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PubMed2003

TBX5 nuclear localization is mediated by dual cooperative intramolecular signals.

The TBX5 transcription factor is required for normal cardiogenesis, and human TBX5 mutations cause congenital heart defects. Previous studies have shown that TBX5 can localize to cellular nuclei during embryogenesis and have suggested that altered nuclear localization may contribute to disease pathogenesis. Current analyses suggest that TBX5 nuclear localization is not uniform during organogenesis. To determine the biochemical mechanisms underlying TBX5 nuclear import, we performed site-directed mutagenesis of human TBX5. We identified two distinct nuclear localization signals in TBX5, one monopartite and one bipartite. While each is insufficient to promote complete TBX5 nuclear localization, they act cooperatively to do so. These sequences are evolutionarily conserved and have cognates in other T-box gene family members.

Active Transport, Cell Nucleus↗
PubMed2004

Stat5a is tyrosine phosphorylated and nuclear localized in a high proportion of human breast cancers.

Signal transducers and activators of transcription (STATs) are latent cytoplasmic transcription factors that are activated and translocated into the nucleus after phosphorylation at a conserved tyrosine residue. Mouse model studies have demonstrated that activated Stat5a acts as a critical survival factor for normal, preneoplastic and malignant mammary epithelial cells. Very limited information is available, however, on the expression, tyrosine phosphorylation status and nuclear localization of Stat5a in human breast cancers. In our study, the pattern of Stat5a cellular localization was analyzed by immunohistochemistry in a series of 83 randomly selected primary human breast adenocarcinomas. Immunoprecipitation/Western blotting and immunohistochemistry assays employing different phospho-specific antibodies verified Stat5a tyrosine phosphorylation status. Stat5a was nuclear localized and tyrosine phosphorylated in 59 of 78 (76%) breast cancers examined; 38 of 78 (49%) demonstrated Stat5a nuclear localization in more than 25% of the breast cancer cells within the adenocarcinomas. Nuclear localized Stat5a was associated positively with increased levels of histologic differentiation (p = 0.03). A statistically significant positive association with p27 nuclear localization also was identified (p = 0.05). No relationship was found between nuclear localized Stat5a and menopausal status, tumor size, ploidy, percentage of cells in S-phase, lymph node metastases, ER, ErbB2, nuclear localized p21 or nuclear localized Stat5b/Stat3. As its role in human breast cancer progression and response to therapy is defined, Stat5a could become a new molecular target for breast cancer therapy.

Adenocarcinoma↗
PubMed1972

Nuclear localization of immunoglobulins in renal biopsies of patients with lupus nephritis.

Immunofluorescent evaluation of renal biopsies from 19 patients with lupus nephritis revealed nuclear localization of immunoglobulins (IgG and IgM) in 6 patients. Homogeneous, nuclear rim and speckled patterns of nuclear localization were observed. The extent of localization varied, with only occasional nuclei fluorescing in 1 case, whereas approximately 50% of the nuclei exhibited fluorescence in the most extreme case. The phenomenon of nuclear localization of immunoglobulins was not observed in immunofluorescent studies of 225 renal biopsies from patients with conditions other than lupus nephritis. The possibility that nuclear localization of immunoglobulins occurred artifactually in the 6 patients was considered and was discounted by determining antinuclear antibody titers on serum obtained concurrently with the renal biopsy Nuclear localization was not confined to areas of histologically evident parenchymal destruction, indicating that antinuclear antibodies do not react only with nuclear antigens after tissue breakdown, but may gain access to intracellular antigens prior to cell dissolution.

Adult↗
PubMed1989

Identification of a human protein that interacts with nuclear localization signals.

Through a series of label transfer experiments, we have identified a HeLa cell nuclear protein that interacts with nuclear localization signals (NLSs). The protein has a molecular weight of 66,000 and an isoelectric point of approximately 6. It associates with a synthetic peptide that contains the SV-40 T antigen NLS peptide but not with an analogous peptide in which an asparagine is substituted for an essential lysine (un-NLS peptide). In addition to these peptides, several proteins have been tested as label donors. With the proteins, there is a correlation between nuclear localization (assayed with lysolecithin-permeabilized cells) and label transfer to the 66-kD protein. The NLS peptide (but not the un-NLS peptide) competes with the proteins in label transfer experiments, but neither wheat germ agglutinin nor ATP has an effect. These results suggest that the 66-kD protein functions as an NLS receptor in the first step of nuclear localization. In the course of this work, we have observed that the Staphylococcus aureus protein A is a strongly karyophilic protein. Its dramatic nuclear localization properties suggest that it may have multiple copies of an NLS.

Amino Acid Sequence↗
PubMed1994

Efficient nuclear localization of the Ad5 E1A 12S protein is necessary for immortalization but not cotransformation of primary epithelial cells.

The Ad5 E1A 12S gene encodes a nuclear protein that can immortalize and cooperate with other oncoproteins to transform primary epithelial cells. Expression of the first exon is necessary for the activation of cellular proliferation, immortalization, and transformation. The second exon is necessary for the maintenance of cellular proliferation, immortalization, and the induction of an epithelial cell growth factor but is dispensable for cotransformation with T24 ras. Expression of the second exon is also necessary for efficient nuclear localization of the 12S polypeptide. A five-amino acid nuclear localization signal (NLS), Lys-Arg-Pro-Arg-Pro is encoded by the last 15 nucleotides of the second exon. To determine the role of subcellular localization in immortalization and transformation, a mutational analysis of the second exon and the 12S NLS was undertaken. The results from our analysis indicate that regions of the second exon outside the NLS affect nuclear localization. These regions necessary for efficient nuclear localization of the 12S protein are coincident with regions necessary for immortalization. In contrast, these regions are dispensable for cotransformation with T24 ras. The importance of the positively charged amino acids of the NLS in signal function and immortalization is varied. Lys 239 is the most critical residue, followed by Arg 240 and Arg 242, respectively. These data indicate that efficient nuclear localization of 12S is necessary for immortalization but not cotransformation with T24 ras.

Adenovirus E1A Proteins↗
PubMed2004

Regulation of subcellular localization of the antiproliferative protein Tob by its nuclear export signal and bipartite nuclear localization signal sequences.

Tob, a member of the Tob and BTG antiproliferative protein family, plays an important role in many cellular processes including cell proliferation. In this study, we have addressed molecular mechanisms regulating subcellular localization of Tob. Treatment with leptomycin B, an inhibitor of nuclear export signal (NES) receptor, resulted in a change in subcellular distribution of Tob from its pan-cellular distribution to nuclear accumulation, indicating the existence of NES in Tob. Our results have then identified an N-terminal region (residues 2-14) of Tob as a functional NES. They have also shown that Tob has a functional, bipartite nuclear localization signal (NLS) in residues 18-40. Thus, Tob is shuttling between the nucleus and the cytoplasm by its NES and NLS. To examine a possible relationship between subcellular distribution of Tob and its function, we exogenously added a strong NLS sequence or a strong NES sequence or both to Tob. The obtained results have demonstrated that the strong NLS-added Tob has a much weaker activity to inhibit cell cycle progression from G0/G1 to S phase. These results suggest that cytoplasmic localization or nucleocytoplasmic shuttling is important for the antiproliferative function of Tob.

3T3 Cells↗
PubMed1998

The cAMP-dependent protein kinase site (Ser312) enhances dorsal nuclear import through facilitating nuclear localization sequence/importin interaction.

Control over the nuclear import of transcription factors (TFs) represents a level of gene regulation integral to cellular processes such as differentiation and transformation. The Drosophila TF Dorsal shares with other rel TF family members the fact that it contains a phosphorylation site for the cAMP-dependent protein kinase (PKA) 22 amino acids N-terminal to the nuclear localization signal (NLS) at amino acids 335-340. This study examines for the first time the nuclear import kinetics of Dorsal fusion proteins in rat hepatoma cells in vivo and in vitro. Nuclear uptake was found to be not only NLS-dependent, but also strongly dependent on the PKA site, whereby substitution of Ser312 by either Ala or Glu using site-directed mutagenesis severely reduced nuclear accumulation. Exogenous cAMP or PKA catalytic subunit significantly enhanced the nuclear import of wild-type proteins both in vivo and in vitro. Using a direct binding assay, the molecular basis of PKA site enhancement of Dorsal fusion protein nuclear import was determined to be PKA site-mediated modulation of NLS recognition by the importin 58/97 complex. The physiological relevance of these results is supported by the observation that Drosophila embryos expressing PKA site Dorsal mutant variants were impaired in development. We conclude that the Dorsal NLS and PKA site constitute a phosphorylation-regulated NLS essential to Dorsal function and able to function in heterologous mammalian cell systems, where phosphorylation modulates the affinity of NLS recognition by importin.

Animals↗
PubMed1998

Effect of laminin on the nuclear localization of nucleolin in rat intestinal epithelial IEC-6 cells.

Laminin is a major component of extracellular matrix. The mechanism of action of laminin on cell proliferation, differentiation, and migration is not fully understood. In this study, we investigated the role of extracellular matrix, especially laminin, on the cellular localization of the nuclear protein, nucleolin, and on cell proliferation. Immunofluorescent and western blot analysis indicated that nucleolin was translocated most efficiently to the nucleus in the small intestinal rat epithelial cell line (IEC-6) when cultured on laminin-coated plates. Specifically, nucleolin was observed predominantly in cytoplasm in the cells cultured without laminin. In contrast, nuclear localization was observed in the cells cultured on laminin. This effect of laminin on nucleolin translocation was time-dependent. Laminin was also observed to stimulate proliferation of IEC-6 cells in serum free medium. Our results suggest that laminin alters the distribution of nucleolin which may be an early signal for cell proliferation.

Animals↗
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↗
PubMedDate not supplied

Localization of a sequence motif complementary to the nuclear localization signal in proteasomes from Thermoplasma acidophilum by immunoelectron microscopy.

A sequence motif complementary to the nuclear localization signal (NLS) has been localized in proteasomes from Thermoplasma acidophilum by immunoelectron microscopy using sequence-specific antibodies. The antibodies were generated in two different ways: by immunization with a carrier-coupled peptide and by isolation of the sequence-specific antibody from an immune serum against native proteasomes using a peptide-affinity column. The sequence specificity of the isolated antibody was confirmed by a PEPSCAN-ELISA performed on overlapping nonapeptides deduced from the sequence of the alpha-subunit of the Thermoplasma proteasome. Compared to the antibody induced by the carrier-coupled peptide this antibody fraction showed a much higher affinity for native proteasomes. The attachment site of the Fab portion of the antibody to the proteasome was mapped by electron microscopy in conjunction with image processing. The antibody was found to bind to the periphery of the two outer "disks" of the proteasome complex formed by the alpha-subunits.

Amino Acid Sequence↗
PubMed1999

Kap104p-mediated nuclear import. Nuclear localization signals in mRNA-binding proteins and the role of Ran and Rna.

Kap104p is a Saccharomyces cerevisiae nuclear import receptor for two essential mRNA-binding proteins, Nab2p and Nab4p/Hrp1p. We demonstrate direct binding of Kap104p to each of these substrates. We have defined the nuclear localization signals in both Nab2p and Nab4p/Hrp1p by Kap104p binding in vitro and KAP104-dependent nuclear import in vivo. The nuclear localization signals map to similar arginine/glycine-rich RNA-binding domains in both proteins and are thus termed rg-nuclear localization signals to distinguish them from classical nuclear localization signals. We also demonstrate that Kap104p, like other known beta-karyopherins (or importins), interacts directly with the small GTPase Ran/Gsp1. However, unlike other known import factors, Ran binding is not sufficient to mediate release of substrates from Kap104p; efficient Ran-GTP-mediated substrate release requires RNA. Also, addition of Kap104p to Nab2p and Nab4p/Hrp1p prebound to single-stranded DNA-cellulose stimulated release of both proteins from the resin. We suggest a simple cycle in which Nab2p and Nab4p/Hrp1p, upon import, are released in the nucleus at sites of transcription by the concerted action of Ran-GTP and binding to newly synthesized mRNA. The resulting ribonucleoprotein complexes are exported to the cytoplasm, where Kap104p rebinds to Nab2p and Nab4p/Hrp1p, contributing to their release from mRNA.

Amino Acid Sequence↗
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↗
PubMed1996

The bovine papillomavirus type 1 E2 transactivator and repressor proteins use different nuclear localization signals.

The E2 gene of bovine papillomavirus type 1 encodes at least three nuclear phosphoproteins that regulate viral transcription and DNA replication. All three proteins have a common C-terminal domain that has DNA-binding and dimerization activities. A basic region in this domain forms an alpha helix which makes direct contact with the DNA target. In this study, it is shown that in addition to its role in DNA binding, this basic region functions as a nuclear localization signal both in the E2 DNA-binding domain and in a heterologous protein. Deletion of this signal sequence resulted in increased accumulation of the E2 transactivator and repressor proteins in the cytoplasm, but nuclear localization was not eliminated. In the full-length transactivator protein, another signal, present in the N-terminal transactivation domain, is used for transport to the nucleus, and the C-terminal nuclear localization signal(s) are masked. The use of different nuclear localization signals could potentially allow differential regulation of the subcellular localization of the E2 transactivator and repressor proteins at some stage in the viral life cycle.

Amino Acid Sequence↗
PubMed1990

Signal transduction by steroid hormones: nuclear localization is differentially regulated in estrogen and glucocorticoid receptors.

The glucocorticoid receptor accumulates in nuclei only in the presence of bound hormone, whereas the estrogen receptor has been reported to be constitutively nuclear. To investigate this distinction, we compared the nuclear localization domains of the two receptors and the capacity of their respective hormone-binding regions to regulate nuclear localization activity. As with the glucocorticoid receptor, we showed that the human estrogen receptor contained a nuclear localization signal between the DNA-binding and hormone-binding regions (amino acids 256-303); however, in contrast to the glucocorticoid receptor, the estrogen receptor lacked a second nuclear localization domain within the hormone-binding region. Moreover, the hormone-binding domain of the unliganded estrogen receptor failed to regulate nuclear localization signals, although it efficiently regulated other receptor functions. We conclude that the two receptors employ a common mechanism for signal transduction involving a novel "inactivation" function, but that they differ in their control of nuclear localization. Thus, despite the strong relatedness of the estrogen and glucocorticoid receptors in structure and activity, certain differences in their properties could have important functional implications.

Amino Acid Sequence↗
PubMed2002

Basic residues within the Kruppel zinc finger DNA binding domains are the critical nuclear localization determinants of EKLF/KLF-1.

EKLF/KLF-1 is an erythroid-restricted transcription factor essential for expression of the adult beta-globin gene. EKLF/KLF-1 is a 358-amino acid nuclear protein with an amino-terminal proline-rich domain and a carboxyl-terminal DNA binding domain. The nuclear localization signal (NLS) of EKLF/KLF-1 has not been empirically determined. We generated a series of epitope-tagged deletion and point mutants and assessed their subcellular localization. Our results delimit the NLS to the 83-amino acid (amino acids 276-358) DNA binding domain that consists of three Kruppel zinc fingers. All three zinc fingers are necessary for efficient nuclear localization; deletion of any individual finger results in cytoplasmic accumulation. Fusion of the three zinc fingers to green fluorescent protein (GFP) targeted GFP to the nucleus, demonstrating that the zinc finger domain is sufficient for nuclear localization. EKLF/KLF-1 containing histidine to alanine mutations that disrupt the structure of all three fingers retains appropriate nuclear localization, indicating that neither the tertiary structure of the zinc fingers nor specific DNA binding are necessary for nuclear localization. We demonstrate that basic residues within the fingers are the critical determinants for nuclear localization; mutations of these basic residues to alanine resulted in cytoplasmic mislocalization. The basic residues of all mammalian Kruppel zinc fingers are highly conserved; therefore we propose that these basic residues are a common NLS shared by all Kruppel family members.

Active Transport, Cell Nucleus↗
PubMed1995

Inhibition of nuclear translocation of transcription factor NF-kappa B by a synthetic peptide containing a cell membrane-permeable motif and nuclear localization sequence.

To control agonist-induced nuclear translocation of transcription factor kappa B (NF-kappa B) in intact cells, cell-permeable synthetic peptides were devised. Their import into intact cells was dependent on a hydrophobic region selected from the signal peptide sequences and was verified by their inaccessibility to extracellular proteases and by confocal laser scanning microscopy. When a cell-permeable peptide carried a functional cargo representing the nuclear localization sequence of NF-kappa B p50, it inhibited in a concentration-dependent manner nuclear translocation of NF-kappa B in cultured endothelial and monocytic cells stimulated with lipopolysaccharide or tumor necrosis factor-alpha. Synthetic peptide analogues with deleted hydrophobic cell membrane-permeable motif or with a mutated nuclear localization sequence were inactive. Cell membrane-permeable peptides were not cytotoxic within the concentration range used in these experiments. These results suggest that cell-permeable synthetic peptides carrying a functional cargo can be applied to control signal transduction-dependent subcellular traffic of transcription factors mediating the cellular responses to different agonists. Moreover, this approach can be used to study other intracellular processes involving proteins with functionally distinct domains.

3T3 Cells↗
PubMed1999

The carboxyl terminus of interferon-gamma contains a functional polybasic nuclear localization sequence.

Cytokines such as interferon-gamma (IFN-gamma), which utilize the well studied JAK/STAT pathway for nuclear signal transduction, are themselves translocated to the nucleus. The exact mechanism for the nuclear import of IFN-gamma or the functional role of the nuclear translocation of ligand in signal transduction is unknown. We show in this study that nuclear localization of IFN-gamma is driven by a simple polybasic nuclear localization sequence (NLS) in its COOH terminus, as verified by its ability to specify nuclear import of a heterologous protein allophycocyanin (APC) in standard import assays in digitonin-permeabilized cells. Similar to other nuclear import signals, we show that a peptide representing amino acids 95-132 of IFN-gamma (IFN-gamma(95-132)) containing the polybasic sequence 126RKRKRSR132 was capable of specifying nuclear uptake of the autofluorescent protein, APC, in an energy-dependent fashion that required both ATP and GTP. Nuclear import was abolished when the above polybasic sequence was deleted. Moreover, deletions immediately NH2-terminal of this sequence did not affect the nuclear import. Thus, the sequence 126RKRKRSR132 is necessary and sufficient for nuclear localization. Furthermore, nuclear import was strongly blocked by competition with the cognate peptide IFN-gamma(95-132) but not the peptide IFN-gamma(95-125), which is deleted in the polybasic sequence, further confirming that the NLS properties were contained in this sequence. A peptide containing the prototypical polybasic NLS sequence of the SV40 large T-antigen was also able to inhibit the nuclear import mediated by IFN-gamma(95-132). This observation suggests that the NLS in IFN-gamma may function through the components of the Ran/importin pathway utilized by the SV40 T-NLS. Finally, we show that intact IFN-gamma, when coupled to APC, was also able to mediate its nuclear import. Again, nuclear import was blocked by the peptide IFN-gamma(95-132) and the SV40 T-NLS peptide, suggesting that intact IFN-gamma was also transported into the nucleus through the Ran/importin pathway. Previous studies have suggested a direct intracellular role for IFN-gamma in the induction of its biological activities. Based on our data in this study, we suggest that a key intracellular site of interaction of IFN-gamma is the one with the nuclear transport mechanism that occurs via the NLS in the COOH terminus of IFN-gamma.

3T3 Cells↗
PubMed2000

Minimal requirements for the nuclear localization of p27(Kip1), a cyclin-dependent kinase inhibitor.

p27(Kip1) is a cyclin-dependent kinase inhibitor, and its nuclear localization is a prerequisite for it to function as a cell cycle regulator. In the present study, the minimal requirement for the nuclear localization signal (NLS) of p27(Kip1) was determined by analyzing the localization of various mutants of p27(Kip1) tagged with green fluorescent protein (GFP) in HeLa cells and porcine aortic endothelial cells. Wild-type p27(Kip1) exclusively localized into nucleus, while GFP alone localized in both cytosol and nucleus. A comparison of various truncation mutants revealed residues 153-166 to be the minimal region necessary for nuclear localization. However, a fusion of this region to GFP showed cytoplasmic retention in addition to nuclear localization, thus suggesting that some extension flanking this region is required to achieve a full function of NLS. The site-directed mutation of the full-length p27(Kip1) therefore showed that four basic residues (K153, R154, K165, R166), especially R166, play a critical role in the nuclear localization of p27(Kip1).

Amino Acid Sequence↗