Identification of an acquired JAK2 mutation in polycythemia vera

Abstract

Polycythemia vera (PV) is a human clonal hematological disorder. The molecular etiology of the disease has not been identified. PV hematopoietic progenitor cells exhibit hypersensitivity to growth factors and cytokines, suggesting possible abnormalities in protein-tyrosine kinases and phosphatases. By sequencing the entire coding regions of cDNAs of candidate enzymes, we identified a G:C--> T:A point mutation of the JAK2 tyrosine kinase in 20 of 24 PV blood samples but none in 12 normal samples. The mutation has varying degrees of heterozygosity and is apparently acquired. It changes conserved Val(617) to Phe in the pseudokinase domain of JAK2 that is known to have an inhibitory role. The mutant JAK2 has enhanced kinase activity, and when overexpressed together with the erythropoietin receptor in cells, it caused hyperactivation of erythropoietin-induced cell signaling. This gain-of-function mutation of JAK may explain the hypersensitivity of PV progenitor cells to growth factors and cytokines. Our study thus defines a molecular defect of PV.

Fig. 1

Identification of JAK mutation in cDNAs and genomic DNAs. The data show sequencing of JAK2 PCR products from cDNAs (from both 5′ and 3′ directions) and the correspondent genomic DNAs (from 3′ side only) of one normal and 4 PV mononuclear cell samples. The mutation point is underlined. Percentages of the mutant in the total PCR products were calculated based on the average signals of nucleotides A and C in the sequencing analysis.

Fig. 2

. Structural analyses of the JAK2 mutant. Shown is a schematic structure of JAK2 together with sequence alignment of the JAK family PTKs in the region where the V⁶¹⁷-to-F mutation (underlined) occurs in PV JAK2.

Fig. 3

JAK2^V617F has enhanced tyrosine kinase activity. JAK2 and JAK2^V617F were immuno-purified from HeLa cells transfected with pcDNA3-JAK2 or JAK2^V617F. Kinase activity assays were performed with GST-JAK2CT as a substrate in the presence of ATP-Mg²⁺. Tyrosine phosphorylation was detected by using anti-phosphotyrosine antibody and the protein levels of JAK2/JAK2^V617F and GST-JAK2CT, by anti-JAK and anti-GST, respectively.

Fig 4

JAK2^V617F causes hyperactivation of EPO-induced cell signaling. HeLa cells were co-transfected with EPOR and JAK2 or JAK2^V617F. Cells were treated with 40 units/ml of EPO for 15 min and then extracted. Cell extracts were subjected to Western blotting analyses with indicated antibodies. Additional data indicate that the protein levels of STAT5, Akt, and ERK1/2 were not altered by the cell transfection (data not shown).