scholarly journals Cytoplasmic Sequestration of Rel Proteins by IκBα Requires CRM1-Dependent Nuclear Export

2000 ◽  
Vol 20 (6) ◽  
pp. 2269-2284 ◽  
Author(s):  
Winnie F. Tam ◽  
Linda H. Lee ◽  
Laura Davis ◽  
Ranjan Sen
Keyword(s):  
Nuclear Export ◽  
Mammalian Cells ◽  
Regulatory Function ◽  
Main Function ◽  
Cell Cytoplasm ◽  
Leptomycin B ◽  
Major Site ◽  
Cos Cells ◽  
Nuclear Export Receptor ◽  
Nuclear Relocation

ABSTRACT Rel and IκB protein families form a complex cellular regulatory network. A major regulatory function of IκB proteins is to retain Rel proteins in the cell cytoplasm. In addition, IκB proteins have also been postulated to serve nuclear functions. These include the maintenance of inducible NF-κB-dependent gene transcription, as well as termination of inducible transcription. We show that IκBα shuttles between the nucleus and the cytoplasm, utilizing the nuclear export receptor CRM1. A CRM1-binding export sequence was identified in the N-terminal domain of IκBα but not in that of IκBβ or IκBɛ. By reconstituting major aspects of NF-κB–IκB sequestration in yeast, we demonstrate that cytoplasmic retention of p65 (also called RelA) by IκBα requires Crm1p-dependent nuclear export. In mammalian cells, inhibition of CRM1 by leptomycin B resulted in nuclear localization of cotransfected p65 and IκBα in COS cells and enhanced nuclear relocation of endogenous p65 in T cells. These observations suggest that the main function of IκBα is that of a nuclear export chaperone rather than a cytoplasmic tether. We propose that the nucleus is the major site of p65-IκBα association, from where these complexes must be exported in order to create the cytoplasmic pool.

A global survey of CRM1-dependent nuclear export sequences in the human deubiquitinase family

2011 ◽  
Vol 441 (1) ◽  
pp. 209-217 ◽  
Author(s):  
Iraia García-Santisteban ◽  
Sonia Bañuelos ◽  
Jose A. Rodríguez
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Site Directed Mutagenesis ◽  
The Novel ◽  
Sequence Motifs ◽  
Leptomycin B ◽  
Specific Amino Acid ◽  
Green Fluorescent ◽  
Nuclear Export Receptor

The mechanisms that regulate the nucleocytoplasmic localization of human deubiquitinases remain largely unknown. The nuclear export receptor CRM1 binds to specific amino acid motifs termed NESs (nuclear export sequences). By using in silico prediction and experimental validation of candidate sequences, we identified 32 active NESs and 78 inactive NES-like motifs in human deubiquitinases. These results allowed us to evaluate the performance of three programs widely used for NES prediction, and to add novel information to the recently redefined NES consensus. The novel NESs identified in the present study reveal a subset of 22 deubiquitinases bearing motifs that might mediate their binding to CRM1. We tested the effect of the CRM1 inhibitor LMB (leptomycin B) on the localization of YFP (yellow fluorescent protein)- or GFP (green fluorescent protein)-tagged versions of six NES-bearing deubiquitinases [USP (ubiquitin-specific peptidase) 1, USP3, USP7, USP21, CYLD (cylindromatosis) and OTUD7B (OTU-domain-containing 7B)]. YFP–USP21 and, to a lesser extent, GFP–OTUD7B relocated from the cytoplasm to the nucleus in the presence of LMB, revealing their nucleocytoplasmic shuttling capability. Two sequence motifs in USP21 had been identified during our survey as active NESs in the export assay. Using site-directed mutagenesis, we show that one of these motifs mediates USP21 nuclear export, whereas the second motif is not functional in the context of full-length USP21.

scholarly journals Leptomycin B, an Inhibitor of the Nuclear Export Receptor CRM1, Inhibits COX-2 Expression

2002 ◽  
Vol 278 (5) ◽  
pp. 2773-2776 ◽  
Author(s):  
Byeong-Churl Jang ◽  
Ursula Muñoz-Najar ◽  
Ji-Hye Paik ◽  
Kevin Claffey ◽  
Minoru Yoshida ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Leptomycin B ◽  
Cox 2 ◽  
Nuclear Export Receptor

scholarly journals Structural and Functional Analysis of the RNA Transport Element, a Member of an Extensive Family Present in the Mouse Genome

2005 ◽  
Vol 79 (4) ◽  
pp. 2356-2365 ◽  
Author(s):  
Sergey Smulevitch ◽  
Daniel Michalowski ◽  
Andrei S. Zolotukhin ◽  
Ralf Schneider ◽  
Jenifer Bear ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Minimal Element ◽  
Structural Features ◽  
Rna Transport ◽  
Immunodeficiency Virus ◽  
Responsive Element ◽  
Nuclear Export Receptor ◽  
Intracisternal A Particle

ABSTRACT We previously identified an RNA transport element (RTE), present in a subclass of rodent intracisternal A particle retroelements (F. Nappi, R. Schneider, A. Zolotukhin, S. Smulevitch, D. Michalowski, J. Bear, B. Felber, and G. Pavlakis, J. Virol. 75:4558-4569, 2001), that is able to replace Rev-responsive element regulation in human immunodeficiency virus type 1. RTE-directed mRNA export is mediated by a still-unknown cellular factor(s), is independent of the CRM1 nuclear export receptor, and is conserved among vertebrates. Here we show that this RTE folds into an extended RNA secondary structure and thus does not resemble any known RTEs. Computer searches revealed the presence of 105 identical elements and more than 3,000 related elements which share at least 70% sequence identity with the RTE and which are found on all mouse chromosomes. These related elements are predicted to fold into RTE-like structures. Comparison of the sequences and structures revealed that the RTE and related elements can be divided into four groups. Mutagenesis of the RTE revealed that the minimal element contains four internal stem-loops, which are indispensable for function in mammalian cells. In contrast, only part of the element is essential to mediate RNA transport in microinjected Xenopus laevis oocyte nuclei. Importantly, the minimal RTE able to promote RNA transport has key structural features which are preserved in all the RTE-related elements, further supporting their functional importance. Therefore, RTE function depends on a complex secondary structure that is important for the interaction with the cellular export factor(s).

scholarly journals The Mobile FG Nucleoporin Nup98 Is a Cofactor for Crm1-dependent Protein Export

2010 ◽  
Vol 21 (11) ◽  
pp. 1885-1896 ◽  
Author(s):  
Masahiro Oka ◽  
Munehiro Asally ◽  
Yoshinari Yasuda ◽  
Yutaka Ogawa ◽  
Taro Tachibana ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Nuclear Protein ◽  
Mutational Analysis ◽  
Specific Inhibitor ◽  
Protein Export ◽  
Dependent Manner ◽  
Repeat Region ◽  
Leptomycin B ◽  
Dependent Protein ◽  
Nuclear Export Receptor

Nup98 is a mobile nucleoporin that forms distinct dots in the nucleus, and, although a role for Nup98 in nuclear transport has been suggested, its precise function remains unclear. Here, we show that Nup98 plays an important role in Crm1-mediated nuclear protein export. Nuclear, but not cytoplasmic, dots of EGFP-tagged Nup98 disappeared rapidly after cell treatment with leptomycin B, a specific inhibitor of the nuclear export receptor, Crm1. Mutational analysis demonstrated that Nup98 physically and functionally interacts with Crm1 in a RanGTP-dependent manner through its N-terminal phenylalanine-glycine (FG) repeat region. Moreover, the activity of the Nup98-Crm1 complex was modulated by RanBP3, a known cofactor for Crm1-mediated nuclear export. Finally, cytoplasmic microinjection of anti-Nup98 inhibited the Crm1-dependent nuclear export of proteins, concomitant with the accumulation of anti-Nup98 in the nucleus. These results clearly demonstrate that Nup98 functions as a novel shuttling cofactor for Crm1-mediated nuclear export in conjunction with RanBP3.

scholarly journals A nuclear export sequence promotes CRM1-dependent targeting of the nucleoporin Nup214 to the nuclear pore complex

2021 ◽  
Vol 134 (6) ◽  
Author(s):  
Mohamed Hamed ◽  
Birgit Caspar ◽  
Sarah A. Port ◽  
Ralph H. Kehlenbach
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Export ◽  
Nuclear Pore ◽  
Wild Type ◽  
Leptomycin B ◽  
Nuclear Export Sequence ◽  
Binding Partners ◽  
Dependent Protein ◽  
Nuclear Export Receptor ◽  
Cytoplasmic Side

ABSTRACT Nup214 is a major nucleoporin on the cytoplasmic side of the nuclear pore complex with roles in late steps of nuclear protein and mRNA export. It interacts with the nuclear export receptor CRM1 (also known as XPO1) via characteristic phenylalanine-glycine (FG) repeats in its C-terminal region. Here, we identify a classic nuclear export sequence (NES) in Nup214 that mediates Ran-dependent binding to CRM1. Nup214 versions with mutations in the NES, as well as wild-type Nup214 in the presence of the selective CRM1 inhibitor leptomycin B, accumulate in the nucleus of Nup214-overexpressing cells. Furthermore, physiological binding partners of Nup214, such as Nup62 and Nup88, are recruited to the nucleus together with Nup214. Nuclear export of mutant Nup214 can be rescued by artificial nuclear export sequences at the C-terminal end of Nup214, leading also to a correct localization of Nup88. Our results suggest a function of the Nup214 NES in the biogenesis of the nuclear pore complex and/or in terminal steps of CRM1-dependent protein export.

Nuclear pore localization and nucleocytoplasmic transport of eIF-5A: evidence for direct interaction with the export receptor CRM1

1999 ◽  
Vol 112 (14) ◽  
pp. 2369-2380 ◽  
Author(s):  
O. Rosorius ◽  
B. Reichart ◽  
F. Kratzer ◽  
P. Heger ◽  
M.C. Dabauvalle ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Mammalian Cells ◽  
Direct Interaction ◽  
Nucleocytoplasmic Transport ◽  
Cellular Protein ◽  
Initiation Factor ◽  
Immunogold Electron Microscopy ◽  
Nuclear Pore ◽  
Nuclear Export Receptor

Eukaryotic initiation factor 5A (eIF-5A) is the only cellular protein known to contain the unusual amino acid hypusine. The exact in vivo function of eIF-5A, however, is to date unknown. The finding that eIF-5A is an essential cofactor of the human immunodeficiency virus type 1 (HIV-1) Rev RNA transport factor suggested that eIF-5A is part of a specific nuclear export pathway. In this study we used indirect immunofluorescence and immunogold electron microscopy to demonstrate that eIF-5A accumulates at nuclear pore-associated intranuclear filaments in mammalian cells and Xenopus oocytes. We are able to show that eIF-5A interacts with the general nuclear export receptor, CRM1. Furthermore, microinjection studies in somatic cells revealed that eIF-5A is transported from the nucleus to the cytoplasm, and that this nuclear export is blocked by leptomycin B. Our data demonstrate that eIF-5A is a nucleocytoplasmic shuttle protein.

scholarly journals Localized RanGTP Accumulation Promotes Microtubule Nucleation at Kinetochores in Somatic Mammalian Cells

2008 ◽  
Vol 19 (5) ◽  
pp. 1873-1882 ◽  
Author(s):  
Liliana Torosantucci ◽  
Maria De Luca ◽  
Giulia Guarguaglini ◽  
Patrizia Lavia ◽  
Francesca Degrassi
Keyword(s):  
Nuclear Export ◽  
Mammalian Cells ◽  
Nuclear Export Signal ◽  
Spindle Assembly ◽  
Microtubule Nucleation ◽  
Leptomycin B ◽  
Data Support ◽  
Assembly Pathways ◽  
Export Signal ◽  
In Cells

Centrosomes are the major sites for microtubule nucleation in mammalian cells, although both chromatin- and kinetochore-mediated microtubule nucleation have been observed during spindle assembly. As yet, it is still unclear whether these pathways are coregulated, and the molecular requirements for microtubule nucleation at kinetochore are not fully understood. This work demonstrates that kinetochores are initial sites for microtubule nucleation during spindle reassembly after nocodazole. This process requires local RanGTP accumulation concomitant with delocalization from kinetochores of the hydrolysis factor RanGAP1. Kinetochore-driven microtubule nucleation is also activated after cold-induced microtubule disassembly when centrosome nucleation is impaired, e.g., after Polo-like kinase 1 depletion, indicating that dominant centrosome activity normally masks the kinetochore-driven pathway. In cells with unperturbed centrosome nucleation, defective RanGAP1 recruitment at kinetochores after treatment with the Crm1 inhibitor leptomycin B activates kinetochore microtubule nucleation after cold. Finally, nascent microtubules associate with the RanGTP-regulated microtubule-stabilizing protein HURP in both cold- and nocodazole-treated cells. These data support a model for spindle assembly in which RanGTP-dependent abundance of nucleation/stabilization factors at centrosomes and kinetochores orchestrates the contribution of the two spindle assembly pathways in mammalian cells. The complex of RanGTP, the export receptor Crm1, and nuclear export signal-bearing proteins regulates microtubule nucleation at kinetochores.

scholarly journals Involvement of CRM1, a nuclear export receptor, in mRNA export in mammalian cells and fission yeast

1999 ◽  
Vol 4 (5) ◽  
pp. 291-297 ◽  
Author(s):  
Mina Watanabe ◽  
Makoto Fukuda ◽  
Minoru Yoshida ◽  
Mitsuhiro Yanagida ◽  
Eisuke Nishida
Keyword(s):  
Fission Yeast ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Mrna Export ◽  
Nuclear Export Receptor

scholarly journals CRM1 promotes capsid disassembly and nuclear envelope translocation of adenovirus independently of its export function

2021 ◽  
Author(s):  
Floriane Lagadec ◽  
Irene Carlon-Andres ◽  
Jessica Ragues ◽  
Sarah Port ◽  
Harald Wodrich ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Microtubule Organizing Center ◽  
Leptomycin B ◽  
Pore Complexes ◽  
Nuclear Export Receptor ◽  
Mitotic Cells ◽  
Capsid Disassembly

After receptor-mediated endocytosis and endosomal escape, adenoviral capsids can travel via microtubule organizing centers to the nuclear envelope. Upon capsid disassembly, viral genome import into nuclei of interphase cells then occurs through nuclear pore complexes, involving the nucleoporins Nup214 and Nup358. Import also requires the activity of the classic nuclear export receptor CRM1, as it is blocked by the selective inhibitor leptomycin B. We have now used artificially enucleated as well as mitotic cells to analyze the role of an intact nucleus in different steps of the viral life cycle. In enucleated U2OS cells, viral capsids traveled to the microtubule organizing center, whereas their removal from this complex was blocked, suggesting that this step required nuclear factors. In mitotic cells, on the other hand, CRM1 promoted capsid disassembly and genome release, suggesting a role of this protein that does not require intact nuclear envelopes or nuclear pore complexes and is distinct from its function as a nuclear export receptor. Similar to enucleation, inhibition of CRM1 by leptomycin B also leads to an arrest of adenoviral capsids at the microtubule organizing center. In a small-scale screen using leptomycin B-resistant versions of CRM1, we identified a mutant, CRM1 W142A P143A, that is compromised with respect to adenoviral capsid disassembly, both in interphase and in mitotic cells. Strikingly, this mutant is capable of exporting cargo proteins out of the nucleus of living cells or digitonin-permeabilized cells, pointing to a role of the mutated region that is not directly linked to nuclear export. IMPORTANCE A role of nucleoporins and of soluble transport factors in adenoviral genome import into the nucleus of infected cells in interphase has previously been established. The nuclear export receptor CRM1 promotes genome import, but its precise function is not known. Using enucleated and mitotic cells, we showed that CRM1 does not simply function by exporting a crucial factor out of the nucleus that would then trigger capsid disassembly and genome import. Instead, CRM1 has an export-independent role, a notion that is also supported by a mutant, CRM1 W142A P143A, which is export-competent but deficient in viral capsid disassembly, both in interphase and in mitotic cells.

scholarly journals Inhibition of XPO-1 Mediated Nuclear Export through the Michael-Acceptor Character of Chalcones

2021 ◽  
Vol 14 (11) ◽  
pp. 1131
Author(s):  
Marta Gargantilla ◽  
José López-Fernández ◽  
Maria-Jose Camarasa ◽  
Leentje Persoons ◽  
Dirk Daelemans ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Viral Infections ◽  
Nuclear Export Signal ◽  
Thiol Groups ◽  
Tumor Resistance ◽  
Leptomycin B ◽  
Covalent Interaction ◽  
Chalcone Derivatives ◽  
Binding Cleft ◽  
Nuclear Export Receptor

The nuclear export receptor exportin-1 (XPO1, CRM1) mediates the nuclear export of proteins that contain a leucine-rich nuclear export signal (NES) towards the cytoplasm. XPO1 is considered a relevant target in different human diseases, particularly in hematological malignancies, tumor resistance, inflammation, neurodegeneration and viral infections. Thus, its pharmacological inhibition is of significant therapeutic interest. The best inhibitors described so far (leptomycin B and SINE compounds) interact with XPO1 through a covalent interaction with Cys528 located in the NES-binding cleft of XPO1. Based on the well-established feature of chalcone derivatives to react with thiol groups via hetero-Michael addition reactions, we have synthesized two series of chalcones. Their capacity to react with thiol groups was tested by incubation with GSH to afford the hetero-Michael adducts that evolved backwards to the initial chalcone through a retro-Michael reaction, supporting that the covalent interaction with thiols could be reversible. The chalcone derivatives were evaluated in antiproliferative assays against a panel of cancer cell lines and as XPO1 inhibitors, and a good correlation was observed with the results obtained in both assays. Moreover, no inhibition of the cargo export was observed when the two prototype chalcones 9 and 10 were tested against a XPO1-mutated Jurkat cell line (XPO1C528S), highlighting the importance of the Cys at the NES-binding cleft for inhibition. Finally, their interaction at the molecular level at the NES-binding cleft was studied by applying the computational tool CovDock.

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