The objective of the proposed project is to elucidate the identity and precise order of protein phosphorylation events which determine in part, if not completely, the fate of T cells exposed to stimulatory conditions which result in different phenotypic traits. Multiple types of cell surface receptors sense changes in the extracellular environment via interaction with their complementary ligands. A frequent result is the signal-induced, reversible, site-specific phosphorylation of selected cellular proteins. Integration of the phosphorylation signals induced by different receptors is hypothesized to be a determinant of control of the physiological consequences of receptor-ligand interactions. The study of the response of T cells to altered peptide ligands is a model system in experimental immunology that has already provided evidence that a T cell, through its receptors, can interpret subtle structural changes in receptor ligands and respond with phenotypes as fundamentally different as cell proliferation and anergy. The identity and sequence of induction of phosphorylation events which correlate with the control of a proliferative or anergic T cell response will be further elucidated by a unique combination of mass spectrometry and protein engineering. First, novel mass spectrometry-based technology will be used to localize phosphorylated residues within polypeptide chains at unprecedented sensitivity. Second, proteins with specificity for a particular phosphopeptide sequence will be engineered and selected from phage displaying a degenerate library of phosphopeptide-binding proteins. These reagents will be used to quantitatively probe the concurrent changes in the state of phosphorylation of signaling proteins which correlate with a distinctive cell fate. An understanding of the biochemical events which control the fate of T cells is of intense interest for disciplines as diverse as experimental immunology, autoimmune disease and cancer. Both the results obtained by this study and the experimental approach based on emerging technologies will add significantly to the understanding of the mechanisms which control fundamental processes such as differentiation, cell proliferation and anergy.
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