Tyrosine 201 Is Critical for Transformation Mediated by JAK2V617F

Abstract 626

The JAK2V617F mutation is associated with most cases of Ph-negative myeloproliferative neoplasms (MPNs). In normal hematopoietic cells, JAK2 becomes activated upon cytokine stimulation. The JAK2V617F protein is a constitutively activated tyrosine kinase that leads to activation of several intracellular signaling pathways and transformation of hematopoietic cells to growth factor-independence. Previous studies have shown several tyrosine residues within JAK2 are phosphorylated upon growth factor stimulation. However, the role of these tyrosine residues in signaling and transformation mediated by JAK2V617F remains unclear. Tyrosine 201 lies within a YXX(L/V) motif, which is a potential binding site for Src homology 2 (SH2) domain containing protein(s). Here, we sought to determine the role of tyrosine 201 in JAK2V617F-induced hematopoietic transformation by introducing a tyrosine-to-phenylalanine point mutation (Y201F) at this site. We observed that Y201F mutation significantly inhibited the cytokine-independent cell growth and induced apoptosis in Ba/F3-EpoR cells expressing JAK2V617F. The Y201F mutation also resulted in significant inhibition of JAK2V617F-mediated transformation of hematopoietic cell lines and bone marrow progenitors in vitro. Biochemical analyzes revealed that Y201F mutation almost completely inhibited the phosphorylation/activation of JAK2V617F. We found that Shp2 binds to the Y201 site of JAK2 and mediates the interaction between JAK2V617F and EpoR. In addition, we observed constitutive phosphorylation of Erk1/2 and p70S6 kinase mediated by JAK2V617F was inhibited by the Y201F mutation. Treatment of Ba/F3-EpoR-JAK2V617F cells with JAK inhibitor I or mTOR inhibitor rapamycin inhibited the phosphorylation of p70S6 kinase. Moreover, inhibition of p70S6 kinase by rapamycin treatment (0.5-1 nM) significantly impaired the proliferation and clonogenic growth of Ba/F3-EpoR-JAK2V617F cells. Together, these results suggest that p70S6 kinase is a downstream target of JAK2V617F, and mTOR inhibitor could be useful in treating JAK2V617F-evoked MPN. Finally, using a bone marrow transduction/transplantation approach, we found that Y201 plays an important role in efficient induction of MPN mediated by JAK2V617F.