Thrombopoietin (Tpo), which binds to its specific receptor, the Mpl protein, is the major cytokine regulator of megakaryopoiesis and circulating platelet number. Tpo binding to Mpl triggers activation of Janus kinase 2 (Jak2) and phosphorylation of the receptor, as well as activation of several intracellular signalling cascades that mediate cellular responses. Three tyrosine (Y) residues in the C-terminal region of the Mpl intracellular domain have been implicated as sites of phosphorylation required for regulation of major Tpo-stimulated signalling pathways: Mpl-Y565, Mpl-Y599 and Mpl-Y604. Here, we have introduced mutations in the mouse germline and report a consistent physiological requirement for Mpl-Y599, mutation of which resulted in thrombocytopenia, deficient megakaryopoiesis, low hematopoietic stem cell (HSC) number and function, and attenuated responses to myelosuppression. We further show that in models of myeloproliferative neoplasms (MPN), where Mpl is required for pathogenesis, thrombocytosis was dependent on intact Mpl-Y599. In contrast, Mpl-Y565 was required for negative regulation of Tpo responses; mutation of this residue resulted in excess megakaryopoiesis at steady-state and in response to myelosuppression, and exacerbated thrombocytosis associated with MPN.

血小板生成素 (Tpo) 与其特定受体 Mpl 蛋白结合，是巨核细胞生成和循环血小板数量的主要细胞因子调节剂。Tpo 与 Mpl 结合会触发 Janus 激酶 2 (Jak2) 的激活和受体的磷酸化，以及介导细胞反应的几种细胞内信号级联的激活。Mpl 胞内结构域 C 端区域的三个酪氨酸 (Y) 残基被认为是调节主要 Tpo 刺激信号通路所需的磷酸化位点：Mpl-Y565、Mpl-Y599 和 Mpl-Y604。在这里，我们在小鼠种系中引入了突变，并报告了对 Mpl-Y599 的一致生理需求，其突变导致血小板减少、巨核细胞生成缺陷、造血干细胞 (HSC) 数量和功能低下，以及对骨髓抑制的反应减弱。我们进一步表明，在骨髓增生性肿瘤（MPN）模型中，Mpl是发病机制所必需的，血小板增多依赖于完整的Mpl-Y599。相比之下，Mpl-Y565 是 Tpo 反应负调节所必需的；该残基的突变导致稳态时巨核细胞生成过多，并对骨髓抑制作出反应，并加剧与 MPN 相关的血小板增多症。