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Results for “Nuclear Localization Signals”

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PubMed2000

Finding nuclear localization signals.

A variety of nuclear localization signals (NLSs) are experimentally known although only one motif was available for database searches through PROSITE. We initially collected a set of 91 experimentally verified NLSs from the literature. Through iterated 'in silico mutagenesis' we then extended the set to 214 potential NLSs. This final set matched in 43% of all known nuclear proteins and in no known non-nuclear protein. We estimated that >17% of all eukaryotic proteins may be imported into the nucleus. Finally, we found an overlap between the NLS and DNA-binding region for 90% of the proteins for which both the NLS and DNA-binding regions were known. Thus, evolution seemed to have used part of the existing DNA-binding mechanism when compartmentalizing DNA-binding proteins into the nucleus. However, only 56 of our 214 NLS motifs overlapped with DNA-binding regions. These 56 NLSs enabled a de novo prediction of partial DNA-binding regions for approximately 800 proteins in human, fly, worm and yeast.

Animals↗

PubMed1999

A nuclear localization signal can enhance both the nuclear transport and expression of 1 kb DNA.

Although the entry of DNA into the nucleus is a crucial step of non-viral gene delivery, fundamental features of this transport process have remained unexplored. This study analyzed the effect of linear double stranded DNA size on its passive diffusion, its active transport and its NLS-assisted transport. The size limit for passive diffusion was found to be between 200 and 310 bp. DNA of 310-1500 bp entered the nuclei of digitonin treated cells in the absence of cytosolic extract by an active transport process. Both the size limit and the intensity of DNA nuclear transport could be increased by the attachment of strong nuclear localization signals. Conjugation of a 900 bp expression cassette to nuclear localization signals increased both its nuclear entry and expression in microinjected, living cells.

Amino Acid Sequence↗

PubMed1996

The conserved ELK-homeodomain of KNOTTED-1 contains two regions that signal nuclear localization.

Nuclear localization serves as a regulatory mechanism in the activity of several transcription factors. KNOTTED-1 (Kn1) is a homeodomain protein likely to regulate vegetative development in maize. At least twelve genes related to Kn1 are known in maize and six in Arabidopsis. Ectopic expression of the maize, rice and Arabidopsis Kn1-related genes have been shown to alter cell fate determination. In this paper, we study the nuclear localization capabilities of the Kn1 homeodomain and the proximal amino acid residues (the ELK region) which is highly conserved among Kn1-related homeodomain proteins. The ELK homeodomain (ELK-HD) of Kn1 was fused to the reporter gene uidA encoding the bacterial enzyme beta-glucuronidase (GUS) and transformed into tobacco and onion cells. Quantitation of GUS activity in nuclear and total protein extracts from transgenic tobacco revealed a highly localized GUS activity in the nucleus for the ELK-HD/GUS fusion protein, as compared to the basal level of GUS activity in the nucleus for the GUS only protein. The ELK-HD/GUS transformants showed no unusual characteristics, thus indicating that expression of the putative Kn1 DNA-binding domain fused to GUS may be insufficient to create a dominant negative phenotype. Histochemical analysis of the onion epidermal cells transfected by particle bombardment demonstrated that greater than 50 % of the transformed onion epidermal cells showed higher levels of GUS staining in the nucleus relative to the cytoplasm. Deletion analysis of the ELK-HD revealed that the Kn1 homeodomain comprising the three predicted alpha-helices and the conserved ELK domain can each function independently as nuclear localization signals.

Amino Acid Sequence↗

PubMed1996

Identification of the nuclear localization signal of mouse DNA primase: nuclear transport of p46 subunit is facilitated by interaction with p54 subunit.

DNA polymerase alpha-primase is a replication enzyme necessary for DNA replication in all eukaryotes. Mouse DNA primase is composed of two subunits: a 46 kDa protein (p46), which is the catalytic subunit capable of RNA primer synthesis, and a 54 kDa protein (p54), whose physiological role is not clear. To understand the structure-function relationship of DNA primase, we set out to characterize these two subunits individually or in combination using a cDNA expression system in mammalian cultured cells, and determined the subcellular distribution of ectopically expressed DNA primase. The p54 expressed in COS-1 cells after transfection was predominantly localized in the nucleus, whereas p46 was retained in the cytoplasm as shown by indirect immunofluorescence analysis. Using several mutant proteins with deletions or substitutions as well as chimeric constructs, we identified the nuclear localization signal of p54 as RIRKKLR, encoded near the amino terminus (residues 6-12). Furthermore, co-expression of both p46 and p54 subunits markedly altered the subcellular distribution of p46; co-expressed p46 was transported into the nucleus as efficiently as p54. These results demonstrate that p54 has a nuclear localization signal and is able to be translocated into the nucleus independently of DNA polymerase alpha subunits. In contrast, p46 lacks a nuclear localization signal, and its nuclear translocation is facilitated by interaction with p54. We present here first evidence for a novel role of p54 in the nuclear translocation process, and a piggy-back binding transport mechanism of mouse DNA primase.

3T3 Cells↗

PubMed2000

Two nuclear localization signals are required for nuclear translocation of nuclear factor 1-A.

Nuclear factor 1 (NF1) proteins are encoded by at least four genes (NF1-A, B, C, X). Although DNA-binding and the transcription regulation domains of these proteins are well characterized, the nuclear localization signals (NLSs) are still unknown in all NF1s. We have identified two NLSs in NF1-A, and both are required for full translocation to the nucleus, although one of them itself has a partial translocation ability. These two NLSs are conserved in all four NF1s. Interestingly, three isoforms of NF1-A (NF1-A1, A2, A4) have two NLSs and translocate completely to the nucleus. In contrast, NF1-A3 lacks the second NLS and partially stays in the cytoplasm. Since NF1s construct homodimer and heterodimer, these findings indicate the differential regulations of the NF1 translocation.

Amino Acid Sequence↗

PubMed1999

A peptide nucleic acid-nuclear localization signal fusion that mediates nuclear transport of DNA.

We have combined a peptide nucleic acid (PNA) with the SV40 core nuclear localization signal (NLS), to create a bifunctional PNA-NLS peptide. The PNA-NLS peptide increased the nuclear uptake of oligonucleotides and enhanced the transfection efficacy of plasmids. Gene expression from an enhanced green fluorescent protein plasmid and a lacZ plasmid was preserved when hybridized to PNA-NLS. In combination with the transfection agent polyethyleneimine, we have improved both the nuclear translocation of fluorescence-marked oligonucleotides, and the efficacy of plasmid transfection, up to eightfold. The technique obviates the use of cumbersome coupling procedures of the vector due to DNA-PNA duplex formation or displacement of the antisense plasmid DNA strand by a PNA molecule.

3T3 Cells↗

PubMed1999

A bipartite nuclear localization signal is required for p53 nuclear import regulated by a carboxyl-terminal domain.

Abnormal p53 cellular localization has been considered to be one of the mechanisms that could inactivate p53 function. To understand the regulation of p53 cellular trafficking, we have previously identified two p53 domains involved in its localization. A basic domain, Lys(305)-Arg(306), is required for p53 nuclear import, and a carboxyl-terminal domain, namely the cytoplasmic sequestration domain (CSD) from residues 326-355, could block the nuclear import of Lys(305) or Arg(306) mutated p53. To characterize further the function of these two domains, we demonstrate in this report that the previously described major nuclear localization signal works together with Lys(305)-Arg(306) to form a bipartite and functional nuclear localization sequence (NLS) for p53 nuclear import. The CSD could block the binding of p53 to the NLS receptor, importin alpha, and reduce the efficiency of p53 nuclear import in MCF-7, H1299, and Saos-2 cells. The blocking effect of the CSD is not due to the enhancement of nuclear export or oligomerization of the p53. These results indicate that the CSD can regulate p53 nuclear import by controlling access of the NLS to importin alpha binding.

Amino Acid Sequence↗

PubMed2000

Molecular cloning of a novel importin alpha homologue from rice, by which constitutive photomorphogenic 1 (COP1) nuclear localization signal (NLS)-protein is preferentially nuclear imported.

Nuclear import of proteins that contain classical nuclear localization signals (NLS) is initiated by importin alpha, a protein that recognizes and binds to the NLS in the cytoplasm. In this paper, we have cloned a cDNA for a novel importin alpha homologue from rice which is in addition to our previously isolated rice importin alpha1a and alpha2, and we have named it rice importin alpha1b. In vitro binding and nuclear import assays using recombinant importin alpha1b protein demonstrate that rice importin alpha1b functions as a component of the NLS-receptor in plant cells. Analysis of the transcript levels for all three rice importin alpha genes revealed that the genes were not only differentially expressed but that they also responded to dark-adaptation in green leaves. Furthermore, we also show that the COP1 protein bears a bipartite-type NLS and its nuclear import is mediated preferentially by the rice importin alpha1b. These data suggest that each of the different rice importin alpha proteins carry distinct groups of nuclear proteins, such that multiple isoforms of importin alpha contribute to the regulation of plant nuclear protein transport.

Amino Acid Sequence↗

PubMed1997

Nuclear localization signals: a driving force for nuclear transport of plasmid DNA in zebrafish.

Nuclear localization signals (NLSs) are short peptides required for nuclear transport of karyophilic proteins. We review in this paper how the nuclear targeting property of NLS peptides has been taken advantage of to enhance the efficiency of nuclear uptake of transgene DNA in zebrafish and how it may improve the efficiency of transgenesis in this species. Synthetic NLS peptides can bind to plasmid DNA by ionic interactions. Cytoplasmic injection of DNA-NLS complexes in zebrafish eggs enhances the rate and the amount of plasmid DNA taken up by embryonic nuclei. Nuclear import of DNA-NLS complexes has been duplicated in vitro and exhibits energetic and cytosolic requirements similar to those for nuclear protein import. Furthermore, binding NLSs to DNA increases expression frequency of the transgene. We suggest that NLS peptides may constitute a valuable tool to improve the efficiency of transgenesis in zebrafish and other species.

Animals↗

PubMed1992

Nuclear localization signal(s) required for nuclear targeting of the maize regulatory protein Opaque-2.

The maize regulatory protein Opaque-2 (O2) localizes to the nucleus in both maize and tobacco cells. Here we show that in-frame carboxy- and amino-terminal fusions of O2 to reporter protein beta-glucuronidase (GUS) were sufficient to direct GUS to the nucleus in transgenic tobacco plants and in transiently transformed onion cells. Two independent regions of O2 containing 135 and 149 amino acids were identified that were able to redirect GUS to the nucleus in both systems. A quantitative biochemical analysis of GUS in nuclei isolated from transgenic tobacco plants revealed that the second region was more efficient than the first one. The precise location of nuclear localization signals (NLSs) was determined using an onion transformation system. The first NLS was located between residues 101 and 135 and had the structure of a simian virus 40 NLS. The second NLS was located in the basic, DNA binding domain (between residues 223 and 254) and had a bipartite structure. The presence of one of the O2 NLSs in the basic domain is in complete agreement with similar findings of NLSs in the basic domain of three other basic/leucine zipper proteins, suggesting that this domain may be bifunctional. The effect of amino- versus carboxy-terminal GUS fusions is discussed.

Allium↗

PubMed2003

Alternative splicing disrupts a nuclear localization signal in spleen tyrosine kinase that is required for invasion suppression in breast cancer.

Spleen tyrosine kinase (Syk) is a candidate tumor (metastasis) suppressor that is highly expressed in mammary epithelial cells. Loss of Syk expression through promoter hypermethylation is associated with increased invasiveness in a subset of breast cancer. Here, we show that in addition to full-length Syk [Syk(L)], an alternatively spliced variant, Syk(S), is frequently expressed in breast cancer cells. Syk(S) is identical to Syk(L), except that it lacks 23 amino acid residues (deletion) within the interdomain B (IDB) of Syk. We also show that the aberrant expression of Syk(S) occurs frequently in primary breast tumors but never in matched normal mammary tissues, suggesting a contribution of Syk(S) to mammary tumor progression. Expression of Syk(L) suppressed breast cancer cell invasiveness. In contrast, Syk(S) expression did not affect the cell invasion potential. This differential phenotypic response is accompanied by their different subcellular localization. Immunocytochemical studies and nuclear and cytoplasmic fractionation experiments indicated that Syk(L) could enter the nucleus, whereas Syk(S) was located exclusively in the cytoplasm. Five basic residues in deletion were found to be critical in determining Syk(L) nuclear transport and invasion suppression activity; mutations completely excluded Syk(L) from the nucleus and blocked Syk(L)-inducible invasion suppression. Moreover, IDB acted as an autonomous nuclear localization signal to facilitate nuclear transport of a heterologous protein. Thus, the IDB of Syk(L) contains a nuclear localization signal that is responsible for Syk(L) nuclear translocation. The correlation of the nuclear localization and invasion suppression function of Syk(L) indicated that nuclear Syk possesses biological activities associated with tumor suppression in mammary epithelial cells.

Alternative Splicing↗

PubMed2003

NLSdb: database of nuclear localization signals.

NLSdb is a database of nuclear localization signals (NLSs) and of nuclear proteins. NLSs are short stretches of residues mediating transport of nuclear proteins into the nucleus. The database contains 114 experimentally determined NLSs that were obtained through an extensive literature search. Using 'in silico mutagenesis' this set was extended to 308 experimental and potential NLSs. This final set matched over 43% of all known nuclear proteins and matches no currently known non-nuclear protein. NLSdb contains over 6000 predicted nuclear proteins and their targeting signals from the PDB and SWISS-PROT/TrEMBL databases. The database also contains over 12 500 predicted nuclear proteins from six entirely sequenced eukaryotic proteomes (Homo sapiens, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana and Saccharomyces cerevisiae). NLS motifs often co-localize with DNA-binding regions. This observation was used to also annotate over 1500 DNA-binding proteins. NLSdb can be accessed via the web site: http://cubic.bioc.columbia.edu/db/NLSdb/.

Active Transport, Cell Nucleus↗

PubMed1998

Nuclear import of protein kinase C occurs by a mechanism distinct from the mechanism used by proteins with a classical nuclear localization signal.

Protein kinase C does not have any known nuclear localization signal but, nevertheless, is redistributed from the cytoplasm to the nucleus upon various stimuli. In NIH 3T3 fibroblasts stimulation with phorbol ester leads to a translocation of protein kinase C alpha to the plasma membrane and into the cell nucleus. We compared the mechanism of protein kinase C alpha's transport into the nucleus with the transport mechanism of a protein with a classical nuclear localization signal at several steps. To this end, we co-microinjected fluorescently labeled bovine serum albumin to which a nuclear localization signal peptide was coupled, together with substances interfering with conventional nuclear protein import. Thereafter, the distribution of both the nuclear localization signal-bearing reporter protein and protein kinase C alpha was analyzed in the same cells. We can show that, in contrast to the nuclear localization signal-dependent transport, the phorbol ester-induced transport of protein kinase C alpha is not affected by microinjection of antibodies against the nuclear import factor p97/importin/karyopherin beta or microinjection of non-hydrolyzable GTP-analogs. This suggests that nuclear import of protein kinase C alpha is independent of p97/importin/karyopherin beta and independent of GTP. At the nuclear pore there are differences between the mechanisms too, since nuclear transport of protein kinase C alpha cannot be inhibited by wheat germ agglutinin or an antibody against nuclear pore complex proteins. Together these findings demonstrate that the nuclear import of protein kinase C alpha occurs by a mechanism distinct from the one used by classical nuclear localization signal-bearing proteins at several stages.

3T3 Cells↗

PubMed1997

Nuclear localization signal binding protein from Arabidopsis mediates nuclear import of Agrobacterium VirD2 protein.

T-DNA nuclear import is a central event in genetic transformation of plant cells by Agrobacterium. Presumably, the T-DNA transport intermediate is a single-stranded DNA molecule associated with two bacterial proteins, VirD2 and VirE2, which most likely mediate the transport process. While VirE2 cooperatively coats the transported single-stranded DNA, VirD2 is covalently attached to its 5' end. To better understand the mechanism of VirD2 action, a cellular receptor for VirD2 was identified and its encoding gene cloned from Arabidopsis. The identified protein, designated AtKAPalpha, specifically bound VirD2 in vivo and in vitro. VirD2-AtKAPalpha interaction was absolutely dependent on the carboxyl-terminal bipartite nuclear localization signal sequence of VirD2. The deduced amino acid sequence of AtKAPalpha was homologous to yeast and animal nuclear localization signal-binding proteins belonging to the karyopherin alpha family. Indeed, AtKAPalpha efficiently rescued a yeast mutant defective for nuclear import. Furthermore, AtKAPalpha specifically mediated transport of VirD2 into the nuclei of permeabilized yeast cells.

Amino Acid Sequence↗

PubMed1999

Appropriately spaced nuclear localizing signals are necessary for efficient nuclear import of nonnuclear proteins.

To deliver nonnuclear proteins into the nucleus, we have examined the locations and number of nuclear localizing signals by use of simian virus 40 large T-antigen (SV40Ta) and yeast enhanced green fluorescent protein (yEGFP) in Saccharomyces cerevisiae as a model system. When only one SV40Ta was added to either the N- or C-terminus of yEGFP, the fluorescence of yEGFP was detected in both the nucleus and the cytoplasm. When two SV40Ta signals were added, one to the N-terminus and one to the C-terminus of yEGFP (SV40Ta-yEGFP-SV40Ta), the fluorescence of yEGFP was localized in only the nucleus. When the presequence of cytochrome oxidase subunit IV (pCOXIV) was inserted between the SV40Ta and the N-terminus of yEGFP (SV40Ta-pCOXIV-yEGFP-SV40Ta) in this construct, the fluorescence was located in both the nucleus and the cytoplasm, suggesting that the increased distance between the two SV40Ta signals decreased the efficiency of transport into the nucleus. When an additional SV40Ta signal was inserted between pCOXIV and yEGFP (SV40Ta-pCOXIV-SV40Ta-yEGFP), the fluorescence was localized only in the nucleus, indicating that two SV40Ta signals spaced by pCOXIV of 28 amino acid residues forming an alpha-helix are potent in transporting yEGFP into the nucleus. These results indicate that two SV40Ta signals spaced appropriately are essential for the efficient transport of the nonnuclear protein into the nucleus.

Amino Acid Sequence↗

PubMed1992

Hormonal regulation of the nuclear localization signals of the human glucocorticosteroid receptor.

Nuclear localization of the rat glucocorticosteroid receptor (rGR) transiently expressed in COS-7 cells appears to be mediated by two nuclear localization signals, NL1 and NL2, in a hormone-dependent mechanism. We investigated the intracellular distribution of the human GR (hGR) expressed in COS-7 cells, by a different immunohistochemical technique involving immunostaining of cell pellet sections, thus avoiding the use of cell permeabilizing agents and allowing rigorous comparison between successive experiments. With a large set of hGR mutants, we could define determinants of the hGR nuclear localization and compare them with those previously reported for rGR. Our study demonstrated two hormone-dependent nuclear localization signals. NL1 activity, overlapping the DNA-binding domain (DBD)-hinge boundary, was repressed by the unliganded ligand-binding domain (LBD), even if the repressed NL1 retained a residual potency to target hGR in the nucleus. Structure/function analysis suggested a bipartite structure of NL1, analogous to that of other nuclear targeting signals (the carboxy-terminal part of DBD between amino acids 478 and 487 and the beginning of the hinge region which includes a basic amino acid stretch between 491 and 498). Upon hormone binding, NL2, located in the LBD, was activated, but was unable by itself to sustain full nuclear localization, which required the derepressed NL1 activity. Only two sequences in the LBD, localized between amino acids 600 and 626 and from amino acid 696 up to the carboxyl-terminal amino acid 777, respectively, were found to inhibit NL1 activity. As previously reported, efficient nuclear retention, mandatory for gene expression, did not required DNA-binding activity. The controversial intracellular localization of the unliganded form of hGR and the role of hsp90 in cytoplasmic localization are further discussed.

Cell Line↗

PubMed1995

Identification of the nuclear localization signal of rat liver CTP:phosphocholine cytidylyltransferase.

CTP:phosphocholine cytidylyltransferase (CT) is a major regulatory enzyme in phosphatidylcholine synthesis in mammalian cells. CT is found in both soluble and particulate forms, both of which are nuclear. We report here the identification of a 21-residue sequence at the amino terminus of CT, 8KVNSRKRRKEVPGPNGATEED28, which was sufficient to direct beta-galactosidase into the cell nucleus. Further deletions from either end of this sequence greatly reduced the nuclear localization of beta-galactosidase. Deletions of amino acids within the nuclear localization signal or of the entire signal disrupted CT nuclear localization, but CT was not completely excluded from the nucleus. Clones of stable transfectants of the nuclear localization signal-deficient CT expressed in Chinese hamster ovary (CHO) 58 cells, which is temperature-sensitive for growth and CT activity, were isolated and characterized. The deletion mutants were active under the same conditions as the wild-type enzyme. Despite the difference in subcellular location from wild-type CT, the nuclear localization mutants were fully able to complement the CT-deficient cell line CHO 58 for both growth and choline incorporation into phosphatidylcholine at the nonpermissive temperature. The mobility of the mutant enzymes on SDS gels was altered relative to the mobility of wild-type CT; however, the extent of phosphorylation of the mutant enzymes was decreased only slightly. Thus, the distribution of CT in both cytoplasm and nucleus, rather than exclusively nucleus, has little effect on the ability of CT to function in growing CHO cells.

Amino Acid Sequence↗

PubMed2001

The C-terminal region of PTHrP, in addition to the nuclear localization signal, is essential for the intracrine stimulation of proliferation in vascular smooth muscle cells.

PTHrP is secreted by most cell types. In addition to a paracrine/autocrine role, PTHrP has "intracrine" actions, entering the nuclear compartment under the direction of a classic bipartite nuclear localization signal. In vascular smooth muscle cells, nuclear entry stimulates mitogenesis. In the current study, we sought to more precisely define the regions of PTHrP required for the activation of mitogenesis in vascular smooth muscle cells. PTHrP deletion mutants missing large regions [i.e. the signal peptide, N terminus (1--36), mid region (38--86), nuclear localization signal, C terminus (108--139), or combinations of the above] were expressed in A-10 vascular smooth muscle cells. The consequences on nuclear localization and proliferation were examined. Deletion of the nuclear localization signal prevented nuclear entry and slowed proliferation. Deletion of the highly conserved N terminus or mid region had no impact on nuclear localization or on proliferation. Deletion of the C terminus had no deleterious effect on nuclear localization but dramatically reduced proliferation. Thus, the nuclear localization signal is both necessary and sufficient for nuclear localization of PTHrP. In contrast, activation of proliferation in vascular smooth muscle cells requires both an intact nuclear localization signal and an intact C terminus. Whereas the nuclear localization signal is required for nuclear entry, the C terminus may serve a trans-activating function to stimulate mitogenesis once inside the nucleus of vascular smooth muscle cells.

Amino Acid Sequence↗