Erythropoietin receptor signaling in erythropoiesis

描述（由申请人提供）：促红细胞生成素受体（EpoR）是哺乳动物红细胞生成的主要调节因子。它缺乏内在的催化活性，并依赖于Janus激酶2（JAK 2）进行信号转导。响应Epo的EpoR二聚化激活JAK 2激酶活性，其进而磷酸化EpoR胞质结构域中的酪氨酸残基。这些磷酸酪氨酸募集信号效应物以触发信号转导。EpoR或JAK 2的异常激活可导致白血病。我们的研究结果表明，JAK 2是一个重要的亚基的EpoR和EpoR/JAK 2复合物是一个功能实体。关于EpoR/JAK 2复合物的生物化学结构，或者该复合物在配体刺激后如何被激活，知之甚少。此外，突变体EpoR基因敲入动物的结果表明，下游效应子与EpoR胞质结构域中的磷酸酪氨酸的结合对于EpoR诱导的红细胞生成不是必需的。因此，我们假设，新的JAK 2底物红细胞生成的关键存在下游的EpoR/JAK 2复合物。该提案旨在描述调节关键Epo信号实体EpoR/JAK 2复合物的机制，并表征对红细胞生成中EpoR功能至关重要的新型JAK 2底物。具体目标是：1.表征EpoR/JAK 2复合物。我们发现，JAK 2的N-末端结构域，由PERM和SH 2结构域组成，是EpoR在细胞表面结合和表达所必需的。我们将描述PERM和SH 2结构域之间的相互作用，这是正确的JAK 2功能所必需的。我们还将使用一种新的基于蛋白酶敏感性的“蛋白质足迹”方法来绘制EpoR和JAK 2 N-末端结构域之间的全局相互作用表面。2.确定JAK 2激活的分子机制。我们将鉴定和表征JAK 2中调节其活性所需的特定残基。我们的研究结果确定了JAK 2激活所必需的四个EpoR残基，其中三个定义了细胞因子受体之间保守的基序。我们将使用化学保护和交联来鉴定这些关键EpoR残基的JAK 2接触位点。3.鉴定和表征EpoR功能所必需的新型JAK 2底物。我们将通过体内免疫沉淀-质谱和体外全基因组表达筛选来鉴定新型JAK 2底物。这些底物将在细胞系和原代红系祖细胞中使用逆转录病毒介导的过表达和siRNA介导的耗竭来表征它们在红细胞生成中的作用。从这些研究中获得的信息将导致对EpoR信号传导如何调节红细胞生成以及致癌EpoR和JAK 2激活如何诱导白血病的更全面的理解。

DESCRIPTION (provided by applicant): The erythropoietin receptor (EpoR) is the primary regulator of mammalian erythropoiesis. It lacks intrinsic catalytic activity and relies on Janus Kinase 2 (JAK2) for signal transduction. EpoR dimerization in response to Epo activates JAK2 kinase activity, which in turn phosphorylates tyrosine residues in the EpoR cytoplasmic domain. These phospho-tyrosines recruit signaling effectors to trigger signal transduction. Aberrant activation of the EpoR or JAK2 can lead to leukemia. Our results show that JAK2 is an essential subunit of the EpoR and that the EpoR/JAK2 complex is a functional entity. Little is known about the biochemical structure of the EpoR/JAK2 complex, or how this complex becomes activated upon ligand stimulation. Moreover, results from mutant EpoR knock-in animals suggest that binding of downstream effectors to phospho-tyrosines in the EpoR cytoplasmic domain is not essential for Epo-induced erythropoiesis. We therefore hypothesize that novel JAK2 substrates critical for erythropoiesis exist downstream of the EpoR/JAK2 complex. This proposal aims to delineate the mechanisms regulating the key Epo signaling entity, the EpoR/JAK2 complex, and to characterize novel JAK2 substrates that are vital to EpoR function in erythropoiesis. The specific aims are to: 1. Characterize the EpoR/JAK2 complex. We showed that the N-terminal domain of JAK2, consisting of a PERM and an SH2 domain, is necessary and sufficient for EpoR binding and expression at the cell surface. We will characterize the interaction between the PERM and the SH2 domain that is required for proper JAK2 function. We will also use a novel "protein footprinting" method based on protease sensitivity to map the global interacting surface between the EpoR and the JAK2 N-terminal domain. 2. Determine the molecular mechanism of JAK2 activation. We will identify and characterize specific residues in JAK2 that are required to regulate its activity. Our results identified four EpoR residues essential for JAK2 activation, three of which define a motif conserved among cytokine receptors. We will identify the JAK2 contact sites of these key EpoR residues using chemical protection and cross-linking. 3. Identify and characterize novel JAK2 substrates essential for EpoR function. We will identify novel JAK2 substrates by in vivo immunoprecipitation-mass spectrometry and in vitro genome-wide expression screen. These substrates will be characterized for their roles in erythropoiesis using retrovirus-mediated overexpression and siRNA-mediated depletion in cell lines and in primary erythroid progenitor cells. Information gained from these studies will lead to a more comprehensive understanding of how EpoR signaling regulates erythropoiesis and how oncogenic EpoR and JAK2 activation induces leukemia.