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Beak and feather disease virus (BFDV) is a single-stranded circular DNA icosahedral virus that belongs to the Circoviridae family. This virus is the causative pathogen of beak and feather disease, which leads to feather loss, malformed claws, and immunosuppression of psittacine birds. Our study produced BFDV virus-like particles (VLPs) including capsid proteins, mutant Cap proteins (Cap ΔNLS54, Cap ΔNLS62, Cap C228S, and Cap ΔNES) and chimeric Cap proteins carrying the epitope (amino acid residues 64-70) of the replication-associated protein (R-Cap, Cap-R, R-Cap ΔNLS54, and Cap ΔNLS54-R). All of the aforementioned VLPs were observed via transmission electron microscopy and verified through immunogold labeling. The nuclear localization sequence (NLS) of the Cap protein was identified between amino acid residues 55-62. Nuclear export of the Cap protein depended on the nuclear export sequence (NES). All VLPs except Cap ΔNLS62 and Cap ΔNES entered the cells 2 h post-infection (hpi) and were shuttled into the nucleus at 8 hpi. Wheat germ agglutinin (WGA) blocked the nuclear entry of Cap proteins at 8 hpi and the nuclear export of Cap proteins at 16 hpi was inhibited by leptomycin B. The nuclear entry of Cap protein was inhibited by importin α and importin β inhibitors, as well as NLS peptides. Moreover, the interactions of Cap proteins and Cap VLPs with both importin α and importin β were characterized via the GST pull-down and immunofluorescence assays. These interactions were blocked by the presence of importin α and importin β inhibitors, as well as NLS peptides. Therefore, our study is the first to describe the precise position of the NLS of the BFDV Cap protein and the interaction of Cap protein with importin α and importin β in vitro.

Neuronal Per-Arnt-Sim (PAS) domain-containing protein 4 (NPAS4) is a basic helix-loop-helix (bHLH)-PAS transcription factor first discovered in neurons in the neuronal layer of the mammalian hippocampus and later discovered in pancreatic β-cells. NPAS4 has been proposed as a therapeutic target not only for depression and neurodegenerative diseases associated with synaptic dysfunction but also for type 2 diabetes and pancreas transplantation. The ability of bHLH-PAS proteins to fulfil their function depends on their intracellular trafficking, which is regulated by specific sequences, i.e. the nuclear localization signal (NLS) and the nuclear export signal (NES). However, until now, no study examining the subcellular localization signals of NPAS4 has been published. We show here that Rattus norvegicus NPAS4 was not uniformly localized in the nuclei of COS-7 and N2a cells 24 h after transfection. Additionally, cytoplasmic localization of NPAS4 was leptomycin B-sensitive. We demonstrate that NPAS4 possesses a unique arrangement of localization signals. Its bHLH domain contains an overlapping NLS and NES. We observed that its PAS-2 domain contains an NLS, an NES, and a second, proximally located, putative NLS. Moreover, the C terminus of NPAS4 contains two active NESs that overlap with a putative NLS. Our data indicate that glucose concentration could be one of the factors influencing NPAS4 localization. The presence of multiple localization signals and the differentiated localization of NPAS4 suggest a precise, multifactor-dependent regulation of NPAS4 trafficking, potentially crucial for its ability to act as a cellular stress sensor and transcription factor.

Background and Aims Heme oxygenase 1 (HO-1) is mainly regulated by the redox-sensitive transcription factor, namely nuclear factor erythroid 2-related factor 2 (Nrf2). We previously found a physically-made gold nanoparticle (GNP) can affect migration, adhesion, and proliferation of rat aortic vascular smooth muscle cells (VSMCs). This study was sought to investigate whether the GNP can affect HO-1 expression level in VSMCs. Methods Cellular fractionation, Western blotting, and immunofluorescence microscopy were used to determine Nrf2 translocation and phosphorylation. SiRNA interference was used to examine role of Nrf2 in GNP-induced HO-1 expression. Results The GNP concentration- and time-dependently enhanced HO-1 protein and mRNA expression; however, the mRNA induction was declined after 16 h treatment. The GNP treatment caused Nrf2 expression level and phosphorylation. In addition, it induced cytosolic Nrf2 translocation into nucleus. The HO-1 induction was inhibited by a ROS scavenger N-acetylcysteine (NAC), thiol-containing antioxidants (glutathione [GSH] and dithiothreitol [DTT]), JNK and p38 MAPK inhibitors, and nuclear transport inhibitor leptomycin. Meanwhile, the GNP-induced Nrf2 translocation (activation) was also reduced by NAC, JNK and p38 MAPK inhibitors, and nuclear transport inhibitor. Intriguingly, the GNP only enhanced activation of p38 MAPK but not JNK1/2. Finally, introduction of Nrf2 siRNA to cells to knockdown Nrf2 expression significantly inhibited GNP-induced HO-1 protein expression. Conclusions This study elucidates the action mechanism that the naked physically-made GNP can enhance HO-1 expression in rat aortic VSMCs by inducing Nrf2 expression and phosphorylation and translocation into nucleus. The Nrf2 activation is mediated through a redox-related reaction and p38 MAPK activation.

// Zhen-Yu She 1 and Wan-Xi Yang 1 1 The Sperm Laboratory, College of Life Sciences, Zhejiang University, Hangzhou 310058, China Correspondence to: Wan-Xi Yang, email: wxyang@spermlab.org Keywords: SOX14, HMG box, nuclear localization signal, nuclear transport, nuclear export Received: November 23, 2016 Accepted: January 11, 2017 Published: February 07, 2017 Abstract The nucleocytoplasmic shuttling of SOX transcription factors play a crucial role in the regulation of SOX protein functions during development. In this study, we have demonstrated two nuclear localization signals in the HMG box of Eriocheir sinensis SOX14A and SOX14B. These two conserved nuclear localization signals mediate nuclear transport. The N-termini nuclear localization signal mediates the calmodulin-dependent pathway and the C-termini nuclear localization signal interacts with the importin-β pathway. The targeted deletion of nuclear localization signals of SOX14A/B dramatically inhibits the nuclear accumulation. We have first time revealed a non-classic nuclear export signal in the HMG box of E. sinensis SOX14A/B proteins is responds to leptomycin B. E. sinensis SOX14A/B is transported from the nucleus to the cytoplasm via a CRM1-dependent nuclear export pathway. And E. sinensis SOX14A/B are not belong to the subgroup E SOX proteins. Furthermore, these findings could shed a light on the mechanisms involved in the nuclear export of SOX proteins. The imperfect nuclear export signal on other SOX proteins, rather than just those of the SOXE group, may also be functional for nuclear export.

The present study was conducted aiming to evaluate the in vitro and in vivo anti-parasitic efficacy of an isolated compound against the ciliate Cryptocaryon irritans. The compound was previously isolated from fermentation products of Streptomyces sp. CJK17 and designated as SFrD. Toxicity of the compound SFrD against the fish hosts (Larimichthys crocea) was also tested and its chemical structure was elucidated. The obtained results showed that the compound has potent anti-parasitic efficacy with the 10 min-, 1 h-, 2 h-, 3 h- and 4 h-LC50 (95% Confidence Intervals) of 6.8 (6.5-7.1), 3.9 (2.8-5.0), 3.3 (2.6-4.0), 2.7 (2.3-3.1) and 2.5 (2.2-2.8) mg L(-1) against theronts of C. irritans and the 6h-LC50 (95% CI) of 3.0 (2.8-3.2) mg L(-1) against the tomonts, respectively. Exposure of the compound SFrD remarkably reduced the mortality of fish infected with C. Irritans, from 100% in the control group to 61.7% and 38.3% in groups of 3.1 mg L(-1) and 6.3 mg L(-1), respectively. In the test of exposing fish to 40 mg L(-1) compound SFrD for 24h, no visible effects were observed affecting the normal behavior or any macroscopic changes. By spectrum analysis (EI-MS, (1)H NMR and (13)C NMR), the compound SFrD was identified as Leptomycin B. This study firstly demonstrated that Leptomycin B has potent anti-parasitic efficacy against ciliates in cultured marine fish.

West Nile virus (WNV) is a single-stranded, positive sense RNA virus of the family Flaviviridae and is a significant pathogen of global medical importance. Flavivirus replication is known to be exclusively cytoplasmic, but we show here for the first time that access to the nucleus of the WNV strain Kunjin (WNVKUN ) RNA-dependent RNA polymerase (protein NS5) is central to WNVKUN virus production. We show that treatment of cells with the specific nuclear export inhibitor leptomycin B (LMB) results in increased NS5 nuclear accumulation in WNVKUN -infected cells and NS5-transfected cells, indicative of nucleocytoplasmic shuttling under normal conditions. We used site-directed mutagenesis to identify the nuclear localisation sequence (NLS) responsible for WNVKUN NS5 nuclear targeting, observing that mutation of this NLS resulted in exclusively cytoplasmic accumulation of NS5 even in the presence of leptomycin B. Introduction of NS5 NLS mutations into FLSDX, an infectious clone of WNVKUN , resulted in lethality, suggesting that the ability of NS5 to traffic into the nucleus in integral to WNVKUN replication. This study thus shows for the first time that NLS-dependent trafficking into the nucleus during infection of WNVKUN NS5 is critical for viral replication. Excitingly, specific inhibitors of NS5 nuclear import reduce WNVKUN virus production, proving the principle that inhibition of WNVKUN NS5 nuclear import is a viable therapeutic avenue for antiviral drug development in the future.

The t(10;11) chromosomal translocation gives rise to the CALM-AF10 fusion gene and is found in patients with aggressive and difficult-to-treat hematopoietic malignancies. CALM-AF10-driven leukemias are characterized by HOXA gene up-regulation and a global reduction in H3K79 methylation. DOT1L, the H3K79 methyltransferase, interacts with the octapeptide/leucine zipper domain of AF10, and this region has been shown to be necessary and sufficient for CALM-AF10-mediated transformation. However, the precise role of CALM in leukemogenesis remains unclear. Here, we show that CALM contains a nuclear export signal (NES) that mediates cytoplasmic localization of CALM-AF10 and is necessary for CALM-AF10-dependent transformation. Fusions of the CALM NES (NES(CALM)-AF10) or NES motifs from heterologous proteins (ABL1, Rev, PKIA, APC) in-frame with AF10 are sufficient to immortalize murine hematopoietic progenitors in vitro. The CALM NES is essential for CALM-AF10-dependent Hoxa gene up-regulation and aberrant H3K79 methylation, possibly by mislocalization of DOT1L. Finally, we observed that CALM-AF10 leukemia cells are selectively sensitive to inhibition of nuclear export by Leptomycin B. These findings uncover a novel mechanism of leukemogenesis mediated by the nuclear export pathway and support further investigation of the utility of nuclear export inhibitors as therapeutic agents for patients with CALM-AF10 leukemias.

Histone deacetylase 4 (HDAC4) and its paralogs, HDAC5, -7, and -9 (all members of class IIa), possess multiple phosphorylation sites crucial for 14-3-3 binding and subsequent nuclear export. cAMP signaling stimulates nuclear import of HDAC4 and HDAC5, but the underlying mechanisms remain to be elucidated. Here we show that cAMP potentiates nuclear localization of HDAC9. Mutation of an SP motif conserved in HDAC4, -5, and -9 prevents cAMP-stimulated nuclear localization. Unexpectedly, this treatment inhibits phosphorylation at the SP motif, indicating an inverse relationship between the phosphorylation event and nuclear import. Consistent with this, leptomycin B-induced nuclear import and adrenocorticotropic hormone (ACTH) treatment result in the dephosphorylation at the motif. Moreover, the modification synergizes with phosphorylation at a nearby site, and similar kinetics was observed for both phosphorylation events during myoblast and adipocyte differentiation. These results thus unravel a previously unrecognized mechanism whereby cAMP promotes dephosphorylation and differentially regulates multisite phosphorylation and the nuclear localization of class IIa HDACs.

The capsid structural protein of the New World alphavirus, Venezuelan equine encephalitis virus (VEEV), interacts with the host nuclear transport proteins importin α/β1 and CRM1. Novel selective inhibitor of nuclear export (SINE) compounds, KPT-185, KPT-335 (verdinexor), and KPT-350, target the host’s primary nuclear export protein, CRM1, in a manner similar to the archetypical inhibitor Leptomycin B. One major limitation of Leptomycin B is its irreversible binding to CRM1; which SINE compounds alleviate because they are slowly reversible. Chemically inhibiting CRM1 with these compounds enhanced capsid localization to the nucleus compared to the inactive compound KPT-301, as indicated by immunofluorescent confocal microscopy. Differences in extracellular versus intracellular viral RNA, as well as decreased capsid in cell free supernatants, indicated the inhibitors affected viral assembly, which led to a decrease in viral titers. The decrease in viral replication was confirmed using a luciferase-tagged virus and through plaque assays. SINE compounds had no effect on VEEV TC83_Cm, which encodes a mutated form of capsid that is unable to enter the nucleus. Serially passaging VEEV in the presence of KPT-185 resulted in mutations within the nuclear localization and nuclear export signals of capsid. Finally, SINE compound treatment also reduced the viral titers of the related eastern and western equine encephalitis viruses, suggesting that CRM1 maintains a common interaction with capsid proteins across the New World alphavirus genus., Author Summary Our data demonstrate that novel selective inhibitor of nuclear export (SINE) compounds reduced viral replication of three related New World alphaviruses, VEEV, EEEV, and WEEV, indicating that CRM1 is instrumental to their life cycle. The novel CRM1 inhibitors have a large selective index and represent a potential pan-antiviral therapeutic that targets the host’s transport proteins, which are hijacked by the New World alphaviruses.

Lentiviral genomic RNAs are encapsidated by the viral Gag protein during virion assembly. The intracellular location of the initial Gag-RNA interaction is unknown. We previously observed feline immunodeficiency virus (FIV) Gag accumulating at the nuclear envelope during live-cell imaging, which suggested that trafficking of human immunodeficiency virus type 1 (HIV-1) and FIV Gag may differ. Here we analyzed the nucleocytoplasmic transport properties of both Gag proteins. We discovered that inhibition of the CRM1 nuclear export pathway with leptomycin B causes FIV Gag but not HIV-1 Gag to accumulate in the nucleus. Virtually all FIV Gag rapidly became intranuclear when the CRM1 export pathway was blocked, implying that most if not all FIV Gag normally undergoes nuclear cycling. In FIV-infected feline cells, some intranuclear Gag was detected in the steady state without leptomycin B treatment. When expressed individually, the FIV matrix (MA), capsid (CA), and nucleocapsid-p2 (NC-p2) domains were not capable of mediating leptomycin B-sensitive nuclear export of a fluorescent protein. In contrast, CA-NC-p2 did mediate nuclear export, with MA being dispensable. We conclude that HIV-1 and FIV Gag differ strikingly in a key intracellular trafficking property. FIV Gag is a nuclear shuttling protein that utilizes the CRM1 nuclear export pathway, while HIV-1 Gag is excluded from the nucleus. These findings expand the spectrum of lentiviral Gag behaviors and raise the possibility that FIV genome encapsidation may initiate in the nucleus.

The Rev protein is essential for the replication of lentiviruses. Rev is a shuttling protein that transports unspliced and partially spliced lentiviral RNAs from the nucleus to the cytoplasm via the nucleopore. To transport these RNAs, the human immunodeficiency virus type 1 (HIV-1) Rev uses the karyopherin β family importin β and CRM1 proteins that interact with the Rev nuclear localization signal (NLS) and nuclear exportation signal (NES), respectively. Recently, we reported the presence of new types of bipartite NLS and nucleolar localization signal (NoLS) in the bovine immunodeficiency virus (BIV) Rev protein. Here we report the characterization of the nuclear import and export pathways of BIV Rev. By using an in vitro nuclear import assay, we showed that BIV Rev is transported into the nucleus by a cytosolic and energy-dependent importin α/β classical pathway. Results from glutathione S -transferase (GST) pulldown assays that showed the binding of BIV Rev with importins α3 and α5 were in agreement with those from the nuclear import assay. We also identified a leptomycin B-sensitive NES in BIV Rev, which indicates that the protein is exported via CRM1 like HIV-1 Rev. Mutagenesis experiments showed that the BIV Rev NES maps between amino acids 109 to 121 of the protein. Remarkably, the BIV Rev NES was found to be of the cyclic AMP (cAMP)-dependent protein kinase inhibitor (PKI) type instead of the HIV-1 Rev type. In summary, our data showed that the nuclear import mechanism of BIV Rev is novel among Rev proteins characterized so far in lentiviruses.

During apoptosis the Golgi apparatus undergoes irreversible fragmentation. In part, this results from caspase-mediated cleavage of several high molecular weight coiled-coil proteins, termed golgins. These include GM130, golgin 160, and the Golgi vesicle tethering protein p115, whose caspase cleavage generates a C-terminal fragment (CTF) of 205 residues. Here we demonstrate that early during apoptosis, following the rapid cleavage of p115, endogenous CTF translocated to the cell nucleus and its nuclear import was required to enhance the apoptotic response. Expression of a series of deletion constructs identified a putative α-helical region of 26 amino acids, whose expression alone was sufficient to induce apoptosis; deletion of these 26 residues from the CTF diminished its proapoptotic activity. This region contains several potential SUMOylation sites and co-expression of SUMO together with the SUMO ligase, UBC9, resulted in SUMOylation of the p115 CTF. Significantly, when cells were treated with drugs that induce apoptosis, SUMOylation enhanced the efficiency of p115 cleavage and the kinetics of apoptosis. A construct in which a nuclear export signal was fused to the N terminus of p115 CTF accumulated in the cytoplasm and surprisingly, its expression did not induce apoptosis. In contrast, treatment of cells expressing this chimera with the antibiotic leptomycin induced its translocation into the nucleus and resulted in the concomitant induction of apoptosis. These results demonstrate that nuclear import of the p115 CTF is required for it to stimulate the apoptotic response and suggest that its mode of action is confined to the nucleus.

SUMOylation is a form of post-translational modification shown to control nuclear transport. Krüppel-like factor 5 (KLF5) is an important mediator of cell proliferation and is primarily localized to the nucleus. Here we show that mouse KLF5 is SUMOylated at lysine residues 151 and 202. Mutation of these two lysines or two conserved nearby glutamates results in the loss of SUMOylation and increased cytoplasmic distribution of KLF5, suggesting that SUMOylation enhances nuclear localization of KLF5. Lysine 151 is adjacent to a nuclear export signal (NES) that resembles a consensus NES. The NES in KLF5 directs a fused green fluorescence protein to the cytoplasm, binds the nuclear export receptor CRM1, and is inhibited by leptomycin and site-directed mutagenesis. SUMOylation facilitates nuclear localization of KLF5 by inhibiting this NES activity, and enhances the ability of KLF5 to stimulate anchorage-independent growth of HCT116 colon cancer cells. A survey of proteins whose nuclear localization is regulated by SUMOylation reveals that SUMOylation sites are frequently located in close proximity to NESs. A relatively common mechanism for SUMOylation to regulate nucleocytoplasmic transport may lie in the interplay between neighboring NES and SUMOylation motifs.

Overexpression of the proto-oncogene TRIP-Br2 (SERTAD2) has been shown to induce E2F activity and promote tumorigenesis, whereas ablation of TRIP-Br2 arrests cell proliferation. Timely degradation of many cell cycle regulators is fundamental to the maintenance of proper cell cycle progression. Here we report novel mechanism(s) that govern the tight regulation of TRIP-Br2 levels during cell cycle progression. TRIP-Br2 was observed to be a short-lived protein in which the expression level peaks at the G(1)/S boundary. TRIP-Br2 accumulated in cells treated with 26 S proteasome inhibitors. Co-immunoprecipitation studies revealed that TRIP-Br2 forms ubiquitin conjugates. In silico analysis identified a putative leucine-rich nuclear export signal (NES) motif that overlaps with the PHD-Bromo interaction domain in the acidic C-terminal transactivation domain (TAD) of TRIP-Br2. This NES motif is highly conserved in widely divergent species and in all TRIP-Br family members. TRIP-Br2 was shown to be stabilized in G(2)/M phase cells through nuclear entrapment, either by deletion of the acidic C-terminal TAD, which includes the NES motif, or by leptomycin B-mediated inhibition of the CRM1-dependent nuclear export machinery. Mutation of leucine residue 238 of this NES motif abolished the interaction between CRM1 and TRIP-Br2, as well as the nuclear export of TRIP-Br2 and its subsequent 26 S proteasome-dependent degradation. These data suggest that CRM1-mediated nuclear export may be required for the proper execution of ubiquitin-proteasome-dependent degradation of TRIP-Br2.

Glycogen synthase kinase 3 (GSK-3) is implicated in neuronal death through a causal role, and precise mechanisms have not been unambiguously defined. We show that short hairpin RNA (shRNA) knockdown of GSK-3beta, but not GSK-3alpha, protects cerebellar granule neurons from trophic-deprivation-induced death. Using compartment-targeted inhibitors of the Wnt-regulated GSK-3 pool, NLS-FRAT1, NES-FRAT1, and axin-GSK-3-interacting domain (axin-GID), we locate proapoptotic GSK-3 action to the cytosol and regulation of Bim protein turnover despite constitutive cycling of GSK-3 between the cytosol and nucleus, revealed by leptomycin B. We examine the importance of Ser21/9 (GSK-3alpha/beta) phosphorylation on proapoptotic GSK-3 function. Neurons isolated from GSK-3alpha/beta(S21A/S9A) knock-in mice survive normally and are fully sensitive to trophic-deprivation-induced death. Nonetheless, inhibition of GSK-3 catalytic activity with lithium or SB216763 protects GSK-3alpha/beta(S21A/S9A) neurons from death. This indicates that dephosphorylation of GSK-3beta/Ser9 and GSK-3alpha/Ser21 is insufficient for GSK-3 proapoptotic function and that another level of regulation is required. Gel filtration reveals a stress-induced loss of neuronal GSK-3beta from a high-molecular-mass complex with a concomitant decrease in axin-bound GSK-3beta. These data imply that Wnt-regulated GSK-3beta plays a nonredundant role in trophic-deprivation-induced death of neurons.

Germ-line mutations of the BRCA2 gene cause inherited susceptibility to breast and ovarian cancers. BRCA2 contains two nuclear localization signals, predominantly localizes in the nucleus and plays significant roles in DNA double-strand break repair. Recently, we reported that BRCA2 localizes to the centrosomes during the S and early M phases of the cell cycle. In this study, for the first time, we identified a functional nuclear export sequence (NES1; (1383)DLSDLTFLEVA(1393)) in BRCA2. The green fluorescent protein (GFP)-NES1 fusion protein was localized in the cytoplasm and could be blocked by the chromosomal region maintenance 1-specific export inhibitor leptomycin B. Mutation of a leucine residue in the NES1 motif to alanine (L1384A) resulted in both cytoplasmic and nuclear localization of the GFP-NES1 fusion protein and a nuclear accumulation of ectopic full-length BRCA2-FLAG. Moreover, treatment of cells with leptomycin B decreased centrosomal localization of BRCA2. Finally, by microinjection of an anti-BRCA2 antibody into the cytoplasm of HeLa S3 cells, we found that depletion of normal BRCA2 proteins in the cytoplasm leads to centrosome amplification and binucleated cells. Our results suggest that disruption of the NES function by genetic changes results in deregulation of BRCA2 export, which ultimately leads to centrosome disorder.

Receptor-bound and endocytosed fibroblast growth factor-1 (FGF-1) is able to cross the vesicle membrane and translocate to cytosol and nucleus. This suggests an intracellular role of FGF-1, which also signals by activating transmembrane FGF receptors. Phosphorylation of internalized FGF-1 by nuclear protein kinase C delta induces rapid export from the nuclei by a leptomycin B-sensitive pathway. In the present work, we have searched for and identified a Leu-rich nuclear export sequence (NES) at the C terminus of FGF-1 required for its nuclear export and able to confer nuclear export activity to a reporter protein in an in vivo system. Mutants where hydrophobic amino acids within the NES were exchanged for alanine exhibited reduced or abolished nuclear export. As demonstrated in co-immunoprecipitation experiments, a complex containing FGF-1, exportin-1, and its co-factor Ran-GTP, was formed in vitro. Formation of this complex in vivo was demonstrated by a peroxisomal targeting assay. Formation of the FGF-1-exportin-1-Ran-GTP complex in vitro as well as nuclear export of FGF-1 in vivo was dependent on phosphorylation of FGF-1, and it was abolished by leptomycin B. The FGF-1 NES was found to be situated along a beta-strand, which has not been reported before, since NESs usually are alpha-helical.

The tight junction (TJ) protein ZO-2 changes its subcellular distribution according to the state of confluency of the culture. Thus in confluent monolayers, it localizes at the TJ region whereas in sparse cultures it concentrates at the nucleus. The canine sequence of ZO-2 displays four putative nuclear export signals (NES), two at the second PDZ domain (NES-0 and NES-1) and the rest at the GK region (NES-2 and NES-3). The functionality of NES-0 and NES-3 was unknown, hence here we have explored it with a nuclear export assay, injecting into the nucleus of MDCK cells peptides corresponding to the ZO-2 NES sequences chemically coupled to ovalbumin. We show that both NES-0 and NES-3 are functional and sensitive to leptomycin B. We also demonstrate that NES-1, previously characterized as a non functional NES, is rendered capable of nuclear export upon the acquisition of a negative charge at its Ser369 residue. Experiments performed injecting at the nucleus WT and mutated ZO-2-GST fusion proteins revealed the need of both NES-0 and NES-1, and NES-2 and NES-3 for attaining an efficient nuclear exit of the respective amino and middle segments of ZO-2. Moreover, the transfection of MDCK cells with full-length ZO-2 revealed that the mutation of any of the NES present in the molecule was sufficient to induce nuclear accumulation of the protein.

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.

The three isoforms of human Ins(1,4,5)P3 3-kinase (IP3K) show remarkable differences in their intracellular targeting. Whereas predominant targeting to the cytoskeleton and endoplasmic reticulum has been shown for IP3K-A and IP3K-B, rat IP3K-C shuttles actively between the nucleus and cytoplasm. In the present study we examined the expression and intracellular localisation of endogenous IP3K-C in different mammalian cell lines using an isoform-specific antibody. In addition, human IP3K-C, showing remarkable differences to its rat homologue in the N-terminal targeting domain, was tagged with EGFP and used to examine active transport mechanisms into and out of the nucleus. We found both a nuclear import activity residing in its N-terminal domain and a nuclear export activity sensitive to treatment with leptomycin B. Different from the rat isoform, an exportin 1-dependent nuclear export site of the human enzyme resides outside the N-terminal targeting domain in the catalytic enzyme domain. A phylogenetic survey of vertebrate IP3K sequences indicates that in each of the three isoforms a nuclear export signal has evolved in the catalytic domain either de novo (IP3K-A) or as a substitute for an earlier evolved corresponding N-terminal signal (IP3K-B and IP3K-C). In higher vertebrates, and in particular in primates, re-export of nuclear IP3K activity may be guaranteed by the mechanism discovered.

The mammalian target of rapamycin (mTOR) regulates nutrient-dependent cell growth and proliferation through cytoplasmic targets, such as S6 kinase 1 (S6K1). Consistent with its main function in the cytoplasm, mTOR is predominantly cytoplasmic. However, previously we have found that mTOR shuttles between the nucleus and cytoplasm, and we have proposed that the nucleocytoplasmic shuttling of mTOR is required for the maximal activation of S6K1. The intrinsic signals directing mTOR nuclear transport and the underlying mechanisms are unknown. In this study we initially set out to identify nuclear export signals in mTOR. A systematic scan of the mTOR sequence revealed 16 peptides conforming to the canonical leucine-rich nuclear export signal, of which 3 were found by reporter assays to contain leptomycin B-sensitive and leucine-dependent nuclear export activity. Unexpectedly, mTOR proteins with those conserved leucines mutated to alanines were unable to enter the nucleus. Further investigation revealed that the L982A/L984A and L1287A/L1289A mutations likely induced a global structural change in mTOR, whereas the L545A/L547A mutation directly impaired the nuclear import of the protein, potentially regulated by a nucleocytoplasmic shuttling signal. The loss of nuclear import was accompanied by the significantly reduced ability of the L545A/L547A mutant to activate S6K1 in cells. Most importantly, when nuclear import was restored in the L545A/L547A mutant by the addition of an exogenous nuclear import signal, signaling to S6K1 was rescued. Taken together, our observations suggest the existence of a nuclear shuttling signal in mTOR and provide definitive evidence for the requirement of mTOR nuclear import in its cytoplasmic signaling to S6K1.

Subcellular compartmentalization and dynamic movements of steroid receptors are major steps in executing their transcription regulatory function. Though significant progress has been made in understanding the mechanisms underlying nuclear import of NLS-bearing proteins, our general and mechanistic understanding about the nuclear export processes has begun to emerge only recently. The discovery of most commonly utilized CRM1/exportin1 dependent nuclear export pathway is attributed to a potent nuclear export inhibitor leptomycin B that helped dissecting this and other nuclear export pathways. Simultaneously, utilization of green fluorescent protein (GFP)-tagged intracellular steroid receptors has contributed to not only resolving controversial issue of subcellular localization of unliganded hormone receptors but also provided further insight into finer details of receptor dynamics in living cells. With judicious use of leptomycin B and expression of GFP-tagged receptors in living cells, existence of exportin1/CRM1 independent pathway(s), nuclear export signals and receptors for bi-directional translocation that are unique to steroid receptor trafficking have been specified. Currently, we appear to be arriving at a consensus that steroid/nuclear receptors follow dynamic nucleocytoplasmic processes that deviate from the ones commonly utilized by majority of other proteins.

The bovine herpesvirus 1 (BHV-1) tegument protein VP22 is predominantly localized in the nucleus after viral infection. To analyze subcellular localization in the absence of other viral proteins, a plasmid expressing BHV-1 VP22 fused to enhanced yellow fluorescent protein (EYFP) was constructed. The transient expression of VP22 fused to EYFP in COS-7 cells confirmed the predominant nuclear localization of VP22. Analysis of the amino acid sequence of VP22 revealed that it does not have a classical nuclear localization signal (NLS). However, by constructing a series of deletion derivatives, we mapped the nuclear targeting domain of BHV-1 VP22 to amino acids (aa) 121 to 139. Furthermore, a 4-aa motif, 130 PRPR 133 , was able to direct EYFP and an EYFP dimer (dEYFP) or trimer (tEYFP) predominantly into the nucleus, whereas a deletion or mutation of this arginine-rich motif abrogated the nuclear localization property of VP22. Thus, 130 PRPR 133 is a functional nonclassical NLS. Since we observed that the C-terminal 68 aa of VP22 mediated the cytoplasmic localization of EYFP, an analysis was performed on these C-terminal amino acid sequences, and a leucine-rich motif, 204 LDRMLKSAAIRIL 216 , was detected. Replacement of the leucines in this putative nuclear export signal (NES) with neutral amino acids resulted in an exclusive nuclear localization of VP22. Furthermore, this motif was able to localize EYFP and dEYFP in the cytoplasm, and the nuclear export function of this NES could be blocked by leptomycin B. This demonstrates that this leucine-rich motif is a functional NES. These data represent the first identification of a functional NLS and NES in a herpesvirus VP22 homologue.

Sex steroids exert anti-apoptotic effects on osteoblasts/osteocytes but exert pro-apoptotic effects on osteoclasts, in both cases requiring activation of the extracellular signal-regulated kinases (ERKs). To explain the mechanistic basis of this divergence, we searched for differences in the kinetics of phosphorylation and/or in the subcellular localization of ERKs in response to 17beta-estradiol in the two cell types. In contrast to its transient effect on ERK phosphorylation in osteocytic cells (return to base line by 30 min), 17beta-estradiol-induced ERK phosphorylation in osteoclasts was sustained for at least 24 h following exposure to the hormone. Conversion of sustained ERK phosphorylation to transient, by means of cholera toxin-induced activation of the adenylate cyclase/cAMP/protein kinase A pathway, abrogated the pro-apoptotic effect of 17beta-estradiol on osteoclasts. Conversely, prolongation of ERK activation in osteocytes, by means of leptomycin B-induced inhibition of ERK export from the nucleus or overexpression of a green fluorescent protein-ERK2 mutant that resides permanently in the nucleus, converted the anti-apoptotic effect of 17beta-estradiol to a pro-apoptotic one. These findings indicate that the kinetics of ERK phosphorylation and the length of time that phospho-ERKs are retained in the nucleus are responsible for pro-versus anti-apoptotic effects of estrogen on different cell types of bone and perhaps their many other target tissues.

ZO-2 is a MAGUK protein that in confluent epithelial sheets localizes at tight junctions (TJ) whereas in sparse cultures accumulates in clusters at the nucleus. Here, we have characterized several nuclear properties of ZO-2. We observe that ZO-2 is present in the nuclear matrix and co-immunoprecipitates with lamin B(1) and actin from the nuclei of sparse cultures. We show that ZO-2 presents several NLS at its amino region, that when deleted, diminish the nuclear import of the ZO-2 amino segment and impair the ability of the region to regulate the transcriptional activity of promoters controlled by AP-1. Several RS repeats are detected in the ZO-2 amino segment, however, their deletion does not preclude the display of a speckled nuclear pattern. ZO-2 displays two putative NES. However, only the second one appears to be functional, as when conjugated to ovalbumin (OV), it is able to translocate this protein from the nucleus to the cytoplasm in a leptomycin B-sensitive way.

The nucleocytoplasmic transport processes are mediated by soluble transport factors constantly navigating between nuclear and cytoplasmic compartments. Our understanding about nuclear export of general ‘nuclear import factors’ that deliver the cargo to the nucleus is still fragmentary. Utilizing green fluorescent protein tagged glucocorticoid receptor (GR) and relA as our working model and with judicious use of LMB, we show in living cells that all the soluble components of the nuclear import machinery exit nucleus via exportin1/CRM1 independent pathway(s).

In situ detection and visualization of S-nitrosylated proteins following chemical derivatization: identification of Ran GTPase as a target for S-nitrosylation.Ckless K, Reynaert NL, Taatjes DJ, Lounsbury KM, van der Vliet A, Janssen-Heininger Y.Department of Pathology, University of Vermont, Burlington, VT 05405, USA. kckless@zoo.uvm.eduThe formation of S-nitrosylated proteins is a nitric oxide-dependent post-translational modification important in signal transduction, yet the in situ detection of S-nitrosylated proteins remains problematic. In this study, we adapted a recently developed biotin derivatization approach to visualize S-nitrosylated proteins in intact cells. This strategy circumvents the use of antibodies directed against S-nitrosocysteine, which may have problematic specificity, due to epitope instability. Endogenous protein S-nitrosylation could be observed in intact cells and in mouse lung sections using fluorophore-conjugated streptavidin and confocal microscopy, and was enhanced by S-nitrosothiols and reduced following treatment with the nitric oxide synthase inhibitor, L-N-monomethyl arginine. Intriguingly, protein S-nitrosylation was detected mainly in the nuclear compartment of cells under baseline conditions and was enhanced when nuclear export was blocked with leptomycin B. We also determined that the small GTPase Ran, a key regulator of nucleocytoplasmic transport, is a target for S-nitrosylation. These findings demonstrate that biotin derivatization is a useful approach to detect S-nitrosylated proteins in situ in cellular compartments or tissues, and will be useful in the assessment of altered S-nitrosylation in pathological conditions.

The aryl hydrocarbon receptor (AhR) mediates the toxicologic and carcinogenic properties of 2,3,7,8-tetrachlorodibenzo-p-dioxin. In the cytoplasm, the AhR is complexed with a dimer of hsp90, and the hepatitis B virus X-associated protein 2 (XAP2). Most studies that have examined the ability of XAP2 to modulate the AhR have characterized the mouse receptor (mAhR). However, the amino acid sequence of mAhR is significantly different from human AhR (hAhR) in the carboxy terminal half of the protein, and this could lead to differences in the behavior of the two receptors. mAhR-yellow fluorescent protein (YFP) and hAhR-YFP were used to compare nucleocytoplasmic shuttling properties and the ability of XAP2 to modulate their activity. As reported previously, mAhR localized predominantly in the nucleus and was redistributed to the cytoplasm by coexpression of XAP2 in COS-1 cells. Leptomycin B treatment revealed that XAP2 blocked mAhR-YFP translocation to the nucleus in the absence of ligand. In contrast, hAhR-YFP localized predominantly in the cytoplasm, and coexpression of XAP2 did not affect this localization, and did not block nuclear accumulation in the presence of leptomycin B. An XAP2 fusion protein with a nuclear localization signal fused to the carboxy terminus (XAP2-NLS) was utilized to test whether this protein could drag the AhR into the nucleus. Coexpression of mAhR-YFP and XAP2-NLS caused cytoplasmic localization of the mAhR, while hAhR-YFP was partially localized in the nucleus, suggesting that XAP2 remains bound to the hAhR during nucleocytoplasmic shuttling. The presence of XAP2 in the ligand-bound hAhR complex enhanced the rate of nuclear translocation but repressed transcriptional activity. Together, these results suggest that the hAhR differs biochemically from the mAhR.

The human IMPs (insulin-like growth factor II mRNA-binding proteins) belong to a vertebrate zipcode-binding protein family consisting of two RNA recognition motifs and four K homology domains and have been implicated in cytoplasmic mRNA localization, turnover and translational control. In the present study, we show that IMP1 is capable of translocating into nuclei of NIH 3T3 fibroblasts and its immunoreactivity is present in the nuclei of human spermatogenic cells. IMP1 does not contain a simple import signal, but nuclear entry was facilitated by disruption of RNA binding and cytoplasmic granule formation. IMP1 contains two NESs (nuclear export signals) within the RNA-binding K homology domains 2 and 4. The former is a leucine-rich leptomycin B-sensitive NES, whereas the latter is a leptomycin B-insensitive NES. Taken together, these results indicate that IMP1 may attach to its target mRNAs in the nucleus and thereby define the cytoplasmic fate of the transcripts.

C to U editing of the nuclear apolipoprotein B (apoB) transcript is mediated by a core enzyme containing a catalytic deaminase, apobec-1, and an RNA binding subunit, apobec-1 complementation factor (ACF). ACF expression is predominantly nuclear, including mutant proteins with deletions of a putative nuclear localization signal. We have now identified a novel 41-residue motif (ANS) in the auxiliary domain of ACF that functions as an authentic nuclear localization signal. ANS-green fluorescence protein and ANS-beta-galactosidase chimeras were both expressed exclusively in the nucleus, whereas wild-type chimeras or an ACF deletion mutant lacking the ANS were cytoplasmic. Nuclear accumulation of ACF is transcription-dependent, temperature-sensitive, and reversible, features reminiscent of a shuttling protein. ACF relocates to the cytoplasm after actinomycin D treatment, an effect blocked by the CRM1 inhibitor leptomycin B. Heterokaryon assays confirmed directly that ACF shuttles in vivo. ACF binds to the protein carrier, transportin 2 in vivo, and colocalizes to the nucleus as determined by confocal microscopy. Co-immunoprecipitation experiments revealed that transportin 2 binds directly to the ANS motif. These data suggest that directed nuclear localization and compartmentalization of the core complex of the apoB RNA editing enzyme is regulated through a dominant targeting sequence (ANS) contained within ACF.

Steroid hormone receptors are ligand-stimulated transcription factors that modulate gene transcription by recruiting coregulators to gene promoters. Subcellular localization and dynamic movements of transcription factors have been shown to be one of the major means of regulating their transcriptional activity. In the present report we describe the subcellular localization and the dynamics of intracellular trafficking of steroid receptor coactivator 1 (SRC-1). After its synthesis in the cytoplasm, SRC-1 is imported into the nucleus, where it activates transcription and is subsequently exported back to the cytoplasm. In both the nucleus and cytoplasm, SRC-1 is localized in speckles. The characterization of SRC-1 nuclear localization sequence reveals that it is a classic bipartite signal localized in the N-terminal region of the protein, between amino acids 18 and 36. This sequence is highly conserved within the other members of the p160 family. Additionally, SRC-1 nuclear export is inhibited by leptomycin B. The region involved in its nuclear export is localized between amino acids 990 and 1038. It is an unusually large domain differing from the classic leucine-rich NES sequences. Thus SRC-1 nuclear export involves either an alternate type of NES or is dependent on the interaction of SRC-1 with a protein, which is exported through the crm1/exportin pathway. Overall, the intracellular trafficking of SRC-1 might be a mechanism to regulate the termination of hormone action, the interaction with other signaling pathways in the cytoplasm and its degradation.

beta-Arrestin1 and beta-arrestin2 play a key role in the regulation of G protein-coupled receptor-mediated signaling, whereas the subcellular distribution of beta-arrestin1 and beta-arrestin2 has been shown to be quite different. In this study, we found that although both beta-arrestin1 and beta-arrestin2 are able to interact with ubiquitin-protein isopeptide ligase (E3) Mdm2, only expression of beta-arrestin2 leads to the relocalization of Mdm2 from the nucleus to the cytoplasm. Further study reveals that beta-arrestin2 but not beta-arrestin1 shuttles between the cytoplasm and nucleus in a leptomycin B-sensitive manner. A hydrophobic amino acid-rich region (VXXXFXXLXL) at the C terminus of beta-arrestin2 was further demonstrated to serve as a nuclear export signal responsible for the extranuclear localization of beta-arrestin2. In the corresponding region of beta-arrestin1, there is a single amino acid difference (Glu instead of Leu in beta-arrestin2), and mutation of Glu to Leu conferred to beta-arrestin1 similar subcellular distribution to that of beta-arrestin2. Moreover, data from a series of deletion mutations demonstrated that the N domain (residues 1-185) was indispensable for the nuclear localization of both beta-arrestins, and the results from a Val to Asp point mutation in the N domain also supported this notion. In addition, our data showed that nucleocytoplasmic shuttling of beta-arrestin2 was required, via protein/protein interaction, for the cytoplasmic relocalization of Mdm2 and JNK3, another well known beta-arrestin2-binding protein. Our study thus suggests that both the nuclear export signal motif and the N domain of beta-arrestins are critical for the regulation of their subcellular localization and that beta-arrestin2 may modulate the function of its binding partners such as Mdm2 and JNK3 by alteration of their subcellular distribution.

GNL3L is an evolutionarily conserved high molecular weight GTP binding nucleolar protein belonging to HSR1-MMR1 subfamily of GTPases. The present investigation reveals that GNL3L is a nucleo-cytoplasmic shuttling protein and its export from the nucleus is sensitive to Leptomycin B. Deletion mutagenesis reveals that the C-terminal domain (amino acids 501-582) is necessary and sufficient for the export of GNL3L from the nucleus and the exchange of hydrophobic residues (M567, L570 and 572) within the C-terminal domain impairs this process. Results from the protein-protein interaction analysis indicate that GNL3L interaction with CRM1 is critical for its export from the nucleus. Ectopic expression of GNL3L leads to lesser accumulation of cells in the 'G2/M' phase of cell cycle whereas depletion of endogenous GNL3L results in 'G2/M' arrest. Interestingly, cell cycle analysis followed by BrdU labeling assay indicates that significantly increased DNA synthesis occurs in cells expressing nuclear export defective mutant (GNL3L?NES) compared to the wild type or nuclear import defective GNL3L. Furthermore, increased hyperphosphorylation of Rb at Serine 780 and the upregulation of E2F1, cyclins A2 and E1 upon ectopic expression of GNL3L?NES results in faster 'S' phase progression. Collectively, the present study provides evidence that GNL3L is exported from the nucleus in CRM1 dependent manner and the nuclear localization of GNL3L is important to promote 'S' phase progression during cell proliferation. � 2015 Thoompumkal et al.

Signal-induced activation of the transcription factor serum response factor (SRF) requires alterations in actin dynamics. SRF activity can be inhibited by ectopic expression of β-actin, either because actin itself participates in SRF regulation or as a consequence of cytoskeletal perturbations. To distinguish between these possibilities, we studied actin mutants. Three mutant actins, G13R, R62D, and a C-terminal VP16 fusion protein, were shown not to polymerize in vivo, as judged by two-hybrid, immunofluorescence, and cell fractionation studies. These actins effectively inhibited SRF activation, as did wild-type actin, which increased the G-actin level without altering the F:G-actin ratio. Physical interaction between SRF and actin was not detectable by mammalian or yeast two-hybrid assays, suggesting that SRF regulation involves an unidentified cofactor. SRF activity was not blocked upon inhibition of CRM1-mediated nuclear export by leptomycin B. Two actin mutants were identified, V159N and S14C, whose expression favored F-actin formation and which strongly activated SRF in the absence of external signals. These mutants seemed unable to inhibit SRF activity, because their expression did not reduce the absolute level of G-actin as assessed by DNase I binding. Taken together, these results provide strong evidence that G-actin, or a subpopulation of it, plays a direct role in signal transduction to SRF.

Summary In Xenopus oocytes, 5S rRNA is exported out of the nucleus in the context of two ribonucleoprotein complexes (RNPs): complexed with transcription factor IIIA as the 7S RNP or as the 5S RNP with ribosomal protein L5. 5S rRNA-containing RNP export takes place at a slow rate in comparison to that of nuclear export signal-containing proteins and the U1 snRNP. Using oocyte microinjection assays we found that the export of 5S RNPs requires nuclear RanGTP and RanGTP hydrolysis and is leptomycin B-sensitive, indicating the process is mediated by the export receptor CRM1. A novel nuclear export signal motif is characterised in a region of L5 also possessing a nuclear import signal, thus identifying a shuttling domain for this protein. This same motif in L5 is found to be required for interaction with CRM1 in vitro and for export in vivo.

Gpn1 and Gpn3 are GTPases individually required for nuclear targeting of RNA polymerase II. Here we show that whereas Gpn3-EYFP distributed between the cytoplasm and cell nucleus, it was mainly cytoplasmic when coexpressed with Gpn1-Flag. Gpn3-Flag retained Gpn1-EYFP in the cytoplasm. However, Gpn3-EYFP/Gpn1-Flag nucleocytoplasmic shuttling was revealed after inhibiting nuclear export with leptomycin B. All Gpn3-EYFP coimmunoprecipitated with Gpn1-Flag, and all Gpn1-EYFP with Gpn3-Flag. Importantly, most endogenous Gpn1 and Gpn3 also associate. Gpn1–Gpn3 interaction was essential to maintain steady-state protein levels of both GTPases. We propose that most Gpn1 and Gpn3 associate, are mobilized, and function as a protein complex.

TAF(II)105 is a sub-stoichiometric subunit of TFIID important for activation of anti-apoptotic genes and B cell specific genes by the transcription factors NF-kappaB and OCA-B. This subunit is highly enriched in B and T lymphocytes, and its expression is regulated at a posttranscriptional level. In the present study we investigated the subcellular localization of TAF(II)105. In normal B cells, a significant portion of native TAF(II)105 protein is found in the cytoplasm. Treatment of these cells with B cell-specific stimuli decreased the level of cytoplasmic TAF(II)105. In adherent cultured cells, TAF(II)105 is predominantly nuclear; however, a small fraction of the cells showed either cytoplasmic or homogenous distribution of TAF(II)105. Analysis of different TAF(II)105 mutants and green fluorescence protein fusion proteins identified a region composed of two adjacent sequences displaying nuclear export activity, suggesting that nuclear export of TAF(II)105 is mediated by a composite nuclear export signal. TAF(II)105 nuclear export signal is leptomycin B-resistant indicating that it belongs to a CRM1-independent nuclear export pathway. These results reveal a novel mode of regulation of a specialized component of the general transcription apparatus that may affect the transcription of its target genes.

The presence of the class II transactivator (CIITA) activates the transcription of all MHC class II genes. Previously, we reported that deletion of a carboxyl-terminal nuclear localization signal (NLS) results in the cytoplasmic localization of CIITA and one form of the type II bare lymphocyte syndrome. However, further sequential carboxyl-terminal deletions of CIITA resulted in mutant forms of the protein that localized predominantly to the nucleus, suggesting the presence of one or more additional NLS in the remaining sequence. We identified a 10-aa motif at residues 405–414 of CIITA that contains strong residue similarity to the classical SV40 NLS. Deletion of this region results in cytoplasmic localization of CIITA and loss of transactivation activity, both of which can be rescued by replacement with the SV40 NLS. Fusion of this sequence to a heterologous protein results in its nuclear translocation, confirming the identification of a NLS. In addition to nuclear localization sequences, CIITA is also controlled by nuclear export. Leptomycin B, an inhibitor of export, blocked the nuclear to cytoplasmic translocation of CIITA; however, leptomycin did not alter the localization of the NLS mutant, indicating that this region mediates only the rate of import and does not affect CIITA export. Several candidate nuclear export sequences were also found in CIITA and one affected the export of a heterologous protein. In summary, we have demonstrated that CIITA localization is balanced between the cytoplasm and nucleus due to the presence of NLS and nuclear export signal sequences in the CIITA protein.

Rous sarcoma virus (RSV), a simple retrovirus, needs to export unspliced viral RNA from the nucleus to the cytoplasm, circumventing the host cell restriction on cytoplasmic expression of intron-containing RNA. The cytoplasmic accumulation of full-length viral RNA is promoted by two cis -acting direct repeat (DR) elements that flank the src gene; at least one copy of the DR sequence is necessary for viral replication. We show here that the DR mediates export of a reporter construct from the nucleus, suggesting it is a constitutive transport element (CTE). In contrast, human immunodeficiency virus type 1 (HIV-1) and other complex retroviruses encode accessory proteins, Rev or Rex, which promote export of incompletely spliced viral transcripts. This RNA export pathway is CRM1 dependent and can be blocked by the cytotoxic agent leptomycin B. We show here that DR-mediated export is CRM1 independent, suggesting that RSV uses a different export pathway from that of HIV-1 and other complex retroviruses. The simian retroviruses have a CTE which interacts with the cellular Tap export protein. However, we were unable to detect binding of the RSV DR RNA to Tap, suggesting it may use a different export pathway from that of the simian retroviruses. These data suggest that the RSV DR element uses a novel nucleocytoplasmic export pathway.

The mammalian transcription activator Nrf2 plays critical roles in executing oxidative stress response by binding to the regulatory DNA sequence Maf recognition element. Bach2 is an Nrf2-related transcription repressor and a tissue-specific partner of the Maf oncoprotein family. We show here how Bach2 is regulated by an oxidative stress-sensitive conditional nuclear export. In cultured cells, Bach2 was localized in cytoplasm through its C-terminal evolutionarily conserved cytoplasmic localization signal (CLS). The CLS directed leptomycin B-sensitive nuclear export of reporter proteins, suggesting its dependence on the nuclear exporter Crm1/exportin 1. However, the CLS sequence does not bear a resemblance to the leucine-rich class of nuclear export signal, and mutagenesis analysis indicated that a stretch of nonhydrophobic amino acids is essential for its activity. Oxidative stressors aborted the CLS activity and induced nuclear accumulation of Bach2. Whereas oxidative stress is known to activate MARE-dependent transcription, overexpression of Bach2 in cultured cells silenced the inducibility of MARE. The results suggest that Bach2 mediates nucleocytoplasmic communication to couple oxidative stress and transcription repression in mammalian cells.

The Rev protein of human immunodeficiency virus is a nuclear shuttling protein that promotes nuclear export of mRNAs that encode the viral structural proteins Gag, Pol, and Env. Rev binds to a highly structured RNA motif, the Rev-responsive element (RRE), that is present in all Rev-responsive viral transcripts and facilitates their entry into a nuclear export pathway by recruiting cellular export factors. In mammalian and yeast cells, the principal export receptor engaged by Rev has been identified as the importin/transportin family member CRM1/exportin 1. CRM1 binds directly to a leucine-rich nuclear export sequence (NES) present in Rev, and similar motifs have been identified in a variety of cellular nuclear shuttling proteins. We and our colleagues previously demonstrated that, in transfected Drosophila cells, HIV-1 Rev is fully functional and promotes expression of the viral envelope glycoprotein. We now demonstrate that the fundamental mechanism of Rev action in insect cells is identical to that observed in the mammalian systems. In particular, we show that Drosophila cells express a leptomycin B-sensitive homologue of human CRM1 that supports Rev-dependent gene expression and is required for nuclear export of NES-containing proteins in insect cells.

We have previously observed, using a green fluorescent protein (GFP) fusion system, that PLC-delta1 is localized mainly at the plasma membrane and in the cytosol, whereas little is present in the nucleus in Madin-Darby canine kidney cells (Fujii, M., Ohtsubo, M., Ogawa, T., Kamata, H., Hirata, H., and Yagisawa, H. (1999) Biochem. Biophys. Res. Commun. 254, 284-291). Herein, we demonstrate that PLC-delta1 has a functional nuclear export signal (NES) sequence in amino acid residues 164-177 of the EF-hand domain. The fluorescence of NES-disrupted GFP/PLC-delta1 expressed in Madin-Darby canine kidney cells was present not only at the plasma membrane and in the cytosol but also in the nucleus. Moreover, treatment with leptomycin B, a specific inhibitor of NES-dependent nuclear export, resulted in the accumulation of GFP/PLC-delta1 in the nucleus. A site-directed mutant containing a pleckstrin homology domain, which does not bind inositol 1,4,5-trisphosphate and cannot hydrolyze phosphatidylinositol 4,5-bisphosphate in vitro, accumulated in the nucleus to a much greater extent than wild-type GFP/PLC-delta1 after treatment with leptomycin B. These results suggest that PLC-delta1 is shuttled between the cytoplasm and the nucleus; its nuclear export is dependent on the leucine-rich NES sequence and its active nuclear import is regulated by an unidentified signal(s).

The adenovirus early region 1A (E1A) protein promotes cell immortalization and transformation by mediating the activities of key cellular regulators. The repressor element 1-silencing transcription factor (REST), which is a major neuronal and tumor suppressor, was previously found mainly in the cytoplasm rather than in the nuclei of adenovirus-transformed rodent cells (22). We now demonstrate that the loss of REST in the nucleus is due to its rapid degradation by the ubiquitin-proteasome system. Only nuclear REST, but not its cytoplasmic counterpart, was ubiquitinated and degraded. REST degradation was blocked by the ubiquitination inhibitor PYR-41 and the proteasome inhibitor MG-132 but not by the nuclear export inhibitor leptomycin B. REST degradation required both of its two C-terminal degrons that are recognized by the ubiquitin ligase SCF β-TrCP , since deletion or mutation of either degron eliminated degradation. Importantly, E1A was shown to mediate REST ubiquitination and degradation by upregulating β-TrCP. Knockdown of E1A in virus-transformed cells reduced both β-TrCP and ubiquitination of nuclear REST. In contrast, when expressed in HeLa cells, E1A enhanced the degradation of nuclear REST. Reconstitution of REST in virus-transformed cells negatively affected E1A-mediated cell proliferation and anchorage-independent growth. These data strongly indicate that E1A stimulates ubiquitination and proteolysis of REST in the nucleus, thereby abolishing the tumor suppressor functions of REST.

The influenza A virus matrix 1 protein (M1) shuttles between the cytoplasm and the nucleus during the viral life cycle and plays an important role in the replication, assembly, and budding of viruses. Here, a leucine-rich nuclear export signal (NES) was identified specifically for the nuclear export of the M1 protein. The predicted NES, designated the Flu-A-M1 NES, is highly conserved among all sequences from the influenza A virus subtype, but no similar NES motifs are found in the M1 sequences of influenza B or C viruses. The biological function of the Flu-A-M1 NES was demonstrated by its ability to translocate an enhanced green fluorescent protein (EGFP)-NES fusion protein from the nucleus to the cytoplasm in transfected cells, compared to the even nuclear and cytoplasmic distribution of EGFP. The translocation of EGFP-NES from the nucleus to the cytoplasm was not inhibited by leptomycin B. NES mutations in M1 caused a nuclear retention of the protein and an increased nuclear accumulation of NEP during transfection. Indeed, as shown by rescued recombinant viruses, the mutation of the NES impaired the nuclear export of M1 and significantly reduced the virus titer compared to titers of wild-type viruses. The NES-defective M1 protein was retained in the nucleus during infection, accompanied by a lowered efficiency of the nuclear export of viral RNPs (vRNPs). In conclusion, M1 nuclear export was specifically dependent on the Flu-A-M1 NES and critical for influenza A virus replication.

The Ro 60-kDa autoantigen, a ring-shaped RNA-binding protein, traffics between the nucleus and cytoplasm in vertebrate cells. In some vertebrate nuclei, Ro binds misfolded noncoding RNAs and may function in quality control. In the cytoplasm, Ro binds noncoding RNAs called Y RNAs. Y RNA binding blocks a nuclear accumulation signal, retaining Ro in the cytoplasm. Following UV irradiation, this signal becomes accessible, allowing Ro to accumulate in nuclei. To investigate how other cellular components influence the function and subcellular location of Ro, we identified several proteins that copurify with the mouse Ro protein. Here, we report that the zipcode-binding protein ZBP1 influences the subcellular localization of both Ro and the Y3 RNA. Binding of ZBP1 to the Ro/Y3 complex increases after UV irradiation and requires the Y3 RNA. Despite the lack of an identifiable CRM1-dependent export signal, nuclear export of Ro is sensitive to the CRM1 inhibitor leptomycin B. In agreement with a previous report, we find that ZBP1 export is partly dependent on CRM1. Both Ro and Y3 RNA accumulate in nuclei when ZBP1 is depleted. Our data indicate that ZBP1 may function as an adapter to export the Ro/Y3 RNA complex from nuclei.

In previous study, we have identified a nuclear localization signal (NLS) and a nucleolar localization signal (NoLS) in bovine herpesvirus-1 (BHV-1) infected cell protein 27 (BICP27), which targets predominantly to the nucleolus. Furthermore, the C-terminal 300 amino acid residues targets exclusively to the cytoplasm, suggesting that BICP27 might contain a nuclear export signal (NES). Amino acid sequence analysis revealed that there is a cluster of leucine-rich residues resembling a NES. Heterokaryon assays demonstrated that BICP27 is capable of shuttling between the nucleus and the cytoplasm of the BHV-1 infected, BICP27 and BICP27-EYFP transfected cells. Deletion mutant analysis revealed that this property is attributed to the leucine-rich NES 299LEELCAARRLSL310. Moreover, the functional NES could mediate transport of a monomer EYFP and a dimer EYFP to the cytoplasm. The nucleocytoplasmic shuttling of BICP27 and the nuclear export of NES-EYFP and NES-dEYFP could be blocked by leptomycin LMB, an inhibitor of the chromosomal region maintenance 1 (CRM1), which is the receptor for exportin-1-dependent nuclear export. In addition, the nuclear import of BICP27 was inhibited by a dominant negative Ran-GTP, namely Ran-GTP Q69L, indicating that BICP27 localized to the nucleus by means of a classic Ran dependent nuclear import mechanism. In conclusion, these results demonstrate that BICP27 shuttles between the nucleus and the cytoplasm by the functional NES and NLS through a CRM1-dependent nuclear export pathway and a Ran dependent nuclear import pathway.

The effect of staurosporine, a potent microbial inhibitor of protein kinases, on the cell cycle of cultured fibroblast cells was investigated. A low concentration of staurosporine (1–10 ng/ml) blocked the cell cycle of rat 3Y1 fibroblasts at the early G1 phase within 2 h after serum stimulation. On the other hand, a higher concentration of the drug (100 ng/ml) caused the specific G2 block. Both of these blocks were reversible. After release from the G2 block, highly synchronous transition to M phase was observed and both nuclear and cell divisions were completed within 180 min. This reversible G2 block showed a clear contrast to those by the other G2 arresters, trichostatin A and leptomycin B, which formed proliferative tetraploid cells after release by entering the cells into a new S phase without passage through M phase. The presence of trichostatin A or leptomycin B did not interfere with this synchronous progression through G2 M phases, suggesting that the arrest point of staurosporine was present in late G2 phase following those of trichostatin A and leptomycin B.

Ras GTPase controls the normal cell growth through binding with an array of effector molecules, such as Raf and PI3-kinase in a GTP-dependent manner. RASSF2, a member of the Ras association domain family, is known to be involved in the suppression of cell growth and is frequently down-regulated in various tumor tissues by promoter hypermethylation. In the present study, we demonstrate that RASSF2 shuttles between nucleus and cytoplasm by a signal-mediated process and its export from the nucleus is sensitive to leptomycin B. Amino acids between 240 to 260 in the C-terminus of RASSF2 harbor a functional nuclear export signal (NES), which is necessary and sufficient for efficient export of RASSF2 from the nucleus. Substitution of conserved Ile254, Val257 and Leu259 within the minimal NES impaired RASSF2 export from the nucleus. In addition, wild type but not the nuclear export defective RASSF2 mutant interacts with export receptor, CRM-1 and exported from the nucleus. Surprisingly, we observed nucleolar localization for the nuclear export defective mutant suggesting the possibility that RASSF2 may localize in different cellular compartments transiently in a cell cycle dependent manner and the observed nuclear localization for wild type protein may be due to faster export kinetics from the nucleolus. Furthermore, our data suggest that RASSF2 is specifically phosphorylated by MAPK/ERK-2 and the inhibitors of MAPK pathway impair the phosphorylation and subsequently block the export of RASSF2 from the nucleus. These data clearly suggest that ERK-2 mediated phosphorylation plays an important role in regulating the nucleo-cytoplasmic shuttling of RASSF2. Interestingly, nuclear import defective mutant of RASSF2 failed to induce cell cycle arrest at G1/S phase and apoptosis suggesting that RASSF2 regulates cell growth in a nuclear localization dependent manner. Collectively, these data provided evidence for the first time that MAPK/ERK-2 mediated phosphorylation regulates nucleo-cytoplasmic transport and cell growth arrest activity of RASSF2. Taken together, the present study suggests that active transport between nucleus and cytoplasm may constitute an important regulatory mechanism for RASSF2 function. � 2009 Elsevier Inc. All rights reserved.

The herpes simplex virus type 1 tegument protein known as VP13/14, or hUL47, localizes to the nucleus and binds RNA. Using fluorescence loss in photobleaching analysis, we show that hUL47 undergoes nucleocytoplasmic shuttling during infection. We identify the hUL47 nuclear export signal (NES) as a C-terminal 10-residue hydrophobic peptide and measure its efficiency relative to that of the classical human immunodeficiency virus type 1 Rev NES. Finally, we show that the hUL47 NES is sensitive to the inhibitor of CRM1-mediated nuclear export leptomycin B. Hence, hUL47 joins a growing list of virus-encoded RNA-binding proteins that use CRM1 to exit the nucleus.

Nucleocytoplasmic translocation constitutes a foundation for nuclear proteins to exert their proper functions and hence for various biological reactions to occur normally in eukaryotic cells. We reported previously that EZI/Zfp467, a 12 zinc finger motif-containing protein, localizes predominantly in the nucleus, yet the underlying mechanism still remains elusive. Here we constructed a series of mutant forms of EZI and examined their subcellular localization. The results delineated a non-canonical nuclear localization signal in the region covering the 9th to the 12th zinc fingers, which was necessary for nuclear accumulation of EZI as well as sufficient to confer nuclear localizing ability to a heterologous protein. We also found that the N-terminal domain of EZI is necessary for its nuclear export, the process of which was not sensitive to the CRM1 inhibitor leptomycin B. An interaction proteomics approach and the following co-immunoprecipitation experiments identified the nuclear import receptor importin-7 as a molecule that associated with EZI and, importantly, short interfering RNA-mediated knockdown of importin-7 expression completely abrogated nuclear accumulation of EZI. Taken together, these results identify EZI as a novel cargo protein for importin-7 and demonstrate a nucleocytoplasmic shuttling mechanism that is mediated by importin-7-dependent nuclear localization and CRM1-independent nuclear export.