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). LAT is a 36-38kD integral membrane protein. We have been able to purify this molecule, and have cloned the cDNA that encodes it. We 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. Two independently derived mutants of the Jurkat human T cell line have been shown to be deficient in LAT. TCR crosslinking in these cells fails to result in any indication of T cell activation. However, reconstitution of these cells with LAT completely restores TCR-mediated activation, thus demonstrating the importance of this molecule for TCR signaling. LAT-deficient mice have also been 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. Current studies on LAT function 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. 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 has been followed and the dynamics of this process is under study.
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