Molecular Mechanisms of JAK2V617F Oncogenic Activation: The JAK2- V617F Mutant Utilizes the SH2 Domain for Constitutive Kinase Activity in the Presence of Unstimulated Heterodimeric Cytokine Receptor.

The JAK2-V617F mutation has been reported in the majority of MPDs including PV, ET, and IMF. This mutation leads to the constitutive activation of the JAK2 tyrosine kinase activity and overexpression of JAK2V617F renders hematopoietic cell lines growth factor-independent. However, the molecular mechanism leading to constitutive activation of JAK2V617F is largely unclear and the requirement of homodimeric or heterodimeric cytokine receptors needs to be determined. Here we show that oncogenic JAK2-V617F requires an intact SH2 domain for constitutive kinase activity. To this end we mutated the conserved arginine 426 within the SH2 domain to a lysine. Ba/F3 cells expressing JAK2V617F grew IL-3-independent and showed constitutive activation of JAK2, STAT5, and ERK1/2. In contrast, introduction of the SH2 mutation in JAK2V617F abrogated both transformation as well as constitutive activation of downstream signaling pathways. Accordingly, reconstitution of JAK2 mutants in a JAK2-negative cell line with IL-3R co-expression revealed reduced activation of JAK2 when the SH2 domain was mutated. It has been reported that JAK2 binding to homodimeric type I cytokine receptor may facilitate JAK2V617F-mediated transformation. Interestingly, co-expression of the homodomeric EpoR with SH2 mutated JAK2V617F rescues the phenotype indicating that the SH2 domain is required for JAK2 signaling in the presence of heterodimeric but not homodimeric cytokine receptors. Membrane localization studies showed equal membrane distribution of SH2-mutated and unmutated JAK2-V617F indicating that the SH2 domain mutation does not affect subcellular distribution of JAK2. However, co-IP experiments revealed a possible role for the SH2 domain in the dimerization and transphosphorylation of JAK2. Consequently, reduced transphosphorylation was seen in IL-3R- but not in EpoR-expressing cells. In a BM transplantation model we found that an intact SH2 domain in JAK2V617F was required for the induction of a MPD-like disease. Thus, our results points to an important role of the SH2 domain for the constitutive activation of JAK2V617F in cells expressing heterodimeric cytokine receptors.