Janus kinase 2 activation mechanisms revealed by analysis of suppressing mutations

Publication date: Available online 6 August 2018

Source: Journal of Allergy and Clinical Immunology

Author(s): Henrik M. Hammarén, Anniina T. Virtanen, Bobin George Abraham, Heidi Peussa, Stevan R. Hubbard, Olli Silvennoinen

Abstract

Background

Janus kinases (JAK1–3, TYK2) mediate cytokine signals in the regulation of hematopoiesis and immunity. JAK2 clinical mutations cause myeloproliferative neoplasms and leukemia and the mutations strongly concentrate in the regulatory pseudokinase domain, JAK homology 2, JH2. Current clinical JAK inhibitors target the tyrosine kinase domain and lack mutation- and pathway-selectivity.

Objective

To characterize mechanisms and differences for pathogenic and cytokine-induced JAK2 activation to enable design of novel selective JAK inhibitors.

Methods

Systematic analysis of JAK2 activation requirements using structure-guided mutagenesis, cell signaling assays, microscopy, and biochemical analysis.

Results

Distinct structural requirements identified for activation of different pathogenic mutations. Specifically, the predominant JAK2 mutation V617F is the most sensitive to structural perturbations in multiple JH2 elements (C helix (αC), SH2-JH2 linker and ATP-binding site). In contrast, activation of K539L is resistant to most perturbations. Normal cytokine signaling shows distinct differences in activation requirements: JH2 ATP-binding site mutations have only a minor effect on signaling, while JH2 αC mutations reduce homomeric (JAK2-JAK2) EPO signaling, and almost completely abrogate heteromeric (JAK2-JAK1) IFNγ signaling, potentially by disrupting a dimerization interface on JH2.

Conclusions

These results suggest that therapeutic approaches targeting the JH2 ATP-binding site and αC could be effective in inhibiting most pathogenic mutations. JH2 ATP-site targeting have potential for reduced side-effects by retaining EPO and IFNγ functions. Simultaneously, however, we identify the JH2 αC interface as a potential target for pathway-selective JAK inhibitors in diseases with unmutated JAK2, thus providing new insights for the development of novel pharmacological interventions.

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