scholarly journals Regulation of RelA Subcellular Localization by a Putative Nuclear Export Signal and p50

1999 ◽  
Vol 19 (10) ◽  
pp. 7088-7095 ◽  
Author(s):  
Edward W. Harhaj ◽  
Shao-Cong Sun
Keyword(s):  
Subcellular Localization ◽  
Nuclear Export ◽  
Nuclear Translocation ◽  
Nuclear Export Signal ◽  
Nuclear Factor Κb ◽  
Nuclear Accumulation ◽  
Physical Interaction ◽  
Cytoplasmic Localization ◽  
Heterologous Proteins ◽  
Export Signal

ABSTRACT Nuclear factor κB (NF-κB) represents a family of dimeric DNA binding proteins, the pleotropic form of which is a heterodimer composed of RelA and p50 subunits. The biological activity of NF-κB is controlled through its subcellular localization. Inactive NF-κB is sequestered in the cytoplasm by physical interaction with an inhibitor, IκBα. Signal-mediated IκBα degradation triggers the release and subsequent nuclear translocation of NF-κB. It remains unknown whether the NF-κB shuttling between the cytoplasm and nucleus is subjected to additional steps of regulation. In this study, we demonstrated that the RelA subunit of NF-κB exhibits strong cytoplasmic localization activity even in the absence of IκBα inhibition. The cytoplasmic distribution of RelA is largely mediated by a leucine-rich sequence homologous to the recently characterized nuclear export signal (NES). This putative NES is both required and sufficient to mediate cytoplasmic localization of RelA as well as that of heterologous proteins. Furthermore, the cytoplasmic distribution of RelA is sensitive to a nuclear export inhibitor, leptomycin B, suggesting that RelA undergoes continuous nuclear export. Interestingly, expression of p50 prevents the cytoplasmic expression of RelA, leading to the nuclear accumulation of both RelA and p50. Together, these results suggest that the nuclear and cytoplasmic shuttling of RelA is regulated by both an intrinsic NES-like sequence and the p50 subunit of NF-κB.

scholarly journals Cytoplasmic Localization of Wis1 MAPKK by Nuclear Export Signal Is Important for Nuclear Targeting of Spc1/Sty1 MAPK in Fission Yeast

2002 ◽  
Vol 13 (8) ◽  
pp. 2651-2663 ◽  
Author(s):  
Aaron Ngocky Nguyen ◽  
Aminah D. Ikner ◽  
Mitsue Shiozaki ◽  
Sasha M. Warren ◽  
Kazuhiro Shiozaki
Keyword(s):  
Fission Yeast ◽  
Nuclear Export ◽  
Nuclear Translocation ◽  
Nuclear Export Signal ◽  
Mapk Cascade ◽  
Mitogen Activated Protein Kinase ◽  
Docking Site ◽  
Cytoplasmic Localization ◽  
Nuclear Targeting ◽  
Export Signal

Mitogen-activated protein kinase (MAPK) cascade is a ubiquitous signaling module that transmits extracellular stimuli through the cytoplasm to the nucleus; in response to activating stimuli, MAPKs translocate into the nucleus. Mammalian MEK MAPK kinases (MAPKKs) have in their N termini an MAPK-docking site and a nuclear export signal (NES) sequence, which are known to play critical roles in maintaining ERK MAPKs in the cytoplasm of unstimulated cells. Herein, we show that the Wis1 MAPKK of the stress-activated Spc1 MAPK cascade in fission yeast also has a MAPK-docking site and an NES sequence in its N-terminal domain. Unexpectedly, an inactivating mutation to the NES of chromosomal wis1 + does not affect the subcellular localization of Spc1 MAPK, whereas this NES mutation disturbs the cytoplasmic localization of Wis1. However, when Wis1 is targeted to the nucleus by fusing to a nuclear localization signal sequence, stress-induced nuclear translocation of Spc1 is abrogated, indicating that cytoplasmic Wis1 is required for nuclear transport of Spc1 upon stress. Moreover, we have observed that a fraction of Wis1 translocates into the nucleus in response to stress. These results suggest that cytoplasmic localization of Wis1 MAPKK by its NES is important for stress signaling to the nucleus.

A Putative NES Mediates Cytoplasmic Localization of Apoptin in Normal Cells

2004 ◽  
Vol 36 (12) ◽  
pp. 817-823 ◽  
Author(s):  
Qing-Ming Wang ◽  
Guo-Cai Fan ◽  
Ji-Zhong Chen ◽  
Hui-Peng Chen ◽  
Fu-Chu He
Keyword(s):  
Tumor Cells ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Chicken Anemia Virus ◽  
Nuclear Accumulation ◽  
Cytoplasmic Localization ◽  
Normal Cells ◽  
Localization Signal ◽  
Anemia Virus ◽  
Export Signal

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.

scholarly journals A Nuclear Export Signal and Phosphorylation Regulate Dok1 Subcellular Localization and Functions

2006 ◽  
Vol 26 (11) ◽  
pp. 4288-4301 ◽  
Author(s):  
Yamei Niu ◽  
François Roy ◽  
Frédéric Saltel ◽  
Charlotte Andrieu-Soler ◽  
Wen Dong ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Specific Inhibitor ◽  
Adaptor Protein ◽  
Cell Spreading ◽  
Nuclear Accumulation ◽  
Serum Starvation ◽  
Cytoplasmic Localization ◽  
Export Signal

ABSTRACT Dok1 is believed to be a mainly cytoplasmic adaptor protein which down-regulates mitogen-activated protein kinase activation, inhibits cell proliferation and transformation, and promotes cell spreading and cell migration. Here we show that Dok1 shuttles between the nucleus and cytoplasm. Treatment of cells with leptomycin B (LMB), a specific inhibitor of the nuclear export signal (NES)-dependent receptor CRM1, causes nuclear accumulation of Dok1. We have identified a functional NES (348LLKAKLTDPKED359) that plays a major role in the cytoplasmic localization of Dok1. Src-induced tyrosine phosphorylation prevented the LMB-mediated nuclear accumulation of Dok1. Dok1 cytoplasmic localization is also dependent on IKKβ. Serum starvation or maintaining cells in suspension favor Dok1 nuclear localization, while serum stimulation, exposure to growth factor, or cell adhesion to a substrate induce cytoplasmic localization. Functionally, nuclear NES-mutant Dok1 had impaired ability to inhibit cell proliferation and to promote cell spreading and cell motility. Taken together, our results provide the first evidence that Dok1 transits through the nucleus and is actively exported into the cytoplasm by the CRM1 nuclear export system. Nuclear export modulated by external stimuli and phosphorylation may be a mechanism by which Dok1 is maintained in the cytoplasm and membrane, thus regulating its signaling functions.

scholarly journals A Novel Heterodimerization Domain, CRM1, and 14-3-3 Control Subcellular Localization of the MondoA-Mlx Heterocomplex

2002 ◽  
Vol 22 (24) ◽  
pp. 8514-8526 ◽  
Author(s):  
Alanna L. Eilers ◽  
Eleanor Sundwall ◽  
Monica Lin ◽  
April A. Sullivan ◽  
Donald E. Ayer
Keyword(s):  
Subcellular Localization ◽  
Nuclear Export ◽  
Leucine Zipper ◽  
Sequence Similarity ◽  
Nuclear Export Signal ◽  
Nuclear Accumulation ◽  
Cytoplasmic Localization ◽  
Terminal Domain ◽  
N Terminus ◽  
Dimerization Interface

ABSTRACT Among members of the bHLHZip family of transcriptional regulators, MondoA and Mlx have the unique property of cytoplasmic localization. We have proposed that MondoA-Mlx heterodimers accumulate in the nucleus in response to extracellular cues. Our previous work implicated heterodimerization between MondoA and Mlx and a conserved domain in the N terminus of MondoA as important determinants of MondoA-Mlx subcellular localization. MondoA and Mlx share sequence similarity in their bHLHZip domains and C termini. Here we show that for both MondoA and Mlx, this C-terminal domain has cytoplasmic localization activity that is required by the protein monomers to accumulate in the cytoplasm. This C-terminal domain is also a novel dimerization interface that functions independently of the leucine zipper to mediate heterotypic interactions between MondoA and Mlx. Dimerization between MondoA and Mlx inactivates the cytoplasmic localization activity of their C termini and is necessary for the heterocomplex to accumulate in the nucleus. MondoA-Mlx heterodimers, while poised for nuclear entry, are retained in the cytoplasm by conserved domains in the N terminus of MondoA. Mondo conserved regions (MCRs) II and III contribute to cytoplasmic localization of MondoA-Mlx by functioning as a CRM1-dependent nuclear export signal and as a novel binding site for 14-3-3 family members, respectively. We propose that the nuclear accumulation of MondoA and Mlx is a two-step process. First, heterodimerization abolishes the cytoplasmic localization activity of their C termini. Second, an extracellular signal(s) must overcome the cytoplasmic localization function imparted by CRM1 and 14-3-3 binding to the N terminus of MondoA.

scholarly journals OsWRKY62 and OsWRKY76 interact with Importin α1s for Negative Regulation of Defensive Responses in Nucleus

2021 ◽  
Author(s):  
Xiaohui Xu ◽  
Han Wang ◽  
Jiqin Liu ◽  
Shuying Han ◽  
Miaomiao Lin ◽  
...  
Keyword(s):  
Amino Acids ◽  
Nuclear Localization ◽  
Nuclear Export ◽  
Nuclear Translocation ◽  
Nuclear Export Signal ◽  
Defense Responses ◽  
Nuclear Localization Signals ◽  
Defensive Responses ◽  
Export Signal ◽  
Positively Charged

Abstract Background: OsWRKY62 and OsWRKY76, two close members of WRKY transcription factors, function together as transcriptional repressors. OsWRKY62 is predominantly localized in the cytosol. What are the regulatory factors for OsWRKY62 nuclear translocation?Results: In this study, we characterized they interacted with rice importin, OsIMα1a and OsIMα1b, for nuclear translocation. Chimeric OsWRKY62.1-GFP, which is predominantly localized in the cytoplasm, was translocated to the nucleus of Nicotiana benthamiana leaf cells in the presence of OsIMα1a or OsIMαDIBB1a lacking the auto-inhibitory importin β-binding domain. OsIMαDIBB1a interacted with the WRKY domain of OsWRKY62.1, which has specific bipartite positively charged concatenated amino acids functioning as a nuclear localization signal. Similarly, we found that OsIMαDIBB1a interacted with the AvrPib effector of rice blast fungus Magnaporthe oryzae, which contains a scattered distribution of positively charged amino acids. Furthermore, we identified a nuclear export signal in OsWRKY62.1 that inhibited nuclear transportation. Overexpression of OsIMα1a or OsIMα1b enhanced resistance to M. oryzae, whereas knockout mutants decreased resistance to the pathogen. However, overexpressing both OsIMα1a and OsWRKY62.1 were slightly more susceptible to M. oryzae than OsWRKY62.1 alone. Ectopic overexpression of OsWRKY62.1 with an extra nuclear export signal compromised the enhanced susceptibility of OsWRKY62.1 to M. oryzae.Conclusion: These results indicated that OsWRKY62 localization is a consequence of competition binding between rice importins and exportins. OsWRKY62, OsWRKY76, and AvrPib effector translocate to nucleus in association with importin α1s through new types of nuclear localization signals for negatively regulating defense responses.

p65 controls NF-κB activity by regulating cellular localization of IκBβ

2011 ◽  
Vol 434 (2) ◽  
pp. 253-263 ◽  
Author(s):  
Taras Valovka ◽  
Michael O. Hottiger
Keyword(s):  
Dna Binding ◽  
Nuclear Export ◽  
Cellular Localization ◽  
Nuclear Export Signal ◽  
Nuclear Factor Κb ◽  
Binding Activity ◽  
Cellular Processes ◽  
Dna Binding Activity ◽  
Localization Signal ◽  
Export Signal

NF-κB (nuclear factor κB) controls diverse cellular processes and is frequently misregulated in chronic immune diseases or cancer. The activity of NF-κB is regulated by IκB (inhibitory κB) proteins which control nuclear–cytoplasmic shuttling and DNA binding of NF-κB. In the present paper, we describe a novel role for p65 as a critical regulator of the cellular localization and functions of NF-κB and its inhibitor IκBβ. In genetically modified p65−/− cells, the localization of ectopic p65 is not solely regulated by IκBα, but is largely dependent on the NLS (nuclear localization signal) and the NES (nuclear export signal) of p65. Furthermore, unlike IκBα, IκBβ does not contribute to the nuclear export of p65. In fact, the cellular localization and degradation of IκBβ is controlled by the p65-specific NLS and NES. The results of our present study also reveal that, in addition to stimulus-induced redistribution of NF-κB, changes in the constitutive localization of p65 and IκBβ specifically modulate activation of inflammatory genes. This is a consequence of differences in the DNA-binding activity and signal responsiveness between the nuclear and cytoplasmic NF-κB–IκBβ complexes. Taken together, the findings of the present study indicate that the p65 subunit controls transcriptional competence of NF-κB by regulating the NF-κB/IκBβ pathway.

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

2004 ◽  
Vol 78 (10) ◽  
pp. 5358-5367 ◽  
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.

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

2007 ◽  
Vol 28 (1) ◽  
pp. 422-434 ◽  
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.

scholarly journals Subcellular localization of CrmA: identification of a novel leucine-rich nuclear export signal conserved in anti-apoptotic serpins

2003 ◽  
Vol 373 (1) ◽  
pp. 251-259 ◽  
Author(s):  
Jose A. RODRIGUEZ ◽  
Simone W. SPAN ◽  
Frank A. E. KRUYT ◽  
Giuseppe GIACCONE
Keyword(s):  
Subcellular Localization ◽  
Nuclear Export ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Nuclear Export Signal ◽  
Cowpox Virus ◽  
Amino Acid Region ◽  
Localization Signal ◽  
Induced Apoptosis ◽  
Export Signal

The cowpox virus-encoded anti-apoptotic protein cytokine response modifier A (CrmA) is a member of the serpin family that specifically inhibits the cellular proteins caspase 1, caspase 8 and granzyme B. In this study, we have used Flag- and yellow fluorescent protein (YFP)-tagged versions of CrmA to investigate the mechanisms that regulate its subcellular localization. We show that CrmA can actively enter and exit the nucleus and we demonstrate the role of the nuclear export receptor CRM1 in this shuttling process. CrmA contains a novel leucine-rich nuclear export signal (NES) that is functionally conserved in the anti-apoptotic cellular serpin PI-9. Besides this leucine-rich export signal, additional sequences mapping to a 103-amino-acid region flanking the NES contribute to the CRM1-dependent nuclear export of CrmA. Although YFP-tagged CrmA is primarily located in the cytoplasm, shifting its localization to be predominantly nuclear by fusion of a heterologous nuclear localization signal did not impair its ability to prevent Fas-induced apoptosis. We propose that nucleocytoplasmic shuttling would allow CrmA to efficiently target cellular pro-apoptotic proteins not only in the cytoplasm, but also in the nucleus, and thus to carry out its anti-apoptotic function in both compartments.

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

2017 ◽  
Vol 216 (11) ◽  
pp. 3471-3484 ◽  
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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