Although intriguing data have come to the forefront regarding a role for cytoskeleton in signal transduction in other cell systems, study of the these molecules in T-cells has lagged. The investigator's studies provide the first direct evidence that T-cell antigen receptor (TCR) polypeptides containing an activation motif (ITAM), including, but not limited to zeta chain, associate with actin cytoskeleton upon cell activation. This association was confirmed using chimeric constructs with deletions in zeta ITAMs, showing that these motifs are necessary for cytoskeletal association. The evidence that zeta cytoskeleton association is of physiological relevance are: 1) association is developmentally regulated; 2) motifs and critical tyrosines involved in signaling are also involved in zeta cytoskeleton association; and 3) disruption of zeta cytoskeletal association is correlated with decrease IL-2 production. These data have led the investigator to hypothesize that zeta complexes compartmentalize and anchor activated enzymes critical for T-cell signal transduction.
Specific Aim 1 will map the region of zeta that binds to actin, using T-cell hybridomas expressing mutated chimeric zeta chains. The region of interest are each of the three ITAMs which are necessary and sufficient for downstream signal transduction, and the two tyrosines in each ITAM, which are critical for binding SH2-containing PTKs. The investigator will use a novel in vitro assay to reconstitute zeta actin binding, an assay which preliminary data suggest recapitulated in vivo binding.
Specific Aim 2 will use the in vitro assay as well as immunoprecipitation, overlay immunoblots, in vitro kinase assays, and fishing experiments using chimeric zeta constructs in mutant and wild type murine and human T-cells or cell lines to identify signaling molecules and cytoskeletal proteins involved in zeta cytoskeleton binding.
Specific Aim 3 will address the physiologic relevance of zeta cytoskeleton association in T-cell activation, apoptosis, anergy, and development, using: 1) inhibitors of zeta cytoskeleton association and actin polymerization; 2) cell lines and transgenic mice expressing mutant receptor chains or signaling molecules; and 3) cell lines and transgenic mice expressing a particular agonist or antagonist MHC-peptide complex and its specific TCR. This research will provide critical insights into a largely unexplored area of T-cell signal transduction by addressing the role of cytoskeleton, specifically actin, as an active player in T-cell activation. The investigator will address the role of cytoskeleton in regulating signaling in situations where the TCR must serve a different function, e.g., under conditions of anergy (when signaling must be inhibited) or at particular stages of development (when signaling must determine survival). These data will have relevance to both autoimmunity and cancer in addressing questions of dysregulation of inhibitory and/or survival signals.
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