WIP in TCR signaling and actin reorganization

描述(由申请人提供)： Wiskott-Aldrich综合征，一种原发免疫缺陷，细胞骨架完整性 造血细胞的数量受到影响，是由WASP基因突变引起的。我们有 鉴定出编码一种新的WASP相互作用蛋白(WIP)的基因。在静息细胞中，WIP形成一个 并抑制其肌动蛋白成核活性的诱导。 Cdc42-GTP。我们最近培育了WIP-/-小鼠。尽管WIP-/-T淋巴细胞发育 正常情况下，它们不能增殖、分泌IL-2、增加其F-肌动蛋白含量或延伸 T细胞受体结扎后的突起。此外，他们在接触方面也很缺乏 含抗CD3/I-CAM1的脂双层和B细胞的形成 超抗原。与WIP在肌动蛋白重组中的关键作用一致，WIP-/-T细胞显示出 它们组装皮质下肌动蛋白细丝网络的能力存在严重缺陷。 我们的初步数据表明，WIP与ZAP-70招募a的接头蛋白CrkL结合 在TCR接洽之后，CRKL/WIP-WASP复合体到TCR，并且WIP随后被 被PKCtheta磷酸化，并与WASP脱离。我们的假设是一个 CrkL/WIP/WASP复合体被招募到脂筏中的TCR，其中WIP被磷酸化 PKCtheta。这将从WIP中释放WASP，使其可由CDC42-GTP激活到 启动局部肌动蛋白聚合，这对于RAFT的稳定性和效率是必不可少的 信号分子在T细胞免疫突触(IS)中的浓缩和整合。 其结果是转录因子持续激活，从而产生最佳的IL-2基因 表达与T细胞增殖的关系。我们建议对在制品生产的三步机制模型进行检验 在T细胞中的功能：1)分析WIP-/-T细胞形成IS的能力，在IS中，关键信号分子集中在脂筏中，导致IL-2基因的激活。我们会 用抗CD3包被的小球、MHC-II类肽/ICAM-1双层和 抗原提呈细胞；脂筏的组成和稳定性；以及持续激活 调节IL-2基因的Ca++动员、信号中间产物和转录因子 在WIP-/-T细胞中表达。2)剖析缺乏肌动蛋白结合的WIP结构域的作用，CrkL TCR信号中的结合或WASP结合位点。我们将研究WIP-/-T细胞中的TCR信号 重组RAG2-/-小鼠T细胞中WIP突变体的体外逆转录病毒重组 用携带WIP突变转基因的WIP-/-造血干细胞和来自WIP-/-的T细胞 用突变的WIP转基因重组的小鼠。3)研究CRKL在细胞周期中的作用 WIP和WASP在TCR和TCR信号中的招募。我们将审查招聘情况 在SLP-76缺陷的T细胞和表达优势的T细胞中WIP-WASP复合体的表达 我们将检测CrkL-/-T细胞中的TCR信号。4)测试 PKCtheta使WIP磷酸化导致WASP从WIP解离的假说 以及它从抑制中释放。我们将研究TCR后基于肌动蛋白的细胞骨架变化 在PKCtheta-/-小鼠中，关键的磷酸化残基(S)在WIP中的身份扰动 WIP的磷酸化对CDC42-GTP驱动的激活的影响 WASP.所获得的结果将有助于阐明TCR和肌动蛋白细胞骨架之间的联系，将为更好地了解细胞激活以及在免疫缺陷疾病、自身免疫和癌症方面的潜在重要应用提供帮助。

DESCRIPTION (provided by the Applicant): The Wiskott-Aldrich syndrome, a primary immunodeficiency in which the cytoskeletal integrity of hematopoietic cells is affected, is caused by a mutation in the WASP gene. We have identified a gene encoding a novel WASP Interacting Protein (WIP). In resting cells, WIP forms a complex with WASP/NWASP and inhibits the induction of their actin nucleating activity by Cdc42-GTP. We recently generated WIP-/- mice. Although WIP-/- T lymphocytes develop normally, they fail to proliferate, secrete IL-2, increase their F-actin content, or extend protrusions following T cell receptor ligation. Furthermore, they are deficient in contact formation with anti-CD3/I-CAM1-containing lipid bilayers and with B cells presenting superantigen. Consistent with a key role for WIP in actin reorganization, WIP-/- T cells display a profound defect in their ability to assemble subcortical actin filament networks. Our preliminary data suggests that WIP binds to the adapter protein CrkL that ZAP-70 recruits a CrkL/WIP-WASP complex to the TCR following TCR engagement, and that WIP is subsequently phosphorylated by PKCtheta and disengages from WASP. Our hypothesis is that a CrkL/WIP/WASP complex is recruited to the TCR in lipid rafts where WIP is phosphorylated by PKCtheta. This releases WASP from WIP, making it available for activation by Cdc42-GTP to initiate localized actin polymerization, which is essential for raft stability and for efficient concentration and integration of signaling molecules in the T cell immunological synapse (IS). The outcome is sustained activation of transcription factors that lead to optimal IL-2 gene expression and T cell proliferation. We propose to test this three step mechanistic model of WIP function in T cells by: 1) Analyzing the ability of WIP-/- T cells to form an IS, in which key signaling molecules are concentrated in lipids rafts leading to activation of the IL-2 gene. We will examine IS formation with anti-CD3 coated beads, MHC class II-peptide/ICAM-1 bilayers and antigen presenting cells; composition and stability of lipids rafts; and sustained activation of Ca++ mobilization, signaling intermediates and transcription factors that regulate IL-2 gene expression in WIP-/- T cells. 2) Dissecting the role of WIP domains that lack actin binding, CrkL binding, or WASP binding sites in TCR signaling. We will examine TCR signaling in WIP-/- T cells retrovirally reconstituted in vitro with WIP mutants, in T cells from RAG2-/- mice reconstituted with WIP-/- hemopoietic stem cells bearing WIP mutant transgenes and in T cells from WIP-/- mice reconstituted with mutant WIP transgenes. 3) Examining the role of CrkL in the recruitment of WIP and WASP to the TCR and in TCR signaling. We will examine the recruitment of the WIP-WASP complex in SLP-76 deficient T cells and in T cells that express dominant negative CrkL mutants and we will examine TCR signaling in CrkL-/- T cells. 4) Testing the hypothesis that phosphorylation of WIP by PKCtheta leads to the dissociation of WASP from WIP and its release from inhibition. We will study actin-based cytoskeletal changes following TCR ligation in PKCtheta-/- mice, the identity of key phosphorylated residue(s) in WIP that perturb WASP binding, and examine the effect of WIP phosphorylation on Cdc42-GTP driven activation of WASP. The results obtained will help clarify the link between the TCR and the actin cytoskeleton, will provide a better understanding of cell activation and potential important applications for immunodeficiency diseases, autoimmunity and cancer.