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Biochim Biophys Acta. 2014 Oct;1844(10):1835-41. doi: 10.1016/j.bbapap.2014.07.003. Epub 2014 Aug 7.

The JH2 domain and SH2-JH2 linker regulate JAK2 activity: A detailed kinetic analysis of wild type and V617F mutant kinase domains.

Author information

1
Department of Hematology, Erasmus MC, Rotterdam, The Netherlands.
2
Institute of Biomedical Technology, School of Medicine, University of Tampere, 33014 Tampere, Finland.
3
PamGene International BV, 5200 BJ 's-Hertogenbosch, The Netherlands.
4
Institute of Biomedical Technology, School of Medicine, University of Tampere, 33014 Tampere, Finland; Department of Internal Medicine, Tampere University Hospital, 33520 Tampere, Finland. Electronic address: olli.silvennoinen@uta.fi.
5
PamGene International BV, 5200 BJ 's-Hertogenbosch, The Netherlands. Electronic address: rhilhorst@pamgene.com.

Abstract

JAK2 tyrosine kinase regulates many cellular functions. Its activity is controlled by the pseudokinase (JH2) domain by still poorly understood mechanisms. The V617F mutation in the pseudokinase domain activates JAK2 and causes myeloproliferative neoplasms. We conducted a detailed kinetic analysis of recombinant JAK2 tyrosine kinase domain (JH1) and wild-type and V617F tandem kinase (JH1JH2) domains using peptide microarrays to define the functions of the kinase domains. The results show that i) JAK2 follows a random Bi-Bi reaction mechanism ii) JH2 domain restrains the activity of the JH1 domain by reducing the affinity for ATP and ATP competitive inhibitors iii) V617F decreases affinity for ATP but increases catalytic activity compared to wild-type and iv) the SH2-JH2 linker region participates in controlling activity by reducing the affinity for ATP.

KEYWORDS:

JAK2 recombinant protein; K(m); Kinetic mechanism; Multiplex kinetic assay; Peptide microarray; V(max)

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