ROLE OF RAS IN T-CELL FATE DETERMINATION

RAS 在 T 细胞命运决定中的作用

• 批准号: 6349903
• 负责人: Jose Alberola-Ila
• 金额: $ 27.03万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6349903
• 关键词: CD4 molecule; CD8 molecule; T lymphocyte; biological signal transduction; cell differentiation; genetic mapping; genetically modified animals; guanine nucleotide binding protein; helper T lymphocyte; laboratory mouse; mitogen activated protein kinase; mutant; phosphatidylinositol 3 kinase

I have proposed a set of experiments aimed to understand the intracellular signals that control cell fate determination at a defined point in T cell development, when immature double positive cells are screened for the reactivity of their TcR. The underlying hypothesis, based on our previous results, is that alternative cell fate decisions are driven by qualitatively different signaling events. In the first aim, we investigate in more detail the role of Ras and its effectors in positive selection using our previously generated dominant negative Ras mice in combination with transgenic mice that express a hypersensitive variant of MAPK, to address whether MAPK is the only Ras effector important for positive selection. In case it is not, we propose a series of experiments that, using a retroviral infection system, test different Ras effector mutants for their ability to rescue dnRas. Using the results obtained in these experiments, we will define in more detail the components of the Ras signaling pathway involved in positive selection and try to identify genes induced by Ras and involved in positive selection. The second aim is directed at understanding the contribution of different signaling pathways to the commitment to either the CD4 or CD8 lineages. The experiments focus on the role of Lck in this process, and test whether the Ras/MAPK cascade is a downstream target of Lck in this process using biochemical and genetic approaches. The third aim of this grant proposes to analyze the role of PI 3-K in T cell development by generating transgenic mice expressing dominant negative and constitutively active mutants of this enzyme. We address the possible role of PI 3-K in positive and negative selection using transgenic models. Given the role of PI 3-K in cell survival we will analyze whether it affects thymocyte survival, using both in vivo and in vitro approaches. These studies should provide us with a better understanding of the mechanisms that control positive and negative selection during T cell development, and, more widely, could provide clues as to how differential signaling through the TCR is achieved. Having the different mice strains will allow a direct comparison of their phenotype, as well as open the possibility of performing genetic experiments to unravel the relationships among the different signal transduction pathways during T cell development.

我提出了一组实验，旨在了解在T细胞发育的一个确定点上控制细胞命运决定的细胞内信号，当未成熟的双阳性细胞被筛选其TcR的反应性时。基于我们之前的结果，潜在的假设是，不同的细胞命运决定是由质量不同的信号事件驱动的。在第一个目标中，我们更详细地研究了Ras及其效应物在正选择中的作用，使用我们之前产生的显性阴性Ras小鼠与表达MAPK超敏感变体的转基因小鼠结合，以解决MAPK是否是唯一对正选择重要的Ras效应物。如果不是，我们提出了一系列的实验，使用逆转录病毒感染系统，测试不同的Ras效应突变体拯救dnRas的能力。利用这些实验获得的结果，我们将更详细地定义参与正选择的Ras信号通路的组成部分，并试图识别由Ras诱导并参与正选择的基因。第二个目标是了解不同信号通路对CD4或CD8谱系的贡献。本实验主要关注Lck在这一过程中的作用，并通过生化和遗传学的方法检测Ras/MAPK级联是否为Lck在这一过程中的下游靶点。该基金的第三个目标是通过产生表达该酶显性阴性和组成活性突变体的转基因小鼠来分析PI 3-K在T细胞发育中的作用。我们利用转基因模型探讨了PI 3-K在正选择和负选择中的可能作用。鉴于PI 3-K在细胞存活中的作用，我们将使用体内和体外方法分析它是否影响胸腺细胞存活。这些研究将使我们更好地理解T细胞发育过程中控制阳性和阴性选择的机制，并且更广泛地说，可以为如何通过TCR实现差异信号传导提供线索。拥有不同的小鼠品系将允许对其表型进行直接比较，并打开进行遗传实验以揭示T细胞发育过程中不同信号转导途径之间关系的可能性。