SEARCH · PubMed Health

Results for “Nuclear Localization Signals”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 127 records · Page 7Linked to original sources

PubMed2003

A nonconventional nuclear localization signal within the UL84 protein of human cytomegalovirus mediates nuclear import via the importin alpha/beta pathway.

The open reading frame UL84 of human cytomegalovirus encodes a multifunctional regulatory protein which is required for viral DNA replication and binds with high affinity to the immediate-early transactivator IE2-p86. Although the exact role of pUL84 in DNA replication is unknown, the nuclear localization of this protein is a prerequisite for this function. To investigate whether the activities of pUL84 are modulated by cellular proteins we used the Saccharomyces cerevisiae two-hybrid system to screen a cDNA-library for interacting proteins. Strong interactions were found between pUL84 and four members of the importin alpha protein family. These interactions could be confirmed in vitro by pull down experiments and in vivo by coimmunoprecipitation analysis from transfected cells. Using in vitro transport assays we showed that the pUL84 nuclear import required importin alpha, importin beta, and Ran, thus following the classical importin-mediated import pathway. Deletion mutagenesis of pUL84 revealed a domain of 282 amino acids which is required for binding to the importin alpha proteins. Its function as a nuclear localization signal (NLS) was confirmed by fusion to heterologous proteins. Although containing a cluster of basic amino acids similar to classical NLSs, this cluster did not contain the NLS activity. Thus, a complex structure appears to be essential for importin alpha binding and import activity.

Active Transport, Cell Nucleus↗

PubMed1995

A single serine mutation on the nuclear localization signal of hepatitis B virus core protein abolishes the inhibition of nuclear transport by surface proteins.

The nuclear localization signal of hepatitis B virus core protein contained three SPRRR motifs. Substitution of a single serine residue on the third SPRRR motif enhanced significantly the nuclear localization of core protein. By performing immunofluorescence staining assays and subcellular fractionation experiments, it was demonstrated that although nuclear transport of the wild type core protein was markedly suppressed by co-expression of hepatitis B virus surface proteins, nuclear transport of this serine mutant protein was not affected. These results indicated that the serine residue in the SPRRR motifs played an important role in regulating the nuclear transport of core protein and the interaction between core and surface proteins.

Animals↗

PubMed1992

Intracellular distribution of a nuclear localization signal binding protein.

The transport of proteins into the nucleus requires the recognition of a nuclear localization signal sequence. Several proteins that interact with these sequences have been identified, including one of about 66 kDa. We have prepared antibodies that recognize the 66-kDa nuclear localization signal binding protein (NLSBP) and inhibit nuclear localization in vitro. By immunofluorescence, it is seen that the NLSBP is predominantly cytoplasmic and is distributed peripherally around the nucleus and the microtubule organizing center. There is also a weak punctate staining of the surface of the nucleus. Methanol-fixed cells can also be stained directly with fluorescently labeled karyophilic proteins. These stains reveal the same cytoplasmic structures as anti-NLSBP. The expression of the NLSBP is growth dependent. When cells grown to confluence are examined, the cytoplasmic staining is greatly reduced, leaving the punctate nuclear staining as the predominant feature. In serum-starved cells, very little staining of either the cytoplasm or the nucleus can be seen. Upon simulation by the addition of serum, the original cytoplasmic and nuclear envelope staining is restored. Cells grown in the presence of colchicine or taxol have an altered NLSBP distribution but apparently normal cytoplasmic nuclear transport.

Amino Acid Sequence↗

PubMed2006

Identification of multiple nuclear localization signals in murine Elf3, an ETS transcription factor.

We investigated nuclear localization signal (NLS) determinants within the AT-hook and ETS DNA-binding domains of murine Elf3 (mElf3), a member of the subfamily of epithelium-specific ETS transcription factors. Deletion mutants containing the AT-hook, ETS domain or both localized strictly in the nucleus, suggesting that these individual domains contain independent NLS motif(s). Within the AT-hook domain, four basic residues (244KRKR247) were critical for strong NLS activity, and two potent bipartite NLS motifs (236-252 and 249-267) were sufficient for nuclear import of mElf3, although less efficient than the full domain. In addition, one stretch of basic residues (318KKK320) within the ETS domain appears to be essential for mElf3 nuclear localization. Taken together, mElf3 contains multiple NLS motifs, which may function cooperatively to effect efficient nuclear transport.

Active Transport, Cell Nucleus↗

PubMed2004

Identification and characterization of a functional nuclear localization signal in the HIV-1 integrase interactor LEDGF/p75.

Human lens epithelium-derived growth factor (LEDGF)/p75 protein forms a specific nuclear complex with human immunodeficiency virus type 1 (HIV-1) integrase and is essential for nuclear localization and chromosomal association of the viral protein. We now studied nuclear import of LEDGF/p75 in live and semipermeabilized cells. We show that nuclear import of LEDGF/p75 is GTP-, Ran-, importin-alpha/beta-, and energy-dependent and that the protein competes with the canonical SV40 large T antigen nuclear localization signal (NLS) for nuclear import receptors. We identified the NLS of LEDGF/p75 through deletion analysis and site-directed mutagenesis. The LEDGF/p75 NLS, 148GRKRKAEKQ156, belongs to the canonical SV40-like family. Fusion of this short peptide to the amino terminus of Escherichia coli beta-galactosidase rendered the fusion protein nuclear, confirming that the LEDGF/p75 NLS is transferable. Moreover, a single amino acid change in the NLS was sufficient to exclude the mutant LEDGF/p75 protein from the nucleus and abolish nuclear import of HIV-1 integrase.

Alternative Splicing↗

PubMed1997

Mutational analysis of human prothymosin alpha reveals a bipartite nuclear localization signal.

Mutants of human prothymosin alpha with impaired ability to inhibit yeast Saccharomyces cerevisiae. cerevisiae cell growth were characterized. Two types of prothymosin alpha-inactivating mutations were observed. Mutations that belong to the first type compromised the nuclear entry of prothymosin alpha by affecting its nuclear localization signal. Analysis of subcellular distribution of GFP-prothymosin alpha fusions revealed a bipartite nuclear localization signal that is both necessary and sufficient for nuclear import of the protein in human cells. Mutations of the second type abrogated the inhibitory action of prothymosin alpha through an unknown mechanism, without influencing the nuclear import of the protein.

Amino Acid Sequence↗

PubMed1995

Interaction of nucleolar protein B23 with peptides related to nuclear localization signals.

Nucleolar protein B23 is a putative ribosome assembly factor with a high affinity for peptides containing nuclear localization signals (NLSs). The interactions of various NLS-containing peptides with two B23 isoforms (B23.1 and B23.2) were examined using equilibrium dialysis and Scatchard analyses. The KD for protein B23 binding to a peptide containing the SV40 T-antigen NLS sequence was approximately 1 microM with a stoichiometry of 1:1 (peptide:protein). No significant differences were seen between the two B23 isoforms in their affinities for any of the peptides tested. Binding by a reverse sequence SV40 T-NLS peptide showed a nonlinear Scatchard plot: this peptide was unable displace the correct sequence peptide, suggesting that the reverse sequence peptide binds to a different site on the protein. A peptide containing the sequence required for nucleolar localization of the HIV-1 Rev protein had an affinity for B23 approximately 10-fold greater than that of the SV40 T-NLS. However, with a sequence sufficient only for Rev location in the nucleoplasm, the affinity for B23 was diminished to a level between that of the longer Rev sequence and the SV40 T-NLS. In competition binding assays, the Rev NLS peptide was able to displace the SV40 T NLS, indicating that both peptides bind to the same site on protein B23. There was no detectable binding to protein B23 by a peptide containing the bipartite NLS of nucleoplasmin. Phosphorylation of protein B23 by casein kinase II enhanced its affinity for the SV40 T- and Rev-derived peptides approximately 2-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed2002

Importin alpha binds to an unusual bipartite nuclear localization signal in the heterogeneous ribonucleoprotein type I.

The heterogeneous nuclear ribonucleoprotein (hnRNP) type I, a modulator of alternative splicing, localizes in the nucleoplasm of mammalian cells and in a discrete perinucleolar structure. HnRNP I contains a novel type of bipartite nuclear localization signal (NLS) at the N-terminus of the protein that we have previously named nuclear determinant localization type I (NLD-I). Recently, a neural counterpart of hnRNP I has been identified that contains a putative NLS with two strings of basic amino acids separated by a spacer of 30 residues. In the present study we show that the neural hnRNP I NLS is necessary and sufficient for nuclear localization and represents a variant of the novel bipartite NLS present in the NLD-I domain. Furthermore, we demonstrate that the NLD-I is transported into the nucleus by cytoplasmic factor(s) with active transport modality. Binding assays using recombinant importin alpha show an interaction with NLD-I similar to that of SV40 large T antigen NLS. Deletion analysis indicates that both stretches of basic residues are necessary for binding to importin alpha. The above experimental results lead to the conclusion that importin alpha acts as cytoplasmic receptor for proteins characterized by a bipartite NLS signal that extends up to 37 residues.

Active Transport, Cell Nucleus↗

PubMedDate not supplied

Coupling of nuclear localization signals to plasmid DNA and specific interaction of the conjugates with importin alpha.

The nuclear localization signal (NLS) of the SV40 large T antigen efficiently induces nuclear targeting of proteins. We have developed a chemical strategy for covalent coupling of NLS peptides to plasmid DNA. A p-azido-tetrafluoro-benzyl-NLS peptide conjugate was synthesized. This conjugate was used to covalently associate NLS peptides to plasmid DNA by photoactivation. Reporter gene was expressed after transfection of the plasmid-NLS conjugates in NIH 3T3 cells. The conjugates interacted specifically with the NLS-receptor importin alpha, but plasmid-NLS conjugates were not detected in the nucleus, by fluorescence microscopy, after cytoplasmic microinjection.

3T3 Cells↗

PubMed2005

In vitro expression, monoclonal antibody and bioactivity for capsid protein of porcine circovirus type II without nuclear localization signal.

We expressed firstly the Capsid protein gene defecting the nuclear localization signal (NLS) of Porcine circovirus type II (PCV2) in Escherichia coli as a fusion protein with glutathione S-transferase (rGST-dCap protein). The purified rGST-dCap protein and the recombinant NLS-defected Cap protein of PCV2 (rdCap protein) from the purified rGST-dCap protein reacted specifically with swine antiserum to PCV2. Furthermore, the obtained monoclonal antibodies (mAbs) to rdCap protein were shown to bind to PCV2 particles replicated in PK15 cell and capsid protein (Cap protein) of PCV2 expressed in PK15 cells, respectively. mAbs to rdCap protein also revealed the neutralizing ability to PCV2 particles. These results demonstrated that rGST-dCap protein expressed in E. coli was folded correctly or at least partly, and mAbs to rdCap protein possessed the binding epitopes of PCV2 particles whereas mAbs 4C4 and 3F6 to rdCap protein remained the neutralization epitope of PCV2 particle, showing a possibility of neutralizing mAb to rdCap protein as an immnuotherapeutic agent and a potential of rGST-dCap protein as a vaccine antigen or serodiagnostic reagent.

Animals↗

PubMed1998

Backbone cyclic peptide, which mimics the nuclear localization signal of human immunodeficiency virus type 1 matrix protein, inhibits nuclear import and virus production in nondividing cells.

Here, we describe an application of the backbone cyclic (BC) proteinomimetic approach to the design and the synthesis of a BC peptide which functionally mimics the nuclear localization signal (NLS) region of the human immunodeficiency virus type 1 matrix protein (HIV-1 MA). On the basis of the NMR structure of HIV-1 MA, a library of BC peptides was designed and screened for the ability to inhibit nuclear import of NLS-BSA in digitonin-permeabilized HeLa and Colo-205 cultured cells. The screening yielded a lead compound (IC50 = 3 microM) which was used for the design of a second library. This library led to the discovery of a highly potent BC peptide, designated BCvir, with an IC50 value of 35 nM. This inhibitory potency is compared to a value of 12 microM exhibited by the linear parent HIV-1 MA NLS peptide. BCvir also reduced HIV-1 production by 75% in infected nondividing cultured human T-cells and was relatively resistant to tryptic digestion. These properties make BCvir a potential candidate for the development of a novel class of antiviral drugs which will be based on blocking nuclear import of viral genomes.

Anti-HIV Agents↗

PubMed2000

Functional characterization of nuclear localization signals in yeast Sm proteins.

In mammals, nuclear localization of U-snRNP particles requires the snRNA hypermethylated cap structure and the Sm core complex. The nature of the signal located within the Sm core proteins is still unknown, both in humans and yeast. Close examination of the sequences of the yeast SmB, SmD1, and SmD3 carboxyl-terminal domains reveals the presence of basic regions that are reminiscent of nuclear localization signals (NLSs). Fluorescence microscopy studies using green fluorescent protein (GFP)-fusion proteins indicate that both yeast SmB and SmD1 basic amino acid stretches exhibit nuclear localization properties. Accordingly, deletions or mutations in the NLS-like motifs of SmB and SmD1 dramatically reduce nuclear fluorescence of the GFP-Sm mutant fusion alleles. Phenotypic analyses indicate that the NLS-like motifs of SmB and SmD1 are functionally redundant: each NLS-like motif can be deleted without affecting yeast viability whereas a simultaneous deletion of both NLS-like motifs is lethal. Taken together, these findings suggest that, in the doughnut-like structure formed by the Sm core complex, the carboxyl-terminal extensions of Sm proteins may form an evolutionarily conserved basic amino acid-rich protuberance that functions as a nuclear localization determinant.

Alleles↗

PubMed1997

Rapid targeting of plasmid DNA to zebrafish embryo nuclei by the nuclear localization signal of SV40 T antigen.

Binding SV40 T antigen nuclear localization signals (NLSs) to plasmid DNA promotes transgene expression following injection of DNA-NLS complexes into the cytoplasm of zebrafish eggs. We now demonstrate that NLS peptides mediate import of DNA from the cytoplasm into embryo nuclei, under conditions in which naked DNA is not imported. Plasmid DNA was localized by polymerase chain reaction (PCR) in isolated nuclei, and relative amounts were quantified by densitometry. Binding DNA to NLSs, but not to nuclear-import-deficient peptides, promoted rapid targeting of DNA-NLS complexes to nuclei, and transport across the nuclear envelope. Import of DNA-NLS complexes was competed by co-injected albumin-NLS conjugates. NLS, but not reverse NLS, was detected on blots of nuclei probed with 32P-labeled DNA. The results suggest that NLS-mediated DNA transfer into nuclei may constitute a valuable tool for several gene transfer applications.

Adenosine Triphosphate↗

PubMed2004

Computer-identified nuclear localization signal in exon 1A of the transporter DMT1 is essentially ineffective in nuclear targeting.

Divalent metal transporter 1 (DMT1; also called DCT1, Nramp2, or SLC11A2) has multiple isoforms that localize differently in many cell types. DMT1 +IRE species (encoded by mRNA with an iron-responsive element) are limited to the plasma membrane and cytosolic vesicles. In neural cells, -IRE isoforms of DMT1 (encoded by mRNA lacking an IRE) localize to the nucleus, plasma membrane, and cytosolic vesicles. In considering nuclear compartmentalization of -IRE isoforms, we hypothesized that the newly identified exon 1A in the N-terminus of this transporter might contain a nuclear localization signal. DNA constructs starting with exon 1A and ending with exons encoding alternative isoforms were made and transiently transfected into HEK293T and PC12 cells as well as rat sympathetic neurons. None of the constructs appeared in the nucleus despite the presence of exon 1A. Antibody specific for exon 1A was also used in both immunostaining and Western blots to investigate localization of exon 1A expressed both endogenously and ectopically in cells. Again, nuclear localization of DMT1 containing exon 1A was not observed. Our data suggest that exon 1A is neither sufficient nor necessary for DMT1 to appear in the nucleus.

Animals↗

PubMed2000

A distinct nuclear localization signal in the N terminus of Smad 3 determines its ligand-induced nuclear translocation.

Smad proteins are intracellular mediators of transforming growth factor beta (TGF-beta) and related cytokines and undergo ligand-induced nuclear translocation. Here we describe the identification of a nuclear localization signal (NLS) in the N-terminal region of Smad 3, the major Smad protein involved in TGF-beta signaling. An NLS-like basic motif (Lys(40)-Lys-Leu-Lys-Lys(44)), conserved among all pathway-specific Smad proteins, not only is responsible for constitutive nuclear localization of the isolated Smad 3 MH1 domain but also is crucial for Smad 3 nuclear import in response to ligand. Mutations in this motif completely abolished TGF-beta-induced nuclear translocation but had no impact on ligand-induced phosphorylation of Smad 3, complex formation with Smad 4, or specific binding to DNA. Hence Smad 3 proteins with NLS mutations are dominant-negative inhibitors of TGF-beta-induced transcriptional activation. Smad 4, which cannot translocate into the nucleus in the absence of Smad 3 or another pathway-specific Smad, contains a Glu in place of the last Lys in this motif. Smad 3 harboring the same mutation (K44E) does not undergo ligand-induced nuclear import. Conversely, the isolated Smad 4 MH1 domain does not accumulate in the nucleus but becomes nuclear enriched when Glu(49) is replaced with Lys. We propose that this highly conserved five-residue NLS motif determines ligand-induced nuclear translocation of all pathway-specific Smads.

Activin Receptors, Type I↗

PubMed2002

Nuclear translocation of plk1 mediated by its bipartite nuclear localization signal.

Polo-like kinase 1 (Plk1), a mammalian ortholog of Drosophila Polo, is a serine-threonine protein kinase implicated in the regulation of multiple aspects of mitosis. The protein level, activity, and localization of Plk1 change during the cell cycle, and its proper subcellular localization is thought to be crucial for its function. Although localization of Plk1 to the centrosome has been established, nuclear localization or nucleocytoplasmic translocation of Plk1 has not been fully addressed. Here we show that Plk1 accumulates in both the nucleus and the cytoplasm in addition to its localization to the centrosome during S and G(2) phases. Our results identify a conserved region in the kinase domain of Plk1 (residues 134-146) as a functional bipartite nuclear localization signal (NLS) sequence that regulates nuclear translocation of Plk1. The identified NLS is necessary and sufficient for directing nuclear localization of Plk1. This bipartite NLS has an unusually short spacer sequence between two clusters of basic amino acids but is sensitive to RanQ69L, a dominant negative form of Ran, similar to ordinary bipartite NLS. Remarkably, the expression of an NLS-disrupted mutant of Plk1 during S phase was found to arrest the cells in G(2) phase. These results suggest that the bipartite NLS-dependent nuclear localization of Plk1 before mitosis is important for ensuring normal cell cycle progression.

Amino Acid Sequence↗

PubMed1999

Gene delivery: a single nuclear localization signal peptide is sufficient to carry DNA to the cell nucleus.

Translocation of exogenous DNA through the nuclear membrane is a major concern of gene delivery technologies. To take advantage of the cellular import machinery, we have synthesized a capped 3.3-kbp CMVLuciferase-NLS gene containing a single nuclear localization signal peptide (PKKKRKVEDPYC). Transfection of cells with the tagged gene remained effective down to nanogram amounts of DNA. Transfection enhancement (10- to 1,000-fold) as a result of the signal peptide was observed irrespective of the cationic vector or the cell type used. A lysine to threonine mutation of the third NLS amino acid completely abolished these remarkable features, suggesting importin-mediated translocation. Our hypothesis is that the 3-nm-wide DNA present in the cytoplasm is initially docked to and translocated through a nuclear pore by the nuclear import machinery. As DNA enters the nucleus, it is quickly condensed into a chromatin-like structure, which provides a mechanism for threading the remaining worm-like molecule through the pore. A single NLS signal is thus sufficient, whereas many signals on a gene would actually inhibit entry, the same DNA molecule being threaded through adjacent pores.

Biological Transport↗

PubMed2003

Characterization of the bipartite nuclear localization signal of protein LANA2 from Kaposi's sarcoma-associated herpesvirus.

LANA2 is a nuclear latent protein detected exclusively in Kaposi's sarcoma-associated herpesvirus-infected B cells. The protein inhibits p53-dependent transactivation and apoptosis, suggesting an important role in the transforming activity of the virus. To explore the molecular mechanisms of its nuclear localization, fusion proteins of green fluorescent protein (EGFP) and deletion constructs of LANA2 were expressed in HeLa cells. Only the fragment comprising amino acid residues 355-440 of LANA2 localized in the cell nucleus. This fragment contains two closely located basic domains and forms a putative bipartite nuclear localization signal (NLS). The putative LANA2 NLS was able to target EGFP to the nucleus consistently. Site-directed mutation analyses demonstrated that LANA2 contains a functional bipartite NLS between amino acid positions 367 and 384. In addition, analysis of cells transfected with a cytoplasmic LANA2 mutant revealed that an appropriate subcellular localization may be crucial to regulate p53 activity.

Active Transport, Cell Nucleus↗