In the present study, we identified an acquired mutation of JAK2 in 83% of PV samples that we analyzed. This mutation causes replacement of a key valine residue by phenylalanine in the pseudokinase domain of JAK2 that has an inhibitory role. Structural analysis predicts gain-of-function phenotype. Our further study by overexpressing the mutant enzyme demonstrated the mutant enzyme had enhanced kinase activity and caused hyperactivation of signal transduction pathways downstream of EPOR. We thus defined a molecular defect of the PV disease. This should have major implications in the diagnosis and treatment of this disease. Increasing evidence has shown that tyrosine kinases are excellent targets for cancer drug therapies. JAK2 thus becomes a potential target for developing therapeutic drugs to treat PV.
JAK2 is a PTK involved in signaling pathways by members of the single chain receptors (e.g. EPOR, TPOR, GHR, PRLR), the IL-3 receptor family (IL-3R, IL-5R and GM-CSF-R), the gp130 receptor family and the class II receptor cytokine family (19
). JAK2 knockout mice exhibited an embryonic lethal phenotype, dying on day 12.5 of gestation due to a failure in definitive erythropoiesis (21
). This is similar to what occurs in EPO−/−
), indicating that JAK2 has pivotal functions for signal transduction initiated by growth factors and cytokines required in definitive erythropoiesis. A primary growth factor involved in erythropoiesis is EPO (24
). Its receptor, EPOR, does not possess kinase activity, but it is associated with the latent JAK2. Binding of EPO to EPOR activates JAK2 and results in activation of PI3K/Akt, STAT5, and ERKs (25
). Growth and expansion of erythroid progenitor cells absolutely requires the presence of EPO. However, some PV erythroid progenitor cells possess the ability to grow autonomously, namely, in the absence of EPO (1
). Earlier studies have demonstrated no abnormality in EPOR in PV cells, suggesting hyperactivation in downstream signaling components. In this regard, it is not a surprise that we found gain-of-function mutation of JAK2 in PV. In fact, earlier cytogenetic studies also support a possible involvement of JAK2 in PV. The JAK2 gene is localized to chromosome 9p24. Trisomies of chromosomes 9 are common manifestations of PV and are found in 6.6% of PV cases (26
). In this study, we detected mutation of JAK2 at Val617
in 20 out of 24 samples analyzed. Those without such a JAK2 mutation might also involve activation of the enzyme through a different mechanism.
The JAK family PTKs have a unique domain structure with a kinase domain (JH1) adjacent to a pseudokinase domain (JH2) (12
). The JH2 domain lacks essential amino acid residues conserved in active protein kinases. The third Gly of the canonical GXGXXG motif in subdomain I is replaced by Thr; the conserved Asp in the DxxxxN motif in subdomain VIB is replaced by Ala; DFG in subdomain VII becomes DPG (see ). The lack of these key residues would make the pseudokinase domain catalytically inactive, and this has been confirmed. In fact, the pseudokinase domain plays a key regulatory role by inhibiting the kinase activity of JH1. The V617
-to-F mutation occurs in the C-terminal side of beta sheet 4 in the subdmain III of the pseudokinase domain. This may cause major conformational changes and thereby disrupt the inhibitory effects. This appears to be the case based on our kinase activity analyses ().
PV is a clonal disorder and the defect is intrinsic to the progenitor cells. The hypersensitivity of PV progenitor cells to growth factors/cytokines and the autonomous growth of some populations of erythroid progenitor cells in the absence of growth factors suggest hyperactivation of downstream signaling pathways. Indeed, an earlier study demonstrated that peripheral granulocytes from some PV patients have constitutive STAT3 DNA binding activity (27
), and a recent study showed systematic increased phosphorylation of Akt/PKB in purified erythroid colony-forming cells from PV patients (28
). As a signal transducer immediately downstream of EPOR, enhanced JAK2 activity due to its mutation would cause hyperactivation of further downstream signaling components thereby causing abnormal cell growth. By co-expressing JAK2 with EPOR in a model cell line, our study demonstrated that the JAK2 mutant indeed causes hyperactivition of signal transduction pathways induced by EPO (). We have also expressed the JAK2 mutant in primary CD34+
hematopoietic progenitor cells from normal blood by using retrovirus-mediated gene transfer as previously described (11
). However, this failed to produce a significant effect on the growth and development of these cells in vitro
(data not shown). One possible explanation is that gain-of-function mutation of JAK2 was not sufficient to transform primary cells. This implies that other abnormalities such as loss of tumor suppressors may be associated with PV.