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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↗
PubMed2000

Regulation of expanded polyglutamine protein aggregation and nuclear localization by the glucocorticoid receptor.

Spinobulbar muscular atrophy and Huntington's disease are caused by polyglutamine expansion in the androgen receptor and huntingtin, respectively, and their pathogenesis has been associated with abnormal nuclear localization and aggregation of truncated forms of these proteins. Here we show, in diverse cell types, that glucocorticoids can up- or down-modulate aggregation and nuclear localization of expanded polyglutamine polypeptides derived from the androgen receptor and huntingtin through specific regulation of gene expression. Wild-type glucocorticoid receptor (GR), as well as C-terminal deletion derivatives, suppressed the aggregation and nuclear localization of these polypeptides, whereas mutations within the DNA binding domain and N terminus of GR abolished this activity. Surprisingly, deletion of a transcriptional regulatory domain within the GR N terminus markedly increased aggregation and nuclear localization of the expanded polyglutamine proteins. Thus, aggregation and nuclear localization of expanded polyglutamine proteins are regulated cellular processes that can be modulated by a well-characterized transcriptional regulator, the GR. Our findings suggest approaches to study the molecular pathogenesis and selective neuronal degeneration of polyglutamine expansion diseases.

Animals↗
PubMed2006

Structural and functional analyses of Kaposi sarcoma-associated herpesvirus ORF57 nuclear localization signals in living cells.

Kaposi sarcoma-associated herpesvirus (KSHV) ORF57 is a multifunctional, nuclear protein involved in post-transcriptional regulation of a subset of viral genes during lytic replication. Three nuclear localization signals (NLSs), NLS1 (amino acids (aa 101-107), NLS2 (aa 121-130), and NLS3 (aa 143-152), were identified in the N terminus of the ORF57 protein, and each of the three represents a short stretch of basic amino acid residues. Disruption of all three NLSs prevented localization of ORF57 in the nucleus. Insertion of individual NLSs into a heterologous cytoplasmic protein converted it into a nuclear protein, confirming that each NLS functions independently and is sufficient to promote protein nuclear localization. Although it exhibits a function similar to that of Epstein-Barr virus EB2 in promoting KSHV ORF59 expression, KSHV ORF57 differs from the herpes simplex virus ICP27 protein, and its function could be disrupted by point mutations of single or two NLSs in random combination, despite the proper localization of the mutant protein in the nucleus. The dysfunctional ORF57 containing NLS mutations also had low affinity with ORF59 RNA and the RNA export factor REF. However, the REF binding of ORF57 in vivo appeared to have no effect on ORF57-mediated enhancement of ORF59 expression. Thus, the three NLSs identified in ORF57 provide at least two functions, nuclear localization of ORF57 and up-regulation of ORF59 expression.

Active Transport, Cell Nucleus↗
PubMed2000

The catalytic subunit of herpes simplex virus type 1 DNA polymerase contains a nuclear localization signal in the UL42-binding region.

The herpes simplex virus type 1 DNA polymerase consists of a catalytic subunit (POL or UL30) and a processivity factor (UL42). The POL/UL42 interaction, which occurs through the extreme C-terminus of POL, is essential for HSV-1 replication and thus represents a valid target for drug inhibition. We recently showed (A. Loregian et al. (1999) Proc. Natl. Acad. Sci. USA 96, 5221-5226) that an oligopeptide corresponding to the 27 C-terminal amino acids of POL, when delivered into herpes simplex virus type 1-infected cells by a protein carrier, was able to localize into the nucleus and to inhibit viral replication by disruption of the POL/UL42 interaction. In this report, to further characterize the 27 mer (Pol peptide), we investigated whether its nuclear localization was due to the presence of a nuclear localization signal. By testing the ability of the Pol peptide to localize the beta-galactosidase, a normally cytoplasmic protein, to the nucleus, we confirmed that the Pol peptide contained a functional nuclear localization signal, corresponding to the RRMLHR motif. This sequence proved not only necessary but also sufficient for nuclear localization, because its substitution with a six-alanine stretch prevented nuclear translocation of the beta-galactosidase-Pol peptide fusion. Site-directed mutagenesis experiments on this revealed that both the three basic arginines and the two hydrophobic residues Met and Leu were crucial for nuclear targeting. Finally, functionally equivalent sequences were also found in the C-terminus of the catalytic subunits of human cytomegalovirus (RRLHL) and of equine herpesvirus-1 DNA polymerase (RRILH).

Amino Acid Sequence↗
PubMed2005

Localization of human T-cell lymphotropic virus type II Tax protein is dependent upon a nuclear localization determinant in the N-terminal region.

Human T-cell lymphotropic viruses (HTLV) types I and II are closely related oncogenic retroviruses that have been associated with lymphoproliferative and neurological disorders. The proviral genome encodes a trans-regulatory Tax protein that activates viral genes and upregulates various cellular genes involved in both cell growth and transformation. Tax proteins of HTLV-I (Tax-I) and HTLV-II (Tax-II) exhibit more than 77% aa homology and expression of either Tax-I or Tax-II is sufficient for immortalization of cultured T lymphocytes. Tax-I shuttles from the nucleus to the cytoplasm and accumulates within the nucleus, whereas Tax-II is found mainly in the cytoplasm. In the present study we have used recombinant vectors to analyze the size and structure of the nuclear localization domain within the Tax-II protein sequence. The Tax-II protein was expressed in HeLa cells either as the complete protein, or regions thereof, that were individually fused to the green fluorescent protein (GFP). Immunoblot analysis of the fused Tax-II products confirmed their expression and size. Fluorescence microscopy studies indicated that the complete Tax-II as well as N-truncated forms presented a punctuate cytoplasmic distribution and that a nuclear localization determinant is confined to within the first 60 aa of Tax-II. Accordingly, site directed mutagenesis and deletion of specific sequences within the first 60 aa showed that the nuclear determinant lies within the first 41 residues of Tax-II. These results point to a direct involvement of the amino-terminal residues of Tax-II protein in determining its nuclear functionality.

Amino Acid Sequence↗
PubMed2007

The B2 domain of VIVIPAROUS1 is bi-functional and regulates nuclear localization and transactivation.

The transcriptional regulator VIVIPA-ROUS1 (VP1) is composed of four functional domains that control different aspects of gene expression during seed development. The B2 domain is required for its role as a transcriptional activator, functioning at the site of transcription and/or for its transport into the nucleus. Previous work showed that the B2 domain was required for transactivation of the Em promoter. We demonstrate that VP1::GFP localizes to the nucleus of barley (Hordeum vulgare) aleurone cells, but when B2 is deleted, nuclear accumulation is lost. However, the B2 domain itself is not sufficient for nuclear localization of GFP::GUS. Using point mutagenesis on the putative NLS within B2, we show that the VP1::GFP still accumulates in the nucleus. Utilizing a comparative approach, through the alignment of B2 domains from various VP1/ABI3 proteins, oincluding the ABI3 orthologs from Physcomitrella patens, revealed the involvement of other conserved amino acids. Mutating VP1 at the conserved threonine on the N-terminal side of the putative NLS and at a conserved arginine-glutamine-arginine sequence on the C-terminal side prevented nuclear localization of VP1. A single amino acid change, from alanine to threonine, within this NLS found in the Arabidopsis abi3-7 mutant prevents transcription of AtEm1 and AtEm6 in vivo. We show that this same mutation in VP1 prevents transactivation of the Em-GUS reporter in barley aleurone but does not interfere with nuclear localization. Our data demonstrate that the B2 domain of VP1 is bifunctional in nature regulating both nuclear localization and transactivation.

Amino Acid Sequence↗
PubMed2004

NLS-dependent nuclear localization of p120ctn is necessary to relieve Kaiso-mediated transcriptional repression.

The Armadillo catenin p120(ctn) regulates cadherin adhesive strength at the plasma membrane and interacts with the novel BTB/POZ transcriptional repressor Kaiso in the nucleus. The dual localization of p120(ctn) at cell-cell junctions and in the nucleus suggests that its nucleocytoplasmic trafficking is tightly regulated. Here we report on the identification of a specific and highly basic nuclear localization signal (NLS) in p120(ctn). The functionality of the NLS was validated by its ability to direct the nuclear localization of a heterologous beta-galactosidase-GFP fusion protein. Mutating two key positively charged lysines to neutral alanines in the NLS of full-length p120(ctn) inhibited both p120(ctn) nuclear localization as well as the characteristic p120(ctn)-induced branching phenotype that correlates with increased cell migration. However, while these findings and others suggested that nuclear localization of p120(ctn) was crucial for the p120(ctn)-induced branching phenotype, we found that forced nuclear localization of both wild-type and NLS-mutated p120(ctn) did not induce branching. Recently, we also found that one role of p120(ctn) was to regulate Kaiso-mediated transcriptional repression. However, it remained unclear whether p120(ctn) sequestered Kaiso in the cytosol or directly inhibited Kaiso transcriptional activity in the nucleus. Using minimal promoter assays, we show here that the regulatory effect of p120(ctn) on Kaiso transcriptional activity requires the nuclear translocation of p120(ctn). Therefore, an intact NLS in p120(ctn) is requisite for its first identified regulatory role of the transcriptional repressor Kaiso.

Alanine↗
PubMed1998

Ataxin-1 nuclear localization and aggregation: role in polyglutamine-induced disease in SCA1 transgenic mice.

Transgenic mice carrying the spinocerebellar ataxia type 1 (SCA1) gene, a polyglutamine neurodegenerative disorder, develop ataxia with ataxin-1 localized to aggregates within cerebellar Purkinje cells nuclei. To examine the importance of nuclear localization and aggregation in pathogenesis, mice expressing ataxin-1[82] with a mutated NLS were established. These mice did not develop disease, demonstrating that nuclear localization is critical for pathogenesis. In a second series of transgenic mice, ataxin-1[77] containing a deletion within the self-association region was expressed within Purkinje cells nuclei. These mice developed ataxia and Purkinje cell pathology similar to the original SCA1 mice. However, no evidence of nuclear ataxin-1 aggregates was found. Thus, although nuclear localization of ataxin-1 is necessary, nuclear aggregation of ataxin-1 is not required to initiate pathogenesis in transgenic mice.

Animals↗
PubMed2004

Phospholipid scramblase 1 contains a nonclassical nuclear localization signal with unique binding site in importin alpha.

Nuclear import of proteins containing a classical nuclear localization signal (NLS) is an energy-dependent process that requires the heterodimer importin alpha/beta. Three to six basic contiguous arginine/lysine residues characterize a classical NLS and are thought to form a basic patch on the surface of the import cargo. In this study, we have characterized the NLS of phospholipid scramblase 1 (PLSCR1), a lipid-binding protein that enters the nucleus via the nonclassical NLS (257)GKISKHWTGI(266). This import sequence lacks a contiguous stretch of positively charged residues, and it is enriched in hydrophobic residues. We have determined the 2.2 A crystal structure of a complex between the PLSCR1 NLS and the armadillo repeat core of vertebrate importin alpha. Our crystallographic analysis reveals that PLSCR1 NLS binds to armadillo repeats 1-4 of importin alpha, but its interaction partially overlaps the classical NLS binding site. Two PLSCR1 lysines occupy the canonical positions indicated as P2 and P5. Moreover, we present in vivo evidence that the critical lysine at position P2, which is essential in other known NLS sequences, is dispensable in PLSCR1 NLS. Taken together, these data provide insight into a novel nuclear localization signal that presents a distinct motif for binding to importin alpha.

Algorithms↗
PubMed1995

Cyclin-dependent kinase site-regulated signal-dependent nuclear localization of the SW15 yeast transcription factor in mammalian cells.

Control over the nuclear transport of transcription factors (TFs) represents a level of gene regulation integral to cellular processes such as differentiation, transformation and signal transduction. The Saccharomyces cerevisiae TF SWI5 is excluded from the nucleus in a cell cycle-dependent fashion, mediated by phosphorylation by the cyclin-dependent kinase (cdk) CDC28. Nuclear entry occurs in G1. beta-galactosidase fusion proteins carrying SWI5 amino acids 633-682, including the nuclear localization sequence (NLS: Lys-Lys-Tyr-Glu-Asn-Val-Val-Ile-Lys-Arg-Ser-Pro-Arg-Lys-Arg-Gly-Arg-Pro- Arg-Lys655) were analyzed for subcellular localization in appropriate temperature-sensitive yeast strains blocked in G1 or G2/M using indirect immunofluorescence, and for nuclear import kinetics in living rat hepatoma or Vero African green monkey kidney cells microinjected with fluorescently labeled bacterially expressed protein and quantitative confocal laser microscopy. Cell cycle-dependent nuclear localization in yeast was both NLS and cdk site-dependent, whereby mutation of the cdk site serines (Ser646 and Ser664) to alanine resulted in constitutive nuclear localization. In mammalian cells, the SWI5 fusion proteins were similarly transported to the nucleus in an NLS-dependent fashion, while the mutation to Ala of the cdk site serines increased the maximal level of nuclear accumulation from about 1- to over 8-fold. We suggest that phosphorylation at the cdk sites inhibits nuclear transport of SWI5, consistent with our previous observations for the inhibition of SV40 large tumor antigen nuclear transport by phosphorylation by the cdk cdc2. The results indicate for the first time that a yeast NLS and, fascinatingly, its regulatory mechanisms are functional in higher eukaryotes, implying the universal nature of regulatory signals for protein transport to the nucleus.

Amino Acid Sequence↗
PubMed1994

Nuclear localization signals also mediate the outward movement of proteins from the nucleus.

Several nuclear proteins, including steroid hormone receptors, have been shown to shuttle continuously between the nucleus and the cytoplasm. The mechanism of entry of proteins into the nucleus is well documented, whereas the mechanism of their outward movement into the cytoplasm is not understood. We have grafted the nuclear localization signals of the progesterone receptor or the simian virus 40 large tumor antigen onto beta-galactosidase. These additions were shown to impart to the protein the ability to shuttle between the nucleus and the cytoplasm. Microinjected proteins devoid of a nuclear localization signal were unable to exit from the nucleus. The same nuclear localization signals are thus involved in both the inward and the outward movement of proteins through the nuclear membrane. We also show that although the nuclear import requires energy, the nuclear export does not. These results suggest that the nucleocytoplasmic shuttling may be a general phenomenon for nuclear proteins that could possibly undergo modifications in the cytoplasm and exert some biological activities there. These conclusions also imply that at least part of the cellular machinery involved in the nuclear import of proteins may function bidirectionally.

Amino Acid Sequence↗
PubMed2004

Impairment of SHOX nuclear localization as a cause for Léri-Weill syndrome.

We report the characterization of the nuclear localization signal (NLS) of the short stature homeobox gene SHOX. Mutations within the SHOX gene cause Léri-Weill dyschondrosteosis (LWD) and Langer mesomelic dysplasia (LD) as well as idiopathic short stature (ISS). Furthermore, haploinsufficiency of SHOX has also been implicated in Turner syndrome. SHOX has been shown to be a cell-type-specific transcriptional activator that localizes to the nucleus. The SHOX protein contains a central homeodomain that together with its transactivation domain regulates the transcription of its target sequences within the nucleus. The sequences for its nuclear localization have not been identified yet. Experimental characterization of SHOX-NLS by deletion mapping identified a non-classic type basic signal, AKCRK, in the recognition helix of the homeodomain. Fusion of this stretch of five amino acids to a cytoplasmic reporter protein resulted in its nuclear translocation. Functional analysis of a missense mutation R173C (C517T) affecting the identified SHOX-NLS in two families with LWS and LD showed that the mutated SHOX protein is unable to enter the nucleus. Conversely, we can demonstrate that insertion of the identified signal adjacent to the mutant site can restore its nuclear translocation. These results establish impairment of nuclear localization as a mechanistic basis for SHOX-related diseases.

Amino Acid Sequence↗
PubMed2006

Nuclear localization of coactivator RAC3 is mediated by a bipartite NLS and importin alpha3.

The nuclear receptor coactivator RAC3 (also known as SRC-3/ACTR/AIB1/p/CIP/TRAM-1) belongs to the p160 coactivator family, which are involved in several physiological processes and diseases. Here we have investigated how RAC3 is translocated into the nucleus and show that it is mediated through a bipartite NLS and importin alpha3. This bipartite NLS is located within the conserved bHLH domain, and its mutation abolished nuclear localization. The NLS is also sufficient to cause nuclear import of EGFP, and the activity requires basic amino acids within the NLS. RAC3 binds strongly to importin alpha3, which also depends on the basic amino acids. Functionally, RAC3 cytoplasmic mutant loses its ability to enhance transcription, suggesting that nuclear localization is essential for coactivator function. Together, these results reveal a previous unknown mechanism for nuclear translocation of p160 coactivators and a critical function of the conserved bHLH within the coactivator.

Active Transport, Cell Nucleus↗
PubMed2004

The bipartite nuclear localization sequence of Rpn2 is required for nuclear import of proteasomal base complexes via karyopherin alphabeta and proteasome functions.

26 S proteasomes fulfill final steps in the ubiquitin-dependent degradation pathway by recognizing and hydrolyzing ubiquitylated proteins. As the 26 S proteasome mainly localizes to the nucleus in yeast, we addressed the question how this 2-MDa multisubunit complex is imported into the nucleus. 26 S proteasomes consist of a 20 S proteolytically active core and 19 S regulatory particles, the latter composed of two subcomplexes, namely the base and lid complexes. We have shown that 20 S core particles are translocated into the nucleus as inactive precursor complexes via the classic karyopherin alphabeta import pathway. Here, we provide evidence that nuclear import of base and lid complexes also depends on karyopherin alphabeta. Potential classic nuclear localization sequences (NLSs) of base subunits were analyzed. Rpn2 and Rpt2, a non-ATPase subunit and an ATPase subunit of the base complex, harbor functional NLSs. The Rpt2 NLS deletion yielded wild type localization. However, the deletion of the Rpn2 NLS resulted in improper nuclear proteasome localization and impaired proteasome function. Our data support the model by which nuclear 26 S proteasomes are assembled from subcomplexes imported by karyopherin alphabeta.

Amino Acid Sequence↗
PubMed1996

Subcellular distribution and phosphorylation of the nuclear localization signal binding protein, NBP60.

We previously purified a nuclear localization signal binding protein, NBP60, from rat liver (1993, J. Biochem. 113, 308-313). In this study, the subcellular localization of NBP60 was examined using anti-NBP60. Most NBP60 was found to be localized in the nuclear envelope fraction of rat liver obtained on cell fractionation followed by immunoblotting. Staining of the nuclei of cultured cells by the antibody was observed on immunofluorescence microscopy. NBP60 was widely detected in rat nuclear fractions prepared from other tissues and also in nuclei of cultured cells derived from other species. It was shown by immunoelectron microscopy that most NBP60 is present in the nuclear envelope and at least some of that is present on nuclear pore complexes. Although NBP60 was localized in the nuclear envelope in interphase cells, it diffused into the cytoplasm in the mitotic phase. The purified NBP60 was highly phosphorylated by a cdc2 mitotic kinase, whereas nuclear pore proteins p144, p62, p60, and p54 were not phosphorylated by the kinase directly. NBP60 was also phosphorylated by protein kinase A, calmodulin-dependent protein kinase II, and casein kinase II. The phosphorylation of NBP60 by cdc2 kinase and/or the other kinases may be related to the change in the protein's location during the mitotic phase.

Animals↗
PubMed1991

A yeast protein that binds nuclear localization signals: purification localization, and antibody inhibition of binding activity.

Short stretches of amino acids, termed nuclear localization sequences (NLS), can mediate assembly of proteins into the nucleus. Proteins from the yeast, Saccharomyces cerevisiae, have been identified that specifically recognize nuclear localization peptides (Silver, P., I. Sadler, and M. A. Osborne. 1989. J. Cell Biol. 109:983-989). We now further define the role of one of these NLS-binding proteins in nuclear protein localization. The NLS-binding protein of 70-kD molecular mass can be purified from salt extracts of nuclei. Antibodies raised against the NLS-binding protein localized the protein mainly to the nucleus with minor amounts in the cytoplasm. These antibodies also inhibited the association of NLS-protein conjugates with nuclei. Incubation of nuclei with proteases coupled to agarose removed NLS-binding protein activity. Extracts enriched for NLS-binding proteins can be added back to salt or protease-treated nuclei to restore NLS-binding activity. These results suggest that the first step of nuclear protein import can be reconstituted in vitro.

Amino Acid Sequence↗
PubMed2003

Two bipartite NLSs mediate constitutive nuclear localization of Mcm10.

Some components of the pre-replicative complex (pre-RC) are in the nucleus only during the G1 phase, while others are constitutively nuclear throughout the cell cycle. A nuclear localization signal (NLS) mediating the G1-specific nuclear localization has been characterized. We report here the identification of bipartite NLSs in constitutively nuclear pre-RC components, including the origin recognition complex, Mcm10 and Noc3. Interestingly, in a subgroup of these pre-RC components, two putative NLSs were found in each protein. To examine whether multiple NLSs are required for these proteins to be nuclear throughout the cell cycle, we characterized the two NLSs in Mcm10. We show that at least one of the two NLSs is required for directing Mcm10 into the nucleus and that either of them is sufficient to bring the green fluorescence protein into the nucleus. Thus, Mcm10 contains two functional NLSs and either is sufficient for the constitutive nuclear localization of Mcm10.

Blotting, Western↗
PubMed2007

The Hsp90 inhibitor, 17-AAG, prevents the ligand-independent nuclear localization of androgen receptor in refractory prostate cancer cells.

BACKGROUND: Androgen receptor (AR) is the key molecule in androgen-refractory prostate cancer. Despite androgen ablative conditions, AR remains active and is necessary for the growth of androgen-refractory prostate cancer cells. Nuclear localization of AR is a prerequisite for its transcriptional activation. We examined AR localization in androgen-dependent and androgen-refractory prostate cancer cells. METHODS AND RESULTS: We demonstrate increased nuclear localization of a GFP-tagged AR in the absence of hormone in androgen-refractory C4-2 cells compared to parental androgen-sensitive human prostate cancer LNCaP cells. Analysis of AR mutants impaired in ligand-binding indicates that the nuclear localization of AR in C4-2 cells is truly androgen-independent. The hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), inhibits basal PSA expression and disrupts the ligand-independent nuclear localization of AR at doses much lower than required to inhibit androgen-induced nuclear import. CONCLUSIONS: Hsp90 is a key regulator of ligand-independent nuclear localization and activation of AR in androgen-refractory prostate cancer cells.

Benzoquinones↗