A nuclear export signal in the N-terminal regulatory domain of Ikappa Balpha controls cytoplasmic localization of inactive NF-kappa B/Ikappa Balpha complexes

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.

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Visualization of Myc/Max/Mad Family Dimers and the Competition for Dimerization in Living Cells

Molecular and Cellular Biology ◽

10.1128/mcb.24.10.4294-4308.2004 ◽

2004 ◽

Vol 24 (10) ◽

pp. 4294-4308 ◽

Cited By ~ 92

Author(s):

Asya V. Grinberg ◽

Chang-Deng Hu ◽

Tom K. Kerppola

Keyword(s):

Nuclear Export ◽

Leucine Zipper ◽

Nuclear Export Signal ◽

Living Cells ◽

Bimolecular Fluorescence Complementation ◽

Cytoplasmic Localization ◽

Amino Acid Residues ◽

Helix Loop Helix ◽

Cell Growth And Proliferation ◽

Export Signal

ABSTRACT Myc and Mad family proteins play opposing roles in the control of cell growth and proliferation. We have visualized the subcellular locations of complexes formed by Myc/Max/Mad family proteins using bimolecular fluorescence complementation (BiFC) analysis. Max was recruited to different subnuclear locations by interactions with Myc versus Mad family members. Complexes formed by Max with Mxi1, Mad3, or Mad4 were enriched in nuclear foci, whereas complexes formed with Myc were more uniformly distributed in the nucleoplasm. Mad4 was localized to the cytoplasm when it was expressed separately, and Mad4 was recruited to the nucleus through dimerization with Max. The cytoplasmic localization of Mad4 was determined by a CRM1-dependent nuclear export signal located near the amino terminus. We compared the relative efficiencies of complex formation among Myc, Max, and Mad family proteins in living cells using multicolor BiFC analysis. Max formed heterodimers with the basic helix-loop-helix leucine zipper (bHLHZIP) domain of Myc (bMyc) more efficiently than it formed homodimers. Replacement of two amino acid residues in the leucine zipper of Max reversed the relative efficiencies of homo- and heterodimerization in cells. Surprisingly, Mad3 formed complexes with Max less efficiently than bMyc, whereas Mad4 formed complexes with Max more efficiently than bMyc. The distinct subcellular locations and the differences between the efficiencies of dimerization with Max indicate that Mad3 and Mad4 are likely to modulate transcription activation by Myc at least in part through distinct mechanisms.

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A Putative NES Mediates Cytoplasmic Localization of Apoptin in Normal Cells

Acta Biochimica et Biophysica Sinica ◽

10.1093/abbs/36.12.817 ◽

2004 ◽

Vol 36 (12) ◽

pp. 817-823 ◽

Cited By ~ 16

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.

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Cytoplasmic Localization of Mitogen-activated Protein Kinase Kinase Directed by Its NH2-terminal, Leucine-rich Short Amino Acid Sequence, Which Acts as a Nuclear Export Signal

Journal of Biological Chemistry ◽

10.1074/jbc.271.33.20024 ◽

1996 ◽

Vol 271 (33) ◽

pp. 20024-20028 ◽

Cited By ~ 242

Author(s):

Makoto Fukuda ◽

Isamu Gotoh ◽

Yukiko Gotoh ◽

Eisuke Nishida

Keyword(s):

Amino Acid ◽

Protein Kinase ◽

Nuclear Export ◽

Nuclear Export Signal ◽

Mitogen Activated Protein Kinase ◽

Cytoplasmic Localization ◽

Mitogen Activated Protein ◽

Short Amino Acid Sequence ◽

Export Signal ◽

Protein Kinase Kinase

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

Molecular and Cellular Biology ◽

10.1128/mcb.01817-05 ◽

2006 ◽

Vol 26 (11) ◽

pp. 4288-4301 ◽

Cited By ~ 20

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.19.10.7088 ◽

1999 ◽

Vol 19 (10) ◽

pp. 7088-7095 ◽

Cited By ~ 75

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.

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