DOCSLIB.ORG

Bub1 Is Required for Kinetochore Localization of Bubr1, Cenp-E, Cenp-F and Mad2, and Chromosome Congression

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Bub1 Is Required for Kinetochore Localization of Bubr1, Cenp-E, Cenp-F and Mad2, and Chromosome Congression Research Article 1577 Bub1 is required for kinetochore localization of BubR1, Cenp-E, Cenp-F and Mad2, and chromosome congression Victoria L. Johnson, Maria I. F. Scott, Sarah V. Holt, Deema Hussein and Stephen S. Taylor* School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester M13 9PT, UK *Author for correspondence (e-mail: [email protected]) Accepted 24 November 2003 Journal of Cell Science 117, 1577-1589 Published by The Company of Biologists 2004 doi:10.1242/jcs.01006 Summary During mitosis, the recruitment of spindle-checkpoint- Repression of Bub1 increases the number of cells with associated proteins to the kinetochore occurs in a defined lagging chromosomes at metaphase, suggesting that Bub1 order. The protein kinase Bub1 localizes to the kinetochore plays a role in chromosome congression. However, very early during mitosis, followed by Cenp-F, BubR1, repression of Bub1 does not appear to compromise spindle Cenp-E and finally Mad2. Using RNA interference, we have checkpoint function either during normal mitosis or in investigated whether this order of binding reflects a level of response to spindle damage. This raises the possibility that, dependency in human somatic cells. Specifically, we show in the absence of Bub1, other mechanisms contribute to that Bub1 plays a key role in the assembly of checkpoint spindle checkpoint function. proteins at the kinetochore, being required for the subsequent localization of Cenp-F, BubR1, Cenp-E and Supplemental data available online Mad2. In contrast to studies in Xenopus, we also show that BubR1 is not required for kinetochore localization of Bub1. Key words: Kinetochore, Mitosis, Bub1, Aurora B Introduction kinetochores undergo a maturation process upon entry into Chromosome segregation in eukaryotes is mediated by a mitosis. Whereas a number of proteins, including Cenp-A, microtubule spindle apparatus. In addition to the spindle, Cenp-C and Cenp-I, localize to the centromere region kinetochores are essential for successful chromosome throughout the cell cycle (Liu et al., 2003; Palmer et al., 1987; segregation. Kinetochores are large complex protein structures Tomkiel et al., 1994), many other proteins only localize to that assemble at the centromeric regions of each sister chromatid kinetochores transiently during mitosis. These include motor and perform three key functions (Nicklas, 1997; Rieder and proteins such as cytoplasmic dynein (Echeverri et al., 1996) Salmon, 1998). First, kinetochores attach chromosomes to the and Cenp-E (Yen et al., 1992), and other proteins such as Cenp- spindle. Second, kinetochores co-ordinate microtubule dynamics F (Liao et al., 1995), ZW10, ROD (Chan et al., 2000) and Hec1 to allow chromosomes to move along the spindle. Third, (Martin-Lluesma et al., 2002). The spindle checkpoint proteins kinetochores generate the ‘wait’ signal that prevents anaphase Bub1, Bub3, Mad1, Mad2, Mps1 and a Mad3/Bub1-related onset until all the chromosomes are correctly aligned on the protein kinase called BubR1, also only localize to kinetochores spindle. This signal forms part of the spindle checkpoint during mitosis (Musacchio and Hardwick, 2002; Shah and mechanism, a highly conserved cell cycle checkpoint that Cleveland, 2000). Consistent with a role in monitoring ensures accurate chromosome segregation (Jallepalli and chromosome alignment, the levels of these latter proteins, Lengauer, 2001; Musacchio and Hardwick, 2002). including Mad2, Bub1 and BubR1, diminishes following Electron microscopy studies show that vertebrate microtubule capture and/or bi-orientation (Chan et al., 1998; kinetochores are trilaminar structures that sit back-to-back on Chen et al., 1996; Taylor and McKeon, 1997). Another group top of the chromatin (Biggins and Walczak, 2003; Cleveland of proteins that localize transiently during mitosis includes et al., 2003). The inner kinetochore plate, directly adjacent to INCENP, Aurora B and Survivin (Adams et al., 2001a; the centromeric heterochromatin, is separated from an outer Bischoff and Plowman, 1999). At the onset of anaphase, these plate by a middle layer. Microtubules embed into the outer proteins relocate from the chromosomes to the spindle and are kinetochore, beyond which extends a fibrous corona. This hence termed chromosome passenger proteins (Earnshaw and trilaminar structure is not visible during interphase. Rather, an Bernat, 1991). Although these proteins do not localize to amorphous ball-like structure called the pre-kinetochore lies kinetochores – in vertebrates, they localize to the inner adjacent to the centromeric heterochromatin (Rieder, 1982). centromere region – they do appear to play a role in This suggests that kinetochores undergo an assembly process kinetochore assembly. Specifically, inhibition of Aurora B or or morphogenesis upon entry into mitosis, maturing from the Survivin inhibits recruitment of several kinetochore proteins pre-kinetochore to a trilaminar structure. including BubR1, Cenp-E and Mad2 (Carvalho et al., 2003; Light microscopy studies are consistent with the notion that Ditchfield et al., 2003; Hauf et al., 2003; Lens et al., 2003). 1578 Journal of Cell Science 117 (8) Proteins that associate transiently with kinetochores in Generation of reagents to detect Mad2 in human cells mitosis are not recruited simultaneously in human cells. To create a sheep polyclonal anti-Mad2 antibody and a DLD-1- Rather, there appears to be a defined order of assembly. derived cell line expressing a Myc-tagged Mad2 fusion protein, a Specifically, two independent co-staining studies show that human Mad2 cDNA was generated by reverse-transcription Bub1 is recruited to kinetochores very early in prophase, polymerase chain reaction (RT-PCR) amplification of HeLa cell followed by Cenp-F and then BubR1, and finally with Cenp-E mRNA using the SuperScript One-Step RT-PCR system being recruited in mid- to late prometaphase (Jablonski et al., (Invitrogen), subcloned and sequenced. For antibody production, the 1998; Taylor et al., 2001). One model to explain this order of cDNA was cloned into pGEX-4T3 (Pharmacia). A glutathione-S- transferase/Mad2 fusion protein was then expressed, purified and used assembly is that recruitment of the latter proteins is dependent for immunization as described previously (Taylor et al., 2001). The on the prior recruitment of the early ones. Thus, as with the anti-Mad2 antibody SM2.2 was then affinity purified following assembly of bacteriophage capsids (Casjens and King, 1974), standard procedures. perhaps proteins recruited early create binding sites that The DLD-1 Myc-tagged Mad2 cell line was created using the Flp- facilitate the sequential binding of others. Consistent with this In system (Invitrogen). Briefly, a Flp-In host cell line was created notion, Bub1 binds Cenp-F in a two-hybrid assay (Chan et al., by integrating a single Flp recombination target (FRT) recombination 1998), BubR1 co-precipitates Cenp-E (Chan et al., 1998; Yao site into the DLD-1 genome. The Mad2 cDNA was then cloned into et al., 2000) and Bub1 co-precipitates BubR1 (Taylor et al., a pcDNA5/FRT-based Myc-tagged vector and co-transfected into the DLD-1 FRT line along with a plasmid expressing the FLP 2001). However, owing to the lack of suitable mammalian kinetochore assembly assays, whether this temporal order recombinase (pOG44, Invitrogen) using LipofectAMINE Plus (Invitrogen). Cells were selected in 400 µg ml–1 Hygromycin B reflects an underlying dependency remains to be determined. (Roche) and colonies were pooled and expanded. Xenopus egg extracts do however provide a tractable kinetochore assembly assay. Immunodepletion of Bub1 from egg extracts prevents kinetochore localization of BubR1, Immunocytochemistry Mad2, Mad1 and Cenp-E (Sharp-Baker and Chen, 2001). In Immunofluorescence analysis was basically done as described (Taylor addition, immunodepletion of BubR1 prevents recruitment of et al., 2001). With the exception of the anti-Mad2 antibody SM2.2 Bub1, Mad2, Mad1 and Cenp-E (Chen, 2002). However, this (see above), all other antibodies have been described previously observation seems to be at odds with the order observed in (Ditchfield et al., 2003; Hussein and Taylor, 2002; Taylor et al., 2001; Tighe et al., 2001). Briefly, cells were fixed in 1% paraformaldehyde, mammalian cells: because Bub1 binds kinetochores before washed in PBS plus 0.1% Triton X-100 (PBST), blocked in PBST BubR1 in mammalian cells, one might predict that recruitment plus 5% non-fat dried milk then stained with the following primary of Bub1 is not dependent on BubR1. One possible explanation antibodies: ACA (human anti-centromere, 1:1000); SB1.3 (sheep anti- for this difference is that, in order to facilitate many rapid cell Bub1, 1:1000); 4B12 (mouse anti-Bub1, 1:10); SBR1.1 (sheep anti- divisions, the Xenopus embryo stockpiles pre-assembled BubR1, 1:1000); 5F9 (mouse anti-BubR1, 1:50); RCE.1, (rabbit anti- kinetochore complexes. Indeed, it is not clear whether there is Cenp-E, 1:2000); SCF.1 (sheep anti-Cenp-F, 1:1000); AIM-1 (mouse a temporal order of recruitment upon entry into mitosis in the anti-Aurora B, Transduction Laboratories, 1:200); TAT-1 (mouse anti- Xenopus system or whether all the transient kinetochore tubulin, 1:500); RAA.1 (rabbit anti-Aurora A, 1:10,000); rabbit anti- proteins are recruited simultaneously. Therefore, it is at present phospho-histone H3, (Upstate Biotechnology, 1:200). Following unclear whether the observations derived from the Xenopus in washes, cells were
Load more

Recommended publications
  • Mad1 Kinetochore Recruitment by Mps1-Mediated Phosphorylation of Bub1 Signals the Spindle Checkpoint
    Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press Mad1 kinetochore recruitment by Mps1-mediated phosphorylation of Bub1 signals the spindle checkpoint Nitobe London1,2 and Sue Biggins1,3 1Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA; 2Molecular and Cellular Biology Program, University of Washington/Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA The spindle checkpoint is a conserved signaling pathway that ensures genomic integrity by preventing cell division when chromosomes are not correctly attached to the spindle. Checkpoint activation depends on the hierarchical recruitment of checkpoint proteins to generate a catalytic platform at the kinetochore. Although Mad1 kinetochore localization is the key regulatory downstream event in this cascade, its receptor and mechanism of recruitment have not been conclusively identified. Here, we demonstrate that Mad1 kinetochore association in budding yeast is mediated by phosphorylation of a region within the Bub1 checkpoint protein by the conserved protein kinase Mps1. Tethering this region of Bub1 to kinetochores bypasses the checkpoint requirement for Mps1-mediated kinetochore recruitment of upstream checkpoint proteins. The Mad1 interaction with Bub1 and kinetochores can be reconstituted in the presence of Mps1 and Mad2. Together, this work reveals a critical mechanism that determines kinetochore activation of the spindle checkpoint. [Keywords: kinetochore; spindle checkpoint; Mps1 kinase; Mad1; Bub1 kinase] Supplemental material is available for this article. Received October 25, 2013; revised version accepted December 13, 2013. Successful eukaryotic cell division requires accurate the conserved checkpoint proteins Mps1, Bub1, Bub3, chromosome segregation, which relies on correct attach- Mad1, and Mad2 as well as the Mad3 homolog BubRI in ments of spindle microtubules to chromosomes.
    [Show full text]
  • Evolution, Expression and Meiotic Behavior of Genes Involved in Chromosome Segregation of Monotremes
    G C A T T A C G G C A T genes Article Evolution, Expression and Meiotic Behavior of Genes Involved in Chromosome Segregation of Monotremes Filip Pajpach , Linda Shearwin-Whyatt and Frank Grützner * School of Biological Sciences, The University of Adelaide, Adelaide, SA 5005, Australia; fi[email protected] (F.P.); [email protected] (L.S.-W.) * Correspondence: [email protected] Abstract: Chromosome segregation at mitosis and meiosis is a highly dynamic and tightly regulated process that involves a large number of components. Due to the fundamental nature of chromosome segregation, many genes involved in this process are evolutionarily highly conserved, but duplica- tions and functional diversification has occurred in various lineages. In order to better understand the evolution of genes involved in chromosome segregation in mammals, we analyzed some of the key components in the basal mammalian lineage of egg-laying mammals. The chromosome passenger complex is a multiprotein complex central to chromosome segregation during both mitosis and meio- sis. It consists of survivin, borealin, inner centromere protein, and Aurora kinase B or C. We confirm the absence of Aurora kinase C in marsupials and show its absence in both platypus and echidna, which supports the current model of the evolution of Aurora kinases. High expression of AURKBC, an ancestor of AURKB and AURKC present in monotremes, suggests that this gene is performing all necessary meiotic functions in monotremes. Other genes of the chromosome passenger complex complex are present and conserved in monotremes, suggesting that their function has been preserved Citation: Pajpach, F.; in mammals.
    [Show full text]
  • Centromere RNA Is a Key Component for the Assembly of Nucleoproteins at the Nucleolus and Centromere
    Downloaded from genome.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press Letter Centromere RNA is a key component for the assembly of nucleoproteins at the nucleolus and centromere Lee H. Wong,1,3 Kate H. Brettingham-Moore,1 Lyn Chan,1 Julie M. Quach,1 Melisssa A. Anderson,1 Emma L. Northrop,1 Ross Hannan,2 Richard Saffery,1 Margaret L. Shaw,1 Evan Williams,1 and K.H. Andy Choo1 1Chromosome and Chromatin Research Laboratory, Murdoch Childrens Research Institute & Department of Paediatrics, University of Melbourne, Royal Children’s Hospital, Parkville 3052, Victoria, Australia; 2Peter MacCallum Research Institute, St. Andrew’s Place, East Melbourne, Victoria 3002, Australia The centromere is a complex structure, the components and assembly pathway of which remain inadequately defined. Here, we demonstrate that centromeric ␣-satellite RNA and proteins CENPC1 and INCENP accumulate in the human interphase nucleolus in an RNA polymerase I–dependent manner. The nucleolar targeting of CENPC1 and INCENP requires ␣-satellite RNA, as evident from the delocalization of both proteins from the nucleolus in RNase-treated cells, and the nucleolar relocalization of these proteins following ␣-satellite RNA replenishment in these cells. Using protein truncation and in vitro mutagenesis, we have identified the nucleolar localization sequences on CENPC1 and INCENP. We present evidence that CENPC1 is an RNA-associating protein that binds ␣-satellite RNA by an in vitro binding assay. Using chromatin immunoprecipitation, RNase treatment, and “RNA replenishment” experiments, we show that ␣-satellite RNA is a key component in the assembly of CENPC1, INCENP, and survivin (an INCENP-interacting protein) at the metaphase centromere.
    [Show full text]
  • Mitotic Arrest Deficient 2 Expression Induces Chemosensitization to a DNA-Damaging Agent, Cisplatin, in Nasopharyngeal Carcinoma Cells
    Research Article Mitotic Arrest Deficient 2 Expression Induces Chemosensitization to a DNA-Damaging Agent, Cisplatin, in Nasopharyngeal Carcinoma Cells Hiu Wing Cheung,1 Dong-Yan Jin,2 Ming-tat Ling,1 Yong Chuan Wong,1 Qi Wang,1 Sai Wah Tsao,1 and Xianghong Wang1 Departments of 1Anatomy and 2Biochemistry, Faculty of Medicine, University of Hong Kong, Hong Kong, China Abstract mitotic checkpoint control, may be associated with tumorigenesis Recently, mitotic arrest deficient 2 (MAD2)–mediated spindle as well as cancer progression. Several regulators of the mitotic checkpoint have been identified checkpoint is shown to induce mitotic arrest in response to DNA damage, indicating overlapping roles of the spindle and most of them are localized to the kinetochore, which is checkpoint and DNA damage checkpoint. In this study, we connected to both the chromosome and the spindle (1). One of investigated if MAD2 played a part in cellular sensitivity to them, mitotic arrest deficient 2 (MAD2), is thought to be a key DNA-damaging agents, especially cisplatin, and whether it was component for a functional mitotic checkpoint because it is regulated through mitotic checkpoint. Using nine nasopha- required for generating the ‘‘wait’’ signal in response to microtubule ryngeal carcinoma (NPC) cell lines, we found that decreased disruption (1). Deletion or down-regulation of MAD2 leads to MAD2 expression was correlated with cellular resistance to mitotic checkpoint inactivation and chromosomal instability (4–6). cisplatin compared with the cell lines with high levels of Down-regulation of MAD2 has also been reported in human MAD2. Exogenous MAD2 expression in NPC cells also cancers such as lung (7), breast (8), nasopharyngeal (9), and ovarian conferred sensitivity to DNA-damaging agents especially carcinomas (10).
    [Show full text]
  • 1 Spindle Assembly Checkpoint Is Sufficient for Complete Cdc20
    Spindle assembly checkpoint is sufficient for complete Cdc20 sequestering in mitotic control Bashar Ibrahim Bio System Analysis Group, Friedrich-Schiller-University Jena, and Jena Centre for Bioinformatics (JCB), 07743 Jena, Germany Email: [email protected] Abstract The spindle checkpoint assembly (SAC) ensures genome fidelity by temporarily delaying anaphase onset, until all chromosomes are properly attached to the mitotic spindle. The SAC delays mitotic progression by preventing activation of the ubiquitin ligase anaphase-promoting complex (APC/C) or cyclosome; whose activation by Cdc20 is required for sister-chromatid separation marking the transition into anaphase. The mitotic checkpoint complex (MCC), which contains Cdc20 as a subunit, binds stably to the APC/C. Compelling evidence by Izawa and Pines (Nature 2014; 10.1038/nature13911) indicates that the MCC can inhibit a second Cdc20 that has already bound and activated the APC/C. Whether or not MCC per se is sufficient to fully sequester Cdc20 and inhibit APC/C remains unclear. Here, a dynamic model for SAC regulation in which the MCC binds a second Cdc20 was constructed. This model is compared to the MCC, and the MCC-and-BubR1 (dual inhibition of APC) core model variants and subsequently validated with experimental data from the literature. By using ordinary nonlinear differential equations and spatial simulations, it is shown that the SAC works sufficiently to fully sequester Cdc20 and completely inhibit APC/C activity. This study highlights the principle that a systems biology approach is vital for molecular biology and could also be used for creating hypotheses to design future experiments. Keywords: Mathematical biology, Spindle assembly checkpoint; anaphase promoting complex, MCC, Cdc20, systems biology 1 Introduction Faithful DNA segregation, prior to cell division at mitosis, is vital for maintaining genomic integrity.
    [Show full text]
  • Kinetochores, Microtubules, and Spindle Assembly Checkpoint
    Review Joined at the hip: kinetochores, microtubules, and spindle assembly checkpoint signaling 1 1,2,3 Carlos Sacristan and Geert J.P.L. Kops 1 Molecular Cancer Research, University Medical Center Utrecht, 3584 CG Utrecht, The Netherlands 2 Center for Molecular Medicine, University Medical Center Utrecht, 3584 CG Utrecht, The Netherlands 3 Cancer Genomics Netherlands, University Medical Center Utrecht, 3584 CG Utrecht, The Netherlands Error-free chromosome segregation relies on stable and cell division. The messenger is the SAC (also known as connections between kinetochores and spindle microtu- the mitotic checkpoint) (Figure 1). bules. The spindle assembly checkpoint (SAC) monitors The transition to anaphase is triggered by the E3 ubiqui- such connections and relays their absence to the cell tin ligase APC/C, which tags inhibitors of mitotic exit cycle machinery to delay cell division. The molecular (CYCLIN B) and of sister chromatid disjunction (SECURIN) network at kinetochores that is responsible for microtu- for proteasomal degradation [2]. The SAC has a one-track bule binding is integrated with the core components mind, inhibiting APC/C as long as incorrectly attached of the SAC signaling system. Molecular-mechanistic chromosomes persist. It goes about this in the most straight- understanding of how the SAC is coupled to the kineto- forward way possible: it assembles a direct and diffusible chore–microtubule interface has advanced significantly inhibitor of APC/C at kinetochores that are not connected in recent years. The latest insights not only provide a to spindle microtubules. This inhibitor is named the striking view of the dynamics and regulation of SAC mitotic checkpoint complex (MCC) (Figure 1).
    [Show full text]
  • MAD2 Expression in Oral Squamous Cell Carcinoma and Its Relationship to Tumor Grade and Proliferation
    ANTICANCER RESEARCH 34: 7021-7028 (2014) MAD2 Expression in Oral Squamous Cell Carcinoma and its Relationship to Tumor Grade and Proliferation CLARA RIZZARDI1, LUCIO TORELLI2, MANUELA SCHNEIDER3, FABIOLA GIUDICI4, LORENZO ZANDONA’1, MATTEO BIASOTTO5, ROBERTO DI LENARDA5 and MAURO MELATO6 1Unit of Pathology, Department of Medical, Surgical and Health Sciences, University of Trieste, Trieste, Italy; 2Department of Mathematics and Earth Science, University of Trieste, Trieste, Italy; 3Unit of Pathology, ASS n.2 “Isontina”, Gorizia, Italy; 4Department of Medical, Surgical and Health Sciences, University of Trieste, Trieste, Italy; 5Unit of Odontology and Stomatology, Department of Medical, Surgical and Health Sciences, University of Trieste, Trieste, Italy; 6Scientific Research Institute and Hospital for Pediatrics “Burlo Garofolo”, Trieste, Italy Abstract. Background: Defects in the cell-cycle surveillance might contribute to the chromosomal instability observed in mechanism, called the spindle checkpoint, might contribute human cancers. Molecular analysis of the genes involved in to the chromosomal instability observed in human cancers, the spindle checkpoint has revealed relatively few genetic including oral squamous cell carcinoma. MAD2 and BUBR1 alterations, suggesting that the spindle checkpoint are key components of the spindle checkpoint, whose role in impairment frequently found in many human cancers might oral carcinogenesis and clinical relevance still need to be result from mutations in as yet unidentified checkpoint genes elucidated. Materials and Methods: We analyzed the or altered expression of known checkpoint genes. A better expression of MAD2 in 49 cases of oral squamous cell understanding of this mechanism might provide valuable carcinoma by immunohistochemistry and compared the insights into CIN and facilitate the design of novel findings with clinicopathological parameters, proliferative therapeutic approaches to treat cancer.
    [Show full text]
  • Phosphorylation of Threonine 3 on Histone H3 by Haspin Kinase Is Required for Meiosis I in Mouse Oocytes
    ß 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 5066–5078 doi:10.1242/jcs.158840 RESEARCH ARTICLE Phosphorylation of threonine 3 on histone H3 by haspin kinase is required for meiosis I in mouse oocytes Alexandra L. Nguyen, Amanda S. Gentilello, Ahmed Z. Balboula*, Vibha Shrivastava, Jacob Ohring and Karen Schindler` ABSTRACT a lesser extent. However, it is not known whether haspin is required for meiosis in oocytes. To date, the only known haspin substrates Meiosis I (MI), the division that generates haploids, is prone to are threonine 3 of histone H3 (H3T3), serine 137 of macroH2A and errors that lead to aneuploidy in females. Haspin is a kinase that threonine 57 of CENP-T (Maiolica et al., 2014). Knockdown or phosphorylates histone H3 on threonine 3, thereby recruiting Aurora inhibition of haspin in mitotically dividing tissue culture cell kinase B (AURKB) and the chromosomal passenger complex lines reveal that phosphorylation of H3T3 is essential for the (CPC) to kinetochores to regulate mitosis. Haspin and AURKC, an alignment of chromosomes at the metaphase plate (Dai and AURKB homolog, are enriched in germ cells, yet their significance Higgins, 2005; Dai et al., 2005), regulation of chromosome in regulating MI is not fully understood. Using inhibitors and cohesion (Dai et al., 2009) and establishing a bipolar spindle (Dai overexpression approaches, we show a role for haspin during MI et al., 2009). In mitotic metaphase, phosphorylation of H3T3 is in mouse oocytes. Haspin-perturbed oocytes display abnormalities restricted to kinetochores, and this mark signals recruitment of the in chromosome morphology and alignment, improper kinetochore– chromosomal passenger complex (CPC) (Dai et al., 2005; Wang microtubule attachments at metaphase I and aneuploidy at et al., 2010; Yamagishi et al., 2010).
    [Show full text]
  • A Single Amino Acid Change Converts Aurora-A Into Aurora-B-Like Kinase in Terms of Partner Specificity and Cellular Function
    A single amino acid change converts Aurora-A into Aurora-B-like kinase in terms of partner specificity and cellular function Jingyan Fua, Minglei Biana, Junjun Liub, Qing Jianga, and Chuanmao Zhanga,1 aThe Education Ministry Key Laboratory of Cell Proliferation and Differentiation and the State Key Laboratory of Bio-membrane and Membrane Bio-engineering, College of Life Sciences, Peking University, Beijing 100871, China; and bDepartment of Biological Sciences, California State Polytechnic University, Pomona, CA 91768 Edited by Don W. Cleveland, University of California at San Diego, La Jolla, CA, and approved March 5, 2009 (received for review January 25, 2009) Aurora kinase-A and -B are key regulators of the cell cycle and the original Aurora-A localization at the spindle. Moreover, tumorigenesis. It has remained a mystery why these 2 Aurora Aurora-AG198N prevented the chromosome misalignment and kinases, although highly similar in protein sequence and structure, cell premature exit from mitosis caused by Aurora-B knock- are distinct in subcellular localization and function. Here, we report down, indicating functional substitution for Aurora-B in cell the striking finding that a single amino acid residue is responsible cycle regulation. for these differences. We replaced the Gly-198 of Aurora-A with the equivalent residue Asn-142 of Aurora-B and found that in HeLa Results cells, Aurora-AG198N was recruited to the inner centromere in Aurora-AG198N Colocalizes with Aurora-B at Centromere and Midzone. metaphase and the midzone in anaphase, reminiscent of the We fused the mutants Aurora-AG198N and Aurora-BN142G (Fig. Aurora-B localization. Moreover, Aurora-AG198N compensated for 1A) with GFP and transiently expressed them in HeLa cells the loss of Aurora-B in chromosome misalignment and cell prema- [supporting information (SI) Fig.
    [Show full text]
  • Bub1 Positions Mad1 Close to KNL1 MELT Repeats to Promote Checkpoint Signalling
    ARTICLE Received 14 Dec 2016 | Accepted 3 May 2017 | Published 12 June 2017 DOI: 10.1038/ncomms15822 OPEN Bub1 positions Mad1 close to KNL1 MELT repeats to promote checkpoint signalling Gang Zhang1, Thomas Kruse1, Blanca Lo´pez-Me´ndez1, Kathrine Beck Sylvestersen1, Dimitriya H. Garvanska1, Simone Schopper1, Michael Lund Nielsen1 & Jakob Nilsson1 Proper segregation of chromosomes depends on a functional spindle assembly checkpoint (SAC) and requires kinetochore localization of the Bub1 and Mad1/Mad2 checkpoint proteins. Several aspects of Mad1/Mad2 kinetochore recruitment in human cells are unclear and in particular the underlying direct interactions. Here we show that conserved domain 1 (CD1) in human Bub1 binds directly to Mad1 and a phosphorylation site exists in CD1 that stimulates Mad1 binding and SAC signalling. Importantly, fusion of minimal kinetochore-targeting Bub1 fragments to Mad1 bypasses the need for CD1, revealing that the main function of Bub1 is to position Mad1 close to KNL1 MELTrepeats. Furthermore, we identify residues in Mad1 that are critical for Mad1 functionality, but not Bub1 binding, arguing for a direct role of Mad1 in the checkpoint. This work dissects functionally relevant molecular interactions required for spindle assembly checkpoint signalling at kinetochores in human cells. 1 The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark. Correspondence and requests for materials should be addressed to G.Z.
    [Show full text]
  • Synergistic Effects of Bubr1 and P53 Deficiency in Tumor Formation
    University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Masters Theses Graduate School 5-2006 Synergistic Effects of BubR1 and p53 Deficiency in umorT Formation Walter Guy Wiles University of Tennessee - Knoxville Follow this and additional works at: https://trace.tennessee.edu/utk_gradthes Part of the Life Sciences Commons Recommended Citation Wiles, Walter Guy, "Synergistic Effects of BubR1 and p53 Deficiency in umorT Formation. " Master's Thesis, University of Tennessee, 2006. https://trace.tennessee.edu/utk_gradthes/1836 This Thesis 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 Masters Theses 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 thesis written by Walter Guy Wiles entitled "Synergistic Effects of BubR1 and p53 Deficiency in umorT Formation." I have examined the final electronic copy of this thesis for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Master of Science, with a major in Biochemistry and Cellular and Molecular Biology. Sundar Venkatachalam, Major Professor We have read this thesis and recommend its acceptance: Ranjan Ganguly, Ana Kitazono 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 thesis written by Walter Guy Wiles IV entitled “Synergistic Effects of BubR1 and p53 Deficiency in Tumor Formation.” I have examined the final electronic copy of this thesis for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Master of Science, with a major in Biochemistry and Cellular and Molecular Biology.
    [Show full text]
  • The Closed Form of Mad2 Is Bound to Mad1 and Cdc20 at Unattached Kinetochores
    bioRxiv preprint doi: https://doi.org/10.1101/305763; this version posted April 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. The closed form of Mad2 is bound to Mad1 and Cdc20 at unattached kinetochores. Gang Zhang1,2,3 and Jakob Nilsson1 1 The Novo Nordisk Foundation Center for Protein Research, Faculty of health and medical sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark 2 Cancer Institute, The Affiliated Hospital of Qingdao University, Qingdao, Shandong 266061, China 3 Qingdao Cancer Institute, Qingdao, Shandong 266061, China For correspondence: [email protected] or [email protected] Keywords: Mad2, Cdc20, Kinetochore, Spindle Assembly Checkpoint, Mad1 1 bioRxiv preprint doi: https://doi.org/10.1101/305763; this version posted April 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. ABSTRACT The spindle assembly checkpoint (SAC) ensures accurate chromosome segregation by delaying anaphase onset in response to unattached kinetochores. Anaphase is delayed by the generation of the mitotic checkpoint complex (MCC) composed of the checkpoint proteins Mad2 and BubR1/Bub3 bound to the protein Cdc20. Current models assume that MCC production is catalyzed at unattached kinetochores and that the Mad1/Mad2 complex is instrumental in the conversion of Mad2 from an open form (O-Mad2) to a closed form (C-Mad2) that can bind to Cdc20.
    [Show full text]