DOCSLIB.ORG

Insights Into BTB-Cul3 Ubiquitin Ligases from the Structures of SPOP-Substrate Complexes Min Zhuang University of Tennessee Health Science Center

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Insights Into BTB-Cul3 Ubiquitin Ligases from the Structures of SPOP-Substrate Complexes Min Zhuang University of Tennessee Health Science Center University of Tennessee Health Science Center UTHSC Digital Commons Theses and Dissertations (ETD) College of Graduate Health Sciences 5-2009 Insights into BTB-Cul3 Ubiquitin Ligases from the Structures of SPOP-Substrate Complexes Min Zhuang University of Tennessee Health Science Center Follow this and additional works at: https://dc.uthsc.edu/dissertations Part of the Amino Acids, Peptides, and Proteins Commons, Enzymes and Coenzymes Commons, Medical Biochemistry Commons, and the Medical Cell Biology Commons Recommended Citation Zhuang, Min , "Insights into BTB-Cul3 Ubiquitin Ligases from the Structures of SPOP-Substrate Complexes" (2009). Theses and Dissertations (ETD). Paper 328. http://dx.doi.org/10.21007/etd.cghs.2009.0386. This Dissertation is brought to you for free and open access by the College of Graduate Health Sciences at UTHSC Digital Commons. It has been accepted for inclusion in Theses and Dissertations (ETD) by an authorized administrator of UTHSC Digital Commons. For more information, please contact [email protected]. Insights into BTB-Cul3 Ubiquitin Ligases from the Structures of SPOP- Substrate Complexes Document Type Dissertation Degree Name Doctor of Philosophy (PhD) Program Biomedical Sciences Track Cell Biology and Biochemistry Research Advisor Brenda A. Schulman, Ph.D. Committee Mary-Ann Bjornsti, Ph.D. Lawrence M. Pfeffer, Ph.D. Martine F. Roussel, Ph.D. Stephen W. White, Ph.D. DOI 10.21007/etd.cghs.2009.0386 This dissertation is available at UTHSC Digital Commons: https://dc.uthsc.edu/dissertations/328 Insights into BTB-Cul3 Ubiquitin Ligases from the Structures of SPOP-Substrate Complexes A Dissertation Presented for The Graduate Studies Council The University of Tennessee Health Science Center In Partial Fulfillment Of the Requirements for the Degree Doctor of Philosophy From The University of Tennessee By Min Zhuang May 2009 Copyright © 2009 by Min Zhuang All rights reserved ii DEDICATION To my parents, Jianhua Zhuang, Fengying Zhou and my husband, Haopeng Wang for their love and support iii ACKNOWLEDGEMENTS First, I would like to sincerely thank my mentor, Dr. Brenda A. Schulman, who opened the door to science for me, shared her experience, showed me an interesting world and, most importantly, helped me to better know myself. Brenda is an amazing scientist, from whom I learned a lot. I thank Brenda for teaching me to think critically and solve problems systematically, which will benefit me in the long term. Brenda is also a lovely person, as long as you do not break the -80 °C fridge in the lab. We had a wonderful vacation together, which is one of my best personal experiences with her. Also, I would like to thank other committee members: Drs. Mary-Ann Bjornsti, Lawrence Pfeffer, Martine Roussel and Steve White for their assistance and time over the past four years. I’m so lucky to have had their advices at the early stage of my career. My thanks are also given to Dr. Wade Harper (Harvard Medical School) for his insights into the project, Dr. Kevin White and Dr. Jiang Liu (the University of Chicago) for their contributions of in vivo experiments, Dr. Michal Hammel (Lawrence Berkeley National Laboratory) for SAXS analysis, Dr. Amanda Nourse and Brett Waddell (Hartwell Center) for protein interaction study and Dr. Jianmin Wang (Hartwell Center) for bioinformatics, without whose collaboration this work would not be complete. In addition, I want to thank Dr. Charles Ross and Dr. Darcie Miller of St. Jude Department of Structural Biology for remote data collection and software assistance. Further, I would like to thank all the members of the Schulman lab, past and present, for their friendship and encouragement. I want to thank Danny Huang, who was practically my mentor for my first six months in the lab and has been a great friend and a role model of mine; Brett Waddell, who was so patient to train me when I barely understood English several years ago and who recently assisted me with tons of BIACORE experiments after he joined Hartwell Center; Helen Walden, who gave the lab the feel of family and accompanied me for my first synchrotron trip and led me to the world of crystallography; Dave Duda, who has been a source of technical expertise and has answered thousands of my “quick questions”; David Miller, who provides tremendous support to build the great environment for my studies; Jing Wang, who is always cheerful and a motivation for me to do physical exercise; Laura Borg, maybe Laura Scott now, whose birthday is the closest to mine and whose Thanksgiving cookies are always sweet; Danny Scott and Billy Dye, who helped me with my presentation skills and helped me better understand my project; Catherine Regni, who was a great desk neighbor and helped me with my proposal; Hari Kamadurai, who encouraged me to keep going; Judith Souphron and Asad Taherbhoy, fellow graduate students, with whom I shared a lot of things in common; Allison Williams and Woody Hunt, who brought so much fun to the lab; Steven Seyedin, who sent me directions in San Francisco during my interview; Allison Voss, who provided me a protein sample she made; and Shelia Bozeman, who has the warmest hug and fed me well with tons of donuts and candies. I want to thank Danny Scott and Dave Duda again for their proofreading of this dissertation. iv Finally, I would like to thank all my family members and friends who have given to me their love and support, especially my parents Jianhua Zhuang and Fengying Zhou, who have provided me invaluable educational opportunities; and my husband Haopeng Wang, who believed in me and supported me during the inevitable stressful and frustrating moments. v ABSTRACT Cullin-Ring ubiquitin ligases (CRLs) are E3 complexes that specifically recognize substrates through substrate adaptors. In the largest CRL subfamily, Cul3 binds a BTB domain, and a protein-interaction domain such as MATH recruits substrates for ubiquitination. Here we present biochemical and structural analyses of the MATH and BTB domain containing protein, SPOP, which regulates diverse signaling pathways. First, we identified a conserved SPOP Binding Consensus (SBC) motif in the transcriptional regulator Ci, the protein phosphatase Puc, and the chromatin component MacroH2A. The SBC motif specifically binds the MATH domain of SPOP, and is required for Puc ubiquitination in vitro and in vivo. Structural determination and analysis of SPOPMATH in complex with peptides encompassing the SBC motif revealed the molecular basis for recognition of diverse substrates by SPOP. Second, the dimeric BTB domain of SPOP assembles into a dimer with Cul3, an interaction that is facilitated by a helical motif that we term 3-box due to resemblance to F- and SOCS-boxes in other cullin-based E3s. 3-box is also found in the BTB protein Gigaxonin and is predicted in a subset of BTB proteins. Furthermore, structures of nearly full-length SPOP constructs indicate flexibility between the MATH and BTB domains, potentially allowing regulation of diverse substrates. Those such as Puc with multiple SBCs may functionally interact with a single SPOP dimer. All together, this study provides a molecular understanding of how MATH-BTB proteins recruit substrates to Cul3, and how their dimerization and structural variability may facilitate recognition and ubiquitination of diverse substrates. vi TABLE OF CONTENTS CHAPTER 1. INTRODUCTION .............................................................................1 1.1 UBIQUITIN LIGASES AND SUBGROUPS ........................................................1 1.2 CULLIN RING LIGASE MODULES ..................................................................3 1.2.1 Composition of CRLs .................................................................................3 1.2.2 Structure of CRLs.......................................................................................5 1.2.3 Regulation of CRLs by NEDD8 Modification.............................................7 1.2.4 CRLs Dimerization.....................................................................................7 1.2.5 CRLs Substrate Recognition .......................................................................8 1.3 CUL3 SUBSTRATE ADAPTORS: BTB PROTEINS...........................................9 1.3.1 BTB Domain: the Versatile Protein-Protein Interaction Domain .................9 1.3.2 Structures of BTB Domains ........................................................................9 1.3.3 BTB Protein Subfamilies ..........................................................................10 1.3.4 MATH-BTBs: the Largest BTB Subfamily...............................................10 1.4 SPOP: THE BEST-STUDIED MAMMALIAN MATH-BTB..............................10 1.5 OUTLINES.........................................................................................................11 CHAPTER 2. EXPERIMENTAL PROCEDURES ...............................................13 2.1 IDENTIFICATION OF WORKABLE PROTEIN FRAGMENTS.......................13 2.2 STRUCTURE DETERMINATION OF SPOPBTB+ ..............................................16 2.2.1 Cloning and Purification of SPOPBTB+ ......................................................16 2.2.2 Crystallization of SPOPBTB+ ......................................................................17 2.2.3 Data Collection of SPOPBTB+ ....................................................................18 2.2.4 Phase Determination of SPOPBTB+ ............................................................18 2.2.5 Crystallographic Refinement of SPOPBTB+
Load more

Recommended publications
  • Transcriptome Analyses of Rhesus Monkey Pre-Implantation Embryos Reveal A
    Downloaded from genome.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press Transcriptome analyses of rhesus monkey pre-implantation embryos reveal a reduced capacity for DNA double strand break (DSB) repair in primate oocytes and early embryos Xinyi Wang 1,3,4,5*, Denghui Liu 2,4*, Dajian He 1,3,4,5, Shengbao Suo 2,4, Xian Xia 2,4, Xiechao He1,3,6, Jing-Dong J. Han2#, Ping Zheng1,3,6# Running title: reduced DNA DSB repair in monkey early embryos Affiliations: 1 State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, China 2 Key Laboratory of Computational Biology, CAS Center for Excellence in Molecular Cell Science, Collaborative Innovation Center for Genetics and Developmental Biology, Chinese Academy of Sciences-Max Planck Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China 3 Yunnan Key Laboratory of Animal Reproduction, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, China 4 University of Chinese Academy of Sciences, Beijing, China 5 Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming, Yunnan 650204, China 6 Primate Research Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, 650223, China * Xinyi Wang and Denghui Liu contributed equally to this work 1 Downloaded from genome.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press # Correspondence: Jing-Dong J. Han, Email: [email protected]; Ping Zheng, Email: [email protected] Key words: rhesus monkey, pre-implantation embryo, DNA damage 2 Downloaded from genome.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press ABSTRACT Pre-implantation embryogenesis encompasses several critical events including genome reprogramming, zygotic genome activation (ZGA) and cell fate commitment.
    [Show full text]
  • Genomic Analysis of the TRIM Family Reveals Two Groups of Genes with Distinct Evolutionary Properties
    BMC Evolutionary Biology BioMed Central Research article Open Access Genomic analysis of the TRIM family reveals two groups of genes with distinct evolutionary properties Marco Sardiello1, Stefano Cairo1,3, Bianca Fontanella1,4, Andrea Ballabio1,2 and Germana Meroni*1 Address: 1Telethon Institute of Genetics and Medicine (TIGEM), Via P. Castellino 111, 80131 Naples, Italy, 2Department of Pediatrics, Federico II University, Via Sergio Pansini 5, 80131 Naples, Italy, 3Unite d'Oncogenèse et Virologie Moléculaire, Batiment Lwoff, Institut Pasteur, 28 rue de Dr. Roux, 75724 Paris Cedex 15, France and 4Department of Pharmaceutical Sciences, University of Salerno, 84084 Fisciano (SA), Italy Email: Marco Sardiello - [email protected]; Stefano Cairo - [email protected]; Bianca Fontanella - [email protected]; Andrea Ballabio - [email protected]; Germana Meroni* - [email protected] * Corresponding author Published: 1 August 2008 Received: 11 September 2007 Accepted: 1 August 2008 BMC Evolutionary Biology 2008, 8:225 doi:10.1186/1471-2148-8-225 This article is available from: http://www.biomedcentral.com/1471-2148/8/225 © 2008 Sardiello et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: The TRIM family is composed of multi-domain proteins that display the Tripartite Motif (RING, B-box and Coiled-coil) that can be associated with a C-terminal domain. TRIM genes are involved in ubiquitylation and are implicated in a variety of human pathologies, from Mendelian inherited disorders to cancer, and are also involved in cellular response to viral infection.
    [Show full text]
  • 14321 NAE1/APPBP1 (D9I4Z) Rabbit Mab
    Revision 1 C 0 2 - t NAE1/APPBP1 (D9I4Z) Rabbit mAb a e r o t S Orders: 877-616-CELL (2355) [email protected] 1 Support: 877-678-TECH (8324) 2 3 Web: [email protected] 4 www.cellsignal.com 1 # 3 Trask Lane Danvers Massachusetts 01923 USA For Research Use Only. Not For Use In Diagnostic Procedures. Applications: Reactivity: Sensitivity: MW (kDa): Source/Isotype: UniProt ID: Entrez-Gene Id: WB H M R Mk Endogenous 60 Rabbit IgG Q13564 8883 Product Usage Information 2. Huang, D.T. et al. (2005) Mol Cell 17, 341-50. 3. Liakopoulos, D. et al. (1998) EMBO J 17, 2208-14. Application Dilution 4. Gong, L. and Yeh, E.T. (1999) J Biol Chem 274, 12036-42. 5. Wada, H. et al. (2000) J Biol Chem 275, 17008-15. Western Blotting 1:1000 6. Sakata, E. et al. (2007) Nat Struct Mol Biol 14, 167-8. 7. Kawakami, T. et al. (2001) EMBO J 20, 4003-12. Storage 8. Podust, V.N. et al. (2000) Proc Natl Acad Sci USA 97, 4579-84. 9. Wu, K. et al. (2002) J Biol Chem 277, 516-27. Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% 10. Amir, R.E. et al. (2002) J Biol Chem 277, 23253-9. glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody. 11. Herrmann, J. et al. (2007) Circ Res 100, 1276-91. 12. Walden, H. et al. (2003) Mol Cell 12, 1427-37.
    [Show full text]
  • Is APP Key to the Appbp1 Pathway?
    Open Access Full Text Article Austin Alzheimer’s and Parkinson’s Disease A Austin Publishing Group Review Article Cycle on Wheels: Is APP Key to the AppBp1 Pathway? Chen Y1,2*, Neve RN4, Zheng H3, Griffin WST1,2, Barger SW1,2 and Mrak RE5 Abstract 1Department of Geriatrics, University of Arkansas for Alzheimer’s disease (AD) is the gradual loss of the cognitive function due Medical Sciences, USA to neuronal death. Currently no therapy is available to slow down, reverse 2Department of Neurobiology and Developmental or prevent the disease. Here we analyze the existing data in literature and Sciences, University of Arkansas for Medical Sciences, hypothesize that the physiological function of the Amyloid Precursor Protein USA (APP) is activating the AppBp1 pathway and this function is gradually lost during 3Huffington Center on Aging, Baylor College of Medicine, the progression of AD pathogenesis. The AppBp1 pathway, also known as the USA neddylation pathway, activates the small ubiquitin-like protein nedd8, which 4Department of Brain and Cognitive Sciences, covalently modifies and switches on Cullin ubiquitin ligases, which are essential Massachusetts Institute of Technology, USA in the turnover of cell cycle proteins. Here we discuss how APP may activate the 5Department of Pathology, University of Toledo Health AppBp1 pathway, which downregulates cell cycle markers and protects genome Sciences Campus, USA integrity. More investigation of this mechanism-driven hypothesis may provide *Corresponding author: Chen Y, Department insights into disease treatment and prevention strategies. of Geriatrics and Department of Neurobiology and Keywords: APP; Alzheimer’s disease; Ubiquitination; Neddylation; Cell Developmental Sciences, University of Arkansas for cycle Medical Sciences, Little Rock, AR 72205, USA Received: September 02, 2014; Accepted: September 29, 2014; Published: September 30, 2014 Abbreviations at least 18 proteins have been identified to bind AICD [25-27].
    [Show full text]
  • APPBP1 (NAE1) Mouse Monoclonal Antibody [Clone ID: OTI1E10] Product Data
    OriGene Technologies, Inc. 9620 Medical Center Drive, Ste 200 Rockville, MD 20850, US Phone: +1-888-267-4436 [email protected] EU: [email protected] CN: [email protected] Product datasheet for TA804384 APPBP1 (NAE1) Mouse Monoclonal Antibody [Clone ID: OTI1E10] Product data: Product Type: Primary Antibodies Clone Name: OTI1E10 Applications: IHC, WB Recommended Dilution: WB 1:2000, IHC 1:150 Reactivity: Human, Mouse, Rat Host: Mouse Isotype: IgG1 Clonality: Monoclonal Immunogen: Human recombinant protein fragment corresponding to amino acids 1-274 of human NAE1 (NP_001018170) produced in E.coli. Formulation: PBS (PH 7.3) containing 1% BSA, 50% glycerol and 0.02% sodium azide. Concentration: 1 mg/ml Purification: Purified from mouse ascites fluids or tissue culture supernatant by affinity chromatography (protein A/G) Conjugation: Unconjugated Storage: Store at -20°C as received. Stability: Stable for 12 months from date of receipt. Predicted Protein Size: 50.4 kDa Gene Name: NEDD8 activating enzyme E1 subunit 1 Database Link: NP_001018170 Entrez Gene 84019 RatEntrez Gene 234664 MouseEntrez Gene 8883 Human Q13564 This product is to be used for laboratory only. Not for diagnostic or therapeutic use. View online » ©2021 OriGene Technologies, Inc., 9620 Medical Center Drive, Ste 200, Rockville, MD 20850, US 1 / 3 APPBP1 (NAE1) Mouse Monoclonal Antibody [Clone ID: OTI1E10] – TA804384 Background: The protein encoded by this gene binds to the beta-amyloid precursor protein. Beta-amyloid precursor protein is a cell surface protein with signal-transducing properties, and it is thought to play a role in the pathogenesis of Alzheimer's disease. In addition, the encoded protein can form a heterodimer with UBE1C and bind and activate NEDD8, a ubiquitin-like protein.
    [Show full text]
  • The Human Dcn1-Like Protein DCNL3 Promotes Cul3 Neddylation at Membranes
    The human Dcn1-like protein DCNL3 promotes Cul3 neddylation at membranes Nathalie Meyer-Schallera, Yang-Chieh Choub,c, Izabela Sumaraa, Dale D. O. Martind, Thimo Kurza, Nadja Kathedera, Kay Hofmanne, Luc G. Berthiaumed, Frank Sicherib,c, and Matthias Petera,1 aInstitute of Biochemistry, Eidgeno¨ssiche Technische Hochschule, 8093 Zurich, Switzerland; bCenter for Systems Biology, Samuel Lunenfeld Research Institute, Toronto, ON, Canada M5G 1X5; cDepartment of Molecular Genetics, University of Toronto, Toronto, ON, Canada M5S 1A8; dDepartment of Cell Biology, University of Alberta, Edmonton, AB, Canada T6G 2H7; and eBioinformatics Group, Miltenyi Biotec, 51429 Bergisch-Gladbach, Germany Edited by Michael Rape, University of California, Berkeley, CA, and accepted by the Editorial Board June 9, 2009 (received for review December 9, 2008) Cullin (Cul)-based E3 ubiquitin ligases are activated through the enzyme and promotes Nedd8 conjugation through formation of attachment of Nedd8 to the Cul protein. In yeast, Dcn1 (defective this complex (14, 15). Human cells harbor 5 Dcn1-like proteins in Cul neddylation 1 protein) functions as a scaffold-like Nedd8 termed DCNL1–DCNL5 (also named DCUN1D 1–5 for defec- E3-ligase by interacting with its Cul substrates and the Nedd8 E2 tive in Cul neddylation 1 domain-containing protein 1–5) (Fig. Ubc12. Human cells express 5 Dcn1-like (DCNL) proteins each S1). These DCNLs have distinct amino-terminal domains, but containing a C-terminal potentiating neddylation domain but dis- share a conserved C-terminal potentiating neddylation (PONY) tinct amino-terminal extensions. Although the UBA-containing domain, which in yeast Dcn1 is necessary and sufficient for Cul DCNL1 and DCNL2 are likely functional homologues of yeast Dcn1, neddylation in vivo and in vitro (14).
    [Show full text]
  • And CAND1-Dependent Remodelling of the Budding Yeast SCF Complex
    ARTICLE Received 31 Jan 2013 | Accepted 20 Feb 2013 | Published 27 Mar 2013 DOI: 10.1038/ncomms2628 OPEN CSN- and CAND1-dependent remodelling of the budding yeast SCF complex Aleksandra Zemla1, Yann Thomas1, Sylwia Kedziora1, Axel Knebel1, Nicola T Wood1, Gwenae¨l Rabut2 & Thimo Kurz1 Cullin–RING ligases (CRLs) are ubiquitin E3 enzymes with variable substrate-adaptor and -receptor subunits. All CRLs are activated by modification of the cullin subunit with the ubiquitin-like protein Nedd8 (neddylation). The protein CAND1 (Cullin-associated-Nedd8- dissociated-1) also promotes CRL activity, even though it only interacts with inactive ligase complexes. The molecular mechanism underlying this behaviour remains largely unclear. Here, we find that yeast SCF (Skp1–Cdc53–F-box) Cullin–RING complexes are remodelled in a CAND1-dependent manner, when cells are switched from growth in fermentable to non-fermentable carbon sources. Mechanistically, CAND1 promotes substrate adaptor release following SCF deneddylation by the COP9 signalosome (CSN). CSN- or CAND1- mutant cells fail to release substrate adaptors. This delays the formation of new complexes during SCF reactivation and results in substrate degradation defects. Our results shed light on how CAND1 regulates CRL activity and demonstrate that the cullin neddylation– deneddylation cycle is not only required to activate CRLs, but also to regulate substrate specificity through dynamic substrate adaptor exchange. 1 Scottish Institute for Cell Signalling, Protein Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, UK. 2 CNRS, Universite´ Rennes 1, Institut de Ge´ne´tique et De´veloppement de Rennes, 2 avenue du Professeur Le´on Bernard, CS 34317, Rennes Cedex 35043, France.
    [Show full text]
  • Multiple Ser/Thr-Rich Degrons Mediate the Degradation of Ci/Gli by the Cul3-HIB/SPOP E3 Ubiquitin Ligase
    Multiple Ser/Thr-rich degrons mediate the degradation of Ci/Gli by the Cul3-HIB/SPOP E3 ubiquitin ligase Qing Zhanga,1,2, Qing Shia,1, Yongbin Chena,1, Tao Yuea, Shuang Lia, Bing Wanga, and Jin Jianga,b,3 Departments of aDevelopmental Biology and bPharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390 Communicated by Gary Struhl, Columbia University College of Physicians and Surgeons, New York, NY, October 19, 2009 (received for review September 8, 2009) The Cul3-based E3 ubiquitin ligases regulate many cellular pro- morphogenetic furrow (MF), where HIB acts together with Cul3 cesses using a large family of BTB domain–containing proteins as to degrade Ci, thereby limiting the duration of Hh signaling (11, their target recognition components, but how they recognize 12, 14). The Cul3-HIB regulatory circuit appears to be con- targets remains unknown. Here we identify and characterize served, because Gli proteins such as Gli2 and Gli3 can be degrons that mediate the degradation of the Hedgehog pathway degraded by HIB when expressed in Drosophila, and the mam- transcription factor cubitus interruptus (Ci)/Gli by Cul3-Hedghog– malian homolog of HIB, SPOP, can functionally replace HIB in induced MATH and BTB domain–containing protein (HIB)/SPOP. Ci degrading Ci (11). uses multiple Ser/Thr (S/T)-rich motifs that bind HIB cooperatively How Cul3-based E3 ligases recognize their substrates is to mediate its degradation. We provide evidence that both HIB and unknown, and the specific degrons in their target proteins Ci form dimers/oligomers and engage in multivalent interactions, remain to be identified for individual BTB proteins that function which underlies the in vivo cooperativity among individual HIB- as target-recognition components.
    [Show full text]
  • Kelch-Like Protein 2 Mediates Angiotensin II–With No Lysine 3 Signaling in the Regulation of Vascular Tonus
    BASIC RESEARCH www.jasn.org Kelch-Like Protein 2 Mediates Angiotensin II–With No Lysine 3 Signaling in the Regulation of Vascular Tonus Moko Zeniya, Nobuhisa Morimoto, Daiei Takahashi, Yutaro Mori, Takayasu Mori, Fumiaki Ando, Yuya Araki, Yuki Yoshizaki, Yuichi Inoue, Kiyoshi Isobe, Naohiro Nomura, Katsuyuki Oi, Hidenori Nishida, Sei Sasaki, Eisei Sohara, Tatemitsu Rai, and Shinichi Uchida Department of Nephrology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan ABSTRACT Recently, the kelch-like protein 3 (KLHL3)–Cullin3 complex was identified as an E3 ubiquitin ligase for with no lysine (WNK) kinases, and the impaired ubiquitination of WNK4 causes pseudohypoaldosteronism type II (PHAII), a hereditary hypertensive disease. However, the involvement of WNK kinase regulation by ubiquitination in situations other than PHAII has not been identified. Previously, we identified the WNK3–STE20/SPS1-related proline/alanine-rich kinase–Na/K/Cl cotransporter isoform 1 phosphoryla- tion cascade in vascular smooth muscle cells and found that it constitutes an important mechanism of vascular constriction by angiotensin II (AngII). In this study, we investigated the involvement of KLHL proteins in AngII-induced WNK3 activation of vascular smooth muscle cells. In the mouse aorta and mouse vascular smooth muscle (MOVAS) cells, KLHL3 was not expressed, but KLHL2, the closest homolog of KLHL3, was expressed. Salt depletion and acute infusion of AngII decreased KLHL2 and increased WNK3 levels in the mouse aorta. Notably, the AngII-induced changes in KLHL2 and WNK3 expression occurred within minutes in MOVAS cells. Results of KLHL2 overexpression and knockdown experiments in MOVAS cells confirmed that KLHL2 is the major regulator of WNK3 protein abundance.
    [Show full text]
  • Filamins but Not Janus Kinases Are Substrates of the Asb2a Cullin-Ring E3 Ubiquitin Ligase in Hematopoietic Cells
    Filamins but Not Janus Kinases Are Substrates of the ASB2a Cullin-Ring E3 Ubiquitin Ligase in Hematopoietic Cells Isabelle Lamsoul1,2, Monique Erard1,2, Peter F. M. van der Ven3, Pierre G. Lutz1,2* 1 CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), 205 route de Narbonne BP64182, F-31077 Toulouse, France, 2 Universite´ de Toulouse, UPS, IPBS, F- 31077 Toulouse, France, 3 Department of Molecular Cell Biology, Institute of Cell Biology, University of Bonn, Bonn, Germany Abstract The ASB2a protein is the specificity subunit of an E3 ubiquitin ligase complex involved in hematopoietic differentiation and is proposed to exert its effects by regulating the turnover of specific proteins. Three ASB2a substrates have been described so far: the actin-binding protein filamins, the Mixed Lineage Leukemia protein, and the Janus kinases 2 and 3. To determine the degradation of which substrate drives ASB2a biological effects is crucial for the understanding of ASB2a functions in hematopoiesis. Here, we show that neither endogenous nor exogenously expressed ASB2a induces degradation of JAK proteins in hematopoietic cells. Furthermore, we performed molecular modeling to generate the first structural model of an E3 ubiquitin ligase complex of an ASB protein bound to one of its substrates. Citation: Lamsoul I, Erard M, van der Ven PFM, Lutz PG (2012) Filamins but Not Janus Kinases Are Substrates of the ASB2a Cullin-Ring E3 Ubiquitin Ligase in Hematopoietic Cells. PLoS ONE 7(8): e43798. doi:10.1371/journal.pone.0043798 Editor: Zhengqi Wang, Emory University, United States of America Received March 21, 2012; Accepted July 26, 2012; Published August 20, 2012 Copyright: ß 2012 Lamsoul et al.
    [Show full text]
  • Cullin 5 Regulates Cortical Layering by Modulating the Speed and Duration of Dab1-Dependent Neuronal Migration
    5668 • The Journal of Neuroscience, April 21, 2010 • 30(16):5668–5676 Cellular/Molecular Cullin 5 Regulates Cortical Layering by Modulating the Speed and Duration of Dab1-Dependent Neuronal Migration Sergi Simó, Yves Jossin, and Jonathan A. Cooper Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109 Themultilayeredmammalianneocortexdevelopsbythecoordinatedimmigrationanddifferentiationofcellsthatareproducedatdistant sites. Correct layering requires an extracellular protein, Reelin (Reln), an intracellular signaling molecule, Disabled-1 (Dab1), and an E3 ubiquitinligase,Cullin-5(Cul5).RelnactivatesDab1,whichisthendegradedbyCul5.HerewetestwhetherCul5regulatesneuronlayering by affecting Dab1 stability or other mechanisms. We find that a stabilized mutant Dab1, which resists Cul5-dependent degradation, causes a similar phenotype to Cul5 deficiency. Moreover, Cul5 has no effect when Dab1 is absent. The effects of Cul5 and Dab1 are cell autonomous, and Cul5 regulates movement of early as well as late cortical neurons. Removing Cul5 increases the speed at which neurons migrate through the cortical plate by reducing the time spent stationary and increasing the speed of individual steps. These results show thatCul5regulatesneuronlayeringbystimulatingDab1degradationandthatCul5controlsmigrationspeedandstoppingpoint,andthey demonstrate the importance of negative feedback in signaling during cortical development. Introduction (Pinto-Lord et al., 1982; Dulabon et al., 2000; Sanada et al., 2004). The mammalian
    [Show full text]
  • NAE1 (APPBP1) Antibody (C-Term) Purified Rabbit Polyclonal Antibody (Pab) Catalog # Ap7273b
    10320 Camino Santa Fe, Suite G San Diego, CA 92121 Tel: 858.875.1900 Fax: 858.622.0609 NAE1 (APPBP1) Antibody (C-term) Purified Rabbit Polyclonal Antibody (Pab) Catalog # AP7273b Specification NAE1 (APPBP1) Antibody (C-term) - Product Information Application WB, IHC-P,E Primary Accession Q13564 Other Accession Q9Z1A5, Q8VBW6, Q4R3L6, NP_003896 Reactivity Human Predicted Monkey, Mouse, Rat Host Rabbit Clonality Polyclonal Isotype Rabbit Ig Calculated MW 60246 Antigen Region 430-459 NAE1 (APPBP1) Antibody (C-term) - Additional Western blot analysis of anti-APPBP1 Information Antibody (C-term) (Cat.#AP7273b) in mouse brain tissue lysates (35ug/lane). Gene ID 8883 APPBP1(arrow) was detected using the purified Pab. Other Names NEDD8-activating enzyme E1 regulatory subunit, Amyloid beta precursor protein-binding protein 1, 59 kDa, APP-BP1, Amyloid protein-binding protein 1, Proto-oncogene protein 1, NAE1, APPBP1 Target/Specificity This NAE1 (APPBP1) antibody is generated from rabbits immunized with a KLH conjugated synthetic peptide between 430-459 amino acids from the C-terminal region of human NAE1 (APPBP1). Dilution WB~~1:1000 IHC-P~~1:50~100 Western blot analysis of APP-BP1(arrow) using rabbit polyclonal APP-BP1 Antibody Format (C-term) (Cat.#AP7273b). 293 cell lysates (2 Purified monoclonal antibody supplied in ug/lane) either nontransfected (Lane 1) or PBS with 0.09% (W/V) sodium azide. This transiently transfected with the APP-BP1 antibody is purified through a protein G gene (Lane 2) (Origene Technologies). column, followed by dialysis against PBS. Storage Maintain refrigerated at 2-8°C for up to 2 Page 1/2 10320 Camino Santa Fe, Suite G San Diego, CA 92121 Tel: 858.875.1900 Fax: 858.622.0609 weeks.
    [Show full text]