Deciphering the Role of WASP Family Proteins in T Cell Function

我们最近开发了模型系统来研究Rho GTP酶的哺乳动物细胞功能（例如， Cdc 42）和Wiskott-Aldrich综合征蛋白（例如，WASP、N-WASP、WAVE）-两个相互作用的 整合传入细胞表面信号以介导细胞骨架变化的蛋白质家族。黄蜂是 当被Rho家族GTP酶（Cdc 42/Rac）和磷酸肌醇激活时， 直接与Arp 2/3复合物结合，导致肌动蛋白组装。在这种情况下，细胞形状的协调 通过细胞骨架的变化，需要这样的不同性质，如细胞间接触，淋巴细胞 激活和趋化性。WASP的激活也受到与其他几种蛋白质相互作用的调节 包括WASP相互作用蛋白（WASP）、Sh 3结构域蛋白（例如Nek）和Srk家族激酶（例如， Hck）。我们已经采用基因靶向来产生Rho家族GTdR Cdc 42缺陷的小鼠， 以及几个WASP家族成员：WASP，N-WASP和WAVE-2。N-WASP-和WAVE-2- 缺陷导致早期胚胎死亡，而WASP缺陷（WKO）小鼠是存活的和可生育的， 淋巴细胞发育正常，但有信号和细胞骨架异常。我们还 产生了ES细胞克隆，这些克隆含有一种激活的WASP敲入突变， 描述了一种新的免疫缺陷，X连锁中性粒细胞增多症（XLN）。因为N-WASP和WAVE-2 缺陷是不可行的，需要选择性靶向这些等位基因来评估这些缺陷的作用。 淋巴细胞中的蛋白质。为此，我们已经产生了具有N-WASP的小鼠， 在白细胞中失活，我们目前正在产生WAVE 2中具有类似突变的小鼠。我们有 在T细胞中产生WASP/N-WASP双敲除（DKO）小鼠，并与Raif Geha博士合作， （项目1），WASP/MDKO小鼠;两种品系均与更严重的信号传导和细胞骨架相关。 与WKO小鼠相比，T细胞异常。这种新型试剂的集合，其中 各种WASP家族成员可以单独或组合地在细胞或小鼠中失活， 为我们正在进行的剖析WASP家庭成员在以下方面的作用的目标提供了强有力的基础： 白细胞功能我们建议：A1）剖析WASP的独特作用和 WASP/N-WASP在白细胞发育和功能中的作用A2）界定宪法积极分子的作用 T细胞中WASP功能的突变。

We have recently developed model systems to study the mammalian cellular function of Rho GTPases (e.g., Cdc42) and the Wiskott-Aldrich syndrome proteins (e.g., WASP, N-WASP, WAVEs) - two interacting families of proteins that integrate incoming cell surface signals to mediate cytoskeletal change. WASPs are cytoplasmic proteins that when activated by Rho-family GTPases (Cdc42/Rac) and phosphoinositides directly bind to the Arp2/3 complex, resulting in actin assembly. In this context, coordination of cell shape through cytoskeletal change is required for such diverse properties as cell-cell contact, lymphocyte activation, and chemotaxis. WASP activation is also regulated by interaction with several other proteins including the WASP-interacting protein (WIP), Sh3 domain proteins (e.g. Nek) and Srk family kinases (e.g., Hck). We have employed gene-targeting to generate mice deficient for the Rho-family GTPase Cdc42 as well as several WASP family members: WASP, N-WASP, and WAVE-2. N-WASP- and WAVE-2- deficiencies result in early embryonic lethality whereas WASP-deficient (WKO) mice are viable and fertile, with lymphocytes that develop normally but have signaling and cytoskeletal abnormalities. We have also generated ES cells clones that contained an activated WASP knock-in mutation associated with the recently described novel immunodeficiency, X-linked Neutropenia (XLN). Because N-WASP and WAVE-2 deficiencies were not viable, selective targetings of these alleles were required to assess the role of these proteins in lymphocytes. To this end, we have generated mice with N-WASP that can be conditionally inactivated in leukocytes, and we are currently generating mice with similar mutations in WAVE2. We have generated WASP/N-WASP double knock-out (DKO) mice in T cells and, in collaboration with Dr. Raif Geha (Project 1), WASP/WIP DKO mice; both strains are associated with more severe signaling and cytoskeletal abnormalities in T cells when compared to WKO mice. This collection of novel reagents, in which the various WASP-family members can be inactivated, singularly or in combination and in either cells or mice, provides a powerful basis for our ongoing goals of dissecting the roles of WASP family members in leukocyte function. We propose: A1) To dissect the unique role of WASP and the combined role of WASP/N-WASP in leukocyte development and function. A2) To define the role of constitutively-active mutations in WASP function in T cells.