Nuclear Import of the U1A Splicesome Protein Is Mediated by Importin α/β and Ran in Living Mammalian Cells

Hieda, Miki; Tachibana, Taro; Fukumoto, Masahiro; Yoneda, Yoshihiro

doi:10.1074/jbc.m008299200

Cited by 17 publications

(15 citation statements)

References 45 publications

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“…The structures of all plasmids were confirmed by restriction enzyme analysis and, in most cases, by DNA sequence analysis across crucial regions. pCGN/Duplin, pBJ-Myc/ Duplin, pEF-BOS-HA/hTcf-4E, pSP-Myc/Duplin, pGEX-GFP, and pGEX/SV40NLS-GFP were constructed as described (19,40,46).…”

Section: Methodsmentioning

confidence: 99%

Nuclear Localization of Duplin, a β-Catenin-binding Protein, Is Essential for Its Inhibitory Activity on the Wnt Signaling Pathway

Kobayashi

Kishida

Fukui

et al. 2002

Journal of Biological Chemistry

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Duplin binds to ␤-catenin and inhibits the Wnt signaling pathway, thereby leading to repression of the ␤-catenin-mediated transactivation and Xenopus axis formation. To find an additional function of Duplin, yeast two-hybrid screening was carried out. Importin ␣ was isolated as a binding protein of Duplin. Importin ␣ bound directly to basic amino acid clusters of Duplin. Although Duplin was present in the nucleus, deletion of the basic amino acid clusters (Duplin ⌬500 -584 ) retained Duplin in the cytoplasm. Duplin ⌬500 -584 bound to ␤-catenin as efficiently as wild-type Duplin, but it neither repressed Wntdependent Tcf transcriptional activation in mammalian cells nor showed ventralization in Xenopus embryos. The Duplin mutant without a ␤-catenin-binding region lost the ability to inhibit the Wnt-dependent Tcf activation, but retained its ventralizing activity. Furthermore, Duplin not only suppressed ␤-catenin-dependent axis duplication and expression of siamois, a Wnt-regulated gene, but also inhibited siamois-dependent axis duplication. These results indicate that Duplin is translocated to the nucleus by interacting with importin ␣, and that nuclear localization is essential for the function of Duplin. Moreover, Duplin has an additional activity of inhibiting the Wnt signaling pathway by affecting the downstream ␤-catenin target genes.

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Section: Methodsmentioning

confidence: 99%

Nuclear Localization of Duplin, a β-Catenin-binding Protein, Is Essential for Its Inhibitory Activity on the Wnt Signaling Pathway

Kobayashi

Kishida

Fukui

et al. 2002

Journal of Biological Chemistry

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“…To decrease the factors remaining in the cytoplasm after permeabilization, the cells were incubated for 10 min at 4°C and washed twice with TB. The cells were then incubated at 30°C for 30 min with the testing solution, which was TB containing 2% bovine serum albumin, GST-U69(21-39)-GFP (10 pmol), each importin-␣ (10 pmol or as indicated in the figure legends), importin-␤ (10 pmol), Ran-GDP (40 pmol), NTF2 (5 pmol), and an ATP regeneration system (1 mM ATP, 20 U of creatine kinase, 5 mM phosphocreatine), with or without Q69L Ran-GTP (40 pmol), in a 10-l sample volume, as described previously (14). After the incubation, the cells were washed twice with TB and fixed with 3.7% formaldehyde in TB at room temperature.…”

Section: Cells and Virusesmentioning

confidence: 99%

Characterization of the Human Herpesvirus 6 U69 Gene Product and Identification of Its Nuclear Localization Signal

Isegawa¹,

Miyamoto²,

Yasuda³

et al. 2008

J Virol

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To elucidate the function of the U69 protein kinase of human herpesvirus 6 (HHV-6) in vivo, we first analyzed its subcellular localization in HHV-6-infected Molt 3 cells by using polyclonal antibodies against the U69 protein. Immunofluorescence studies showed that the U69 signal localized to the nucleus in a mesh-like pattern in both HHV-6-infected and HHV6-transfected cells. A computer program predicted two overlapping classic nuclear localization signals (NLSs) in the N-terminal region of the protein; this NLS motif is highly conserved in the N-terminal region of most of the herpesvirus protein kinases examined to date. An N-terminal deletion mutant form of the protein failed to enter the nucleus, whereas a fusion protein of green fluorescent protein (GFP) and/or glutathione S-transferase (GST) and the U69 N-terminal region was transported into the nucleus, demonstrating that the predicted N-terminal NLSs of the protein actually function as NLSs. The nuclear transport of the GST-GFP fusion protein containing the N-terminal NLS of U69 was inhibited by wheat germ agglutinin and by the Q69L Ran-GTP mutant, indicating that the U69 protein is transported into the nucleus from the cytoplasm via classic nuclear transport machinery. A cell-free import assay showed that the nuclear transport of the U69 protein was mediated by importin ␣/␤ in conjunction with the small GTPase Ran. When the import assay was performed with a low concentration of each importin-␣ subtype, NPI2/importin-␣7 elicited more efficient transport activity than did Rch1/importin-␣1 or Qip1/importin-␣3. These results suggest a relationship between the localization of NPI2/importin-␣7 and the cell tropism of HHV-6.

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“…Transfection into HeLa and HEK293T cells was performed by means of an Effectene Transfection Reagent Kit according to the manufacturer's recommended protocol (Qiagen, Hilden, Germany). Ehrlich cytosolic extract was prepared as described previously (Hieda et al, 2001).…”

Section: Plasmid Constructionmentioning

confidence: 99%

Phosphorylation of RanGAP1 Stabilizes Its Interaction with Ran and RanBP1

Takeda

Hieda

Katahira

et al. 2005

Cell Struct. Funct.

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ABSTRACT. Ran is a nuclear Ras-like GTPase that is required for various nuclear events including the bi-directional transport of proteins and ribonucleoproteins through the nuclear pore complex, spindle formation, and reassembly of the nuclear envelope. One of the key regulators of Ran is RanGAP1, a Ran specific GTPase activating protein. The question of whether a mechanism exists for controlling nucleocytoplasmic transport through the regulation of RanGAP1 activity continues to be debated. Here we show that RanGAP1 is phosphorylated in vivo and in vitro. Serine-358 ( 358 S) was identified as the major phosphorylation site, by MALDI-TOF-MS spectrometry. Site directed mutagenesis at this position abolished the phosphorylation. Experiments using purified recombinant kinase and specific inhibitors such as DRB and apigenin strongly suggest that casein kinase II (CK2) is the responsible kinase. Although the phosphorylation of 358 S of RanGAP1 did not significantly alter its GAP activity, the phosphorylated wild type RanGAP1, but not a mutant harboring a mutation at the phosphorylation site 358 S, efficiently formed a stable ternary complex with Ran and RanBP1 in vivo, suggesting that the 358 S phosphorylation of RanGAP1 affects the Ran system.

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Nuclear Import of the U1A Splicesome Protein Is Mediated by Importin α/β and Ran in Living Mammalian Cells

Cited by 17 publications

References 45 publications

Nuclear Localization of Duplin, a β-Catenin-binding Protein, Is Essential for Its Inhibitory Activity on the Wnt Signaling Pathway

Nuclear Localization of Duplin, a β-Catenin-binding Protein, Is Essential for Its Inhibitory Activity on the Wnt Signaling Pathway

Characterization of the Human Herpesvirus 6 U69 Gene Product and Identification of Its Nuclear Localization Signal

Phosphorylation of RanGAP1 Stabilizes Its Interaction with Ran and RanBP1

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