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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.
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.
To define the effects of pp60v-src activity at different intracellular sites, we have constructed chimeric molecules which target the pp60v-src kinase to specific intracellular locations. pp60v-src was targeted to the nucleus by insertion of the SV40 large T antigen nuclear localization signal. Nuclear pp60v-src was active as a tyrosine kinase and phosphorylated nuclear proteins at tyrosine. However, cells expressing the nuclear pp60v-src were phenotypically normal by a number of criteria, and nuclear src kinase did not induce the expression of an mRNA (CEF-4) whose induction is characteristic of transformation by wild-type v-src. pp60v-src was targeted to perinuclear membranes by fusion to rat growth hormone and vesicular stomatitis G protein sequences. Cells expressing this chimeric molecule were phenotypically normal by most criteria. However the perinuclear src protein did induce elevated levels of CEF-4 mRNA, indicating that the v-src kinase expressed at this site induces partial transformation. The v-src and activated c-src kinases were targeted to adhesion plaques by fusion to the talin-binding sequence of vinculin. Cells expressing these fusion proteins were transformed by morphological, physiological and biochemical criteria, although the foci induced by these viruses were distinct from those induced by wild-type v-src. A chimeric protein which targeted c-src to adhesion plaques was not transforming. Thus targeting pp60src to adhesion plaques, although not sufficient to activate the transforming capacity of c-src, is sufficient to allow transformation by v-src.
Smad1 mediates signaling by bone morphogenetic proteins (BMPs). In the resting state, Smad1 is found in both the nucleus and cytosol. BMP addition triggers Smad1 serine phosphorylation, binding of Smad4, and its accumulation in the nucleus. Mutations in the Smad1 N-terminal basic nuclear localization signal (NLS)-like motif, conserved among all Smad proteins, eliminated its ligand-induced nuclear translocation without affecting its other functions, including DNA binding and complex formation with Smad4. Addition of leptomycin B, an inhibitor of nuclear export, induced rapid nuclear accumulation of Smad1, whereas overexpression of CRM1, the receptor for nuclear export, resulted in Smad1 re-localization to the cytoplasm and inhibition of BMP-induced nuclear accumulation. Thus, in addition to the NLS, Smad1 also contains a functional nuclear export signal (NES). We identified a leucine-rich NES motif in the C terminus of Smad1; its disruption led to constitutive Smad1 nuclear distribution. Reporter gene activation assays demonstrated that both the NLS and NES are required for optimal transcriptional activation by Smad1. Despite its constitutive nuclear accumulation, a Smad1 NES mutant did not display higher basal reporter gene activity. We conclude that Smad1 is under constant nucleocytoplasmic shuttling conferred by its NLS and NES; nuclear accumulation after ligand-induced phosphorylation represents a change in the balance of the activities of these opposing signals and is essential for transcriptional activation.
Human papillomavirus (HPV) type 16 E6 (16E6) is an oncogenic, multifunctional nuclear protein that induces p53 degradation and perturbs normal cell cycle control, leading to immortalization and transformation of infected keratinocytes and epithelial cells. Although it is unclear how 16E6 disrupts the epigenetic profile of host genes, its presence in the nucleus is a key feature. The present report describes intrinsic properties of 16E6 that influence its nuclear import in living cells. When the coding region of full-length 16E6 was inserted in frame into the C terminus of green fluorescent protein (GFP), it effectively prevented the 16E6 pre-mRNA from being spliced and led to the expression of a GFP-E6 fusion which localized predominantly to the nucleus. Further studies identified three novel nuclear localization signals (NLSs) in 16E6 that drive the protein to accumulate in the nucleus. We found that all three NLS sequences are rich in positively charged basic residues and that point mutations in these key residues could abolish the retention of 16E6 in the nucleus as well as the p53 degradation and cell immortalization activities of the protein. When inserted into corresponding regions of low-risk HPV type 6 E6, the three NLS sequences described for 16E6 functioned actively in converting the normally cytoplasmic HPV type 6 E6 into a nuclear protein. The separate NLS sequences, however, appear to play different roles in nuclear import and retention of HPV E6. The discovery of three unique NLS sequences in 16E6 provides new insights into the nuclear association of 16E6 which may reveal other novel activities of this important oncogenic protein.
The order Nidovirales, which includes the arteriviruses and coronaviruses, incorporate a cytoplasmic replication scheme; however, the nucleocapsid (N) protein of several members of this group localizes to the nucleolus suggesting that viral proteins influence nuclear processes during replication. The relatively small, 123 amino acid, N protein of porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus, presents an ideal model system for investigating the properties and mechanism of N protein nucleolar localization. The PRRSV N protein is found in both cytoplasmic and nucleolar compartments during infection and after transfection of gene constructs that express N-enhanced green fluorescent protein (EGFP) fusion proteins. Experiments using oligopeptides, truncated polypeptides and amino acid-substituted proteins have identified several domains within PRRSV N protein that participate in nucleo-cytoplasmic shuttling, including a cryptic nuclear localization signal (NLS) called NLS-1, a functional NLS (NLS-2), a nucleolar localization sequence (NoLS), as well as a possible nuclear export signal (NES). The purpose of this paper is to review our current understanding of PRRSV N protein shuttling and propose a shuttling scheme regulated by RNA binding and post-translational modification.
Although phosphatase and tensin homologue deleted on chromosome 10 (PTEN) localization in the nucleus and cytoplasm is established, the mechanism is unknown. PTEN is a tumor suppressor phosphatase that causes cell cycle arrest and/or apoptosis. Nuclear-cytoplasmic compartmentalization may be a novel mechanism in regulating these events. PTEN does not contain a traditional nuclear localization sequence (NLS); however, we identified putative NLS-like sequences, which we analyzed by site-directed mutagenesis and localization studies in MCF-7 cells. Two double site mutations exhibited nuclear localization defects. Furthermore, unlike wild-type PTEN, double NLS mutant PTEN did not interact with major vault protein (MVP), a previously hypothesized nuclear-cytoplasmic transport protein. We conclude that these two NLS-like sequences are required for PTEN nuclear import that is mediated by MVP. Further, we show that this MVP-mediated nuclear import is independent of PTEN phosphorylation and of the lipid and protein phosphatase activities of PTEN.
Fragile X syndrome is a frequent cause of mental retardation resulting from the absence of FMRP, the protein encoded by the FMR1 gene. FMRP is an RNA-binding protein of unknown function which is associated with ribosomes. To gain insight into FMRP function, we performed immunolocalization analysis of FMRP truncation and fusion constructs which revealed a nuclear localization signal (NLS) in the amino terminus of FMRP as well as a nuclear export signal (NES) encoded by exon 14. A 17 amino acid peptide containing the FMRP NES, which closely resembles the NES motifs recently described for HIV-1 Rev and PKI, is sufficient to direct nuclear export of a microinjected protein conjugate. Sucrose gradient analysis shows that FMRP ribosome association is RNA-dependent and FMRP is found in ribonucleoprotein (RNP) particles following EDTA treatment. These data are consistent with nascent FMRP entering the nucleus to assemble into mRNP particles prior to export back into the cytoplasm and suggests that fragile X syndrome may result from altered translation of transcripts which normally bind to FMRP.
High mobility group 2 (HMG2) protein is ubiquitously distributed in the nucleus of higher eukaryotic cells. Accumulation of an HMG2-beta-galactosidase fusion protein expressed in COS-7 cells suggested active transport of HMG2 protein into the nucleus after translation in the cytoplasm. Deletion analysis of the HMG2 sequence in the HMG2-beta-galactosidase fusion protein indicated that basic regions interspaced with the long DNA-binding sequence in HMG2, called the HMG1/2 box, are necessary for the nuclear accumulation of HMG2. The close configuration of basic regions at both ends of the DNA-binding sequence in the tertiary structure may function as the nuclear localization signal. This novel nuclear localization signal structure is different from typical ones such as the single or bipartite basic cluster in many nuclear proteins. A portion of the HMG2 molecule remained in the cytoplasm after translation. Interspecies heterokaryon assay demonstrated that the acidic carboxyl terminus of HMG2 was necessary for retention of the protein in the nucleus.
We have cloned and characterized a novel isoform of the skeletal muscle LIM protein 1 (SLIM1), designated SLIMMER. SLIM1 contains an N-terminal single zinc finger followed by four LIM domains. SLIMMER is identical to SLIM1 over the first three LIM domains but contains a novel C-terminal 96 amino acids with three potential bipartite nuclear localization signals, a putative nuclear export sequence, and 27 amino acids identical to the RBP-J binding region of KyoT2, a murine isoform of SLIM1. SLIM1 localized to the cytosol of Sol8 myoblasts and myotubes. SLIMMER was detected in the nucleus of myoblasts and, following differentiation into myotubes, was exclusively cytosolic. Recombinant green fluorescent protein-SLIM1 localized to the cytoplasm and associated with focal adhesions and actin filaments in COS-7 cells, while green fluorescent protein-SLIMMER was predominantly nuclear. SLIMMER truncation mutants revealed that the first nuclear localization signal mediates nuclear localization. The addition of the proposed nuclear export sequence decreased the level of exclusively nuclear expression and increased cytosolic SLIMMER expression in COS-7 cells. The leucine-rich nuclear export signal was required for the export of SLIMMER from the nucleus of myoblasts to the cytoplasm of myotubes. Collectively, these results suggest distinct roles for SLIM1 and SLIMMER in focal adhesions and nuclear-cytoplasmic communication.
Human T-cell leukemia virus type 1 and type 2 (HTLV-1/2) are related retroviruses that infect T-lymphocytes. Whereas HTLV-1 infection can cause leukemia, HTLV-2 has not been demonstrated to be the agent of a hematological malignant disease. Nevertheless, the virally encoded Tax-1 and Tax-2 transactivators display a high percentage of similarity. Tax-1 is a shuttling protein that contains a noncanonical nuclear localization signal as well as a nuclear export signal. The presence of the nuclear localization signal and the nuclear export signal domains in the Tax-2 sequence has not been determined. The distribution of Tax-2 in infected cells is not known but has been assumed to be similar to that of Tax-1. By using a Tax-2-specific antibody, we report here that Tax-2 is located predominantly in the cytoplasm of the HTLV-2 immortalized or transformed infected T-cells. These results were confirmed after transient transfection of untagged Tax-1 and Tax-2 constructs, histidine tag Tax1/Tax2, GFP-Tax, and Tax-GFP fusion constructs in several cell lines. We show that this unanticipated localization is not due to a default in the Tax-2 nuclear localization signal functions nor to major differences in Tax-2 versus Tax-1 binding to the IKKgamma/NEMO protein. In addition, we demonstrate that inhibiting the proteasome results in a relocalization of Tax-1 in the cytoplasm, similar to that of Tax-2. By using a series of Tax-1/Tax-2 chimeras, we determined that the minimal domain that is necessary for Tax-2 peculiar distribution encompasses amino acids 90-100. Finally, we show a high correlation between intracellular localization of Tax and their NF-kappaB or CREB transactivating ability.
The gamma-tubulin complex acts as a nucleation unit for microtubule assembly. It remains unknown, however, how spatial and temporal regulation of the complex activity affects microtubule-mediated cellular processes. Alp4 is one of the essential components of the S. pombe gamma-tubulin complex. We show here that overproduction of a carboxy-terminal form of Alp4 (Alp4C) and its derivatives tagged to a nuclear localization signal or to a nuclear export signal affect localization of gamma-tubulin complexes and induces novel phenotypes that reflect distinct functions of nuclear and cytoplasmic gamma-tubulin complexes. Nuclear Alp4C induces a Wee1-dependent G2 delay, reduces the levels of the gamma-tubulin complex at the spindle pole body, and results in defects in mitotic progression including spindle assembly, cytoplasmic microtubule disassembly, and chromosome segregation. In contrast, cytoplasmic Alp4C induces oscillatory nuclear movement and affects levels of cell polarity markers, Bud6 and Tip1, at the cell ends. These results demonstrate that regulation of nuclear gamma-tubulin complex activity is essential for cell cycle progression through the G2/M boundary and M phase, whereas regulation of cytoplasmic gamma-tubulin complex activity is important for nuclear positioning and cell polarity control during interphase.
In plants, the chromatin-associated high mobility group (HMG) proteins occur in two subfamilies termed HMGA and HMGB. The HMGA proteins are characterized by the presence of four AT-hook DNA binding motifs, and the HMGB proteins contain an HMG box DNA binding domain. As architectural factors, the HMG proteins appear to be involved in the regulation of transcription and other DNA-dependent processes. We have examined the subcellular localization of Arabidopsis thaliana HMGA, HMGB1, and HMGB5, revealing that they localize to the cell nucleus. They display a speckled distribution pattern throughout the chromatin of interphase nuclei, whereas none of the proteins associate with condensed mitotic chromosomes. HMGA is targeted to the nucleus by a monopartite nuclear localization signal, while efficient nuclear accumulation of HMGB1/5 requires large portions of the basic N-terminal part of the proteins. The acidic C-terminal domain interferes with nucleolar targeting of HMGB1. Fluorescence recovery after photobleaching experiments revealed that HMGA and HMGB proteins are extremely dynamic in the nucleus, indicating that they bind chromatin only transiently before moving on to the next site, thereby continuously scanning the genome for targets. By contrast, the majority of histone H2B is basically immobile within the nucleus, while linker histone H1.2 is relatively mobile.
The low cytoplasmic and high nuclear concentration of the GTP-bound form of Ran provides directionality for both nuclear protein import and export. Both import and export factors bind RanGTP directly, yet this interaction produces opposite effects; in the former case, RanGTP binding induces nuclear cargo release, whereas in the latter, RanGTP binding induces nuclear cargo assembly. Therefore, nuclear import and export receptors and their protein recognition sites are predicted to be distinct. Nevertheless, the approximately 38-amino acid M9 sequence present in heterogeneous nuclear ribonucleoprotein A1 has been reported to serve as both a nuclear localization signal and a nuclear export signal, even though only one protein, the nuclear import factor transportin, has been shown to bind M9 directly. We have used a combination of mutational randomization followed by selection for transportin binding to exhaustively define amino acids in M9 that are critical for transportin binding in vivo. As expected, the resultant approximately 12-amino acid transportin-binding consensus sequence is also predictive of nuclear localization signal activity. Surprisingly, however, this extensive mutational analysis failed to dissect M9 nuclear localization signal and nuclear export signal function. Nevertheless, transportin appears unlikely to be the M9 export receptor, as RanGTP can be shown to block M9 binding by transportin not only in vitro, but also in the nucleus in vivo. This analysis therefore predicts the existence of a nuclear export receptor distinct from transportin that nevertheless shares a common protein-binding site on heterogeneous nuclear ribonucleoprotein A1.
A striking characteristic of mRNA export factors is that they shuttle continuously between the cytoplasm and the nucleus. This shuttling is mediated by specific factors interacting with peptide motifs called nuclear export signals (NES) and nuclear localization signals. We have identified a novel CRM-1-independent transferable NES and two nuclear localization signals in the Epstein-Barr virus mRNA export factor EB2 (also called BMLF1, Mta, or SM) localized at the N terminus of the protein between amino acids 61 and 146. We have also found that a previously described double NES (amino acids 213-236) does not mediate the nuclear shuttling of EB2, but is an interaction domain with the cellular export factor REF in vitro. This newly characterized REF interaction domain is essential for EB2-mediated mRNA export. Accordingly, in vivo, EB2 is found in complexes containing REF as well as the cellular factor TAP. However, these interactions are RNase-sensitive, suggesting that the RNA is an essential component of these complexes.
In the present study, we investigated the antisense properties of conjugate oligonucleotides (ODNs) inhibiting human telomerase activity. Conjugate oligonucleotides assembled with signal peptides, artificially designed peptides, amines and sugars were synthesized by solid phase fragment condensation (SPFC) in sufficient yields. Conjugate ODNs showed a high resistance to nuclease degradation and sufficient binding affinity to target RNA, comparatively rapid and sufficient intracellular delivery and specific localization controlled by signal peptides (nuclear localization signals, NLS; nuclear export signals, NES). ODN-NLS conjugates demonstrated high antisense inhibitory effects against human telomerase activity into the nucleus (e. g, phosphorothioate conjugate inhibited the telomerase activity over 95%), whereas ODN-NES conjugates inhibited target mRNA expression into the cytoplasm.
The regulation of nuclear protein transport by phosphorylation plays a central role in gene expression in eukaryotic cells. We previously showed that nuclear import of SV40 large tumor antigen (T-ag) fusion proteins is regulated by the CcN motif, comprising phosphorylation sites for casein kinase II and the cyclin-dependent kinase cdc2, together with the nuclear localization signal. Regulation of nuclear uptake by CcN motif kinase sites also holds true for the yeast transcription factor SWI5 and the Xenopus nuclear phosphoprotein nucleoplasmin. To test directly whether a kinase site other than those of the CcN motif could regulate nuclear import of T-ag, the CcN motif casein kinase II site, which markedly increases the rate of T-ag nuclear import, was replaced by a consensus site for the cAMP-dependent protein kinase (PK-A) using site-directed mutagenesis. The resultant fusion protein could be specifically phosphorylated by PK-A in vitro and in cell extracts. Nuclear import of the fluorescently labeled protein was analyzed in the HTC rat hepatoma cell line both in vivo (microinjected cells) and in vitro (mechanically perforated cells) in the presence and the absence of cAMP and/or PK-A catalytic subunit using confocal laser scanning microscopy. In vitro PK-A-prephosphorylated protein was also tested. All results indicated that the rate of nuclear import was increased by phosphorylation at the PK-A site (2-5-fold), demonstrating that kinases other than those of the CcN motif can regulate nuclear import in response to stimulatory signals. The phosphorylation-regulated nuclear localization signal derived here represents an important first step toward developing a signal conferring inducible nuclear targeting of molecules of interest.
Expression of the recombinase proteins RAG-1 and RAG-2 is discordant: while RAG-1 is relatively long lived, RAG-2 is degraded periodically at the G(1)-S transition. Destruction of RAG-2 is mediated by a conserved interval in the recombination-dispensable region. The need for RAG-2 to reaccumulate in the nucleus at each cell division suggested the existence of an intrinsic RAG-2 nuclear localization signal (NLS). RAG-1 or RAG-2, expressed individually, is a nuclear protein. A screen for proteins that bind the recombination-dispensable region of RAG-2 identified the nuclear transport protein Importin 5. Mutation of residues 499 to 508 in RAG-2 abolished Importin 5 binding, nuclear accumulation, and periodic degradation of RAG-2. The Importin 5 binding site overlaps an NLS, defined by mutagenesis. RAG-1 rescued the localization of degradation-defective, RAG-2 NLS mutants; this required an intact RAG-1 NLS. Mutations in RAG-2 that abolish intrinsic nuclear accumulation but spare periodic degradation impaired recombination in cycling cells; induction of quiescence restored recombination to wild-type levels. Recombination defects were correlated with a cell cycle-dependent defect in the ability of RAG-1 to rescue localization of the RAG-2 mutants. These results suggest that the intrinsic RAG-2 NLS functions in the nuclear uptake of RAG-2 following its reexpression in cycling cells.