title: Three Tyrosine Residues in the Erythropoietin Receptor Are Essential for Janus Kinase 2 V617F Mutant-induced Tumorigenesis.
abstract: The 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.
authors.affiliation: From the Division of Hygienic Chemistry, Faculty of Pharmacy, Keio University, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512.
authors.forename: Fumihito
authors.initials: F
authors.lastname: Ueda
authors.affiliation: the Division of Structural Biochemistry, Department of Biochemistry, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke-shi, Tochigi-ken 329-0498, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Tago
authors.affiliation: From the Division of Hygienic Chemistry, Faculty of Pharmacy, Keio University, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512.
authors.forename: Hiroomi
authors.initials: H
authors.lastname: Tamura
authors.affiliation: From the Division of Hygienic Chemistry, Faculty of Pharmacy, Keio University, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512. Electronic address: tago-mg@pha.keio.ac.jp.
authors.forename: Megumi
authors.initials: M
authors.lastname: Funakoshi-Tago
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000235:genetics / Q000378:metabolism*; D019337:Hematologic Neoplasms / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D053614:Janus Kinase 2 / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D020125:Mutation, Missense; D009196:Myeloproliferative Disorders / Q000235:genetics / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017467:Receptors, Erythropoietin / Q000235:genetics / Q000378:metabolism*; D050799:STAT5 Transcription Factor / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D009363:Neoplasm Proteins; D017467:Receptors, Erythropoietin; D050799:STAT5 Transcription Factor; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2
keywords: Janus kinase (JAK); Janus kinase 2 (JAK2); STAT transcription factor; V617F mutant; erythropoietin; erythropoietin receptor (EpoR); mutant; myeloproliferative neoplasms (MPN); signal transducer and activator of transcription 5 (STAT5); tumor