Movement of Ezrin and CD43 During T Cell Signaling

在T细胞与抗原呈递细胞相互作用过程中，细胞骨架重塑驱动膜蛋白和信号分子的重新分配，从而影响T细胞信号转导的结果。尽管人们对T细胞APC界面上细胞表面蛋白和信号分子的浓度非常感兴趣，但从界面上主动去除其他蛋白也起着重要作用。从细胞接触部位移除的一种蛋白质是CD43，这是一种大的、高度糖基化的蛋白质，被认为是T细胞激活的屏障。我们的数据表明，CD43和其他蛋白质从细胞接触部位的去除是由ezrin介导的，ezrin是一种在许多细胞类型中将细胞表面蛋白与肌动蛋白细胞骨架连接起来的蛋白质。本研究的重点是阐明在T细胞极化过程中调控ezrin和CD43运动的机制。在Aim 1中，将在T细胞中表达不能与ezrin相互作用的CD43突变体和ezrin的显性负突变体，以询问CD43的运动是否需要与ezrin相互作用。ezrin和CD43从T细胞APC接触部位转移的机制将通过可视化这些蛋白的GFP和rfp标记版本在活T细胞中的移动来评估。最后，ezrin也控制其他货物蛋白运动的可能性将被解决。在Aim 2中，我们将测试ezrin和CD43突变体对偶联物形成、肌动蛋白重塑和SMAC形成的影响。T细胞激活的早期事件，包括酪氨酸磷酸化、钙离子通量和激活标记的表达，也将在表达ezrin突变体的细胞中进行测试，并且CD43作为T细胞激活的可移动屏障的假设将被研究。在Aim 3中，将研究ezrin运动的调节。PKCtheta和rho激酶抑制剂将测试其抑制ezrin磷酸化和运动的能力，ezrin点突变体将用于识别分子上参与激活、寡聚和货物结合的关键调控位点。综上所述，这些研究将解决ezrin、CD43和其他蛋白质从T细胞中移除的机制：APC接触位点，以及这种分子重排对T细胞功能的影响。

During the interaction of T cells with antigen pre4senting cells, cytoskeletal remodeling drives the redistribution of membrane proteins and signaling molecules, thereby affecting the outcome of T cell signaling. Although there has been much interest in the concentration of cell surface proteins and signaling molecules at the T cell: APC interface, the active removal of other proteins from the interface also plays an important role. One protein that is removed from the cell: cell contact site is CD43, a large, highly glycosylated protein thought to function as a barrier to T cell activation. Our data suggest that the removal of CD43 and other proteins from the cell: cell contact site is mediated by ezrin, a protein that links cell surface proteins to the actin cytoskeleton in many cell types. This proposal focuses on elucidating the mechanisms responsible for regulating the movement of ezrin and CD43 during T cell polarization. In Aim 1, mutants of CD43 incapable of interacting with ezrin and dominant negative mutants of ezrin will be expressed in T cells to ask if interaction with ezrin is required for CD43 movement. The mechanisms by which ezrin and CD43 are moved away from the T cell: APC contact site will be assessed by visualizing the movements of GFP and RFP-tagged versions of these proteins in living T cells. Finally, the possibility that ezrin also controls the movement of other cargo proteins will be addressed. In Aim 2, the effects of ezrin and CD43 mutants on conjugate formation, actin remodeling and SMAC formation will be tested. Early events of T cell activation, including tyrosine phosphorylation, Ca++ flux, and expression of activation markers will also be tested in cells expressing ezrin mutants, and the hypothesis that CD43 functions as a moveable barrier to T cell activation will be investigated. In Aim 3, the regulation of ezrin movement will be investigated. Inhibitors of PKCtheta and Rho-kinase will be tested for their ability to inhibit ezrin phosphorylation and movement, and ezrin point mutants will be used to identify the key regulatory sites on the molecule involved in activation, oligomerization and cargo binding. Taken together, these studies will address the mechanism by which ezrin, CD43 and other proteins are removed from the T cell: APC contact site, and the consequences of this molecular rearrangement for T cell function.