Engagement of multicomponent immunoreceptors such as the T cell antigen receptor (TCR) results in rapid activation of multiple protein tyrosine kinases (PTKs) including Lck, Fyn, ZAP-70 and Itk. These PTKs then phosphorylate a number of enzymes and adapter molecules involved in a complex signaling cascade. Our studies have focused on a critical substrate of the PTKs, LAT (linker for activation of T cells), a 36-38kD integral membrane protein. LAT is a critical transmembrane adaptor protein. We have performed studies to characterize how LAT is phosphorylated and binds a number of critical signaling molecules, thus bringing these other adaptor molecules and enzymes to the plasma membrane in the vicinity of the activated TCR. Biochemical, biophysical, genetic and microscopic techniques are currently employed to study the characteristics of LAT-based signaling complexes.Studies on LAT function in vivo have focused on the role of individual tyrosine residues of the molecule. Tyrosine to phenylalanine mutations have been introduced into the murine germline. Interestingly when one particular tyrosine at position 136 is replaced by phenylalanine, the enzyme phospholipase-C gamma is not activated, thymocyte development is partially blocked and a striking immunoproliferative disease ensues within a month of birth. These mice have been used to study how failure of phospholipase-C activation affects intrathymic development of T cells, and in particular, the processes of positive and negative selection.Members of the laboratory have studied LAT function by analyzing the binding of recombinant adapter molecules to phosphopeptides synthesized from the LAT sequence. Using state-of-the-art biophysical techniques we have demonstrated binding cooperativity in the interaction of LAT and LAT-binding proteins. Moreover we have demonstrated that LAT-based signaling complexes themselves can oligomerize. This result explains data observed in many signaling systems.In addition to specific studies of the LAT molecule the laboratory has developed new methods of visualizing T cell activation using confocal microscopy. Many of the signaling molecules involved in the early TCR-coupled activation process have been tagged with fluorescent markers and expressed in T cells. The group has used these methods to observe the process of assembly of signaling molecules into signaling clusters at the site of T cell activation. We have determined using imaging and biophysical methods that cluster formation is highly cooperative. New insights into the binding properties of phospholipase-C gamma and into the activation of actin polymerization have been reported. Additionally we have demonstrated that signaling complexes can be endocytosed in a clathrin-independent manner. This results suggests an additional mode of signal down-regulation following TCR activation.
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