Skip to main content
SpringerLink
Log in

Analyze the SUMOylation of IKK γ/NEMO During Genotoxic Stress

  • Protocol
  • First Online:
NF-κB Transcription Factors

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2366))

  • 1068 Accesses

Abstract

SUMOylation is an important posttranslational modification of substrate proteins that regulates their functions in a variety of cellular processes including epigenetic and transcriptional regulation of gene expression, genomic stability, DNA repair, subcellular translocation, and protein turnover. The critical roles of SUMOylation in regulating NF-κB signaling is exemplified by the findings that it regulates IκBα stability, transactivity of RelA and RelB, as well as initiating the export of nuclear DNA damage signal to cytoplasmic IKK complex through NEMO SUMOylation. Detection of SUMOylated protein is technically challenging due to only a small fraction of substrate proteins is SUMOylated and this process is also reversible by highly active SUMO-deconjugating enzymes. In this protocol, we outline a method for detecting SUMOylation of NEMO in mammalian cells treated by genotoxic agents.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Perkins ND (2006) Post-translational modifications regulating the activity and function of the nuclear factor kappa B pathway. Oncogene 25(51):6717–6730. https://doi.org/10.1038/sj.onc.1209937

    Article  CAS  PubMed  Google Scholar 

  2. Mahajan R, Delphin C, Guan T, Gerace L, Melchior F (1997) A small ubiquitin-related polypeptide involved in targeting RanGAP1 to nuclear pore complex protein RanBP2. Cell 88(1):97–107. https://doi.org/10.1016/s0092-8674(00)81862-0

    Article  CAS  PubMed  Google Scholar 

  3. Matunis MJ, Coutavas E, Blobel G (1996) A novel ubiquitin-like modification modulates the partitioning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex. J Cell Biol 135(6 Pt 1):1457–1470. https://doi.org/10.1083/jcb.135.6.1457

    Article  CAS  PubMed  Google Scholar 

  4. Mabb AM, Miyamoto S (2007) SUMO and NF-kappaB ties. Cell Mol Life Sci 64(15):1979–1996. https://doi.org/10.1007/s00018-007-7005-2

    Article  CAS  PubMed  Google Scholar 

  5. Celen AB, Sahin U (2020) Sumoylation on its 25th anniversary: mechanisms, pathology, and emerging concepts. FEBS J 287(15):3110–3140. https://doi.org/10.1111/febs.15319

    Article  CAS  PubMed  Google Scholar 

  6. Pichler A, Gast A, Seeler JS, Dejean A, Melchior F (2002) The nucleoporin RanBP2 has SUMO1 E3 ligase activity. Cell 108(1):109–120. https://doi.org/10.1016/s0092-8674(01)00633-x

    Article  CAS  Google Scholar 

  7. Kagey MH, Melhuish TA, Wotton D (2003) The polycomb protein Pc2 is a SUMO E3. Cell 113(1):127–137. https://doi.org/10.1016/s0092-8674(03)00159-4

    Article  CAS  PubMed  Google Scholar 

  8. Schmidt D, Muller S (2002) Members of the PIAS family act as SUMO ligases for c-Jun and p53 and repress p53 activity. Proc Natl Acad Sci U S A 99(5):2872–2877. https://doi.org/10.1073/pnas.052559499

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Desterro JM, Rodriguez MS, Hay RT (1998) SUMO-1 modification of IkappaBalpha inhibits NF-kappaB activation. Mol Cell 2(2):233–239. https://doi.org/10.1016/s1097-2765(00)80133-1

    Article  CAS  PubMed  Google Scholar 

  10. Vatsyayan J, Qing G, Xiao G, Hu J (2008) SUMO1 modification of NF-kappaB2/p100 is essential for stimuli-induced p100 phosphorylation and processing. EMBO Rep 9(9):885–890. https://doi.org/10.1038/embor.2008.122

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Leidner J, Voogdt C, Niedenthal R, Möller P, Marienfeld U, Marienfeld RB (2014) SUMOylation attenuates the transcriptional activity of the NF-κB subunit RelB. J Cell Biochem 115(8):1430–1440. https://doi.org/10.1002/jcb.24794

    Article  CAS  PubMed  Google Scholar 

  12. Liu J, Tao X, Zhang J, Wang P, Sha M, Ma Y, Geng X, Feng L, Shen Y, Yu Y, Wang S, Fang S, Shen Y (2016) Small ubiquitin-related modifier 1 is involved in hepatocellular carcinoma progression via mediating p65 nuclear translocation. Oncotarget 7(16):22206–22218. https://doi.org/10.18632/oncotarget.8066

    Article  PubMed  PubMed Central  Google Scholar 

  13. Huang TT, Wuerzberger-Davis SM, Wu ZH, Miyamoto S (2003) Sequential modification of NEMO/IKKgamma by SUMO-1 and ubiquitin mediates NF-kappaB activation by genotoxic stress. Cell 115(5):565–576

    Article  CAS  Google Scholar 

  14. Chen ZJ, Parent L, Maniatis T (1996) Site-specific phosphorylation of IkappaBalpha by a novel ubiquitination-dependent protein kinase activity. Cell 84(6):853–862

    Article  CAS  Google Scholar 

  15. Hayden MS, Ghosh S (2004) Signaling to NF-kappaB. Genes Dev 18(18):2195–2224. https://doi.org/10.1101/gad.1228704

    Article  CAS  PubMed  Google Scholar 

  16. Wu ZH, Shi Y, Tibbetts RS, Miyamoto S (2006) Molecular linkage between the kinase ATM and NF-kappaB signaling in response to genotoxic stimuli. Science 311(5764):1141–1146. https://doi.org/10.1126/science.1121513

    Article  CAS  PubMed  Google Scholar 

  17. Kunz K, Piller T, Muller S (2018) SUMO-specific proteases and isopeptidases of the SENP family at a glance. J Cell Sci 131(6). https://doi.org/10.1242/jcs.211904

  18. Hay RT (2007) SUMO-specific proteases: a twist in the tail. Trends Cell Biol 17(8):370–376. https://doi.org/10.1016/j.tcb.2007.08.002

    Article  CAS  PubMed  Google Scholar 

  19. Mabb AM, Wuerzberger-Davis SM, Miyamoto S (2006) PIASy mediates NEMO sumoylation and NF-kappaB activation in response to genotoxic stress. Nat Cell Biol 8(9):986–993. https://doi.org/10.1038/ncb1458

    Article  CAS  PubMed  Google Scholar 

  20. Lee MH, Mabb AM, Gill GB, Yeh ET, Miyamoto S (2011) NF-kappaB induction of the SUMO protease SENP2: a negative feedback loop to attenuate cell survival response to genotoxic stress. Mol Cell 43(2):180–191. https://doi.org/10.1016/j.molcel.2011.06.017

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Janssens S, Tinel A, Lippens S, Tschopp J (2005) PIDD mediates NF-kappaB activation in response to DNA damage. Cell 123(6):1079–1092. https://doi.org/10.1016/j.cell.2005.09.036

    Article  CAS  PubMed  Google Scholar 

  22. Kfoury Y, Setterblad N, El-Sabban M, Zamborlini A, Dassouki Z, El Hajj H, Hermine O, Pique C, de Thé H, Saïb A, Bazarbachi A (2011) Tax ubiquitylation and SUMOylation control the dynamic shuttling of Tax and NEMO between Ubc9 nuclear bodies and the centrosome. Blood 117(1):190–199. https://doi.org/10.1182/blood-2010-05-285742

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Radiation Oncology, University of Tennessee Health Science Center, Memphis, TN, USA

    Zhao-Hui Wu

  2. Department of Pathology and Laboratory Medicine, University of Tennessee Health Science Center, Memphis, TN, USA

    Zhao-Hui Wu

  3. McArdle Laboratory for Cancer Research, Department of Oncology, University of Wisconsin-Madison, Madison, WI, USA

    Shigeki Miyamoto

Authors
  1. Zhao-Hui Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shigeki Miyamoto
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Shigeki Miyamoto .

Editor information

Editors and Affiliations

  1. Centre for Molecular Immunology and Inflammation, Department of Immunology and Inflammation, Imperial College London, London, UK

    Guido Franzoso

  2. Department of Biotechnological and Applied Clinical Sciences, University of L’Aquila, L’Aquila, Italy

    Francesca Zazzeroni

Rights and permissions

Reprints and permissions

Copyright information

© 2021 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Wu, ZH., Miyamoto, S. (2021). Analyze the SUMOylation of IKK γ/NEMO During Genotoxic Stress. In: Franzoso, G., Zazzeroni, F. (eds) NF-κB Transcription Factors. Methods in Molecular Biology, vol 2366. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1669-7_11

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1669-7_11

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1668-0

  • Online ISBN: 978-1-0716-1669-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation