The Constitutive Activation of Jak2-V617F Is Mediated by a π Stacking Mechanism Involving Phenylalanines 595 and 617

Somatic mutations in the Jak2 allele that lead to constitutive kinase activation of the protein have been identified in human disease conditions such as the myeloproliferative neoplasms (MPNs). The most common mutation in these patients is a V617F substitution mutation, which is believed to play a causative role in the MPN pathogenesis. As such, identifying the molecular basis for the constitutive activation of Jak2-V617F is important for understanding its clinical implications and potential treatment. Here, we hypothesized that conversion of residue 617 from Val to Phe resulted in the formation of novel pi stacking interactions with neighboring Phe residues. To test this, we first examined the Jak2 structure via molecular modeling and identified a potential pi stacking interaction between F594, F595, and F617. Disruption of this interaction through site-directed mutagenesis impaired Jak2 autophosphorylation, Jak2-dependent gene transcription, and in vitro kinase activity of the Jak2-V617F protein. Further, substitution of F594 and F595 with Trp did not affect Jak2 function significantly, but replacement with charged residues did, showing the importance of aromaticity and hydropathy index conservation at these positions. Using molecular dynamics (MD) simulations, we found that the pi stacking interaction between residues 595 and 617 in the Jak2-V617F protein was of much greater energy and conformed to the properties of pi stacking, relative to the Jak2-WT or Jak2-V617F/F594A/F595A. In summary, we have identified a pi stacking interaction between F595 and F617 that is specific to and is critical for the constitutive activation of Jak2-V617F.