DOCSLIB.ORG

Cell-Cycle Dependent Phosphorylation of Yeast Pericentrin Regulates Γ-Tusc

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Cell-Cycle Dependent Phosphorylation of Yeast Pericentrin Regulates Γ-Tusc 1 Cell-cycle dependent phosphorylation of yeast pericentrin regulates -TuSC- 2 mediated microtubule nucleation 3 4 Tien-chen Lin1,3, Annett Neuner1, Yvonne Schlosser1, Annette Scharf2, Lisa Weber2 and 5 Elmar Schiebel1,* 6 1-Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Allianz, 7 Im Neuenheimer Feld 282, 69120 Heidelberg, Germany 8 2-Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Allianz, 9 Z-MS, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany 10 3-The Hartmut Hoffmann-Berling International Graduate School of Molecular and Cellular 11 Biology (HBIGS), University of Heidelberg, Heidelberg, 69120, Germany 12 13 *Correspondence: [email protected] 14 Key words: pericentrin, Spc110, Spc98, spindle pole body, Mps1, Cdk1, gamma-tubulin 15 complex, phosphorylation, mitosis, microtubules, oligomerization 16 17 Summary 18 Budding yeast Spc110, a member of -tubulin complex receptor family (-TuCR), recruits - 19 tubulin complexes to microtubule (MT) organizing centers (MTOCs). Biochemical studies 20 suggest that Spc110 facilitates higher-order -tubulin complex assembly (Kollman et al., 21 2010). Nevertheless the molecular basis for this activity and the regulation are unclear. 22 Here we show that Spc110 phosphorylated by Mps1 and Cdk1 activates -TuSC 23 oligomerization and MT nucleation in a cell cycle dependent manner. Interaction between 24 the N-terminus of the -TuSC subunit Spc98 and Spc110 is important for this activity. 25 Besides the conserved CM1 motif in -TuCRs (Sawin et al., 2004), a second motif that we 26 named Spc110/Pcp1 motif (SPM) is also important for MT nucleation. The activating Mps1 27 and Cdk1 sites lie between SPM and CM1 motifs. Most organisms have both SPM-CM1 28 (Spc110/Pcp1/PCNT) and CM1-only (Spc72/Mto1/Cnn/CDK5RAP2/ myomegalin) types of 29 -TuCRs. The two types of -TuCRs contain distinct but conserved C-terminal MTOC 30 targeting domains. 31 32 Highlights 33 1. Phosphorylation of Spc110 N-terminal domain (N-Spc110) regulates -TuSC 34 oligomerization. 35 2. Both SPM and CM1 are required for N-Spc110 to activate -TuSC oligomerization. 36 3. The conserved N-Spc98 is required for Spc110-mediated oligomerization. 37 4. -TuCRs can be categorized into CM1-only (Spc72/Mto1/Cnn/CDK5RAP2) and 38 SPM-CM1 (Spc110/Pcp1/PCNT) type of receptors. 39 5. While the SPM-CM1 type is targeted via the PACT domain to centrosomes/SPBs, 40 the CM1-only type has either MASC (Mto1 and Spc72 C-terminus) or a CM2 motif 41 for MTOC targeting. 42 Introduction 43 The budding yeast spindle consists of ~40 microtubules (MTs) that extend between the two 44 opposed spindle pole bodies (SPBs). Because of the closed mitosis in yeast, the SPBs 45 remain embedded in the nuclear membrane throughout mitosis. Cell-cycle regulated 46 spindle assembly begins in S-phase with nucleation of MTs onto the surface of the newly 47 assembled SPB. As soon as MTs emanate from both SPBs, they interdigitate (pole to pole 48 MTs) or attach to the kinetochores (pole to kinetochore MTs) at the end of S phase, forming 49 the bipolar spindle (O'Toole et al., 1999). 50 The -tubulin complex is the core player in MT nucleation. In budding yeast Saccharomyces 51 cerevisiae, two molecules of -tubulin (Tub4) assemble together with one molecule of 52 Spc97 (ortholog of human GCP2) and Spc98 (ortholog of human GCP3) into a tetrameric - 53 tubulin small complex (-TuSC), which is conserved in all eukaryotes (Geissler et al., 1996; 54 Guillet et al., 2011; Knop et al., 1997). The purified -TuSC of budding yeast is able to self- 55 oligomerize into symmetric ring-like structures under low salt buffer conditions. The 56 diameter and the pitch of the -TuSC ring resemble that of MT cylinder, suggesting that the 57 -TuSC ring functions as a MT nucleation template. However, the in vitro nucleation activity 58 of the -TuSC assemblies remained poor, presumably because of the suboptimal spacing 59 of every second Tub4 within the -TuSC ring that blocks interaction with tubulin in the MT 60 cylinder (Choy et al., 2009; Kollman et al., 2010; Kollman et al., 2008). The concept of- 61 TuSC oligomerization is further supported by in vivo measurements of budding yeast - 62 tubulin complex components on detached single MT nucleation sites. The - 63 tubulin:Spc97:Spc98 ratio was 2.4:1.0:1.3 with a total of ~17 -tubulin molecules per 64 nucleation site (Erlemann et al., 2012), suggesting a slight excess of -tubulin and Spc98 65 molecules over Spc97 in the assembled MT nucleation site. 66 In most eukaryotic organisms, such as fission yeast, Drosophila, Xenopus and human, 67 multiple -TuSCs further assemble with additional GCP family members (GCP4-6) into the 68 larger -tubulin ring complex (-TuRC) (Anders et al., 2006; Gunawardane et al., 2000; 69 Murphy et al., 2001; Zhang et al., 2000). However these additional GCP proteins are not 70 encoded in the budding yeast genome. 71 Various proteins are involved in the recruitment of -tubulin complexes to microtubule 72 organizing centers (MTOCs) such as centrosomes and SPBs. The small protein Mozart1 is 73 encoded in most eukaryotic genomes except the one of budding yeast (Teixido-Travesa et 74 al., 2012). In Schizosaccharomyces pombe and Arabidopsis thaliana Mozart1 interacts with 75 the GCP3 subunit of -tubulin complexes (Batzenschlager et al., 2013; Dhani et al., 2013; 76 Janski et al., 2012; Masuda et al., 2013; Nakamura et al., 2012). In S. pombe Mozart1 77 appears important for the -TuSC recruitment to SPBs (Dhani et al., 2013; Masuda et al., 78 2013). 79 Besides Mozart1, a group of conserved proteins called -tubulin complex receptors (- 80 TuCRs) are required for targeting -tubulin complexes to MTOCs. Most of them carry a 81 highly conserved centrosomin motif 1 (CM1) that interacts with GCP subunits of -tubulin 82 complexes (Sawin et al., 2004). How Mozart1 and -TuCRs cooperate is not understood. 83 However, in budding yeast cells that lack a Mozart1 gene, -TuCRs are the sole factors 84 responsible for -TuSC recruitment to SPBs. Spc110 is the budding yeast homolog of 85 pericentrin (PCNT) and functions as -TuCRs at the nuclear side of the SPB (Knop and 86 Schiebel, 1997; Sundberg and Davis, 1997). The N-terminal Spc110 encompasses the 87 CM1 that interacts with the Spc98 subunit of -TuSC (Fong et al., 2008; Knop and Schiebel, 88 1997; Nguyen et al., 1998; Samejima et al., 2008; Sawin et al., 2004; Vinh et al., 2002; 89 Zhang and Megraw, 2007). In addition, the N-terminal region of Spc110 is phosphorylated 90 in a cell-cycle dependent manner. Phospho-Spc110 appears as cells progress from S 91 phase, continues accumulating during mitosis and vanishes at the anaphase onset 92 (Friedman et al., 1996; Stirling and Stark, 1996). Spc110 phosphorylation accounts for the 93 impact of Cdk1 and Mps1 kinases on spindle dynamics (Friedman et al., 2001; Huisman et 94 al., 2007; Liang et al., 2013). However, a clear understanding behind this observation is 95 lacking. Interestingly, when -TuSC and an N-terminal fragment of Spc110 (amino acids 1- 96 220 of Spc110; Spc1101-220) were co-expressed in insect cells, a filament-like -TuSC- 97 Spc1101-220 complex formed. The nucleation capacity of this purified -TuSC-Spc1101-220 98 complex exceeded that of the -TuSC alone (Kollman et al., 2010). Thus, Spc1101-220 99 influences -TuSC properties with yet unclear mechanism. 100 Here we have tested the possibility that phosphorylation of the -TuCR Spc110 regulates 101 MT nucleation by inducing -TuSC oligomerization. Single particle analysis of -TuSC 102 incubated with phospho-mimetic Spc110 mutant proteins showed that Mps1 and Cdk1 103 promoted MT nucleation through Spc110 phosphorylation. Phosphorylated Spc110 and the 104 interaction with the N-terminal domain of Spc98 induce -TuSC oligomerization. In addition, 105 bioinformatic analysis revealed a conserved region around T18, that we named 106 Spc110/Pcp1 motif (SPM). SPM and CM1 motifs are both important for -TuSC binding and 107 oligomerization. A comparison of -TuCRs for the presence of SPM and CM1, identified 108 SPM-CM1 (Spc110, Pcp1, PCNT) and CM1-only types of -TuCRs (Spc72, Mto1, Cnn, 109 CDK5RAP2, myomegalin) in most organisms. While the SPM-CM1 type of -TuCRs carries 110 the PACT domain and is targeted only to the centrosome or the nuclear side of the SPB, 111 the CM1-only type of -TuCRs has either a MASC (Mto1 and Spc72 C-terminus) (Samejima 112 et al., 2010) or a CM2 motif and is recruited to, centrosomes, the cytoplasmic side of the 113 SPB or acentrosomal MTOCs. 114 115 Results 116 117 Phosphorylation of N-Spc110 at Mps1 and Cdk1 sites is required for efficient 118 interaction with -TuSC 119 To test whether Spc1101-220 phosphorylation promoted -TuSC ring formation, we purified 120 GST-Spc1101-220 (named Spc1101-220) from both E. coli and the baculovirus expression 121 system. Spc1101-220 encompasses Cdk1 and Mps1 phosphorylation sites and the 122 conserved CM1 motif (Figure 1A). Because of the post-translational modification 123 machinery, Spc1101-220 purified from insect cells harboured phosphorylations on S60/T68 124 and S36/S91 (Figure 1—figure supplement 1A-D), that correspond to established Mps1 and 125 Cdk1 sites, respectively (Figure 1A) (Friedman et al., 2001; Huisman et al., 2007). In 126 contrast, Spc1101-220 was not phosphorylated when purified from E. coli. 127 We incubated purified -TuSC with either the phosphorylated Spc1101-220 from insect cells 128 (Spc1101-220-P) or the non-phosphorylated Spc1101-220 from E. coli and then analyzed the 129 protein complexes by gel filtration. Spc110 dimerizes via the coiled-coli region in the centre 130 of the protein (Kilmartin et al., 1993; Muller et al., 2005).
Load more

Recommended publications
  • Mutations in CDK5RAP2 Cause Seckel Syndrome Go¨ Khan Yigit1,2,3,A, Karen E
    ORIGINAL ARTICLE Mutations in CDK5RAP2 cause Seckel syndrome Go¨ khan Yigit1,2,3,a, Karen E. Brown4,a,Hu¨ lya Kayserili5, Esther Pohl1,2,3, Almuth Caliebe6, Diana Zahnleiter7, Elisabeth Rosser8, Nina Bo¨ gershausen1,2,3, Zehra Oya Uyguner5, Umut Altunoglu5, Gudrun Nu¨ rnberg2,3,9, Peter Nu¨ rnberg2,3,9, Anita Rauch10, Yun Li1,2,3, Christian Thomas Thiel7 & Bernd Wollnik1,2,3 1Institute of Human Genetics, University of Cologne, Cologne, Germany 2Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany 3Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany 4Chromosome Biology Group, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, London, W12 0NN, UK 5Department of Medical Genetics, Istanbul Medical Faculty, Istanbul University, Istanbul, Turkey 6Institute of Human Genetics, Christian-Albrechts-University of Kiel, Kiel, Germany 7Institute of Human Genetics, Friedrich-Alexander University Erlangen-Nuremberg, Erlangen, Germany 8Department of Clinical Genetics, Great Ormond Street Hospital for Children, London, WC1N 3EH, UK 9Cologne Center for Genomics, University of Cologne, Cologne, Germany 10Institute of Medical Genetics, University of Zurich, Schwerzenbach-Zurich, Switzerland Keywords Abstract CDK5RAP2, CEP215, microcephaly, primordial dwarfism, Seckel syndrome Seckel syndrome is a heterogeneous, autosomal recessive disorder marked by pre- natal proportionate short stature, severe microcephaly, intellectual disability, and Correspondence characteristic facial features. Here, we describe the novel homozygous splice-site Bernd Wollnik, Center for Molecular mutations c.383+1G>C and c.4005-9A>GinCDK5RAP2 in two consanguineous Medicine Cologne (CMMC) and Institute of families with Seckel syndrome. CDK5RAP2 (CEP215) encodes a centrosomal pro- Human Genetics, University of Cologne, tein which is known to be essential for centrosomal cohesion and proper spindle Kerpener Str.
    [Show full text]
  • Supplemental Information Proximity Interactions Among Centrosome
    Current Biology, Volume 24 Supplemental Information Proximity Interactions among Centrosome Components Identify Regulators of Centriole Duplication Elif Nur Firat-Karalar, Navin Rauniyar, John R. Yates III, and Tim Stearns Figure S1 A Myc Streptavidin -tubulin Merge Myc Streptavidin -tubulin Merge BirA*-PLK4 BirA*-CEP63 BirA*- CEP192 BirA*- CEP152 - BirA*-CCDC67 BirA* CEP152 CPAP BirA*- B C Streptavidin PCM1 Merge Myc-BirA* -CEP63 PCM1 -tubulin Merge BirA*- CEP63 DMSO - BirA* CEP63 nocodazole BirA*- CCDC67 Figure S2 A GFP – + – + GFP-CEP152 + – + – Myc-CDK5RAP2 + + + + (225 kDa) Myc-CDK5RAP2 (216 kDa) GFP-CEP152 (27 kDa) GFP Input (5%) IP: GFP B GFP-CEP152 truncation proteins Inputs (5%) IP: GFP kDa 1-7481-10441-1290218-1654749-16541045-16541-7481-10441-1290218-1654749-16541045-1654 250- Myc-CDK5RAP2 150- 150- 100- 75- GFP-CEP152 Figure S3 A B CEP63 – – + – – + GFP CCDC14 KIAA0753 Centrosome + – – + – – GFP-CCDC14 CEP152 binding binding binding targeting – + – – + – GFP-KIAA0753 GFP-KIAA0753 (140 kDa) 1-496 N M C 150- 100- GFP-CCDC14 (115 kDa) 1-424 N M – 136-496 M C – 50- CEP63 (63 kDa) 1-135 N – 37- GFP (27 kDa) 136-424 M – kDa 425-496 C – – Inputs (2%) IP: GFP C GFP-CEP63 truncation proteins D GFP-CEP63 truncation proteins Inputs (5%) IP: GFP Inputs (5%) IP: GFP kDa kDa 1-135136-424425-4961-424136-496FL Ctl 1-135136-424425-4961-424136-496FL Ctl 1-135136-424425-4961-424136-496FL Ctl 1-135136-424425-4961-424136-496FL Ctl Myc- 150- Myc- 100- CCDC14 KIAA0753 100- 100- 75- 75- GFP- GFP- 50- CEP63 50- CEP63 37- 37- Figure S4 A siCtl
    [Show full text]
  • Genetic and Genomic Analysis of Hyperlipidemia, Obesity and Diabetes Using (C57BL/6J × TALLYHO/Jngj) F2 Mice
    University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Nutrition Publications and Other Works Nutrition 12-19-2010 Genetic and genomic analysis of hyperlipidemia, obesity and diabetes using (C57BL/6J × TALLYHO/JngJ) F2 mice Taryn P. Stewart Marshall University Hyoung Y. Kim University of Tennessee - Knoxville, [email protected] Arnold M. Saxton University of Tennessee - Knoxville, [email protected] Jung H. Kim Marshall University Follow this and additional works at: https://trace.tennessee.edu/utk_nutrpubs Part of the Animal Sciences Commons, and the Nutrition Commons Recommended Citation BMC Genomics 2010, 11:713 doi:10.1186/1471-2164-11-713 This Article is brought to you for free and open access by the Nutrition at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Nutrition Publications and Other Works by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. Stewart et al. BMC Genomics 2010, 11:713 http://www.biomedcentral.com/1471-2164/11/713 RESEARCH ARTICLE Open Access Genetic and genomic analysis of hyperlipidemia, obesity and diabetes using (C57BL/6J × TALLYHO/JngJ) F2 mice Taryn P Stewart1, Hyoung Yon Kim2, Arnold M Saxton3, Jung Han Kim1* Abstract Background: Type 2 diabetes (T2D) is the most common form of diabetes in humans and is closely associated with dyslipidemia and obesity that magnifies the mortality and morbidity related to T2D. The genetic contribution to human T2D and related metabolic disorders is evident, and mostly follows polygenic inheritance. The TALLYHO/ JngJ (TH) mice are a polygenic model for T2D characterized by obesity, hyperinsulinemia, impaired glucose uptake and tolerance, hyperlipidemia, and hyperglycemia.
    [Show full text]
  • Autosomal Recessive Primary Microcephaly
    Mahmood et al. Orphanet Journal of Rare Diseases 2011, 6:39 http://www.ojrd.com/content/6/1/39 REVIEW Open Access Autosomal recessive primary microcephaly (MCPH): clinical manifestations, genetic heterogeneity and mutation continuum Saqib Mahmood1, Wasim Ahmad2 and Muhammad J Hassan3* Abstract Autosomal Recessive Primary Microcephaly (MCPH) is a rare disorder of neurogenic mitosis characterized by reduced head circumference at birth with variable degree of mental retardation. In MCPH patients, brain size reduced to almost one-third of its original volume due to reduced number of generated cerebral cortical neurons during embryonic neurogensis. So far, seven genetic loci (MCPH1-7) for this condition have been mapped with seven corresponding genes (MCPH1, WDR62, CDK5RAP2, CEP152, ASPM, CENPJ, and STIL) identified from different world populations. Contribution of ASPM and WDR62 gene mutations in MCPH World wide is more than 50%. By and large, primary microcephaly patients are phenotypically indistinguishable, however, recent studies in patients with mutations in MCPH1, WDR62 and ASPM genes showed a broader clinical and/or cellular phenotype. It has been proposed that mutations in MCPH genes can cause the disease phenotype by disturbing: 1) orientation of mitotic spindles, 2) chromosome condensation mechanism during embryonic neurogenesis, 3) DNA damage- response signaling, 4) transcriptional regulations and microtubule dynamics, 5) certain unknown centrosomal mechanisms that control the number of neurons generated by neural precursor cells. Recent discoveries of mammalian models for MCPH have open up horizons for researchers to add more knowledge regarding the etiology and pathophysiology of MCPH. High incidence of MCPH in Pakistani population reflects the most probable involvement of consanguinity.
    [Show full text]
  • Molecular Genetics of Microcephaly Primary Hereditary: an Overview
    brain sciences Review Molecular Genetics of Microcephaly Primary Hereditary: An Overview Nikistratos Siskos † , Electra Stylianopoulou †, Georgios Skavdis and Maria E. Grigoriou * Department of Molecular Biology & Genetics, Democritus University of Thrace, 68100 Alexandroupolis, Greece; [email protected] (N.S.); [email protected] (E.S.); [email protected] (G.S.) * Correspondence: [email protected] † Equal contribution. Abstract: MicroCephaly Primary Hereditary (MCPH) is a rare congenital neurodevelopmental disorder characterized by a significant reduction of the occipitofrontal head circumference and mild to moderate mental disability. Patients have small brains, though with overall normal architecture; therefore, studying MCPH can reveal not only the pathological mechanisms leading to this condition, but also the mechanisms operating during normal development. MCPH is genetically heterogeneous, with 27 genes listed so far in the Online Mendelian Inheritance in Man (OMIM) database. In this review, we discuss the role of MCPH proteins and delineate the molecular mechanisms and common pathways in which they participate. Keywords: microcephaly; MCPH; MCPH1–MCPH27; molecular genetics; cell cycle 1. Introduction Citation: Siskos, N.; Stylianopoulou, Microcephaly, from the Greek word µικρoκεϕαλi´α (mikrokephalia), meaning small E.; Skavdis, G.; Grigoriou, M.E. head, is a term used to describe a cranium with reduction of the occipitofrontal head circum- Molecular Genetics of Microcephaly ference equal, or more that teo standard deviations
    [Show full text]
  • Changing the Name of the NBPF/DUF1220 Domain to the Olduvai Domain [Version 2; Peer Review: 3 Approved]
    F1000Research 2018, 6:2185 Last updated: 31 AUG 2021 OPINION ARTICLE Changing the name of the NBPF/DUF1220 domain to the Olduvai domain [version 2; peer review: 3 approved] Previously titled: A proposal to change the name of the NBPF/DUF1220 domain to the Olduvai domain James M. Sikela 1, Frans van Roy2,3 1Department of Biochemistry and Molecular Genetics, Human Medical Genetics and Neuroscience Programs, University of Colorado School of Medicine, Aurora, CO, 80045, USA 2Department of Biomedical Molecular Biology, Ghent University, Ghent, 9052, Belgium 3VIB-UGent Center for Inflammation Research, Ghent, 9052, Belgium v2 First published: 28 Dec 2017, 6:2185 Open Peer Review https://doi.org/10.12688/f1000research.13586.1 Latest published: 17 Jul 2018, 6:2185 https://doi.org/10.12688/f1000research.13586.2 Reviewer Status Invited Reviewers Abstract We are jointly proposing a new name for a protein domain of 1 2 3 approximately 65 amino acids that has been previously termed NBPF or DUF1220. Our two labs independently reported the initial studies of version 2 this domain, which is encoded almost entirely within a single gene (revision) report report family. The name Neuroblastoma Breakpoint Family (NBPF) was 17 Jul 2018 applied to this gene family when the first identified member of the family was found to be interrupted in an individual with version 1 neuroblastoma. 28 Dec 2017 report report report Prior to this discovery, the Pfam database had termed the domain DUF1220, denoting it as one of many protein domains of unknown f unction. It has been Pfam’s intention to use “DUF” nomenclature to 1.
    [Show full text]
  • A Newly Identified Myomegalin Isoform Functions in Golgi Microtubule Organization and ER–Golgi Transport
    ß 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 4904–4917 doi:10.1242/jcs.155408 RESEARCH ARTICLE A newly identified myomegalin isoform functions in Golgi microtubule organization and ER–Golgi transport Zhe Wang, Chao Zhang and Robert Z. Qi* ABSTRACT the formation of ER–Golgi cargo carriers that associate with dynein–dynactin to move along microtubules towards the Golgi The Golgi of mammalian cells is known to be a major microtubule- (Presley et al., 1997; Scales et al., 1997; Watson et al., 2005). organizing site that requires microtubules for its organization and The Golgi serves as a major microtubule-organizing center protein trafficking. However, the mechanisms underlying the (Chabin-Brion et al., 2001; Efimov et al., 2007; Miller et al., 2009; microtubule organization of the Golgi remain obscure. We used Rivero et al., 2009). For example, almost half of all cellular immunoprecipitation coupled with mass spectrometry to identify a microtubules originate from the Golgi in human retinal pigment widely expressed isoform of the poorly characterized muscle protein epithelial RPE1 cells (Efimov et al., 2007). Moreover, microtubule myomegalin. This newly identified isoform, myomegalin variant 8 nucleation at the Golgi does not require centrosomes, and it (MMG8), localized predominantly to cis-Golgi networks by interacting depends instead on c-tubulin (Efimov et al., 2007), the principal with AKAP450 (also known as AKAP9), and this interaction with microtubule nucleator in cells, which exists in the form of c-tubulin AKAP450 was required for the stability of both proteins. Disrupting complexes (cTuCs). The cis-Golgi proteins AKAP450 (also known MMG8 expression affected endoplasmic reticulum (ER)-to-Golgi as AKAP9, AKAP350, CG-NAP and hyperion) and GMAP210 trafficking and caused Golgi fragmentation.
    [Show full text]
  • Supplementary Data
    Supplementary Fig. 1 A B Responder_Xenograft_ Responder_Xenograft_ NON- NON- Lu7336, Vehicle vs Lu7466, Vehicle vs Responder_Xenograft_ Responder_Xenograft_ Sagopilone, Welch- Sagopilone, Welch- Lu7187, Vehicle vs Lu7406, Vehicle vs Test: 638 Test: 600 Sagopilone, Welch- Sagopilone, Welch- Test: 468 Test: 482 Responder_Xenograft_ NON- Lu7860, Vehicle vs Responder_Xenograft_ Sagopilone, Welch - Lu7558, Vehicle vs Test: 605 Sagopilone, Welch- Test: 333 Supplementary Fig. 2 Supplementary Fig. 3 Supplementary Figure S1. Venn diagrams comparing probe sets regulated by Sagopilone treatment (10mg/kg for 24h) between individual models (Welsh Test ellipse p-value<0.001 or 5-fold change). A Sagopilone responder models, B Sagopilone non-responder models. Supplementary Figure S2. Pathway analysis of genes regulated by Sagopilone treatment in responder xenograft models 24h after Sagopilone treatment by GeneGo Metacore; the most significant pathway map representing cell cycle/spindle assembly and chromosome separation is shown, genes upregulated by Sagopilone treatment are marked with red thermometers. Supplementary Figure S3. GeneGo Metacore pathway analysis of genes differentially expressed between Sagopilone Responder and Non-Responder models displaying –log(p-Values) of most significant pathway maps. Supplementary Tables Supplementary Table 1. Response and activity in 22 non-small-cell lung cancer (NSCLC) xenograft models after treatment with Sagopilone and other cytotoxic agents commonly used in the management of NSCLC Tumor Model Response type
    [Show full text]
  • Downloaded 18 July 2014 with a 1% False Discovery Rate (FDR)
    UC Berkeley UC Berkeley Electronic Theses and Dissertations Title Chemical glycoproteomics for identification and discovery of glycoprotein alterations in human cancer Permalink https://escholarship.org/uc/item/0t47b9ws Author Spiciarich, David Publication Date 2017 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California Chemical glycoproteomics for identification and discovery of glycoprotein alterations in human cancer by David Spiciarich A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Chemistry in the Graduate Division of the University of California, Berkeley Committee in charge: Professor Carolyn R. Bertozzi, Co-Chair Professor David E. Wemmer, Co-Chair Professor Matthew B. Francis Professor Amy E. Herr Fall 2017 Chemical glycoproteomics for identification and discovery of glycoprotein alterations in human cancer © 2017 by David Spiciarich Abstract Chemical glycoproteomics for identification and discovery of glycoprotein alterations in human cancer by David Spiciarich Doctor of Philosophy in Chemistry University of California, Berkeley Professor Carolyn R. Bertozzi, Co-Chair Professor David E. Wemmer, Co-Chair Changes in glycosylation have long been appreciated to be part of the cancer phenotype; sialylated glycans are found at elevated levels on many types of cancer and have been implicated in disease progression. However, the specific glycoproteins that contribute to cell surface sialylation are not well characterized, specifically in bona fide human cancer. Metabolic and bioorthogonal labeling methods have previously enabled enrichment and identification of sialoglycoproteins from cultured cells and model organisms. The goal of this work was to develop technologies that can be used for detecting changes in glycoproteins in clinical models of human cancer.
    [Show full text]
  • E-Mutpath: Computational Modelling Reveals the Functional Landscape of Genetic Mutations Rewiring Interactome Networks
    bioRxiv preprint doi: https://doi.org/10.1101/2020.08.22.262386; this version posted August 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. e-MutPath: Computational modelling reveals the functional landscape of genetic mutations rewiring interactome networks Yongsheng Li1, Daniel J. McGrail1, Brandon Burgman2,3, S. Stephen Yi2,3,4,5 and Nidhi Sahni1,6,7,8,* 1Department oF Systems Biology, The University oF Texas MD Anderson Cancer Center, Houston, TX 77030, USA 2Department oF Oncology, Livestrong Cancer Institutes, Dell Medical School, The University oF Texas at Austin, Austin, TX 78712, USA 3Institute For Cellular and Molecular Biology (ICMB), The University oF Texas at Austin, Austin, TX 78712, USA 4Institute For Computational Engineering and Sciences (ICES), The University oF Texas at Austin, Austin, TX 78712, USA 5Department oF Biomedical Engineering, Cockrell School of Engineering, The University oF Texas at Austin, Austin, TX 78712, USA 6Department oF Epigenetics and Molecular Carcinogenesis, The University oF Texas MD Anderson Science Park, Smithville, TX 78957, USA 7Department oF BioinFormatics and Computational Biology, The University oF Texas MD Anderson Cancer Center, Houston, TX 77030, USA 8Program in Quantitative and Computational Biosciences (QCB), Baylor College oF Medicine, Houston, TX 77030, USA *To whom correspondence should be addressed. Nidhi Sahni. Tel: +1 512 2379506; Email: [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.22.262386; this version posted August 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder.
    [Show full text]
  • CDK5RAP2 Functions in Centrosome to Spindle Pole Attachment and DNA Damage Response
    JCB: Article CDK5RAP2 functions in centrosome to spindle pole attachment and DNA damage response Alexis R. Barr,1,2 John V. Kilmartin,3 and Fanni Gergely1,2 1Cancer Research UK Cambridge Research Institute, Cambridge CB2 0RE, England, UK 2Department of Oncology, University of Cambridge, Cambridge CB2 3RA, England, UK 3Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH, England, UK he centrosomal protein, CDK5RAP2, is mutated fail to recruit specific PCM components that mediate in primary microcephaly, a neurodevelopmental attachment to spindle poles. Furthermore, we show that Tdisorder characterized by reduced brain size. The the CNN1 domain enforces cohesion between parental Drosophila melanogaster homologue of CDK5RAP2, centrioles during interphase and promotes efficient DNA centrosomin (Cnn), maintains the pericentriolar matrix damage–induced G2 cell cycle arrest. Because mitotic (PCM) around centrioles during mitosis. In this study, we spindle positioning, asymmetric centrosome inheritance, demonstrate a similar role for CDK5RAP2 in vertebrate and DNA damage signaling have all been implicated cells. By disrupting two evolutionarily conserved domains in cell fate determination during neurogenesis, our find- of CDK5RAP2, CNN1 and CNN2, in the avian B cell line ings provide novel insight into how impaired CDK5RAP2 DT40, we find that both domains are essential for link- function could cause premature depletion of neural stem ing centrosomes to mitotic spindle poles. Although struc- cells and thereby microcephaly. turally intact, centrosomes lacking the CNN1 domain Introduction The centrosome consists of a pair of centrioles surrounded by 1995; Merdes et al., 1996, 2000; Gordon et al., 2001; Quintyne the pericentriolar matrix (PCM). In most animal cells, it is the and Schroer, 2002; Silk et al., 2009).
    [Show full text]
  • And an Imatinib-Responsive Myeloproliferative Disorder1
    [CANCER RESEARCH 64, 2673–2676, April 15, 2004] Advances in Brief NIN, a Gene Encoding a CEP110-Like Centrosomal Protein, Is Fused to PDGFRB in a Patient with a t(5;14)(q33;q24) and an Imatinib-Responsive Myeloproliferative Disorder1 Jose´L. Vizmanos,1 Francisco J. Novo,1 Jose´P. Roma´n,1 E. Joanna Baxter,2 Idoya Lahortiga,1 Marı´a J. Larra´yoz,1 Marı´a D. Odero,1 Pilar Giraldo,3 Marı´a J. Calasanz,1 and Nicholas C. P. Cross2 1Department of Genetics, University of Navarra, Pamplona, Spain; 2Wessex Regional Genetics Laboratory, Salisbury and Human Genetics Division, University of Southampton, Southampton, United Kingdom; and 3Haematology Service, Miguel Servet Hospital, Zaragoza, Spain Abstract mesylate (4–7) although this drug is inactive against FGFR1 fusions (8). Consequently, identification of patients with PDGFRB rearrange- We describe a new PDGFRB fusion associated with a t(5;14)(q33;q24) ments is very important for their clinical management. Rearrangement in a patient with a longstanding chronic myeloproliferative disorder with of PDGFRB was first described in patients with the t(5;12)(q31;p12), eosinophilia. After confirmation of PDGFRB involvement and definition of the chromosome 14 breakpoint by fluorescence in situ hybridization, leading to the formation of the ETV6-PDGFRB fusion (9). Subse- candidate partner genes were selected on the basis of the presence of quently, PDGFRB has been found fused to CEV14/TRIP11 by the predicted oligomerization domains believed to be an essential feature of t(5;14)(q31;p12) (Ref. 10); HIP1 by the t(5;7)(q33;q11.2) (Ref.
    [Show full text]