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PubMed2006

A regulated nucleocytoplasmic shuttle contributes to Bright's function as a transcriptional activator of immunoglobulin genes.

Bright/ARID3a has been implicated in mitogen- and growth factor-induced up-regulation of immunoglobulin heavy-chain (IgH) genes and in E2F1-dependent G1/S cell cycle progression. For IgH transactivation, Bright binds to nuclear matrix association regions upstream of certain variable region promoters and flanking the IgH intronic enhancer. While Bright protein was previously shown to reside within the nuclear matrix, we show here that a significant amount of Bright resides in the cytoplasm of normal and transformed B cells. Leptomycin B, chromosome region maintenance 1 (CRM1) overexpression, and heterokaryon experiments indicate that Bright actively shuttles between the nucleus and the cytoplasm in a CRM1-dependent manner. We mapped the functional nuclear localization signal to the N-terminal region of REKLES, a domain conserved within ARID3 paralogues. Residues within the C terminus of REKLES contain its nuclear export signal, whose regulation is primarily responsible for Bright shuttling. Growth factor depletion and cell synchronization experiments indicated that Bright shuttling during S phase of the cell cycle leads to an increase in its nuclear abundance. Finally, we show that shuttle-incompetent Bright point mutants, even if sequestered within the nucleus, are incapable of transactivating an IgH reporter gene. Therefore, regulation of Bright's cellular localization appears to be required for its function.

Active Transport, Cell Nucleus↗
PubMed1998

The influenza virus NEP (NS2 protein) mediates the nuclear export of viral ribonucleoproteins.

Nuclear import and export of viral nucleic acids is crucial for the replication cycle of many viruses, and elucidation of the mechanism of these steps may provide a paradigm for understanding general biological processes. Influenza virus replicates its RNA genome in the nucleus of infected cells. The influenza virus NS2 protein, which had no previously assigned function, was shown to mediate the nuclear export of virion RNAs by acting as an adaptor between viral ribonucleoprotein complexes and the nuclear export machinery of the cell. A functional domain on the NS2 with characteristics of a nuclear export signal was mapped: it interacts with cellular nucleoporins, can functionally replace the effector domain of the human immunodeficiency virus type 1 (HIV-1) Rev protein and mediates rapid nuclear export when cross-linked to a reporter protein. Microinjection of anti-NS2 antibodies into infected cells inhibited nuclear export of viral ribonucleoproteins, suggesting that the Rev-like NS2 mediates this process. Therefore, we have renamed this Rev-like factor the influenza virus nuclear export protein or NEP. We propose a model by which NEP acts as a protein adaptor molecule bridging viral ribonucleoproteins and the nuclear pore complex.

Amino Acid Sequence↗
PubMed2005

Temporal and spatial control of nucleophosmin by the Ran-Crm1 complex in centrosome duplication.

Centrosome duplication is tightly controlled during faithful cell division, and unnecessary reduplication can lead to supernumerary centrosomes and multipolar spindles that are associated with most human cancer cells. In addition to nucleocytoplasmic transport, the Ran-Crm1 network is involved in regulating centrosome duplication to ensure the formation of a bipolar spindle. Here, we discover that nucleophosmin (NPM) may be a Ran-Crm1 substrate that controls centrosome duplication. NPM contains a functional nuclear export signal (NES) that is responsible for both its nucleocytoplasmic shuttling and its association with centrosomes, which are Ran-Crm1-dependent as they are sensitive to Crm1-specific nuclear export inhibition, either by leptomycin B (LMB) or by the expression of a Ran-binding protein, RanBP1. Notably, LMB treatment induces premature centrosome duplication in quiescent cells, which coincides with NPM dissociation from centrosomes. Moreover, deficiency of NPM by RNA interference results in supernumerary centrosomes, which can be reversed by reintroducing wild-type but not NES-mutated NPM. Mutation of a potential proline-dependent kinase phosphorylation site at residue 95, from threonine to aspartic acid (T95D) within the NES motif, abolishes NPM association and inhibition of centrosome duplication. Our results are consistent with the hypothesis that the Ran-Crm1 complex may promote a local enrichment of NPM on centrosomes, thereby preventing centrosome reduplication.

Active Transport, Cell Nucleus↗
PubMed2003

Caspase-dependent cleavage of c-Abl contributes to apoptosis.

The nonreceptor tyrosine kinase c-Abl may contribute to the regulation of apoptosis. c-Abl activity is induced in the nucleus upon DNA damage, and its activation is required for execution of the apoptotic program. Recently, activation of nuclear c-Abl during death receptor-induced apoptosis has been reported; however, the mechanism remains largely obscure. Here we show that c-Abl is cleaved by caspases during tumor necrosis factor- and Fas receptor-induced apoptosis. Cleavage at the very C-terminal region of c-Abl occurs mainly in the cytoplasmic compartment and generates a 120-kDa fragment that lacks the nuclear export signal and the actin-binding region but retains the intact kinase domain, the three nuclear localization signals, and the DNA-binding domain. Upon caspase cleavage, the 120-kDa fragment accumulates in the nucleus. Transient-transfection experiments show that cleavage of c-Abl may affect the efficiency of Fas-induced cell death. These data reveal a novel mechanism by which caspases can recruit c-Abl to the nuclear compartment and to the mammalian apoptotic program.

Active Transport, Cell Nucleus↗
PubMed1998

Nuclear-cytoplasmic shuttling of C-ABL tyrosine kinase.

The ubiquitously expressed nonreceptor tyrosine kinase c-Abl contains three nuclear localization signals, however, it is found in both the nucleus and the cytoplasm of proliferating fibroblasts. A rapid and transient loss of c-Abl from the nucleus is observed upon the initial adhesion of fibroblasts onto a fibronectin matrix, suggesting the possibility of nuclear export [Lewis, J., Baskaran, R. , Taagepera, S., Schwartz, M. & Wang, J. (1996) Proc. Natl. Acad. Sci. USA 93, 15174-15179]. Here we show that the C terminus of c-Abl does indeed contain a functional nuclear export signal (NES) with the characteristic leucine-rich motif. The c-Abl NES can functionally complement an NES-defective HIV Rev protein (RevDelta3NI) and can mediate the nuclear export of glutathione-S-transferase. The c-Abl NES function is sensitive to the nuclear export inhibitor leptomycin B. Mutation of a single leucine (L1064A) in the c-Abl NES abrogates export function. The NES-mutated c-Abl, termed c-Abl NES(-), is localized exclusively to the nucleus. Treatment of cells with leptomycin B also leads to the nuclear accumulation of wild-type c-Abl protein. The c-Abl NES(-) is not lost from the nucleus when detached fibroblasts are replated onto fibronectin matrix. Taken together, these results demonstrate that c-Abl shuttles continuously between the nucleus and the cytoplasm and that the rate of nuclear import and export can be modulated by the adherence status of fibroblastic cells.

3T3 Cells↗
PubMed2004

Cdc42-dependent nuclear translocation of non-receptor tyrosine kinase, ACK.

Ras signals for the transformation of mammalian cells are apparently transduced through Rho GTPases. The Rho GTPase family member Cdc42 generates independent signals that regulate the rearrangement of the actin cytoskeleton and the transcription of genes. However, the molecular mechanism of signal transduction from Cdc42 to the nucleus remains to be understood. The non-receptor tyrosine kinases ACK-1 and ACK-2 have been found to bind specifically to Cdc42. In this paper we studied whether ACKs transduce Cdc42 signals to the nucleus directly, or through other cytoplasmic proteins. Using immunocytochemistry and Western blot analysis, we found a nuclear localization of ACKs in semi-confluent glioblastoma (U251) cells, as opposed to a cytosolic localization in confluent cells. In agreement with the nuclear localization, a putative nuclear export signal was identified in ACK-1 and ACK-2. Furthermore, the interaction of Cdc42 with ACKs was shown to be essential for the nuclear localization of ACKs. Overexpression of ACK42 (a Cdc42 binding domain of ACK) inhibited cell growth and movement, indicating that Cdc42 signals are transduced to the nucleus through ACKs. This is the first report providing evidence of a novel role for ACKs in transducing Cdc42 signals directly to the nucleus.

Actins↗
PubMed2002

Nucleocytoplasmic distribution of human RNA-editing enzyme ADAR1 is modulated by double-stranded RNA-binding domains, a leucine-rich export signal, and a putative dimerization domain.

The human RNA-editing enzyme adenosine deaminase that acts on RNA (ADAR1) is expressed in two versions. A longer 150-kDa protein is interferon inducible and can be found both in the nucleus and cytoplasm. An amino-terminally truncated 110-kDa version, in contrast, is constitutively expressed and predominantly nuclear. In the absence of transcription, however, the shorter protein is also cytoplasmic and thus displays the hallmarks of a shuttling protein. The nuclear localization signal (NLS) of human hsADAR1 is atypical and overlaps with its third double-stranded RNA-binding domain (dsRBD). Herein, we identify regions in hsADAR1 that interfere with nuclear localization and mediate cytoplasmic accumulation. We show that interferon-inducible hsADAR1 contains a Crm1-dependent nuclear export signal in its amino terminus. Most importantly, we demonstrate that the first dsRBD of hsADAR1 interferes with nuclear localization of a reporter construct containing dsRBD3 as an active NLS. The same effect can be triggered by several other, but not all dsRBDs. Active RNA binding of either the inhibitory dsRBD1 or the NLS bearing dsRBD3 is required for cytoplasmic accumulation. Furthermore, hsADAR1's dsRBD1 has no effect on other NLSs, suggesting RNA-mediated cross talk between dsRBDs, possibly leading to masking of the NLS. A model, incorporating these findings is presented. Finally, we identify a third region located in the C terminus of hsADAR1 that also interferes with nuclear accumulation of this protein.

Active Transport, Cell Nucleus↗
PubMed1998

Direct triggering of the type I interferon system by virus infection: activation of a transcription factor complex containing IRF-3 and CBP/p300.

It has been hypothesized that certain viral infections directly activate a transcription factor(s) which is responsible for the activation of genes encoding type I interferons (IFNs) and interferon-stimulated genes (ISGs) via interferon regulatory factor (IRF) motifs present in their respective promoters. These events trigger the activation of defense machinery against viruses. Here we demonstrate that IRF-3 transmits a virus-induced signal from the cytoplasm to the nucleus. In unstimulated cells, IRF-3 is present in its inactive form, restricted to the cytoplasm due to a continuous nuclear export mediated by nuclear export signal, and it exhibits few DNA-binding properties. Virus infection but not IFN treatment induces phosphorylation of IRF-3 on specific serine residues, thereby allowing it to complex with the co-activator CBP/p300 with simultaneous nuclear translocation and its specific DNA binding. We also show that a dominant-negative mutant of IRF-3 could inhibit virus-induced activation of chromosomal type I IFN genes and ISGs. These findings suggest that IRF-3 plays an important role in the virus-inducible primary activation of type I IFN and IFN-responsive genes.

Amino Acid Sequence↗
PubMed2001

Localization of human Cdc25C is regulated both by nuclear export and 14-3-3 protein binding.

Entry into mitosis requires activation of the Cdc2 protein kinase by the Cdc25C protein phosphatase. The interactions between Cdc2 and Cdc25C are negatively regulated throughout interphase and in response to G2 checkpoint activation. This is accomplished in part by maintaining the Cdc25 phosphatase in a phosphorylated form that binds 14-3-3 proteins. Here we report that 14-3-3 binding regulates the intracellular trafficking of Cdc25C. Although primarily cytoplasmic, Cdc25C accumulated in the nuclei of leptomycin B (LMB)-treated cells, indicating that Cdc25C is actively exported out of the nucleus. A mutant of Cdc25C that is unable to bind 14-3-3 was partially nuclear in the absence of LMB and its nuclear accumulation was greatly enhanced by LMB-treatment. A nuclear export signal (NES) was identified within the amino terminus of Cdc25C. Although mutation of the NES did not effect 14-3-3 binding, it did cause nuclear accumulation of Cdc25C. These results demonstrate that 14-3-3 binding is dispensable for the nuclear export of Cdc25C. However, complete nuclear accumulation of Cdc25C required loss of both NES function and 14-3-3 binding and this was accomplished both pharmacologically and by mutation. These findings suggest that the nuclear export of Cdc25C is mediated by an intrinsic NES and that 14-3-3 binding negatively regulates nuclear import.

14-3-3 Proteins↗
PubMed2001

Borna disease virus nucleoprotein requires both nuclear localization and export activities for viral nucleocytoplasmic shuttling.

Nuclear transport of viral nucleic acids is crucial to the life cycle of many viruses. Borna disease virus (BDV) belongs to the order Mononegavirales and replicates its RNA genome in the nucleus. Previous studies have suggested that BDV nucleoprotein (N) and phosphoprotein (P) have important functions in the nuclear import of the viral ribonucleoprotein (RNP) complexes via their nuclear targeting activity. Here, we showed that BDV N has cytoplasmic localization activity, which is mediated by a nuclear export signal (NES) within the sequence. Our analysis using deletion and substitution mutants of N revealed that NES of BDV N consists of a canonical leucine-rich motif and that the nuclear export activity of the protein is mediated through the chromosome region maintenance protein-dependent pathway. Interspecies heterokaryon assay indicated that BDV N shuttles between the nucleus and cytoplasm as a nucleocytoplasmic shuttling protein. Furthermore, interestingly, the NES region overlaps a binding site to the BDV P protein, and nuclear export of a 38-kDa form of BDV N is prevented by coexpression of P. These results suggested that BDV N has two contrary activities, nuclear localization and export activity, and plays a critical role in the nucleocytoplasmic transport of BDV RNP by interaction with other viral proteins.

Amino Acid Sequence↗
PubMed1998

Nuclear export of cyclin B1 and its possible role in the DNA damage-induced G2 checkpoint.

M-phase-promoting factor (MPF), a complex of cdc2 and a B-type cyclin, is a key regulator of the G2/M cell cycle transition. Cyclin B1 accumulates in the cytoplasm through S and G2 phases and translocates to the nucleus during prophase. We show here that cytoplasmic localization of cyclin B1 during interphase is directed by its nuclear export signal (NES)-dependent transport mechanism. Treatment of HeLa cells with leptomycin B (LMB), a specific inhibitor of the NES-dependent transport, resulted in nuclear accumulation of cyclin B1 in G2 phase. Disruption of an NES which has been identified in cyclin B1 here abolished the nuclear export of this protein, and consequently the NES-disrupted cyclin B1 when expressed in cells accumulated in the nucleus. Moreover, we show that expression of the NES-disrupted cyclin B1 or LMB treatment of the cells is able to override the DNA damage-induced G2 checkpoint when combined with caffeine treatment. These results suggest a role of nuclear exclusion of cyclin B1 in the DNA damage-induced G2 checkpoint.

Animals↗
PubMed2004

A CRM1-dependent nuclear export pathway is involved in the regulation of IRF-5 subcellular localization.

Interferon regulatory factors (IRFs) are involved in gene regulation in many biological processes including the antiviral, growth regulatory, and immune modulatory functions of the interferon system. Several studies have demonstrated that IRF-3, IRF-5, and IRF-7 specifically contribute to the innate antiviral response to virus infection. It has been reported that virus-specific phosphorylation leads to IRF-5 nuclear localization and up-regulation of interferon, cytokine, and chemokine gene expression. Two nuclear localization signals have been identified in IRF-5, both of which are sufficient for nuclear translocation and retention in virus-infected cells. In the present study, we demonstrate that a CRM1-dependent nuclear export pathway is involved in the regulation of IRF-5 subcellular localization. IRF-5 possesses a functional nuclear export signal (NES) that controls dynamic shuttling between the cytoplasm and the nucleus. The NES element is dominant in unstimulated cells and results in the predominant cytoplasmic localization of IRF-5. Mutation of two leucine residues in the NES motif to alanine, or three adjacent Ser/Thr residues to the phosphomimetic Asp, results in constitutively nuclear IRF-5 and suggests that phosphorylation of adjacent Ser/Thr residues may contribute to IRF-5 nuclear accumulation in virus-induced cells. IKK-related kinases TBK1 and IKKepsilon have been shown to phosphorylate and activate IRF-3 and IRF-7, leading to the production of type 1 interferons and the development of a cellular antiviral state. We examined the phosphorylation and activation of IRF-5 by TBK1 and IKKepsilon kinases. Although IRF-5 is phosphorylated by IKKepsilon and TBK1 in co-transfected cells, the phosphorylation of IRF-5 did not lead to IRF-5 nuclear localization or activation.

Active Transport, Cell Nucleus↗
PubMed2000

The MDM2 RING-finger domain is required to promote p53 nuclear export.

MDM2 can bind to p53 and promote its ubiquitination and subsequent degradation by the proteasome. Current models propose that nuclear export of p53 is required for MDM2-mediated degradation, although the function of MDM2 in p53 nuclear export has not been clarified. Here we show that MDM2 can promote the nuclear export of p53 in transiently transfected cells. This activity requires the nuclear-export signal (NES) of p53, but not the NES of MDM2. A mutation within the MDM2 RING-finger domain that inhibits p53 ubiquitination also inhibits the ability of MDM2 to promote p53 nuclear export. Finally, inhibition of nuclear export stabilizes wild-type p53 and leads to accumulation of ubiquitinated p53 in the nucleus. Our results indicate that MDM2-mediated ubiquitination, or other activities associated with the RING-finger domain, can stimulate the export of p53 to the cytoplasm through the activity of the p53 NES.

Active Transport, Cell Nucleus↗
PubMed2000

Identification of novel Saccharomyces cerevisiae proteins with nuclear export activity: cell cycle-regulated transcription factor ace2p shows cell cycle-independent nucleocytoplasmic shuttling.

Nuclear export of proteins containing leucine-rich nuclear export signals (NESs) is mediated by the NES receptor CRM1/Crm1p. We have carried out a yeast two-hybrid screen with Crm1p as a bait. The Crm1p-interacting clones were subscreened for nuclear export activity in a visual assay utilizing the Crm1p-inhibitor leptomycin B (LMB). This approach identified three Saccharomyces cerevisiae proteins not previously known to have nuclear export activity. These proteins are the 5' RNA triphosphatase Ctl1p, the cell cycle-regulated transcription factor Ace2p, and a protein encoded by the previously uncharacterized open reading frame YDR499W. Mutagenesis analysis show that YDR499Wp contains an NES that conforms to the consensus sequence for leucine-rich NESs. Mutagenesis of Ctl1p and Ace2p were unable to identify specific NES residues. However, a 29-amino-acid region of Ace2p, rich in hydrophobic residues, contains nuclear export activity. Ace2p accumulates in the nucleus at the end of mitosis and activates early-G(1)-specific genes. We now provide evidence that Ace2p is nuclear not only in late M-early G(1) but also during other stages of the cell cycle. This feature of Ace2p localization explains its ability to activate genes such as CUP1, which are not expressed in a cell cycle-dependent manner.

Active Transport, Cell Nucleus↗
PubMed2004

Tryptophan 621 and serine 667 residues of Daxx regulate its nuclear export during glucose deprivation.

The cellular target responsible for the nuclear export of Daxx has been identified as chromosomal region maintenance 1 (CRM1), which is a carrier protein for nuclear export and a receptor for the nuclear export signal (NES) of Daxx. Binding of Daxx to CRM1 was increased early during glucose deprivation and then gradually decreased. This interaction was inhibited by leptomycin B, a specific inhibitor of CRM1-dependent nuclear export. Substitution of the serine 667 amino acid residue of Daxx with alanine reduced the interaction with CRM1 during glucose deprivation, suggesting that the phosphorylation of Ser-667 is required for its binding to CRM1 and for its subsequent nuclear export. Data from coupled transcription-translation studies reveal that the NES (amino acids 565-575) of Daxx is a binding site for CRM1. Interestingly, constitutive export of Daxx has occurred by replacement of the tryptophan 621 Daxx residue with alanine. These results suggest that this tryptophan residue plays a key role in masking the NES of Daxx from its receptor, CRM1, in the resting state, whereas phosphorylation of serine 667 would release the NES, which could then be recognized by the CRM1.

Active Transport, Cell Nucleus↗
PubMed1998

[Signal transductions by the MAP kinase cascades].

The classical mitogen-activated protein(MAP) kinase cascade is one of the central intracellular signaling pathways that play a crucial role in cell proliferation, cell differentiation, cell transformation, and many other cellular responses. Two novel MAP kinase cascades, the SAPK/JNK cascade and the p38/MPK2 cascade, were identified, and were shown to function in various stress responses and apoptotic processes. Intracellular distribution of classical MAP kinase kinase (MAPKK/MEK) is regulated by its nuclear export signal (NES) which may function to suppress malignant cell transformation. CRM1 protein has been identified as a receptor for leucine-rich NES. CRM1 binds to CAN/NUP214, one of nucleopore proteins, which has been suggested to be involved in myeloid leukemia. Thus, the nuclear export system may be by somehow related to cancer development.

Animals↗
PubMed1999

Nuclear export in plants. Use of geminivirus movement proteins for a cell-based export assay.

The nuclear export of proteins and RNAs has been studied in heterokaryons or by microinjecting test substrates into nuclei of HeLa cells or Xenopus oocytes. We have previously shown that the two movement proteins BR1 and BL1 encoded by the plant pathogenic squash leaf curl virus act in a coordinated manner to facilitate virus cell-to-cell movement and that one of these (BR1) is a nuclear shuttle protein. By using a novel in vivo cell-based assay for nuclear export in which nuclear-localized BR1 is trapped by BL1 and redirected to the cortical cytoplasm, we demonstrate that residues 177 to 198 of BR1 contain a leucine-rich nuclear export signal (NES) of the type found in the Rev protein encoded by the human immunodeficiency virus and in Xenopus TFIIIA. We further show that the TFIIIA NES can functionally replace the NES of BR1 in both nuclear export and viral infectivity. These findings suggest that this basic pathway for nuclear export is highly conserved among plant and animal cells and in yeast.

Amino Acid Sequence↗
PubMed1999

Aryl hydrocarbon receptor imported into the nucleus following ligand binding is rapidly degraded via the cytosplasmic proteasome following nuclear export.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that dimerizes with the AHR nuclear translocator protein to mediate gene regulation. However, the AHR protein is rapidly depleted in vitro and in vivo following exposure to ligands. The purpose of the studies in this report was to characterize the mechanism of AHR degradation and determine the consequence of blocking the degradation process. Western blot and immunological analysis of rat smooth muscle (A7), murine Hepa-1, and human HepG2 cells show that ligand-induced degradation of AHR is blocked when the proteasome is inhibited by MG-132. AHR degradation is also blocked in Hepa-1 and HepG2 cells when nuclear export is inhibited with leptomycin B. Mutation of a putative nuclear export signal present in the AHR results in the accumulation of AHR in the nucleus and reduced levels of degradation following ligand exposure. In addition, inhibition of AHR degradation results in an increase in the concentration of AHR.AHR nuclear translocator complexes associated with DNA and extends the duration that the complex resides in the nucleus. These findings show that nuclear export and degradation of the AHR protein are two additional steps in the AHR-mediated signal transduction pathway and suggest novel areas for regulatory control.

Animals↗