T CELL RECEPTOR COUPLED SIGNALING AND APOPTOSIS

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We have recently obtained evidence for a novel signalling pathway coupled to TCR ligation by MTV superantigen an high concentrations of peptide which leads to transient activation and apoptosis. This signalling cascade may be associated with MAP2 kinase-dependent activation of phospholipase A2 (PLA2) and lipoxygenase which in turn catalyzes the generation of reactive oxygen intermediates (R01s). We also outline evidence suggesting that this pathway may regulate gene expression via activation of th p50/52 component of the NF-kB complex. In Specific Aim 1, we propose experiments to systematically define the kinetics of phospholipase A2 (PLA2) activation of HPETE and release of hydroxyl radicals (OH). In Specific Aim 2, we propose to define the proximal events which couple the TCR to PLA2 activation. This will include an initial assessment of the role of MAP-2 kinase in PLA2 activation, followed by experimental analysis of the potential roles of p21 Ras-dependent pathways that may connect these events with TCR ligation. Delineation of this pathway will allow us to map the interaction between this pathway and CD28-dependent signals which inhibit the apoptotic response following TCR ligation. In Specific Aim 3, we propose experiments to further define the distal biochemical events which may result in activation of NF-kB. In Specific Aim 4, we will determine whether generation of hydroxyl radicals is necessary and sufficient for T-cell apoptosis or whether these represent intermediates in a signalling pathway which results in transcriptional activation of gene products responsible for cell death.