Role of SHP-1 in T cell activation and development

描述(申请人提供)：酪氨酸磷酸化是关键 正常细胞生长、分化和死亡的调节机制。这个 任何蛋白质上酪氨酸磷酸化的稳定水平由以下因素决定 他反对蛋白质酪氨酸激酶和磷酸酶的作用。SHP-1是一种 蛋白酪氨酸磷酸酶，已被证明与阴性有关 细胞因子、生长因子和细胞因子诱导的信号事件的调节 抗原。小鼠的SHP-1基因突变引起蛾子(Me/Me) 表型。Me等位基因纯合的小鼠，导致缺乏 任何可检测到的SHP-1蛋白，都会在11 造血血统。这些小鼠为体外结合提供了一个有价值的工具 生化分析与体内和体外生物学研究。我们的远距离飞行 目的是了解SHP-1如何影响细胞的生长和分化 造血细胞。本申请书将试图阐明牵涉其中 SHP-1在未成熟和成熟细胞功能中的表达。 最近，我们和其他人已经证明了SHP-1在T细胞中的明确作用 发展。然而，其潜在的分子机制仍不清楚。在……里面 第一个目标，我们建议使用胎儿胸腺器官来解决这个问题 培养(FTOC)。对Me/Me：DO11.10胸腺细胞的分析表明SHP-1有助于 在正选择和负选择中设置TCR信号阈值。的研究 这些小鼠的FTOCs将被应用于跟踪胸腺T细胞的发育 细节。特别是，我们建议通过以下方式挽救蛾子的表型 从Me/Me重组FTOC：DO11.10野生型及突变型SHP-1小鼠 通过逆转录病毒基因转移。 虽然SHP-1对TCR介导的信号转导具有负调控作用，但其 其作用机制尚不明确。最近，一种专门的 膜微结构域(称为脂筏)因其 对TCR信令的重要性。的几个关键球员的木筏本地化 已有研究表明，T细胞的早期激活对信号转导至关重要。我们的 工作假说是SHP-1定位于木筏，这 本地化对于它的功能是重要的。事实上，我们观察到有一小部分 SHP-1基因定位于木筏。在第二个目标中，我们建议研究 SHP-1‘S定位于筏子的机制及其功能后果。 在第三个目标中，我们将检验SHP-1在 改变了多肽配体的信号。而SHP-1的S在T细胞发育中的作用 虽然已被认识到，但对其在成熟细胞中的作用却知之甚少。我们将使用 解决这一问题的几种方法，重点是激动剂/部分 激动剂/拮抗剂信号。我们将尝试描述对以下方面的反应 野生型和SHP-1缺陷的CD4+DO11.10的多肽配体改变 TCR表达株和CD8+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.