Several Phenylalanine-Glycine Motives in the Nucleoporin Nup214 Are Essential for Binding of the Nuclear Export Receptor CRM1

Roloff, Stephanie; Spillner, Christiane; Kehlenbach, Ralph H.

doi:10.1074/jbc.m112.433243

Cited by 32 publications

(42 citation statements)

References 60 publications

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“…3B). This indicated that the C-terminal region of Nup214 can function as a dominant negative mutant of endogenous Nup214, as reported previously (39). From these observations, we conclude that SET-Nup214 and DEK-Nup214 change the subcellular localizations of endogenous proteins harboring NES by inhibiting the function of endogenous Nup214.…”

Section: Resultssupporting

confidence: 61%

“…In addition, in cells expressing DEK-Nup214, we could not find clear accumulation of NXF1 at the sites where DEK-Nup214 was accumulated. It was reported previously that the FG repeat region of Nup214 plays crucial roles in binding with NTRs (31,32,39,42). Therefore, we next examined the importance of the FG repeat region of the fusion proteins for the changes in the localization of XPO1 and NFX1 by using the deletion mutant of SET-Nup214 termed SET-Nup214(1637), which lacks the FG repeat region of SET-Nup214.…”

Section: Resultsmentioning

confidence: 99%

“…The export of both mRNAs and proteins is severely reduced when the level of Nup214 expression is decreased (36)(37)(38), and ectopic overexpression of truncated Nup214 causes the accumulation of NES proteins in the nucleus in mammals (39). On the other hand, in Drosophila spp., nup214 deletion enhances the export of green fluorescent protein (GFP) fused with the NES (40).…”

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confidence: 99%

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Leukemia-Associated Nup214 Fusion Proteins Disturb the XPO1-Mediated Nuclear-Cytoplasmic Transport Pathway and Thereby the NF-κB Signaling Pathway

Saito

Cigdem

Okuwaki

et al. 2016

Molecular and Cellular Biology

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c Nuclear-cytoplasmic transport through nuclear pore complexes is mediated by nuclear transport receptors. Previous reports have suggested that aberrant nuclear-cytoplasmic transport due to mutations or overexpression of nuclear pore complexes and nuclear transport receptors is closely linked to diseases. Nup214, a component of nuclear pore complexes, has been found as chimeric fusion proteins in leukemia. Among various Nup214 fusion proteins, SET-Nup214 and DEKNup214 have been shown to be engaged in tumorigenesis, but their oncogenic mechanisms remain unclear. In this study, we examined the functions of the Nup214 fusion proteins by focusing on their effects on nuclear-cytoplasmic transport. We found that SET-Nup214 and DEK-Nup214 interact with exportin-1 (XPO1)/CRM1 and nuclear RNA export factor 1 (NXF1)/TAP, which mediate leucine-rich nuclear export signal (NES)-dependent protein export and mRNA export, respectively. SET-Nup214 and DEK-Nup214 decreased the XPO1-mediated nuclear export of NES proteins such as cyclin B and proteins involved in the NF-B signaling pathway by tethering XPO1 onto nuclear dots where Nup214 fusion proteins are localized. We also demonstrated that SET-Nup214 and DEK-Nup214 expression inhibited NF-B-mediated transcription by abnormal tethering of the complex containing p65 and its inhibitor, IB, in the nucleus. These results suggest that SETNup214 and DEK-Nup214 perturb the regulation of gene expression through alteration of the nuclear-cytoplasmic transport system. Biological macromolecules are transported between the nucleus and the cytoplasm in response to extracellular signals. Transport of molecules with a molecular mass greater than 40 kDa does not occur by simple diffusion but is generally facilitated by nuclear transport receptors (NTRs) through nuclear pore complexes (NPCs) embedded in the nuclear envelope (1-3). Controlled nuclear-cytoplasmic transport plays important roles in maintaining cellular integrity in eukaryotic cells. It is reported that aberrant subcellular localization of some proteins is associated with various cancer cases (4). p53 has nuclear localization signals (NLSs) and nuclear export signals (NESs), and the accumulation of p53 in the cytoplasm has been reported to be a prognostic indicator in cancer (5). The nuclear factor B (NF-B) transcription factor is observed mainly in the cytoplasm in normal cells, whereas in many cancer cells, it is localized largely in the nucleus (6).In addition to aberrant subcellular localization of proteins in cancer, mutations of genes encoding NTRs and NPC components are found in various types of cancer (7). Mutations of exportin-5 (XPO5) and exportin-1 (XPO1)/CRM1, members of export receptors (8-10), are found in solid cancer and leukemia, respectively. Four nucleoporins-Nup98, Nup214/CAN, Nup358/ RanBP2, and Tpr-have been reported to form chimeric proteins by chromosomal translocations mainly in leukemia (11-15). Nup214, located at the cytoplasmic filament of the NPC, interacts with NTRs to control macromolecular transport....

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Section: Resultssupporting

confidence: 61%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Leukemia-Associated Nup214 Fusion Proteins Disturb the XPO1-Mediated Nuclear-Cytoplasmic Transport Pathway and Thereby the NF-κB Signaling Pathway

Saito

Cigdem

Okuwaki

et al. 2016

Molecular and Cellular Biology

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“…Third, there may be some redundancy in the roles of FG-Nups, but this redundancy may be necessary, for example, to guarantee that some FG-Nups can bind critical NTRs that carry crucial macromolecules cross the NPC in any situation. [35][36][37] Another evolutionary feature present in FG-Nups in higher eukaryotic cells is the post-transcription modification. For example, in metazoans, FG-Nups are often glycosylated via O-linked-N-acetyL-glucosamine transferase, but functions of this modification in cells remain unknown.…”

Section: Fg-nups In Yeast Cellsmentioning

confidence: 99%

“…6 It is also possible that, at least in some cases, a cargo-bound NTR that binds to multiple FG-Nups would enhance their avidity of the interactions, which is likely necessary for their ultimate transporting success. 36,58 On the other hand, multiple FG-repeats of each FG-Nup could cope to ease the heavy transporting tasks and also provide cells with some necessary and some redundant functions. Sequence alignment of FG-Nups from 4 species of yeast identified small stretches of conserved AAs (6-11 AAs) (called 'islands') among divergent FG-domains.…”

Section: Multiple Binding Sites Of Ntrs For Fg-nupsmentioning

confidence: 99%

The selective permeability barrier in the nuclear pore complex

Goryaynov

Yang

2016

Nucleus

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The nuclear pore complex (NPC) mediates the shuttle transport of macromolecules between the nucleus and cytoplasm in eukaryotic cells. The permeability barrier formed by intrinsically disordered phenylalanine-glycine-rich nucleoporins (FG-Nups) in the NPC functions as the critical selective control for nucleocytoplasmic transport. Signal-independent small molecules (< 40 kDa) passively diffuse through the pore, but passage of large cargo molecules is inhibited unless they are chaperoned by nuclear transport receptors (NTRs). NTRs are capable of interacting with FG-Nups and guide the cargos to cross the barrier by facilitated diffusion. The native conformation of the FGNups permeability barrier and the competition among multiple NTRs interacting with this barrier in the native NPCs are the 2 core questions still being highly debated in the field. Recently, we applied high-speed super-resolution fluorescence microscopy to map out the natural structure of the FGNups barrier and determined the competition among multiple NTRs as they interact with the barrier in the native NPCs. In this extra-view article, we will review the current understanding in the configuration and function of FG-Nups barrier and highlight the new evidence obtained recently to answer the core questions in nucleocytoplasmic transport.

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Analysis of CRM1-Dependent Nuclear Export in Permeabilized Cells

Kehlenbach

Port

2016

Methods in Molecular Biology

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Nuclear protein import and export assays in permeabilized cells have been instrumental for the identification of transport factors and for the molecular characterization of nucleocytoplasmic transport pathways. Our original assay to quantitatively analyze CRM1-dependent export was based on stably transfected cells expressing GFP-NFAT. We now present a simplified version of the assay using transiently transfected cells expressing GFP-NFAT or GFP-snurportin1 as a fluorescent export cargo and mCherry-emerin as a marker protein for transfected cells. CRM1- and Ran-dependent export is recapitulated in digitonin-permeabilized cells and quantified by flow cytometry. The assay should be applicable to other combinations of cargo and marker proteins.

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Several Phenylalanine-Glycine Motives in the Nucleoporin Nup214 Are Essential for Binding of the Nuclear Export Receptor CRM1

Cited by 32 publications

References 60 publications

Leukemia-Associated Nup214 Fusion Proteins Disturb the XPO1-Mediated Nuclear-Cytoplasmic Transport Pathway and Thereby the NF-κB Signaling Pathway

Leukemia-Associated Nup214 Fusion Proteins Disturb the XPO1-Mediated Nuclear-Cytoplasmic Transport Pathway and Thereby the NF-κB Signaling Pathway

The selective permeability barrier in the nuclear pore complex

Analysis of CRM1-Dependent Nuclear Export in Permeabilized Cells

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