RESUMEN
The Ras effector NORE1 is frequently silenced in primary adenocarcinomas, although the significance of this silencing for tumorigenesis is unclear. Here we show that NORE1 induces polyubiquitination and proteasomal degradation of oncoprotein HIPK1 by facilitating its interaction with the Mdm2 E3 ubiquitin ligase. Endogenous HIPK1 is stabilized in Nore1-deficient mouse embryonic fibroblasts, and depletion of HIPK1 in NORE1-silenced lung adenocarcinoma cells inhibits anchorage-independent cell growth and tumour formation in nude mice. These findings indicate that the control of HIPK1 stability by Mdm2-NORE1 has a major effect on cell behaviour, and epigenetic inactivation of NORE1 enables adenocarcinoma formation in vivo through HIPK1 stabilization.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas Oncogénicas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteolisis , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteínas ras/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Línea Celular Tumoral , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Poliubiquitina/metabolismo , Unión Proteica , Saccharomyces cerevisiae/metabolismo , UbiquitinaciónRESUMEN
By defining the chromosomal breakpoint of a balanced t(10;12) translocation from a subject with Kallmann syndrome and scanning genes in its vicinity in unrelated hypogonadal subjects, we have identified WDR11 as a gene involved in human puberty. We found six patients with a total of five different heterozygous WDR11 missense mutations, including three alterations (A435T, R448Q, and H690Q) in WD domains important for ß propeller formation and protein-protein interaction. In addition, we discovered that WDR11 interacts with EMX1, a homeodomain transcription factor involved in the development of olfactory neurons, and that missense alterations reduce or abolish this interaction. Our findings suggest that impaired pubertal development in these patients results from a deficiency of productive WDR11 protein interaction.
Asunto(s)
Cromosomas Humanos Par 10/genética , Proteínas de Homeodominio/genética , Hipogonadismo/genética , Síndrome de Kallmann/genética , Proteínas de la Membrana/genética , Proteínas Proto-Oncogénicas/genética , Pubertad/genética , Factores de Transcripción/genética , Translocación Genética/genética , Adolescente , Animales , Humanos , Immunoblotting , Inmunohistoquímica , Inmunoprecipitación , Hibridación in Situ , Hibridación Fluorescente in Situ , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Análisis por Micromatrices , Mutación Missense/genética , Polimorfismo de Nucleótido Simple/genética , Proteínas Proto-Oncogénicas/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Técnicas del Sistema de Dos Híbridos , Pez CebraRESUMEN
The RASSF family proteins were identified as tumor suppressors in a variety of human cancers, and evidenced distinct subcellular localization patterns among their subfamilies and isoforms. In this study, we showed that NORE1A was exported actively via its nuclear export signal (NES) in the C-terminus (residues 372-379). Substitutions of three lysine residues of NORE1A NES to alanines (L372, 376, 379A) showed its localization to the dot structures of the nucleus, which was similar to the NORE1A localizations observed after the administration to cells of Leptomycin B, a nuclear export inhibitor. The NORE1A NES mutant inhibited caspase-mediated apoptosis, whereas wild-type NORE1A induced caspase-3 activation. Furthermore, the NORE1A NES mutant did not co-localize with GFP-MST1, the direct downstream target of NORE1A. These results show that the nuclear export of NORE1A via NES is involved in the NORE1A-mediated induction of apoptosis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Apoptosis , Proteínas de Unión al GTP Monoméricas/metabolismo , Transporte Activo de Núcleo Celular , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas Reguladoras de la Apoptosis , Línea Celular , Chlorocebus aethiops , Citoplasma/metabolismo , Eliminación de Gen , Humanos , Ratones , Proteínas de Unión al GTP Monoméricas/genéticaRESUMEN
The processing of amyloid precursor protein (APP) by gamma-secretase generates the APP intracellular domain (AICD), which functions as a transcriptional factor for target gene activation following localization into the nucleus. In this study, we demonstrate that AICD could be modified via covalent conjugation with Nedd8, a ubiquitin-like protein. Domain analysis and site-directed substitution of neddylation sites showed that multiple lysine residues of the APP C-terminal C99 fragment including AICD were acceptor sequences for Nedd8 conjugation. AICD-mediated transcriptional activation was inhibited by Nedd8 conjugation. Furthermore, the transcriptional activity of the neddylation-defective AICD mutant was not altered by Nedd8 expression. Nedd8 conjugation of AICD inhibited its interaction with Fe65, and consequently resulted in the impairment of AICD-Fe65-Tip60 complex formation for the transcriptional activation of the target gene. These results illustrate the regulatory mechanisms by which AICD transcriptional activity might be regulated via covalent conjugation with Nedd8.