Role for Vav Family Proteins in T Lymphocyte Activation

Vav 家族蛋白在 T 淋巴细胞激活中的作用

• 批准号: 7059423
• 负责人: WOJCIECH A SWAT
• 金额: $ 37.35万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7059423
• 关键词: T cell receptor; T lymphocyte; biological signal transduction; cell differentiation; cell proliferation; clone cells; developmental immunology; enzyme activity; gene complementation; gene mutation; genetically modified animals; guanine nucleotide exchange factors; laboratory mouse; leukocyte activation /transformation; phosphatidylinositol 3 kinase; phosphorylation; protein structure function

DESCRIPTION (provided by applicant): The Vav family proteins are thought to function as molecular adaptors and guanine-nucleotide exchange factors orchestrating signaling downstream of antigen receptors in lymphocytes. Previous efforts to elucidate Vav function in lymphocyte development and activation in vivo have been hampered by the redundancy among individual members of the Vav family, which comprises 3 highly homologous proteins. As a part of our preliminary studies for this proposal we generated and characterized for the first time mice lacking all 3 Vav proteins which allowed us to demonstrate the essential function of the entire Vav family as well as to conclusively define the limits of functional redundancy among the individual isoforms in the lymphoid lineage. However, the exact mechanism of Vav function remains elusive. To facilitate biochemical analyses of Vav function, in Aim 1 we propose to use Vav-deficient antigen-specific Jurkat T cells engineered to express a murine alpha/beta TCR, the 3.L2, and a murine CD4. Since 3.L2 TCR-ligands are soluble I-Ek MHC molecules covalently linked to antigenic peptides of graded potency, this system allows both the subtlety of antigen activation (by altered peptide ligands) but also the convenience of biochemical analysis possible when using a transformed tissue culture cell line. Using J.3.L2 system we will determine the effects of a large panel of Vav mutants in multiple signaling pathways emanating from the TCR. These Vav mutants were motivated by structural features which implicitly incorporate several distinct hypotheses about Vav mechanism. In Aim 2, we propose to use a novel Vav null-hematopoietic stem cell complementation (Vav nulI-HSCC)assay developed for rapid analyses of the effects of mutant Vav proteins in T cell development and activation. Using this assay, we will determine the structural basis for Vav protein function in T lymphocytes in vivo. Based on results of these experiments, selected Vav mutants will be introduced into murine germ line using Vav exon-replacement (knock-in). In Aim 3 we will examine the major unanswered questions surrounding the regulation of Vav function in vivo by carrying out complex analyses, which will build on the strength of a large panel of Vav mutants examined by approaches established in Aims 1 and 2.

描述（由申请人提供）：VAV家族蛋白被认为充当分子适配器和鸟嘌呤核苷酸交换因子，策划了淋巴细胞中抗原受体下游的信号传导。先前阐明VAV在淋巴细胞发育和体内激活中的VAV功能的努力受到VAV家族各个成员的冗余的阻碍，VAV家族的各个成员包括3种高度同源蛋白。作为我们对该提案的初步研究的一部分，我们首次产生和表征了所有3种VAV蛋白的小鼠，这使我们能够证明整个VAV家族的基本功能，并最终定义了功能冗余的限制淋巴谱系中的个体同工型。 但是，VAV功能的确切机制仍然难以捉摸。为了促进VAV功能的生化分析，在AIM 1中，我们建议使用工程设计的VAV缺陷抗原特异性的Jurkat T细胞来表达鼠α/beta TCR，3.L2和鼠CD4。由于3.L2 TCR配体是可溶的I-EK MHC分子，该分子与分级效力的抗原肽共价链接，因此该系统既允许抗原激活的微妙（通过改变的肽配体）的微妙性，也允许在使用变换的生物学分析时使用变换的便利性。组织培养细胞系。使用J.3.L2系统，我们将确定来自TCR发出的多个信号通路中的大型VAV突变体的影响。这些VAV突变体是由结构特征激励的，这些特征隐含了关于VAV机制的几种不同的假设。 在AIM 2中，我们建议使用一种新型的VAV无毛 - 莫大律干细胞互补（VAV NULI-HSCC）分析，用于快速分析突变体VAV蛋白在T细胞发育和激活中的影响。使用此测定，我们将确定体内T淋巴细胞中VAV蛋白功能的结构基础。根据这些实验的结果，使用VAV外显子替代（敲入）将选定的VAV突变体引入鼠类种生殖系。 在AIM 3中，我们将通过进行复杂的分析来研究围绕体内VAV功能调节功能的主要问题，该分析将建立在AIM 1和2中建立的方法检查的大型VAV突变体的强度上。