Movement of Ezrin and CD43 During T Cell Signaling

在T细胞与抗原前体细胞相互作用的过程中，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。虽然细胞表面蛋白和信号分子在T细胞：APC界面的浓度引起了人们的极大兴趣，但其他蛋白质从界面上的主动去除也起到了重要作用。从细胞：细胞接触部位移除的一种蛋白质是CD43，一种被认为是T细胞激活障碍的高度糖化的大蛋白质。我们的数据表明，CD43和其他蛋白质从细胞：细胞接触部位的移除是由Ezrin介导的，Ezrin是一种将细胞表面蛋白与许多细胞类型的肌动蛋白细胞骨架联系起来的蛋白质。这项建议侧重于阐明在T细胞极化过程中调节Ezrin和CD43运动的机制。在目标1中，CD43不能与Ezrin相互作用的突变体和Ezrin的显性阴性突变体将在T细胞中表达，以询问CD43是否需要与Ezrin相互作用才能运动。Ezrin和CD43从T细胞：APC接触部位移开的机制将通过可视化活的T细胞中这些蛋白质的GFP和RFP标记版本的运动来评估。最后，还将讨论Ezrin还控制其他货物蛋白运动的可能性。在目标2中，将测试Ezrin和CD43突变体对偶联形成、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，并将研究CD43作为T细胞激活的可移动屏障的假设。在目标3中，将研究Ezrin运动的调节。PKCtheta和Rho-Kinase的抑制剂将被测试它们抑制Ezrin磷酸化和运动的能力，Ezrin点突变将被用来确定参与激活、寡聚和货物结合的分子上的关键调控位点。综上所述，这些研究将解决Ezrin、CD43和其他蛋白质从T细胞：APC接触部位被移除的机制，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.