RESUMEN
Like the type I interferons (IFNs), the recently discovered cytokine IFN-λ displays antiviral, antiproliferative, and proapoptotic activities, mediated by a heterodimeric IFN-λ receptor complex composed of a unique IFN-λR1 chain and the IL-10R2 chain. However, the molecular mechanism of the IFN-λ-regulated pathway remains unclear. In this study, we newly identified RAN-binding protein M (RanBPM) as a binding partner of IFN-λR1. The interaction between RanBPM and IFN-λR1 was identified with a glutathione S-transferase pull-down assay and coimmunoprecipitation experiments. IFN-λ1 stimulates this interaction and affects the cellular distribution of RanBPM. However, the interaction between RanBPM and IFN-λR1 does not correlate with their conserved TRAF6-binding sites. Furthermore, we also found that RanBPM is a scaffolding protein with a modulatory function that regulates the activities of IFN-stimulated response elements. Therefore, RanBPM plays a novel role in the IFN-λ-regulated signaling pathway.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Citoesqueleto/metabolismo , Interleucinas/metabolismo , Proteínas Nucleares/metabolismo , Receptores de Citocinas/metabolismo , Transducción de Señal/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Sitios de Unión , Línea Celular Transformada , Línea Celular Tumoral , Proteínas del Citoesqueleto/genética , Células HEK293 , Humanos , Inmunoprecipitación , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/metabolismo , Interferones , Péptidos y Proteínas de Señalización Intracelular , Proteínas Nucleares/genética , Unión Proteica , Receptores de Citocinas/genética , Receptores de Interferón , Elementos de Respuesta , Transducción de Señal/genética , Factor 6 Asociado a Receptor de TNF/metabolismo , UbiquitinaciónRESUMEN
Ectopically expressed Ring1 and YY1 binding protein (RYBP) induces tumor cell apoptosis through promoting the formation of the death-inducing signaling complex (DISC) in the cytoplasm. However, transiently overexpressed as well as endogenous RYBP in tumor tissues were observed to be mainly located in the nucleus while that in adjacent non-tumor tissues distributed majorly in the cytoplasm. Currently, we do not know the nuclear localization signals and biological function of different subcellular location of RYBP. In this study, we employed bioinformatic analysis, deletion, point mutation, enhanced green fluorescence protein (EGFP) fusion and others, to investigate the elements responsible for RYBP nuclear import and to explore the anti-tumor activities of cytoplasm- and nuclear-located RYBP. Herein, we identified three functional monopartite nuclear localization signals (NLSs), all of which located at the N-terminus of RYBP. Through four basic amino acid replacements within the NLSs, we obtained a cytoplasm-located RYBP mutant (RYBPmut). Compared with wild-type counterpart, RYBPmut exhibited more potent abilities to bind to caspase 8, to prevent MDM2-mediated polyubiquitination and degradation of p53, thereby leading to its stabilization. Further investigation revealed that, in contrast to its wild type, RYBPmut showed more potentials to inhibit tumor cell proliferation and to induce apoptosis, in both p53-dependent and -independent manner. Collectively, our current study revealed the molecular mechanism responsible for RYBP nuclear translocation, and provided evidences to support that RYBPmut could be a more promising candidate agent for cancer treatment.