Differential Biological Properties and Signaling Effects of Different JAK2 Alleles

JAK2, a member of the Janus family of non-receptor protein tyrosine kinases, acts as an important mediator of cytokine receptor signaling and plays a critical role in hematopoiesis. Somatic mutations in JAK2 have been identified in a spectrum of myeloproliferative disorders (MPD). A specific point mutation (V617F) in the pseudokinase domain (JH2) of JAK2 has been identified in most patients with polycythemia vera (PV) and 50–60% patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). Although JAK2V617F is the predominant allele associated with PV, ET and PMF, other activating alleles of JAK2 also have been identified in distinct MPD. Somatic mutations in the exon 12 (linker region between JH2 and JH3) of JAK2 are associated with a distinct type of PV characterized by isolated erythrocytosis. Another activating mutation (T875N) in the kinase domain (JH1) of JAK2 has been identified in a cell line derived from acute megakaryoblastic leukemia (AMKL) patient, and expression of JAK2T875N in murine bone marrow induces a MPD with features of AMKL. Thus, different activating JAK2 alleles are associated with different disease phenotypes. The basis for the differences in the in vivo effects of different JAK2 alleles remains unclear. To understand the biological and signaling effects of these JAK2 mutants, we have generated Ba/F3-EpoR cell lines expressing JAK2WT, JAK2V617F, JAK2 exon 12 mutant K539L or JAK2T875N. Expression of JAK2V617F, JAK2K539L or JAK2T875N in Ba/F3-EpoR cells resulted in IL-3 independence, whereas JAK2WT failed to grow in absence of IL-3. We observed significantly increased proliferation of JAK2T875N expressing cells compared to cells expressing either JAK2V617F or JAK2K539L in the absence of cytokine. Moreover, expression of JAK2T875N caused increased transformation of Ba/F3-EpoR cells in a colony assay. Biochemical analysis revealed that the kinase activity of JAK2K539L was higher than JAK2V617F but comparable to JAK2T875N. More importantly, significant differences in signaling downstream of these JAK2 mutants were observed. Expression of JAK2T875N resulted in increased phosphorylation of Stat3, Stat5, Gab2, Akt, and Erk/MAPK and induction of Pim1 and Pim2 compared to JAK2V617F and JAK2K539L mutants. Although JAK2K539L exhibited more kinase activity than JAK2V617F but it was less potent in activation of downstream signaling molecules, such as Stat5, Gab2, Erk/MAPK. Variable association of mutant JAK2 with EpoR was also observed, with JAK2K539L being the most and JAK2T875N being the least associated with EpoR. These results strongly suggest that the biological properties and signaling effects of JAK2 alleles are different, and they may interact differentially with cytokine receptor, which might contribute to the differences in the disease phenotype.