Movement of Ezrin and CD43 During T Cell Signaling

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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.