Uncoupling JAK2 V617F activation from cytokine-induced signaling by modulation of JH2 αC helix.

Leroy, Emilie [UCL] Dusa, Alexandra [UCL] Colau, Didier [UCL] Motamedi, Amir Cahu, Xavier [UCL] Mouton, Céline [UCL] Huang, Lily J Shiau, Andrew K Constantinescu, Stefan N. [UCL] et al.[show all]

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.