Movement of Ezrin and CD43 During T Cell Signaling

在T细胞与抗原提呈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布，从而影响T细胞信号转导的结果。 虽然在T细胞：APC界面上的细胞表面蛋白和信号分子的浓度一直很感兴趣，但从界面上主动去除其他蛋白也起着重要作用。 从细胞中移除的一种蛋白质：细胞接触位点是CD43，一种被认为是T细胞活化屏障的大的高度糖基化蛋白质。我们的数据表明，CD43和其他蛋白质从细胞中的去除：细胞接触位点是由ezrin介导的，ezrin是一种在许多细胞类型中将细胞表面蛋白质连接到肌动蛋白细胞骨架的蛋白质。 该提案的重点是阐明负责调节ezrin和CD43在T细胞极化过程中的运动的机制。 在目的1中，不能与ezrin相互作用的CD43突变体和ezrin的显性阴性突变体将在T细胞中表达，以询问CD43移动是否需要与ezrin相互作用。 ezrin和CD43从T细胞：APC接触位点移开的机制将通过观察活T细胞中这些蛋白质的GFP和RFP标记形式的移动来评估。 最后，ezrin也控制其他货物蛋白的运动的可能性将得到解决。 在目的2中，将测试ezrin和CD43突变体对缀合物形成、肌动蛋白重塑和SMAC形成的影响。 还将在表达ezrin突变体的细胞中测试T细胞活化的早期事件，包括酪氨酸磷酸化、Ca++通量和活化标志物的表达，并将研究CD43作为T细胞活化的可移动屏障的假设。 在目标3中，将研究ezrin运动的调节。 将测试PKC θ和Rho激酶的抑制剂抑制ezrin磷酸化和移动的能力，并且ezrin点突变体将用于鉴定参与活化、寡聚化和货物结合的分子上的关键调控位点。 总之，这些研究将解决ezrin，CD43和其他蛋白质从T细胞：APC接触位点去除的机制，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.