RESUMO
The decision between T cell activation and tolerance is governed by the spatial and temporal integration of diverse molecular signals and events occurring downstream of TCR and costimulatory or coinhibitory receptor engagement. The PI3K-protein kinase B (PKB; also known as Akt) signaling pathway is a central axis in mediating proximal signaling events of TCR and CD28 engagement in T cells. Perturbation of the PI3K-PKB pathway, or the loss of negative regulators of T cell activation, such as the E3 ubiquitin ligase Cbl-b, have been reported to lead to increased susceptibility to autoimmunity. In this study, we further examined the molecular pathway linking PKB and Cbl-b in murine models. Our data show that the protein kinase GSK-3, one of the first targets identified for PKB, catalyzes two previously unreported phosphorylation events at Ser476 and Ser480 of Cbl-b. GSK-3 inactivation by PKB abrogates phosphorylation of Cbl-b at these two sites and results in reduced Cbl-b protein levels. We further show that constitutive activation of PKB in vivo results in a loss of tolerance that is mediated through the downregulation of Cbl-b. Altogether, these data indicate that the PI3K-PKB-GSK-3 pathway is a novel regulatory axis that is important for controlling the decision between T cell activation and tolerance via Cbl-b.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Quinase 3 da Glicogênio Sintase/fisiologia , Tolerância Imunológica/fisiologia , Ativação Linfocitária/fisiologia , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Subpopulações de Linfócitos T/enzimologia , Sequência de Aminoácidos , Animais , Autoimunidade/fisiologia , Ativação Enzimática , Regulação da Expressão Gênica/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fosfatidilinositol 3-Quinases/fisiologia , Fosforilação , Fosfosserina/metabolismo , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/fisiologia , Alinhamento de Sequência , Transdução de Sinais/fisiologia , Especificidade da Espécie , Organismos Livres de Patógenos Específicos , Subpopulações de Linfócitos T/imunologiaRESUMO
Inflammation following tissue damage promotes lymphocyte recruitment, tissue remodeling, and wound healing while maintaining self tolerance. Endogenous signals associated with tissue damage and cell death have been proposed to initiate and instruct immune responses following injury. In this study, we have examined the effects of elevated levels of a candidate endogenous danger signal, heat shock cognate protein 70 (hsc70), on stimulation of inflammation and autoimmunity following cell damage. We find that damage to pancreatic beta cells expressing additional cytosolic hsc70 leads to an increased incidence of diabetes in a transgenic mouse model. Steady-state levels of activated APC and T cell populations in the draining lymph node were enhanced, which further increased following streptozotocin-induced beta cell death. In addition, proinflammatory serum cytokines, and lymphocyte recruitment were increased in hsc70 transgenic mice. Islet Ag-specific T cells underwent a greater extent of proliferation in the lymph nodes of mice expressing hsc70 following beta cell damage, suggesting elevated Ag presentation following release of Ag in the presence of hsc70. These findings suggest that an elevated content of hsc70 in cells undergoing necrotic or apoptotic cell death can increase the extent of sterile inflammation and increase the susceptibility to autoimmunity.
Assuntos
Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Experimental/patologia , Proteínas de Choque Térmico HSC70/biossíntese , Proteínas de Choque Térmico HSC70/genética , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/patologia , Animais , Doenças Autoimunes/epidemiologia , Doenças Autoimunes/genética , Doenças Autoimunes/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Bovinos , Morte Celular/genética , Morte Celular/imunologia , Movimento Celular/genética , Movimento Celular/imunologia , Citosol/imunologia , Citosol/metabolismo , Diabetes Mellitus Experimental/epidemiologia , Diabetes Mellitus Experimental/genética , Modelos Animais de Doenças , Incidência , Ilhotas Pancreáticas/metabolismo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , RatosRESUMO
T lymphocyte activation signals regulate the expression and transactivation function of retinoid X receptor (RXR) alpha through an interplay of complex signaling cascades that are not yet fully understood. We show that cellular Ser/Thr protein phosphatases (PPs) play an important role in mediating these processes. Inhibitors specific for PP1 and PP2A decreased basal expression of RXR alpha RNA and protein in T lymphocyte leukemia Jurkat cells and prevented activation-induced RXR alpha accumulation in these cells. In addition, these inhibitors attenuated the RXR responsive element (RXRE)-dependent transcriptional activation in transient transfection assays. Inhibitors of calcineurin (CN), by contrast, did not have any effect on the basal RXR alpha expression and even augmented activation-induced RXR alpha expression. Expression of a dominant-active (DA) mutant of CN together with a DA mutant of protein kinase C (PKC)theta;, a novel PKC isoform, significantly increased RXRE-dependent transcription. Expression of catalytically inactive PKC theta; or a dominant-negative mutant of PKC theta; failed to synergize with CN and did not increase RXRE-dependent transcription. Expression of a DA mutant of PKC alpha or treatment with PMA was found to attenuate PKC theta; and CN synergism. We conclude that PP1, PP2A, and CN regulate levels and transcriptional activation function of RXR alpha in T cells. In addition, CN synergizes with PKC theta; to induce RXRE-dependent activation, a cooperative function that is antagonized by the activation of the conventional PKC alpha isoform. Thus, PKC theta; and PKC alpha may function as positive and negative modulators, respectively, of CN-regulated RXRE-dependent transcription during T cell activation.