We seek to understand the spatial reorganization during chemokine-induced lymphocyte polarization and elucidate the biochemical basis for that reorganization using a combination of microscopic, biochemical and molecular genetic approaches. We study cytoskeleton primarily in purified human peripheral blood T cells which are the biologically relevant cell type and have a distinctive differentiation state not well modeled by in vitro cell lines. This fills an important gap because, due to their small size and other technical considerations, PBT have not been well studied by cell biologists. Our studies elucidate a major participant in cytoskeletal organization of lymphocytes that has been largely ignored: vimentin intermediate filaments. We have discovered a cage-like organization of vimentin intermediate filaments in resting T cells and demonstrate that this system is a major contributor to lymphocyte resistance to deformation, previously ascribed to other filament systems. We have discovered that plectin, best known in epithelial cells, is expressed in lymphocytes and that this huge protein is a key contributor to their intermediate filament organization. Furthermore we demonstrate that Vimentin, Plectin and Fodrin are physically interconnected in lymphocytes (therefore a VPF assembly) and condense into the uropod within 1 min of chemokine stimulation; this assembly spans all the way from the nucleus (constitutive lamin B association) to the plasma membrane (where fodrin localizes), filling in an important piece in the tensegrity model of cellular architecture. We have come to appreciate the central role played by serine/ threonine phosphorylation cascades in cytoskeletal reorganization. Understanding individual phosphorylation / dephosphorylation events is a powerful way to dissect this complex process, as illustrated by our studies of moesin phospho-Thr-558 (pT558), one of 5 phosphorylation sites we have identified in the chemokine response. Microvilli are critical for leukocyte-endothelial adhesion; their loss precedes transmigration, but the molecular basis was unknown. We hypothesized that chemokine stimulation would induce rapid loss of microvilli, and that regulation of moesin, a key component of microvilli, would be involved. Our studies confirm that prediction and implicate chemokine-induced dephosphorylation of pT558 as the cause of microvillus loss and of moesin dissociation from the cytoskeleton. This is the first such precise identification in lymphocyte responses to chemokines of an early phosphorylation event and its molecular consequences. Serine/threonine phosphorylation cascades are critical both to cytoskeletal reorganization and to antigen-specific stimulation of T cells. Mechanism-based understanding of these processes depends on understanding 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. Although PKC-theta has become a 'superstar' for its role in T cell receptor signaling, we predicted that PKC-theta would have broader relevance, including regulation of cytoskeleton. We have undertaken a systematic structure-function analysis of PKC-theta. Our approach emphasizes careful analysis of evolutionary sequence conservation and mutagenesis-based hypothesis testing. Our studies reveal important functional similarities with PKC-delta and provide new insights into the roles of PKC-theta in receptor-mediated stimulation of hematopoietic cells. For example, we demonstrate that PKC-theta is expressed in mast cells, and participates in their FcERI-mediated signal transduction. Moreover, our recent studies implicate PKC-theta in the regulation of moesin dephosphorylation described above. We have established an informative set of functional assays relevant to T lymphocytes and a number of novel structural hypotheses; testing our hypotheses in these functional assays promises to elucidate the structural basis for PKC-theta's functions in T cells. Our studies demonstrate that PKC-theta is phosphorylated at its activation loop, its turn motif and its hydrophobic motif. Functional analysis of the importance of this phosphorylation demonstrates that activation loop is essential for kinase activity and for in vivo NFkB induction by CD3/CD28, while turn motif phosphorylation plays a negative regulatory role in NFkB induction in vivo.
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