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1.
J Recept Signal Transduct Res ; 30(6): 430-43, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20945976

RESUMEN

CONTEXT: Although ZAP-70 is required for T-cell development, it's unclear how this kinase controls both positive and negative selection. OBJECTIVE AND METHODS: Using OT-I pre-selection thymocytes and a panel of peptide major histocompatibility complex (pMHC) ligands of defined affinity, the recruitment, phosphorylation and activity of ZAP-70 was determined at the interface with antigen-presenting cells (APCs). RESULTS: pMHC ligands promoting negative selection induce a discrete elevation of ZAP-70 recruitment, phosphorylation and enzymatic activity in the thymocyte:APCs interface. DISCUSSION: The quantity of ZAP-70 kinase activity per cell is a key parameter controlling the fate of a developing thymocyte since partial inhibition of ZAP-70 kinase activity converted negative into positive selection. Surprisingly, the amount of ZAP-70 enzymatic activity observed during negative selection is not controlled by differential phosphorylation of the ZAP-70 protein but rather by the total amount of T-cell receptor and co-associated ZAP-70 recruited to the thymocyte:APC interface. CONCLUSIONS: These data provide evidence that a burst of ZAP-70 activity initiates the signaling pathways for negative selection.


Asunto(s)
Activación de Linfocitos/inmunología , Transducción de Señal/inmunología , Linfocitos T/inmunología , Proteína Tirosina Quinasa ZAP-70/metabolismo , Animales , Células Presentadoras de Antígenos/inmunología , Ligandos , Complejo Mayor de Histocompatibilidad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/fisiología , Proteína Tirosina Quinasa ZAP-70/genética
2.
Curr Top Microbiol Immunol ; 334: 31-46, 2009.
Artículo en Inglés | MEDLINE | ID: mdl-19521680

RESUMEN

The use of appropriate fluorescent proteins has allowed the use of FRET microscopy for investigation of intermolecular interactions in living cells. This method has the advantage of both being dynamic and of working at the subcellular level, so that the time and place where proteins interact can be visualized. We have used FRET microscopy to analyze the interactions between the T cell antigen receptor and the coreceptors CD4 and CD8. This chapter reviews data on how these coreceptors are recruited to the immunological synapse, and how they interact when the T cell is stimulated by different ligands.


Asunto(s)
Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/inmunología , Animales , Presentación de Antígeno , Humanos , Nanotubos , Linfocitos T/metabolismo
3.
J Immunol ; 180(12): 8211-21, 2008 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-18523287

RESUMEN

The CD8 coreceptor contributes to the recognition of peptide-MHC (pMHC) ligands by stabilizing the TCR-pMHC interaction and enabling efficient signaling initiation. It is unclear though, which structural elements of the TCR ensure a productive association of the coreceptor. The alpha-chain connecting peptide motif (alpha-CPM) is a highly conserved sequence of eight amino acids in the membrane proximal region of the TCR alpha-chain. TCRs lacking the alpha-CPM respond poorly to low-affinity pMHC ligands and are unable to induce positive thymic selection. In this study we show that CD8 participation in ligand binding is compromised in T lineage cells expressing mutant alpha-CPM TCRs, leading to a slight reduction in apparent affinity; however, this by itself does not explain the thymic selection defect. By fluorescence resonance energy transfer microscopy, we found that TCR-CD8 association was compromised for TCRs lacking the alpha-CPM. Although high-affinity (negative-selecting) pMHC ligands showed reduced TCR-CD8 interaction, low-affinity (positive-selecting) ligands completely failed to induce molecular approximation of the TCR and its coreceptor. Therefore, the alpha-CPM of a TCR is an important element in mediating CD8 approximation and signal initiation.


Asunto(s)
Antígenos CD8/metabolismo , Antígenos CD8/fisiología , Fragmentos de Péptidos/fisiología , Receptores de Antígenos de Linfocitos T alfa-beta/fisiología , Transducción de Señal/inmunología , Secuencias de Aminoácidos/genética , Animales , Antígenos CD8/química , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Línea Celular , Humanos , Hibridomas , Ratones , Ratones Noqueados , Ratones Transgénicos , Técnicas de Cultivo de Órganos , Fragmentos de Péptidos/química , Receptores de Antígenos de Linfocitos T alfa-beta/química , Transducción de Señal/genética , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo
4.
EMBO J ; 23(24): 4760-9, 2004 Dec 08.
Artículo en Inglés | MEDLINE | ID: mdl-15538381

RESUMEN

Butyrate response factor (BRF1) belongs to the Tis11 family of CCCH zinc-finger proteins, which bind to mRNAs containing an AU-rich element (ARE) in their 3' untranslated region and promote their deadenylation and rapid degradation. Independent signal transduction pathways have been reported to stabilize ARE-containing transcripts by a process thought to involve phosphorylation of ARE-binding proteins. Here we report that protein kinase B (PKB/Akt) stabilizes ARE transcripts by phosphorylating BRF1 at serine 92 (S92). Recombinant BRF1 promoted in vitro decay of ARE-containing mRNA (ARE-mRNA), yet phosphorylation by PKB impaired this activity. S92 phosphorylation of BRF1 did not impair ARE binding, but induced complex formation with the scaffold protein 14-3-3. In vivo and in vitro data support a model where PKB causes ARE-mRNA stabilization by inactivating BRF1 through binding to 14-3-3.


Asunto(s)
Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Estabilidad del ARN , ARN Mensajero/metabolismo , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Proteínas 14-3-3/metabolismo , Animales , Genes Reporteros , Insulina/metabolismo , Ratones , Células 3T3 NIH , Fosforilación , Unión Proteica , Proteínas Proto-Oncogénicas c-akt , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/metabolismo , Factores Asociados con la Proteína de Unión a TATA/genética
5.
EMBO J ; 21(17): 4709-18, 2002 Sep 02.
Artículo en Inglés | MEDLINE | ID: mdl-12198173

RESUMEN

To identify regulators of AU-rich element (ARE)-dependent mRNA turnover we have followed a genetic approach using a mutagenized cell line (slowC) that fails to degrade cytokine mRNA. Accordingly, a GFP reporter construct whose mRNA is under control of the ARE from interleukin-3 gives an increased fluorescence signal in slowC. Here we describe rescue of slowC by a retroviral cDNA library. Flow cytometry allowed us to isolate revertants with reconstituted rapid mRNA decay. The cDNA was identified as butyrate response factor-1 (BRF1), encoding a zinc finger protein homologous to tristetraprolin. Mutant slowC carries frame-shift mutations in both BRF1 alleles, whereas slowB with intermediate decay kinetics is heterozygous. By use of small interfering (si)RNA, independent evidence for an active role of BRF1 in mRNA degradation was obtained. In transiently transfected NIH 3T3 cells, BRF1 accelerated mRNA decay and antagonized the stabilizing effect of PI3-kinase, while mutation of the zinc fingers abolished both function and ARE-binding activity. This approach, which identified BRF1 as an essential regulator of ARE-dependent mRNA decay, should also be applicable to other cis-elements of mRNA turnover.


Asunto(s)
Regiones no Traducidas 3'/genética , Proteínas de Unión al ADN , Estabilidad del ARN , ARN Mensajero/metabolismo , Factor de Transcripción TFIIIB , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Células 3T3 , Animales , Factor 1 de Respuesta al Butirato , Clonación Molecular , Codón sin Sentido , Citocinas/genética , ADN Complementario/genética , Fibrosarcoma/química , Fibrosarcoma/patología , Mutación del Sistema de Lectura , Genes Reporteros , Prueba de Complementación Genética , Humanos , Proteínas Inmediatas-Precoces/análisis , Proteínas Inmediatas-Precoces/química , Ratones , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/aislamiento & purificación , Inhibidores de las Quinasa Fosfoinosítidos-3 , ARN Interferente Pequeño , ARN no Traducido/metabolismo , Proteínas de Saccharomyces cerevisiae , Relación Estructura-Actividad , Fracciones Subcelulares/química , Factores Asociados con la Proteína de Unión a TATA , Factores de Transcripción/aislamiento & purificación , Transfección , Tristetraprolina , Células Tumorales Cultivadas/química , Dedos de Zinc/genética
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