Role of SHP-1 in T cell activation and development

SHP-1 在 T 细胞激活和发育中的作用

• 批准号: 6749457
• 负责人: Ulrike Lorenz
• 金额: $ 33.3万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6749457
• 关键词: T cell receptor; T lymphocyte; biological signal transduction; cell age; cell growth regulation; cell line; cell membrane; cytotoxic T lymphocyte; embryo /fetus tissue /cell culture; enzyme activity; enzyme mechanism; interleukin 2; laboratory mouse; leukocyte activation /transformation; ligands; peptides; phenotype; protein localization; protein tyrosine phosphatase; receptor expression; thymus; tissue /cell culture; transfection; viral rescue

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.

描述（由申请人提供）：酪氨酰磷酸化是关键 正常细胞生长、分化和死亡的调节机制。这 任何蛋白质酪氨酰磷酸化的稳态水平由下式确定 他对抗蛋白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负面影响有关 调节细胞因子、生长因子和诱导的信号事件 抗原。小鼠 SHP-1 基因突变导致受害 (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，都会显示出 11 种疾病的全部症状 造血谱系。这些小鼠提供了体外结合的宝贵工具 体内和离体生物学研究的生化测定。我们的长距离 目标是了解 SHP-1 如何影响生长和分化 造血细胞。该应用程序将尝试阐明参与 SHP-1 在未成熟和成熟细胞功能中的作用。 最近，我们和其他人已经证明了 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 第一个目标，我们建议使用胎儿胸腺器官来解决这个问题 文化（FTOC）。 me/me 的分析：DO11.10 胸腺细胞表明 SHP-1 有帮助 设置正向和负向选择中的 TCR 信号阈值。研究 这些小鼠的 FTOC 将用于追踪胸腺 T 细胞的发育 细节。特别是，我们建议通过以下方式拯救受害表型： 从具有野生型和 SHP-1 突变体的 me/me:DO11.10 小鼠中重建 FTOC 通过逆转录病毒基因转移。 虽然 SHP-1 对 TCR 介导的信号传导具有负调节作用，但其 作用机制尚未确定。最近，一种专门的 膜微区（称为脂筏）因其 TCR 信号传导的重要性。几个主要参与者的筏本地化 早期 T 细胞激活已被证明对于信号传导至关重要。我们的 工作假设是 SHP-1 定位于筏，并且这 本地化对其功能来说很重要。事实上，我们观察到一小部分 SHP-1 的定位于筏。在第二个目标中，我们建议研究 SHP-1 定位于筏的机制及其功能后果。 在第三个目标中，我们将检验 SHP-1 在以下方面发挥作用的假设： 改变肽配体信号传导。虽然 SHP-1 在 T 细胞发育中的作用已 尽管人们已经认识到它在成熟细胞中的作用，但人们对它的作用知之甚少。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 CD4+ DO11.10 中肽配体的改变 TCR 表达系以及 CD8+ 细胞毒性 T 细胞克隆。