Nucleocytoplasmic shuttling by human immunodeficiency virus type 1 Vpr

Human immunodeficiency virus type 1 (HIV-1) is capable of infecting nondividing cells such as macrophages because the viral preintegration complex is able to actively traverse the limiting nuclear pore due to the redundant and possibly overlapping nuclear import signals present in Vpr, matrix, and integrase. We have previously recognized the presence of at least two distinct and novel nuclear import signals residing within Vpr that, unlike matrix and integrase, bypass the classical importin alpha/beta-dependent signals and do not require energy or a RanGTP gradient. We now report that the carboxy-terminal region of Vpr (amino acids 73 to 96) contains a bipartite nuclear localization signal (NLS) composed of multiple arginine residues. Surprisingly, when the leucine-rich Vpr(1-71) fragment, previously shown to harbor an NLS, or full-length Vpr is fused to the C terminus of a green fluorescent protein-pyruvate kinase (GFP-PK) chimera, the resultant protein is almost exclusively detected in the cytoplasm. However, the addition of leptomycin B (LMB), a potent inhibitor of CRM1-dependent nuclear export, produces a shift from a cytoplasmic localization to a nuclear pattern, suggesting that these Vpr fusion proteins shuttle into and out of the nucleus. Studies of nuclear import with GFP-PK-Vpr fusion proteins in the presence of LMB reveals that both of the leucine-rich alpha-helices are required for effective nuclear uptake and thus define a unique NLS. Using a modified heterokaryon analysis, we have localized the Vpr nuclear export signal to the second leucine-rich helix, overlapping a portion of the amino-terminal nuclear import signal. These studies thus define HIV-1 Vpr as a nucleocytoplasmic shuttling protein.     

Characterization of a novel transferable CRM-1-independent nuclear export signal in a herpesvirus tegument protein that shuttles between the nucleus and cytoplasm

A new group of nucleocytoplasmic shuttling proteins has recently been identified in the structural proteins encoded by several alphaherpesvirus UL47 genes. Nuclear import and export signals for the bovine herpesvirus type 1 UL47 protein (VP8 or bUL47) have been described previously. Here, we study the trafficking of bUL47 in detail and identify an import signal different from that shown before. It comprises a 20-residue N-terminal peptide that is fully transferable and targets a large, normally cytosolic protein to the nucleus. A conserved RRPRRS motif within this peptide was shown to be essential but not sufficient for nuclear targeting. Using interspecies heterokaryon assays, we further demonstrate that the export activity of the published leucine-rich nuclear export signal (NES) is also transferable to a large protein but is functionally weak compared to the activity of the HIV-1 Rev NES. We show that nuclear export dictated by this bUL47 NES is sensitive to leptomycin B (LMB) and therefore dependent on the export receptor CRM-1. However, nuclear export of full-length bUL47 is fully resistant to LMB, suggesting the presence of an additional NES. We go on to identify a second NES in bUL47 within a 28-residue peptide that is in close proximity to but entirely separable from the N-terminal import signal, and we use fluorescence loss in photobleaching to confirm its activity. This NES is resistant to leptomycin B, and therefore utilizes an export receptor other than CRM-1. As this new sequence bears little similarity to other export signals so far defined, we suggest it may be involved in bUL47 export from the nucleus via a novel cellular receptor.     

Histone deacetylase 4 possesses intrinsic nuclear import and export signals

Nucleocytoplasmic trafficking of histone deacetylase 4 (HDAC4) plays an important role in regulating its function, and binding of 14-3-3 proteins is necessary for its cytoplasmic retention. Here, we report the identification of nuclear import and export sequences of HDAC4. While its N-terminal 118 residues modulate the nuclear localization, residues 244 to 279 constitute an authentic, strong nuclear localization signal. Mutational analysis of this signal revealed that three arginine-lysine clusters are necessary for its nuclear import activity. As for nuclear export, leucine-rich sequences located in the middle part of HDAC4 do not function as nuclear export signals. By contrast, a hydrophobic motif (MXXLXVXV) located at the C-terminal end serves as a nuclear export signal that is necessary for cytoplasmic retention of HDAC4. This motif is required for CRM1-mediated nuclear export of HDAC4. Furthermore, binding of 14-3-3 proteins promotes cytoplasmic localization of HDAC4 by both inhibiting its nuclear import and stimulating its nuclear export. Unlike wild-type HDAC4, a point mutant with abrogated MEF2-binding ability remains cytoplasmic upon exogenous expression of MEF2C, supporting the notion that direct MEF2 binding targets HDAC4 to the nucleus. Therefore, HDAC4 possesses intrinsic nuclear import and export signals for its dynamic nucleocytoplasmic shuttling, and association with 14-3-3 and MEF2 proteins affects such shuttling and thus directs HDAC4 to the cytoplasm and the nucleus, respectively.     

Analysis of nuclear targeting activities of transport signals in the human immunodeficiency virus Rev protein

The human immunodeficiency Rev protein shuttles between the nucleus and cytoplasm, while accumulating to high levels in the nucleus. Rev has a nuclear localization signal (NLS; AA 35-50) with an arginine-rich motif (ARM) that interacts with importin beta and a leucine-rich nuclear export signal (NES; AA 75-84) recognized by CRM1/exportin 1. Here we explore nuclear targeting activities of the transport signals of Rev. GFP tagging and quantitative fluorescence microscopy were used to study the localization behavior of Rev NLS/ARM mutants under conditions inhibiting the export of Rev. Rev mutant M5 was actively transported to the nucleus, despite its known failure to bind importin beta. Microinjection of transport substrates with Rev-NES peptides revealed that the Rev-NES has both nuclear import and export activities. Replacement of amino acid residues "PLER" (77-80) of the NES with alanines abolished bidirectional transport activity of the Rev-NES. These results indicate that both transport signals of Rev have nuclear import capabilities and that the Rev NLS has more than one nuclear targeting activity. This suggests that Rev is able to use various routes for nuclear entry rather than depending on a single pathway.     

Nuclear import and export signals in control of Nrf2

Nrf2 binds to the antioxidant response element and regulates expression and antioxidant induction of a battery of chemopreventive genes. In this study, we have identified nuclear import and export signals of Nrf2 and show that the nuclear import and export of Nrf2 is regulated by antioxidants. We demonstrate that Nrf2 contains a bipartite nuclear localization signal (NLS) and a leucine-rich nuclear export signal, which regulate Nrf2 shuttling in and out of the nucleus. Immunofluorescence and immunoblot analysis revealed that Nrf2 accumulates in the nucleus within 15 min of antioxidant treatment and is exported out of nucleus by 8 h after treatment. Nrf2 mutant lacking the NLS failed to enter the nucleus and displayed diminished expression and induction of the downstream NAD(P)H:quinone oxidoreductase 1 gene. The Nrf2 NLS sequence, when fused to green fluorescence protein, resulted in the nuclear accumulation of green fluorescence protein, indicating that this signal sequence was sufficient to direct nuclear localization of Nrf2. A nuclear export signal (NES) was characterized in the C terminus of Nrf2, the deletion of which caused Nrf2 to accumulate predominantly in the nucleus. The Nrf2 NES was sensitive to leptomycin B and could function as an independent export signal when fused to a heterologous protein. Further studies demonstrate that NES-mediated nuclear export of Nrf2 is required for degradation of Nrf2 in the cytosol. These results led to the conclusion that Nrf2 localization between cytosol and nucleus is controlled by both nuclear import and export of Nrf2, and the overall distribution of Nrf2 is probably the result from a balance between these two processes. Antioxidants change this balance in favor of nuclear accumulation of Nrf2, leading to activation of chemopreventive proteins. Once this is achieved, Nrf2 exits the nucleus for binding to INrf2 and degradation.     

The carboxyl terminus of RNA helicase A contains a bidirectional nuclear transport domain

Human RNA helicase A was recently identified to be a shuttle protein which interacts with the constitutive transport element (CTE) of type D retroviruses. Here we show that a domain of 110 amino acids at the carboxyl terminus of helicase A is both necessary and sufficient for nuclear localization as well as rapid nuclear export of glutathione S-transferase fusion proteins. The import and export activities of this domain overlap but are separable by point mutations. This bidirectional nuclear transport domain (NTD) has no obvious sequence homology to previously identified nuclear import or export signals. However, the Ran-dependent nuclear import of NTD was efficiently competed by excess amounts of the nuclear localization signal (NLS) peptide from simian virus 40 large T antigen, suggesting that import is mediated by the classical NLS pathway. The nuclear export pathway accessed by NTD is insensitive to leptomycin B and thus is distinct from the leucine-rich nuclear export signal pathway mediated by CRM1.     

A nuclear transport signal in mammalian target of rapamycin is critical for its cytoplasmic signaling to S6 kinase 1

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.     

Leptomycin B-sensitive nuclear export of MAPKAP kinase 2 is regulated by phosphorylation.

To study the intracellular localization of MAPKAP kinase 2 (MK2), which carries a putative bipartite nuclear localization signal (NLS), we constructed a green fluorescent protein-MAPKAP kinase 2 fusion protein (GFP-MK2). In transfected cells, this protein is located predominantly in the nucleus; unexpectedly, upon stress, it rapidly translocates to the cytoplasm. This translocation can be blocked by the p38 MAP kinase inhibitor SB203580, indicating its regulation by phosphorylation. Molecular mimicry of MK2 phosphorylation at T317 in GFP-MK2 led to a mutant which is located almost exclusively in the cytoplasm of the cell, whereas the mutant T317A shows no stress-induced redistribution. Since leptomycin B, which inhibits the interaction of exportin 1 with the Rev-type leucine-rich nuclear export signal (NES), blocks stress-dependent translocation of GFP-MK2, it is supposed that phosphorylation-induced export of the protein causes the translocation. We have identified the region responsible for nuclear export in MK2 which is partially overlapping with and C-terminal to the autoinhibitory motif. This region contains a cluster of hydrophobic amino acids in the characteristic spacing of a leucine-rich Rev-type NES which is necessary to direct GFP-MK2 to the cytoplasm. However, unlike the Rev-type NES, this region alone is not sufficient for nuclear export. The data obtained indicate that MK2 contains a constitutively active NLS and a stress-regulated signal for nuclear export. Keywords: nuclear export/nuclear import/protein phosphorylation/signal transduction/stress response     

Qualitative highly divergent nuclear export signals can regulate export by the competition for transport cofactors in vivo

Nucleo-cytoplasmic transport of proteins is mediated by nuclear export signals, identified in various proteins executing heterologous biological functions. However, the molecular mechanism underlying the orchestration of export is only poorly understood. Using microinjection of defined recombinant export substrates, we now demonstrate that leucine-rich nuclear export signals varied dramatically in determining the kinetics of export in vivo . Thus, nuclear export signals could be kinetically classified which correlated with their affinities for CRM1-containing export complexes in vitro . Strikingly, cotransfection experiments revealed that proteins containing a fast nuclear export signal inhibited export and the biological activity of proteins harboring a slower nuclear export signal in vivo . The affinity for export complexes seems therefore predominantly controlled by the nuclear export signal itself, even in the context of the complete protein in vivo . Overexpression of FG-rich repeats of nucleoporins affected a medium nuclear export signal containing protein to the same extent as a fast nuclear export signal containing protein, indicating that nucleoporins appear not to contribute significantly to nuclear export signal-specific export regulation. Our results imply a novel mode for controlling the biological activity of shuttle proteins already by the composition of the nuclear export signal itself.     

Phospholipase C-delta1 contains a functional nuclear export signal sequence.

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).     

Members of the tristetraprolin family of tandem CCCH zinc finger proteins exhibit CRM1-dependent nucleocytoplasmic shuttling.

Members of the tristetraprolin (TTP) family of CCCH tandem zinc finger proteins can bind directly to certain types of AU-rich elements (AREs) in mRNA. Experiments in TTP-deficient mice have shown that TTP is involved in the physiological destabilization of at least two cytokine mRNAs, those encoding tumor necrosis factor alpha and granulocyte-macrophage colony-stimulating factor. The two other known mammalian members of the TTP family, CMG1 and TIS11D, also contain ARE-binding CCCH tandem zinc finger domains and can also destabilize ARE-containing mRNAs. To investigate the effects of primary sequence on the subcellular localization of these proteins, we constructed green fluorescent protein fusions with TTP, CMG1, and TIS11D; these were predominantly cytoplasmic when expressed in 293 or HeLa cells. Deletion and mutation analyses revealed functional nuclear export signals in the amino terminus of TTP and in the carboxyl termini of CMG1 and TIS11D. This type of leucine-rich nuclear export signal interacts with the nuclear export receptor CRM1; abrogation of CRM1 activity resulted in nuclear accumulation of TTP, CMG1, and TIS11D. These proteins are thus nucleocytoplasmic shuttling proteins and rely on CRM1 for their export from the nucleus. Although TTP, CMG1, and TIS11D lack known nuclear import sequences, mapping experiments revealed that their nuclear accumulation required an intact tandem zinc finger domain but did not require RNA binding ability. These findings suggest possible roles for nuclear import and export in the regulation of cellular TTP, CMG1, and TIS11D activity.     

Nuclear Import and Export Signals Control the Subcellular Localization of Nurr1 Protein in Response to Oxidative Stress

Orphan receptor Nurr1 participates in the acquisition and maintenance of the dopaminergic cell phenotype, modulation of inflammation, and cytoprotection, but little is known about its regulation. In this study, we report that Nurr1 contains a bipartite nuclear localization signal (NLS) within its DNA binding domain and two leucine-rich nuclear export signals (NES) in its ligand binding domain. Together, these signals regulate Nurr1 shuttling in and out of the nucleus. Immunofluorescence and immunoblot analysis revealed that Nurr1 is mostly nuclear. A Nurr1 mutant lacking the NLS failed to enter the nucleus. The Nurr1 NLS sequence, when fused to green fluorescent protein, led to nuclear accumulation of this chimeric protein, indicating that this sequence was sufficient to direct nuclear localization of Nurr1. Furthermore, two NES were characterized in the ligand binding domain, whose deletion caused Nurr1 to accumulate predominantly in the nucleus. The Nurr1 NES was sensitive to CRM1 and could function as an independent export signal when fused to green fluorescent protein. Sodium arsenite, an agent that induces oxidative stress, promoted nuclear export of ectopically expressed Nurr1 in HEK293T cells, and the antioxidant N-acetylcysteine rescued from this effect. Similarly, in dopaminergic MN9D cells, arsenite induced the export of endogenous Nurr1, resulting in the loss of expression of Nurr1-dependent genes. This study illustrates that Nurr1 shuttling between the cytosol and nucleus is controlled by specific nuclear import and export signals and that oxidative stress can unbalance the distribution of Nurr1 to favor its cytosolic accumulation.     

Regulation of nucleocytoplasmic trafficking of transcription factor OREBP/TonEBP/NFAT5

The osmotic response element-binding protein (OREBP), also known as tonicity enhancer-binding protein (TonEBP) or NFAT5, regulates the hypertonicity-induced expression of a battery of genes crucial for the adaptation of mammalian cells to extracellular hypertonic stress. The activity of OREBP/TonEBP is regulated at multiple levels, including nucleocytoplasmic trafficking. OREBP/TonEBP protein can be detected in both the cytoplasm and nucleus under isotonic conditions, although it accumulates exclusively in the nucleus or cytoplasm when subjected to hypertonic or hypotonic challenges, respectively. Using immunocytochemistry and green fluorescent protein fusions, the protein domains that determine its subcellular localization were identified and characterized. We found that OREBP/TonEBP nuclear import is regulated by a nuclear localization signal. However, under isotonic conditions, nuclear export of OREBP/TonEBP is mediated by a CRM1-dependent, leucine-rich canonical nuclear export sequence (NES) located in the N terminus. Disruption of NES by site-directed mutagenesis yielded a mutant OREBP/TonEBP protein that accumulated in the nucleus under isotonic conditions but remained a target for hypotonicity-induced nuclear export. More importantly, a putative auxiliary export domain distal to the NES was identified. Disruption of the auxiliary export domain alone is sufficient to abolish the nuclear export of OREBP/TonEBP induced by hypotonicity. By using bimolecular fluorescence complementation assay, we showed that CRM1 interacts with OREBP/TonEBP, but not with a mutant protein deficient in NES. Our findings provide insight into how nucleocytoplasmic trafficking of OREBP/TonEBP is regulated by changes in extracellular tonicity.     

Nucleocytoplasmic shuttling of receptor-interacting protein 3 (RIP3): identification of novel nuclear export and import signals in RIP3

Receptor-interacting protein 3 (RIP3), a member of the RIP Ser/Thr kinase family, has been characterized as a pro-apoptotic protein involved in the tumor necrosis factor receptor-1 signaling pathway. In this study, we have mapped a minimal region of RIP3 sufficient for apoptosis induction to a fragment of 31 amino acids in length. This minimal region also functions as an unconventional nuclear localization signal sufficient to confer the import of full-length RIP3 to the nucleus to trigger apoptosis, suggesting that RIP3 is able to play an apoptosis-inducing role in the nucleus. In addition, we have characterized two novel leucine-rich nuclear export signals (NESs) that are responsible for the nuclear export of RIP3 to the cytoplasm via a chromosome region maintenance 1 (CRM1)-dependent pathway and an extra leucine-rich NES in the N terminus of RIP3 that contributes to the cytoplasmic distribution in a CRM1-independent manner. Thus, we provide the first evidence that RIP3 acts a nucleocytoplasmic shuttling protein, which presents a possible link between death receptor signaling and nuclear apoptosis.     

A novel transferable nuclear export signal mediates CRM1-independent nucleocytoplasmic shuttling of the human cytomegalovirus transactivator protein pUL69.

The best studied nuclear export processes are mediated by classical leucine-rich nuclear export signals that specify recognition by the CRM1 export receptor. However, details concerning alternative nuclear export signals and pathways are beginning to emerge. Within the family of Herpesviridae, a set of homologous regulatory proteins that are exemplified by the ICP27 of herpes simplex virus were described recently as nucleocytoplasmic shuttling proteins. Here we report that pUL69 of the beta-herpesvirus human cytomegalovirus is a nuclear protein that is able to shuttle between the nucleus and the cytoplasm independently of virus-encoded cofactors. In contrast to proteins containing a leucine-rich export signal, the shuttling activity of pUL69 was not affected by leptomycin B, indicating that pUL69 trafficking is not mediated by the export receptor CRM1. Importantly, we identified and characterized a novel type of transferable, leptomycin B-insensitive export signal that is distinct from other export signals described previously and is required for pUL69-mediated activation of gene expression. These data suggest that pUL69 is exported via a novel nuclear export pathway, based on a so far unique nuclear export signal of 28 amino acids.