Skip to main content
SpringerLink
Log in

Multiple Site-Specific Phosphorylation of IDPs Monitored by NMR

  • Protocol
  • First Online:
Intrinsically Disordered Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2141))

Abstract

In line with their high accessibility, disordered proteins are exquisite targets of kinases. Eukaryotic organisms use the so-called intrinsically disordered proteins (IDPs) or intrinsically disordered regions of proteins (IDRs) as molecular switches carrying intracellular information tuned by reversible phosphorylation schemes. Solvent-exposed serines and threonines are abundant in IDPs, and, consistently, kinases often modify disordered regions of proteins at multiple sites. In this context, nuclear magnetic resonance (NMR) spectroscopy provides quantitative, residue-specific information that permits mapping of phosphosites and monitoring of their individual kinetics. Hence, NMR monitoring emerges as an in vitro approach, complementary to mass-spectrometry or immuno-blotting, to characterize IDP phosphorylation comprehensively. Here, we describe in detail generic protocols for carrying out NMR monitoring of IDP phosphorylation, and we provide a number of practical insights that improve handiness and reproducibility of this method.

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

Access this chapter

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 219.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 299.99
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

Similar content being viewed by others

References

  1. Hornbeck PV, Kornhauser JM, Latham V et al (2019) 15 years of PhosphoSitePlus®: integrating post-translationally modified sites, disease variants and isoforms. Nucleic Acids Res 47:D433–D441. https://doi.org/10.1093/nar/gky1159

    Article  CAS  PubMed  Google Scholar 

  2. Huang K-Y, Lee T-Y, Kao H-J et al (2019) dbPTM in 2019: exploring disease association and cross-talk of post-translational modifications. Nucleic Acids Res 47:D298–D308. https://doi.org/10.1093/nar/gky1074

    Article  CAS  PubMed  Google Scholar 

  3. Vlastaridis P, Kyriakidou P, Chaliotis A et al (2017) Estimating the total number of phosphoproteins and phosphorylation sites in eukaryotic proteomes. GigaScience 6:1–11. https://doi.org/10.1093/gigascience/giw015

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Yu K, Zhang Q, Liu Z et al (2019) qPhos: a database of protein phosphorylation dynamics in humans. Nucleic Acids Res 47:D451–D458. https://doi.org/10.1093/nar/gky1052

    Article  CAS  PubMed  Google Scholar 

  5. Wright PE, Dyson HJ (2015) Intrinsically disordered proteins in cellular signalling and regulation. Nat Rev Mol Cell Biol 16:18–29. https://doi.org/10.1038/nrm3920

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Schweiger R, Linial M (2010) Cooperativity within proximal phosphorylation sites is revealed from large-scale proteomics data. Biol Direct 5:6. https://doi.org/10.1186/1745-6150-5-6

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Sharma K, D’Souza RCJ, Tyanova S et al (2014) Ultradeep human phosphoproteome reveals a distinct regulatory nature of Tyr and Ser/Thr-based signaling. Cell Rep 8:1583–1594. https://doi.org/10.1016/j.celrep.2014.07.036

    Article  CAS  PubMed  Google Scholar 

  8. Theillet F-X, Binolfi A, Frembgen-Kesner T et al (2014) Physicochemical properties of cells and their effects on intrinsically disordered proteins (IDPs). Chem Rev 114:6661–6714. https://doi.org/10.1021/cr400695p

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Landry CR, Freschi L, Zarin T, Moses AM (2014) Turnover of protein phosphorylation evolving under stabilizing selection. Front Genet 5:245. https://doi.org/10.3389/fgene.2014.00245

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Nishi H, Shaytan A, Panchenko AR (2014) Physicochemical mechanisms of protein regulation by phosphorylation. Front Genet 5:270. https://doi.org/10.3389/fgene.2014.00270

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Levy ED, Michnick SW, Landry CR (2012) Protein abundance is key to distinguish promiscuous from functional phosphorylation based on evolutionary information. Phil Trans R Soc B 367:2594–2606. https://doi.org/10.1098/rstb.2012.0078

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Wu R, Haas W, Dephoure N et al (2011) A large-scale method to measure absolute protein phosphorylation stoichiometries. Nat Methods 8:677–683. https://doi.org/10.1038/nmeth.1636

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Kanshin E, Pascariu M, Tyers M et al (2018) Combined enrichment/enzymatic approach to study tightly clustered multisite phosphorylation on Ser-rich domains. J Proteome Res 17:3050–3060. https://doi.org/10.1021/acs.jproteome.8b00205

    Article  CAS  PubMed  Google Scholar 

  14. Riley NM, Coon JJ (2016) Phosphoproteomics in the age of rapid and deep proteome profiling. Anal Chem 88:74–94. https://doi.org/10.1021/acs.analchem.5b04123

    Article  CAS  PubMed  Google Scholar 

  15. Humphrey SJ, Karayel O, James DE, Mann M (2018) High-throughput and high-sensitivity phosphoproteomics with the EasyPhos platform. Nat Protoc 13:1897–1916. https://doi.org/10.1038/s41596-018-0014-9

    Article  CAS  PubMed  Google Scholar 

  16. Theillet F-X, Smet-Nocca C, Liokatis S et al (2012) Cell signaling, post-translational protein modifications and NMR spectroscopy. J Biomol NMR 54:217–236. https://doi.org/10.1007/s10858-012-9674-x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Theillet F-X, Rose HM, Liokatis S et al (2013) Site-specific NMR mapping and time-resolved monitoring of serine and threonine phosphorylation in reconstituted kinase reactions and mammalian cell extracts. Nat Protoc 8:1416–1432. https://doi.org/10.1038/nprot.2013.083

    Article  CAS  PubMed  Google Scholar 

  18. Smith MJ, Marshall CB, Theillet F-X et al (2015) Real-time NMR monitoring of biological activities in complex physiological environments. Curr Opin Struct Biol 32:39–47. https://doi.org/10.1016/j.sbi.2015.02.003

    Article  CAS  PubMed  Google Scholar 

  19. Cordier F, Chaffotte A, Terrien E et al (2012) Ordered phosphorylation events in two independent cascades of the PTEN C-tail revealed by NMR. J Am Chem Soc 134:20533–20543. https://doi.org/10.1021/ja310214g

    Article  CAS  PubMed  Google Scholar 

  20. Mylona A, Theillet F-X, Foster C et al (2016) Opposing effects of Elk-1 multisite phosphorylation shape its response to ERK activation. Science 354:233–237. https://doi.org/10.1126/science.aad1872

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Bienkiewicz EA, Lumb KJ (1999) Random-coil chemical shifts of phosphorylated amino acids. J Biomol NMR 15:203–206

    Article  CAS  Google Scholar 

  22. Liokatis S, Stützer A, Elsässer SJ et al (2012) Phosphorylation of histone H3 Ser10 establishes a hierarchy for subsequent intramolecular modification events. Nat Struct Mol Biol 19:819–823. https://doi.org/10.1038/nsmb.2310

    Article  CAS  PubMed  Google Scholar 

  23. Hendus-Altenburger R, Pedraz-Cuesta E, Olesen CW et al (2016) The human Na(+)/H(+) exchanger 1 is a membrane scaffold protein for extracellular signal-regulated kinase 2. BMC Biol 14:31. https://doi.org/10.1186/s12915-016-0252-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Getz EB, Xiao M, Chakrabarty T et al (1999) A comparison between the sulfhydryl reductants tris(2-carboxyethyl)phosphine and dithiothreitol for use in protein biochemistry. Anal Biochem 273:73–80. https://doi.org/10.1006/abio.1999.4203

    Article  CAS  PubMed  Google Scholar 

  25. Schanda P, Kupče Ē, Brutscher B (2005) SOFAST-HMQC experiments for recording two-dimensional deteronuclear correlation spectra of proteins within a few seconds. J Biomol NMR 33:199–211. https://doi.org/10.1007/s10858-005-4425-x

    Article  CAS  PubMed  Google Scholar 

  26. Theillet F-X, Binolfi A, Liokatis S et al (2011) Paramagnetic relaxation enhancement to improve sensitivity of fast NMR methods: application to intrinsically disordered proteins. J Biomol NMR 51:487–495. https://doi.org/10.1007/s10858-011-9577-2

    Article  CAS  PubMed  Google Scholar 

  27. Lee C-R, Park Y-H, Kim Y-R et al (2013) Phosphorylation-dependent mobility shift of proteins on SDS-PAGE is due to decreased binding of SDS. Bull Kor Chem Soc 34:2063–2066. https://doi.org/10.5012/bkcs.2013.34.7.2063

    Article  CAS  Google Scholar 

  28. Kjaergaard M, Brander S, Poulsen FM (2011) Random coil chemical shift for intrinsically disordered proteins: effects of temperature and pH. J Biomol NMR 49:139–149. https://doi.org/10.1007/s10858-011-9472-x

    Article  CAS  PubMed  Google Scholar 

  29. Azatian SB, Kaur N, Latham MP (2019) Increasing the buffering capacity of minimal media leads to higher protein yield. J Biomol NMR 73:11–17. https://doi.org/10.1007/s10858-018-00222-4

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Kinoshita E, Kinoshita-Kikuta E, Koike T (2009) Separation and detection of large phosphoproteins using Phos-tag SDS-PAGE. Nat Protoc 4:1513–1521. https://doi.org/10.1038/nprot.2009.154

    Article  CAS  PubMed  Google Scholar 

  31. Hansen RE, Winther JR (2009) An introduction to methods for analyzing thiols and disulfides: reactions, reagents, and practical considerations. Anal Biochem 394:147–158. https://doi.org/10.1016/j.ab.2009.07.051

    Article  CAS  PubMed  Google Scholar 

  32. Liu P, O'Mara BW, Warrack BM et al (2010) A tris (2-carboxyethyl) phosphine (TCEP) related cleavage on cysteine-containing proteins. J Am Soc Mass Spectrom 21:837–844. https://doi.org/10.1016/j.jasms.2010.01.016

    Article  CAS  PubMed  Google Scholar 

  33. Miller CJ, Turk BE (2018) Homing in: mechanisms of substrate targeting by protein kinases. Trends Biochem Sci 43:380–394. https://doi.org/10.1016/j.tibs.2018.02.009

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Horn H, Schoof EM, Kim J et al (2014) KinomeXplorer: an integrated platform for kinome biology studies. Nat Meth 11:603–604. https://doi.org/10.1038/nmeth.2968

    Article  CAS  Google Scholar 

  35. Patrick R, Horin C, Kobe B et al (2016) Prediction of kinase-specific phosphorylation sites through an integrative model of protein context and sequence. BBA Proteins Proteomics 1864:1599–1608. https://doi.org/10.1016/j.bbapap.2016.08.001

    Article  CAS  PubMed  Google Scholar 

  36. Palmeri A, Ferrè F, Helmer-Citterich M (2014) Exploiting holistic approaches to model specificity in protein phosphorylation. Front Genet 5:315. https://doi.org/10.3389/fgene.2014.00315

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Bai Y, Milne JS, Mayne L, Englander SW (1993) Primary structure effects on peptide group hydrogen exchange. Proteins 17:75–86. https://doi.org/10.1002/prot.340170110

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Becher I, Savitski MM, Savitski MF et al (2013) Affinity profiling of the cellular kinome for the nucleotide cofactors ATP, ADP, and GTP. ACS Chem Biol 8:599–607. https://doi.org/10.1021/cb3005879

    Article  CAS  PubMed  Google Scholar 

  39. Smet-Nocca C, Launay H, Wieruszeski J-M et al (2013) Unraveling a phosphorylation event in a folded protein by NMR spectroscopy: phosphorylation of the Pin1 WW domain by PKA. J Biomol NMR 55:323–337. https://doi.org/10.1007/s10858-013-9716-z

    Article  CAS  PubMed  Google Scholar 

  40. Platzer G, Okon M, McIntosh LP (2014) pH-dependent random coil 1H, 13C, and 15N chemical shifts of the ionizable amino acids: a guide for protein pK a measurements. J Biomol NMR 60:109–129. https://doi.org/10.1007/s10858-014-9862-y

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgement

This work was supported by CNRS, CEA, University Paris South and ENS Paris-Saclay, by the French Infrastructure for Integrated Structural Biology (https://www.structuralbiology.eu/networks/FRISBI, grant number ANR-10-INSB-05-01, Acronym FRISBI), and by the French National Research Agency (ANR; research grant ANR-14-ACHN-0015). We thank the protein production facility of the Institut Curie for providing the purified kinase Plk1.

Author information

Authors and Affiliations

  1. Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, 91198, France

    Manon Julien, Chafiaa Bouguechtouli, Ania Alik, Rania Ghouil, Sophie Zinn-Justin & François-Xavier Theillet

Authors
  1. Manon Julien
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chafiaa Bouguechtouli
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ania Alik
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rania Ghouil
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sophie Zinn-Justin
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. François-Xavier Theillet
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to François-Xavier Theillet .

Editor information

Editors and Affiliations

  1. Structural Biology and NMR Laboratory, Department of Biology, University of Copenhagen, København N, Denmark

    Birthe B. Kragelund

  2. Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Copenhagen, Denmark

    Karen Skriver

Rights and permissions

Reprints and permissions

Copyright information

© 2020 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Julien, M., Bouguechtouli, C., Alik, A., Ghouil, R., Zinn-Justin, S., Theillet, FX. (2020). Multiple Site-Specific Phosphorylation of IDPs Monitored by NMR. In: Kragelund, B.B., Skriver, K. (eds) Intrinsically Disordered Proteins. Methods in Molecular Biology, vol 2141. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0524-0_41

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0524-0_41

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0523-3

  • Online ISBN: 978-1-0716-0524-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation