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: 6890472
• 负责人: WOJCIECH A SWAT
• 金额: $ 38.25万
• 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/6890472
• 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接头和鸟嘌呤核苷酸交换因子，协调淋巴细胞中抗原受体下游的信号传导。先前阐明 Vav 在体内淋巴细胞发育和激活中的功能的努力因 Vav 家族各个成员之间的冗余而受到阻碍，该家族由 3 种高度同源的蛋白质组成。作为该提案初步研究的一部分，我们首次生成并表征了缺乏所有 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，并最终确定淋巴谱系中各个亚型之间功能冗余的限制。 然而，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，在目标 1 中，我们建议使用 Vav 缺陷型抗原特异性 Jurkat T 细胞，该细胞经过改造可表达小鼠 α/β TCR、3.L2 和小鼠 CD4。由于 3.L2 TCR 配体是与分级效力的抗原肽共价连接的可溶性 I-Ek MHC 分子，因此该系统既可以实现抗原激活的微妙性（通过改变肽配体），又可以在使用转化的组织培养细胞系时方便地进行生化分析。使用 J.3.L2 系统，我们将确定一大组 Vav 突变体对 TCR 发出的多个信号通路的影响。这些 Vav 突变体是由结构特征驱动的，这些结构特征隐含地包含了关于 Vav 机制的几个不同的假设。 在目标 2 中，我们建议使用一种新型 Vav null 造血干细胞互补 (Vav nulI-HSCC) 检测，用于快速分析突变 Vav 蛋白在 T 细胞发育和激活中的影响。使用该测定，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，将使用 Vav 外显子替换（敲入）将选定的 Vav 突变体引入小鼠种系。 在目标 3 中，我们将通过进行复杂的分析来研究围绕体内 Vav 功能调节的主要未解答问题，这将建立在目标 1 和 2 中建立的方法检查的大量 Vav 突变体的基础上。