title: JAK2 V617F constitutive activation requires JH2 residue F595: a pseudokinase domain target for specific inhibitors.
abstract: The JAK2 V617F mutation present in over 95% of Polycythemia Vera patients and in 50% of Essential Thrombocythemia and Primary Myelofibrosis patients renders the kinase constitutively active. In the absence of a three-dimensional structure for the full-length protein, the mechanism of activation of JAK2 V617F has remained elusive. In this study, we used functional mutagenesis to investigate the involvement of the JH2 alphaC helix in the constitutive activation of JAK2 V617F. We show that residue F595, located in the middle of the alphaC helix of JH2, is indispensable for the constitutive activity of JAK2 V617F. Mutation of F595 to Ala, Lys, Val or Ile significantly decreases the constitutive activity of JAK2 V617F, but F595W and F595Y are able to restore it, implying an aromaticity requirement at position 595. Substitution of F595 to Ala was also able to decrease the constitutive activity of two other JAK2 mutants, T875N and R683G, as well as JAK2 K539L, albeit to a lower extent. In contrast, the F595 mutants are activated by erythropoietin-bound EpoR. We also explored the relationship between the dimeric conformation of EpoR and several JAK2 mutants. Since residue F595 is crucial to the constitutive activation of JAK2 V617F but not to initiation of JAK2 activation by cytokines, we suggest that small molecules that target the region around this residue might specifically block oncogenic JAK2 and spare JAK2 wild-type.
authors.affiliation: Ludwig Institute for Cancer Research Ltd., Brussels, Belgium.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Dusa
authors.forename: Céline
authors.initials: C
authors.lastname: Mouton
authors.forename: Christian
authors.initials: C
authors.lastname: Pecquet
authors.forename: Murielle
authors.initials: M
authors.lastname: Herman
authors.forename: Stefan N
authors.initials: SN
authors.lastname: Constantinescu
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D053614:Janus Kinase 2 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D017386:Sequence Homology, Amino Acid
chemical_list: D047428:Protein Kinase Inhibitors; D053614:Janus Kinase 2