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. 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. We have added a major new element to our recent discovery that chemokine rapidly induces moesin dephosphorylation in T-lymphocytes. We demonstrate that the small G-protein Rac1 plays a fundamental role in mediating the global T-lymphocyte response to chemokine, and specifically it mediates chemokine-induced microvillar breakdown. The supporting evidence includes: first, chemokine induces Rac1 activation within 5 s via a signaling pathway that involves Galpha(i). Second, constitutively active Rac1 mediates microvilli disintegration. Third, blocking Rac1 function by cell permeant C-terminal """"""""Trojan"""""""" peptides corresponding to Rac1 (but not Rac2, Rho, or Cdc42) blocks microvillar loss induced by the chemokine stromal cell-derived factor 1alpha (SDF-1alpha). Furthermore, we demonstrate that the molecular mechanism of Rac1 action involves dephosphorylation-induced inactivation of the ezrin/radixin/moesin (ERM) family of actin regulators; such inactivation is known to detach the membrane from the underlying actin cytoskeleton, thereby facilitating disassembly of actin-based peripheral processes. Specifically, ERM dephosphorylation is induced by constitutively active Rac1 and stromal cell-derived factor 1alpha-induced ERM dephosphorylation is blocked by either the dominant negative Rac1 construct or by Rac1 C-terminal peptides. Importantly, the basic residues at the C terminus of Rac1 are critical to Rac1's participation in ERM dephosphorylation and in microvillar retraction. Together, these data elucidate new roles for Rac1 in early signal transduction and cytoskeletal rearrangement of T lymphocytes responding to chemokine. Our current evidence indicates that Rac1 is not one of multiple parallel signaling pathways, but rather that Rac1 is singularly important in mediating diverse downstream effects of chemokine signaling. Protein phosphorylation is a central part of normal cell function as well as to carcinogenesis / metastasis. We have made major progress in characterizing fundamental processes that regulate kinase specificity, including: the precise peptide-specificity of AGC kinases, and the control of their recruitment. Despite hundreds of protein kinases in a cell, phosphorylation of any particular site is mediated by at most a few kinases. Such specificity of phosphorylation is achieved in part by preferences of different kinases for different patterns of amino acids surrounding the phosphorylation site, i.e. peptide specificity. We have developed novel synthetic-peptide-based strategies to characterize more precisely than previously possible the peptide specificity of basophilic kinases. These determinations have allowed us to make much accurate predictions than previously possible of phosphorylation sites for PKC isoforms (80-90% specificity and sensitivity). Of particular importance, analysis of 124 experimentally determinedPKC sites from the literature demonstrates a very strong role of peptide specificity in many of those sites. One important proof of principal of these predictive approaches is our prediction/validation of a functionally important phosphorylation site on the tyrosine phosphatase SHP-1, which is an important negative regulator of diverse signaling pathways in hematopoietic cells. Our studies demonstrate that this site is phosphorylated early TCR-mediated activation of lymphocytes. Moreover, phosphorylation of this site prevents nuclear translocation of SHP-1. We have demonstrated that PKC-theta phosphorylates SHP-1 in vitro, and in co-transfected cells and regulates its nuclear translocation.
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