Three Tyrosine Residues in the Erythropoietin Receptor Are Essential for Janus Kinase 2 V617F Mutant-induced Tumorigenesis

Abstract

Edited by Alex TokerThe erythropoietin receptor (EpoR) regulates development of blood cells, and its full activation normally requires the cytokine erythropoietin (Epo). In the case of myeloproliferative neoplasms (MPN), Epo-independent signaling through EpoR can be caused by a point mutation, V617F, in the EpoR-interacting tyrosine kinase Janus kinase 2 (JAK2). In cells expressing the JAK2 V617F mutant, eight tyrosine residues in the intracellular domain of EpoR are phosphorylated, but the functional role of these phosphorylations in oncogenic signaling is incompletely understood. Here, to evaluate the functional consequences of the phosphorylation of these tyrosine residues, we constructed an EpoR-8YF mutant in which we substituted all eight tyrosine residues with phenylalanine. Co-expression of EpoR-8YF with the JAK2 V617F mutant failed to induce cytokine-independent cell proliferation and tumorigenesis, indicating that JAK2-mediated EpoR phosphorylation is the reason for JAK2 V617F mutant-induced oncogenic signaling. An exhaustive mutational analysis of the eight EpoR tyrosine residues indicated that three of these residues, Tyr-343, Tyr-460, and Tyr-464, are required for the JAK2 V617F mutant to exhibit its oncogenic activity. We also showed that phosphorylation at these three residues was necessary for full activation of the transcription factor STAT5, which is a critical downstream factor of JAK2 V617F-induced oncogenic signaling. In contrast, Epo stimulation could moderately stimulate the proliferation of cells expressing wild type JAK2 and EpoR-8YF, suggesting that the requirement of the phosphorylation of these three tyrosine residues seems to be specific for the oncogenic proliferation provoked by V617F mutation. Collectively, these results have revealed that phosphorylation of Tyr-343, Tyr-460, and Tyr-464 in EpoR underlies JAK2 V617F mutant-induced tumorigenesis. We propose that the targeted disruption of this pathway has therapeutic utility for managing MPN.The proliferation and differentiation of hematopoietic cells are regulated by various cytokines that act through their specific receptors (1, 2). Erythropoietin (Epo) 2 is a pleiotropic cytokine that exhibits hematopoietic functions such as the development of erythrocytes through the erythropoietin receptor (EpoR) (3). Once Epo binds to EpoR, the non-receptor tyrosine kinase, Janus kinase 2 (JAK2), which interacts with EpoR, is activated. Activated JAK2 then immediately phosphorylates multiple tyrosine residues in the cytoplasmic domain of EpoR (4).EpoR contains the following eight tyrosine residues in its intracellular domain that are phosphorylated by JAK2: Tyr-343, Tyr-401, Tyr-429, Tyr-431, Tyr-443, Tyr-460, Tyr-464, and Tyr-479. The JAK2-induced phosphorylation of these tyrosine residues in EpoR provides a platform for the recruitment and activation of signaling mediators and is critical for Epo-induced cellular proliferation (3, 5, 6). Previous studies clarified that the transcription factor, signal transducer and activator of transcript...

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