Skip to main content
SpringerLink
Log in

Detection of Post-translational Modifications on MYC

  • Protocol
  • First Online:
The Myc Gene

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2318))

Abstract

Detection of post-translational modifications in c-Myc is an invaluable tool in assessing Myc status, particularly in cancer. However, it can be challenging to detect these modifications. The evaluation of phosphorylation status of c-Myc can also be challenging with the current commercially available phosphorylation sensitive antibodies. Here, we describe protocols for the immunoprecipitation of endogenous c-Myc to probe for phosphorylation status, as well as the detection of ubiquitination and SUMOylation on c-Myc. We will also discuss the challenges of detecting phosphorylated c-Myc in formalin-fixed paraffin-embedded tissues by immunofluorescence and describe a protocol using a new rat monoclonal antibody we have generated suitable for this purpose.

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. Meyer N, Penn LZ (2008) Reflecting on 25 years with MYC. Nat Rev Cancer 8:976–990

    Article  CAS  Google Scholar 

  2. Sears RC (2004) The life cycle of C-myc: from synthesis to degradation. Cell Cycle 3:1133–1137

    Article  CAS  Google Scholar 

  3. Yeh E, Cunningham M, Arnold H, Chasse D, Monteith T, Ivaldi G, Hahn WC, Stukenberg PT, Shenolikar S, Uchida T, Counter CM, Nevins JR, Means AR, Sears R (2004) A signalling pathway controlling c-Myc degradation that impacts oncogenic transformation of human cells. Nat Cell Biol 6:308–318

    Article  CAS  Google Scholar 

  4. Wang X, Cunningham M, Zhang X, Tokarz S, Laraway B, Troxell M, Sears RC (2011) Phosphorylation regulates c-Myc's oncogenic activity in the mammary gland. Cancer Res 71:925–936

    Article  CAS  Google Scholar 

  5. Hemann MT, Bric A, Teruya-Feldstein J, Herbst A, Nilsson JA, Cordon-Cardo C, Cleveland JL, Tansey WP, Lowe SW (2005) Evasion of the p53 tumour surveillance network by tumour-derived MYC mutants. Nature 436:807–811

    Article  CAS  Google Scholar 

  6. Zhang X, Farrell AS, Daniel CJ, Arnold H, Scanlan C, Laraway BJ, Janghorban M, Lum L, Chen D, Troxell M, Sears R (2012) Mechanistic insight into Myc stabilization in breast cancer involving aberrant Axin1 expression. Proc Natl Acad Sci U S A 109:2790–2795

    Article  CAS  Google Scholar 

  7. Tibbitts DC, Escamilla-Powers JR, Zhang X, Sears RC (2012) Studying c-Myc serine 62 phosphorylation in leukemia cells: concern over antibody cross-reactivity. Blood 119:5334–5335

    Article  CAS  Google Scholar 

  8. Chen Y, Sun XX, Sears RC, Dai MS (2019) Writing and erasing MYC ubiquitination and SUMOylation. Genes Dis 6:359–371

    Article  CAS  Google Scholar 

  9. Farrell AS, Pelz C, Wang X, Daniel CJ, Wang Z, Su Y, Janghorban M, Zhang X, Morgan C, Impey S, Sears RC (2013) Pin1 regulates the dynamics of c-Myc DNA binding to facilitate target gene regulation and oncogenesis. Mol Cell Biol 33:2930–2949

    Article  CAS  Google Scholar 

  10. Sears R, Leone G, DeGregori J, Nevins JR (1999) Ras enhances Myc protein stability. Mol Cell 3:169–179

    Article  CAS  Google Scholar 

  11. Sears R, Nuckolls F, Haura E, Taya Y, Tamai K, Nevins JR (2000) Multiple Ras-dependent phosphorylation pathways regulate Myc protein stability. Genes Dev 14:2501–2514

    Article  CAS  Google Scholar 

  12. Su Y, Pelz C, Huang T, Torkenczy K, Wang X, Cherry A, Daniel CJ, Liang J, Nan X, Dai MS, Adey A, Impey S, Sears RC (2018) Post-translational modification localizes MYC to the nuclear pore basket to regulate a subset of target genes involved in cellular responses to environmental signals. Genes Dev 32:1398–1419

    Article  CAS  Google Scholar 

  13. Welcker M, Orian A, Jin J, Grim JE, Harper JW, Eisenman RN, Clurman BE (2004) The Fbw7 tumor suppressor regulates glycogen synthase kinase 3 phosphorylation-dependent c-Myc protein degradation. Proc Natl Acad Sci U S A 101:9085–9090

    Article  CAS  Google Scholar 

  14. Yada M, Hatakeyama S, Kamura T, Nishiyama M, Tsunematsu R, Imaki H, Ishida N, Okumura F, Nakayama K, Nakayama KI (2004) Phosphorylation-dependent degradation of c-Myc is mediated by the F-box protein Fbw7. EMBO J 23:2116–2125

    Article  CAS  Google Scholar 

  15. Arnold HK, Zhang X, Daniel CJ, Tibbitts D, Escamilla-Powers J, Farrell A, Tokarz S, Morgan C, Sears RC (2009) The Axin1 scaffold protein promotes formation of a degradation complex for c-Myc. EMBO J 28:500–512

    Article  CAS  Google Scholar 

  16. Arnold HK, Sears RC (2006) Protein phosphatase 2A regulatory subunit B56alpha associates with c-myc and negatively regulates c-myc accumulation. Mol Cell Biol 26:2832–2844

    Article  CAS  Google Scholar 

  17. Sun XX, Sears RC, Dai MS (2015) Deubiquitinating c-Myc: USP36 steps up in the nucleolus. Cell Cycle 14:3786–3793

    Article  CAS  Google Scholar 

  18. Sun XX, He X, Yin L, Komada M, Sears RC, Dai MS (2015) The nucleolar ubiquitin-specific protease USP36 deubiquitinates and stabilizes c-Myc. Proc Natl Acad Sci U S A 112:3734–3739

    Article  CAS  Google Scholar 

  19. Sun XX, Chen Y, Su Y, Wang X, Chauhan KM, Liang J, Daniel CJ, Sears RC, Dai MS (2018) SUMO protease SENP1 deSUMOylates and stabilizes c-Myc. Proc Natl Acad Sci U S A 115:10983–10988

    Article  CAS  Google Scholar 

  20. Dang CV (2012) MYC on the path to cancer. Cell 149:22–35

    Article  CAS  Google Scholar 

  21. Yoshida GJ (2018) Emerging roles of Myc in stem cell biology and novel tumor therapies. J Exp Clin Cancer Res 37:173

    Article  Google Scholar 

  22. Link JM, Ota S, Zhou ZQ, Daniel CJ, Sears RC, Hurlin PJ (2012) A critical role for Mnt in Myc-driven T-cell proliferation and oncogenesis. Proc Natl Acad Sci U S A 109:19685–19690

    Article  CAS  Google Scholar 

  23. de Alboran IM, O'Hagan RC, Gartner F, Malynn B, Davidson L, Rickert R, Rajewsky K, DePinho RA, Alt FW (2001) Analysis of C-MYC function in normal cells via conditional gene-targeted mutation. Immunity 14:45–55

    Article  Google Scholar 

Download references

Acknowledgments

The authors would like to thank Julienne R. Escamilla-Powers, Deanne Tibbits and Hugh K. Arnold for helpful discussion. Authors also thank the OHSU Pancreas Tissue Registry and support from the Brenden-Colson Center for Pancreatic Care in providing patient tumor tissue under IRB # 3609 and 3330. This work was supported by Department of Defense Breast Cancer Research Program Award BC061306, Susan. G. Komen Breast Cancer Foundation Awards BCTR0201697 and BCTR0706821, and National Institutes of Health grants R01 CA129040, CA100855 and CA196228 to R.C.S., R01 CA186241 to M.-S.D. and R.C.S. and R01 GM130604 to M.-S.D.

Author information

Authors and Affiliations

  1. Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR, USA

    Colin J. Daniel, Xiao-Xin Sun, Yingxiao Chen, Xiaoli Zhang, Mu-Shui Dai & Rosalie C. Sears

  2. Knight Cancer Institute, Oregon Health and Science University, Portland, OR, USA

    Mu-Shui Dai & Rosalie C. Sears

Authors
  1. Colin J. Daniel
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiao-Xin Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yingxiao Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xiaoli Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mu-Shui Dai
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Rosalie C. Sears
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Mu-Shui Dai or Rosalie C. Sears .

Editor information

Editors and Affiliations

  1. Vall d’Hebron Institute of Oncology (VHIO), Vall d’Hebron Barcelona Hospital Campus, Barcelona, Spain

    Laura Soucek

  2. Vall d’Hebron Institute of Oncology (VHIO), Vall d’Hebron Barcelona Hospital Campus, Barcelona, Spain

    Jonathan Whitfield

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

Daniel, C.J., Sun, XX., Chen, Y., Zhang, X., Dai, MS., Sears, R.C. (2021). Detection of Post-translational Modifications on MYC. In: Soucek, L., Whitfield, J. (eds) The Myc Gene. Methods in Molecular Biology, vol 2318. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1476-1_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1476-1_5

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1475-4

  • Online ISBN: 978-1-0716-1476-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation