Home LiteratureArticle Details
PMID: 19091944 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

Analysis of nondegradative protein ubiquitylation with a monoclonal antibody specific for lysine-63-linked polyubiquitin.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 105 ·No. 51 ·2008-12-23 ·Pages 20197-202

Wang H, Matsuzawa A, Brown SA, Zhou J, Guy CS, Tseng PH, Forbes K, Nicholson TP, Sheppard PW, Häcker H, Karin M, Vignali DA

Abstract

Modification of proteins by the addition of lysine (K)-63-linked polyubiquitin (polyUb) chains is suggested to play important roles in a variety of cellular events, including DNA repair, signal transduction, and receptor endocytosis. However, identifying such modifications in living cells is complex and cumbersome. We have generated a monoclonal antibody (mAb) that specifically recognizes K63-linked polyUb, but not any other isopeptide-linked (K6, K11, K27, K29, K33, or K48) polyUb or monoubiquitin. We demonstrate the sensitivity and specificity of this K63Ub-specific mAb to detect K63Ub-modified proteins in cell lysates by Western blotting and in cells by immunofluorescence, and K63Ub-modified TRAF6 and MEKK1 in vitro and ex vivo. This unique mAb will facilitate the analysis of K63-linked polyubiquitylation ex vivo and presents a strategy for the generation of similar reagents against other forms of polyUb.

MeSH Terms
Antibodies, Monoclonal Antibody Specificity Clinical Laboratory Techniques Lysine Polyubiquitin/analysis,immunology,isolation & purification Protein Processing, Post-Translational Ubiquitination
Chemicals
Antibodies, Monoclonal Polyubiquitin Lysine
Authors & Affiliations
12 authors, click to expand affiliations / ORCID
Wang Haopeng
Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN 38105-2794, USA.
Matsuzawa Atsushi
Brown Scott A
Zhou JingRan
Guy Cliff S
Tseng Ping-Hui
Forbes Karen
Nicholson Thomas P
Sheppard Paul W
Häcker Hans
Karin Michael
Vignali Dario A A
References (53)
53 references, click to expand
  1. Novel multiubiquitin chain linkages catalyzed by the conjugating enzymes E2EPF and RAD6 are recognized by 26 S proteasome subunit 5.
    J Biol Chem. 1996 Feb 2;271(5):2823-31 PMID: 8576261
  2. Kinase MEKK1 is required for CD40-dependent activation of the kinases Jnk and p38, germinal center formation, B cell proliferation and antibody production.
    Nat Immunol. 2007 Jan;8(1):57-63 PMID: 17143273
  3. Ubiquitylation of MEKK1 inhibits its phosphorylation of MKK1 and MKK4 and activation of the ERK1/2 and JNK pathways.
    J Biol Chem. 2003 Jan 17;278(3):1403-6 PMID: 12456688
  4. Certain pairs of ubiquitin-conjugating enzymes (E2s) and ubiquitin-protein ligases (E3s) synthesize nondegradable forked ubiquitin chains containing all possible isopeptide linkages.
    J Biol Chem. 2007 Jun 15;282(24):17375-86 PMID: 17426036
  5. The PHD domain of MEKK1 acts as an E3 ubiquitin ligase and mediates ubiquitination and degradation of ERK1/2.
    Mol Cell. 2002 May;9(5):945-56 PMID: 12049732
  6. Distinct regulation of Ubc13 functions by the two ubiquitin-conjugating enzyme variants Mms2 and Uev1A.
    J Cell Biol. 2005 Aug 29;170(5):745-55 PMID: 16129784
  7. Solution conformation of Lys63-linked di-ubiquitin chain provides clues to functional diversity of polyubiquitin signaling.
    J Biol Chem. 2004 Feb 20;279(8):7055-63 PMID: 14645257
  8. Mms2-Ubc13 covalently bound to ubiquitin reveals the structural basis of linkage-specific polyubiquitin chain formation.
    Nat Struct Mol Biol. 2006 Oct;13(10):915-20 PMID: 16980971
  9. Control of AMPK-related kinases by USP9X and atypical Lys(29)/Lys(33)-linked polyubiquitin chains.
    Biochem J. 2008 Apr 15;411(2):249-60 PMID: 18254724
  10. Key function for the Ubc13 E2 ubiquitin-conjugating enzyme in immune receptor signaling.
    Nat Immunol. 2006 Sep;7(9):962-70 PMID: 16862162
  11. Lingering mysteries of ubiquitin-chain assembly.
    Cell. 2006 Jan 13;124(1):27-34 PMID: 16413479
  12. TAB2 and TAB3 activate the NF-kappaB pathway through binding to polyubiquitin chains.
    Mol Cell. 2004 Aug 27;15(4):535-48 PMID: 15327770
  13. Noncanonical MMS2-encoded ubiquitin-conjugating enzyme functions in assembly of novel polyubiquitin chains for DNA repair.
    Cell. 1999 Mar 5;96(5):645-53 PMID: 10089880
  14. Involvement of polyubiquitin chains via specific chain linkages in stress response in mammalian cells.
    Biol Pharm Bull. 2005 Dec;28(12):2315-8 PMID: 16327172
  15. Monoubiquitinated H2B is associated with the transcribed region of highly expressed genes in human cells.
    Nat Cell Biol. 2008 Apr;10(4):483-8 PMID: 18344985
  16. Intracellular protein degradation: from a vague idea, through the lysosome and the ubiquitin-proteasome system, and onto human diseases and drug targeting (Nobel lecture).
    Angew Chem Int Ed Engl. 2005 Sep 19;44(37):5944-67 PMID: 16142822
  17. Ubiquitination on nonlysine residues by a viral E3 ubiquitin ligase.
    Science. 2005 Jul 1;309(5731):127-30 PMID: 15994556
  18. Regeneration of peroxiredoxins by p53-regulated sestrins, homologs of bacterial AhpD.
    Science. 2004 Apr 23;304(5670):596-600 PMID: 15105503
  19. The tumour suppressor CYLD negatively regulates NF-kappaB signalling by deubiquitination.
    Nature. 2003 Aug 14;424(6950):801-5 PMID: 12917691
  20. A ubiquitin mutant with specific defects in DNA repair and multiubiquitination.
    Mol Cell Biol. 1995 Mar;15(3):1265-73 PMID: 7862120
  21. Preparation and application of polyclonal and monoclonal sequence-specific anti-phosphoamino acid antibodies.
    Curr Protoc Protein Sci. 2004 Feb;Chapter 13:Unit 13.6 PMID: 18429250
  22. Antigen-inducible, H-2-restricted, interleukin-2-producing T cell hybridomas. Lack of independent antigen and H-2 recognition.
    J Exp Med. 1981 May 1;153(5):1198-214 PMID: 6166712
  23. Cell cycle-regulated modification of the ribosome by a variant multiubiquitin chain.
    Cell. 2000 Jul 7;102(1):67-76 PMID: 10929714
  24. Itch/AIP4 mediates Deltex degradation through the formation of K29-linked polyubiquitin chains.
    EMBO Rep. 2006 Nov;7(11):1147-53 PMID: 17028573
  25. The BRCA1/BARD1 heterodimer assembles polyubiquitin chains through an unconventional linkage involving lysine residue K6 of ubiquitin.
    J Biol Chem. 2003 Sep 12;278(37):34743-6 PMID: 12890688
  26. Proteasome-mediated degradation of p21 via N-terminal ubiquitinylation.
    Cell. 2003 Oct 3;115(1):71-82 PMID: 14532004
  27. Atypical ubiquitin chains: new molecular signals. 'Protein Modifications: Beyond the Usual Suspects' review series.
    EMBO Rep. 2008 Jun;9(6):536-42 PMID: 18516089
  28. Site-specific Lys-63-linked tumor necrosis factor receptor-associated factor 6 auto-ubiquitination is a critical determinant of I kappa B kinase activation.
    J Biol Chem. 2007 Feb 9;282(6):4102-12 PMID: 17135271
  29. De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-kappaB signalling.
    Nature. 2004 Aug 5;430(7000):694-9 PMID: 15258597
  30. Mapping of the inducible IkappaB phosphorylation sites that signal its ubiquitination and degradation.
    Mol Cell Biol. 1996 Apr;16(4):1295-304 PMID: 8657102
  31. Characterization of a mutant cell line that does not activate NF-kappaB in response to multiple stimuli.
    Mol Cell Biol. 1997 Mar;17(3):1441-9 PMID: 9032271
  32. Quantitative analysis of in vitro ubiquitinated cyclin B1 reveals complex chain topology.
    Nat Cell Biol. 2006 Jul;8(7):700-10 PMID: 16799550
  33. CYLD is a deubiquitinating enzyme that negatively regulates NF-kappaB activation by TNFR family members.
    Nature. 2003 Aug 14;424(6950):793-6 PMID: 12917689
  34. Lysine 63-polyubiquitination guards against translesion synthesis-induced mutations.
    PLoS Genet. 2006 Jul;2(7):e116 PMID: 16789823
  35. Specificity in Toll-like receptor signalling through distinct effector functions of TRAF3 and TRAF6.
    Nature. 2006 Jan 12;439(7073):204-7 PMID: 16306937
  36. A proteomics approach to understanding protein ubiquitination.
    Nat Biotechnol. 2003 Aug;21(8):921-6 PMID: 12872131
  37. Control of spontaneous and damage-induced mutagenesis by SUMO and ubiquitin conjugation.
    Nature. 2003 Sep 11;425(6954):188-91 PMID: 12968183
  38. TAK1 is a ubiquitin-dependent kinase of MKK and IKK.
    Nature. 2001 Jul 19;412(6844):346-51 PMID: 11460167
  39. Characterization of polyubiquitin chain structure by middle-down mass spectrometry.
    Anal Chem. 2008 May 1;80(9):3438-44 PMID: 18351785
  40. The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction.
    Physiol Rev. 2002 Apr;82(2):373-428 PMID: 11917093
  41. p53 pathway in renal cell carcinoma is repressed by a dominant mechanism.
    Cancer Res. 2004 Mar 15;64(6):1951-8 PMID: 15026329
  42. Mechanism of ubiquitin-chain formation by the human anaphase-promoting complex.
    Cell. 2008 May 16;133(4):653-65 PMID: 18485873
  43. The deubiquitinating enzyme ataxin-3, a polyglutamine disease protein, edits Lys63 linkages in mixed linkage ubiquitin chains.
    J Biol Chem. 2008 Sep 26;283(39):26436-43 PMID: 18599482
  44. Role of protein ubiquitylation in regulating endocytosis of receptor tyrosine kinases.
    Oncogene. 2004 Mar 15;23(11):2057-70 PMID: 15021893
  45. Trabid, a new positive regulator of Wnt-induced transcription with preference for binding and cleaving K63-linked ubiquitin chains.
    Genes Dev. 2008 Feb 15;22(4):528-42 PMID: 18281465
  46. Requirement for cotolerogenic gene products in the clonal deletion of I-E reactive T cells.
    Science. 1990 Feb 23;247(4945):964-7 PMID: 1968289
  47. Essential cytoplasmic translocation of a cytokine receptor-assembled signaling complex.
    Science. 2008 Aug 1;321(5889):663-8 PMID: 18635759
  48. Distinct monoubiquitin signals in receptor endocytosis.
    Trends Biochem Sci. 2003 Nov;28(11):598-603 PMID: 14607090
  49. Mechanism of processing of the NF-kappa B2 p100 precursor: identification of the specific polyubiquitin chain-anchoring lysine residue and analysis of the role of NEDD8-modification on the SCF(beta-TrCP) ubiquitin ligase.
    Oncogene. 2004 Apr 1;23(14):2540-7 PMID: 14676825
  50. The structure of the CYLD USP domain explains its specificity for Lys63-linked polyubiquitin and reveals a B box module.
    Mol Cell. 2008 Feb 29;29(4):451-64 PMID: 18313383
  51. Proteasome-independent functions of ubiquitin in endocytosis and signaling.
    Science. 2007 Jan 12;315(5809):201-5 PMID: 17218518
  52. A tandem affinity tag for two-step purification under fully denaturing conditions: application in ubiquitin profiling and protein complex identification combined with in vivocross-linking.
    Mol Cell Proteomics. 2006 Apr;5(4):737-48 PMID: 16432255
  53. Protein degradation and protection against misfolded or damaged proteins.
    Nature. 2003 Dec 18;426(6968):895-9 PMID: 14685250
Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
1091-6490
DOI
10.1073/pnas.0810461105
Published
2008-12-23
Epub
2008-00-17
Pages
20197-202
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC2629300
Subset
IM
Grants
NIAID NIH HHS · AI043477 · United States
NIAID NIH HHS · AI52199 · United States
NIAID NIH HHS · R01 AI052199 · United States
NIAID NIH HHS · R21 AI052199 · United States
NIAID NIH HHS · R56 AI052199 · United States
NCI NIH HHS · CA21765 · United States
NCI NIH HHS · P30 CA021765 · United States
NIAID NIH HHS · R01 AI043477 · United States
External Links
PubMed source DOI: 10.1073/pnas.0810461105 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: product@genelibs.com

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2