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PubMed2001

Nucleo-cytoplasmic trafficking of metal-regulatory transcription factor 1 is regulated by diverse stress signals.

The metal-regulatory transcription factor 1 (MTF-1) is a key regulator of heavy metal-induced transcription of metallothionein I and II and other genes in mammals and other metazoans. Transcriptional activation of genes by MTF-1 is mediated through binding to metal-responsive elements of consensus TGCRCNC in the target gene promoters. In an attempt to further clarify the mechanisms by which certain external signals activate MTF-1 and in turn modulate gene transcription, we show here that human MTF-1 has a dual nuclear and cytoplasmic localization in response to diverse stress stimuli. MTF-1 contains a consensus nuclear localization signal located just N-terminal to the first zinc finger that contributes to but is not essential for nuclear import. MTF-1 also harbors a leucine-rich, nuclear export signal. Under resting conditions, the nuclear export signal is required for cytoplasmic localization of MTF-1 as indicated by mutational analysis and transfer to the heterologous green fluorescent protein. Export from the nucleus was inhibited by leptomycin B, suggesting the involvement of the nuclear export protein CRM1. Our results further show that in addition to the heavy metals zinc and cadmium, heat shock, hydrogen peroxide, low extracellular pH (pH 6.0), inhibition of protein synthesis by cycloheximide, and serum induce nuclear accumulation of MTF-1. However, heavy metals alone (and not the other stress conditions) induce a significant transcriptional response via metal-responsive element promoter sequences, implying that nuclear import of MTF-1 is necessary but not sufficient for transcriptional activation. Possible roles for nuclear import under non-metal stress conditions are discussed.

Cadmium↗
PubMed1997

Interactions between HIV Rev and nuclear import and export factors: the Rev nuclear localisation signal mediates specific binding to human importin-beta.

The human immunodeficiency virus type 1 (HIV-1) Rev protein binds to unspliced HIV-1 pre-mRNA and exports it from the nucleus. Rev itself can "shuttle" between the nucleus and cytoplasm. This bi-directional transport is mediated by two specific Rev sequences: a nuclear localisation signal (NLS), which overlaps the RNA-binding domain, and a distinct nuclear export signal (NES). In this study we characterised new monoclonal antibodies that bind different epitopes of Rev, including the import and export sequences. In RNA bandshift assays, we observed that formation of a multimeric complex between Rev and its target RNA completely masks the Rev NLS, whereas the NES remains readily accessible. We then tested for signal-mediated interactions between Rev and different nuclear transport receptors, using mutations in the Rev NES or NLS to control for specificity. Extensive biochemical analyses did not reveal any direct NES-dependent interaction between Rev (free or RNA-bound) and the previously proposed export co-factors, human RIP/Rab and eIF-5A. By contrast, similar tests showed that Rev binds directly via its arginine-rich NLS to the human nuclear import receptor, importin-beta. This interaction was highly specific and was abolished by mutation in the Rev NLS. Importin-beta did not bind to the RNA-bound form of Rev, providing a mechanism to ensure that Rev is imported only following release of its RNA cargo. Unlike many NLS-containing proteins that bind stably to an importin-alpha/beta heterodimer, the binding of Rev to importin-beta was actually blocked by importin-alpha receptor. Our findings suggest that Rev and importin-alpha bind (via an arginine-rich sequence) to a similar region on importin-beta. In addition, we show that the complex between Rev and importin-beta can be dissociated by the nuclear Ran GTPase, but only when Ran is in the GTP-bound form. The series of interactions we describe provide a novel pathway for the import of Rev across the nuclear pore complex, and a mechanism for its release into the nucleoplasm.

Amino Acid Sequence↗
PubMed1998

Transport of macromolecules between the nucleus and the cytoplasm.

Nuclear transport is an energy-dependent process mediated by saturable receptors. Import and export receptors are thought to recognize and bind to nuclear localization signals or nuclear export signals, respectively, in the transported molecules. The receptor-substrate interaction can be direct or mediated by an additional adapter protein. The transport receptors dock their cargoes to the nuclear pore complexes (NPC) and facilitate their translocation through the NPC. After delivering their cargoes, the receptors are recycled to initiate additional rounds of transport. Because a transport event for a cargo molecule is unidirectional, the transport receptors engage in asymmetric cycles of translocation across the NPC. The GTPase Ran acts as a molecular switch for receptor-cargo interaction and imparts directionality to the transport process. Recently, the combined use of different in vitro and in vivo approaches has led to the characterization of novel import and export signals and to the identification of the first nuclear import and export receptors.

Biological Transport↗
PubMed1995

Identification of a signal for rapid export of proteins from the nucleus.

Active nuclear import of protein is controlled by nuclear localization signals (NLSs), but nuclear export is not understood well. Nuclear trafficking of the catalytic (C) subunit of cAMP-dependent protein kinase (cAPK) is critical for regulation of gene expression. The heat-stable inhibitor (PKl) of cAPK contains a nuclear export signal (NES) that triggers rapid, active net extrusion of the C-PKl complex from the nucleus. This NES (residues 35-49), fused or conjugated to heterologous proteins, was sufficient for rapid nuclear export. Hydrophobic residues were critical. The NES is a slightly weaker signal than the SV40 NLS. A sequence containing only residues 37-46, LALKLAGLDI, is also sufficient for nuclear export. This is an example of a protein-based NES having no obvious association with RNA. A similar sequence, LQLPPLERLTL, from Rev, an RNA-binding protein of HIV-1, also is an NES.

Amino Acid Sequence↗
PubMed2005

Nuclear export is evolutionarily conserved in CVC paired-like homeobox proteins and influences protein stability, transcriptional activation, and extracellular secretion.

Homeodomain transcription factors control a variety of essential cell fate decisions during development. To understand the developmental regulation by these transcription factors, we describe here the molecular analysis of paired-like CVC homeodomain protein (PLC-HDP) trafficking. Complementary experimental approaches demonstrated that PLC-HDP family members are exported by the Crm1 pathway and contain an evolutionary conserved leucine-rich nuclear export signal. Importantly, inactivation of the nuclear export signal enhanced protein stability, resulting in increased transactivation of transfected reporters and decreased extracellular secretion. In addition, PLC-HDPs harbor a conserved active nuclear import signal that could also function as a protein transduction domain. In our study, we characterized PLC-HDPs as mobile nucleocytoplasmic shuttle proteins with the potential for unconventional secretion and intercellular transfer. Nucleocytoplasmic transport may thus represent a conserved control mechanism to fine-tune the transcriptional activity of PLC-HDPs prerequisite for regulating and maintaining the complex expression pattern during development.

Active Transport, Cell Nucleus↗
PubMed1996

Nuclear export of late HIV-1 mRNAs occurs via a cellular protein export pathway.

The Rev protein of HIV-1 is essential for the nuclear export of incompletely spliced viral mRNAs. This action depends on the mutationally defined Rev activation domain, which both binds the nucleoporin-like human cellular cofactor Rab/hRIP and also functions as a nuclear export signal. Protein kinase inhibitor alpha (PKI) also contains a potent nuclear export signal. However, PKI plays no role in nuclear RNA export and instead induces the nuclear export of a specific protein target, the catalytic subunit of cAMP-dependent protein kinase. Here, it is demonstrated that the nuclear export signal of PKI not only binds the Rab/hRIP cofactor specifically but also can effectively substitute for the Rev activation domain in mediating the nuclear export of HIV-1 mRNAs. We conclude that HIV-1 Rev and PKI act through an identical nuclear export pathway and that Rev, rather than using a dedicated RNA export pathway, is instead acting as an adaptor that allows viral mRNAs to access a cellular protein export pathway.

Amino Acid Sequence↗
PubMed2005

Camptothecin induces nuclear export of prohibitin preferentially in transformed cells through a CRM-1-dependent mechanism.

Prohibitin is a growth-suppressive protein that has multiple functions in the nucleus and the mitochondria. Our earlier studies had shown that prohibitin represses the activity of E2F transcription factors while enhancing p53-mediated transcription. At the same time, prohibitin has been implicated in mediating the proper folding of mitochondrial proteins. We had found that treatment of cells with camptothecin, a topoisomerase 1 inhibitor, led to the export of prohibitin and p53 from the nucleus to the mitochondria. Here we show that the camptothecin-induced export of prohibitin occurs preferentially in transformed cell lines, but not in untransformed or primary cells. Cells that did not display the translocation of prohibitin were refractive to the apoptotic effects of camptothecin. The translocation was mediated by a putative nuclear export signal at the C-terminal region of prohibitin; fusion of the nuclear export signal (NES) of prohibitin to green fluorescence protein led to its export from the nucleus. Leptomycin B could inhibit the nuclear export of prohibitin showing that it was a CRM-1-dependent event driven by Ran GTPase. Confirming this, prohibitin was found to physically interact with CRM-1, and this interaction was significantly higher in transformed cells. Delivery of a peptide corresponding to the NES of prohibitin prevented the export of prohibitin to cytoplasm and protected cells from apoptosis. These results suggest that the regulated translocation of prohibitin from the nucleus to the mitochondria facilitates its pleiotropic functions and might contribute to its anti-proliferative and tumor suppressive properties.

Active Transport, Cell Nucleus↗
PubMed2006

Nucleocytoplasmic shuttling of phospholipase C-delta1: a link to Ca2+.

Phosphoinositides (PIs) and proteins involved in the PI signaling pathway are distributed in the nucleus as well as at the plasma membrane and in the cytoplasm, although their nuclear localization mechanisms have not been clarified in detail. Generally, proteins that shuttle between the cytoplasm and nucleus contain nuclear localization signal (NLS) and nuclear export signal (NES) sequences for nuclear import and export, respectively. They bind to specific carrier proteins of the importin/exportin family and are transported to and from the nucleus. Thus there is a steady state shuttling of the cargo molecules to and from the nucleus, and the shift in equilibrium determines their nuclear or cytoplasmic localization. Our previous studies have shown that phospholipase C (PLC)-delta1, regarded as having cytoplasmic- or plasma membrane-bound localization, accumulates in the nucleus when its NES sequence is disrupted. In addition, a cluster of positively charged residues on the surface of the catalytic barrel is important for nuclear import. In quiescent cells, the shuttling equilibrium seems to be shifted to the nuclear export of PLCdelta1. In this review, recent findings regarding the molecular machineries and mechanisms of the nucleocytoplasmic shuttling of PLCdelta1 will be discussed. It is important to know when and how they are regulated. A shift in the equilibrium in a certain stage of the cell cycle or by external stimuli is possible and resulting changes in the intra-nuclear environments (or architectures) may alter proliferation and differentiation patterns. Evidences support the idea that an increase in the levels of intracellular Ca2+ shifts the equilibrium to the nuclear import of PLCdelta1. A myriad of external stimuli have also been reported to change the nuclear PI metabolism following accelerated accumulation in the nucleus of other phospholipases such as phospholipase A2 and phospholipase D in addition to PLC isoforms such as PLCbeta1 and PLCgamma1. The consequence of the nuclear accumulation of PLC is also discussed.

Active Transport, Cell Nucleus↗
PubMed2006

Both carboxy-terminus NES motif and mutated tryptophan(s) are crucial for aberrant nuclear export of nucleophosmin leukemic mutants in NPMc+ AML.

We recently identified aberrant cytoplasmic expression of nucleophosmin (NPM) as the immunohistochemical marker of a large subgroup of acute myeloid leukemia (AML) (about one-third of adult AML) that is characterized by normal karyotype and mutations occurring at the exon-12 of the NPM gene. In this paper, we have elucidated the molecular mechanism underlying the abnormal cytoplasmic localization of NPM. All 29 AML-associated mutated NPM alleles so far identified encode abnormal proteins which have acquired at the C-terminus a nuclear export signal (NES) motif and lost both tryptophan residues 288 and 290 (or only the residue 290) which determine nucleolar localization. We show for the first time that both alterations are crucial for NPM mutant export from nucleus to cytoplasm. In fact, the cytoplasmic accumulation of NPM is blocked by leptomycin-B and ratjadones, specific exportin-1/Crm1-inhibitors, and by reinsertion of tryptophan residues 288 and 290, which respectively relocate NPM mutants in the nucleoplasm and nucleoli. NPM leukemic mutants in turn recruit the wild-type NPM from nucleoli to nucleoplasm and cytoplasm. These findings indicate that potential therapeutic strategies aimed to retarget NPM to its physiological sites will have to overcome 2 obstacles, the new NES motif and the mutated tryptophan(s) at the NPM mutant C-terminus.

Active Transport, Cell Nucleus↗
PubMed1998

Karyopherins and kissing cousins.

In eukaryotic cells, a regulated flux of molecules between the cytoplasm and the nucleus maintains two very different environments while allowing the controlled exchange of macromolecules necessary for their individual functions. Molecules entering or leaving the nucleus use nuclear localization signals or nuclear export signals to pass through selective channels in the nuclear envelope formed by nuclear pore complexes. The recognition of signal-bearing cargo, its interaction with the nuclear pore complex and its translocation through the pore complex are mediated by soluble transport factors. Recently, the list of potential transport factors has grown rapidly, suggesting a previously unanticipated level of complexity for nuclear transport.

Animals↗
PubMed2000

Nucleocytoplasmic protein traffic and its significance to cell function.

In eukaryotic cells, cell functions are maintained in an orderly manner through the continuous traffic of various proteins between the cell nucleus and the cytoplasm. The nuclear import and export of proteins occurs through nuclear pore complexes and typically requires specific signals: the nuclear localization signal and nuclear export signal, respectively. The transport pathways have been found to be highly divergent, but are known to be largely mediated by importin beta-like transport receptor family molecules. These receptor molecules bind to and carry their cargoes directly or via adapter molecules. A small GTPase Ran ensures the directionality of nuclear transport by regulating the interaction between the receptors and their cargoes through its GTP/GDP cycle. Moreover, it has been recently elucidated how the transport system is involved in various functions of cell physiology, such as cell cycle control.

Active Transport, Cell Nucleus↗
PubMed2001

MKP-7, a novel mitogen-activated protein kinase phosphatase, functions as a shuttle protein.

Mitogen-activated protein kinase (MAPK) phosphatases (MKPs) negatively regulate MAPK activity. In the present study, we have identified a novel MKP, designated MKP-7, and mapped it to human chromosome 12p12. MKP-7 possesses a long C-terminal stretch containing both a nuclear export signal and a nuclear localization signal, in addition to the rhodanese-like domain and the dual specificity phosphatase catalytic domain, both of which are conserved among MKP family members. When expressed in mammalian cells MKP-7 protein was localized exclusively in the cytoplasm, but this localization became exclusively nuclear following leptomycin B treatment or introduction of a mutation in the nuclear export signal. These findings indicate that MKP-7 is the first identified leptomycin B-sensitive shuttle MKP. Forced expression of MKP-7 suppressed activation of MAPKs in COS-7 cells in the order of selectivity, JNK p38 > ERK. Furthermore, a mutant form MKP-7 functioned as a dominant negative particularly against the dephosphorylation of JNK, suggesting that MKP-7 works as a JNK-specific phosphatase in vivo. Co-immunoprecipitation experiments and histological analysis suggested that MKP-7 determines the localization of MAPKs in the cytoplasm.

Active Transport, Cell Nucleus↗
PubMed2006

Antiviral properties of combination peptides of HIV-1 Rev NLS and NES.

Nuclear translocation signal has been identified as a mediator of protein shuttling between nuclear and cytoplasm. Here we report that the combination of peptides from nuclear localization signal (NLS) and nuclear export signal (NES) of HIV-1 Rev have an antiviral activity against the Herpes virus of turkey and Marek's disease virus serotype 1.

Amino Acid Sequence↗
PubMed2003

Identification of novel nuclear export and nuclear localization-related signals in human heat shock cognate protein 70.

Heat shock cognate protein 70 (Hsc70) serves nuclear transport of several proteins as a molecular chaperone. We have recently identified a novel variant of human Hsc70, heat shock cognate protein 54 (Hsc54), that lacks amino acid residues 464-616 in the protein binding and variable domains of Hsc70. In the present study, we examined nucleocytoplasmic localization of Hsc70 and Hsc54 by using green fluorescent protein (GFP) fusions. GFP-Hsc70 is localized in both the cytoplasm and the nucleus at 37 degrees C and accumulated into the nucleolus/nucleus after heat shock, whereas GFP-Hsc54 always remained exclusively in the cytoplasm under these conditions. Mutation studies indicated that 20 amino acid residues of nuclear localization-related signals, which are missing in Hsc54 but are retained in Hsc70, are required for proper nuclear localization of Hsc70. We further found that Hsc54 contains a functional leucine-rich nuclear export signal (NES, (394)LDVTPLSL(401)) which is differently situated from the previously proposed NES in Saccharomyces cerevisiae Ssb1p. The cytoplasmic localization of Hsc54 was impaired by a mutation in NES as well as by a nuclear export inhibitor, leptomycin B, suggesting that Hsc54 is actively exported from the nucleus to the cytoplasm through a CRM1-dependent mechanism. In contrast, the nucleocytoplasmic localization of Hsc70 was not affected by the same mutation of NES or leptomycin B. These results suggest that the nuclear localization-related signal could functionally mask NES leading to prolonged retention of Hsc70 in the nucleus. An additional mechanism for unmasking the NES may regulate nucleocytoplasmic trafficking of Hsc70.

Active Transport, Cell Nucleus↗
PubMed2003

Nuclear export of the APC tumour suppressor controls beta-catenin function in transcription.

The adenomatous polyposis coli (APC) protein is inactivated in most colorectal tumours. APC loss is an early event in tumorigenesis, and causes an increase of nuclear beta-catenin and its transcriptional activity. This is thought to be the driving force for tumour progression. APC shuttles in and out of the nucleus, but the functional significance of this has been controversial. Here, we show that APC truncations are nuclear in colorectal cancer cells and adenocarcinomas, and this correlates with loss of centrally located nuclear export signals. These signals confer efficient nuclear export as measured directly by fluorescence loss in photobleaching (FLIP), and they are critical for the function of APC in reducing the transcriptional activity of beta-catenin in complementation assays of APC mutant colorectal cancer cells. Importantly, targeting a functional APC construct to the nucleus causes a striking nuclear accumulation of beta-catenin without changing its transcriptional activity. Our evidence indicates that the rate of nuclear export of APC, rather than its nuclear import or steady-state levels, determines the transcriptional activity of beta-catenin.

Active Transport, Cell Nucleus↗
PubMed1998

HNS, a nuclear-cytoplasmic shuttling sequence in HuR.

Proteins are transported into and out of the cell nucleus via specific signals. The two best-studied nuclear transport processes are mediated either by classical nuclear localization signals or nuclear export signals. There also are shuttling sequences that direct the bidirectional transport of RNA-binding proteins. Two examples are the M9 sequence in heterogeneous nuclear ribonucleoprotein A1 and the heterogeneous nuclear ribonucleoprotein K shuttling domain (KNS) sequence in heterogeneous nuclear ribonucleoprotein K, both of which appear to contribute importantly to the export of mRNA to the cytoplasm. HuR is an RNA-binding protein that can stabilize labile mRNAs containing AU-rich elements in their 3' untranslated regions and has been shown to shuttle between the nucleus and cytoplasm (18, 19). We have identified in HuR a shuttling sequence that also possess transcription-dependent nuclear localization signal activity. We propose that HuR first may bind AU-rich element-containing mRNAs in the nucleus and then escort them through the nuclear pore, providing protection during and after export to the cytoplasmic compartment.

3T3 Cells↗
PubMed2006

Nucleocytoplasmic shuttling of the NAD+-dependent histone deacetylase SIRT1.

Sir2 (silent information regulator 2) is an NAD(+)-dependent histone deacetylase that contributes to longevity in yeast. SIRT1, a mammalian Sir2 ortholog, deacetylates histones and various transcription factors, including p53, FOXO proteins, and peroxisome proliferator-activated receptor-gamma. We found that its subcellular localization varied in different tissues of the adult mouse. Some subsets of neurons predominantly expressed SIRT1 in the cytoplasm, but ependymal cells expressed it in both the nucleus and cytoplasm. On the other hand, spermatocytes expressed SIRT1 only in the nucleus. Cardiomyocytes in the day 12.5 mouse embryo expressed SIRT1 exclusively in the nucleus, but in the adult heart, they expressed it in both the cytoplasm and nucleus. C2C12 myoblast cells expressed SIRT1 in the nucleus, but it localized to the cytoplasm after differentiation. LY294002, an inhibitor of phosphoinositide 3-hydroxykinase, strongly inhibited the nuclear localization of SIRT1 in undifferentiated C2C12 cells. In a heterokaryon assay, SIRT1 shuttled between the nucleus and cytoplasm, and leptomycin B, an inhibitor of CRM1-mediated nuclear exportation, inhibited this shuttling. Two nuclear localization signals and two nuclear export signals were identified by deletion and site-directed mutation analyses. Overexpressed nuclear (but not cytoplasmic or dominant-negative) SIRT1 enhanced the deacetylation of histone H3 in C2C12 cells. Moreover, only the nuclear form suppressed the apoptosis of C2C12 cells induced by antimycin A, an oxidative stressor. These findings indicate that nucleocytoplasmic shuttling is a novel regulatory mechanism of SIRT1, which may participate in differentiation and in inhibition of cell death.

Active Transport, Cell Nucleus↗
PubMed1999

Disruption of the YRB2 gene retards nuclear protein export, causing a profound mitotic delay, and can be rescued by overexpression of XPO1/CRM1.

Disruption of the YRB2 gene encoding a nuclear Ran-binding protein homologous to Yrb1p/RanBP1 makes Saccharomyces cerevisiae cold sensitive for colony-formation, but not for growth in liquid medium. Schizosaccharomyces pombe Hba1p, which is homologous to Saccharomyces cerevisiae Yrb2p, rescued the cold sensitivity of Deltayrb2 cells. When released from an alpha factor block, Deltayrb2 cells underwent a prolonged delay at the short spindle stage of mitosis with a normal level of Clb/p34(CDC28) kinase activity, but there was no chromosome loss, this being consistent with the finding that Deltayrb2 was synthetic lethal with neither Deltamad1 nor Deltamad3. The cold sensitive colony-formation of Deltayrb2 cells was rescued by both XPO1/CRM1 and GSP1, but not CDC5, carried on a multicopy vector. XPO1/CRM1 rescued Deltayrb2 even in a single copy. Consistent with such a tight functional interaction, Xpo1p/Crm1p directly bound to Yrb2p, but not Yrb1p, and Deltayrb2 cells were found to have a defect in nuclear export signal (NES)-dependent nuclear protein export. From these results together, the ability of Xpo1/Crm1p to export NES-proteins is suggested to be enhanced by both Yrb2p and Gsp1p, and thereby disruption of YRB2 retards nuclear protein export, resulting in the mitotic delay.

Amino Acid Sequence↗