Different JAK2 mutations are associated with different hematologic disorders, but the basis for various pathologies associated with different JAK2 mutants has remained unclear. Previous studies suggested that all disease-associated JAK2 mutants have increased basal catalytic activity. We have found that disease-associated JAK2 mutants differ in their catalytic activity, and their effects on downstream signaling also significantly vary. However, catalytic activity alone cannot explain all mutant JAK2-associated pathology. We observed that different disease-associated JAK2 mutants have differential ability to transform erythroid progenitors, which could be due to their differential interaction with the erythropoietin receptor.
Murine models of JAK2V617F, JAK2K539L and JAK2T875N have been reported. We and other investigators have shown that physiologic expression of JAK2V617F in murine hematopoietic progenitors resulted in a PV-like disease characterized by marked increase in hemoglobin and hematocrit, increased RBC, leukocytosis, neutrophilia, thrombocytosis and splenomegaly [24
]. Expression of JAK2 exon 12 mutant JAK2K539L resulted in erythrocytosis (increased RBC, hemoglobin and hematocrit [31
], whereas expression of JAK2T875N in a murine bone marrow transplant (BMT) model induced a MPN with a megakaryocyte phenotype, including megakaryocytic hyperplasia, impaired megakaryocyte polyploidization and increased reticulin fibrosis in the bone marrow and spleen [33
]. JAK2T875N transplanted mice also exhibited an increase in hematocrit without any significant increase in platelets [33
]. Previously, it has been observed that BMT approach has inherent deficiency in modeling the thrombocytosis phenotype in mice. Several BMT models for JAK2V617F have been reported; although these mice models showed polycythemia phonotype but they did not show any increase in platelets [39
]. In contrast, transgenic or knock-in mice models of Jak2V617F reproducibly produced all the features of PV including erythrocytosis, leukocytosis and thrombocytosis [24
]. Thus, the failure to observe thrombocytosis in JAK2T875N transplanted mice does not mean that the mutant is not capable of inducing AMKL. It is also possible that JAK2T875N mutation is one of several genetic events required for development of AMKL. Better mouse models, such as transgenic or knock-in mouse models, of JAK2T875N would determine if JAK2T875N is sufficient to cause AMKL. However, Studies by Mercher and colleagues have suggested that JAK2T875N may have a more prominent effect on megakaryocytic lineage compared with JAK2V617F [33
]. In addition, compared with JAK2V617F, JAK2K539L and JAK2T875N mutants did not induce a striking leukocytosis [31
]. The results from these mice models indicate that different JAK2 mutants perturb hematopoietic signaling in a different manner.
We used Ba/F3-EpoR cells expressing JAK2V617F, JAK2K539L and JAK2T875N mutants to compare the biochemical and signaling properties of JAK2 mutants. In addition, we expressed these JAK2 mutants in murine BM and compared their ability to transform erythroid progenitors. We observed that JAK2 kinase domain (JH1) mutant JAK2T875N has higher basal kinase activity than the JH2 domain mutant JAK2V617F or the JH2–JH3 linker domain mutant JAK2K539L (). Autophosphorylation of JAK2 mutants as determined by immunoblotting also matched with the results of in vitro
kinase assays (, ). Several signaling molecules/pathways, such as, Stat5, Stat3, Pim-1, Pim-2, Shp2, Gab2, Akt and Erk, which are inducibly regulated by growth factors or cytokines in hematopoietic cells, are constitutively activated in cells expressing JAK2 mutants. JAK2T875N expression resulted in significantly more activation/induction of these signaling molecules/pathways compared with the JAK2V617F or JAK2K539L mutant (). Haan et al., and Elliott et al., also have compared the effects of these JAK2 mutants by expressing in HEK293 cells [42
]. However, they did not observe any significant difference in activation of the JAK2 mutants and their downstream signaling. It is possible that the effects of these JAK2 mutants on 293 cells and hematopoietic cells could be different. Moreover, excessive overexpression of the JAK2 mutants (as usually occurs in 293 cells) might not allow us to see the differences in their catalytic activity and downstream signaling pathways. Also, previous studies did not compare the effects of these JAK2 mutants on hematopoietic progenitors. Our studies suggest that different JAK2 mutants may have differential ability to transform hematopoietic progenitors. Although JAK2T875N exhibited significantly more basal catalytic activity than the JAK2V617F or JAK2K539L mutant (), it was less potent in transforming erythroid progenitors in the BM (). Thus, our data show that transforming ability is not directly proportional to the catalytic (kinase) activity of the JAK2 mutants. Similar results were observed previously with the Noonan syndrome- and leukemia-associated PTPN11 mutants, in which catalytic (phosphatase) activity of PTPN11 mutants did not correlate with their transforming ability and associated pathology [44
Although all disease-associated JAK2 mutants have increased basal kinase activity, the mechanisms underlying the constitutive activation of these different JAK2 mutants remain elusive. Based on the computer modeling, it was suggested that V617 residue in the JH2 domain directly interacts with the JH1 kinase activation loop to keep in an inactive conformation [45
]. The V617F mutation disrupts this critical interaction between JH2 and JH1 domains and results in constitutive activation of the JAK2 kinase [45
]. The K539 residue lies within a region linking JH2 and JH3 domains. Several residues within this linker region are believed to be important to support the local conformation near V617 [46
]. The K539L mutation distorts the local conformation surrounding V617 and causes significant changes in the JH1/JH2 interface [46
]. It has been found that residues within the linker region between JH2 and JH3 domains are essential for interaction with EpoR and JAK2 activity [38
]. The T875 residue lies within the β2–β3 loop of the JH1 kinase domain [33
]. It was proposed that T875N mutation could potentially disrupt the JH1/JH2 interface [33
]. Computer based models only helped to make some predictions on the structural effects of JAK2 mutants. However, crystal structure of full-length JAK2 would be required to better understand the mechanism of constitutive activation of the JAK2 mutants and why some mutants are more active than others.
Previous studies have demonstrated the requirement of homodimeric type I cytokine receptor expression in JAK2V617F-mediated transformation of hematopoietic cells [25
]. Hematopoietic cell transformation by other JAK2 mutants (JAK2K539L, JAK2T875N) also requires the presence of homodimeric type I cytokine receptor [31
]. This raises the possibility that differential expression of homodimeric type I cytokine receptors in different hematopoietic lineages and different levels of interaction of the JAK2 mutants with the type I cytokine receptor may modulate the disease phenotype. Indeed, we observed that PV and idiopathic erythrocytosis associated JAK2 mutants, JAK2V617F and JAK2K539L, were more potent at transforming erythroid progenitors and exhibited significantly more interaction with the EpoR compared with the AMKL-associated JAK2T875N mutant (, ). We also have tried to analyze the interaction of different JAK2 mutants with the EpoR in murine bone marrow cells. However, available EpoR antibodies failed to efficiently pull down endogenous EpoR from the primary bone marrow cells (data not shown). Nevertheless, we have provided evidence that different JAK2 mutants have differences in their ability to interact with the EpoR.
In conclusion, our studies show that disease-associated JAK2 mutants have differences in the kinase activity, and their biological and signaling properties may also differ. However, the extent of activation of the JAK2 kinase alone cannot predict the disease phenotype of a JAK2 mutant. We have found evidence that disease-associated JAK2 mutants have distinct intrinsic transforming properties, which could be due to differential activation of downstream signaling pathways and their specific interaction with certain cytokine receptor. High-resolution crystal structure of full-length JAK2 is yet to be resolved. Structural insights into the effects of different JAK2 mutations would shed light in further understanding the relationship between JAK2 activation, intracellular signaling and pathology.