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[Large subunit of translation initiation factor--3 p170 contains potentially functional nuclear localization signals].

Eukaryotic translation factors and their subunits can have independent cellular functions, including regulation of nuclear events. We analyzed primary structure of p170 large subunit of human translation initiation factor eIF3 and found four potential bipartite nuclear localization signals (NLS). Then we studied whether these NLS were functional, that is were able to direct protein to cell nucleus. Complementary DNA of p170 fragments were expressed in cultured CV-1 and Cos-1 green monkey cells, and localization of fused with GFP proteins was determined by fluorescent microscopy. We established that p170 molecule possessed at least two functional NLS which determined nuclear localization of p170 fragments. At the same time more long p170 fragments containing the same functional NLS could be retained in cytoplasm. We speculate that either using specific factors or after limited proteolysis p170 can enter cell nucleus and participate in genome expression regulation. Also we do not exclude the possibility that functioning of p170 in cytoplasm can be regulated by reversible binding of importins to its NLS.

Animals↗
PubMed2005

Effects of 15-deoxy-delta(12,14)-prostaglandin J2 on nuclear localization of GATA-3 in the murine lung in the presence of lipopolysaccharide.

15-Deoxy-delta(12, 14)-prostaglandin J2 (15d-PG J2) is a regulator of a nuclear transcriptional factor, peroxisome proliferator-activated receptor (PPAR)-gamma. A previous study has demonstrated that 15d-PG J2 enhanced acute lung injury induced by lipopolysaccharide (LPS) in mice. 15d-PG J2 induced mucin-producing cells in the bronchial epithelium, especially in the presence of LPS. The present study investigated the effects of 15d-PG J2 on the activation of GATA-3 and Signal Transducer and Activator of Transcription (STAT) 6, important transcriptional factors in mucus secretion, in the lung in the presence or absence of LPS. ICR mice were divided into 4 experimental groups that intratracheally received vehicle, lipopolysaccharide (LPS: 125 microg/kg), 15d-PG J2 (1 mg/kg), or 15d-PG J2 + LPS. The nuclear localization of GATA-3 and phosphorylated STAT 6 was evaluated 2 h after the intratracheal administration. 15d-PG J2 enhanced the nuclear localization of GATA-3 in the presence of LPS, whereas the nuclear localization of phosphorylated STAT 6 was not altered in the groups. These results suggest that the enhancing effects of 15d-PG J2 on the production of mucin-producing cells might be related, at least in part, to the activation of GATA-3.

Animals↗
PubMed2000

Two putative alpha-helical domains of human immunodeficiency virus type 1 Vpr mediate nuclear localization by at least two mechanisms.

To identify the domains of Vpr that are involved nuclear localization, we transfected HeLa cells with a panel of expression vectors that encode mutant Vpr protein with deletions or substitutions within putative domains. Immunofluorescence staining of transfected cells revealed that wild-type Vpr was localized predominantly in the nucleus and the nuclear envelope and certainly in the cytoplasm. Introduction of substitutions or deletions within alphaH1 or alphaH2 resulted, by contrast, in diffuse expression over the entire cell. In addition, double mutations within both of these alpha-helical domains led to the complete absence of Vpr from nuclei. Next, we prepared HeLa cells that express chimeric proteins which consist of the alphaH1 and alphaH2 domains fused individually with green fluorescent protein (GFP) and a Flag tag and extracted them with digitonin and Triton X-100 prior to fixation. Flag-alphaH1-GFP was detected in the nucleus but not in the cytoplasm, while Flag-alphaH2-GFP was retained predominantly in the nucleus and in a small amount in the cytoplasm. The immunostaining patterns were almost eliminated by substitutions in each chimeric protein. Thus, it appeared that the two alpha-helical domains might be involved in nuclear import by binding to certain cellular factors. Taken together, our data suggest that the two putative alpha-helical domains mediate the nuclear localization of Vpr by at least two mechanisms.

Amino Acid Sequence↗
PubMed2006

Calcineurin inhibitors reduce nuclear localization of transcription factor NFAT in UV-irradiated keratinocytes and reduce DNA repair.

Calcineurin inhibitors are drugs used to suppress the immune system by blocking the nuclear localization of the NFAT transcription factor. Systemic use of these drugs is essential to organ transplantation, but comes at the cost of elevated rates of skin cancer. They have been used topically in atopic dermatitis and other skin diseases on the assumption that they avoid the cancer risk by localized use. The results here show that in skin cells and artificial models of human skin, calcineurin inhibitors block UV-induced nuclear localization of NFAT, and significantly reduce repair of cyclobutane pyrimidine dimers induced in DNA. In addition they inhibit apoptosis of UV-irradiated cells. The effect of blocking nuclear localization of NFAT and inhibiting DNA repair should be considered in judging the risk of topical use of calcineurin inhibitors.

Apoptosis↗
PubMed2004

Identification of a nuclear localization signal in OCT4 and generation of a dominant negative mutant by its ablation.

OCT4 plays a critical role in maintaining stem cell pluripotency in a dose-dependent manner by activating and repressing multiple downstream genes. The precise mechanism by which OCT4 achieves these diverse biological functions remains unknown. In this report, we identify and characterize (195)RKRKR as a nuclear localization signal responsible for its localization in the nuclei and required for the transactivation of its target genes. Point mutations within this motif yielded a mutant that localizes randomly throughout the cells and is defective in transactivating target genes. However, restoration of nuclear localization with a heterologous nuclear localization signal failed to rescue its transactivation function, suggesting that this (195)RKRKR motif has additional function in mediating transactivation function. We further demonstrate that this mutant is competent in dimerization with not only itself but also wild type OCT4 and can interfere with the activity of wild type OCT4, thus acting as a dominant negative mutant. Indeed, this mutant can induce the differentiation of P19 cells into trophoblast-like giant cells. These data suggest that this dominant negative form of OCT4 may be a useful tool for modulating the activity of OCT4 in pluripotent cells such as embryonic stem cells to achieve the desired cell types for therapeutic applications.

Amino Acid Motifs↗
PubMed1995

Human foamy virus Bel1 transactivator contains a bipartite nuclear localization determinant which is sensitive to protein context and triple multimerization domains.

The Bel1 protein of human foamy virus is a 300-amino-acid nuclear regulatory protein which transactivates the gene expression directed by the homologous long terminal repeat and the human immunodeficiency virus type 1 long terminal repeat. While previous reports suggested that the single basic domain of Bel1 from residues 211 to 222 and/or 209 to 226 is necessary and sufficient for efficient nuclear localization (L. K. Venkatesh, C. Yang, P. A. Theodorakis, and G. Chinnandurai, J. Virol. 67:161-169, 1993; F. He, J. D. Sun, E. D. Garrett, and B. R. Cullen, J. Virol. 67:1896-1904, 1993), our recent data showed that another basic domain, from amino acid residues 199 to 200, is also required for nuclear localization of Bel1 (C. W. Lee, C. Jun, K. J. Lee, and Y. C. Sung, J. Virol. 68:2708-2719, 1994). To clarify this discrepancy, we constructed various bel1-lacZ chimeric constructs and several linker insertion mutants and determined their subcellular localization. When the region of Bel1 containing basic domains was placed at an internal site of the lacZ gene, the nuclear localization signal (NLS) of Bel1 consisted of two discontinuous basic regions separated by an intervening sequence. Moreover, insertion of specific amino acids between two basic regions disrupted the activity of the Bel1 NLS. On the other hand, Bel1 residues 199 and 200 were not required to direct the Bel1-beta-galactosidase chimeric protein to the nucleus when the Bel1 NLS was appended to the amino terminus of beta-galactosidase. These results indicate that the function of the Bel1 NLS is sensitive to the protein context within which the sequence is present. In addition, we demonstrated that the Bel1 protein forms a multimeric complex in the nuclei of mammalian cells by using a sensitive in vivo protein-protein interaction assay. Mutational analyses revealed that the regions which mediate multimer formation map to three domains of Bel1, i.e., residues 1 to 31, 42 to 82, and 82 to 111. Furthermore, our results show that the region of Bel1 from residues 202 to 226 prevents Bel1 from forming a multimeric complex.

Amino Acid Sequence↗
PubMed2000

Identification of novel residues involved in nuclear localization of a baculovirus polyhedrin protein.

A baculovirus polyhedrin protein has proven to possess a nuclear localization signal (NLS) sequence and a domain required for supramolecular assembly. Here we investigated five Bombyx mori nucleopolyhedrovirus (BmNPV) mutants that did not produce polyhedra. Two of five mutants were generated during routine baculoviral expression vector screening, and three were isolated by treatment with the mutagen 5-bromo-2'-deoxyuridine (BrdU). Marker rescue mapping and nucleotide sequence analysis showed that mutations in the polyhedrin gene caused the altered phenotype of these mutants. Biochemical fractionation indicated that cells infected with these mutants exhibited polyhedrin protein in both the nucleus and the cytoplasm. Electron microscopic observation revealed that polyhedrin produced by these mutants ocurred in both the nucleus and the cytoplasm, but did not form a crystalline lattice. Despite the incompleteness of polyhedrin nuclear localization, the NLSs of the five mutants were unchanged, although some of the mutations occurred within residues just outside of the domain reported to be required for polyhedron assembly (4). This result suggests that (a) the polyhedrin NLS directs polyhedrin to the nucleus, but the efficiency of this localization is regulated by regions other than the NLS (probably, polyhedrin conformation and its association with the nucleus are also involved), and (b) formation of a crystalline lattice may also be determined by several domains within polyhedrin.

Animals↗
PubMed2005

Nuclear localization signal peptides induce molecular delivery along microtubules.

Many essential processes in eukaryotic cells depend on regulated molecular exchange between its two major compartments, the cytoplasm and the nucleus. In general, nuclear import of macromolecular complexes is dependent on specific peptide signals and their recognition by receptors that mediate translocation through the nuclear pores. Here we address the question of how protein products bearing such nuclear localization signals arrive at the nuclear membrane before import, i.e., by simple diffusion or perhaps with assistance of cytoskeletal elements or cytoskeleton-associated motor proteins. Using direct single-particle tracking and detailed statistical analysis, we show that the presence of nuclear localization signals invokes active transport along microtubules in a cell-free Xenopus egg extract. Chemical and antibody inhibition of minus-end directed cytoplasmic dynein blocks this active movement. In the intact cell, where microtubules project radially from the centrosome, such an interaction would effectively deliver nuclear-targeted cargo to the nuclear envelope in preparation for import.

Actins↗
PubMed2003

Nuclear localization of the Hermes transposase depends on basic amino acid residues at the N-terminus of the protein.

For the Hermes transposable element to be mobilized in its eukaryotic host, the transposase, encoded by the element, must make contact with its DNA. After synthesis in the cytoplasm, the transposase has to be actively imported into the nucleus because its size of 70.1 kDa prevents passive diffusion through the nuclear pore. Studies in vitro using transient expression of a Hermes-EGFP fusion protein in Drosophila melanogaster Schneider 2 cells showed the transposase was located predominantly in the nucleus. In silico sequence analysis, however, did not reveal any nuclear localization signal (NLS). To identify the sequence(s) responsible for localization of Hermes transposase in the nucleus, truncated or mutated forms of the transposase were examined for their influence on sub-cellular localization of marker proteins fused to the transposase. Using the same expression system and a GFP-GUS fusion double marker, residues 1-110 were recognized as sufficient, and residues 1-32 as necessary, for nuclear localization. Amino acid K25 greatly facilitated nuclear localization, indicating that at least this basic amino acid plays a significant role in this process. This sequence overlaps the proposed DNA binding region of the Hermes transposase and is not necessarily conserved in all members of the hAT transposable element family.

Amino Acid Sequence↗
PubMed2005

[Nuclear localization region in soluble adenylyl cyclase].

OBJECTIVE: To locate the region responsible for nuclear localization of protein sAC. METHODS: The eukaryotic expression vector of vairous sAC deletion mutants were transfected into Hela cells. The localization of each mutant was observed using confocal microscope. RESULTS: For some mutants, the localization of sAC changed. Deletion of some region made it unable to locate in the nuclear. CONCLUSION: It is possible to figure out that the nucleotide region (739-1038 and 1045-1261) take charge of nuclear localization of sAC.

Adenylyl Cyclases↗
PubMed1992

Nuclear localization signals, but not putative leucine zipper motifs, are essential for nuclear transport of hepatitis delta antigen.

Hepatitis delta antigen (HDAg) is the only known protein of hepatitis delta virus and was previously shown to localize in the nucleoplasm of infected liver cells. In this study, nuclear localization signals of HDAg were defined by expressing various domains of the antigen in both hepatic and nonhepatic cells as beta-galactosidase fusion proteins. A cytochemical staining assay demonstrated that a domain from amino acid residues 35 to 88 of HDAg was able to facilitate transport to the nucleus of the originally cytoplasm-localized protein beta-galactosidase. Two nuclear localization signals, NLS1 and NLS2, which are similar to those of simian virus 40 T antigen and polyomavirus T antigen, respectively, were identified. Either NLS1 or NLS2 alone was sufficient for the nuclear transport of HDAg. However, a fusion protein (N65Z) containing beta-galactosidase and the N-terminal 65 amino acids of HDAg, containing NLS1, was localized exclusively in the cytoplasm and perinuclear region. A possible hydrophobic subdomain between amino acid residues 50 and 65 may block the function of NLS1. Nevertheless, N65Z could enter the nuclei of transfected cells when it was coexpressed with full-length HDAg. Entry into the nucleus may be mediated by the coiled-coil structure rather than the putative leucine zipper motif located between amino acid residues 35 and 65. The existence of two independent nuclear localization signals may ensure the proper functioning of HDAg in the multiplication of delta virus in the nucleus. In addition, two putative casein kinase II sites (SRSE-5 and SREE-126) that may be important in controlling the rate of nuclear transport were found in HDAg.

Amino Acid Sequence↗
PubMed1997

Characterization of a nuclear localization signal of canine parvovirus capsid proteins.

We investigated the abilities of synthetic peptides mimicking the potential nuclear localization signal of canine parvovirus (CPV) capsid proteins to translocate a carrier protein to the nucleus following microinjection into the cytoplasm of A72 cells. Possible nuclear localization sequences were chosen for synthesis from CPV capsid protein sequences (VP1, VP2) on the basis of the presence of clustered basic residues, which is a common theme in most of the previously identified targeting peptides. Nuclear targeting activity was found within the N-terminal residues 4-13 (PAKRARRGYK) of the VP1 capsid protein. While replacement of Arg10 with glycine did not affect the activity, replacement of Lys6, Arg7, or Arg9 with glycine abolished it. The targeting activity was found to residue in a cluster of basic residues, Lys5, Arg7, and Arg9. Nuclear import was saturated by excess of unlabelled peptide conjugates (showing that it was a receptor-mediated process). Transport into the nucleus was an energy-dependent and temperature-dependent process actively mediated by the nuclear pores and inhibited by wheat germ agglutinin.

Adenosine Triphosphate↗
PubMed2006

The nuclear localization signal of mitotic kinesin-like protein Mklp-1: effect on Mklp-1 function during cytokinesis.

The mitotic kinesin-like protein (Mklp-1) localizes in the nucleus during interphase due to the presence of nuclear localization signal(s) [NLS(s)] within its sequence. Here, we mapped two NLSs to be 899SRKRRSST906 and 949KRKKP953 in the tail domain of Mklp-1, and showed that ectopic expression of a mutant Mklp-1 without the NLSs leads to cell cycle arrest at cytokinesis, indicating that the NLSs are necessary for Mklp-1 to execute its normal function during cell division. Furthermore, mutation of two serine residues in the first NLS to aspartic acid, which mimics phosphorylation, attenuated its nuclear localization function, suggesting that the function of this NLS might be regulated by phosphorylation.

Amino Acid Sequence↗
PubMed2004

Regulation of nuclear import by phosphorylation adjacent to nuclear localization signals.

Many important regulatory proteins, including cell cycle regulators and transcription factors, contain a phosphorylation site within or adjacent to a classic nuclear localization signal (NLS) sequence. Previous studies show that the nuclear localization of these cargoes can be regulated by phosphorylation at these sites. It was hypothesized that this phosphorylation regulates the nuclear import of NLS cargo proteins by modulating the interaction of the cargo with the classic nuclear transport receptor, importin alpha. In this study, we utilize in vitro solution binding assays and in vivo analyses to directly test this model. We demonstrate that mimicking phosphorylation at a site adjacent to an NLS decreases the binding affinity of the NLS for importin alpha. This decrease in cargo affinity for importin alpha correlates with a decrease in nuclear accumulation in vivo. Through these analyses, we show that the cell cycle-dependent nuclear import of the Saccharomyces cerevisiae transcription factor Swi6p correlates with a phosphorylation-dependent change in affinity for importin alpha. Furthermore, we present data using the SV40 NLS to suggest that this form of regulation can be utilized to artificially modulate the nuclear import of a cargo, which is usually constitutively targeted to the nucleus. This work defines one molecular mechanism for regulating nuclear import by the classic NLS-mediated transport pathway.

Amino Acid Sequence↗
PubMed2000

The Dlx3 protein harbors basic residues required for nuclear localization, transcriptional activity and binding to Msx1.

The murine Dlx3 protein is a putative transcriptional activator that has been implicated during development and differentiation of epithelial tissue. Dlx3 contains a homeodomain and mutational analysis has revealed two regions, one N-terminal and one C-terminal to the homeodomain, that act as transcriptional activators in a yeast one-hybrid assay. In addition to transactivation, data are presented to demonstrate specific DNA binding and an association between Dlx3 and the Msx1 protein in vitro. Immunohistochemical analysis confirmed coexpression of Dlx3 and Msx1 proteins in the differentiated layers of murine epidermal tissues. Transcription factor function requires nuclear localization. In this study, the intracellular localization of the green fluorescent protein fused to Dlx3 was examined in keratinocytes induced to differentiate by calcium and is shown to localize to the nucleus. A bipartite nuclear localization signal (NLS) was identified by mutational analysis and shown to be sufficient for nuclear localization. This was demonstrated by insertion of the Dlx3 bipartite NLS sequence into a cytoplasmic fusion protein, GFP-keratin 14, which functionally redirected GFP-keratin 14 expression to the nucleus. Further analysis of Dlx3 NLS mutants revealed that the Dlx3 NLS sequences are required for specific DNA binding, transactivation potential and interactions with the Msx1 protein.

Amino Acid Sequence↗
PubMed2006

The unfolded protein response transducer Ire1p contains a nuclear localization sequence recognized by multiple beta importins.

The Ire1p transmembrane receptor kinase/endonuclease transduces the unfolded protein response (UPR) from the endoplasmic reticulum (ER) to the nucleus in Saccharomyces cerevisiae. In this study, we analyzed the capacity of a highly basic sequence in the linker region of Ire1p to function as a nuclear localization sequence (NLS) both in vivo and in vitro. This 18-residue sequence is capable of targeting green fluorescent protein to the nucleus of yeast cells in a process requiring proteins involved in the Ran GTPase cycle that facilitates nuclear import. Mutagenic analysis and importin binding studies demonstrate that the Ire1p linker region contains overlapping potential NLSs: at least one classical NLS (within sequences 642KKKRKR647 and/or 653KKGR656) that is recognized by yeast importin alpha (Kap60p) and a novel betaNLS (646KRGSRGGKKGRK657) that is recognized by several yeast importin beta homologues. Kinetic binding data suggest that binding to importin beta proteins would predominate in vivo. The UPR, and in particular ER stress-induced HAC1 mRNA splicing, is inhibited by point mutations in the Ire1p NLS that inhibit nuclear localization and also requires functional RanGAP and Ran GEF proteins. The NLS-dependent nuclear localization of Ire1p would thus seem to be central to its role in UPR signaling.

Active Transport, Cell Nucleus↗
PubMed1993

Overlapping domains of the heterochromatin-associated protein HP1 mediate nuclear localization and heterochromatin binding.

The Drosophila protein HP1 is a 206 amino acid heterochromatin-associated nonhistone chromosomal protein. Based on the characterization of HP1 to date, there are three properties intrinsic to HP1: nuclear localization, heterochromatin binding, and gene silencing. In this work, we have concentrated on the identification of domains responsible for the nuclear localization and heterochromatin binding properties of HP1. We have expressed a series of beta-galactosidase/HP1 fusion proteins in Drosophila embryos and polytene tissue and have used beta-galactosidase enzymatic activity to identify the subcellular localization of each fusion protein. We have identified two functional domains in HP1: a nuclear localization domain of amino acids 152-206 and a heterochromatin binding domain of amino acids 95-206. Both of these functional domains overlap an evolutionarily conserved COOH-terminal region.

Amino Acid Sequence↗
PubMed1993

Nuclear localization of vertebrate cyclin A correlates with its ability to form complexes with cdk catalytic subunits.

Cyclins control the activities of cyclin-dependent protein kinases (cdks) and hence play a key role in cell cycle regulation. While B-type cyclins associate with p34cdc2 to trigger entry into mitosis, progression through S phase requires cyclin A, presumably in association with p33cdk2. Vertebrate A- and B-type cyclins display strikingly distinct subcellular localizations, but the mechanisms underlying these differential distributions are unknown. Here, we have begun to study the requirements for nuclear localization of cyclin A. We have isolated a cDNA coding for chicken cyclin A and constructed a series of deletion mutants. These were then transfected into HeLa cells, and the subcellular distribution of the mutant cyclin A proteins was determined by indirect immunofluorescence microscopy. In parallel, the cyclin A mutants were assayed for their ability to form complexes with cdk subunits. We found that deletion of more than 100 residues from the N terminus of cyclin A did not impair nuclear localization or cdk subunit binding and kinase activation. In contrast, removal of as few as 15 residues from the C terminus, or deletion of part of the internal cyclin box domain, abolished nuclear localization of cyclin A as well as its ability to bind to and activate cdk subunits. These results suggest that nuclear transport of cyclin A may depend on the formation of multiprotein complexes comprising cdk catalytic subunits.

Amino Acid Sequence↗