ROLE OF VAV AND ITS EFFECTORS IN LYMPHOCYTE ACTIVATION

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We have shown that Vav-deficiency results in defective development and activation of T lymphocytes. More recently, we found that the activation- defects in Vav-deficient murine lymphocytes are associated with defects in reorganization of the actin cytoskeleton, similar to those of human WASp- deficient lymphocytes. Members of the Rho-family of small GTPases, such as Cdc42, have been shown by others to be downstream effectors of Vav in fibroblasts and also to feed into both the cytoskeleton and stress- activated protein kinase cascades. The goal of the proposed work is to elucidate intracellular signaling pathways effected by Vav and its potential downstream effectors in lymphocyte development and activation and, in the context of these studies, to dissect the contribution of cytoskeletal versus mitogen and stress-activated protein kinase pathways. The first aim is to elucidate defects in signaling from the T and B cell antigen receptor in Vav-deficient lymphocytes. A major focus will be to employ Vav+ lymphocytes generated by RAG-2-deficient blastocyst complementation for studies aimed at elucidating potential roles of Vav in regulation of the actin-cytoskeleton, proliferation, and activation- induced cell death. We also propose to generate mice which harbor germline mutations in the Vav gene to facilitate studies of the physiologic consequences of Vav-deficiency on the immune system. The second aim is to dissect specific roles for Vav-protein domains and potential downstream effectors in the Vav-signaling pathway and will be accomplished by carrying out "rescue" experiments which involve introducing wild type or mutant cDNA expression constructs into Vav- deficient ES cells followed by assay via RAG-2-deficient blastocyst complementation. The third aim is to elucidate developmental and functional defects in lymphocytes deficient in potential downstream Vav- effectors, including Cdc42 (which has been functionally linked to both Vav and WASp), as well as particular MAP kinases (MEK-1 and SEK-1). In this aim, a major focus will be to compare and contrast potential phenotypic effects of specific mutations to those observed in the context of Vav- and WASp-deficient lympyhocytes.