Erythropoietin receptor signaling in erythropoiesis

描述(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，依赖Janus Kinase 2(JAK2)进行信号转导。EPO使EPOR二聚化激活JAK2激酶活性，进而磷酸化EPOR胞质结构域中的酪氨酸残基。这些磷酸酪氨酸招募信号效应器来触发信号转导。EPOR或JAK2的异常激活可导致白血病。我们的结果表明，JAK2是EPOR的一个重要亚基，EPOR/JAK2复合体是一个功能实体。对于EPOR/JAK2复合体的生化结构，或者该复合体如何在配体刺激下被激活，人们知之甚少。此外，突变的EPOR敲入动物的结果表明，下游效应物与EPOR胞浆区域的磷酸酪氨酸结合在EPOR胞浆区域对于EPO诱导的红细胞生成不是必需的。因此，我们假设EPOR/JAK2复合体下游存在对红细胞生成至关重要的新的JAK2底物。这项建议旨在描述EPOR/JAK2复合体这一关键的EPO信号实体的调节机制，并表征在EPOR在红细胞生成中起关键作用的新的JAK2底物。具体目的是：1.对EPOR/JAK2复合体进行表征。我们证明了JAK2的N-末端结构域由PERM和SH2结构域组成，是EPOR在细胞表面结合和表达的必要条件和充分条件。我们将描述PERM和SH2结构域之间的相互作用，这是正常的JAK2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JAK2 N末端结构域之间的全球相互作用面。2.确定JAK2激活的分子机制。我们将识别和表征JAK2中调节其活性所需的特定残基。我们的结果鉴定出四个EPOR残基是JAK2激活所必需的，其中三个定义了一个在细胞因子受体中保守的基序。我们将使用化学保护和交联来确定这些关键EPOR残基的JAK2接触位点。3.鉴定和鉴定EPOR所必需的新型JAK2底物。我们将通过体内免疫沉淀-质谱法和体外全基因组表达筛选来鉴定新的JAK2底物。这些底物将通过在细胞系和原代红系祖细胞中通过逆转录病毒介导的过度表达和siRNA介导的耗尽来表征它们在红系生成中的作用。从这些研究中获得的信息将使我们更全面地了解EPOR信号如何调节红细胞生成，以及致癌基因EPOR和JAK2激活如何诱导白血病。

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.