHK2 binding partner and show that their interaction is modulated by DNA damage. B'α binds to the SQ/TQ cluster domain of CHK2, which is a target of ATM phosphorylation. CHK2 is able to bind to many B' subunits as well as the PP2A C subunit, indicating that it can bind to the active PP2A enzyme. The induction of DNA double-strand breaks by ionizing radiation (IR) as well as treatment with doxorubicin causes dissociation of the B'α and CHK2 proteins, however, it does not have an effect on the binding of B'α to CHK1.
Load more

Recommended publications
  • RASSF1A Interacts with and Activates the Mitotic Kinase Aurora-A
    Oncogene (2008) 27, 6175–6186 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $32.00 www.nature.com/onc ORIGINAL ARTICLE RASSF1A interacts with and activates the mitotic kinase Aurora-A L Liu1, C Guo1, R Dammann2, S Tommasi1 and GP Pfeifer1 1Division of Biology, Beckman Research Institute, City of Hope Cancer Center, Duarte, CA, USA and 2Institute of Genetics, University of Giessen, Giessen, Germany The RAS association domain family 1A (RASSF1A) gene tumorigenesis and carcinogen-induced tumorigenesis is located at chromosome 3p21.3 within a specific area of (Tommasi et al., 2005; van der Weyden et al., 2005), common heterozygous and homozygous deletions. RASS- supporting the notion that RASSF1A is a bona fide F1A frequently undergoes promoter methylation-asso- tumor suppressor. However, it is not fully understood ciated inactivation in human cancers. Rassf1aÀ/À mice how RASSF1A is involved in tumor suppression. are prone to both spontaneous and carcinogen-induced The biochemical function of the RASSF1A protein is tumorigenesis, supporting the notion that RASSF1A is a largely unknown. The homology of RASSF1A with the tumor suppressor. However, it is not fully understood how mammalian Ras effector novel Ras effector (NORE)1 RASSF1A is involved in tumor suppression pathways. suggests that the RASSF1A gene product may function Here we show that overexpression of RASSF1A inhibits in signal transduction pathways involving Ras-like centrosome separation. RASSF1A interacts with Aurora-A, proteins. However, recent data indicate that RASSF1A a mitotic kinase. Surprisingly, knockdown of RASS- itself binds to RAS only weakly and that binding to F1A by siRNA led to reduced activation of Aurora-A, RAS may require heterodimerization of RASSF1A and whereas overexpression of RASSF1A resulted in in- NORE1 (Ortiz-Vega et al., 2002).
    [Show full text]
  • HIPK2 and Extrachromosomal Histone H2B Are Separately Recruited by Aurora-B for Cytokinesis
    Oncogene https://doi.org/10.1038/s41388-018-0191-6 ARTICLE HIPK2 and extrachromosomal histone H2B are separately recruited by Aurora-B for cytokinesis 1 1 2 3 2 Laura Monteonofrio ● Davide Valente ● Manuela Ferrara ● Serena Camerini ● Roberta Miscione ● 3 1,2 1 Marco Crescenzi ● Cinzia Rinaldo ● Silvia Soddu Received: 14 November 2017 / Revised: 24 January 2018 / Accepted: 5 February 2018 © The Author(s) 2018. This article is published with open access Abstract Cytokinesis, the final phase of cell division, is necessary to form two distinct daughter cells with correct distribution of genomic and cytoplasmic materials. Its failure provokes genetically unstable states, such as tetraploidization and polyploidization, which can contribute to tumorigenesis. Aurora-B kinase controls multiple cytokinetic events, from chromosome condensation to abscission when the midbody is severed. We have previously shown that HIPK2, a kinase involved in DNA damage response and development, localizes at the midbody and contributes to abscission by phosphorylating extrachromosomal histone H2B at Ser14. Of relevance, HIPK2-defective cells do not phosphorylate H2B 1234567890();,: and do not successfully complete cytokinesis leading to accumulation of binucleated cells, chromosomal instability, and increased tumorigenicity. However, how HIPK2 and H2B are recruited to the midbody during cytokinesis is still unknown. Here, we show that regardless of their direct (H2B) and indirect (HIPK2) binding of chromosomal DNA, both H2B and HIPK2 localize at the midbody independently of nucleic acids. Instead, by using mitotic kinase-specific inhibitors in a spatio-temporal regulated manner, we found that Aurora-B kinase activity is required to recruit both HIPK2 and H2B to the midbody.
    [Show full text]
  • The Aurora B Kinase Activity Is Required for the Maintenance of the Differentiated State of Murine Myoblasts
    Cell Death and Differentiation (2009) 16, 321–330 & 2009 Macmillan Publishers Limited All rights reserved 1350-9047/09 $32.00 www.nature.com/cdd The Aurora B kinase activity is required for the maintenance of the differentiated state of murine myoblasts G Amabile1,2, AM D’Alise1, M Iovino1, P Jones3, S Santaguida4, A Musacchio4, S Taylor5 and R Cortese*,1 Reversine is a synthetic molecule capable of inducing dedifferentiation of C2C12, a murine myoblast cell line, into multipotent progenitor cells, which can be redirected to differentiate in nonmuscle cell types under appropriate conditions. Reversine is also a potent inhibitor of Aurora B, a protein kinase required for mitotic chromosome segregation, spindle checkpoint function, cytokinesis and histone H3 phosphorylation, raising the possibility that the dedifferentiation capability of reversine is mediated through the inhibition of Aurora B. Indeed, here we show that several other well-characterized Aurora B inhibitors are capable of dedifferentiating C2C12 myoblasts. Significantly, expressing drug-resistant Aurora B mutants, which are insensitive to reversine block the dedifferentiation process, indicating that Aurora B kinase activity is required to maintain the differentiated state. We show that the inhibition of the spindle checkpoint or cytokinesis per se is not sufficient for dedifferentiation. Rather, our data support a model whereby changes in histone H3 phosphorylation result in chromatin remodeling, which in turn restores the multipotent state. Cell Death and Differentiation (2009) 16, 321–330; doi:10.1038/cdd.2008.156; published online 31 October 2008 Lineage-restricted cells can be reprogramed to a state Evidence is emerging that the role of Aurora B is not of pluripotency by several different manipulations, including restricted to mitosis and cell division.
    [Show full text]
  • 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.
    [Show full text]
  • Kinase-Targeted Cancer Therapies: Progress, Challenges and Future Directions Khushwant S
    Bhullar et al. Molecular Cancer (2018) 17:48 https://doi.org/10.1186/s12943-018-0804-2 REVIEW Open Access Kinase-targeted cancer therapies: progress, challenges and future directions Khushwant S. Bhullar1, Naiara Orrego Lagarón2, Eileen M. McGowan3, Indu Parmar4, Amitabh Jha5, Basil P. Hubbard1 and H. P. Vasantha Rupasinghe6,7* Abstract The human genome encodes 538 protein kinases that transfer a γ-phosphate group from ATP to serine, threonine, or tyrosine residues. Many of these kinases are associated with human cancer initiation and progression. The recent development of small-molecule kinase inhibitors for the treatment of diverse types of cancer has proven successful in clinical therapy. Significantly, protein kinases are the second most targeted group of drug targets, after the G-protein- coupled receptors. Since the development of the first protein kinase inhibitor, in the early 1980s, 37 kinase inhibitors have received FDA approval for treatment of malignancies such as breast and lung cancer. Furthermore, about 150 kinase-targeted drugs are in clinical phase trials, and many kinase-specific inhibitors are in the preclinical stage of drug development. Nevertheless, many factors confound the clinical efficacy of these molecules. Specific tumor genetics, tumor microenvironment, drug resistance, and pharmacogenomics determine how useful a compound will be in the treatment of a given cancer. This review provides an overview of kinase-targeted drug discovery and development in relation to oncology and highlights the challenges and future potential for kinase-targeted cancer therapies. Keywords: Kinases, Kinase inhibition, Small-molecule drugs, Cancer, Oncology Background Recent advances in our understanding of the fundamen- Kinases are enzymes that transfer a phosphate group to a tal molecular mechanisms underlying cancer cell signaling protein while phosphatases remove a phosphate group have elucidated a crucial role for kinases in the carcino- from protein.
    [Show full text]
  • Epigenetic Regulation of Neurogenesis by Citron Kinase Matthew Irg Genti University of Connecticut - Storrs, [email protected]
    University of Connecticut OpenCommons@UConn Doctoral Dissertations University of Connecticut Graduate School 12-10-2015 Epigenetic Regulation of Neurogenesis By Citron Kinase Matthew irG genti University of Connecticut - Storrs, [email protected] Follow this and additional works at: https://opencommons.uconn.edu/dissertations Recommended Citation Girgenti, Matthew, "Epigenetic Regulation of Neurogenesis By Citron Kinase" (2015). Doctoral Dissertations. 933. https://opencommons.uconn.edu/dissertations/933 Epigenetic Regulation Of Neurogenesis By Citron Kinase Matthew J. Girgenti, Ph.D. University of Connecticut, 2015 Patterns of neural progenitor division are controlled by a combination of asymmetries in cell division, extracellular signals, and changes in gene expression programs. In a screen for proteins that complex with citron kinase (CitK), a protein essential to cell division in developing brain, I identified the histone methyltransferase- euchromatic histone-lysine N-methyltransferse 2 (G9a). CitK is present in the nucleus and binds to positions in the genome clustered around transcription start sites of genes involved in neuronal development and differentiation. CitK and G9a co-occupy these genomic positions in S/G2-phase of the cell cycle in rat neural progenitors, and CitK functions with G9a to repress gene expression in several developmentally important genes including CDKN1a, H2afz, and Pou3f2/Brn2. The study indicates a novel function for citron kinase in gene repression, and contributes additional evidence to the hypothesis that mechanisms that coordinate gene expression states are directly linked to mechanisms that regulate cell division in the developing nervous system. Epigenetic Regulation Of Neurogenesis By Citron Kinase Matthew J. Girgenti B.S., Fairfield University, 2002 M.S., Southern Connecticut State University, 2004 A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the University of Connecticut 2015 ii Copyright by Matthew J.
    [Show full text]
  • Jadomycin B, an Aurora-B Kinase Inhibitor Discovered Through Virtual Screening
    2386 Jadomycin B, an Aurora-B kinase inhibitor discovered through virtual screening Da-Hua Fu,1 Wei Jiang,1 Jian-Ting Zheng,2 Jadomycin B is a new Aurora-B kinase inhibitor worthy of Gui-Yu Zhao,1 Yan Li,1 Hong Yi,1 Zhuo-Rong Li,1 further investigation. [Mol Cancer Ther 2008;7(8):2386–93] Jian-Dong Jiang,1 Ke-Qian Yang,2 Yanchang Wang,3 and Shu-Yi Si1 Introduction In mammals, there are three Aurora kinases, Aurora-A, B, 1Institute of Medicinal Biotechnology, Peking Union Medical College & Chinese Academy of Medical Sciences, and 2Institute and C, which display >60% sequence identity (1). Although of Microbiology, Chinese Academy of Sciences, Beijing, China; the catalytic domains of the three kinases are highly and 3Department of Biomedical Sciences, College of Medicine, conserved, they show distinct subcellular localization and Florida State University, Tallahassee, Florida biological function (2, 3). Aurora-A, which is required for centrosome maturation and separation, localizes to centro- somes from early S phase to late M phase (4). Aurora-B is a Abstract component of chromosomal passenger complex, whose Aurora kinases have emerged as promising targets for function in mitosis has been extensively studied. In cancer therapy because of their critical role in mitosis. addition to Aurora-B, the chromosomal passenger complex These kinases are well-conserved in all eukaryotes, and contains Survivin, Borealin, and inner centromere protein IPL1 gene encodes the single Aurora kinase in budding (5–7). The chromosomal passenger complex associates yeast. In a virtual screening attempt, 22 compounds were with centromeric regions of chromosomes in the early identified from nearly 15,000 microbial natural products as stages of mitosis, but it translocates to microtubules after potential small-molecular inhibitors of human Aurora-B the onset of anaphase.
    [Show full text]
  • Catch and Release: How Do Kinetochores Hook the Right Microtubules During Mitosis?
    TIGS-1107; No. of Pages 10 Review Catch and release: how do kinetochores hook the right microtubules during mitosis? Krishna K. Sarangapani and Charles L. Asbury Department of Physiology & Biophysics, University of Washington, Seattle, WA, USA Sport fishermen keep tension on their lines to prevent microtubule tips, thereby harnessing the dynamics of the hooked fish from releasing. A molecular version of this filaments to produce force and movement. Accurate mitosis angler’s trick, operating at kinetochores, ensures accu- requires all kinetochores to become properly ‘bioriented’, racy during mitosis: the mitotic spindle attaches ran- with replicated sister chromatids attached to opposite domly to chromosomes and then correctly bioriented sides of the spindle (or with homologous chromosomes attachments are stabilized due to the tension exerted on attached to opposite sides during the first meiotic division them by opposing microtubules. Incorrect attachments, of gametogenesis). Dietz [1] was the first to recognize that which lack tension, are unstable and release quickly, chromosomes repeatedly reorient on the spindle, in a trial- allowing another chance for biorientation. Stabilization and-error process that ceases only when proper biorienta- of molecular interactions by tension also occurs in other tion is achieved because only this arrangement is stable physiological contexts, such as cell adhesion, motility, [2,3]. Dietz also suggested a possible cause for this differ- hemostasis, and tissue morphogenesis. Here, we review ential stability: mechanical tension. Bioriented chromo- models for the stabilization of kinetochore attachments somes come under tension and their sister kinetochores with an eye toward emerging models for other force- are stretched apart by opposing spindle forces, whereas activated systems.
    [Show full text]
  • Dual PDK1/Aurora Kinase a Inhibitors Reduce Pancreatic Cancer Cell Proliferation and Colony Formation
    cancers Article Dual PDK1/Aurora Kinase A Inhibitors Reduce Pancreatic Cancer Cell Proliferation and Colony Formation 1, 1, 2 3 3 Ilaria Casari y, Alice Domenichini y, Simona Sestito , Emily Capone , Gianluca Sala , Simona Rapposelli 2 and Marco Falasca 1,* 1 Metabolic Signalling Group, School of Pharmacy and Biomedical Sciences, Curtin Health Innovation Research Institute, Curtin University, Bentley 6102, Australia; [email protected] (I.C.); [email protected] (A.D.) 2 Department of Pharmacy, University of Pisa, Via Bonanno, 6, 56126 Pisa, Italy; [email protected] (S.S.); [email protected] (S.R.) 3 Dipartimento di Scienze Mediche, Orali e Biotecnologiche, University “G. d’Annunzio” di Chieti-Pescara, Center for Advanced Studies and Technology (CAST), 66100 Chieti, Italy; [email protected] (E.C.); [email protected] (G.S.) * Correspondence: [email protected]; Tel.: +61-8-92669712 These authors contributed equally to this work. y Received: 8 October 2019; Accepted: 28 October 2019; Published: 31 October 2019 Abstract: Deregulation of different intracellular signaling pathways is a common feature in cancer. Numerous studies indicate that persistent activation of the phosphoinositide 3-kinase (PI3K) pathway is often observed in cancer cells. 3-phosphoinositide dependent protein kinase-1 (PDK1), a transducer protein that functions downstream of PI3K, is responsible for the regulation of cell proliferation and migration and it also has been found to play a key role in different cancers, pancreatic and breast cancer amongst others. As PI3K is being described to be aberrantly expressed in several cancer types, designing inhibitors targeting various downstream molecules of PI3K has been the focus of anticancer agent development for a long time.
    [Show full text]
  • A Framework for Identification of Actionable Cancer Genome
    A framework for identification of actionable cancer genome dependencies in small cell lung cancer Martin L. Sosa,b,c,d,1,2, Felix Dietleina,b,1, Martin Peifera,b, Jakob Schöttlea,b, Hyatt Balke-Wanta,b, Christian Müllera,b, Mirjam Kokera,b, André Richterse,f, Stefanie Heyncka,b, Florian Malchersa,b, Johannes M. Heuckmanna,b, Danila Seidela,b, Patrick A. Eyersg, Roland T. Ullrichb, Andrey P. Antonchickh, Viktor V. Vintonyakh, Peter M. Schneideri, Takashi Ninomiyaj, Herbert Waldmanne,h, Reinhard Büttnerk, Daniel Rauhe,f, Lukas C. Heukampk, and Roman K. Thomasa,b,k,2 aDepartment of Translational Genomics, University of Cologne, 50931 Cologne, Germany; bMax Planck Institute for Neurological Research, 50931 Cologne, Germany; cHoward Hughes Medical Institute, dDepartment of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158; eChemical Genomics Center of the Max Planck Society, 44227 Dortmund, Germany; fTechnical University Dortmund, D-44221 Dortmund, Germany; gYorkshire Cancer Research (YCR) Institute for Cancer Studies, Cancer Research United Kingdom (CR-UK)/YCR Sheffield Cancer Research Centre, Department of Oncology, University of Sheffield, Sheffield S10 2RX, United Kingdom; hMax Planck Institute of Molecular Physiology, D-44227 Dortmund, Germany; iInstitute of Forensic Medicine, University of Cologne, 50823 Cologne, Germany; jDepartment of Hematology, Oncology, and Respiratory Medicine, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 700-8558 Okayama, Japan; and kInstitute of Pathology, University of Cologne, 50924 Cologne, Germany Edited by Peter K. Vogt, The Scripps Research Institute, La Jolla, CA, and approved September 11, 2012 (received for review April 30, 2012) Small cell lung cancer (SCLC) accounts for about 15% of all lung and “synthetic lethality” have emerged (15–19).
    [Show full text]
  • Function and Regulation of Aurora/Ipl1p Kinase Family in Cell Division
    Cell Research (2003); 13(2):69-81 http://www.cell-research.com Function and regulation of Aurora/Ipl1p kinase family in cell division 1 1 1 1,2, YU WEN KE , ZHEN DOU , JIE ZHANG , XUE BIAO YAO * 1 Laboratory for Cell Dynamics, School of Life Sciences, University of Science and Technology of China, Hefei 230027, China 2 Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA ABSTRACT During mitosis, the parent cell distributes its genetic materials equally into two daughter cells through chromosome segregation, a complex movements orchestrated by mitotic kinases and its effector proteins. Faithful chromosome segregation and cytokinesis ensure that each daughter cell receives a full copy of genetic materials of parent cell. Defects in these processes can lead to aneuploidy or polyploidy. Aurora/Ipl1p family, a class of conserved serine/threonine kinases, plays key roles in chromosome segregation and cytokinesis. This article highlights the function and regulation of Aurora/Ipl1p family in mitosis and provides potential links between aberrant regulation of Aurora/Ipl1p kinases and pathogenesis of human cancer. Key words: Aurora (Ipl1p), mitosis, and cancer. INTRODUCTION among species, the regulatory machinery is conserved from yeast to human. One of most conserved regu- Cell is a fundamental unit of life that is relayed lators is serine/theronine protein kinase superfam- via mitosis. The essence of mitosis is to segregate ily that alters the function of its effectors via protein parental genomes encoded in sister chromatides into phosphorylation. Entry into mitosis is driven by pro- two daughter cells, such that each of them inherits tein kinases while initiation of exit from mitosis is one complete copy of genome.
    [Show full text]
  • Inhibition of Mtor Pathway Sensitizes Acute Myeloid Leukemia Cells to Aurora Inhibitors by Suppression of Glycolytic Metabolism
    Published OnlineFirst September 5, 2013; DOI: 10.1158/1541-7786.MCR-13-0172 Molecular Cancer Cell Death and Survival Research Inhibition of mTOR Pathway Sensitizes Acute Myeloid Leukemia Cells to Aurora Inhibitors by Suppression of Glycolytic Metabolism Ling-Ling Liu1,2, Zi-Jie Long1,3, Le-Xun Wang1,3, Fei-Meng Zheng2, Zhi-Gang Fang1, Min Yan2, Dong-Fan Xu1,3, Jia-Jie Chen1,3, Shao-Wu Wang4,5, Dong-Jun Lin1,3, and Quentin Liu1,2,3,4 Abstract Aurora kinases are overexpressed in large numbers of tumors and considered as potential therapeutic targets. In this study, we found that the Aurora kinases inhibitors MK-0457 (MK) and ZM447439 (ZM) induced polyploidization in acute myeloid leukemia (AML) cell lines. The level of glycolytic metabolism was significantly increased in the polyploidy cells, which were sensitive to glycolysis inhibitor 2-deoxy-D-glucose (2DG), suggesting that polyploidy cells might be eliminated by metabolism deprivation. Indeed, inhibition of mTOR pathway by mTOR inhibitors (rapamycin and PP242) or 2DG promoted not only apoptosis but also autophagy in the polyploidy cells induced by Aurora inhibitors. Mechanically, PP242 or2DGdecreased the level of glucose uptake and lactate production in polyploidy cells as well as the expression of p62/SQSTM1. Moreover, knockdown of p62/ SQSTM1 sensitized cells to the Aurora inhibitor whereas overexpression of p62/SQSTM1 reduced drug efficacy. Thus, our results revealed that inhibition of mTOR pathway decreased the glycolytic metabolism of the polyploidy cells, and increased the efficacy of Aurora kinases inhibitors, providing a novel approach of combination treatment in AML. Mol Cancer Res; 11(11); 1326–36.
    [Show full text]