Role of SHP-1 in T cell activation and development

描述（由申请人提供）：酪氨酰磷酸化是 正常细胞生长、分化和死亡的调节机制。的 任何蛋白质上酪氨酰磷酸化的稳态水平通过 蛋白酪氨酸激酶和磷酸酶的相反作用。SHP-1是一种 蛋白酪氨酸磷酸酶，已被证明是参与负 调节由细胞因子、生长因子和 抗原小鼠SHP-1基因突变导致蛾化（me/me） 表型小鼠的me等位基因纯合子，这导致缺乏 任何可检测到的SHP-1蛋白，都会在11例患者中表现出一系列疾病 造血谱系这些小鼠为体外联合收割机提供了有价值的工具 生物化学测定与体内和离体生物学研究。我们的远程 目的是了解SHP-1如何影响生长和分化， 造血细胞本申请将试图阐明 SHP-1在未成熟和成熟细胞功能中的作用 最近，我们和其他人已经显示了SHP-1在T细胞中的明确作用。 发展然而，潜在的分子机制仍然未知。在 第一个目的，我们建议使用胎儿胸腺器官来解决这个问题 培养物（FTOC）。me/me分析：DO11.10胸腺细胞表明SHP-1有助于 以设置阳性和阴性选择中的TCR信号传导阈值。研究 来自这些小鼠的FTOC将用于跟踪胸腺T细胞发育， 详细特别是，我们建议通过以下方法来拯救蛾虫表型： 从me/me：具有野生型和SHP-1突变体的DO11.10小鼠中重建FTOC 通过逆转录病毒基因转移。 虽然已经显示了SHP-1对TCR介导的信号传导的负调节，但其 作用机制尚待确定。最近，一种专门的 膜微区（称为脂筏）已被公认为其 TCR信号的重要性。几个主要参与者的木筏本地化 已经证明早期T细胞活化对于信号传导是关键的。我们 工作假设是，SHP-1本地化到他筏，这是 定位对其功能很重要。事实上，我们观察到， SHP-1定位到了救生筏上在第二个目标中，我们建议研究 SHP-1在筏上的定位机制及其功能后果。 在第三个目标中，我们将测试SHP-1在以下方面发挥作用的假设： 改变的肽配体信号传导。虽然SHP-1在T细胞发育中的作用 虽然已经被认识到，但它在成熟细胞中的作用却不太清楚。我们将使用 解决这个问题的几种方法，重点是激动剂/部分 激动剂/拮抗剂信号传导。我们将尝试描述对以下问题的反应： 野生型和SHP-1缺陷型CD 4 + DO11.10中改变的肽配体 TCR表达系以及CD 8+细胞毒性T细胞克隆。

DESCRIPTION (provided by applicant): Tyrosyl phosphorylation is a key regulatory mechanism for normal cell growth, differentiation, and death. The steady state level of tyrosyl phosphorylation on any protein is determined by he opposing actions of protein tyrosine kinases and phosphatases. SHP-1 is a protein tyrosine phosphatase that has been shown to be involved in the negative regulation of signaling events induced by cytokines, growth factors and antigens. Mutations in the SHP-1 gene in mice cause the motheaten (me/me) phenotype. Mice homozygous for the me allele, which results in the absence of any detectable SHP-1 protein, display a panoply of disorders in 11 hematopoietic lineages. These mice provide a valuable tool to combine in vitro biochemical assays with in vivo and ex vivo biological studies. Our long range goal is to understand how SHP-1 influences the growth and differentiation of hematopoietic cells. This application will attempt to elucidate the involvement of SHP-1 in immature and mature cell functions. Recently, we and others have shown a clear role for SHP-1 in T-cell development. However, the underlying molecular mechanism remains unknown. In the first Aim, we propose to address this question using fetal thymic organ cultures (FTOCs). Analyses of me/me:DO11.10 thymocytes indicate at SHP-1 helps to set TCR signaling thresholds in positive and negative selection. Studies of FTOCs from these mice will be applied to follow thymic T-cell development in detail. In particular, we propose to rescue the motheaten phenotype by reconstituting FTOC from me/me:DO11.10 mice with wild type and mutants of SHP-1 via retroviral gene transfer. While SHP-1' negative regulation of TCR-mediated signaling has been shown, its mechanism of action ha yet to be defined. Recently, one type of specialized membrane microdomains (referred to as lipid-rafts) has been recognized for its importance for TCR signaling. The rafts localization of several key players of early T-cell activation has be n shown to be critical for signaling. Our working hypothesis is that SHP-1 localizes to he rafts and that this localization is important for its function. Indeed, we observe that a fraction of SHP-1 localizes to the rafts. In the second Aim, we propose to study the mechanism of SHP-1's localization to the rafts and its functional consequences. In the third Aim, we will test the hypothesis that SHP-1 plays a role in altered peptide ligand signaling. While SHP-1's role in T-cell development has been recognized, its role in mature cells is less understood. We will use several approaches to address this question with a focus on agonist/partial agonist/antagonist signaling. We will attempt to characterize the responses to altered peptide ligands in wild type and SHP-1-deficient CD4+ DO11.10 TCR-expressing lines as well as CD8+ cytotoxic T-cell clones.