Ueda Fumihito, Tago Kenji, Tamura Hiroomi, Funakoshi-Tago Megumi
From the Division of Hygienic Chemistry, Faculty of Pharmacy, Keio University, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512.
the Division of Structural Biochemistry, Department of Biochemistry, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke-shi, Tochigi-ken 329-0498, Japan.
J Biol Chem. 2017 Feb 3;292(5):1826-1846. doi: 10.1074/jbc.M116.749465. Epub 2016 Dec 20.
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.
促红细胞生成素受体（EpoR）调节血细胞的发育，其完全激活通常需要细胞因子促红细胞生成素（Epo）。在骨髓增殖性肿瘤（MPN）中，EpoR的Epo非依赖性信号传导可由EpoR相互作用的酪氨酸激酶Janus激酶2（JAK2）中的点突变V617F引起。在表达JAK2 V617F突变体的细胞中，EpoR细胞内结构域中的八个酪氨酸残基被磷酸化，但这些磷酸化在致癌信号传导中的功能作用尚未完全了解。在这里，为了评估这些酪氨酸残基磷酸化的功能后果，我们构建了一个EpoR-8YF突变体，其中我们将所有八个酪氨酸残基替换为苯丙氨酸。EpoR-8YF与JAK2 V617F突变体的共表达未能诱导细胞因子非依赖性细胞增殖和肿瘤发生，表明JAK2介导的EpoR磷酸化是JAK2 V617F突变体诱导致癌信号传导的原因。对八个EpoR酪氨酸残基的详尽突变分析表明，这些残基中的三个，即Tyr-343、Tyr-460和Tyr-464，是JAK2 V617F突变体发挥其致癌活性所必需的。我们还表明，这三个残基的磷酸化对于转录因子STAT5的完全激活是必需的，STAT5是JAK2 V617F诱导的致癌信号传导的关键下游因子。相比之下，Epo刺激可以适度刺激表达野生型JAK2和EpoR-8YF的细胞的增殖，这表明这三个酪氨酸残基磷酸化的需求似乎对V617F突变引发的致癌增殖具有特异性。总的来说，这些结果表明EpoR中Tyr-343、Tyr-460和Tyr-464的磷酸化是JAK2 V617F突变体诱导肿瘤发生的基础。我们提出，靶向破坏该途径对管理MPN具有治疗效用。
