Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

The pseudokinase domain of JAK2 is a dual-specificity protein kinase that negatively regulates cytokine signaling

Nature Structural & Molecular Biology volume 18, pages 971–976 (2011)Cite this article

Subjects

Abstract

Human JAK2 tyrosine kinase mediates signaling through numerous cytokine receptors. The JAK2 JH2 domain functions as a negative regulator and is presumed to be a catalytically inactive pseudokinase, but the mechanism(s) for its inhibition of JAK2 remains unknown. Mutations in JH2 lead to increased JAK2 activity, contributing to myeloproliferative neoplasms (MPNs). Here we show that JH2 is a dual-specificity protein kinase that phosphorylates two negative regulatory sites in JAK2: Ser523 and Tyr570. Inactivation of JH2 catalytic activity increased JAK2 basal activity and downstream signaling. Notably, different MPN mutations abrogated JH2 activity in cells, and in MPN (V617F) patient cells phosphorylation of Tyr570 was reduced, suggesting that loss of JH2 activity contributes to the pathogenesis of MPNs. These results identify the catalytic activity of JH2 as a previously unrecognized mechanism to control basal activity and signaling of JAK2.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Identification of JAK2 JH2 catalytic activity in vitro.
Figure 2: Identification of phosphorylated residues in JAK2 JH2.
Figure 3: Analysis of JAK2 JH2 autophosphorylation and ATP-binding activity.
Figure 4: Analysis of JAK2 signaling in mammalian cells.
Figure 5: Phosphorylation of different JAK2 MPN mutants.

Similar content being viewed by others

References

  1. Gadina, M. et al. Signaling by type I and II cytokine receptors: ten years after. Curr. Opin. Immunol. 13, 363–373 (2001).

    Article  CAS  PubMed  Google Scholar 

  2. Imada, K. & Leonard, W.J. The Jak-STAT pathway. Mol. Immunol. 37, 1–11 (2000).

    Article  CAS  PubMed  Google Scholar 

  3. Silvennoinen, O. et al. Structure of the murine Jak2 protein-tyrosine kinase and its role in interleukin 3 signal transduction. Proc. Natl. Acad. Sci. USA 90, 8429–8433 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Yamaoka, K. et al. The Janus kinases (Jaks). Genome Biol. 5, 253 (2004).

    Article  PubMed  PubMed Central  Google Scholar 

  5. Ishida-Takahashi, R. et al. Phosphorylation of Jak2 on Ser(523) inhibits Jak2-dependent leptin receptor signaling. Mol. Cell. Biol. 26, 4063–4073 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Matsuda, T., Feng, J., Witthuhn, B.A., Sekine, Y. & Ihle, J.N. Determination of the transphosphorylation sites of Jak2 kinase. Biochem. Biophys. Res. Commun. 325, 586–594 (2004).

    Article  CAS  PubMed  Google Scholar 

  7. Mazurkiewicz-Munoz, A.M. et al. Phosphorylation of JAK2 at serine 523: a negative regulator of JAK2 that is stimulated by growth hormone and epidermal growth factor. Mol. Cell. Biol. 26, 4052–4062 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Robertson, S.A. et al. Regulation of Jak2 function by phosphorylation of Tyr317 and Tyr637 during cytokine signaling. Mol. Cell. Biol. 29, 3367–3378 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Feener, E.P., Rosario, F., Dunn, S.L., Stancheva, Z. & Myers, M.G. Jr. Tyrosine phosphorylation of Jak2 in the JH2 domain inhibits cytokine signaling. Mol. Cell. Biol. 24, 4968–4978 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Argetsinger, L.S. et al. Autophosphorylation of JAK2 on tyrosines 221 and 570 regulates its activity. Mol. Cell. Biol. 24, 4955–4967 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Argetsinger, L.S. et al. Tyrosines 868, 966, and 972 in the kinase domain of JAK2 are autophosphorylated and required for maximal JAK2 kinase activity. Mol. Endocrinol. 24, 1062–1076 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Shuai, K. & Liu, B. Regulation of JAK-STAT signalling in the immune system. Nat. Rev. Immunol. 3, 900–911 (2003).

    Article  CAS  PubMed  Google Scholar 

  13. Saharinen, P., Takaluoma, K. & Silvennoinen, O. Regulation of the Jak2 tyrosine kinase by its pseudokinase domain. Mol. Cell. Biol. 20, 3387–3395 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Saharinen, P. & Silvennoinen, O. The pseudokinase domain is required for suppression of basal activity of Jak2 and Jak3 tyrosine kinases and for cytokine-inducible activation of signal transduction. J. Biol. Chem. 277, 47954–47963 (2002).

    Article  CAS  PubMed  Google Scholar 

  15. Saharinen, P., Vihinen, M. & Silvennoinen, O. Autoinhibition of Jak2 tyrosine kinase is dependent on specific regions in its pseudokinase domain. Mol. Biol. Cell 14, 1448–1459 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Haan, C., Behrmann, I. & Haan, S. Perspectives for the use of structural information and chemical genetics to develop inhibitors of Janus kinases. J. Cell. Mol. Med. 14, 504–527 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Baxter, E.J. et al. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet 365, 1054–1061 (2005).

    Article  CAS  PubMed  Google Scholar 

  18. James, C. et al. A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature 434, 1144–1148 (2005).

    Article  CAS  PubMed  Google Scholar 

  19. Kralovics, R. et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N. Engl. J. Med. 352, 1779–1790 (2005).

    Article  CAS  PubMed  Google Scholar 

  20. Chen, M. et al. Complex effects of naturally occurring mutations in the JAK3 pseudokinase domain: evidence for interactions between the kinase and pseudokinase domains. Mol. Cell. Biol. 20, 947–956 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Lucet, I.S. et al. The structural basis of Janus kinase 2 inhibition by a potent and specific pan-Janus kinase inhibitor. Blood 107, 176–183 (2006).

    Article  CAS  PubMed  Google Scholar 

  22. Boggon, T.J., Li, Y., Manley, P.W. & Eck, M.J. Crystal structure of the Jak3 kinase domain in complex with a staurosporine analog. Blood 106, 996–1002 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Boudeau, J., Miranda-Saavedra, D., Barton, G.J. & Alessi, D.R. Emerging roles of pseudokinases. Trends Cell Biol. 16, 443–452 (2006).

    Article  CAS  PubMed  Google Scholar 

  24. Hubbard, S.R. & Till, J.H. Protein tyrosine kinase structure and function. Annu. Rev. Biochem. 69, 373–398 (2000).

    Article  CAS  PubMed  Google Scholar 

  25. Hall, T. et al. Expression, purification, characterization and crystallization of non- and phosphorylated states of JAK2 and JAK3 kinase domain. Protein Expr. Purif. 69, 54–63 (2010).

    Article  CAS  PubMed  Google Scholar 

  26. Scott, L.M. et al. JAK2 exon 12 mutations in polycythemia vera and idiopathic erythrocytosis. N. Engl. J. Med. 356, 459–468 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Bercovich, D. et al. Mutations of JAK2 in acute lymphoblastic leukaemias associated with Down's syndrome. Lancet 372, 1484–1492 (2008).

    Article  CAS  PubMed  Google Scholar 

  28. Zeqiraj, E. & van Aalten, D.M. Pseudokinases-remnants of evolution or key allosteric regulators? Curr. Opin. Struct. Biol. 20, 772–781 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Scheeff, E.D., Eswaran, J., Bunkoczi, G., Knapp, S. & Manning, G. Structure of the pseudokinase VRK3 reveals a degraded catalytic site, a highly conserved kinase fold, and a putative regulatory binding site. Structure 17, 128–138 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Mukherjee, K. et al. CASK functions as a Mg2+-independent neurexin kinase. Cell 133, 328–339 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Eswaran, J. et al. Structure and functional characterization of the atypical human kinase haspin. Proc. Natl. Acad. Sci. USA 106, 20198–20203 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Min, X., Lee, B.H., Cobb, M.H. & Goldsmith, E.J. Crystal structure of the kinase domain of WNK1, a kinase that causes a hereditary form of hypertension. Structure 12, 1303–1311 (2004).

    Article  CAS  PubMed  Google Scholar 

  33. Shi, F., Telesco, S.E., Liu, Y., Radhakrishnan, R. & Lemmon, M.A. ErbB3/HER3 intracellular domain is competent to bind ATP and catalyze autophosphorylation. Proc. Natl. Acad. Sci. USA 107, 7692–7697 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Zeqiraj, E., Filippi, B.M., Deak, M., Alessi, D.R. & van Aalten, D.M. Structure of the LKB1-STRAD-MO25 complex reveals an allosteric mechanism of kinase activation. Science 326, 1707–1711 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Jura, N., Shan, Y., Cao, X., Shaw, D.E. & Kuriyan, J. Structural analysis of the catalytically inactive kinase domain of the human EGF receptor 3. Proc. Natl. Acad. Sci. USA 106, 21608–21613 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Santos, F.P. & Verstovsek, S. JAK2 inhibitors: what's the true therapeutic potential? Blood Rev. 25, 53–63 (2011).

    Article  CAS  PubMed  Google Scholar 

  37. Shevchenko, A., Tomas, H., Havlis, J., Olsen, J.V. & Mann, M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protoc. 1, 2856–2860 (2006).

    Article  CAS  PubMed  Google Scholar 

  38. Thingholm, T.E., Jorgensen, T.J., Jensen, O.N. & Larsen, M.R. Highly selective enrichment of phosphorylated peptides using titanium dioxide. Nat. Protoc. 1, 1929–1935 (2006).

    Article  CAS  PubMed  Google Scholar 

  39. Ye, J. et al. Optimized IMAC-IMAC protocol for phosphopeptide recovery from complex biological samples. J. Proteome Res. 9, 3561–3573 (2010).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank M. Myers (University of Michigan Medical School) for reagents (anti-pSer523 and anti-pTyr570 specific antibodies), E. Koskenalho, P. Kosonen and M. Lehtinen for technical assistance, and the Biocenter Finland protein production platform (V. Hytönen and U. Kiiskinen) for technological support. This study was supported by the Medical Research Council of Academy of Finland (O.S.), the Sigrid Juselius Foundation (O.S.), the Finnish Cancer Foundation (O.S.), the EU Research Training Network ReceptEur (O.S.), Science Center, Competitive Research Funding and Centre of Laboratory Medicine of the Tampere University Hospital (O.S.), the Tampere Tuberculosis Foundation (O.S.), US National Institutes of Health core grant CA016087 (T.A.N.), the Danish Research Agency and the Danish National Research Foundation (Centre for Epigenetics) (C.Y. and O.N.J.).

Author information

Author notes

  1. Jinhua Wu, Clifford Young & Chong-Feng Xu

    Present address: Present addresses: Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA (J.W.); Analytical Development, Biogen Idec, Cambridge, Massachusetts, USA (C.‐F.X.); Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark (C.Y.).,

Authors and Affiliations

  1. Institute of Biomedical Technology, University of Tampere, Tampere, Finland

    Daniela Ungureanu, Tuija Pekkala, Yashavanthi Niranjan & Olli Silvennoinen

  2. Structural Biology Program, Kimmel Center for Biology and Medicine of the Skirball Institute, New York University School of Medicine, New York, New York, USA

    Jinhua Wu, Chong-Feng Xu, Thomas A Neubert & Stevan R Hubbard

  3. Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark

    Clifford Young & Ole N Jensen

  4. Department of Biomedicine, University Hospital Basel, Basel, Switzerland

    Radek C Skoda

  5. Tampere University Hospital, Tampere, Finland

    Olli Silvennoinen

Authors
  1. Daniela Ungureanu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jinhua Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tuija Pekkala
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yashavanthi Niranjan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Clifford Young
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ole N Jensen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Chong-Feng Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Thomas A Neubert
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Radek C Skoda
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Stevan R Hubbard
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Olli Silvennoinen
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

D.U. performed the experiments and wrote the paper. O.S. and S.R.H. designed the experiments and wrote the paper. J.W. performed the in vitro experiments with recombinant proteins. T.P. and Y.N. performed the mutagenesis experiments in mammalian cells. C.Y., O.N.J., T.A.N. and C.-F.X. performed the experiments for MS analysis. R.C.S. performed the experiments for clinical sample analysis.

Corresponding author

Correspondence to Olli Silvennoinen.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10 and Supplementary Methods (PDF 13161 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ungureanu, D., Wu, J., Pekkala, T. et al. The pseudokinase domain of JAK2 is a dual-specificity protein kinase that negatively regulates cytokine signaling. Nat Struct Mol Biol 18, 971–976 (2011). https://doi.org/10.1038/nsmb.2099

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2099

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing