Erythropoietin receptor signaling in erythropoiesis

红细胞生成中的促红细胞生成素受体信号传导

基本信息

批准号：
7911774
负责人：
LILY JUNSHEN HUANG
金额：
$ 39.25万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-09-01 至 2012-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7911774
关键词：
Animals Binding Biochemical Cell Line Cell membrane Cell surface Chemicals Complex Cytokine Receptors Cytoplasmic Tail Defect Dimerization Disease Docking Endoplasmic Reticulum Erythrocytes Erythrocytoses Erythroid Erythroid Progenitor Cells Erythropoiesis Erythropoietin Receptor Event Homo Immunoprecipitation In Vitro Investigation JAK1 gene Janus kinase 2 Knock-in Mouse Lead Ligands Lilium Maps Mass Spectrum Analysis Mediating Methods Modeling Molecular Mus Mutant Strains Mice Mutation Myeloproliferative disease N-terminal Oncogenic Peptide Hydrolases Phosphotransferases Phosphotyrosine Physiological Polycythemia Vera Production Protein Footprinting Protein Tyrosine Kinase Proteins Receptor Signaling Recruitment Activity Research Personnel Retroviridae Role Signal Transduction Signaling Protein Site Small Interfering RNA Structure Surface Therapeutic Agents Tyrosine base crosslink design expression cloning genome-wide in vivo insight leukemia mutant novel overexpression progenitor programs receptor response src Homology Region 2 Domain trafficking

项目摘要

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.

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剂。它缺乏内在催化活性，依赖 Janus 激酶 2 (JAK2) 进行信号转导。 Epo 响应的 EpoR 二聚化会激活 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 的重要亚基，并且 EpoR/JAK2 复合物是一个功能实体。关于 EpoR/JAK2 复合物的生化结构，或者该复合物如何在配体刺激下被激活，人们知之甚少。此外，突变型 EpoR 敲入动物的结果表明，下游效应子与 EpoR 胞质结构域中磷酸酪氨酸的结合对于 Epo 诱导的红细胞生成并不是必需的。因此，我们假设对红细胞生成至关重要的新型 JAK2 底物存在于 EpoR/JAK2 复合物的下游。该提案旨在描述调节关键 Epo 信号实体 EpoR/JAK2 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 JAK2 底物。具体目标是： 1. 表征 EpoR/JAK2 复合物。我们表明，JAK2 的 N 端结构域由 PERM 和 SH2 结构域组成，对于 EpoR 结合和在细胞表面的表达来说是必要且充分的。我们将描述 JAK2 正常功能所需的 PERM 和 SH2 结构域之间的相互作用。我们还将使用一种基于蛋白酶敏感性的新型“蛋白质足迹”方法来绘制 EpoR 和 JAK2 N 端结构域之间的全局相互作用表面。 2.确定JAK2激活的分子机制。我们将鉴定和表征 JAK2 中调节其活性所需的特定残基。我们的结果确定了 JAK2 激活所必需的四个 EpoR 残基，其中三个定义了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识别这些关键 EpoR 残基的 JAK2 接触位点。 3. 识别和表征对于 EpoR 功能至关重要的新型 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型 JAK2 底物。将使用逆转录病毒介导的过表达和 siRNA 介导的细胞系和原代红系祖细胞中的耗竭来表征这些底物在红细胞生成中的作用。从这些研究中获得的信息将有助于更全面地了解 EpoR 信号如何调节红细胞生成以及致癌性 EpoR 和 JAK2 激活如何诱发白血病。