Role for Vav Family Proteins in T Lymphocyte Activation

描述（由申请人提供）：Vav家族蛋白被认为作为分子接头和鸟嘌呤核苷酸交换因子，在淋巴细胞中协调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受到Vav家族成员之间冗余的阻碍，Vav家族成员包括3种高度同源的蛋白质。作为本研究的初步研究的一部分，我们首次生成并鉴定了缺乏所有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，并最终确定淋巴系中单个亚型之间功能冗余的限制。

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.