RESUMO
Similarities in physiological roles of LXR (liver X receptors) and co-repressor RIP140 (receptor-interacting protein 140) in regulating energy homoeostasis and lipid and glucose metabolism suggest that the effects of LXR could at least partly be mediated by recruitment of the co-repressor RIP140. In the present study, we have elucidated the molecular basis for regulation of LXR transcriptional activity by RIP140. LXR is evenly localized in the nucleus and neither the N-terminal domain nor the LBD (ligand-binding domain) is necessary for nuclear localization. Both LXR subtypes, LXRalpha and LXRbeta, interact with RIP140 and co-localize in diffuse large nuclear domains. Interaction and co-localization are dependent on the LBD of the receptor. The C-terminal domain of RIP140 is sufficient for full repressive effect. None of the C-terminal NR (nuclear receptor)-boxes is required for the co-repressor activity, whereas the NR-box-like motif as well as additional elements in the C-terminal region are required for full repressive function. The C-terminal NR-box-like motif is necessary for interaction with LXRbeta, whereas additional elements are needed for strong interaction with LXRalpha. In conclusion, our results suggest that co-repression of LXR activity by RIP140 involves an atypical binding mode of RIP140 and a repression element in the RIP140 C-terminus.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Proteínas Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transcrição Gênica , Proteínas Adaptadoras de Transdução de Sinal/genética , Motivos de Aminoácidos , Animais , Células COS , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Chlorocebus aethiops , Proteínas de Ligação a DNA/genética , Genes Reporter , Humanos , Receptores X do Fígado , Proteínas Nucleares/genética , Proteína 1 de Interação com Receptor Nuclear , Receptores Nucleares Órfãos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Regulation of gene transcription by nuclear receptors involves association with numerous coregulators. Receptor-interacting protein 140 (RIP140) is a corepressor that negatively regulates the ligand-induced activity of several nuclear receptors, including the glucocorticoid receptor (GR). In the present study, we have characterized the role of the intranuclear localization of RIP140 in its corepressor activity. In the absence of ligand-activated GR, RIP140 is localized in small nuclear foci targeted by a 40-amino-acid-long sequence. Although the focus-targeting domain overlaps with a binding sequence for the corepressor CtBP (C-terminal binding protein), interaction with CtBP is not involved in the localization. RIP140 foci do not correspond to PML bodies but partly colocalize with domains harboring the corepressor SMRT. Upon ligand binding, GR and RIP140 are redistributed to large nuclear domains distinct from the RIP140 foci. The redistribution requires regions of RIP140 with corepressor activity, as well as the DNA-binding domain of GR. Furthermore, we show that full RIP140 corepressor activity is contributed both by C-terminal receptor-binding LXXLL motifs and interaction with the CtBP corepressor. In conclusion, our results suggest that the corepressor function of RIP140 is multifaceted and involves binding to nuclear receptors, as well as additional functions mediated by the formation and intranuclear relocalization of a repressive protein complex.
Assuntos
Núcleo Celular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Motivos de Aminoácidos , Animais , Sítios de Ligação , Western Blotting , Células COS , Genes Reporter , Glutationa Transferase/metabolismo , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Ligantes , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Modelos Genéticos , Proteína 1 de Interação com Receptor Nuclear , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
Polyamine-modulated factor 1 (PMF-1) has been reported to interact with NF-E2 related factor 2 (Nrf-2) and activate the polyamine-induced transcription of spermidine/spermine N(1)-acetyltransferase (SSAT) gene. Polyamines are important regulators of cell growth and cell death and have been implicated in glucocorticoid-induced apoptosis. In the present study, we have identified and characterized new functional binding partners for PMF-1. Our results demonstrate that PMF-1 binds to the glucocorticoid receptor (GR). PMF-1 also represses glucocorticoid-induced transcription. Furthermore, we show that PMF-1 has an intrinsic repression activity, which could contribute to the repressive effect on GR. PMF-1 can also interact with the GR corepressor, receptor-interacting protein 140 (RIP140), but does not further enhance the repressive effect of RIP140. Our results suggest that PMF-1 has a broader function in regulation of genes and can contribute to glucocorticoid signaling.
Assuntos
Receptores de Glucocorticoides/antagonistas & inibidores , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células COS , Chlorocebus aethiops , Camundongos , Proteínas Nucleares/metabolismo , Proteína 1 de Interação com Receptor Nuclear , Ativação TranscricionalRESUMO
Glucocorticoids are widely used to treat inflammatory diseases but have a number of side effects that partly are connected to inhibition of cell proliferation. Glucocorticoids mediated their action by binding to the glucocorticoid receptor. In the present study, we have identified by two-hybrid screens the germinal center-associated protein (GANP) and MCM3-associated protein (MCM3AP), a splicing variant of GANP, as glucocorticoid receptor interacting proteins. GANP and MCM3AP can bind to the MCM3 protein involved in initiation of DNA replication. Glutathione-S-transferase-pull-down and co-immunoprecipitation assays showed that the C-terminal domain of GANP, encompassing MCM3AP, interacts with the ligand-binding domain of the glucocorticoid receptor. Characterization of the intracellular localization of GANP revealed that GANP is shuttling between the nucleus and the cytoplasm. Furthermore, we show that glucocorticoids are unable to inhibit DNA replication in HeLa cells overexpressing MCM3AP suggesting a role for both glucocorticoid receptor and GANP/MCM3AP in regulating cell proliferation.