scholarly journals Nuclear Export of NBN Is Required for Normal Cellular Responses to Radiation

2008 ◽  
Vol 29 (4) ◽  
pp. 1000-1006 ◽  
Author(s):  
Christine S. Vissinga ◽  
Tiong C. Yeo ◽  
Sarah Warren ◽  
James V. Brawley ◽  
Jennifer Phillips ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Mammalian Cells ◽  
Cellular Response ◽  
Nuclear Export Signal ◽  
Nijmegen Breakage Syndrome ◽  
Dna Double Strand Breaks ◽  
Gene Encoding ◽  
Mrn Complex ◽  
Nbn Gene

ABSTRACT Nijmegen breakage syndrome arises from hypomorphic mutations in the NBN gene encoding nibrin, a component of the MRE11/RAD50/nibrin (MRN) complex. In mammalian cells, the MRN complex localizes to the nucleus, where it plays multiple roles in the cellular response to DNA double-strand breaks. In the current study, sequences in mouse nibrin required to direct the nuclear localization of the MRN complex were identified by site-specific mutagenesis. Unexpectedly, nibrin was found to contain both nuclear localizing signal (NLS) sequences and a nuclear export signal (NES) sequence whose functions were confirmed by mutagenesis. Both nuclear import and export sequences were active in vivo. Disruption of either the NLS or NES sequences of nibrin significantly altered the cellular distribution of nibrin and Mre11 and impaired survival after exposure to ionizing radiation. Mutation of the NES sequence in nibrin slowed the turnover of phosphorylated nibrin after irradiation, indicating that nuclear export of nibrin may function, in part, to downregulate posttranslationally modified MRN complex components after DNA damage responses are complete.

scholarly journals Constitutive phosphorylation of MDC1 physically links the MRE11–RAD50–NBS1 complex to damaged chromatin

2008 ◽  
Vol 181 (2) ◽  
pp. 227-240 ◽  
Author(s):  
Christoph Spycher ◽  
Edward S. Miller ◽  
Kelly Townsend ◽  
Lucijana Pavic ◽  
Nicholas A. Morrice ◽  
...  
Keyword(s):  
Nijmegen Breakage Syndrome ◽  
Dependent Manner ◽  
Sequence Motifs ◽  
Dna Double Strand Breaks ◽  
Focus Formation ◽  
Strand Breaks ◽  
Mrn Complex ◽  
Interfering Rna

The MRE11–RAD50–Nijmegen breakage syndrome 1 (NBS1 [MRN]) complex accumulates at sites of DNA double-strand breaks (DSBs) in microscopically discernible nuclear foci. Focus formation by the MRN complex is dependent on MDC1, a large nuclear protein that directly interacts with phosphorylated H2AX. In this study, we identified a region in MDC1 that is essential for the focal accumulation of the MRN complex at sites of DNA damage. This region contains multiple conserved acidic sequence motifs that are constitutively phosphorylated in vivo. We show that these motifs are efficiently phosphorylated by caseine kinase 2 (CK2) in vitro and directly interact with the N-terminal forkhead-associated domain of NBS1 in a phosphorylation-dependent manner. Mutation of these conserved motifs in MDC1 or depletion of CK2 by small interfering RNA disrupts the interaction between MDC1 and NBS1 and abrogates accumulation of the MRN complex at sites of DNA DSBs in vivo. Thus, our data reveal the mechanism by which MDC1 physically couples the MRN complex to damaged chromatin.

scholarly journals Nuclear export of misfolded SOD1 mediated by a normally buried NES-like sequence reduces proteotoxicity in the nucleus

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Yongwang Zhong ◽  
Jiou Wang ◽  
Mark J Henderson ◽  
Peixin Yang ◽  
Brian M Hagen ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Defense Mechanism ◽  
Consensus Sequence ◽  
Nuclear Export Signal ◽  
Egg Laying ◽  
Gene Encoding ◽  
Mutant Sod1 ◽  
Misfolded Sod1

Over 170 different mutations in the gene encoding SOD1 all cause amyotrophic lateral sclerosis (ALS). Available studies have been primarily focused on the mechanisms underlying mutant SOD1 cytotoxicity. How cells defend against the cytotoxicity remains largely unknown. Here, we show that misfolding of ALS-linked SOD1 mutants and wild-type (wt) SOD1 exposes a normally buried nuclear export signal (NES)-like sequence. The nuclear export carrier protein CRM1 recognizes this NES-like sequence and exports misfolded SOD1 to the cytoplasm. Antibodies against the NES-like sequence recognize misfolded SOD1, but not native wt SOD1 both in vitro and in vivo. Disruption of the NES consensus sequence relocalizes mutant SOD1 to the nucleus, resulting in higher toxicity in cells, and severer impairments in locomotion, egg-laying, and survival in Caenorhabditis elegans. Our data suggest that SOD1 mutants are removed from the nucleus by CRM1 as a defense mechanism against proteotoxicity of misfolded SOD1 in the nucleus.

scholarly journals NXT1 (p15) Is a Crucial Cellular Cofactor in TAP-Dependent Export of Intron-Containing RNA in Mammalian Cells

2001 ◽  
Vol 21 (7) ◽  
pp. 2545-2554 ◽  
Author(s):  
Brian W. Guzik ◽  
Lyne Levesque ◽  
Susan Prasad ◽  
Yeou-Cherng Bor ◽  
Ben E. Black ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Mammalian Cells ◽  
Nuclear Export Signal ◽  
Chimeric Proteins ◽  
Mutant Proteins ◽  
Rev Response Element ◽  
Hiv Rna ◽  
Cellular Cofactor ◽  
Rna Export

ABSTRACT TAP, the human homologue of the yeast protein Mex67p, has been proposed to serve a role in mRNA export in mammalian cells. We have examined the ability of TAP to mediate export of Rev response element (RRE)-containing human immunodeficiency virus (HIV) RNA, a well-characterized export substrate in mammalian cells. To do this, the TAP gene was fused in frame to either RevM10 or RevΔ78–79. These proteins are nonfunctional Rev mutant proteins that can bind to HIV RNA containing the RRE in vivo but are unable to mediate the export of this RNA to the cytoplasm. However, the fusion of TAP to either of these mutant proteins gave rise to chimeric proteins that were able to complement Rev function. Significantly, cotransfection with a vector expressing NXT1 (p15), an NTF2-related cellular factor that binds to TAP, led to dramatic enhancement of the ability of the chimeric proteins to mediate RNA export. Mutant-protein analysis demonstrated that the domain necessary for nuclear export mapped to the C-terminal region of TAP and required the domain that interacts with NXT1, as well as the region that has been shown to interact with nucleoporins. RevM10-TAP function was leptomycin B insensitive. In contrast, the function of this protein was inhibited by ΔCAN, a protein consisting of part of the FG repeat domain of CAN/Nup214. These results show that TAP can complement Rev nuclear export signal function and redirect the export of intron-containing RNA to a CRM1-independent pathway. These experiments support the role of TAP as an RNA export factor in mammalian cells. In addition, they indicate that NXT1 serves as a crucial cellular cofactor in this process.

scholarly journals Phosphorylation of SDT repeats in the MDC1 N terminus triggers retention of NBS1 at the DNA damage–modified chromatin

2008 ◽  
Vol 181 (2) ◽  
pp. 213-226 ◽  
Author(s):  
Fredrik Melander ◽  
Simon Bekker-Jensen ◽  
Jacob Falck ◽  
Jiri Bartek ◽  
Niels Mailand ◽  
...  
Keyword(s):  
Adaptor Protein ◽  
Nijmegen Breakage Syndrome ◽  
Local Concentration ◽  
Dna Double Strand Breaks ◽  
Phosphate Binding ◽  
Chromosomal Breakage ◽  
Mrn Complex ◽  
N Terminus

DNA double-strand breaks (DSBs) trigger accumulation of the MRE11–RAD50–Nijmegen breakage syndrome 1 (NBS1 [MRN]) complex, whose retention on the DSB-flanking chromatin facilitates survival. Chromatin retention of MRN requires the MDC1 adaptor protein, but the mechanism behind the MRN–MDC1 interaction is unknown. We show that the NBS1 subunit of MRN interacts with the MDC1 N terminus enriched in Ser-Asp-Thr (SDT) repeats. This interaction was constitutive and mediated by binding between the phosphorylated SDT repeats of MDC1 and the phosphate-binding forkhead-associated domain of NBS1. Phosphorylation of the SDT repeats by casein kinase 2 (CK2) was sufficient to trigger MDC1–NBS1 interaction in vitro, and MDC1 associated with CK2 activity in cells. Inhibition of CK2 reduced SDT phosphorylation in vivo, and disruption of the SDT-associated phosphoacceptor sites prevented the retention of NBS1 at DSBs. Together, these data suggest that phosphorylation of the SDT repeats in the MDC1 N terminus functions to recruit NBS1 and, thereby, increases the local concentration of MRN at the sites of chromosomal breakage.

scholarly journals Notch1 regulated autophagy controls survival and suppressor activity of activated murine T-regulatory cells

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Nimi Marcel ◽  
Apurva Sarin
Keyword(s):  
Cell Fate ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Cellular Response ◽  
T Regulatory Cells ◽  
Nuclear Export Signal ◽  
Ectopic Expression ◽  
Notch Pathway ◽  
Regulatory Cells ◽  
Cell Fate Decisions

Cell survival is one of several processes regulated by the Notch pathway in mammalian cells. Here we report functional outcomes of non-nuclear Notch signaling to activate autophagy, a conserved cellular response to nutrient stress, regulating survival in murine natural T-regulatory cells (Tregs), an immune subset controlling tolerance and inflammation. Induction of autophagy required ligand-dependent, Notch intracellular domain (NIC) activity, which controlled mitochondrial organization and survival of activated Tregs. Consistently, NIC immune-precipitated Beclin and Atg14, constituents of the autophagy initiation complex. Further, ectopic expression of an effector of autophagy (Atg3) or recombinant NIC tagged to a nuclear export signal (NIC-NES), restored autophagy and suppressor function in Notch1-/- Tregs. Furthermore, Notch1 deficiency in the Treg lineage resulted in immune hyperactivity, implicating Notch activity in Treg homeostasis. Notch1 integration with autophagy, revealed in these experiments, holds implications for Notch regulated cell-fate decisions governing differentiation.

scholarly journals eEF1A Is a Novel Component of the Mammalian Nuclear Protein Export Machinery

2008 ◽  
Vol 19 (12) ◽  
pp. 5296-5308 ◽  
Author(s):  
Mireille Khacho ◽  
Karim Mekhail ◽  
Karine Pilon-Larose ◽  
Arnim Pause ◽  
Jocelyn Côté ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Nuclear Export Signal ◽  
Point Mutations ◽  
Nuclear Retention ◽  
Von Hippel Lindau ◽  
Trna Species

The cytoplasmic translation factor eEF1A has been implicated in the nuclear export of tRNA species in lower eukaryotes. Here we demonstrate that eEF1A plays a central role in nuclear export of proteins in mammalian cells. TD-NEM (transcription-dependent nuclear export motif), a newly characterized nuclear export signal, mediates efficient nuclear export of several proteins including the von Hippel-Lindau (VHL) tumor suppressor and the poly(A)-binding protein (PABP1) in a manner that is dependent on ongoing RNA polymerase II (RNA PolII)-dependent transcription. eEF1A interacts specifically with TD-NEM of VHL and PABP1 and disrupting this interaction, by point mutations of key TD-NEM residues or treatment with actinomycin D, an inhibitor of RNA PolII-dependent transcription, prevents assembly and nuclear export. siRNA-induced knockdown or antibody-mediated depletion of eEF1A prevents in vivo and in vitro nuclear export of TD-NEM–containing proteins. Nuclear retention experiments and inhibition of the Exportin-5 pathway suggest that eEF1A stimulates nuclear export of proteins from the cytoplasmic side of the nuclear envelope, without entering the nucleus. Together, these data identify a role for eEF1A, a cytoplasmic mediator of tRNA export in yeast, in the nuclear export of proteins in mammalian cells. These results also provide a link between the translational apparatus and subcellular trafficking machinery demonstrating that these two central pathways in basic metabolism can act cooperatively.

scholarly journals Spongiform Neurodegeneration-associated E3 Ligase Mahogunin Ubiquitylates TSG101 and Regulates Endosomal Trafficking

2007 ◽  
Vol 18 (4) ◽  
pp. 1129-1142 ◽  
Author(s):  
Bong Yoon Kim ◽  
James A. Olzmann ◽  
Gregory S. Barsh ◽  
Lih-Shen Chin ◽  
Lian Li
Keyword(s):  
Mammalian Cells ◽  
Growth Factor Receptor ◽  
Endosomal Trafficking ◽  
Small Interfering Rnas ◽  
Gene Encoding ◽  
Endosomal Sorting ◽  
Ubiquitin Protein Ligase ◽  
Downstream Signaling

A null mutation in the gene encoding the putative E3 ubiquitin–protein ligase Mahogunin causes spongiform neurodegeneration, a recessively transmitted prion-like disease in mice. However, no substrates of Mahogunin have been identified, and the cellular role of Mahogunin is unknown. Here, we report the identification of TSG101, a key component of the endosomal sorting complex required for transport (ESCRT)-I, as a specific Mahogunin substrate. We find that Mahogunin interacts with the ubiquitin E2 variant (UEV) domain of TSG101 via its PSAP motif and that it catalyzes monoubiquitylation of TSG101 both in vivo and in vitro. Depletion of Mahogunin by small interfering RNAs in mammalian cells disrupts endosome-to-lysosome trafficking of epidermal growth factor receptor, resulting in prolonged activation of a downstream signaling cascade. Our findings support a role for Mahogunin in a proteasome-independent ubiquitylation pathway and suggest a link between dysregulation of endosomal trafficking and spongiform neurodegeneration.

scholarly journals Characterization of a murine gene encoding a developmentally regulated cytoplasmic dual-specificity mitogen-activated protein kinase phosphatase

2002 ◽  
Vol 364 (1) ◽  
pp. 145-155 ◽  
Author(s):  
Robin J. DICKINSON ◽  
David J. WILLIAMS ◽  
David N. SLACK ◽  
Jill WILLIAMSON ◽  
Ole-Morten SETERNES ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Mammalian Cells ◽  
Critical Role ◽  
Nuclear Export Signal ◽  
Cellular Growth ◽  
Cell Fate Decisions ◽  
Isolation And Characterization ◽  
Dual Specificity ◽  
Mitogen Activated Protein

Mitogen-activated protein kinases (MAPKs) play a vital role in cellular growth control, but far less is known about these signalling pathways in the context of embryonic development. Duration and magnitude of MAPK activation are crucial factors in cell fate decisions, and reflect a balance between the activities of upstream activators and specific MAPK phosphatases (MKPs). Here, we report the isolation and characterization of the murine Pyst3 gene, which encodes a cytosolic dual-specificity MKP. This enzyme selectively interacts with, and is catalytically activated by, the ‘classical’ extracellular signal-regulated kinases (ERKs) 1 and 2 and, to a lesser extent, the stress-activated MAPK p38α. These properties define the ability of this enzyme to dephosphorylate and inactivate ERK1/2 and p38α, but not JNK (c-Jun N-terminal kinase) in vivo. When expressed in mammalian cells, the Pyst3 protein is predominantly cytoplasmic. Furthermore, leptomycin B causes a complete redistribution of the protein to the nucleus, implicating a CRM (chromosomal region maintenance)1/exportin 1-dependent nuclear export signal in determining the subcellular localization of this enzyme. Finally, whole-mount in situ hybridization studies in mouse embryos reveal that the Pyst3 gene is expressed specifically in the placenta, developing liver and in migratory muscle cells. Our results suggest that this enzyme may have a critical role in regulating the activity of MAPK signalling during early development and organogenesis.

scholarly journals A novel LIM protein Cal promotes cardiac differentiation by association with CSX/NKX2-5

2004 ◽  
Vol 164 (3) ◽  
pp. 395-405 ◽  
Author(s):  
Hiroshi Akazawa ◽  
Sumiyo Kudoh ◽  
Naoki Mochizuki ◽  
Noboru Takekoshi ◽  
Hiroyuki Takano ◽  
...  
Keyword(s):  
Heart Development ◽  
Nuclear Export ◽  
Target Genes ◽  
Nuclear Export Signal ◽  
Gene Promoter ◽  
Myocardial Cell ◽  
Cardiac Differentiation ◽  
Lim Protein

The cardiac homeobox transcription factor CSX/NKX2-5 plays an important role in vertebrate heart development. Using a yeast two-hybrid screening, we identified a novel LIM domain–containing protein, named CSX-associated LIM protein (Cal), that interacts with CSX/NKX2-5. CSX/NKX2-5 and Cal associate with each other both in vivo and in vitro, and the LIM domains of Cal and the homeodomain of CSX/NKX2-5 were necessary for mutual binding. Cal itself possessed the transcription-promoting activity, and cotransfection of Cal enhanced CSX/NKX2-5–induced activation of atrial natriuretic peptide gene promoter. Cal contained a functional nuclear export signal and shuttled from the cytoplasm into the nucleus in response to calcium. Accumulation of Cal in the nucleus of P19CL6 cells promoted myocardial cell differentiation accompanied by increased expression levels of the target genes of CSX/NKX2-5. These results suggest that a novel LIM protein Cal induces cardiomyocyte differentiation through its dynamic intracellular shuttling and association with CSX/NKX2-5.

scholarly journals A yeast model system for functional analysis of β-catenin signaling

2002 ◽  
Vol 158 (6) ◽  
pp. 1067-1078 ◽  
Author(s):  
Margaret S. Lee ◽  
Karen A. D'Amour ◽  
Jackie Papkoff
Keyword(s):  
Reporter Gene ◽  
Gene Transcription ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Glycogen Synthase ◽  
Nuclear Export Signal ◽  
Signal Transduction Pathway ◽  
Model System ◽  
Nuclear Accumulation ◽  
Molecular Events

We have developed a novel Saccharomyces cerevisiae model system to dissect the molecular events of β-catenin (β-cat) signaling. Coexpression of mammalian β-cat with TCF4 or LEF1 results in nuclear accumulation of these proteins and a functional complex that activates reporter gene transcription from constructs containing leukocyte enhancer factor (LEF)/T cell factor (TCF) response elements. Reporter transcription is constitutive, requires expression of both β-cat and TCF4 or LEF1, and is not supported by mutated LEF/TCF binding elements or by TCF4 or LEF1 mutants. A cytoplasmic domain of E-cadherin or a functional fragment of adenomatous polyposis coli (APC) protein (APC-25) complexes with β-cat, reduces β-cat binding to TCF4, and leads to increased cytoplasmic localization of β-cat and a reduction in reporter activation. Systematic mutation of putative nuclear export signal sequences in APC-25 decreases APC-25 binding to β-cat and restores reporter gene transcription. Additional β-cat signaling components, Axin and glycogen synthase kinase 3β, form a multisubunit complex similar to that found in mammalian cells. Coexpression of the F-box protein β-transducin repeat-containing protein reduces the stability of β-cat and decreases reporter activation. Thus, we have reconstituted a functional β-cat signal transduction pathway in yeast and show that β-cat signaling can be regulated at multiple levels, including protein subcellular localization, protein complex formation, and protein stability.

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