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PubMed2004

Nucleo-cytoplasmic shuttling of the beet necrotic yellow vein virus RNA-3-encoded p25 protein.

The protein p25 encoded by beet necrotic yellow vein virus (BNYVV) RNA-3 is involved in symptom expression of infected plants. Confocal microscopy analysis of wild-type and mutated p25 fused to GFP and transiently expressed in BY-2 tobacco suspension cells identified a nuclear localization signal (NLS) in the N-terminal part of the protein. Functionality of the NLS was confirmed by pull-down assays using rice and pepper importin-alpha. Furthermore, it was demonstrated that p25 contains a nuclear export sequence sensitive to leptomycin B. The nuclear export signal (NES) was characterized by mutagenesis. A GFP-p25 fusion protein expressed during a BNYVV infection of Chenopodium quinoa leaves had the same subcellular localization as observed during transient expression in BY-2 cells. The symptom phenotype induced by expression of GFP-p25 during infection was similar to that induced by wild-type virus. Studies with mutated derivatives of GFP-p25 revealed that symptom phenotype was altered when the subcellular localization of GFP-p25 was modified.

Active Transport, Cell Nucleus↗
PubMed2004

Cytosolic, nuclear and nucleolar localization signals determine subcellular distribution and activity of the NF-kappaB inducing kinase NIK.

It has been shown previously that the transcription factor NF-kappaB and its inhibitor IkappaBalpha shuttle constitutively between cytosol and nucleus. Moreover, we have recently demonstrated nucleocytoplasmic shuttling of the NF-kappaB-inducing kinase NIK, a component of the NF-kappaB pathway, which is essential for lymph node development and B-cell function. Here we show that nuclear NIK also occurs in nucleoli and that this localization is mediated by a stretch of basic amino acids in the N-terminal part of the protein (R(143)-K-K-R-K-K-K(149)). This motif is necessary and sufficient for nucleolar localization of NIK, as judged by nuclear localization of mutant versions of the full-length protein and the fact that coupling of these seven amino acids to GFP also leads to accumulation in nucleoli. Using fluorescence loss in photobleaching (FLIP) and fluorescence recovery after photobleaching (FRAP) approaches, we demonstrate a dynamic distribution between nucleoli and nucleoplasm and a high mobility of NIK in both compartments. Together with the nuclear export signal in the C-terminal portion of NIK that we have also characterized in detail, the nuclear/nucleolar targeting signals of NIK mediate dynamic circulation of the protein between the cytoplasmic, nucleoplasmic and nucleolar compartments. We demonstrate that nuclear NIK is capable of activating NF-kappaB and that this effect is diminished by nucleolar localization. Thus, subcellular distribution of NIK to different compartments might be a means of regulating the function of this kinase.

Base Sequence↗
PubMed2002

A synthetic HIV-1 Rev inhibitor interfering with the CRM1-mediated nuclear export.

The HIV-1 Rev protein is an essential regulator of the HIV-1 mRNA expression that promotes the export of unspliced and partially spliced mRNA. The export receptor for the leucine-rich nuclear export signal (NES) of Rev has recently been recognized as CRM1. We identified a low molecular weight compound PKF050-638 as an inhibitor of HIV-1 Rev. This drug inhibits in a dose-dependent fashion Rev-dependent mRNA expression in a cellular assay for Rev function. We show that PKF050-638 is an inhibitor of the CRM1-mediated Rev nuclear export. By using a quantitative in vitro CRM1-NES cargo-binding assay, we could demonstrate that PKF050-638 disrupts CRM1-NES interaction. This mode of action is confirmed in cell culture because the drug reversibly interferes with the colocalization of CRM1 and Rev in the nucleolus of the cell. In addition, we prove that the inhibition is through direct interaction of the compound with Cys-539 of CRM1. These effects are similar to those of the known CRM1 inhibitor leptomycin B and suggest that the inhibitory effect of the compound is caused by binding to CRM1 at a similar site. The compound displayed strict structural requirements for its activity, as its enantiomer was inactive in all assays tested. These results show that we identified a drug that interferes with the CRM1-mediated nuclear export of Rev through inhibition of the CRM1-NES complex formation. The reversibility of its binding to CRM1 and its availability through chemical synthesis could make it useful for studying CRM1-mediated export pathways.

Active Transport, Cell Nucleus↗
PubMed2003

Control of intracellular delivery and inhibition of genetic expression by DNA-peptide conjugates.

Various types of DNA-peptide conjugates were synthesized by solid phase fragment condensation (SPFC). DNA-LNS (nuclear localizing signal) peptide conjugate was proved to be delivered and localized into cellular nucleus and exhibited higher antisense inhibitory effect against telomerase than antisense phosphorothioate DNA. In contrast, DNAzymes conjugated with NES (nuclear export signal) peptide was shown to be taken up and localized in cytoplasm. Inhibitory effect of the conjugate DNAzyme against BCR-ABL tyrosine kinase was evaluated to be more significant than the native DNAzyme.

Amino Acid Sequence↗
PubMed2005

Keap1 regulates the oxidation-sensitive shuttling of Nrf2 into and out of the nucleus via a Crm1-dependent nuclear export mechanism.

Keap1 is a negative regulator of Nrf2, a transcription factor essential for antioxidant response element (ARE)-mediated gene expression. We find that Keap1 sequesters Nrf2 in the cytoplasm, not by docking it to the actin cytoskeleton but instead through an active Crm1/exportin-dependent nuclear export mechanism. Deletion and mutagenesis studies identified a nuclear export signal (NES) in the intervening region of Keap1 comprised of hydrophobic leucine and isoleucine residues in agreement with a traditional NES consensus sequence. Mutation of the hydrophobic amino acids resulted in nuclear accumulation of both Keap1 and Nrf2, as did treatment with the drug leptomycin B, which inactivates Crm1/exportin. ARE genes were partially activated under these conditions, suggesting that additional oxidation-sensitive elements are required for full activation of the antioxidant response. Based on these data, we propose a new model for regulation of Nrf2 by Keap1. Under normal conditions, Keap1 and Nrf2 are complexed in the cytoplasm where they are targeted for degradation. Oxidative stress inactivates Keap1's NES, allowing entry of both Keap1 and Nrf2 into the nucleus and transcriptional transactivation of ARE genes.

Active Transport, Cell Nucleus↗
PubMed2000

Human T-cell leukemia virus type 1 Tax shuttles between functionally discrete subcellular targets.

Human T-cell leukemia virus type 1 (HTLV-1) Tax is a nuclear protein with striking pleiotropic functionality. We recently demonstrated that Tax localizes to a multicomponent nuclear speckled structure (Tax speckled structure [TSS]). Here, we examine these structures further and identify a partial overlap of TSS with transcription hot spots. We used a strategy of directed expression via fusion proteins to determine if these transcription sites are the subtargets within TSS required for Tax function. When fused to human immunodeficiency virus type 1 (HIV-1) Tat, the resulting Tat-Tax fusion protein displayed neither a Tat-like nor a Tax-like pattern but rather was targeted specifically to the transcription subsites. The Tat-Tax fusion was able to activate both the HIV-1 long terminal repeat (LTR) and the HTVL-1 LTR at the same level as the individual component; thus, targeting proteins to transcription hot spots was compatible with both Tax and Tat transcription function. In contrast, the fusion with HIV-1 Rev, Rev-Tax, resulted in a pattern of expression that was largely Rev-like (nucleolar and cytoplasmic). The reduced localization of Rev-Tax to transcription sites was reflected in a 10-fold drop in activation of the HTLV-1 LTR. However, there was no loss in the ability of Tax to activate via NF-kappaB. Thus, NF-kappaB-dependent Tax function does not require targeting of Tax to these transcription sites and suggests that activation via NF-kappaB is a cytoplasmic function. Selective mutation of the nuclear localization signal site in the Rev portion resulted in retargeting of Rev-Tax to TSS and subsequent restoration of transcription function, demonstrating that inappropriate localization preceded loss of function. Mutation of the nuclear export signal site in the Rev portion had no effect on transcription, although the relative amount of Rev-Tax in the cytoplasm was reduced. Finally, in explaining how Tax can occupy multiple subcellular sites, we show that Tax shuttles from the nucleus to the cytoplasm in a heterokaryon fusion assay. Thus, pleiotropic functionality by Tax is regulatable via shuttling between discrete subcellular compartments.

Blotting, Western↗
PubMed1998

Analysis of intracellular trafficking and interactions of cytoplasmic HIV-1 Rev mutants in living cells.

The HIV-1 Rev protein is an essential nuclear regulatory viral protein. Rev mutants that are able to block wild-type (WT) Rev activity in trans have been reported and used in antiviral approaches. Not only nuclear but also cytoplasmic Rev mutants were described and suspected to be transdominant by retaining WT Rev in the cytoplasm. To investigate their potential for cytoplasmic retention, we studied the localization, trafficking, and interactions of cytoplasmic Rev mutants containing mutations in the N-terminal multifunctional domain. Using a novel dual-color autofluorescent protein-tagging system, we found that coexpression of the nucleolar blue-tagged WT Rev protein together with green-labeled cytoplasmic Rev mutants did not result in the retention of WT Rev in the cytoplasm but, on the contrary, in colocalization of the mutants to the nucleolus. A combination of mutations abolished the interaction with WT Rev, defining two domains important for Rev protein interaction. The identified domains were also essential for specific Rev responsive element (RRE) RNA binding and nuclear retention. Inactivation of the nuclear export signal shifted the steady-state distribution of the mutants from the cytoplasm to the nucleus, indicating their capability for nucleo-cytoplasmic shuttling. The cytoplasmic mutants were not transdominant compared to the nuclear mutant RevM10BL. These results emphasize that efficient oligomerization with WT Rev combined with RRE-specific RNA binding are prerequisites for effective transdominance.

Cell Line↗
PubMed1996

A nuclear role for the Fragile X mental retardation protein.

Fragile X syndrome results from lack of expression of a functional form of Fragile X mental retardation protein (FMRP), a cytoplasmic RNA-binding protein of uncertain function. Here, we report that FMRP contains a nuclear export signal (NES) that is similar to the NES recently identified in the Rev regulatory protein of human immunodeficiency virus type 1 (HIV-1). Mutation of this FMRP NES results in mis-localization of FMRP to the cell nucleus. The FMRP NES is encoded within exon 14 of the FMR1 gene, thus explaining the aberrant nuclear localization of a natural isoform of FMRP that lacks this exon. The NES of FMRP can substitute fully for the Rev NES in mediating Rev-dependent nuclear RNA export and specifically binds a nucleoporin-like cellular cofactor that has been shown to mediate Rev NES function. Together, these findings demonstrate that the normal function of FMRP involves entry into the nucleus followed by export via a pathway that is identical to the one utilized by HIV-1 Rev. In addition, these data raise the possibility that FMRP could play a role in mediating the nuclear export of its currently undefined cellular RNA target(s).

Amino Acid Sequence↗
PubMed2000

A second calcineurin binding site on the NFAT regulatory domain.

NFATc (a member of the family of nuclear factors of activated T cells) is a transcriptional factor responsible for the Ca(2+)-inducible activation of cytokine genes during the immune response. In resting T cells, NFATc is retained in the cytoplasm by a mechanism that depends on multiple phosphorylations in an N-terminal regulatory domain. Physical interaction with and dephosphorylation by Ca(2+)-activated calcineurin (Cn) allows the protein to enter the nucleus, where it binds to specific sites in cytokine gene promoters. Previous studies had identified a peptide segment in NFATc that binds Cn stably. Here we report the identification of a second Cn-binding element in NFATc, which synergizes with the previously identified element. Although these sequences are conserved in all isoforms of NFAT, we find that the two sites contribute differentially to the overall affinity for Cn in an isoform-dependent manner. The regulatory domain of NFAT also was found to be entirely devoid of structure, both in the phosphorylated and unphosphorylated state. This finding suggests that the NFAT regulatory domain does not undergo phosphorylation-induced conformational switching, but instead requires partner proteins to control accessibility of the NFAT nuclear localization and nuclear export signals.

Amino Acid Sequence↗
PubMed2002

An alternative domain containing a leucine-rich sequence regulates nuclear cytoplasmic localization of protein 4.1R.

In red blood cells, protein 4.1 (4.1R) is an 80-kDa protein that stabilizes the spectrin-actin network and anchors it to the plasma membrane. The picture is more complex in nucleated cells, in which many 4.1R isoforms, varying in size and intracellular location, have been identified. To contribute to the characterization of signals involved in differential intracellular localization of 4.1R, we have analyzed the role the exon 5-encoded sequence plays in 4.1R distribution. We show that exon 5 encodes a leucine-rich sequence that shares key features with nuclear export signals (NESs). This sequence adopts the topology employed for NESs of other proteins and conserves two hydrophobic residues that are shown to be critical for NES function. A 4.1R isoform expressing the leucine-rich sequence binds to the export receptor CRM1 in a RanGTP-dependent fashion, whereas this does not occur in a mutant whose two conserved hydrophobic residues are substituted. These two residues are also essential for 4.1R intracellular distribution, because the 4.1R protein containing the leucine-rich sequence localizes in the cytoplasm, whereas the mutant protein predominantly accumulates in the nucleus. We hypothesize that the leucine-rich sequence in 4.1R controls distribution and concomitantly function of a specific set of 4.1R isoforms.

Amino Acid Sequence↗
PubMed2004

A putative NES mediates cytoplasmic localization of Apoptin in normal cells.

Apoptin, a protein expressed by chicken anemia virus, is found predominantly in the cytoplasm in normal cells, whereas it localizes in the nucleus in transformed and malignant cells. However, the mechanisms that regulate the different subcellular localization of Apoptin in normal and tumor cells have not been fully clarified. In this work, a putative nuclear export signal (NES) in Apoptin was predicted. It was testified that the putative NES (pNES) of Apoptin was not a functional NES, but actually acted as a cytoplasmic retention signal. Deletion of the pNES led to the nuclear accumulation of Apoptin in normal cells. In addition, when a strong nuclear localization signal was introduced into Apoptin, it exclusively translocated to the nucleus in normal cells. These observations indicated that the cytoplasmic localization of Apoptin in normal cells results from the balance between cytoplasmic retention and nuclear import. On the other hand, the pNES was also proved to be necessary for Apoptin multimerization. Mutants lacking the pNES did not form obviously visible globular aggregates in normal or tumor cells.

Active Transport, Cell Nucleus↗
PubMed2001

RelB cellular regulation and transcriptional activity are regulated by p100.

RelB mediates the constitutive nuclear pool of NF-kappaB transcriptional activity in myeloid and lymphoid cells, which is believed to be secondary to its weak interaction with the classical NF-kappaB inhibitor proteins, the IkappaBs. In other cell types, RelB is located in the cytosol, thus suggesting that RelB is also regulated by an inhibitory protein(s). In this study, it is demonstrated that RelB is associated in the cytosol with p100 but not with IkappaBalpha, IkappaBbeta, IkappaBepsilon, nor p105. Its cytosolic control is not affected by stimuli that lead to RelA nuclear translocation, and RelB nuclear localization is prevented by p100, but not by p105 or IkappaBalpha. Structure function analysis p100-RelB interactions indicates that p100 amino acids 623-900 are required for effective interaction and repression of nuclear translocation and RelB driven NF-kappaB-dependent transcription. Moreover, this carboxyl-portion of p100 contains a nuclear export signal(s), which is required for effective retrieval of RelB from the nucleus. Finally, overexpression of NF-kappaB-inducing kinase, a kinase that has recently been shown to induce p100 processing, possibly through IKKalpha activation, causes nuclear translocation of RelB protein. Thus, these studies indicate that p100 is a bone fide inhibitor of RelB and that this transcription factor may be regulated by NF-kappaB-inducing kinase and/or IKKalpha.

Active Transport, Cell Nucleus↗
PubMed1997

The K nuclear shuttling domain: a novel signal for nuclear import and nuclear export in the hnRNP K protein.

Protein import into the nucleus and export from the nucleus are signal-mediated processes that require energy. The nuclear transport process about which the most information is currently available is classical nuclear localization signal (NLS)-mediated nuclear import. However, details concerning the signal-mediated export of proteins and RNAs as well as alternative nuclear import pathways are beginning to emerge. An example of this is the heterogeneous nuclear ribonucleoprotein (hnRNP) A1 protein which, by virtue of its M9 domain, is actively exported from the nucleus and imported into the nucleus via a novel pathway mediated by the recently characterized transportin protein. Here we report that the shuttling hnRNP K protein contains a novel shuttling domain (termed KNS) which has many of the characteristics of M9, in that it confers bi-directional transport across the nuclear envelope. KNS-mediated nuclear import is dependent on RNA polymerase II transcription, and we show that a classical NLS can override this effect. Furthermore, KNS accesses a separate import pathway from either classical NLSs or M9. This demonstrates the existence of a third protein import pathway into the nucleus and thereby defines a new type of nuclear import/export signal.

Amino Acid Sequence↗
PubMed2002

Carboxyl-terminal basic amino acids in the X domain are essential for the nuclear import of phospholipase C delta1.

BACKGROUND: Although phospholipase C (PLC)delta1 containing a functional nuclear export signal (NES) is normally localized at the plasma membrane and in the cytoplasm, it shuttles between the nucleus and the cytoplasm. Since nucleocytoplasmic shuttling of a molecule is generally regulated by a balance between its NES and the nuclear localization signal (NLS), we examined whether PLCdelta1 contains an NLS sequence. RESULTS: A region corresponding to the C terminus of the X domain and the XY-linker, which contains clusters of basic amino acid residues, was essential for the nuclear import of PLCdelta1 in Madin-Darby canine kidney cells. A series of point mutations on lysine residues in this region revealed that K432 and K434 in combination were important for the nuclear import. A short synthetic peptide corresponding to residues 429-442, however, was not able to function as an NLS sequence when they were injected into the cytoplasm in a carrier-conjugated form. Neither a longer peptide equivalent to PLCdelta1 412-498 fused to a protein tag consisting of glutathione S-transferase and green fluorescent protein was imported to the nucleus after microinjection into the cytoplasm. CONCLUSION: The nuclear import of PLCdelta1 requires the C-terminus of the X domain, particularly the amino acid residues K432 and K434, and the XY-linker. The region alone, however, cannot serve as a functional NLS. The machinery for nuclear transport may require additional structural component(s) of the enzyme.

Active Transport, Cell Nucleus↗
PubMed2001

Nuclear localization and shuttling of herpes simplex virus tegument protein VP13/14.

The herpes simplex virus type 1 gene UL47 encodes the tegument proteins referred to collectively as VP13/14, which are believed to be differentially modified forms of the same protein. Here we show that the major product of the UL47 gene during transient expression is VP14, suggesting that some feature of virus infection is required to produce VP13. We have tagged VP13/14 with green fluorescent protein and have demonstrated that the protein is targeted efficiently to the nucleus, where it often localizes in numerous punctate domains. Furthermore, we show that removal of the N-terminal 127 residues of the protein abrogates nuclear accumulation, and we have identified a 14-amino-acid peptide from this region that is sufficient to function as a nuclear targeting signal and transport a heterologous protein to the nucleus. This short peptide contains two runs of four arginine residues, suggesting that the VP13/14 nuclear localization signal may behave in a manner similar to that of the arginine-rich nuclear localization signals of the retrovirus transactivator proteins Tat, Rev, and Rex. In addition, by using heterokaryon assays, we show that VP13/14 is capable of shuttling between the nucleus and cytoplasm of the cell, a property that may be attributed to three leucine-rich stretches in the C-terminal half of the protein that again bear similarity to the nuclear export signals of Rev and Rex. This is the first demonstration of a tegument protein that is specifically targeted to the nucleus, a feature which may be relevant both during virus entry, when VP13/14 enters the cell as a component of the tegument, and at later times, when large amounts of newly synthesized VP13/14 are present within the cell.

3T3 Cells↗
PubMed2006

Delineation and modelling of a nucleolar retention signal in the coronavirus nucleocapsid protein.

Unlike nuclear localization signals, there is no obvious consensus sequence for the targeting of proteins to the nucleolus. The nucleolus is a dynamic subnuclear structure which is crucial to the normal operation of the eukaryotic cell. Studying nucleolar trafficking signals is problematic as many nucleolar retention signals (NoRSs) are part of classical nuclear localization signals (NLSs). In addition, there is no known consensus signal with which to inform a study. The avian infectious bronchitis virus (IBV), coronavirus nucleocapsid (N) protein, localizes to the cytoplasm and the nucleolus. Mutagenesis was used to delineate a novel eight amino acid motif that was necessary and sufficient for nucleolar retention of N protein and colocalize with nucleolin and fibrillarin. Additionally, a classical nuclear export signal (NES) functioned to direct N protein to the cytoplasm. Comparison of the coronavirus NoRSs with known cellular and other viral NoRSs revealed that these motifs have conserved arginine residues. Molecular modelling, using the solution structure of severe acute respiratory (SARS) coronavirus N-protein, revealed that this motif is available for interaction with cellular factors which may mediate nucleolar localization. We hypothesise that the N-protein uses these signals to traffic to and from the nucleolus and the cytoplasm.

Active Transport, Cell Nucleus↗
PubMed2002

CRM1-mediated nuclear export determines the cytoplasmic localization of the antiapoptotic protein Survivin.

Survivin is a member of the inhibitor of apoptosis (IAP) family of negative regulators of programmed cell death that is frequently overexpressed in human tumors. Survivin is not only involved in the regulation of apoptosis, but is also known to play a role in the control of cell cycle progression at the G2/M phase. Survivin is a predominantly cytoplasmic protein expressed in a cell cycle-dependent manner, but the mechanism(s) that determine its nuclear-cytoplasmic localization have not been described. In this study, we report that Survivin is a nuclear shuttling protein that is actively exported from the nucleus via the CRM1-dependent pathway. Nuclear export of Survivin is independent of the export of other shuttling proteins that control the G2/M phase transition, such as cyclin B1 and cdc25. The carboxy-terminal domain of Survivin is both necessary and sufficient for its nuclear export, although this region does not contain a functional leucine-rich nuclear export signal. Differences in the amino acid sequence of this region determine the dramatically different localization of Survivin (in the cytoplasm) and its splicing variant Survivin-DeltaEx3 (in the nucleus). The carboxy-terminal end of Survivin-DeltaEx3 contains a bipartite nuclear localization signal, not present in Survivin, which mediates its strong nuclear accumulation. These data suggest that active transport between the nucleus and cytoplasm may constitute an important regulatory mechanism for Survivin function.

Alternative Splicing↗
PubMed2000

Regulated nuclear-cytoplasmic localization of interferon regulatory factor 3, a subunit of double-stranded RNA-activated factor 1.

Viral double-stranded RNA (dsRNA) generated during the course of infection leads to the activation of a latent transcription factor, dsRNA-activated factor 1 (DRAF1). DRAF1 binds to a DNA target containing the type I interferon-stimulated response element and induces transcription of responsive genes. DRAF1 is a multimeric transcription factor containing the interferon regulatory factor 3 (IRF-3) protein and one of the histone acetyl transferases, CREB binding protein (CBP) or p300 (CBP/p300). In uninfected cells, the IRF-3 component of DRAF1 resides in the cytoplasm. The cytoplasmic localization of IRF-3 is dependent on a nuclear export signal, and we demonstrate IRF-3 recognition by the chromosome region maintenance 1 (CRM1) (also known as exportin 1) shuttling receptor. Following infection and specific phosphorylation, IRF-3 accumulates in the nucleus where it associates with CBP and p300. We identify a nuclear localization signal (NLS) in IRF-3 that is critical for nuclear accumulation. Mutation of the NLS abrogates nuclear localization even following infection. The NLS appears to be active constitutively, but it is recognized by only a subset of importin-alpha shuttling receptors. Evidence is presented to support a model in which IRF-3 normally shuttles between the nucleus and the cytoplasm but cytoplasmic localization is dominant prior to infection. Following infection, phosphorylated IRF-3 can bind to the CBP/p300 proteins resident in the nucleus. We provide the evidence of a role for CBP/p300 binding in the nuclear sequestration of a transcription factor that normally resides in the cytoplasm.

Cell Nucleus↗