Daxx contains two nuclear localization signals and interacts with importin alpha3.
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.
Department of Pharmacology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey, USA.
Percy Luk
PL
Yeung
Liuh-Yow
LY
Chen
Shih-Chieh
SC
Tsai
Aihua
A
Zhang
J Don
JD
Chen
D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells / Q000378:metabolism; D002460:Cell Line / Q000378:metabolism; D002522:Chlorocebus aethiops; D056970:Co-Repressor Proteins; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019913:Nuclear Localization Signals / Q000737:chemistry* / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014162:Transfection; D028901:alpha Karyopherins / Q000378:metabolism* / Q000502:physiology
D048868:Adaptor Proteins, Signal Transducing; D056970:Co-Repressor Proteins; C106985:DAXX protein, human; C106259:KPNA4 protein, human; D018832:Molecular Chaperones; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D028901:alpha Karyopherins