A shift in the salt bridge interaction of residues D620 and E621 mediates the constitutive activation of Jak2-H538Q/K539L


Jak2 mutations in the exon 14 and exon 12 regions that cause constitutive activation have been associated with myeloproliferative neoplasms. We have previously shown that a pi stacking interaction between F617 and F595 is important for the constitutive activation of Jak2-V617F (Gnanasambandan et al., Biochemistry 49:9972–9984, 2010). Here, using a combination of molecular dynamics (MD) simulations and in vitro mutagenesis, we studied the molecular mechanism for the constitutive activation of the Jak2 exon 12 mutation, H538Q/K539L. The activation levels of Jak2-H538Q/K539L were found to be similar to that of Jak2-V617F, and Jak2-H538Q/K539L/V617F. Data from MD simulations indicated a shift in the salt bridge interactions of D620 and E621 with K539 in Jak2-WT to R541 in Jak2-H538Q/K539L. When compared to Jak2-WT, K539A mutation resulted in increased activation, while K539D or K539E mutations diminished Jak2 activation by 50 %. In the context of Jak2-H538Q/K539L, R541A mutation reduced its activation by 50 %, while R541D and R541E mutations returned its activation levels to that of Jak2-WT. Collectively, these results indicate that a shift in the salt bridge interaction of D620 and E621 with K539 in Jak2-WT to R541 in Jak2-H538Q/K539L is critical for constitutive activation of this Jak2 exon 12 mutant.

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Janus kinase


Signal transducers and activators of transcription


4.1 protein/ezrin/radixin/moesin


Myeloproliferative neoplasms


Src homology 2


  1. 1.

    Neubauer H, Cumano A, Muller M, Wu H, Huffstadt U, Pfeffer K (1998) Jak2 deficiency defines an essential developmental checkpoint in definitive hematopoiesis. Cell 93:397–409. doi:10.1016/S0092-8674(00)81168-X

  2. 2.

    Parganas E, Wang D, Stravopodis D, Topham DJ, Marine JC, Teglund S, Vanin EF, Bodner S, Colamonici OR, van Deursen JM, Grosveld G, Ihle JN (1998) Jak2 is essential for signaling through a variety of cytokine receptors. Cell 93:385–395. doi:10.1016/S0092-8674(00)81167-8

  3. 3.

    Lacronique V, Boureux A, Valle VD, Poirel H, Quang CT, Mauchauffé M, Berthou C, Lessard M, Berger R, Ghysdael J, Bernard OA (1997) A TEL-JAK2 fusion protein with constitutive kinase activity in human leukemia. Science 278:1309–1312

  4. 4.

    Peeters P, Raynaud SD, Cools J, Wlodarska I, Grosgeorge J, Philip P, Monpoux F, Van Rompaey L, Baens M, Van den Berghe H, Marynen P (1997) Fusion of TEL, the ETS-variant gene 6 (ETV6), to the receptor-associated kinase JAK2 as a result of t(9;12) in a lymphoid and t(9;15;12) in a myeloid leukemia. Blood 90:2535–2540

  5. 5.

    Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR, Tichelli A, Cazzola M, Skoda RC (2005) A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med 352:1779–1790. doi:10.1056/NEJMoa051113

  6. 6.

    Levine RL, Wadleigh M, Cools J, Ebert BL, Wernig G, Huntly BJ, Boggon TJ, Wlodarska I, Clark JJ, Moore S, Adelsperger J, Koo S, Lee JC, Gabriel S, Mercher T, D’Andrea A, Frohling S, Dohner K, Marynen P, Vandenberghe P, Mesa RA, Tefferi A, Griffin JD, Eck MJ, Sellers WR, Meyerson M, Golub TR, Lee SJ, Gilliland DG (2005) Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell 7:387–397. doi:10.1016/j.ccr.2005.03.023

  7. 7.

    Baxter EJ, Scott LM, Campbell PJ, East C, Fourouclas N, Swanton S, Vassiliou GS, Bench AJ, Boyd EM, Curtin N, Scott MA, Erber WN, Green AR (2005) Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet 365:1054–1061. doi:10.1016/S0140-6736(05)71142-9

  8. 8.

    James C, Ugo V, Le Couedic JP, Staerk J, Delhommeau F, Lacout C, Garcon L, Raslova H, Berger R, Bennaceur-Griscelli A, Villeval JL, Constantinescu SN, Casadevall N, Vainchenker W (2005) A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature 434:1144–1148. doi:10.1038/nature03546

  9. 9.

    Zhao R, Xing S, Li Z, Fu X, Li Q, Krantz SB, Zhao ZJ (2005) Identification of an acquired JAK2 mutation in polycythemia vera. J Biol Chem 280:22788–22792. doi:10.1074/jbc.C500138200

  10. 10.

    Scott LM, Tong W, Levine RL, Scott MA, Beer PA, Stratton MR, Futreal PA, Erber WN, McMullin MF, Harrison CN, Warren AJ, Gilliland DG, Lodish HF, Green AR (2007) JAK2 exon 12 mutations in polycythemia vera and idiopathic erythrocytosis. N Engl J Med 356:459–468. doi:10.1056/NEJMoa065202

  11. 11.

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

  12. 12.

    Ungureanu D, Wu J, Pekkala T, Niranjan Y, Young C, Jensen ON, Xu CF, Neubert TA, Skoda RC, Hubbard SR, Silvennoinen O (2011) The pseudokinase domain of JAK2 is a dual-specificity protein kinase that negatively regulates cytokine signaling. Nat Struct Mol Biol 18:971–976. doi:10.1038/nsmb.2099

  13. 13.

    Dusa A, Mouton C, Pecquet C, Herman M, Constantinescu SN (2010) JAK2 V617F constitutive activation requires JH2 residue F595: a pseudokinase domain target for specific inhibitors. PLoS ONE 5:e11157. doi:10.1371/journal.pone.0011157

  14. 14.

    Lee TS, Ma W, Zhang X, Giles F, Kantarjian H, Albitar M (2009) Mechanisms of constitutive activation of Janus kinase 2-V617F revealed at the atomic level through molecular dynamics simulations. Cancer 115:1692–1700. doi:10.1002/cncr.24183

  15. 15.

    Gnanasambandan K, Magis A, Sayeski PP (2010) The constitutive activation of Jak2-V617F is mediated by a π stacking mechanism involving phenylalanines 595 and 617. Biochemistry 49:9972–9984. doi:10.1021/bi1014858

  16. 16.

    Zhao L, Dong H, Zhang CC, Kinch L, Osawa M, Iacovino M, Grishin NV, Kyba M, Huang LJ (2009) A JAK2 interdomain linker relays Epo receptor engagement signals to kinase activation. J Biol Chem 284:26988–26998. doi:10.1074/jbc.M109.011387

  17. 17.

    Haan C, Behrmann I, Haan S (2010) Perspectives for the use of structural information and chemical genetics to develop inhibitors of Janus kinases. J Cell Mol Med 14:504–527. doi:10.1111/j.1582-4934.2010.01018.x

  18. 18.

    Gnanasambandan K, Sayeski PP (2011) A structure-function perspective of jak2 mutations and implications for alternate drug design strategies: the road not taken. Curr Med Chem 18(30):4659–4673. doi:10.2174/092986711797379267

  19. 19.

    Lindauer K, Loerting T, Liedl KR, Kroemer RT (2001) Prediction of the structure of human Janus kinase 2 (JAK2) comprising the two carboxy-terminal domains reveals a mechanism for autoregulation. Protein Eng 14:27–37

  20. 20.

    Phillips JC, Braun R, Wang W, Gumbart J, Tajkhorshid E, Villa E, Chipot C, Skeel RD, Kale L, Schulten K (2005) Scalable molecular dynamics with NAMD. J Comput Chem 26:1781–1802. doi:10.1002/jcc.20289

  21. 21.

    Zhang L, Hermans J (1996) Hydrophilicity of cavities in proteins. Proteins 24:433–438. doi:10.1002/(SICI)1097-0134(199604)24

  22. 22.

    Brooks BR, Bruccoleri RE, Olafson BD, States DJ, Swaminathan S, Karplus M (1983) Charmm: a program for macromolecular energy, minimization, and dynamics calculations. J Comput Chem 4:187–217

  23. 23.

    Humphrey W, Dalke A, Schulten K (1996) VMD: visual molecular dynamics. J Mol Graph 14(33–8):27–28. doi:10.1016/0263-7855(96)00018-5

  24. 24.

    Passamonti F, Elena C, Schnittger S, Skoda RC, Green AR, Girodon F, Kiladjian JJ, McMullin MF, Ruggeri M, Besses C, Vannucchi AM, Lippert E, Gisslinger H, Rumi E, Lehmann T, Ortmann CA, Pietra D, Pascutto C, Haferlach T, Cazzola M (2011) Molecular and clinical features of the myeloproliferative neoplasm associated with JAK2 exon 12 mutations. Blood 117:2813–2816. doi:10.1182/blood-2010-11-316810

  25. 25.

    Kurzer JH, Argetsinger LS, Zhou YJ, Kouadio JL, O’Shea JJ, Carter-Su C (2004) Tyrosine 813 is a site of JAK2 autophosphorylation critical for activation of JAK2 by SH2-B beta. Mol Cell Biol 24:4557–4570

  26. 26.

    Haan C, Kroy DC, Wuller S, Sommer U, Nocker T, Rolvering C, Behrmann I, Heinrich PC, Haan S (2009) An unusual insertion in Jak2 is crucial for kinase activity and differentially affects cytokine responses. J Immunol 182:2969–2977. doi:10.4049/jimmunol.0800572

  27. 27.

    Huse M, Kuriyan J (2002) The conformational plasticity of protein kinases. Cell 109:275–282. doi:10.1016/S0092867402007419

  28. 28.

    Lee TS, Ma W, Zhang X, Kantarjian H, Albitar M (2009) Structural effects of clinically observed mutations in JAK2 exons 13–15: comparison with V617F and exon 12 mutations. BMC Struct Biol 9:58. doi:10.1186/1472-6807-9-58

  29. 29.

    Wadleigh M, Tefferi A (2010) Preclinical and clinical activity of ATP mimetic JAK2 inhibitors. Clin Adv Hematol Oncol 8:557–563

  30. 30.

    Majumder A, Sayeski PP (2010) Tyrosine-protein kinase Jak2 inhibitors for the treatment of myeloproliferative neoplasms. Drugs Future 35:651–660. doi:10.1358/dof.2010.35.8.1514131

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The PDB code for full-length Jak2 homology model was kindly provided by Dr. Romano Kroemer. We acknowledge the University of Florida High-Performance Computing Center for providing computational resources and support that have contributed to the results from the Molecular Dynamics simulation reported within this paper. URL: We thank Dr. Joe Zhao for the human Jak2 expression plasmid used in this work. This work was supported, in whole or in part, by National Institutes of Health Grant [R01-HL67277], a University of Florida Opportunity Fund Award, and a University of Florida/Moffitt Cancer Center Collaborative Initiative Award.

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Correspondence to Peter P. Sayeski.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Interactions at the JH1-JH2 interface in Jak2-WT Supplementary material 1 (MPG 31.1 mb)

Interactions at the JH1-JH2 interface in Jak2-H538Q/K539L Supplementary material 2 (MPG 30.8 mb)

Interactions at the JH1-JH2 interface in Jak2-V617F Supplementary material 3 (MPG 31.7 mb)

Interactions at the JH1-JH2 interface in Jak2-H538Q/K539L/V617F Supplementary material 4 (MPG 32.3 mb)

Interactions at the JH1-JH2 interface in Jak2-WT Supplementary material 1 (MPG 31.1 mb)

Interactions at the JH1-JH2 interface in Jak2-H538Q/K539L Supplementary material 2 (MPG 30.8 mb)

Interactions at the JH1-JH2 interface in Jak2-V617F Supplementary material 3 (MPG 31.7 mb)

Interactions at the JH1-JH2 interface in Jak2-H538Q/K539L/V617F Supplementary material 4 (MPG 32.3 mb)

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Gnanasambandan, K., Magis, A.T. & Sayeski, P.P. A shift in the salt bridge interaction of residues D620 and E621 mediates the constitutive activation of Jak2-H538Q/K539L. Mol Cell Biochem 367, 125–140 (2012) doi:10.1007/s11010-012-1326-7

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  • Jak2
  • Exon 12 mutation
  • MPNs
  • Salt bridge
  • Simulations
  • Mechanism