RESUMEN
Optineurin is an adaptor protein involved in signal transduction, membrane vesicle trafficking and autophagy. Optineurin expression is induced by cytokines. Previously we have shown that tumor necrosis factor-α activates optineurin promoter through NF-κB-binding site in the core promoter. However, this promoter was not activated by interferon-γ. Here, we report identification of a functional IRF-1-binding site in the first intron of human optineurin gene that mediates interferon-γ-induced activation of the promoter. Optineurin promoter, containing the contiguous intronic sequences with IRF-1 responsive sites, is strongly activated by IRF-1. Mutational inactivation of IRF-1 site resulted in loss of activation of the promoter by interferon-γ and also by IRF-1. We also show that IRF-1 cooperates with NF-κB to activate optineurin promoter. The synergistic effect of these two transcription factors (IRF-1 and NF-κB) may be involved in cooperative induction of optineurin promoter by interferon-γ and tumor necrosis factor-α.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Factor 1 Regulador del Interferón/metabolismo , Interferón gamma/farmacología , Intrones/genética , Regiones Promotoras Genéticas , Factor de Transcripción TFIIIA/genética , Secuencia de Bases , Sitios de Unión/genética , Proteínas de Ciclo Celular , Análisis Mutacional de ADN , Células HeLa , Humanos , Proteínas de Transporte de Membrana , Datos de Secuencia Molecular , FN-kappa B/metabolismo , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Factor de Transcripción TFIIIA/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Rab GTPases regulate various membrane trafficking pathways but the mechanisms by which GTPase-activating proteins recognise specific Rabs are not clear. Rab8 is involved in controlling several trafficking processes, including the trafficking of transferrin receptor from the early endosome to the recycling endosome. Here, we provide evidence to show that TBC1D17, a Rab GTPase-activating protein, through its catalytic activity, regulates Rab8-mediated endocytic trafficking of transferrin receptor. Optineurin, a Rab8-binding effector protein, mediates the interaction and colocalisation of TBC1D17 with Rab8. A non-catalytic region of TBC1D17 is required for direct interaction with optineurin. Co-expression of Rab8, but not other Rabs tested, rescues the inhibition of transferrin receptor trafficking by TBC1D17. The activated GTP-bound form of Rab8 is localised to the tubules emanating from the endocytic recycling compartment. Through its catalytic activity, TBC1D17 inhibits recruitment of Rab8 to the tubules and reduces colocalisation of transferrin receptor and Rab8. Knockdown of optineurin or TBC1D17 results in enhanced recruitment of Rab8 to the tubules. A glaucoma-associated mutant of optineurin, E50K, causes enhanced inhibition of Rab8 by TBC1D17, resulting in defective endocytic recycling of transferrin receptor. Our results show that TBC1D17, through its interaction with optineurin, regulates Rab8-mediated endocytic recycling of transferrin receptor and recruitment of Rab8 to the endocytic recycling tubules. We describe a mechanism of regulating a Rab GTPase by an effector protein (optineurin) that acts as an adaptor to bring together a Rab (Rab8) and its GTPase-activating protein (TBC1D17).
