ROLE OF VAV AND ITS EFFECTORS IN LYMPHOCYTE ACTIVATION

我们已经表明，Vav缺陷导致发育缺陷， 活化T淋巴细胞。 最近，我们发现激活- Vav缺陷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WASp的重组， 缺乏淋巴细胞。 小GTP酶的Rho家族的成员，如 如Cdc 42，已经被其他人证明是Vav的下游效应子， 成纤维细胞，也进入细胞骨架和压力， 激活的蛋白激酶级联。 拟议工作的目标是 阐明细胞内信号通路的影响Vav及其 淋巴细胞发育和活化中的潜在下游效应物 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活化蛋白激酶途径。 第一个目的是阐明T和B细胞信号传导的缺陷 Vav缺陷淋巴细胞中的抗原受体。 一个主要的重点将是 使用由RAG-2缺陷型胚泡产生的Vav+淋巴细胞 补充旨在阐明Vav在以下方面的潜在作用的研究： 肌动蛋白细胞骨架、增殖和活化的调节- 诱导细胞死亡。 我们还建议产生小鼠， Vav基因中的种系突变，以促进对 Vav缺陷对免疫系统的生理后果。 的 第二个目的是剖析Vav蛋白结构域的特定作用， Vav信号通路中的潜在下游效应物， 通过进行“拯救”实验来完成， 将野生型或突变体cDNA表达构建体引入Vav- 缺陷ES细胞，随后通过RAG-2缺陷囊胚进行测定 互补 第三个目标是阐明发展和 淋巴细胞功能缺陷，缺乏潜在的下游Vav- 效应子，包括Cdc 42（其在功能上与Vav 和WASp），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 特定突变对在Vav-和 WASP缺陷型红细胞。

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.