Skip to main content
SpringerLink
Log in

Purification of SOCS (Suppressor of Cytokine Signaling) SH2 Domains for Structural and Functional Studies

  • Protocol
  • First Online:
SH2 Domains

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1555))

Abstract

Src Homology 2 (SH2) domains are protein domains which have a high binding affinity for specific amino acid sequences containing a phosphorylated tyrosine residue. The Suppressors of Cytokine Signaling (SOCS) proteins use an SH2 domain to bind to components of certain cytokine signaling pathways to downregulate the signaling cascade. The recombinantly produced SH2 domains of various SOCS proteins have been used to undertake structural and functional studies elucidating the method of how such targeting occurs. Here, we describe the protocol for the recombinant production and purification of SOCS SH2 domains, with an emphasis on SOCS3.

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 149.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 199.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
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. Endo TA et al (1997) A new protein containing an SH2 domain that inhibits JAK kinases. Nature 387(6636):921–4

    Article  CAS  PubMed  Google Scholar 

  2. Naka T et al (1997) Structure and function of a new STAT-induced STAT inhibitor. Nature 387(6636):924–9

    Article  CAS  PubMed  Google Scholar 

  3. Starr R et al (1997) A family of cytokine-inducible inhibitors of signalling. Nature 387(6636):917–21

    Article  CAS  PubMed  Google Scholar 

  4. Shuai K et al (1993) Polypeptide signalling to the nucleus through tyrosine phosphorylation of Jak and Stat proteins. Nature 366(6455):580–3

    Article  CAS  PubMed  Google Scholar 

  5. Wilks AF et al (1991) Two novel protein-tyrosine kinases, each with a second phosphotransferase-related catalytic domain, define a new class of protein kinase. Mol Cell Biol 11(4):2057–65

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Ihle JN (1995) Cytokine receptor signalling. Nature 377(6550):591–4

    Article  CAS  PubMed  Google Scholar 

  7. Haan C et al (2006) Jaks and cytokine receptors—an intimate relationship. Biochem Pharmacol 72(11):1538–46

    Article  CAS  PubMed  Google Scholar 

  8. Baxter EJ et al (2005) Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet 365(9464):1054–61

    Article  CAS  PubMed  Google Scholar 

  9. James C et al (2005) A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature 434(7037):1144–8

    Article  CAS  PubMed  Google Scholar 

  10. Kralovics R et al (2005) A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med 352(17):1779–90

    Article  CAS  PubMed  Google Scholar 

  11. Levine RL et al (2005) Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell 7(4):387–97

    Article  CAS  PubMed  Google Scholar 

  12. Zhao R et al (2005) Identification of an acquired JAK2 mutation in polycythemia vera. J Biol Chem 280(24):22788–92

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Hilton DJ et al (1998) Twenty proteins containing a C-terminal SOCS box form five structural classes. Proc Natl Acad Sci U S A 95(1):114–9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Babon JJ et al (2008) The SOCS box domain of SOCS3: structure and interaction with the elonginBC-cullin5 ubiquitin ligase. J Mol Biol 381(4):928–40

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Babon JJ et al (2009) The SOCS box encodes a hierarchy of affinities for Cullin5: implications for ubiquitin ligase formation and cytokine signalling suppression. J Mol Biol 387(1):162–74

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Yasukawa H et al (1999) The JAK-binding protein JAB inhibits Janus tyrosine kinase activity through binding in the activation loop. EMBO J 18(5):1309–20

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Sasaki A et al (1999) Cytokine-inducible SH2 protein-3 (CIS3/SOCS3) inhibits Janus tyrosine kinase by binding through the N-terminal kinase inhibitory region as well as SH2 domain. Genes Cells 4(6):339–51

    Article  CAS  PubMed  Google Scholar 

  18. Kershaw NJ et al (2013) SOCS3 binds specific receptor-JAK complexes to control cytokine signaling by direct kinase inhibition. Nat Struct Mol Biol 20(4):469–76

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Hilton DJ et al (1998) Twenty proteins containing a C-terminal SOCS box form five structural classes. Proc Natl Acad Sci U S A 95(1):114–9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Koch CA et al (1991) SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins. Science 252(5006):668–74

    Article  CAS  PubMed  Google Scholar 

  21. Alexander WS (2002) Suppressors of cytokine signalling (SOCS) in the immune system. Nat Rev Immunol 2(6):410–6

    CAS  PubMed  Google Scholar 

  22. Babon JJ et al (2005) Secondary structure assignment of mouse SOCS3 by NMR defines the domain boundaries and identifies an unstructured insertion in the SH2 domain. FEBS J 272(23):6120–30

    Article  CAS  PubMed  Google Scholar 

  23. Babon JJ et al (2006) The structure of SOCS3 reveals the basis of the extended SH2 domain function and identifies an unstructured insertion that regulates stability. Mol Cell 22(2):205–16

    Article  CAS  PubMed  Google Scholar 

  24. Lilie H, Schwarz E, Rudolph R (1998) Advances in refolding of proteins produced in E. coli. Curr Opin Biotechnol 9(5):497–501

    Article  CAS  PubMed  Google Scholar 

  25. Varghese LN et al (2014) Mechanistic insights into activation and SOCS3-mediated inhibition of myeloproliferative neoplasm-associated JAK2 mutants from biochemical and structural analyses. Biochem J 458(2):395–405

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Nicholson SE et al (2000) Suppressor of cytokine signaling-3 preferentially binds to the SHP-2-binding site on the shared cytokine receptor subunit gp130. Proc Natl Acad Sci U S A 97(12):6493–8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Kershaw NJ et al (2014) Reconstruction of an active SOCS3-based E3 ubiquitin ligase complex in vitro: identification of the active components and JAK2 and gp130 as substrates. Growth Factors 32(1):1–10

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Bullock AN et al (2006) Crystal structure of the SOCS2-elongin C-elongin B complex defines a prototypical SOCS box ubiquitin ligase. Proc Natl Acad Sci U S A 103(20):7637–42

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Bullock AN et al (2007) Structure of the SOCS4-ElonginB/C complex reveals a distinct SOCS box interface and the molecular basis for SOCS-dependent EGFR degradation. Structure (London 1993) 15(11):1493–504

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors’ original work was supported by grants from the National Health and Medical Research Council, Australia (program grants 461219, 1016647; The Independent Research Institutes Infrastructure Scheme 361646) the Australian Research Council (future fellowship FT110100169 JJB), the Cancer Council of Victoria (1065180) the National Institutes of Health, Bethesda, MD (Grant RO1CA22556) and Operational Infrastructure Support grants from the State Government of Victoria.

Author information

Authors and Affiliations

  1. The Walter and Eliza Hall Institute of Medical Research, 1G Royal Pde, Parkville, Melbourne, 3052, VIC, Australia

    Nicholas P. D. Liau, Artem Laktyushin & Jeffrey J. Babon

  2. Department of Medical Biology, University of Melbourne, Royal Pde, Melbourne, 3050, VIC, Australia

    Nicholas P. D. Liau, Artem Laktyushin & Jeffrey J. Babon

Authors
  1. Nicholas P. D. Liau
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Artem Laktyushin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jeffrey J. Babon
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jeffrey J. Babon .

Editor information

Editors and Affiliations

  1. Department of Genetics and Genome Sciences, University of Connecticut School of Medicine, Farmington, Connecticut, USA

    Kazuya Machida

  2. Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA

    Bernard A. Liu

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Liau, N.P.D., Laktyushin, A., Babon, J.J. (2017). Purification of SOCS (Suppressor of Cytokine Signaling) SH2 Domains for Structural and Functional Studies. In: Machida, K., Liu, B. (eds) SH2 Domains. Methods in Molecular Biology, vol 1555. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6762-9_10

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-6762-9_10

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6760-5

  • Online ISBN: 978-1-4939-6762-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation