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 a number of 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 protein. LAT is a critical transmembrane adapter protein. We have performed studies to characterize how LAT is phosphorylated and binds a number of 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 three studies that can be viewed as a continuation of previous work. We have already published several studies in which we generated and described mice with SOS1-deficient T cells. Two papers reported on the role of SOS1 in thymocyte development and one confirmed our model showing that SOS1 has both enzymatic and structural functions. This year we demonstrated that SOS1 deficiency in peripheral T cells does not affect Erk activation, but does result in an increase in Akt phosphorylation. We provided data showing that this increase was likely due to enhanced PI3-kinase recruitment to signaling complexes activated by the TCR. These biochemical changes resulted in impaired T cell migration. Our laboratory has extensively studied the LAT adapter molecule and in previous papers we have reported on LAT dynamics and the effect of ubiquitinylation-defect LAT mutant molecules. In T cell lines expression of these mutants enhanced T cell activation. This year we published a report on the transgenic expression of these mutations in normal murine T cells. We found again that T cell function in T cells expressing these mutations could be enhanced. Similarly several in vivo functions such as cytotoxic killing and antibody production were positively affected. However despite these findings, greater T cell effector function expression mediated by the mutant LAT molecules did not have an effect on clearance of certain pathogens or tumors. Our data also suggested that expression of these mutants increased T cell differentiation to a degree that some T functional responses were dampened. Mutation of one LAT tyrosine residue and generation of a knock-in mouse in which all the LAT molecules bear the mutant resulted in a lymphoproliferative disease. We have extensively studied this mouse to understand biochemical mechanisms that underlie the disease. We previously demonstrated that the microRNA, miR-155 is overexpressed as a consequence of the mutation. This year we published a study showing that the cross between the LAT knock-in mouse and a miR-155 deficient mouse resulted in a decrease in lymphoproliferative disease. We showed that the decrease was due to an enhanced level of BIM-mediated T cell apoptosis. Activation of BIM was mediated by elevated FOXO3, which in turn was impacted by two pathways, SHIP-1 regulation of Akt and PDK1 and Pak1-mediated JNK1 activation.
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