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Results for “Nuclear Export Signals”

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At least 19 recordsLinked to original sources

PubMed1998

Mechanisms of progesterone receptor export from nuclei: role of nuclear localization signal, nuclear export signal, and ran guanosine triphosphate.

Steroid hormone receptors are, in most cases, mainly nuclear proteins that undergo a continuous nucleocytoplasmic shuttling. The mechanism of the nuclear export of these proteins remains largely unknown. To approach this problem experimentally in vivo, we have prepared cell lines permanently coexpressing the wild-type nuclear progesterone receptor (PR) and a cytoplasmic receptor mutant deleted of its nuclear localization signal (NLS) [(deltaNLS)PR]. Each receptor species was deleted from the epitope recognized by a specific monoclonal antibody, thus allowing separated observation of the two receptor forms in the same cells. Administration of hormone provoked formation of heterodimers during nucleocytoplasmic shuttling and import of (deltaNLS)PR into the nucleus. Washing out of the hormone allowed us to follow the export of (deltaNLS)PR into the cytoplasm. Microinjection of BSA coupled to a NLS inhibited the export of (deltaNLS)PR. On the contrary, microinjection of BSA coupled to a nuclear export signal (NES) was without effect. Moreover, leptomycin B, which inhibits NES-mediated export, was also without effect. tsBN2 cells contain a thermosensitive RCC1 protein (Ran GTP exchange protein). At the nonpermissive temperature, the nuclear export of (deltaNLS)PR could be observed, whereas the export of NES-BSA was suppressed. Microinjection of GTPgammaS confirmed that the export of (deltaNLS)PR was not dependent on GTP hydrolysis. These experiments show that the nuclear export of PR is not NES mediated but probably involves the NLS. It does not involve Ran GTP, and it is not dependent on the hydrolysis of GTP. The nucleocytoplasmic shuttling of steroid hormone receptors thus appears to utilize mechanisms different from those previously described for some viral, regulatory, and heterogeneous ribonuclear proteins.

Amino Acid Sequence↗

PubMed2000

A comparison of the activity, sequence specificity, and CRM1-dependence of different nuclear export signals.

Nuclear export sequences (NESs) have been identified in many cellular proteins, but it remains unclear how different NESs compare in activity. We describe a sensitive new in vivo export assay which we have used to assess the relative export activity of different types of NES. The most common type of export sequence resembles that first identified in the HIV-1 Rev protein and typically comprises a core of large hydrophobic amino acids that specify recognition by the CRM1 export receptor. We compared 10 previously identified Rev-type NESs in our assay, and whereas all were functional, the relative export activities of these signals varied considerably. We further identified 3 new Rev-type NESs from a computer database search, and each export signal was assigned a score of 1 to 9 and ranked in order of activity (e.g., PKI > c-ABL > Ran-BP1 > FMRP > PML > IkappaB-alpha > hdm2). The weakest NESs were found in the p53 tumor suppressor and the p53-regulated proteins p21 and hdm2, which are all normally localized to the nucleus. All of the Rev-type NESs were inactivated by mutation of key hydrophobic residues and by treatment with the CRM1-specific export inhibitor, leptomycin B. In contrast, a different type of export signal, the KNS shuttling element derived from hnRNP K, exhibited a modest export activity that was insensitive to leptomycin B treatment. KNS thus appears to mediate export via a CRM1-independent pathway. Mutagenesis of the KNS sequence identified, for the first time, specific serines and acidic residues necessary for its export activity, thereby distinguishing KNS from other types of nuclear transport signal. We have shown that different nuclear export signals can vary profoundly in activity and therefore conclude that the nuclear export rate of a specific shuttling protein largely depends on both the strength and the accessibility of its NES.

Amino Acid Sequence↗

PubMed2001

LIM domain protein Trip6 has a conserved nuclear export signal, nuclear targeting sequences, and multiple transactivation domains.

Trip6 is a member of a subfamily of LIM domain proteins, including also zyxin, LPP, Ajuba, and Hic-5, which localize primarily to focal adhesion plaques. However, in this report, we demonstrate that Trip6 is largely in the nucleus in cells treated with leptomycin B, suggesting that Trip6 shuttles between nuclear and cytoplasmic compartments and that nuclear export of Trip6 is dependent on Crm1. Consistent with this finding, we have identified a nuclear export signal (NES) in Trip6, and mutation of this NES also results in sequestration of Trip6 in the nucleus. Addition of the Trip6 NES to the nuclear v-Rel oncoprotein redirects v-Rel to the cytoplasm. Trip6 also has at least two sequences that can direct cytoplasmic beta-galactosidase to the nucleus. Using GAL4 fusion proteins and reporter gene assays, we demonstrate that Trip6 has multiple transactivation domains, including one that appears to overlap with sequences of the NES. In vitro- or in vivo-synthesized Trip6, however, does not bind to DNA-cellulose. Taken together, these results are consistent with Trip6, and other members of this LIM protein family, having a role in relaying signals between focal adhesion plaques and the nucleus.

ATPases Associated with Diverse Cellular Activitie↗

PubMed1999

The human tap nuclear RNA export factor contains a novel transportin-dependent nuclear localization signal that lacks nuclear export signal function.

The human Tap protein mediates the sequence-specific nuclear export of RNAs containing the constitutive transport element and is likely also critical for general mRNA export. Here, we demonstrate that a previously defined arginine-rich nuclear localization signal (NLS) present in Tap acts exclusively via the transportin import factor. Previously, transportin has been shown to mediate the nuclear import of several heterogeneous nuclear ribonucleoproteins, including heterogeneous nuclear ribonucleoprotein (hnRNP) A1, by binding to a sequence element termed M9. Although the Tap NLS and the hnRNP A1 M9 element are shown to compete for transportin binding, they show no sequence homology, and the Tap NLS does not conform to the recently defined M9 consensus. The Tap NLS also differs from M9 in that only the latter is able to act as a nuclear export signal. The Tap NLS is therefore the first member of a novel class of transportin-specific NLSs that lack nuclear export signal function.

Amino Acid Sequence↗

PubMed2003

A novel CRM1-mediated nuclear export signal governs nuclear accumulation of glyceraldehyde-3-phosphate dehydrogenase following genotoxic stress.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein with glycolytic and non-glycolytic functions, including pro-apoptotic activity. GAPDH accumulates in the nucleus after cells are treated with genotoxic drugs, and it is present in a protein complex that binds DNA modified by thioguanine incorporation. We identified a novel CRM1-dependent nuclear export signal (NES) comprising 13 amino acids (KKVVKQASEGPLK) in the C-terminal domain of GAPDH, truncation or mutation of which abrogated CRM1 binding and caused nuclear accumulation of GAPDH. Alanine scanning of the sequence encompassing the putative NES demonstrated at least two regions important for nuclear export. Site mutagenesis of Lys259 did not affect oligomerization but impaired nuclear efflux of GAPDH, indicating that this amino acid residue is essential for proper functioning of this NES. This novel NES does not contain multiple leucine residues unlike other CRM1-interacting NES, is conserved in GAPDH from multiple species, and has sequence similarities to the export signal found in feline immunodeficiency virus Rev protein. Similar sequences (KKVV*7-13PLK) were found in two other human proteins, U5 small nuclear ribonucleoprotein, and transcription factor BT3.

Active Transport, Cell Nucleus↗

PubMed2004

Identification of a nuclear localization signal and nuclear export signal of the umbraviral long-distance RNA movement protein.

The 27 kDa protein encoded by ORF3 of Groundnut rosette virus (GRV) is required for viral RNA protection and movement of viral RNA through the phloem. Localization studies have revealed that this protein is located in nuclei, preferentially targeting nucleoli. We have demonstrated that amino acids (aa) 108-122 of the GRV ORF3 protein contain an arginine-rich nuclear localization signal. Arginine-to-asparagine substitutions in this region decreased the level of the ORF3 protein accumulation in nuclei. A leucine-rich nuclear export signal (NES) was located at aa 148-156 of the GRV ORF3 protein. Leucine-to-alanine substitutions in this region resulted in a dramatic increase in GRV ORF3 protein accumulation in both nuclei and nucleoli. Consistent with this, we also showed that the previously identified NES of BR1 protein of Squash leaf curl virus can functionally replace the leucine-rich region of GRV ORF3 in nuclear export.

Active Transport, Cell Nucleus↗

PubMed2001

DNA binding domains in diverse nuclear receptors function as nuclear export signals.

BACKGROUND: The nuclear receptor superfamily of transcription factors directs gene expression through DNA sequence-specific interactions with target genes. Nuclear import of these receptors involves recognition of a nuclear localization signal (NLS) by importins, which mediate translocation into the nucleus. Nuclear receptors lack a leucine-rich nuclear export signal (NES), and export is insensitive to leptomycin B, indicating that nuclear export is not mediated by Crm1. RESULTS: We set out to define the NES in the glucocorticoid receptor (GR) and to characterize the export pathway. We found that the 69 amino acid DNA binding domain (DBD) of GR, which is unrelated to any known NES, is necessary and sufficient for export. Mutational analysis revealed that a 15 amino acid sequence between the two zinc binding loops in the GR-DBD confers nuclear export to a GFP reporter protein, and alanine-scanning mutagenesis was used to identify the residues within this sequence that are critical for export. The DBD is highly related (41%-88% identity) in steroid, nonsteroid, and orphan nuclear receptors, and we found that the DBDs from ten different nuclear receptors all function as export signals. DBD-dependent nuclear export is saturable, and prolonged nuclear localization of the GR increases its transcriptional activity. CONCLUSIONS: Multiple members of the nuclear receptor superfamily use a common pathway to exit the nucleus. We propose that NLS-mediated import and DBD-mediated export define a shuttling cycle that integrates the compartmentalization and activity of nuclear receptors.

Active Transport, Cell Nucleus↗

PubMed2004

Regulation of subcellular localization of the antiproliferative protein Tob by its nuclear export signal and bipartite nuclear localization signal sequences.

Tob, a member of the Tob and BTG antiproliferative protein family, plays an important role in many cellular processes including cell proliferation. In this study, we have addressed molecular mechanisms regulating subcellular localization of Tob. Treatment with leptomycin B, an inhibitor of nuclear export signal (NES) receptor, resulted in a change in subcellular distribution of Tob from its pan-cellular distribution to nuclear accumulation, indicating the existence of NES in Tob. Our results have then identified an N-terminal region (residues 2-14) of Tob as a functional NES. They have also shown that Tob has a functional, bipartite nuclear localization signal (NLS) in residues 18-40. Thus, Tob is shuttling between the nucleus and the cytoplasm by its NES and NLS. To examine a possible relationship between subcellular distribution of Tob and its function, we exogenously added a strong NLS sequence or a strong NES sequence or both to Tob. The obtained results have demonstrated that the strong NLS-added Tob has a much weaker activity to inhibit cell cycle progression from G0/G1 to S phase. These results suggest that cytoplasmic localization or nucleocytoplasmic shuttling is important for the antiproliferative function of Tob.

3T3 Cells↗

PubMed1995

A nuclear export signal in hnRNP A1: a signal-mediated, temperature-dependent nuclear protein export pathway.

Pre-mRNAs are associated with hnRNPs, and these proteins play important roles in the biogenesis of mRNAs. The hnRNP A1 is one of the most abundant hnRNPs, and although localized primarily in the nucleoplasm, shuttles continuously between the nucleus and the cytoplasm. A 38 amino acid domain within A1, termed M9, which bears no resemblance to classical nuclear localization signal (NLS) sequences, localizes A1 to the nucleus. Here we show that M9 is also a nuclear export signal; placing M9 on a protein that is otherwise restricted to the nucleus, the nucleoplasmin core domain (NPc), efficiently exports it to the cytoplasm in a temperature-dependent manner. In contrast, classical NLSs cannot promote the export of NPc. These findings demonstrate that there is a signal-dependent, temperature-sensitive nuclear export pathway and strengthen the suggestion that A1 and other shuttling hnRNPs function as carriers for RNA during export to the cytoplasm.

Amino Acid Sequence↗

PubMed1997

CRM1 is responsible for intracellular transport mediated by the nuclear export signal.

The discovery of nuclear export signals (NESs) in a number of proteins revealed the occurrence of signal-dependent transport of proteins from the nucleus to the cytoplasm. Although the consensus motif of the NESs has been shown to be a leucine-rich, short amino-acid sequence, its receptor has not been identified. A cytotoxin leptomycin B (LMB) has recently been suggested to inhibit the NES-mediated transport of Rev protein. Here we show that LMB is a potent and specific inhibitor of the NES-dependent nuclear export of proteins. Moreover, we have found a protein of relative molecular mass 110K (p110) in Xenopus oocyte extracts that binds to the intact NES but not to the mutated, non-functional NES. The binding of p110 to NES is inhibited by LMB. We show that p110 is CRM1, which is an evolutionarily conserved protein originally found as an essential nuclear protein in fission yeast and known as a likely target of LMB. We also show that nuclear export of a fission yeast protein, Dsk1, which has a leucine-rich NES, is disrupted in wild-type yeast treated with LMB or in the crm1 mutant. These results indicate that CRM1 is an essential mediator of the NES-dependent nuclear export of proteins in eukaryotic cells.

Animals↗

PubMed1997

Nucleocytoplasmic recycling of the nuclear localization signal receptor alpha subunit in vivo is dependent on a nuclear export signal, energy, and RCC1.

Nuclear protein import requires a nuclear localization signal (NLS) receptor and at least three other cytoplasmic factors. The alpha subunit of the NLS receptor, Rag cohort 1 (Rch1), enters the nucleus, probably in a complex with the beta subunit of the receptor, as well as other import factors and the import substrate. To learn more about which factors and/or events end the import reaction and how the import factors return to the cytoplasm, we have studied nucleocytoplasmic shuttling of Rch1 in vivo. Recombinant Rch1 microinjected into Vero or tsBN2 cells was found primarily in the cytoplasm. Rch1 injected into the nucleus was rapidly exported in a temperature-dependent manner. In contrast, a mutant of Rch1 lacking the first 243 residues accumulated in the nuclei of Vero cells after cytoplasmic injection. After nuclear injection, the truncated Rch1 was retained in the nucleus, but either Rch1 residues 207-217 or a heterologous nuclear export signal, but not a mutant form of residues 207-217, restored nuclear export. Loss of the nuclear transport factor RCC1 (regulator of chromosome condensation) at the nonpermissive temperature in the thermosensitive mutant cell line tsBN2 caused nuclear accumulation of wild-type Rch1 injected into the cytoplasm. However, free Rch1 injected into nuclei of tsBN2 cells at the nonpermissive temperature was exported. These results suggested that RCC1 acts at an earlier step in Rch1 recycling, possibly the disassembly of an import complex that contains Rch1 and the import substrate. Consistent with this possibility, incubation of purified RanGTP and RCC1 with NLS receptor and import substrate prevented assembly of receptor/substrate complexes or stimulated their disassembly.

Animals↗

PubMed2002

Nuclear-localization-signal-dependent and nuclear-export-signal-dependent mechanisms determine the localization of 5-lipoxygenase.

5-Lipoxygenase (5-LO) metabolizes arachidonic acid to leukotriene A4, a key intermediate in leukotriene biosynthesis. To explore the molecular mechanisms of its cell-specific localization, a fusion protein between green fluorescent protein (GFP) and human 5-LO (GFP-5LO) was expressed in various cells. GFP-5LO was localized in the cytosol in HL-60 cells and in both the nucleus and the cytosol in RBL (rat basophilic leukaemia) cells, similarly to the native enzyme in these cells. The localization of GFP fusion proteins for mutant 5-LOs in a putative bipartite nuclear localization signal (NLS), amino acids 638-655, in Chinese hamster ovary (CHO)-K1 and Swiss3T3 cells revealed that this motif is important for the nuclear localization of 5-LO. A GFP fusion protein of this short peptide localized consistently in the nucleus. Leptomycin B, a specific inhibitor of nuclear export signal (NES)-dependent transport, diminished the cytosolic localization of 5-LO in HL-60 cells and that of GFP-5LO in CHO-K1 cells, suggesting that an NES-system might also function in determining 5-LO localization. Analysis of the localization of 5-LO during the cell cycle points to a controlled movement of this enzyme. Thus we conclude that a balance of NLS- and NES-dependent mechanisms determines the cell-type-specific localization of 5-LO, suggesting a nuclear function for this enzyme.

Active Transport, Cell Nucleus↗

PubMed2002

Cytoplasmic localization of Wis1 MAPKK by nuclear export signal is important for nuclear targeting of Spc1/Sty1 MAPK in fission yeast.

Mitogen-activated protein kinase (MAPK) cascade is a ubiquitous signaling module that transmits extracellular stimuli through the cytoplasm to the nucleus; in response to activating stimuli, MAPKs translocate into the nucleus. Mammalian MEK MAPK kinases (MAPKKs) have in their N termini an MAPK-docking site and a nuclear export signal (NES) sequence, which are known to play critical roles in maintaining ERK MAPKs in the cytoplasm of unstimulated cells. Herein, we show that the Wis1 MAPKK of the stress-activated Spc1 MAPK cascade in fission yeast also has a MAPK-docking site and an NES sequence in its N-terminal domain. Unexpectedly, an inactivating mutation to the NES of chromosomal wis1(+) does not affect the subcellular localization of Spc1 MAPK, whereas this NES mutation disturbs the cytoplasmic localization of Wis1. However, when Wis1 is targeted to the nucleus by fusing to a nuclear localization signal sequence, stress-induced nuclear translocation of Spc1 is abrogated, indicating that cytoplasmic Wis1 is required for nuclear transport of Spc1 upon stress. Moreover, we have observed that a fraction of Wis1 translocates into the nucleus in response to stress. These results suggest that cytoplasmic localization of Wis1 MAPKK by its NES is important for stress signaling to the nucleus.

Active Transport, Cell Nucleus↗

PubMed2004

Analysis and prediction of leucine-rich nuclear export signals.

We present a thorough analysis of nuclear export signals and a prediction server, which we have made publicly available. The machine learning prediction method is a significant improvement over the generally used consensus patterns. Nuclear export signals (NESs) are extremely important regulators of the subcellular location of proteins. This regulation has an impact on transcription and other nuclear processes, which are fundamental to the viability of the cell. NESs are studied in relation to cancer, the cell cycle, cell differentiation and other important aspects of molecular biology. Our conclusion from this analysis is that the most important properties of NESs are accessibility and flexibility allowing relevant proteins to interact with the signal. Furthermore, we show that not only the known hydrophobic residues are important in defining a nuclear export signals. We employ both neural networks and hidden Markov models in the prediction algorithm and verify the method on the most recently discovered NESs. The NES predictor (NetNES) is made available for general use at http://www.cbs.dtu.dk/.

Active Transport, Cell Nucleus↗

PubMed2006

The C-terminal SH3 domain of CRKL as a dynamic dimerization module transiently exposing a nuclear export signal.

CRKL plays essential roles in cell signaling. It consists of an N-terminal SH2 domain followed by two SH3 domains. SH2 and SH3N bind to signaling proteins, but the function of the SH3C domain has remained largely enigmatic. We show here that the SH3C of CRKL forms homodimers in protein crystals and in solution. Evidence for dimer formation of full-length CRKL is also presented. In the SH3C dimer, a nuclear export signal (NES) is mostly buried under the domain surface. The same is true for a monomeric SH3C obtained under different crystallization conditions. Interestingly, partial SH3 unfolding, such as occurs upon dimer/monomer transition, produces a fully-accessible NES through translocation of a single beta strand. Our results document the existence of an SH3 domain dimer formed through exchange of the first SH3 domain beta strand and suggest that partial unfolding of the SH3C is important for the relay of information in vivo.

Adaptor Proteins, Signal Transducing↗

PubMed2001

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.

Active Transport, Cell Nucleus↗

PubMed1999

Regulation of RelA subcellular localization by a putative nuclear export signal and p50.

Nuclear factor kappaB (NF-kappaB) represents a family of dimeric DNA binding proteins, the pleotropic form of which is a heterodimer composed of RelA and p50 subunits. The biological activity of NF-kappaB is controlled through its subcellular localization. Inactive NF-kappaB is sequestered in the cytoplasm by physical interaction with an inhibitor, IkappaBalpha. Signal-mediated IkappaBalpha degradation triggers the release and subsequent nuclear translocation of NF-kappaB. It remains unknown whether the NF-kappaB shuttling between the cytoplasm and nucleus is subjected to additional steps of regulation. In this study, we demonstrated that the RelA subunit of NF-kappaB exhibits strong cytoplasmic localization activity even in the absence of IkappaBalpha inhibition. The cytoplasmic distribution of RelA is largely mediated by a leucine-rich sequence homologous to the recently characterized nuclear export signal (NES). This putative NES is both required and sufficient to mediate cytoplasmic localization of RelA as well as that of heterologous proteins. Furthermore, the cytoplasmic distribution of RelA is sensitive to a nuclear export inhibitor, leptomycin B, suggesting that RelA undergoes continuous nuclear export. Interestingly, expression of p50 prevents the cytoplasmic expression of RelA, leading to the nuclear accumulation of both RelA and p50. Together, these results suggest that the nuclear and cytoplasmic shuttling of RelA is regulated by both an intrinsic NES-like sequence and the p50 subunit of NF-kappaB.

Amino Acid Motifs↗

PubMed1997

Inhibition of mRNA export in vertebrate cells by nuclear export signal conjugates.

Leucine-rich nuclear export signals (NESs) are recognized by the NES receptor exportin 1 and are central to the export of multiple shuttling proteins and RNAs. The export of messenger RNA in vertebrates was, however, thought to occur by a different pathway, because inhibition by injection of a synthetic Rev NES conjugate could not be demonstrated. Here we find that peptide conjugates composed of the NES of either protein kinase A inhibitor protein (PKI) or the HIV-1 Rev protein, when coupled to human serum albumin, are potent inhibitors of mRNA and small nuclear RNA export. These results provide direct evidence that mRNA export in vertebrates depends on interactions between an NES and its cognate NES receptors. PKI NES conjugates are significantly more efficient at inhibiting RNA export than are REV NES conjugates, indicating that different NESs may have different abilities to promote protein and RNA export. Surprisingly, an expected control conjugate containing the mutant Rev NES sequence M10 strongly inhibited the export of intronless dihydrofolate reductase mRNA. Nuclear injection of NES peptide conjugates led to mislocalization to the nucleus of 10-20% of the cytoplasmic Ran GTPase-binding protein (RanBP1) indicating that RanBP1 shuttles between the nucleus and the cytoplasm via an NES pathway. These results demonstrate that in vertebrates the export of mRNA, like that of small nuclear RNA, 5S rRNA, and transport factors such as RanBP1, employs NES-mediated molecular machinery.

Amino Acid Sequence↗