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. We previously cloned the cDNA that encodes LAT and have performed studies to characterize how LAT is phosphorylated and binds a number of critical signaling molecules, thus bringing these adaptor molecules and enzymes to the plasma membrane in the vicinity of the activated TCR. Biochemical and biophysical techniques are currently employed to study the physical characteristics of LAT-based signaling complexes. LAT-deficient mice were generated using standard gene targeting procedures. T cell development in these mice is blocked at an early stage within the thymus. Thus this complex developmental pathway is also dependent on LAT. More recent studies on LAT function in vivo focus on the role of individual tyrosine residues. Tyrosine to phenylalanine mutations have been introduced into the murine germline. These studies reveal that within the LAT molecule the four distal tyrosines are required for normal T cell development. Interestingly when one particular tyrosine at position 136 is replaced by phenylalanine, thymocyte developed is partially blocked and a striking immunoproliferative disease develops within a month of birth. These mice are being using to study the mechanism of positive and negative selection. 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. Their recruitment to the site of T cell activation and formation of signaling clusters has been followed. Extensive analyses of the regulation of signaling complexes are underway. Molecules regulating actin polymerization and calcium homeostasis are under investigation using these methods.
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