Apoptin Nuclear Accumulation Is Modulated by a CRM1-Recognized Nuclear Export Signal that Is Active in Normal but not in Tumor Cells

Abstract Apoptin, a protein expressed by chicken anemia virus, is found predominantly in the cytoplasm in normal cells, whereas it localizes in the nucleus in transformed and malignant cells. However, the mechanisms that regulate the different subcellular localization of Apoptin in normal and tumor cells have not been fully clarified. In this work, a putative nuclear export signal (NES) in Apoptin was predicted. It was testified that the putative NES (pNES) of Apoptin was not a functional NES, but actually acted as a cytoplasmic retention signal. Deletion of the pNES led to the nuclear accumulation of Apoptin in normal cells. In addition, when a strong nuclear localization signal was introduced into Apoptin, it exclusively translocated to the nucleus in normal cells. These observations indicated that the cytoplasmic localization of Apoptin in normal cells results from the balance between cytoplasmic retention and nuclear import. On the other hand, the pNES was also proved to be necessary for Apoptin multimerization. Mutants lacking the pNES did not form obviously visible globular aggregates in normal or tumor cells.

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Identification of the Nuclear Export Signal and STAT-Binding Domains of the Nipah Virus V Protein Reveals Mechanisms Underlying Interferon Evasion

Journal of Virology ◽

10.1128/jvi.78.10.5358-5367.2004 ◽

2004 ◽

Vol 78 (10) ◽

pp. 5358-5367 ◽

Cited By ~ 92

Author(s):

Jason J. Rodriguez ◽

Cristian D. Cruz ◽

Curt M. Horvath

Keyword(s):

Amino Acids ◽

Protein Interactions ◽

Nuclear Export ◽

Nipah Virus ◽

Nuclear Export Signal ◽

Hendra Virus ◽

Nuclear Accumulation ◽

V Protein ◽

Binding Domains ◽

Export Signal

ABSTRACT The V proteins of Nipah virus and Hendra virus have been demonstrated to bind to cellular STAT1 and STAT2 proteins to form high-molecular-weight complexes that inhibit interferon (IFN)-induced antiviral transcription by preventing STAT nuclear accumulation. Analysis of the Nipah virus V protein has revealed a region between amino acids 174 and 192 that functions as a CRM1-dependent nuclear export signal (NES). This peptide is sufficient to complement an export-defective human immunodeficiency virus Rev protein, and deletion and substitution mutagenesis revealed that this peptide is necessary for both V protein shuttling and cytoplasmic retention of STAT1 and STAT2 proteins. However, the NES is not required for V-dependent IFN signaling inhibition. IFN signaling is blocked primarily by interaction between Nipah virus V residues 100 to 160 and STAT1 residues 509 to 712. Interaction with STAT2 requires a larger Nipah virus V segment between amino acids 100 and 300, but deletion of residues 230 to 237 greatly reduced STAT2 coprecipitation. Further, V protein interactions with cellular STAT1 is a prerequisite for STAT2 binding, and sequential immunoprecipitations demonstrate that V, STAT1, and STAT2 can form a tripartite complex. These findings characterize essential regions for Henipavirus V proteins that represent potential targets for therapeutic intervention.

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Shuttling Imbalance of MLF1 Results in p53 Instability and Increases Susceptibility to Oncogenic Transformation

Molecular and Cellular Biology ◽

10.1128/mcb.02335-06 ◽

2007 ◽

Vol 28 (1) ◽

pp. 422-434 ◽

Cited By ~ 30

Author(s):

Noriko Yoneda-Kato ◽

Jun-ya Kato

Keyword(s):

Nuclear Export ◽

Nuclear Export Signal ◽

Chromosomal Translocation ◽

Nuclear Accumulation ◽

Tumor Suppressor P53 ◽

Oncogenic Transformation ◽

Early Passage ◽

Leptomycin B ◽

Nuclear Shuttling ◽

Export Signal

ABSTRACT Myeloid leukemia factor 1 (MLF1) stabilizes the activity of the tumor suppressor p53 by suppressing its E3 ubiquitin ligase, COP1, through a third component of the COP9 signalosome (CSN3). However, little is known about how MLF1 functions upstream of the CSN3-COP1-p53 pathway and how its deregulation by the formation of the fusion protein nucleophosmin (NPM)-MLF1, generated by t(3;5)(q25.1;q34) chromosomal translocation, leads to leukemogenesis. Here we show that MLF1 is a cytoplasmic-nuclear-shuttling protein and that its nucleolar localization on fusing with NPM prevents the full induction of p53 by both genotoxic and oncogenic cellular stress. The majority of MLF1 was located in the cytoplasm, but the treatment of cells with leptomycin B rapidly induced a nuclear accumulation of MLF1. A mutation of the nuclear export signal (NES) motif identified in the MLF1 sequence enhanced the antiproliferative activity of MLF1. The fusion of MLF1 with NPM translocated MLF1 to the nucleolus and abolished the growth-suppressing activity. The introduction of NPM-MLF1 into early-passage murine embryonic fibroblasts allowed the cells to escape from cellular senescence at a markedly earlier stage and induced neoplastic transformation in collaboration with the oncogenic form of Ras. Interestingly, disruption of the MLF1-derived NES sequence completely abolished the growth-promoting activity of NPM-MLF1 in murine fibroblasts and hematopoietic cells. Thus, our results provide important evidence that the shuttling of MLF1 is critical for the regulation of cell proliferation and a disturbance in the shuttling balance increases the cell's susceptibility to oncogenic transformation.

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TORC1 signaling exerts spatial control over microtubule dynamics by promoting nuclear export of Stu2

The Journal of Cell Biology ◽

10.1083/jcb.201606080 ◽

2017 ◽

Vol 216 (11) ◽

pp. 3471-3484 ◽

Cited By ~ 5

Author(s):

Babet van der Vaart ◽

Josef Fischböck ◽

Christine Mieck ◽

Peter Pichler ◽

Karl Mechtler ◽

...

Keyword(s):

Nuclear Export ◽

Cell Cycle Progression ◽

Nuclear Export Signal ◽

Nuclear Accumulation ◽

Yeast Saccharomyces Cerevisiae ◽

Cycle Progression ◽

Cytoskeletal Organization ◽

Spindle Positioning ◽

Cellular Processes ◽

Export Signal

The target of rapamycin complex 1 (TORC1) is a highly conserved multiprotein complex that functions in many cellular processes, including cell growth and cell cycle progression. In this study, we define a novel role for TORC1 as a critical regulator of nuclear microtubule (MT) dynamics in the budding yeast Saccharomyces cerevisiae. This activity requires interactions between EB1 and CLIP-170 plus end–tracking protein (+TIP) family members with the TORC1 subunit Kog1/Raptor, which in turn allow the TORC1 proximal kinase Sch9/S6K1 to regulate the MT polymerase Stu2/XMAP215. Sch9-dependent phosphorylation of Stu2 adjacent to a nuclear export signal prevents nuclear accumulation of Stu2 before cells enter mitosis. Mutants impaired in +TIP–TORC1 interactions or Stu2 nuclear export show increased nuclear but not cytoplasmic MT length and display nuclear fusion, spindle positioning, and elongation kinetics defects. Our results reveal key mechanisms by which TORC1 signaling controls Stu2 localization and thereby contributes to proper MT cytoskeletal organization in interphase and mitosis.

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CDK-dependent phosphorylation of Alp7–Alp14 (TACC–TOG) promotes its nuclear accumulation and spindle microtubule assembly

Molecular Biology of the Cell ◽

10.1091/mbc.e13-11-0679 ◽

2014 ◽

Vol 25 (13) ◽

pp. 1969-1982 ◽

Cited By ~ 19

Author(s):

Naoyuki Okada ◽

Takashi Toda ◽

Masayuki Yamamoto ◽

Masamitsu Sato

Keyword(s):

Nuclear Export ◽

Nuclear Export Signal ◽

Coiled Coil ◽

Microtubule Assembly ◽

Nuclear Accumulation ◽

Cyclin Dependent Kinase ◽

Mitotic Entry ◽

Localization Signal ◽

Export Signal ◽

Overexpressed Gene

As cells transition from interphase to mitosis, the microtubule cytoskeleton is reorganized to form the mitotic spindle. In the closed mitosis of fission yeast, a microtubule-associated protein complex, Alp7–Alp14 (transforming acidic coiled-coil–tumor overexpressed gene), enters the nucleus upon mitotic entry and promotes spindle formation. However, how the complex is controlled to accumulate in the nucleus only during mitosis remains elusive. Here we demonstrate that Alp7–Alp14 is excluded from the nucleus during interphase using the nuclear export signal in Alp14 but is accumulated in the nucleus during mitosis through phosphorylation of Alp7 by the cyclin-dependent kinase (CDK). Five phosphorylation sites reside around the nuclear localization signal of Alp7, and the phosphodeficient alp7-5A mutant fails to accumulate in the nucleus during mitosis and exhibits partial spindle defects. Thus our results reveal one way that CDK regulates spindle assembly at mitotic entry: CDK phosphorylates the Alp7–Alp14 complex to localize it to the nucleus.

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CRM1 Mediates the Export of ADAR1 through a Nuclear Export Signal within the Z-DNA Binding Domain

Molecular and Cellular Biology ◽

10.1128/mcb.21.22.7862-7871.2001 ◽

2001 ◽

Vol 21 (22) ◽

pp. 7862-7871 ◽

Cited By ~ 93

Author(s):

Hanne Poulsen ◽

Jakob Nilsson ◽

Christian K. Damgaard ◽

Jan Egebjerg ◽

Jørgen Kjems

Keyword(s):

Nuclear Export ◽

Fluorescent Protein ◽

Protein A ◽

Nuclear Export Signal ◽

Nuclear Accumulation ◽

Adenosine Deaminases ◽

Editing Activity ◽

Green Fluorescent ◽

Export Signal

ABSTRACT RNA editing of specific residues by adenosine deamination is a nuclear process catalyzed by adenosine deaminases acting on RNA (ADAR). Different promoters in the ADAR1 gene give rise to two forms of the protein: a constitutive promoter expresses a transcript encoding (c)ADAR1, and an interferon-induced promoter expresses a transcript encoding an N-terminally extended form, (i)ADAR1. Here we show that (c)ADAR1 is primarily nuclear whereas (i)ADAR1 encompasses a functional nuclear export signal in the N-terminal part and is a nucleocytoplasmic shuttle protein. Mutation of the nuclear export signal or treatment with the CRM1-specific drug leptomycin B induces nuclear accumulation of (i)ADAR1 fused to the green fluorescent protein and increases the nuclear editing activity. In concurrence, CRM1 and RanGTP interact specifically with the (i)ADAR1 nuclear export signal to form a tripartite export complex in vitro. Furthermore, our data imply that nuclear import of (i)ADAR1 is mediated by at least two nuclear localization sequences. These results suggest that the nuclear editing activity of (i)ADAR1 is modulated by nuclear export.

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Nuclear export signal of IkappaBalpha interferes with the Rev-dependent posttranscriptional regulation of human immunodeficiency virus type I

Journal of Cell Science ◽

10.1242/jcs.110.22.2883 ◽

1997 ◽

Vol 110 (22) ◽

pp. 2883-2893

Author(s):

F. Bachelerie ◽

M.S. Rodriguez ◽

C. Dargemont ◽

D. Rousset ◽

D. Thomas ◽

...

Keyword(s):

Dna Binding ◽

Nuclear Export ◽

Nuclear Export Signal ◽

Binding Activity ◽

Nuclear Accumulation ◽

Viral Rnas ◽

Dna Binding Activity ◽

Nf Kappab ◽

Export Signal ◽

Hiv 1

De novo synthesized IkappaBalpha accumulates transiently in the nucleus where it inhibits NF-kappaB-dependent transcription and reduces nuclear NF-kappaB content. A sequence present in the C-terminal domain of IkappaBalpha and homologous to the HIV-1 Rev nuclear export signal (NES) has been recently defined as a functional NES conferring on IkappaBalpha the ability to export IkappaBalpha/NF-kappaB complexes. Rev utilises its RNA-binding activity and NES sequence to promote specifically the transport of unspliced and monospliced viral RNAs to the cytoplasm. The object of this work was to determine if nuclear IkappaBalpha could interfere with Rev-dependent transport of viral RNA from the nucleus to the cytoplasm. We report that accumulation of IkappaBalpha in the cell nucleus blocks viral replication. This effect could be dissociated from the capacity of IkappaBalpha to inhibit NF-kappaB-DNA-binding activity and required a functional IkappaBalpha NES motif. Indeed, mutation of the NES abrogated the capacity of IkappaBalpha to inhibit Rev-dependent mechanisms involved in the replication of either wild-type or NF-kappaB-mutated HIV-1 molecular clones. Nuclear accumulation of a reporter protein tagged with a nuclear localization signal (NLS) and fused to the IkappaBalpha NES motif (NLS-PK-NES) was sufficient to inhibit HIV-1 replication at a post-transcriptional level by specifically blocking the expression of a Rev-dependent gene. Furthermore, in cells pulsed with TNF, a treatment which favors nuclear accumulation of newly synthesized IkappaBalpha, NLS-PK-NES expression promoted sustained accumulation of nuclear NF-kappaB lacking DNA-binding activity. This NES-mediated accumulation of inactive nuclear NF-kappaB is likely the consequence of interference in the IkappaBalpha-mediated export of NF-kappaB. These findings indicate that IkappaBalpha and Rev compete for the same nuclear export pathway and suggest that nuclear accumulation of IkappaBalpha, which would occur during normal physiological cell activation process, may interfere with the Rev-NES-mediated export pathway of viral RNAs, thus inhibiting HIV-1 replication.

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A nuclear export signal is essential for the cytosolic localization of the Ran binding protein, RanBP1.

The Journal of Cell Biology ◽

10.1083/jcb.134.5.1157 ◽

1996 ◽

Vol 134 (5) ◽

pp. 1157-1168 ◽

Cited By ~ 100

Author(s):

S A Richards ◽

K M Lounsbury ◽

K L Carey ◽

I G Macara

Keyword(s):

Nuclear Export ◽

Protein Import ◽

Nuclear Protein ◽

Fluorescent Protein ◽

Binding Protein ◽

Nuclear Export Signal ◽

Cyclin B1 ◽

Nuclear Accumulation ◽

Protein Fusion ◽

Export Signal

RanBP1 is a Ran/TC4 binding protein that can promote the interaction between Ran and beta-importin /beta-karyopherin, a component of the docking complex for nuclear protein cargo. This interaction occurs through a Ran binding domain (RBD). Here we show that RanBP1 is primarily cytoplasmic, but the isolated RBD accumulates in the nucleus. A region COOH-terminal to the RBD is responsible for this cytoplasmic localization. This domain acts heterologously, localizing a nuclear cyclin B1 mutant to the cytoplasm. The domain contains a nuclear export signal that is necessary but not sufficient for the nuclear export of a functional RBD In transiently transfected cells, epitope-tagged RanBP1 promotes dexamethasone-dependent nuclear accumulation of a glucocorticoid receptor-green fluorescent protein fusion, but the isolated RBD potently inhibits this accumulation. The cytosolic location of RanBP1 may therefore be important for nuclear protein import. RanBP1 may provide a key link between the nuclear import and export pathways.

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Facilitated Nucleocytoplasmic Shuttling of the Ran Binding Protein RanBP1

Molecular and Cellular Biology ◽

10.1128/mcb.20.10.3510-3521.2000 ◽

2000 ◽

Vol 20 (10) ◽

pp. 3510-3521 ◽

Cited By ~ 51

Author(s):

Kendra Plafker ◽

Ian G. Macara

Keyword(s):

Nuclear Export ◽

Binding Protein ◽

Nuclear Export Signal ◽

Nuclear Accumulation ◽

Leptomycin B ◽

Simple Diffusion ◽

Permeabilized Cells ◽

C Terminus ◽

Transport Receptors ◽

Export Signal

ABSTRACT The Ran binding protein RanBP1 is localized to the cytosol of interphase cells. A leucine-rich nuclear export signal (NES) near the C terminus of RanBP1 is essential to maintain this distribution. We now show that RanBP1 accumulates in nuclei of cells treated with the export inhibitor, leptomycin B, and collapse of the nucleocytoplasmic Ran:GTP gradient leads to equilibration of RanBP1 across the nuclear envelope. Low temperature prevents nuclear accumulation of RanBP1, suggesting that import does not occur via simple diffusion. Glutathione S-transferase (GST)–RanBP1(1-161), which lacks the NES, accumulates in the nucleus after cytoplasmic microinjection. In permeabilized cells, nuclear accumulation of GST-RanBP1(1-161) requires nuclear Ran:GTP but is not inhibited by a dominant interfering G19V mutant of Ran. Nuclear accumulation is enhanced by addition of exogenous karyopherins/importins or RCC1, both of which also enhance nuclear Ran accumulation. Import correlates with Ran concentration. Remarkably, an E37K mutant of RanBP1 does not import into the nuclei under any conditions tested despite the fact that it can form a ternary complex with Ran and importin β. These data indicate that RanBP1 translocates through the pores by an active, nonclassical mechanism and requires Ran:GTP for nuclear accumulation. Shuttling of RanBP1 may function to clear nuclear pores of Ran:GTP, to prevent premature release of import cargo from transport receptors.

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