Abstract
The discovery that a range of myeloproliferative diseases and leukemias are associated with Janus Kinase (JAK) mutations has highlighted the importance of JAK/STAT signalling in disease and sparked a renewed interest in developing JAK inhibitors. In vitro kinase assays are the most direct and quantitative method to assess mutant forms of JAK for altered enzymatic properties as well as verifying and quantifying the affinity and efficacy of potential inhibitors. Here, we describe protocols for heterologous expression and purification of JAK kinases from insect cells, assays to determine the activity of these purified kinases, and finally inhibition assays to determine the effectiveness of potential inhibitors.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Wilks AF, Harpur AG (1994) Cytokine signal transduction and the JAK family of protein tyrosine kinases. Bioessays 16:313–320
Shuai K, Ziemiecki A, Wilks AF, Harpur AG, Sadowski HB, Gilman MZ, Darnell JE (1993) Polypeptide signalling to the nucleus through tyrosine phosphorylation of Jak and Stat proteins. Nature 366:580–583
Schindler C, Shuai K, Prezioso VR, Darnell JE Jr (1992) Interferon-dependent tyrosine phosphorylation of a latent cytoplasmic transcription factor. Science 257:809–813
Shuai K, Schindler C, Prezioso VR, Darnell JE Jr (1992) Activation of transcription by IFN-gamma: tyrosine phosphorylation of a 91-kD DNA binding protein. Science 258:1808–1812
Shuai K, Horvath CM, Huang LH, Qureshi SA, Cowburn D, Darnell JE Jr (1994) Interferon activation of the transcription factor Stat91 involves dimerization through SH2-phosphotyrosyl peptide interactions. Cell 76:821–828
Wilks AF, Harpur AG, Kurban RR, Ralph SJ, Zurcher G, Ziemiecki A (1991) Two novel protein-tyrosine kinases, each with a second phosphotransferase-related catalytic domain, define a new class of protein kinase. Mol Cell Biol 11:2057–2065
Harpur AG, Andres AC, Ziemiecki A, Aston RR, Wilks AF (1992) JAK2, a third member of the JAK family of protein tyrosine kinases. Oncogene 7:1347–1353
Velazquez L, Fellous M, Stark GR, Pellegrini S (1992) A protein tyrosine kinase in the interferon alpha/beta signaling pathway. Cell 70:313–322
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
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
Bercovich D, Ganmore I, Scott LM, Wainreb G, Birger Y, Elimelech A, Shochat C, Cazzaniga G, Biondi A, Basso G, Cario G, Schrappe M, Stanulla M, Strehl S, Haas OA, Mann G, Binder V, Borkhardt A, Kempski H, Trka J, Bielorei B, Avigad S, Stark B, Smith O, Dastugue N, Bourquin JP, Tal NB, Green AR, Izraeli S (2008) Mutations of JAK2 in acute lymphoblastic leukaemias associated with Down’s syndrome. Lancet 372:1484–1492
Campbell PJ, Scott LM, Buck G, Wheatley K, East CL, Marsden JT, Duffy A, Boyd EM, Bench AJ, Scott MA, Vassiliou GS, Milligan DW, Smith SR, Erber WN, Bareford D, Wilkins BS, Reilly JT, Harrison CN, Green AR (2005) Definition of subtypes of essential thrombocythaemia and relation to polycythaemia vera based on JAK2 V617F mutation status: a prospective study. Lancet 366:1945–1953
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
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
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
Mullighan CG, Zhang J, Harvey RC, Collins-Underwood JR, Schulman BA, Phillips LA, Tasian SK, Loh ML, Su X, Liu W, Devidas M, Atlas SR, Chen IM, Clifford RJ, Gerhard DS, Carroll WL, Reaman GH, Smith M, Downing JR, Hunger SP, Willman CL (2009) JAK mutations in high-risk childhood acute lymphoblastic leukemia. Proc Natl Acad Sci U S A 106:9414–9418
Walters DK, Mercher T, Gu TL, O’Hare T, Tyner JW, Loriaux M, Goss VL, Lee KA, Eide CA, Wong MJ, Stoffregen EP, McGreevey L, Nardone J, Moore SA, Crispino J, Boggon TJ, Heinrich MC, Deininger MW, Polakiewicz RD, Gilliland DG, Druker BJ (2006) Activating alleles of JAK3 in acute megakaryoblastic leukemia. Cancer Cell 10:65–75
Cornejo MG, Kharas MG, Werneck MB, Le Bras S, Moore SA, Ball B, Beylot-Barry M, Rodig SJ, Aster JC, Lee BH, Cantor H, Merlio JP, Gilliland DG, Mercher T (2009) Constitutive JAK3 activation induces lymphoproliferative syndromes in murine bone marrow transplantation models. Blood 113:2746–2754
Lacronique V, Boureux A, Valle VD, Poirel H, Quang CT, Mauchauffe 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
Reiter A, Walz C, Watmore A, Schoch C, Blau I, Schlegelberger B, Berger U, Telford N, Aruliah S, Yin JA, Vanstraelen D, Barker HF, Taylor PC, O’Driscoll A, Benedetti F, Rudolph C, Kolb HJ, Hochhaus A, Hehlmann R, Chase A, Cross NC (2005) The t(8;9)(p22;p24) is a recurrent abnormality in chronic and acute leukemia that fuses PCM1 to JAK2. Cancer Res 65:2662–2667
Bousquet M, Quelen C, De Mas V, Duchayne E, Roquefeuil B, Delsol G, Laurent G, Dastugue N, Brousset P (2005) The t(8;9)(p22;p24) translocation in atypical chronic myeloid leukaemia yields a new PCM1-JAK2 fusion gene. Oncogene 24:7248–7252
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
Babon JJ, Kershaw NJ, Murphy JM, Varghese L, Laktyushin A, Lucet IS, Norton RS, Nicola NA (2012) Immunity 36:239–250
Acknowledgements
This work was made possible through Victorian State Government Operational Infrastructure Support and the Australian Government NHMRC IRIISS. This research was supported by an NHMRC Program Grant (461219), NIH Grant (CA022556), NHMRC CDA (JJB, 516777), ARC Future Fellowship (JMM, FT100100100), and NHMRC Project Grant (1011804).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media New York
About this protocol
Cite this protocol
Babon, J.J., Murphy, J.M. (2013). In Vitro JAK Kinase Activity and Inhibition Assays. In: Nicholson, S., Nicola, N. (eds) JAK-STAT Signalling. Methods in Molecular Biology, vol 967. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-242-1_3
Download citation
DOI: https://doi.org/10.1007/978-1-62703-242-1_3
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-241-4
Online ISBN: 978-1-62703-242-1
eBook Packages: Springer Protocols