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PubMed2004

Activation-induced cytidine deaminase shuttles between nucleus and cytoplasm like apolipoprotein B mRNA editing catalytic polypeptide 1.

Activation-induced cytidine deaminase (AID) is a molecule central to initiating class switch recombination, somatic hypermutation, and gene conversion of Ig genes. However, its mechanism to initiate these genetic alterations is still unclear. AID can convert cytosine to uracil on either mRNA or DNA and is involved in DNA cleavage. Although these events are expected to take place in the nucleus, overexpressed AID was found predominantly in the cytoplasm. Here, we demonstrated that AID is a nucleocytoplasmic shuttling protein with a bipartite nuclear localization signal and a nuclear export signal in its N and C termini, respectively. In addition to previously identified genetic, structural, and biochemical similarities of AID with apolipoprotein B mRNA editing catalytic polypeptide 1, an RNA editing enzyme of ApoB100 mRNA, the present finding provides another aspect to their resemblance, suggesting that both may have homologous reaction mechanisms.

APOBEC-1 Deaminase↗
PubMed1998

Nuclear localization and export signals of the human aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (Ahr) is a ligand-activated transcription factor that binds DNA in the form of a heterodimer with the Ahr nuclear translocator (hypoxia-inducible factor 1beta). We found in this study that Ahr contains both nuclear localization and export signals in the NH2-terminal region. A fusion protein composed of beta-galactosidase and full-length Ahr translocates from the cytoplasm to the nucleus in a ligand-dependent manner. However, a fusion protein lacking the PAS (Per-Ahr nuclear translocator-Sim homology) domain of the Ahr showed strong nuclear localization activity irrespective of the presence or absence of ligand. A minimum bipartite Ahr nuclear localization signal (NLS) consisting of amino acid residues 13-39 was identified by microinjection of fused proteins with glutathione S-transferase-green fluorescent protein. A NLS having mutations in bipartite basic amino acids lost nuclear translocation activity completely, which may explain the reduced binding activity to the NLS receptor, PTAC58. A 21-amino acid peptide (residues 55-75) containing the Ahr nuclear export signal is sufficient to direct nuclear export of a microinjected complex of glutathione S-transferase-Ahr-green fluorescent protein. These findings strongly suggest that Ahr act as a ligand- and signal-dependent nucleocytoplasmic shuttling protein.

Biological Transport↗
PubMed1999

Nucleocytoplasmic protein transport and recycling of Ran.

The active transport of proteins into and out of the nucleus is mediated by specific signals, the nuclear localization signal (NLS) and nuclear export signal (NES), respectively. The best characterized NLS is that of the SV40 large T antigen, which contains a cluster of basic amino acids. The NESs were first identified in the protein kinase inhibitor (PKI) and HIV Rev protein, which are rich in leucine residues. The SV40 T-NLS containing transport substrates are carried into the nucleus by an importin alpha/beta heterodimer. Importin alpha recognizes the NLS and acts as an adapter between the NLS and importin beta, whereas importin beta interacts with importin alpha bound to the NLS, and acts as a carrier of the NLS/importin alpha/beta trimer. It is generally thought that importin alpha and beta are part of a large protein family. The leucine rich NES-containing proteins are exported from the nucleus by one of the importin beta family molecules, CRM1/exportin 1. A Ras-like small GTPase Ran plays a crucial role in both import/export pathways and determines the directionality of nuclear transport. It has recently been demonstrated in living cells that Ran actually shuttles between the nucleus and the cytoplasm and that the recycling of Ran is essential for the nuclear transport. Furthermore, it has been shown that nuclear transport factor 2 (NTF2) mediates the nuclear import of RanGDP. This review largely focuses on the issue concerning the functional divergence of importin alpha family molecules and the role of Ran in nucleocytoplasmic protein transport.

Animals↗
PubMed2006

Nucleo-cytoplasmic shuttling of human Kank protein accompanies intracellular translocation of beta-catenin.

The human Kank protein has a role in controlling the formation of the cytoskeleton by regulating actin polymerization. Besides the cytoplasmic localization as reported before, we observed the nuclear localization of Kank in OS-RC-2 cells. To uncover the mechanism behind this phenomenon, we focused on the nuclear localization signal (NLS) and the nuclear export signal (NES). We found one NLS (NLS1) and two NESs (NES1 and NES2) in the N-terminal region of Kank-L that were absent in Kank-S, and another NLS (NLS2) and NES (NES3) in the common region. These signals were active as mutations introduced into them abolished the nuclear import (for NLS1 and NLS2) or the nuclear export (for NES1 to NES3) of Kank. The localization of Kank in the cells before and after treatment with leptomycin B suggested that the transportation of Kank from the nucleus to the cytoplasm was mediated by a CRM1-dependent mechanism. TOPFLASH reporter assays revealed a positive relationship between the nuclear import of Kank and the activation of beta-catenin-dependent transcription. Kank can bind to beta-catenin and regulate the subcellular distribution of beta-catenin. Based on the findings shown here, we propose that Kank has multiple functions in the cells and plays different roles in the cytoplasm and the nucleus.

Active Transport, Cell Nucleus↗
PubMed2002

A ubiquitin-interacting motif (UIM) is essential for Eps15 and Eps15R ubiquitination.

An important negative control mechanism in the signaling of epidermal growth factor (EGF) is the endocytosis and subsequent degradation of activated EGF receptors. Eps15 and its related partner Eps15R play a key role in clathrin-mediated endocytosis of transmembrane receptors. Upon EGF stimulation of the cell, Eps15 becomes both phosphorylated on tyrosine residues and monoubiquitinated. Although tyrosine phosphorylation of Eps15 has been implicated in EGF receptor internalization, the function of Eps15 ubiquitination is not known. Using a mutational approach, we have found that the second ubiquitin-interacting motif (UIM) of Eps15 and Eps15R is essential for their ubiquitination. This UIM partially overlaps with the recently characterized nuclear export signal in Eps15. We show that these two overlapping motifs have different structural requirements with respect to nuclear export signal versus ubiquitination signal activity. Our data demonstrate that the UIM does not contain the ubiquitin acceptor site but functions as a recruitment site for the ubiquitination machinery leading to the monoubiquitination of both Eps15 and Eps15R.

Active Transport, Cell Nucleus↗
PubMed2006

PINCH-1 expression during early avian embryogenesis: implications for neural crest and heart development.

The invasion of the cardiac neural crest (CNC) into the outflow tract (OFT) and subsequent OFT septation are critical events during vertebrate heart development. We previously had performed four modified differential display (DD) screens in the chick embryo to identify genes that may be involved in CNC and heart development. Full-length sequence of one of the DD clones has been obtained and identified as chick PINCH-1. This particularly interesting new cysteine-histidine-rich protein contains five protein-binding LIM domains (five double zinc fingers), a nuclear localization signal, and a nuclear export signal, allowing it to participate in integrin and growth factor signaling and possibly act as a transcription factor. We show here for the first time that chick PINCH-1 is expressed in neural crest cells, both in the neural fold and cardiac OFT, and is also expressed in mesoderm derived-structures, including the myocardium, during avian embryogenesis. The normal expression pattern and overexpression in neural crest cell explants suggest that PINCH-1 may be a regulator of neural crest cell adhesion and migration.

Amino Acid Sequence↗
PubMed2000

A rev-like NES mediates cytoplasmic localization of HERV-K cORF.

The human endogenous retrovirus K (HERV-K)-encoded protein cORF has recently been shown to be a functional homolog of the HIV Rev protein. Rev-mediated RNA export requires interaction between a leucine-rich nuclear export signal (NES) in Rev and the nuclear export receptor Crm1/exportin1. Like Rev, cORF binds to Crm1 and cORF-mediated RNA export depends on Crm1 activity. Here we document that mutation of the putative NES in cORF results in trapping of the protein in the nucleus, suggesting that the cORF NES functions in analogy to the Rev NES.

Amino Acid Motifs↗
PubMed2005

Development of an autofluorescent translocation biosensor system to investigate protein-protein interactions in living cells.

Protein-protein interactions are crucial for all cellular events. To analyze protein-protein interactions in live mammalian cells, we developed novel protein translocation biosensors composed of glutathione S-transferase, mutants of GFP, and a rational combination of nuclear import and export signals. Nuclear accumulation of the cytoplasmic biosensors served as the reliable indicator, which was induced by the formation of protein complexes and could easily be detected by fluorescence microscopy. The efficacy of the system was systematically investigated by mapping the p53/mdm2 protein interaction interface. Specificity and general applicability of the biosensors were confirmed by studying additional classes of protein interaction domains (IDs), e.g., the leucine zipper IDs of Jun/Fos and the coiled-coil ID of Bcr-Abl in different cell lines. Importantly, we found that, in comparison to protein complementation assays, our system proved highly efficient and reversible and thus suited for the identification of molecular decoys to prevent specific protein-protein interactions in living cells. Reversibility was demonstrated in competition experiments by overexpressing the specific IDs or by the application of a p53/mdm2 protein interaction inhibitor. Thus, besides the convenient mapping of protein IDs in living cells, the modular translocation system has great potential to be employed in numerous cell-based assays for the identification of small-molecule protein interaction inhibitors as potential novel therapeutics.

Animals↗
PubMed1997

The fragile X syndrome.

The fragile X syndrome is caused by the amplification of a polymorphic CGG repeat in the 5' untranslated region of the FMR1 gene and is the most common form of inherited mental retardation. When the repeat is amplified beyond 200 repeat units, the repeat and the FMR1 promoter region are methylated. As a result of this methylation the gene is silenced and no FMR1 gene product (FMRP) is translated. The lack of expression of FMRP in the fragile X syndrome causes the fragile X phenotype. A mouse model for the fragile X syndrome (knockout for FMRP) has been generated to study the pathological mechanisms leading to the symptoms seen in fragile X patients. FMRP is widely expressed in different tissues and localized predominantly in the cytoplasm associated with the 60S ribosomal subunit. The protein has RNA binding properties and possibly shuttles between cytoplasm and nucleus. The target signals necessary for this intracellular transport, like a nuclear location signal and a nuclear export signal, are present in FMRP. FMRP is also able to bind to other proteins by using specific sequence domains present in the protein. The coiled-coil structures formed by these domains are known to be involved in protein-protein interaction. In this review we postulate that FMRP is involved in the transport of RNA and/or proteins from the nucleus to the cytoplasm.

Animals↗
PubMed2000

Ran-mediated nuclear export of the von Hippel-Lindau tumor suppressor protein occurs independently of its assembly with cullin-2.

Inactivating mutations of the von Hippel-Lindau (VHL) tumor suppressor gene cause the VHL cancer syndrome and sporadic renal clear cell carcinoma. VHL engages in a nucleocytoplasmic shuttle, which is required for its function. Here, we pursue our investigation to identify mechanisms by which VHL-green fluorescent protein (VHL-GFP) is exported from the nucleus. We show that nuclear export of VHL-GFP in living cells requires ongoing RNA polymerase II activity, and is mediated by mechanisms that are temperature-sensitive and energy-dependent. In vitro nuclear export of VHL-GFP is inhibited by nuclear pore-specific lectins, requires ATP hydrolysis and polyadenylated mRNAs, and occurs with kinetics that are similar to those of proteins containing a nuclear export signal. Biochemical fractionation has revealed that nuclear export of VHL-GFP occurs by way of a Ran-dependent pathway. Size exclusion column chromatography and deletion mutant analysis suggest that VHL-GFP does not require assembly with one of its associated proteins, cullin-2, to engage in nuclear export. These results demonstrate that nuclear export of VHL-GFP is Ran-mediated and ATP hydrolysis-dependent. They also suggest that sequences outside the elongin C binding box may function as a nuclear export domain, potentially providing a novel role for this region of VHL frequently mutated in renal cell carcinoma.

Adenosine Triphosphate↗
PubMed2004

Role of the tetradecapeptide repeat domain of human histone deacetylase 6 in cytoplasmic retention.

Histone deacetylase 6 (HDAC6) contains tandem catalytic domains and a ubiquitin-binding zinc finger and displays deacetylase activity toward acetylated microtubules. Here we show that unlike its orthologs from Caenorhabditis elegans, Drosophila, and mouse, human HDAC6 possesses a tetradecapeptide repeat domain located between the second deacetylase domain and the C-terminal ubiquitin-binding motif. Related to this structural difference, the cytoplasmic localization of human, but not murine, HDAC6 is resistant to treatment with leptomycin B (LMB). Although it is dispensable for the deacetylase and ubiquitin binding activities of human HDAC6, the tetradecapeptide repeat domain displays acetyl-microtubule targeting ability. Moreover, it forms a unique structure and is required for the LMB-resistant cytoplasmic localization of human HDAC6. Besides the tetradecapeptide repeat domain, human HDAC6 possesses two LMB-sensitive nuclear export signals and a nuclear localization signal. These results thus indicate that the cytoplasmic localization for murine and human HDAC6 proteins is differentially regulated and suggest that the tetradecapeptide repeat domain serves as an important sequence element to stably retain human HDAC6 in the cytoplasm.

Active Transport, Cell Nucleus↗
PubMed2003

Ehp53, an Entamoeba histolytica protein, ancestor of the mammalian tumour suppressor p53.

This paper reports the identification of Ehp53, a p53-like Entamoeba histolytica protein, which binds to the human p53 DNA consensus sequence (oli-p53). Monoclonal antibodies against p53 (Ab-1 and Ab-2) recognized a single 53 kDa spot in two-dimensional gels and inhibited the formation of complexes produced by E. histolytica nuclear extracts and oli-p53. Additionally, E. histolytica gene promoter sequences with high homology to oli-p53 formed complexes with nuclear proteins that were abolished by oli-p53. Ehp53 protein levels increased in UV-irradiated trophozoites. This protein was also detected in Entamoeba moshkovskii and Entamoeba invadens. By confocal microscopy, Ehp53 was located in the nuclei, EhkO organelles and cytoplasm. The Ehp53-encoding gene was cloned and its predicted amino acid sequence showed 30-54 % and 50-57 % homology with important domains of the human and the Drosophila melanogaster p53 proteins, respectively. This homology included the tetramerization domain, the nuclear export signal and a nuclear localization signal. Ehp53 also contains seven of the eight DNA-binding residues and two of the four Zn(2+)-binding sites described for p53. A recombinant Ehp53 was recognized by Ab-2. Ehp53 is believed to be the first p53-like protein found in protozoa and may be the evolutionary ancestor of the mammalian p53.

Amino Acid Sequence↗
PubMed2004

Nup358/RanBP2 attaches to the nuclear pore complex via association with Nup88 and Nup214/CAN and plays a supporting role in CRM1-mediated nuclear protein export.

Nuclear pore complexes (NPCs) traverse the nuclear envelope (NE), providing a channel through which nucleocytoplasmic transport occurs. Nup358/RanBP2, Nup214/CAN, and Nup88 are components of the cytoplasmic face of the NPC. Here we show that Nup88 localizes midway between Nup358 and Nup214 and physically interacts with them. RNA interference of either Nup88 or Nup214 in human cells caused a strong reduction of Nup358 at the NE. Nup88 and Nup214 showed an interdependence at the NPC and were not affected by the absence of Nup358. These data indicate that Nup88 and Nup214 mediate the attachment of Nup358 to the NPC. We show that localization of the export receptor CRM1 at the cytoplasmic face of the NE is Nup358 dependent and represents its empty state. Also, removal of Nup358 causes a distinct reduction in nuclear export signal-dependent nuclear export. We propose that Nup358 provides both a platform for rapid disassembly of CRM1 export complexes and a binding site for empty CRM1 recycling into the nucleus.

Active Transport, Cell Nucleus↗
PubMed2005

Relevance of nuclear localization and functions of RNA helicase A.

RNA helicase A (RHA) regulates gene expression at transcriptional and post-transcriptional levels. It is also known that RHA shuttles between the nucleus and the cytoplasm. RHA possesses nuclear transport domain which functions as a nuclear localization signal (NLS) and nuclear export signal (NES), and predominantly localizes in the nucleus. Here, we identified regions that influence cellular localization of RHA. We showed that double-stranded RNA binding domain I and II (dsRBD I and II) positively localized to the cytoplasm. Mutational analysis revealed that RNA-binding ability was important to localization of dsRBD to the cytoplasm. RHA mutants lacking association with Pol II complexes were partially spilled from the nucleus. Furthermore, a minimal transactivation domain (MTAD), which is minimal region to interact with Pol II complexes, mainly localized to the nucleus in comparison with GFP. Our results indicated that the intracellular localization of RHA might be regulated by not only NLS and NES but also functional domains.

Autoantigens↗
PubMed2000

Signal-dependent nuclear export of a histone deacetylase regulates muscle differentiation.

Members of the myocyte enhancer factor-2 (MEF2) family of transcription factors associate with myogenic basic helix-loop-helix transcription factors such as MyoD to activate skeletal myogenesis. MEF2 proteins also interact with the class II histone deacetylases HDAC4 and HDAC5, resulting in repression of MEF2-dependent genes. Execution of the muscle differentiation program requires release of MEF2 from repression by HDACs, which are expressed constitutively in myoblasts and myotubes. Here we show that HDAC5 shuttles from the nucleus to the cytoplasm when myoblasts are triggered to differentiate. Calcium/calmodulin-dependent protein kinase (CaMK) signalling, which stimulates myogenesis and prevents formation of MEF2-HDAC complexes, also induces nuclear export of HDAC4 and HDAC5 by phosphorylation of these transcriptional repressors. An HDAC5 mutant lacking two CaMK phosphorylation sites is resistant to CaMK-mediated nuclear export and acts as a dominant inhibitor of skeletal myogenesis, whereas a cytoplasmic HDAC5 mutant is unable to block efficiently the muscle differentiation program. Our results highlight a mechanism for transcriptional regulation through signal- and differentiation-dependent nuclear export of a chromatin-remodelling enzyme, and suggest that nucleo-cytoplasmic trafficking of HDACs is involved in the control of cellular differentiation.

Animals↗
PubMed2006

CRM 1-mediated degradation and agonist-induced down-regulation of beta-adrenergic receptor mRNAs.

The beta1-adrenergic receptor (beta1-AR) mRNAs are post-transcriptionally regulated at the level of mRNA stability and undergo accelerated agonist-mediated degradation via interaction of its 3' untranslated region (UTR) with RNA binding proteins, including the HuR nuclear protein. In a previous report [Kirigiti et al. (2001). Mol. Pharmacol. 60:1308-1324], we examined the agonist-mediated down-regulation of the rat beta1-AR mRNAs, endogenously expressed in the rat C6 cell line and ectopically expressed in transfectant hamster DDT1MF2 and rat L6 cells. In this report, we determined that isoproterenol treatment of neonatal rat cortical neurons, an important cell type expressing beta1-ARs in the brain, results in significant decreases in beta1-AR mRNA stability, while treatment with leptomycin B, an inhibitor of the nuclear export receptor CRM 1, results in significant increases in beta1-AR mRNA stability and nuclear retention. UV-crosslinking/immunoprecipitation and glycerol gradient fractionation analyses indicate that the beta1-AR 3' UTR recognize complexes composed of HuR and multiple proteins, including CRM 1. Cell-permeable peptides containing the leucine-rich nuclear export signal (NES) were used as inhibitors of CRM 1-mediated nuclear export. When DDT1MF2 transfectants were treated with isoproterenol and peptide inhibitors, only the co-addition of the NES inhibitor reversed the isoproterenol-induced reduction of beta1-AR mRNA levels. Our results suggest that CRM 1-dependent NES-mediated mechanisms influence the degradation and agonist-mediated down-regulation of the beta1-AR mRNAs.

3' Untranslated Regions↗
PubMed2005

Charge modification at multiple C-terminal lysine residues regulates p53 oligomerization and its nucleus-cytoplasm trafficking.

The basal level of the tumor suppressor p53 is regulated by MDM2-mediated ubiquitination at specific lysines, which leads to p53 nuclear export and degradation. Upon p53 activation, however, these lysines become acetylated by p300/CREB-binding protein. Here we have reported an unexpected finding that p300-mediated acetylation also regulates p53 subcellular localization and can promote cytoplasmic localization of p53. This activity is independent of MDM2 but requires a p53 nuclear export signal and acetylation of multiple lysines by p300. Mechanistically, we showed that conversion of a minimal four of these lysines to alanines but not arginines mimics p300-mediated p53 nuclear export, and these lysine-neutralizing mutations effectively prevent p53 tetramerization, thus exposing the oligomerization-regulated nuclear export signal. Our study suggested a threshold mechanism whereby the degree of acetylation regulates p53 nucleus-cytoplasm trafficking by neutralizing a lysine-dependent charge patch, which in turn, controls oligomerization-dependent p53 nuclear export.

Acetylation↗
PubMed1999

The nuclear exportin Msn5 is required for nuclear export of the Mig1 glucose repressor of Saccharomyces cerevisiae.

BACKGROUND: Mig1 is a transcriptional repressor responsible for glucose repression of many genes in the budding yeast Saccharomyces cerevisiae. Glucose regulates Mig1 function by affecting its phosphorylation, which is catalyzed by the Snf1 protein kinase. Phosphorylation alters the subcellular localization of Mig1, causing it to be nuclear when glucose is present, and cytoplasmic when glucose is absent. RESULTS: Here, we report that Msn5, a member of the importin beta family of nuclear transport receptors, is required to export Mig1 from the nucleus when glucose is removed. Mig1 and Msn5 interacted in a yeast two-hybrid assay. Within the portion of Mig1 that regulates its nuclear transport, we found a region that directed its nuclear export. Within this region, two leucine-rich sequences similar to known nuclear export signals were not required for Mig1 export. The corresponding domain of the yeast Kluyveromyces lactis Mig1 conferred glucose-regulated Msn5-dependent protein export from the nucleus in S. cerevisiae. Sequence comparison with S. cerevisiae Mig1 revealed short patches of homology in K. lactis and K. marxianus Mig1 that might be Msn5-interaction domains. These regions overlapped with the serine residues predicted to be Snf1 phosphorylation sites, suggesting that Msn5 and Snf1 recognize similar sequences in Mig1. Altering these serines abolished glucose-dependent phosphorylation of Mig1 and caused it to be a constitutive repressor that was retained in the nucleus. CONCLUSIONS: Mig1 contains a new nuclear export signal that is phosphorylated by Snf1 upon glucose removal, causing it to be recognized by the nuclear exportin Msn5 and carried out of the nucleus into the cytoplasm where it contributes to derepression of glucose-repressed genes.

Amino Acid Sequence↗