WIP in TCR signaling and actin reorganization

描述（申请人提供）： Wiskott-Aldrich综合征，一种原发性免疫缺陷，其中细胞骨架完整性 造血细胞受到影响，是由WASP基因突变引起的。我们有 鉴定了编码新的WASP相互作用蛋白（WASP Interacting Protein，WASP）的基因。在休眠细胞中， 与WASP/NWASP复合并抑制其肌动蛋白成核活性的诱导， Cdc42-GTP。我们最近培育出了一种新的小鼠。尽管γ-/-T淋巴细胞 正常情况下，它们不能增殖、分泌IL-2、增加它们的F-肌动蛋白含量或延伸， 在T细胞受体连接后突起。此外，他们缺乏联系， 形成含抗CD3/I-CAM 1的脂质双层和B细胞呈递 超抗原与肌动蛋白重组中的关键作用一致，T-/-T细胞显示出一种与肌动蛋白重组相关的功能。 他们在组装皮层下肌动蛋白丝网络的能力方面存在严重缺陷。 我们的初步数据表明，ZAP-70与接头蛋白CrkL结合，ZAP-70招募接头蛋白CrkL。 在TCR接合后，CrkL/WIP-WASP复合物与TCR结合，并且随后 被PKC θ磷酸化并从WASP脱离。我们假设 CrkL/WASP/WASP复合物被募集到脂筏中的TCR，在脂筏中，CrkL/WASP被磷酸化。 PKC θ这将从Cdc42-GTP中释放WASP，使其可用于激活Cdc42-GTP， 引发局部肌动蛋白聚合，这对于筏的稳定性和有效的 T细胞免疫突触（IS）中信号分子的浓度和整合。 结果是持续激活转录因子，导致最佳IL-2基因 表达和T细胞增殖。我们建议测试这三个步骤的机制模型， 在T细胞中的功能：1）分析IFN-/-T细胞形成IS的能力，其中关键信号分子集中在脂筏中，导致IL-2基因的活化。我们将 用抗CD3包被的珠粒、MHC II类肽/ICAM-1双层检查IS形成， 抗原呈递细胞;脂筏的组成和稳定性;以及 调节IL-2基因的Ca~（++）动员、信号中间体和转录因子 表达在γ-/-T细胞。2)分析缺乏肌动蛋白结合的CrkL结构域的作用， TCR信号传导中的WASP结合位点。我们将研究TCR信号在T细胞中的作用。 逆转录病毒在体外用RAG2突变体重建，在来自重建的RAG2-/-小鼠的T细胞中 在携带有突变转基因的造血干细胞和来自造血干细胞的T细胞中， 用突变的转基因重组的小鼠。3)研究CrkL在 在TCR和TCR信号传导中，WASP和WASP的募集。我们将审查招募 在SLP-76缺陷型T细胞和表达显性WIP-WASP复合物的T细胞中， 阴性CrkL突变体，我们将检查CrkL-/-T细胞中的TCR信号传导。4)测试 假设PKC θ对WASP磷酸化导致WASP从WASP上解离 以及从抑制中释放出来。我们将研究TCR后基于肌动蛋白的细胞骨架变化 在PKC θ-/-小鼠中的连接，干扰PKC θ-/-小鼠的PKC β中关键磷酸化残基的身份， WASP结合，并检查Cdc42-GTP磷酸化对Cdc42-GTP驱动的活化的影响 的WASP。获得的结果将有助于澄清TCR和肌动蛋白细胞骨架之间的联系，将提供更好地了解细胞活化和免疫缺陷疾病，自身免疫和癌症的潜在重要应用。

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.