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.
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|Gmyrek, Grzegorz B; Akilesh, Holly M; Graham, Daniel B et al. (2013) Loss of DAP12 and FcR? drives exaggerated IL-12 production and CD8(+) T cell response by CCR2(+) Mo-DCs. PLoS One 8:e76145|
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|Sandoval, Gabriel J; Graham, Daniel B; Gmyrek, Grzegorz B et al. (2013) Novel mechanism of tumor suppression by polarity gene discs large 1 (DLG1) revealed in a murine model of pediatric B-ALL. Cancer Immunol Res 1:426-37|
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|Graham, Daniel B; Akilesh, Holly M; Gmyrek, Grzegorz B et al. (2010) ITAM signaling in dendritic cells controls T helper cell priming by regulating MHC class II recycling. Blood 116:3208-18|
|Stephenson, Linda M; Miller, Brian C; Ng, Aylwin et al. (2009) Identification of Atg5-dependent transcriptional changes and increases in mitochondrial mass in Atg5-deficient T lymphocytes. Autophagy 5:625-35|
|Miletic, Ana V; Graham, Daniel B; Sakata-Sogawa, Kumiko et al. (2009) Vav links the T cell antigen receptor to the actin cytoskeleton and T cell activation independently of intrinsic Guanine nucleotide exchange activity. PLoS One 4:e6599|
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