scholarly journals CDK-dependent phosphorylation of Alp7–Alp14 (TACC–TOG) promotes its nuclear accumulation and spindle microtubule assembly

2014 ◽  
Vol 25 (13) ◽  
pp. 1969-1982 ◽  
Author(s):  
Naoyuki Okada ◽  
Takashi Toda ◽  
Masayuki Yamamoto ◽  
Masamitsu Sato
Keyword(s):  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Coiled Coil ◽  
Microtubule Assembly ◽  
Nuclear Accumulation ◽  
Cyclin Dependent Kinase ◽  
Mitotic Entry ◽  
Localization Signal ◽  
Export Signal ◽  
Overexpressed Gene

As cells transition from interphase to mitosis, the microtubule cytoskeleton is reorganized to form the mitotic spindle. In the closed mitosis of fission yeast, a microtubule-associated protein complex, Alp7–Alp14 (transforming acidic coiled-coil–tumor overexpressed gene), enters the nucleus upon mitotic entry and promotes spindle formation. However, how the complex is controlled to accumulate in the nucleus only during mitosis remains elusive. Here we demonstrate that Alp7–Alp14 is excluded from the nucleus during interphase using the nuclear export signal in Alp14 but is accumulated in the nucleus during mitosis through phosphorylation of Alp7 by the cyclin-dependent kinase (CDK). Five phosphorylation sites reside around the nuclear localization signal of Alp7, and the phosphodeficient alp7-5A mutant fails to accumulate in the nucleus during mitosis and exhibits partial spindle defects. Thus our results reveal one way that CDK regulates spindle assembly at mitotic entry: CDK phosphorylates the Alp7–Alp14 complex to localize it 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.

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 Control of NF–κB transcriptional activation by signal induced proteolysis of IκBα

1999 ◽  
Vol 354 (1389) ◽  
pp. 1601-1609 ◽  
Author(s):  
R. T. Hay ◽  
L. Vuillard ◽  
J. M. P. Desterro ◽  
M. S. Rodriguez
Keyword(s):  
Nuclear Localization ◽  
Transcriptional Activation ◽  
Nuclear Localization Signal ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Cytoplasmic Process ◽  
Leptomycin B ◽  
Rapid Degradation ◽  
Localization Signal ◽  
Export Signal

In unstimulated cells the transcription factor NF–κB is held in the cytoplasm in an inactive state by IκB inhibitor proteins. Ultimately activation of NF–κB is achieved by ubiquitination and proteasome–mediated degradation of IκBα and we have therefore investigated factors which control this proteolysis. Signal–induced degradation of IκBα exposes the nuclear localization signal of NF–κB, thus allowing it to translocate into the nucleus and activate transcription from responsive genes. An autoregulatory loop is established when NF–κB induces expression of the IκBα gene and newly synthesized IκBα accumulates in the nucleus where it negatively regulates NF–κB–dependent transcription. As part of this post–induction repression, the nuclear export signal on IκBα mediates transport of NF–κB–IκBα complexes from the nucleus to the cytoplasm. As nuclear export of IκBα is blocked by leptomycin B this drug was used to examine the effect of cellular location on susceptibility of IκBα to signal–induced degradation. In the presence of leptomycin B, IκBα is accumulated in the nucleus and in this compartment is resistant to signal–induced degradation. Thus signal–induced degradation of IκBα is mainly, if not exclusively a cytoplasmic process. An efficient nuclear export of IκBα is therefore essential for maintaining a low level of IκBα in the nucleus and allowing NF–κB to be transcriptionally active upon cell stimulation. We have detected a modified form of IκBα, conjugated to the small ubiquitin–like protein SUMO–1, which is resistant to signal–induced degradation. SUMO–1 modified IκBα remains associated with NF–κB and thus overexpression of SUMO–1 inhibits the signal–induced activation of NF–κB–dependent transcription. Reconstitution of the conjugation reaction with highly purified proteins demonstrated that in the presence of a novel E1 SUMO–1 activating enzyme, Ubch9 directly conjugated SUMO–1 to IκBα on residues K21 and K22, which are also used for ubiquitin modification. Thus, while ubiquitination targets proteins for rapid degradation, SUMO–1 modification acts antagonistically to generate proteins resistant to degradation.

Nuclear-localization-signal-dependent and nuclear-export-signal-dependent mechanisms determine the localization of 5-lipoxygenase

2002 ◽  
Vol 361 (3) ◽  
pp. 505-514 ◽  
Author(s):  
Hiromi HANAKA ◽  
Takao SHIMIZU ◽  
Takashi IZUMI
Keyword(s):  
Fusion Protein ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Nuclear Export ◽  
Molecular Mechanisms ◽  
Nuclear Export Signal ◽  
Specific Localization ◽  
Localization Signal ◽  
Leukotriene A4 ◽  
Export Signal

5-Lipoxygenase (5-LO) metabolizes arachidonic acid to leukotriene A4, a key intermediate in leukotriene biosynthesis. To explore the molecular mechanisms of its cell-specific localization, a fusion protein between green fluorescent protein (GFP) and human 5-LO (GFP—5LO) was expressed in various cells. GFP—5LO was localized in the cytosol in HL-60 cells and in both the nucleus and the cytosol in RBL (rat basophilic leukaemia) cells, similarly to the native enzyme in these cells. The localization of GFP fusion proteins for mutant 5-LOs in a putative bipartite nuclear localization signal (NLS), amino acids 638–655, in Chinese hamster ovary (CHO)-K1 and Swiss3T3 cells revealed that this motif is important for the nuclear localization of 5-LO. A GFP fusion protein of this short peptide localized consistently in the nucleus. Leptomycin B, a specific inhibitor of nuclear export signal (NES)-dependent transport, diminished the cytosolic localization of 5-LO in HL-60 cells and that of GFP—5LO in CHO-K1 cells, suggesting that an NES-system might also function in determining 5-LO localization. Analysis of the localization of 5-LO during the cell cycle points to a controlled movement of this enzyme. Thus we conclude that a balance of NLS- and NES-dependent mechanisms determines the cell-type-specific localization of 5-LO, suggesting a nuclear function for this enzyme.

scholarly journals Identification of a nuclear localization signal and nuclear export signal of the umbraviral long-distance RNA movement protein

2004 ◽  
Vol 85 (5) ◽  
pp. 1329-1333 ◽  
Author(s):  
Eugene V. Ryabov ◽  
Sang Hyon Kim ◽  
Michael Taliansky
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Viral Rna ◽  
Protein Accumulation ◽  
Long Distance ◽  
Orf3 Protein ◽  
Localization Signal ◽  
Export Signal

The 27 kDa protein encoded by ORF3 of Groundnut rosette virus (GRV) is required for viral RNA protection and movement of viral RNA through the phloem. Localization studies have revealed that this protein is located in nuclei, preferentially targeting nucleoli. We have demonstrated that amino acids (aa) 108–122 of the GRV ORF3 protein contain an arginine-rich nuclear localization signal. Arginine-to-asparagine substitutions in this region decreased the level of the ORF3 protein accumulation in nuclei. A leucine-rich nuclear export signal (NES) was located at aa 148–156 of the GRV ORF3 protein. Leucine-to-alanine substitutions in this region resulted in a dramatic increase in GRV ORF3 protein accumulation in both nuclei and nucleoli. Consistent with this, we also showed that the previously identified NES of BR1 protein of Squash leaf curl virus can functionally replace the leucine-rich region of GRV ORF3 in nuclear export.

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 Nucleocytoplasmic Shuttling Modulates Activity and Ubiquitination-Dependent Turnover of SUMO-Specific Protease 2

2006 ◽  
Vol 26 (12) ◽  
pp. 4675-4689 ◽  
Author(s):  
Yoko Itahana ◽  
Edward T. H. Yeh ◽  
Yanping Zhang
Keyword(s):  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Nucleocytoplasmic Shuttling ◽  
Leptomycin B ◽  
Sumo Protease ◽  
Specific Protease ◽  
Localization Signal ◽  
Modified Proteins ◽  
Export Signal

ABSTRACT Small ubiquitin-related modifier (SUMO) proteins are conjugated to numerous polypeptides in cells, and attachment of SUMO plays important roles in regulating the activity, stability, and subcellular localization of modified proteins. SUMO modification of proteins is a dynamic and reversible process. A family of SUMO-specific proteases catalyzes the deconjugation of SUMO-modified proteins. Members of the Sentrin (also known as SUMO)-specific protease (SENP) family have been characterized with unique subcellular localizations. However, little is known about the functional significance of or the regulatory mechanism derived from the specific localizations of the SENPs. Here we identify a bipartite nuclear localization signal (NLS) and a CRM1-dependent nuclear export signal (NES) in the SUMO protease SENP2. Both the NLS and the NES are located in the nonhomologous domains of SENP2 and are not conserved among other members of the SENP family. Using a series of SENP2 mutants and a heterokaryon assay, we demonstrate that SENP2 shuttles between the nucleus and the cytoplasm and that the shuttling is blocked by mutations in the NES or by treating cells with leptomycin B. We show that SENP2 can be polyubiquitinated in vivo and degraded through proteolysis. Restricting SENP2 in the nucleus by mutations in the NES impairs its polyubiquitination, whereas a cytoplasm-localized SENP2 made by introducing mutations in the NLS can be efficiently polyubiquitinated, suggesting that SENP2 is ubiquitinated in the cytoplasm. Finally, treating cells with MG132 leads to accumulation of polyubiquitinated SENP2, indicating that SENP2 is degraded through the 26S proteolysis pathway. Thus, the function of SENP2 is regulated by both nucleocytoplasmic shuttling and polyubiquitin-mediated degradation.

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