We seek to understand the molecular basis of early activation events in T-lymphocytes in response to physiologically important stimuli; the functional responses most important to our research are T-lymphocyte cytoskeletal reorganization, adhesion, migration, and gene transcription. We emphasize analysis of: 1) changes in serine/threonine phosphorylation as key signaling events which regulate these processes, and 2) the serine/threonine kinases that mediate such phosphorylation. One major project is to understand the spatial reorganization during chemokine-induced polarization of peripheral blood T cells (PBT) and to elucidate the biochemical basis for that reorganization using a combination of microscopic, biochemical and molecular genetic approaches. Of particular importance we have demonstrated that chemokines induce an acute change in the balance between serine-threonine kinases and phosphatases, which we find is required for moesin dephosphorylation, for loss of microvilli and for polarization. This promises to be a major novel signaling element in chemokine signaling; we are exploring the kinases/phosphatases involved and the signaling pathways coupling them to G-protein-coupled receptors. Phosphorylation cascades are critical both to antigen-specific activation of T cells and to chemokine-mediated stimulation. We are investigating serine/threonine phosphorylation in lymphocytes, since it is much less well understood than tyrosine phosphorylation. Mechanism-based understanding of these processes will require detailed knowledge of the kinases involved. The protein kinase C family of serine/threonine kinases has been repeatedly implicated in lymphocyte adhesion and cytoskeletal reorganization, but there is little precise understanding of mechanisms. We have undertaken a systematic structure-function analysis of PKC-theta and have begun to extend that analysis to structure and function of other serine/threonine kinases which have a close evolutionary relationship to theta and are expressed in lymphocytes. We have previously identified three regulatory phosphorylation sites on PKC-theta and now have identified two new ones, whose functional significance is under investigation. Moreover we are investigating in detail the peptide specificity of phosphorylation by different PKC isoforms. Our findings differ strikingly from the prevailing view of PKC specificity, showing greater similarity than expected between some and greater difference than expected between others. We have constructed homology models from which to predict and test hypothesis regarding the structural basis for such similarities and differences. Early results are beginning to confirm our hypothesis that exploiting this understanding will help us decipher the complex phosphorylation cascades involved in T-lymphocyte activation.
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