ROLE OF VAV AND ITS EFFECTORS IN LYMPHOCYTE ACTIVATION

我们已经证明，Vav缺乏会导致发育缺陷和 T淋巴细胞的活化。最近，我们发现激活- Vav缺陷小鼠淋巴细胞的缺陷与 肌动蛋白细胞骨架的重组，类似于人类黄蜂- 淋巴细胞缺乏。Rho-小GTP酶家族的成员，如 已被其他人证明是Vav的下游效应物。 成纤维细胞，也给细胞骨架和应激提供营养- 活化的蛋白激酶级联反应。拟议工作的目标是 Vav及其受体对细胞内信号转导途径的影响 淋巴细胞发育和激活中潜在的下游效应因子 并在这些研究的背景下，剖析 细胞骨架与有丝分裂原和应激激活蛋白激酶途径的比较。 第一个目标是阐明T和B细胞信号的缺陷 Vav缺陷淋巴细胞中的抗原受体。主要的重点将是 利用RAG-2缺陷胚泡产生的Vav+淋巴细胞 对旨在阐明血管活性肠病潜在作用的研究的补充 肌动蛋白-细胞骨架、增殖和激活的调节- 诱导细胞死亡。我们还建议培育出窝藏在 Vav基因的种系突变有助于研究 血管紧张素转换酶缺乏对免疫系统的生理影响。这个 第二个目标是剖析Vav蛋白结构域和 Vav信号通路中潜在的下游效应器，并将 通过进行“救援”实验来完成，这些实验包括 将野生型或突变型表达载体导入Vav-1 利用RAG-2缺陷的囊胚法检测缺陷ES细胞 互补性。第三个目的是阐明发育和 潜在下游Vav-1缺陷淋巴细胞的功能缺陷 效应器，包括Cdc42(已在功能上链接到两个Vav 和黄蜂)，以及特定的MAP激酶(MEK-1和SEK-1)。在这 目的，一个主要的焦点将是比较和对比潜在的表型 特定突变对Vav-和Vav背景下观察到的影响 缺乏黄蜂的淋巴巨噬细胞。

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.