RESUMEN
Unlike the other MAP3Ks, MEKK1 (encoded by Map3k1) contains a PHD motif. To understand the role of this motif, we have created a knockin mutant of mouse Map3k1 (Map3k1(m) (PHD)) with an inactive PHD motif. Map3k1(m) (PHD) ES cells demonstrate that the MEKK1 PHD controls p38 and JNK activation during TGF-ß, EGF and microtubule disruption signalling, but does not affect MAPK responses to hyperosmotic stress. Protein microarray profiling identified the adaptor TAB1 as a PHD substrate, and TGF-ß- or EGF-stimulated Map3k1(m) (PHD) ES cells exhibit defective non-canonical ubiquitination of MEKK1 and TAB1. The MEKK1 PHD binds and mediates the transfer of Lys63-linked poly-Ub, using the conjugating enzyme UBE2N, onto TAB1 to regulate TAK1 and MAPK activation by TGF-ß and EGF. Both the MEKK1 PHD and TAB1 are critical for ES-cell differentiation and tumourigenesis. Map3k1(m) (PHD) (/+) mice exhibit aberrant cardiac tissue, B-cell development, testis and T-cell signalling.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Células Madre Embrionarias/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , MAP Quinasa Quinasa 4/metabolismo , Quinasa 1 de Quinasa de Quinasa MAP/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Ubiquitinación/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencias de Aminoácidos , Animales , Diferenciación Celular/fisiología , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Células Madre Embrionarias/citología , Factor de Crecimiento Epidérmico/genética , MAP Quinasa Quinasa 4/genética , Quinasa 1 de Quinasa de Quinasa MAP/genética , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Ratones Noqueados , Poliubiquitina/genética , Poliubiquitina/metabolismo , Unión Proteica , Factor de Crecimiento Transformador beta/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
The discovery of RNA interference has led to the development of short interfering RNA (siRNA) screening, which has been widely used to study biological pathways. Here, we describe the development and validation of a system suitable to identify cellular genes involved in interferon A2 (IFNA2) promoter activation and interleukin (IL)-8 secretion downstream of MyD88. Forty genes were identified. Five genes were selected for further study. One gene, protein kinase, DNA-activated catalytic polypeptide (PRKDC), was confirmed to play a role in MyD88-induced IFNA2 activation and IL-8 secretion.
Asunto(s)
Proteína Quinasa Activada por ADN/metabolismo , Interferón-alfa/genética , Interleucina-8/metabolismo , Factor 88 de Diferenciación Mieloide/metabolismo , Proteínas Nucleares/metabolismo , Supervivencia Celular , Proteína Quinasa Activada por ADN/genética , Biblioteca de Genes , Células HEK293 , Humanos , Proteínas Nucleares/genética , Regiones Promotoras Genéticas/genética , ARN Interferente Pequeño/genética , Regulación hacia Arriba/genéticaRESUMEN
MAPK signaling is important for T lymphocyte development, homeostasis, and effector responses. To better understand the role of Mekk1 (encoded by Map3k1) in T cells, we conditionally deleted Map3k1 in Lck(Cre/+)Map3k1(f/f) mice, and these display larger iNKT cell populations within the liver, spleen, and bone marrow. Mekk1 signaling controls splenic and liver iNKT cell expansion in response to glycolipid antigen. Lck(Cre/+)Map3k1(f/f) mice have enhanced liver damage in response to glycolipid antigen. Mekk1 regulates Jnk activation in iNKT cells and binds and transfers Lys63-linked poly-ubiquitin onto Carma1. Map3k1 is critical for the regulation of p27(Kip1) (encoded by Cdkn1b).