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

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.

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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.

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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.).

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Authors

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.

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The authors declare no competing financial interests.

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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

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