RESUMO
Polarization of lipid rafts and granules to the site of target contact is required for the development of cell-mediated killing by cytotoxic lymphocytes. We have previously shown that these events require the activation of proximal protein tyrosine kinases. However, the downstream intracellular signaling molecules involved in the development of cell-mediated cytotoxicity remain poorly defined. We report here that a RhoA/ROCK/LIM-kinase axis couples the receptor-initiated protein tyrosine kinase activation to the reorganization of the actin cytoskeleton required for the polarization of lipid rafts and the subsequent generation of cell-mediated cytotoxicity. Pharmacologic and genetic interruption of any element of this RhoA/ROCK/LIM-kinase pathway inhibits both the accumulation of F-actin and lipid raft polarization to the site of target contact and the subsequent delivery of the lethal hit. These data define a specialized role for a RhoA-->ROCK-->LIM-kinase pathway in cytotoxic lymphocyte activation.
Assuntos
Citotoxicidade Imunológica , Proteínas Quinases/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Linfócitos T Citotóxicos/imunologia , Proteína rhoA de Ligação ao GTP/fisiologia , Actinas/metabolismo , Amidas/farmacologia , Animais , Adesão Celular , Polaridade Celular , Células Cultivadas , Testes Imunológicos de Citotoxicidade , Inibidores Enzimáticos/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Quinases Lim , Ativação Linfocitária , Microdomínios da Membrana/ultraestrutura , Camundongos , Mutação , Inibidores de Proteínas Quinases , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Piridinas/farmacologia , Transdução de Sinais , Linfócitos T Citotóxicos/ultraestrutura , Células Tumorais Cultivadas , Quinases Associadas a rho , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP), specifically binds to a region of WASp that is frequently mutated in Wiskott-Aldrich syndrome. Due to the similar phenotypes of WASp- and Vav-deficient T cells, and the putative importance of the WIP/WASp complex in mediating normal signals from the TCR, we investigated the role of WIP in regulating NF-AT/AP-1-mediated gene transcription. We show that WIP has the ability to enhance Vav-mediated activation of NF-AT/AP-1 gene transcription. In addition, we provide evidence that the interaction of WIP with WASp is necessary, but not sufficient for the ability of WIP to regulate NF-AT/AP-1 activity. Finally, we have identified a region in WIP required for its regulation of NF-AT/AP-1 activity. Our data suggests that the WIP-WASp interaction is important for NF-AT/AP-1-mediated gene transcription, and that defects seen in the activation of T cells from WAS patients may be due to the inability of these cells to form a functional WIP/WASp-signaling complex.
