Biochemical Basis of T Cell Activation

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

Engagement of multicomponent immunoreceptors such as the T cell antigen receptor results in rapid recruitment and activation of multiple protein tyrosine kinases (PTKs) including Lck, Fyn, ZAP-70 and Itk. These PTKs then phosphorylate many enzymes and adapter molecules involved in complex signaling cascades. Our studies have focused on a critical substrate of the PTKs, LAT (linker for activation of T cells), a 36-38kD integral membrane adapter protein. We have performed studies to characterize how LAT is phosphorylated and then binds many critical signaling molecules, thus bringing other adapter molecules and enzymes in multimolecular complexes to the plasma membrane in the vicinity of the activated TCR. Biochemical, biophysical, microscopic and genetic techniques are currently employed to study the characteristics of LAT-based signaling complexes and the enzyme pathways that are coupled to and activated at LAT complexes. In the past year we have published studies that focus on different aspects of TCR-mediated signaling. The first was described briefly in the last annual report. We had already demonstrated that LAT-based complexes known as microclusters are formed upon T cell activation and are the site where signaling complexes are formed. We demonstrated that microclusters contain two spatially separate domains, one with the TCR and associated ZAP-70 PTK, and the other with LAT and LAT-bound signaling molecules. A kinetic analysis of the formation of the microclusters revealed that molecules are recruited in a step-wise fashion beginning with ZAP-70 recruitment to the TCR followed by LAT and other signaling proteins and then by molecules involved in microcluster dissociation. Current studies are expanding on these observations to understand other signaling events and to use these results to make new forms of clinically relevant chimeric antigen receptors. In the past year we have published a study that focuses on the functional effects of a critical signaling complex, which we have previously described. The critical enzyme phospholipase-C-gamma1 (PLC-gamma1), when activated cleaves the phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP2) in the plasma membrane of cells to produce diacylglycerol and inositol 3-phosphate (IP3). Both of these products are critical second messengers in activated T cells. In the activated T cells PLC-gamma1 is found in a complex with three adapter proteins, LAT, SLP-76 and Gads at the cell membrane. Previously we have reconstituted a signaling complex containing these four proteins and have demonstrated the binding parameters and thermodynamics for the formation of the tetrameric complex. In the past year we tested the effect of these proteins alone and in complex when they are formed on liposomes containing the appropriate PLC-gamma 1 lipid substrates. We assayed the product of PLC-gamma1 activity by measuring IP3 levels. We demonstrated that activation of the PLC-gamma1 is a cooperative process requiring its recruitment to the liposome membrane, the formation of the LAT-Gads-SLP-76-PLC-gamma1 tetrameric complex and the phosphorylation of a particular tyrosine residue in PLC-gamma1. These studies demonstrate the complex mechanism of activation and regulation of the enzyme.