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PubMed1999

Nuclear transport and transcriptional regulation.

Studies over the past 10 years have provided major insights into the molecular mechanisms responsible for active transport of macromolecules in and out of the nucleus. Nucleocytoplasmic transport pathways correspond to active and signal-mediated processes that involve substrates, adaptors and receptors. Regulation of both nuclear import and nuclear export is mainly exerted at the level of transport complex formation and has emerged as one of the most efficient mechanisms to adapt gene expression to the cell environment by restricting the access of transcriptional regulators to their target genes.

Animals↗
PubMed2002

Hepatitis delta virus ribonucleoproteins shuttle between the nucleus and the cytoplasm.

Hepatitis delta virus (HDV) infection of individuals infected with hepatitis B virus (HBV) is associated with more severe liver damage and an increased risk of fulminant disease. HDV is a single-stranded RNA virus that encodes a single protein, the delta antigen, which is expressed in two forms, small (S-HDAg) and large (L-HDAg). Here we show that although HDV ribonucleoproteins are mainly detected in the nucleus, they are also present in the cytoplasm of cells infected with HDV or transfected with HDV cDNA. Making use of an heterokaryon assay, we demonstrate that HDV ribonucleoproteins shuttle continuously between the nucleus and the cytoplasm. In the absence of HDV RNA, both forms of the delta antigen are retained in the nucleus, whereas in the absence of the delta antigen, HDV RNA is predominantly detected in the cytoplasm. Coexpression of HDV RNA and S-HDAg (which binds to the viral RNA and contains a nuclear localization signal) results in nuclear accumulation of the viral RNA. This suggests that HDV RNA mediates export of viral particles to the cytoplasm whereas the delta antigen triggers their reimport into the nucleus.

3T3 Cells↗
PubMed1997

[Structure and function of influenza virus NS1 and NS2 proteins].

This review discusses the structure and function of the influenza virus NS1 and NS2 proteins. The NS1 is a phosphoprotein and has two nuclear localization signals. In the nucleus, the NS1 interferes with the splicing as well as the nuclear export of cellular mRNAs. In the later time of the infection, the NS1 is present in the cytoplasm and associates with the polysomes. The NS1 binds to the 5'UTR of some viral mRNAs and stimulates translation. The NS2 is a phosphoprotein and binds to the nucleoporin yRip1 and Rab/hRip as well as the M1 protein which associates with the vRNPs. Therefore, the NS2 protein plays an important role in the nuclear export of the vRNPs. Improved technique to genetically manipulate influenza virus allowed us to rescue NS1 and NS2 mutants which are useful for further study.

Gene Expression Regulation, Viral↗
PubMed2006

Induction of hepatitis D virus large antigen translocation to the cytoplasm by hepatitis B virus surface antigens correlates with endoplasmic reticulum stress and NF-kappaB activation.

It is known that hepatitis D virus (HDV) requires hepatitis B virus (HBV) for supplying envelope proteins (HBsAgs) to produce mature virions, and the HDV large antigen (LDAg) is responsible for interacting with HBsAgs. However, the signal molecules involved in the cross-talk between HBsAgs and LDAg have never been reported. It has been previously demonstrated that the small form of HBsAg can facilitate the translocation of HDV large antigen green fluorescent protein (GFP) fusion protein (GFP-LD) from the nucleus to the cytoplasm. In this study, it was confirmed that the small form of HBsAg can facilitate both GFP-LD and authentic LDAg for nuclear export. It was also shown that the three forms of HBsAgs (large, middle and small) induced various rates (from 35.4 to 57.2%) of GFP-LD nuclear export. Since HBsAgs are localized inside the endoplasmic reticulum (ER), this suggests that ER stress possibly initiates the signal for inducing LDAg translocation. This supposition is supported by results that show that around 9% of cells appear with GFP-LD in the cytoplasm after treatment with the ER stress inducers, brefeldin A (BFA) and tunicamycin, in the absence of HBsAg. Western blot and immunofluorescence microscopy results further showed that the activation of NF-kappaB is linked to the ER stress that induces GFP-LD translocation. Combining this with results showing that tumour necrosis factor alpha (TNF-alpha) can also induce GFP-LD translocation, it was concluded that LDAg translocation correlates with ER stress and activation of NF-kappaB. Nevertheless, TNF-alpha-induced GFP-LD translocation was independent of new protein synthesis, suggesting that a post-translational event occurs to GFP-LD to allow translocation.

Active Transport, Cell Nucleus↗
PubMed2006

Nuclear localization of Survivin renders HeLa tumor cells more sensitive to apoptosis by induction of p53 and Bax.

Clinical studies have shown that nuclear expression of the inhibitor of apoptosis protein Survivin in tumor cells predicted a favorable prognosis whereas cytosolic-localized protein caused a decreased overall survival. Therefore Survivin's subcellular localization may be important for its anti-apoptotic capacity. To address this question, we investigated localization and function of Survivin in normal human lung fibroblasts (NHLFs) and HeLa tumor cells. NHLFs of early passages expressed Survivin in the nucleus and were highly sensitive to C2 ceramide, which induces the mitochondrial apoptotic pathway. In contrast, NHLFs at higher passages relocated Survivin to the cytosol and became more resistant to C2 ceramide. Blocking nuclear export of Survivin by leptomycin B in HeLa cells increased susceptibility to C2 ceramide. In addition, transduction of HeLa cells with Survivin fused to a nuclear localization signal augmented basal expression levels of p53 and Bax and enhanced sensitivity for intrinsic apoptosis. Those findings suggest that a predominant nuclear localization of Survivin increases the sensitivity for pro-apoptotic stimuli, whereas nuclear export enables Survivin to fulfill its inhibitor of apoptosis function. A therapeutic intervention which holds Survivin in the nucleus of tumor cells might improve cancer therapy.

Apoptosis↗
PubMed2002

The Saccharomyces cerevisiae Mob2p-Cbk1p kinase complex promotes polarized growth and acts with the mitotic exit network to facilitate daughter cell-specific localization of Ace2p transcription factor.

The Saccharomyces cerevisiae mitotic exit network (MEN) is a conserved signaling network that coordinates events associated with the M to G1 transition. We investigated the function of two S. cerevisiae proteins related to the MEN proteins Mob1p and Dbf2p kinase. Previous work indicates that cells lacking the Dbf2p-related protein Cbk1p fail to sustain polarized growth during early bud morphogenesis and mating projection formation (Bidlingmaier, S., E.L. Weiss, C. Seidel, D.G. Drubin, and M. Snyder. 2001. Mol. Cell. Biol. 21:2449-2462). Cbk1p is also required for Ace2p-dependent transcription of genes involved in mother/daughter separation after cytokinesis. Here we show that the Mob1p-related protein Mob2p physically associates with Cbk1p kinase throughout the cell cycle and is required for full Cbk1p kinase activity, which is periodically activated during polarized growth and mitosis. Both Mob2p and Cbk1p localize interdependently to the bud cortex during polarized growth and to the bud neck and daughter cell nucleus during late mitosis. We found that Ace2p is restricted to daughter cell nuclei via a novel mechanism requiring Mob2p, Cbk1p, and a functional nuclear export pathway. Furthermore, nuclear localization of Mob2p and Ace2p does not occur in mob1-77 or cdc14-1 mutants, which are defective in MEN signaling, even when cell cycle arrest is bypassed. Collectively, these data indicate that Mob2p-Cbk1p functions to (a) maintain polarized cell growth, (b) prevent the nuclear export of Ace2p from the daughter cell nucleus after mitotic exit, and (c) coordinate Ace2p-dependent transcription with MEN activation. These findings may implicate related proteins in linking the regulation of cell morphology and cell cycle transitions with cell fate determination and development.

Active Transport, Cell Nucleus↗
PubMed1996

Export of ribosomal subunits from resealed rat liver nuclear envelopes.

We have previously described the rat liver resealed nuclear envelope model system for the study of the selective import of nuclear proteins, and the export of poly(A)-containing mRNA [Riedel, N., Bachmann, M., Richter, H. & Fasold, H. (1987) Proc. Natl Acad. Sci. USA 83, 3540-3544]. The vesicles still respond to the importin-ATP signal for the uptake of nuclear-location-sequence (NLS)-carrying proteins. During the preparation of the vesicles and extraction of the chromatin from nuclei in cold hypotonic heparin solution, ribosomal subunits may be introduced into these envelopes, and after resealing remain stably included. Efflux from the resealed nuclear envelopes is effected by a cytoplasmatic protein fraction, and strongly enhanced in the presence of ATP. The heterogeneous nuclear RNP (hnRNP) A1, the components of importin, or GTP showed no influence on this export. The ATP-dependent efflux of mRNA is not affected by these cytoplasmic proteins in this model system.

Adenosine Triphosphate↗
PubMed2004

beta-catenin-mediated signaling: a molecular target for early chemopreventive intervention.

Dysregulation of Wnt signaling appears to be a critical event in the formation of intestinal tumors and some other cancers. Accumulating data from preclinical studies strongly suggest that targeted disruption of beta-catenin-mediated TCF signaling is a promising strategy for early chemopreventive intervention, particularly with respect to intestinal tumorigenesis. While the search for potent inhibitors is just getting underway, the ability of several synthetic and naturally occurring agents to decrease the transcriptional activity of a luciferase reporter plasmid under the control of TCF-4 regulatory elements (pTOPFLASH) has been demonstrated already. Additional enthusiasm for this approach is provided by data from several groups, which indicate that sulindac, sulindac sulfone and indomethacin can modulate the subcellular localization of beta-catenin in vivo, resulting in either decreased nuclear compartmentalization or enhanced localization of beta-catenin to the plasma membrane. Although the mechanism by which agents disrupt beta-catenin-mediated TCF signaling remains to be elucidated, possibilities include: (1) physical inhibition of the beta-catenin/TCF complex formation, (2) upregulation of the ubiquitin-mediated proteosomal degradation of beta-catenin, (3) accelerated nuclear export of beta-catenin and (4) enhanced sequestration of beta-catenin by E-cadherin. The common role of beta-catenin in both Wnt signaling and cell adhesion provides a unique opportunity to develop chemopreventive therapies that both prevent the development of cancer and delay tumor progression.

Animals↗
PubMed2005

Identification of a novel Rev-interacting cellular protein.

BACKGROUND: Human cell types respond differently to infection by human immunodeficiency virus (HIV). Defining specific interactions between host cells and viral proteins is essential in understanding how viruses exploit cellular functions and the innate strategies underlying cellular control of HIV replication. The HIV Rev protein is a post-transcriptional inducer of HIV gene expression and an important target for interaction with cellular proteins. Identification of Rev-modulating cellular factors may eventually contribute to the design of novel antiviral therapies. RESULTS: Yeast-two hybrid screening of a T-cell cDNA library with Rev as bait led to isolation of a novel human cDNA product (16.4.1). 16.4.1-containing fusion proteins showed predominant cytoplasmic localization, which was dependent on CRM1-mediated export from the nucleus. Nuclear export activity of 16.4.1 was mapped to a 60 amino acid region and a novel transport signal identified. Interaction of 16.4.1 with Rev in human cells was shown in a mammalian two-hybrid assay and by colocalization of Rev and 16.4.1 in nucleoli, indicating that Rev can recruit 16.4.1 to the nucleus/nucleoli. Rev-dependent reporter expression was inhibited by overexpressing 16.4.1 and stimulated by siRNAs targeted to 16.4.1 sequences, demonstrating that 16.4.1 expression influences the transactivation function of Rev. CONCLUSION: These results suggest that 16.4.1 may act as a modulator of Rev activity. The experimental strategies outlined in this study are applicable to the identification and biological characterization of further novel Rev-interacting cellular factors.

Active Transport, Cell Nucleus↗
PubMed2004

Nuclear export inhibitors and kinase inhibitors identified using a MAPK-activated protein kinase 2 redistribution screen.

Redistribution (BioImage) A/S, Søborg, Denmark) is a novel high-throughput screening technology that monitors translocation of specific protein components of intracellular signaling pathways within intact mammalian cells, using green fluorescent protein as a tag. A single Redistribution assay can be used to identify multiple classes of compounds that act at, or upstream of, the level of the protein target used in the primary screening assay. Such compounds may include both conventional and allosteric enzyme inhibitors, as well as protein-protein interaction modulators. We have developed a series of Redistribution assays to discover and characterize compounds that inhibit tumor necrosis factor-alpha biosynthesis via modulation of the p38 mitogen-activated protein kinase (MAPK) pathway. A primary assay was designed to identify low-molecular-weight compounds that inhibit the activation-dependent nuclear export of the p38 kinase substrate MAPK-activated protein kinase 2 (MK2). Hits from the primary screen were categorized, using secondary assays, either as direct inhibitors of MK2 nuclear export, or as inhibitors of the upstream p38 MAPK pathway. Activity profiles are presented for a nuclear export inhibitor, and a compound that structurally and functionally resembles a known p38 kinase inhibitor. These results demonstrate the utility of Redistribution technology as a pathway screening method for the identification of diverse and novel compounds that are active within therapeutically important signaling pathways.

Active Transport, Cell Nucleus↗
PubMed2005

Endosomal transport of ErbB-2: mechanism for nuclear entry of the cell surface receptor.

The cell membrane receptor ErbB-2 migrates to the nucleus. However, the mechanism of its nuclear translocation is unclear. Here, we report a novel mechanism of its nuclear localization that involves interaction with the transport receptor importin beta1, nuclear pore protein Nup358, and a host of players in endocytic internalization. Knocking down importin beta1 using small interfering RNA oligonucleotides or inactivation of small GTPase Ran by RanQ69L, a dominant-negative mutant of Ran, causes a nuclear transport defect of ErbB-2. Mutation of a putative nuclear localization signal in ErbB-2 destroys its interaction with importin beta1 and arrests nuclear translocation, while inactivation of nuclear export receptor piles up ErbB-2 within the nucleus. Additionally, blocking of internalization by a dominant-negative mutant of dynamin halts its nuclear localization. Thus, the cell membrane-embedded ErbB-2, through endocytosis using the endocytic vesicle as a vehicle, importin beta1 as a driver and Nup358 as a traffic light, migrates from the cell surface to the nucleus. This novel mechanism explains how a receptor tyrosine kinase on the cell surface can be translocated into the nucleus. This pathway may serve as a general mechanism to allow direct communication between cell surface receptors and the nucleus, and our findings thus open a new era in understanding direct trafficking between the cell membrane and nucleus.

Active Transport, Cell Nucleus↗
PubMed2001

Regulation of nucleocytoplasmic trafficking by cell adhesion receptors and the cytoskeleton.

It has become widely accepted that adhesion receptors can either directly activate, or significantly modulate, many of the signaling cascades initiated by circulating growth factors. An interesting recent development is the realization that adhesion receptors and their cytoskeletal partners can regulate the trafficking of signaling proteins between the cytoplasm and nucleus. Cell adhesion molecule control of nucleocytoplasmic trafficking allows adhesion to influence many cell decisions, and highlights the diversity of nuclear import and export mechanisms.

Active Transport, Cell Nucleus↗
PubMed2006

Phosphorylation-dependent regulation of unique nuclear and nucleolar localization signals of LIM kinase 2 in endothelial cells.

LIM kinases (LIMKs) regulate actin dynamics through cofilin phosphorylation and also have a function in the nucleus. Recently we have shown that LIMK2 shuttles between cytoplasm and nucleus in endothelial cells and that nuclear import is inhibited by protein kinase C-mediated phosphorylation of Ser-283. Here we aimed to identify the structural features of LIMK2 responsible for nuclear import. We found that the kinase domain of LIMK2 is localized exclusively in the nucleus and, in contrast to the kinase domain of LIMK1, it accumulated in the nucleolus. Through site-directed mutagenesis, we identified the basic amino acid-rich motif KKRTLRKNDRKKR (amino acids 491-503) as the functional nuclear and nucleolar localization signal of LIMK2. After fusing this motif to enhanced green fluorescent protein, the fusion protein localized exclusively in the nucleus and nucleolus. Mutagenesis studies showed that phosphorylation of Thr-494, a putative protein kinase C phosphorylation site identified within the nuclear localization signal, inhibits nuclear import of the enhanced green fluorescent protein-PDZ kinase domain of LIMK2. After inhibiting nuclear export with leptomycin B, phosphorylation of either Ser-283 or Thr-494 reduced the nuclear import of LIMK2. Phosphorylation of both Ser-283 and Thr-494 sites inhibited nuclear import completely. Our findings identify a unique basic amino acid-rich motif (amino acids 491-503) in LIMK2 which is not present in LIMK1 that serves to target the protein not only to the nucleus but also to the nucleolus. Phosphorylation of Thr-494 within this motif negatively regulates nuclear import of LIMK2.

Active Transport, Cell Nucleus↗
PubMed2002

Unique motif for nucleolar retention and nuclear export regulated by phosphorylation.

By microinjecting purified glutathione S-transferase linked to all or parts of herpes simplex virus type 1 US11 protein into either the nucleus or the cytoplasm, we have demonstrated that this nucleolar protein exhibits a new type of localization signal controlling both retention in nucleoli and export to the cytoplasm. Saturated mutagenesis combined with computer modeling allowed us to draw the fine-structure map of this domain, revealing a new proline-rich motif harboring both activities, which are temperature dependent and regulated by phosphorylation. Finally, crossing the nuclear pore complex from the cytoplasm to the nucleus is an energy-dependent process for US11 protein, while getting to nucleoli through the nucleoplasm is energy independent.

Active Transport, Cell Nucleus↗
PubMed1999

Kinase activity-dependent nuclear export opposes stress-induced nuclear accumulation and retention of Hog1 mitogen-activated protein kinase in the budding yeast Saccharomyces cerevisiae.

Budding yeast adjusts to increases in external osmolarity via a specific mitogen-activated protein kinase signal pathway, the high-osmolarity glycerol response (HOG) pathway. Studies with a functional Hog1-green fluorescent protein (GFP) fusion reveal that even under nonstress conditions the mitogen-activated protein kinase Hog1 cycles between cytoplasmic and nuclear compartments. The basal distribution of the protein seems independent of its activator, Pbs2, and independent of its phosphorylation status. Upon osmotic challenge, the Hog1-GFP fusion becomes rapidly concentrated in the nucleus from which it is reexported after return to an iso-osmotic environment or after adaptation to high osmolarity. The preconditions and kinetics of increased nuclear localization correlate with those found for the dual phosphorylation of Hog1-GFP. The duration of Hog1 nuclear residence is modulated by the presence of the general stress activators Msn2 and Msn4. Reexport of Hog1 to the cytoplasm does not require de novo protein synthesis but depends on Hog1 kinase activity. Thus, at least three different mechanisms contribute to the intracellular distribution pattern of Hog1: phosphorylation-dependent nuclear accumulation, retention by nuclear targets, and a kinase-induced export.

Base Sequence↗
PubMed2006

Local InsP3-dependent perinuclear Ca2+ signaling in cardiac myocyte excitation-transcription coupling.

Previous work showed that calmodulin (CaM) and Ca2+-CaM-dependent protein kinase II (CaMKII) are somehow involved in cardiac hypertrophic signaling, that inositol 1,4,5-trisphosphate receptors (InsP3Rs) in ventricular myocytes are mainly in the nuclear envelope, where they associate with CaMKII, and that class II histone deacetylases (e.g., HDAC5) suppress hypertrophic gene transcription. Furthermore, HDAC phosphorylation in response to neurohumoral stimuli that induce hypertrophy, such as endothelin-1 (ET-1), activates HDAC nuclear export, thereby regulating cardiac myocyte transcription. Here we demonstrate a detailed mechanistic convergence of these 3 issues in adult ventricular myocytes. We show that ET-1, which activates plasmalemmal G protein-coupled receptors and InsP3 production, elicits local nuclear envelope Ca2+ release via InsP3R. This local Ca2+ release activates nuclear CaMKII, which triggers HDAC5 phosphorylation and nuclear export (derepressing transcription). Remarkably, this Ca2+-dependent pathway cannot be activated by the global Ca2+ transients that cause contraction at each heartbeat. This novel local Ca2+ signaling in excitation-transcription coupling is analogous to but separate (and insulated) from that involved in excitation-contraction coupling. Thus, myocytes can distinguish simultaneous local and global Ca2+ signals involved in contractile activation from those targeting gene expression.

Active Transport, Cell Nucleus↗
PubMed1985

Expression of chicken egg white lysozyme by Saccharomyces cerevisiae.

An efficient yeast promoter was isolated using a beta-galactosidase (beta Gal) promoter probe vector. This promoter was then used to express chicken egg white lysozyme in yeast using a complete intron-free lysozyme-coding sequence constructed by in vitro recombination between a cDNA clone lacking the 5' end and the corresponding 5' end from a nuclear DNA clone. The resulting lysozyme is efficiently exported into the growth medium suggesting that the chicken signal sequence is recognized by the yeast secretion process.

Amino Acid Sequence↗
PubMed2002

The regulation of glycogen synthase kinase-3 nuclear export by Frat/GBP.

Previous studies have shown that nuclear levels of glycogen synthase kinase-3 (GSK-3) are dynamically regulated and may affect access of GSK-3 to its substrates. In this study we show that the GSK-3-binding protein Frat/GBP regulates the nuclear export of GSK-3. We show that Frat/GBP contains a nuclear export sequence that promotes its own nuclear export and that of associated GSK-3. Treating cells with leptomycin B increased nuclear levels of endogenous GSK-3 suggesting that an endogenous process targets GSK-3 for nuclear export. To investigate this further, we used two approaches to disrupt the interaction between GSK-3 and endogenous Frat. First we isolated mutants of GSK-3 that selectively interfered with Frat binding and found that these mutants were poorly exported. Second we expressed a peptide that competes with Frat for GSK-3 binding and found that it caused endogenous GSK-3 to accumulate in the nucleus. Together these data suggest that Frat may be the endogenous factor that targets GSK-3 for nuclear export. The dynamic expression patterns of Frat mRNAs together with the role of Frat in mediating GSK-3 nuclear export have important implications for the control of the substrate access of GSK-3 in several signaling pathways.

Active Transport, Cell Nucleus↗