-
[show abstract]
[hide abstract]
ABSTRACT: Daxx plays a major role in several important signaling pathways including transcription and cell death. It has been postulated that Daxx regulates both events from the nucleus; however, the mechanism by which Daxx is localized in the nucleus remains obscure. Here we show that nuclear localization of Daxx is controlled by two independent signals and importin 3. Domain analysis reveals that Daxx contains two separate nuclear localizing domains. Site-directed mutagenesis reveals that the basic aa sequence RLKRK at residues 227-231 (NLS1) is responsible for nuclear localization of N-terminal domain, while aa sequence KKSRKEKK at residues 630-637 (NLS2) is responsible for nuclear localization of the C-terminal domain. Mutations of a NLS consensus sequence RKKRR at residues 391-395 and several other basic aa clusters have no effect on Daxx nuclear localization. In full-length Daxx, NLS1 contributes partially to nuclear localization, while NLS2 plays a major role. Markedly, it is essential to disrupt both NLS1 and NLS2 in order to completely block nuclear localization of the full-length protein and to prevent its association with PML nuclear bodies. Furthermore, Daxx interacts selectively with importin alpha3 through its NLS1 and NLS2 sequences. Conversely, importin alpha3 utilizes two NLS-binding sites for Daxx interaction, suggesting that the importin/mediates nuclear import of Daxx. Finally, we show that nuclear localization of Daxx is essential for its transcriptional effects on GR and p53. Together, these data unveil a molecular mechanism that controls nuclear localization of Daxx and support a nuclear role of Daxx in transcriptional regulation.
Journal of Cellular Biochemistry 03/2008; 103(2):456-70. · 2.87 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The ankyrin repeats cofactor-1 (ANCO-1) was recently identified as a novel nuclear receptor corepressor that regulates receptor-mediated transcription through interactions with p160 coactivators and histone deacetylases. Interestingly, exogenously expressed ANCO-1 is localized at distinct subnuclear domains. The relevance of these subnuclear domains and the mechanisms of nucleocytoplasmic translocation of ANCO-1 have not been determined. We report here the identification of an N-terminal signaling motif that is essential for both nuclear/subnuclear localization and transcription corepressor function of ANCO-1. This N-terminal motif at residues 80-86 of ANCO-1 constitutes a classical nuclear localization signal (NLS1). Disruption of NLS1 causes complete cytoplasmic accumulation of the full-length ANCO-1, and abolishes its corepressor function on receptor-mediated transcription. A second NLS (NLS2) is found at the C-terminal residues 2384-2390; however, its disruption abolishes only nuclear localization of isolated C-terminal fragments. We also identify a leucine-rich nuclear export signal (NES) at residues 2415-2424 of ANCO-1, and show that both the NLSs and NES sequences are capable of mediating nuclear import and export of heterologous protein, respectively. In addition, attachment of the NES sequence to a transcription factor impairs its activation function. These results suggest that ANCO-1 subnuclear localization is regulated by both nuclear import and export signals, and that proper subcellular localization of ANCO-1 is essential for its corepressor function.
Journal of Cellular Biochemistry 09/2007; 101(5):1301-15. · 2.87 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Daxx plays a major role in several important signaling pathways including transcription and cell death. It has been postulated that Daxx regulates both events from the nucleus; however, the mechanism by which Daxx is localized in the nucleus remains obscure. Here we show that nuclear localization of Daxx is controlled by two independent signals and importin 3. Domain analysis reveals that Daxx contains two separate nuclear localizing domains. Site-directed mutagenesis reveals that the basic aa sequence RLKRK at residues 227–231 (NLS1) is responsible for nuclear localization of N-terminal domain, while aa sequence KKSRKEKK at residues 630–637 (NLS2) is responsible for nuclear localization of the C-terminal domain. Mutations of a NLS consensus sequence RKKRR at residues 391–395 and several other basic aa clusters have no effect on Daxx nuclear localization. In full-length Daxx, NLS1 contributes partially to nuclear localization, while NLS2 plays a major role. Markedly, it is essential to disrupt both NLS1 and NLS2 in order to completely block nuclear localization of the full-length protein and to prevent its association with PML nuclear bodies. Furthermore, Daxx interacts selectively with importin α3 through its NLS1 and NLS2 sequences. Conversely, importin α3 utilizes two NLS-binding sites for Daxx interaction, suggesting that the importin/mediates nuclear import of Daxx. Finally, we show that nuclear localization of Daxx is essential for its transcriptional effects on GR and p53. Together, these data unveil a molecular mechanism that controls nuclear localization of Daxx and support a nuclear role of Daxx in transcriptional regulation. J. Cell. Biochem. 103: 456–470, 2008. © 2007 Wiley-Liss, Inc.
Journal of Cellular Biochemistry 07/2007; 103(2):456 - 470. · 2.87 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Members of the p160 nuclear receptor coactivators interact with liganded nuclear receptors to enhance transcription of target genes. Here we identify a novel family of ankyrin repeats containing cofactors (ANCOs) that interact with the p160 coactivators. ANCO-1 binds to the conserved Per-Arnt-Sim (PAS) region of the p160 coactivators. It encodes a large nuclear protein with five ankyrin repeats, and parts of its sequences have been reported as nasopharyngeal carcinoma susceptibility protein and medulloblastoma antigen. Immunofluorescence staining reveals discrete nuclear foci of ANCO-1 that are distinct from known nuclear structures. Intriguingly, ANCO-1 also colocalizes and interacts with histone deacetylases. Transient reporter gene assay shows that ANCO-1 expression inhibits ligand-dependent transactivation by both steroid and nonsteroid nuclear receptors. Taken together, we have identified a novel family of ankyrin repeats containing cofactors that may recruit histone deacetylases to the p160 coactivators/nuclear receptor complex to inhibit ligand-dependent transactivation.
Journal of Biological Chemistry 09/2004; 279(32):33799-805. · 4.77 Impact Factor
