Uncoupling JAK2 V617F activation from cytokine-induced signalling by modulation of JH2 αC helix.
Publication Type:Journal Article
Source:Biochem J, Volume 473, Issue 11, p.1579-91 (2016)
Keywords:Blotting, Western, Cell Line, Tumor, Cell Proliferation, Cytokines, Flow Cytometry, Humans, Janus Kinase 2, Mutation, Phosphorylation, Protein Domains
<p>The mechanisms by which JAK2 is activated by the prevalent pseudokinase (JH2) V617F mutation in blood cancers remain elusive. Via structure-guided mutagenesis and transcriptional and functional assays, we identify a community of residues from the JH2 helix αC, SH2-JH2 linker and JH1 kinase domain that mediate V617F-induced activation. This circuit is broken by altering the charge of residues along the solvent-exposed face of the JH2 αC, which is predicted to interact with the SH2-JH2 linker and JH1. Mutations that remove negative charges or add positive charges, such as E596A/R, do not alter the JH2 V617F fold, as shown by the crystal structure of JH2 V617F E596A. Instead, they prevent kinase domain activation via modulation of the C-terminal residues of the SH2-JH2 linker. These results suggest strategies for selective V617F JAK2 inhibition, with preservation of wild-type function.</p>