RESUMEN
Although all three Vav family members are expressed in T lymphocytes, the role that Vav3 plays in T cell activation is poorly defined. Here we show that, like Vav1, Vav3 undergoes rapid tyrosine phosphorylation after T cell receptor (TCR) cross-linkage and interacts with the adaptor molecules SLP76 and 3BP2 in a SH2-dependent manner. However, depletion of Vav1 but not Vav3 protein by RNA interference affects TCR-mediated IL-2 promoter activity. In contrast, Vav3 function is specifically required for coupling TCR stimulation to serum response element-mediated gene transcription. These data indicate that, although both Vav proteins are biochemically coupled to the TCR, they regulate distinct molecular pathways leading to defined gene transcriptional events.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas Oncogénicas/inmunología , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/inmunología , Regulación de la Expresión Génica/inmunología , Factores de Intercambio de Guanina Nucleótido , Humanos , Células Jurkat , Proteínas Proto-Oncogénicas/inmunología , Proteínas Proto-Oncogénicas c-vav , Transcripción Genética/inmunologíaRESUMEN
RhoG is a low-molecular-weight GTPase highly expressed in lymphocytes that activates gene transcription and promotes cytoskeletal reorganization in vitro. To study the in vivo function of RhoG, we generated mice homozygous for a targeted disruption of the RhoG gene. Despite the absence of RhoG, the development of B and T lymphocytes was unaffected. However, there was an increase in the level of serum immunoglobulin G1 (IgG1) and IgG2b as well as a mild increase of the humoral immune response to thymus-dependent antigens. In addition, B- and T-cell proliferation in response to antigen receptor cross-linking was slightly increased. Although RhoG deficiency produces a mild phenotype, our experiments suggest that RhoG may contribute to the negative regulation of immune responses. The lack of a strong phenotype could indicate a functional redundancy of RhoG with other Rac proteins in lymphocytes.
Asunto(s)
GTP Fosfohidrolasas/deficiencia , GTP Fosfohidrolasas/inmunología , Animales , Antígenos/administración & dosificación , Apoptosis/efectos de la radiación , Linfocitos B/citología , Linfocitos B/enzimología , Linfocitos B/inmunología , Diferenciación Celular , Citocinas/biosíntesis , GTP Fosfohidrolasas/genética , Rayos gamma , Inmunoglobulina G/sangre , Técnicas In Vitro , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Sistema Nervioso/anatomía & histología , Sistema Nervioso/enzimología , Fenotipo , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/citología , Linfocitos T/enzimología , Linfocitos T/inmunología , Linfocitos T/efectos de la radiación , Proteínas de Unión al GTP rhoRESUMEN
RhoG, a member of the Rho family of GTPases, has been implicated as a regulator of the actin cytoskeleton. In this study, we show a novel function for the small GTPase RhoG on the regulation of the interferon-gamma promoter and nuclear factor of activated T cells (NFAT) gene transcription in lymphocytes. Optimal function of RhoG for the expression of these genes requires a calcium signal, normally provided by the antigen receptor. In addition, RhoG potentiation of NFAT requires the indirect activity of Rac and Cdc42; however, pathways distinct from those activated by Rac and Cdc42 mediate RhoG activation of NFAT-dependent transcription. Using effector domain mutants of RhoG we found that its ability to potentiate NFAT-dependent transcription correlates with its capacity to increase actin polymerization, supporting the suggestion that NFAT-dependent transcription is an actin-dependent process. RhoG also promotes T-cell spreading on fibronectin, a property that is independent of its ability to enhance NFAT-dependent transcription. Hence, these results implicate RhoG in leukocyte trafficking and the control of gene expression induced in response to antigen encounter.
Asunto(s)
Actinas/metabolismo , Proteínas de Ciclo Celular , Citoesqueleto/metabolismo , GTP Fosfohidrolasas/metabolismo , Linfocitos/fisiología , Proteínas Nucleares , Actinas/ultraestructura , Linfocitos B/fisiología , Linfocitos B/ultraestructura , Calcio/metabolismo , Células Cultivadas , Citoesqueleto/ultraestructura , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Fibronectinas/metabolismo , GTP Fosfohidrolasas/genética , Regulación de la Expresión Génica , Humanos , Interferón gamma/genética , Células Jurkat , Leucocitos/citología , Leucocitos/metabolismo , Linfocitos/citología , Linfocitos/ultraestructura , Mutación , Factores de Transcripción NFATC , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-vav , Transducción de Señal , Linfocitos T/fisiología , Linfocitos T/ultraestructura , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo , Proteínas de Unión al GTP rhoRESUMEN
Mice lacking all 3 Vav proteins fail to produce significant numbers of recirculating follicular or marginal zone B cells. Those B cells that do mature have shortened lifespans. The constitutive nuclear factor-kappaB (NF-kappaB) activity of resting naive B cells required Vav function and expression of cellular reticuloendotheliosis (c-Rel). Rel-A was reduced in Vav-deficient B cells. Furthermore, expression of the NF-kappaB-regulated antiapoptotic genes A1 and Bcl-2 was reduced in mature Vav-deficient B cells. Overexpression of Bcl-2 restored the number of mature follicular B cells in the spleens of Vav-deficient mice. When activated by B-cell receptor (BCR) cross-linking, Vav-deficient B cells failed to activate NF-kappaB. Vav proteins thus regulate an NF-kappaB-dependent survival signal in naive B cells and are required for NF-kappaB function after BCR cross-linking.
Asunto(s)
Linfocitos B/citología , Regulación de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/fisiología , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-vav/fisiología , Animales , Apoptosis , Western Blotting , Células de la Médula Ósea/citología , Bromodesoxiuridina/farmacología , Núcleo Celular/metabolismo , Proliferación Celular , Separación Celular , Supervivencia Celular , Colorantes/farmacología , Reactivos de Enlaces Cruzados/farmacología , Regulación hacia Abajo , Citometría de Flujo , Factores de Intercambio de Guanina Nucleótido/genética , Ratones , Mutación , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-rel/metabolismo , Proteínas Proto-Oncogénicas c-vav/genética , Receptores de Antígenos de Linfocitos B/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Bazo/citología , Factores de Tiempo , Factor de Transcripción ReIA/metabolismo , TransgenesRESUMEN
B cells from phospholipase C (PLC)gamma2-deficient mice express reduced levels of the pro-survival protein Bcl-2 and show a defect in the development of transitional T3 and marginal zone (MZ) B cells that reflects reduced B cell survival. Introduction of a bcl-2 transgene restored the numbers of MZ, T3 and follicular B cells in PLCgamma2(-/-) mice. Restricting the B cell repertoire in PLCgamma2-deficient mice by the introduction of a BCR transgene resulted in a striking reduction in the number of IgM-positive B cells and a paucity of IgD-expressing cells in the spleen which was also rescued by the bcl-2 transgene. BCR-stimulated ERK and IkappaBalpha phosphorylation were PLCgamma2 dependent, while calcium flux was reduced, but not abrogated, in the absence of PLCgamma2, suggesting an ancillary role for PLCgamma1. The bcl-2 transgene rescued development of PLCgamma2(-/-) B cells and serum IgM levels but did not restore BCR-mediated signaling, proliferation or serum IgG3 levels. These data suggest that PLCgamma2 performs a critical role in B cell development through regulation of survival rather than differentiation.