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Results for “Nuclear Localization Signals”

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PubMed2002

Integrative nuclear FGFR1 signaling (INFS) pathway mediates activation of the tyrosine hydroxylase gene by angiotensin II, depolarization and protein kinase C.

The integrative nuclear FGFR1 signaling (INFS) pathway functions in association with cellular growth, differentiation, and regulation of gene expression, and is activated by diverse extracellular signals. Here we show that stimulation of angiotensin II (AII) receptors, depolarization, or activation protein kinase C (PKC) or adenylate cyclase all lead to nuclear accumulation of fibroblast growth factor 2 (FGF-2) and FGFR1, association of FGFR1 with splicing factor-rich domains, and activation of the tyrosine hydroxylase (TH) gene promoter in bovine adrenal medullary cells (BAMC). The up-regulation of endogenous TH protein or a transfected TH promoter-luciferase construct by AII, veratridine, or PMA (but not by forskolin) is abolished by transfection with a dominant negative FGFR1TK-mutant which localizes to the nucleus and plasma membrane, but not by extracellularly acting FGFR1 antagonists suramin and inositolhexakisphosphate (IP6). Mechanism of TH gene activation by FGF-2 and FGFR1 was further investigated in BAMC and human TE671 cultures. TH promoter was activated by co-transfected HMW FGF-2 (which is exclusively nuclear) but not by cytoplasmic FGF-1 or extracellular FGFs. Promoter transactivation by HMWFGF-2 was accompanied by an up-regulation of FGFR1 specifically in the cell nucleus and was prevented FGFR1(TK-) but not by IP6 or suramin. The TH promoter was also transactivated by co-transfected wild-type FGFR1, which localizes to both to the nucleus and the plasma membrane, and by an exclusively nuclear, soluble FGFR1(SP-/NLS) mutant with an inserted nuclear localization signal. Activation of the TH promoter by nuclear FGFR1 and FGF-2 was mediated through the cAMP-responsive element (CRE) and was associated with induction of CREB- and CBP/P-300-containing CRE complexes. We propose a new model for gene regulation in which nuclear FGFR1 acts as a mediator of CRE transactivation by AII, cell depolarization, and PKC.

Adrenal Medulla↗

PubMed2001

Nuclear import and DNA-binding activity of RFX1. Evidence for an autoinhibitory mechanism.

RFX1 binds and regulates the enhancers of a number of viruses and cellular genes. RFX1 belongs to the evolutionarily conserved RFX protein family that shares a DNA-binding domain and a conserved C-terminal region. In RFX1 this conserved region mediates dimerization, and is followed by a unique C-terminal tail, containing a highly acidic stretch. In HL-60 cells nuclear translocation of RFX1 is regulated by protein kinase C with unknown mechanisms. By confocal fluorescence microscopy, we have identified a nonclassical nuclear localization signal (NLS) at the extreme C-terminus. The adjacent 'acidic region', which showed no independent NLS activity, potentiated the function of the NLS. Subcellular fractionation showed that the tight association of RFX1 with the nucleus is mediated by its DNA-binding domain and enhanced by the dimerization domain. In contrast, the acidic region inhibited nuclear association, by down-regulating the DNA-binding activity of RFX1. These data suggest an autoinhibitory interaction, which may regulate the function of RFX1 at the level of DNA binding. The C-terminal tail thus constitutes a composite localization domain, which on the one hand mediates nuclear import of RFX1, and on the other hand inhibits its association with the nucleus and binding to DNA. The participation of the acidic region in both activities suggests a mechanism by which the nuclear import and DNA-binding activity of RFX1 may be coordinately regulated by phosphorylation by kinases such as PKC.

Animals↗

PubMed1999

The dormancy-related peroxiredoxin anti-oxidant, PER1, is localized to the nucleus of barley embryo and aleurone cells.

Protection against desiccation-induced injury, including damage by reactive oxygen species (ROS), is a necessary component of the genetic programmes active during late seed development. Likewise, protection against ROS respiration by-products is required during seed imbibition and germination. Late embryogenesis abundant (LEA) proteins are proposed to protect seed tissues against desiccation-induced damage. Specifically, the atypical Lea gene Per1 in barley (Hordeum vulgare L.) has been proposed to play a protective role in embryo and aleurone cells against free-radical damage during late seed development and early imbibition. PER1 represents a subgroup of the peroxiredoxin family of thiol-requiring anti-oxidants with one conserved cysteine residue (1-Cys), and displays in vitro anti-oxidant activity. In this work, we use antiserum generated against PER1 to study protein accumulation patterns as well as localization at the tissue, cellular and subcellular level. While previous studies have shown the Per1 transcript to be dormancy-related, we show here that the protein level is maintained in imbibed dormant seeds, but not in non-dormant seeds. Our data identify the location of this seed-specific peroxiredoxin as the nucleus of immature embryos and aleurone layers. Highest levels of protein are detected in nucleoli. In contrast, in mature imbibed dormant seeds, cytosolic levels are comparable to that of the nucleus. A putative nuclear localization signal (NLS) of bipartite nature was identified in the C-terminal end of the PER1 sequence. Protective roles for PER1 in seeds are discussed.

Amino Acid Sequence↗

PubMed2006

SorLA signaling by regulated intramembrane proteolysis.

The single-transmembrane receptor SorLA/LR11 contains binding domains typical for lipoprotein receptors and a VPS10 domain, which binds the neuropeptide head-activator. This undecapeptide enhances proliferation of neuronal precursor cells in a SorLA-dependent manner. Using specific inhibitors we found previously that head activator activates shedding of SorLA by the metalloprotease TACE close to the transmembrane domain releasing the large extra-cellular part of the receptor. Here we show that the remaining COOH-terminal membrane fragment of SorLA is processed by gamma-secretase. Inhibition of gamma-secretase by specific inhibitors or overexpression of dominant negative presenilin mutants and knock out of the presenilin genes led to accumulation of the SorLA membrane fragment and also of full-length SorLA in the membrane. In an in vitro assay we observed the gamma-secretase-dependent release of the two soluble cleavage products, the SorLA cytoplasmic domain and the SorLA beta-peptide. These processing steps are reminiscent of a novel signaling pathway that has been described for the notch receptor. Here, the notch cytoplasmic domain is released into the cytoplasm by the gamma-secretase and migrates to the nucleus where it acts as a transcriptional regulator. In parallel we found that a fusion protein of the released cytoplasmic tail of SorLA with EGFP located to the nucleus only if the nuclear localization signal of SorLA was intact. In a reporter gene assay the cytoplasmic domain of SorLA acted as a transcriptional activator indicating that SorLA might directly regulate transcription after activation by gamma-secretase.

Amino Acid Sequence↗

PubMed2000

Prothymosin alpha fragmentation in apoptosis.

We observed fragmentation of an essential proliferation-related human nuclear protein prothymosin alpha in the course of apoptosis induced by various stimuli. Prothymosin alpha cleavage occurred at the DDVD(99) motif. In vitro, prothymosin alpha could be cleaved at D(99) by caspase-3 and -7. Caspase hydrolysis disrupted the nuclear localization signal of prothymosin alpha and abrogated the ability of the truncated protein to accumulate inside the nucleus. Prothymosin alpha fragmentation may therefore be proposed to disable intranuclear proliferation-related function of prothymosin alpha in two ways: by cleaving off a short peptide containing important determinants, and by preventing active nuclear uptake of the truncated protein.

Apoptosis↗

PubMed2004

Analysis of DNA ligase IV mutations found in LIG4 syndrome patients: the impact of two linked polymorphisms.

LIG4 syndrome patients have hypomorphic mutations in DNA ligase IV. Although four of the five identified patients display immunodeficiency and developmental delay, one patient was developmentally normal. The developmentally normal patient had the same homozygous mutation (R278H) in DNA ligase IV as one of the more severely affected patients, who additionally had two linked polymorphisms. Here, we examine the impact of the mutations and polymorphisms identified in the LIG4 syndrome patients. Examination of recombinant mutant proteins shows that the severity of the clinical features correlates with the level of residual ligase activity. The polymorphisms decrease the activity of DNA ligase IV by approximately 2-fold. When combined with the otherwise mild R278H mutation, the activity is reduced to a level similar to other LIG4 patients who display immunodeficiency and developmental delay. This demonstrates how coupling of a mutation and polymorphism can have a marked impact on protein function and provides an example where a polymorphism may have influenced clinical outcome. Analysis of additional mutational changes in LIG4 syndrome (R580X, R814X and G469E) have led to the identification of a nuclear localization signal in DNA ligase IV and sites impacting upon DNA ligase IV adenylation.

Animals↗

PubMed2004

Subcellular localization of RPB5-mediating protein and its putative functional partner.

We previously identified a novel cellular protein, RPB5-mediating protein (RMP), that retains corepressor activity and functionally antagonizes transcriptional modulation via hepatitis B virus X protein. The subcellular localization of RMP was examined using green fluorescent protein-fused protein forms. We found that a nuclear localization signal (NLS) and a coiled-coil (CC) domain functioning as a cytoplasmic localization signal (CLS) are important for the subcellular localization of RMP. The CLS apparently acts dominantly, since RMP was mostly localized in the cytoplasm with weak and diffuse signals in the nucleus, and the NLS was indispensable for the nuclear localization of RMP only in the absence of the CLS. Using a yeast two-hybrid method, we isolated a putative corepressor, DNA methyltransferase 1-associating protein (DMAP1), which was found to bind to the CC domain of RMP. DMAP1 facilitated the nuclear localization of RMP and the corepressor activity of RMP in a dose-dependent manner by interacting with the CC domain of RMP. These results are discussed in light of a recent paper showing a novel evolutionarily conserved role of URI in the TOR signaling pathway.

Animals↗

PubMed2006

The capsid protein of beak and feather disease virus binds to the viral DNA and is responsible for transporting the replication-associated protein into the nucleus.

Circoviruses lack an autonomous DNA polymerase and are dependent on the replication machinery of the host cell for de novo DNA synthesis. Accordingly, the viral DNA needs to cross both the plasma membrane and the nuclear envelope before replication can occur. Here we report on the subcellular distribution of the beak and feather disease virus (BFDV) capsid protein (CP) and replication-associated protein (Rep) expressed via recombinant baculoviruses in an insect cell system and test the hypothesis that the CP is responsible for transporting the viral genome, as well as Rep, across the nuclear envelope. The intracellular localization of the BFDV CP was found to be directed by three partially overlapping bipartite nuclear localization signals (NLSs) situated between residues 16 and 56 at the N terminus of the protein. Moreover, a DNA binding region was also mapped to the N terminus of the protein and falls within the region containing the three putative NLSs. The ability of CP to bind DNA, coupled with the karyophilic nature of this protein, strongly suggests that it may be responsible for nuclear targeting of the viral genome. Interestingly, whereas Rep expressed on its own in insect cells is restricted to the cytoplasm, coexpression with CP alters the subcellular localization of Rep to the nucleus, strongly suggesting that an interaction with CP facilitates movement of Rep into the nucleus.

Active Transport, Cell Nucleus↗

PubMed1997

The Arabidopsis GAI gene defines a signaling pathway that negatively regulates gibberellin responses.

The Arabidopsis gai mutant allele confers a reduction in gibberellin (GA) responsiveness. Here we report the molecular cloning of GAI and a closely related gene GRS. The predicted GAI (wild-type) and gai (mutant) proteins differ only by the deletion of a 17-amino-acid segment from within the amino-terminal region. GAI and GRS contain nuclear localization signals, a region of homology to a putative transcription factor, and motifs characteristic of transcriptional coactivators. Genetic analysis indicates that GAI is a repressor of GA responses, that GA can release this repression, and that gai is a mutant repressor that is relatively resistant to the effects of GA. Mutations at SPY and GAR2 suppress the gai phenotype, indicating the involvement of GAI, SPY, and GAR2 in a signaling pathway that regulates GA responses negatively. The existence of this pathway suggests that GA modulates plant growth through derepression rather than through simple stimulation.

Alleles↗

PubMed1999

Identification of amino acid sequence in the hinge region of human vitamin D receptor that transfers a cytosolic protein to the nucleus.

The localization of human vitamin D receptor (VDR) in the absence of its ligand 1,25-dihydroxyvitamin D(3) was investigated using chimera proteins fused to green fluorescent protein (GFP) at either the N or C terminus, and the nuclear localization signal (NLS) was identified. Plasmids carrying the fusion proteins were transiently or stably introduced into COS7 cells, and the subcellular distribution of the fusion proteins was examined. GFP-tagged wild-type VDRs were located predominantly in nuclei but with a significant cytoplasmic presence, while GFP alone was equally distributed throughout the cells. 10(-8) M 1,25-dihydroxyvitamin D(3) promoted the nuclear import of VDR in a few hours. To identify the NLS, we constructed several mutated VDRs fused to GFP. Mutant VDRs that did not bind to DNA were also localized predominantly in nuclei, while the deletion of the hinge region resulted in the loss of preference for nucleus. A short segment of 20 amino acids in the hinge region enabled cytoplasmic GFP-tagged alkaline phosphatase to translocate to nuclei. These results indicate that 1) VDR is located predominantly in nuclei with a significant presence in cytoplasm without the ligand and 2) an NLS consisting of 20 amino acids in the hinge region facilitates the transfer of VDR to the nucleus.

Amino Acid Sequence↗

PubMed2003

Karyopherin-alpha2 protein interacts with Chk2 and contributes to its nuclear import.

Chk2 is a nuclear protein kinase involved in the DNA damage-induced ataxia telangiectasia mutated-dependent checkpoint arrest at multiple cell cycle phases. Searching for Chk2-binding proteins by a yeast two-hybrid system, we identified a strong interaction with karyopherin-alpha2 (KPNA-2), a gene product involved in active nuclear import of proteins bearing a nuclear localization signal (NLS). This finding was confirmed by glutathione S-transferase pull-down and co-immunoprecipitation assays. Of the three predicted Chk2 NLSs, located at amino acids 179-182 (NLS-1), 240-256 (NLS-2), and 515-522 (NLS-3), only the latter mediated the interaction with KPNA-2 in the yeast two-hybrid system, and in particular with its C terminus. Unlike mutations in NLS-1 or NLS-2, which left the nuclear localization of Chk2 unaffected, mutations in NLS-3 caused a cytoplasmic relocalization, indicating that the NLS-3 motif acts indeed as NLS for Chk2 in vivo. Finally, co-transfection experiments with green fluorescent protein (GFP)-Chk2 and wild type or mutant KPNA-2 confirmed the role of KPNA-2 in nuclear import of Chk2.

Active Transport, Cell Nucleus↗

PubMed1997

Molecular cloning and cell cycle-dependent expression of mammalian CRM1, a protein involved in nuclear export of proteins.

Crm1 of Schizosaccharomyces pombe, a nuclear protein essential for proliferation and chromosome region maintenance, is a possible target of leptomycin B, an antifungal and antitumor antibiotic with cell cycle-arresting activity. cDNA encoding a human homolog of Crm1 was cloned. Human CRM1 (hCRM1) consisted of 1071 amino acids, of which the sequence showed 52% homology with S. pombe Crm1. hCRM1 weakly complemented the cold-sensitive mutation of S. pombe crm1-809, as did S. pombe crm1+. Overproduction of hCRM1 under the control of a series of nmt1 promoters suppressed cell proliferation in wild-type S. pombe in an expression level-dependent manner. A similar inhibitory effect was also observed for crm1+. Cells overproducing either hCRM1 or S. pombe Crm1 were distinctly larger than uninduced cells and contained compacted and fragmented nuclei. Furthermore, calcofluor staining demonstrated that most of these cells formed two septa per cell and accumulated a large amount of chitin or its related polysaccharides around the septa. Closely similar phenotypes between hCRM1- and S. pombe Crm1-induced cells indicate that the cloned cDNA encodes a functional homolog of S. pombe crm1+. Northern blot analyses with RNAs isolated from synchronized mammalian cells showed that the expression of mammalian CRM1 was initiated in late G1 and reached a peak at G2/M, although its protein level unchanged during the cell cycle. Transient expression of hCRM1 fused to the green fluorescent protein (GFP) in NIH3T3 cells showed that hCRM1 was localized preferentially in the nuclear envelope and was also detectable in the nucleoplasm and the cytoplasm. A crm1 mutation of S. pombe caused nuclear import of a GFP fusion protein containing a nuclear export signal but no change in the distribution of a GFP fusion protein containing a nuclear localization signal. All of these data suggest that CRM1 is a novel cell-cycle regulated gene that is essential for the nuclear export signal-dependent nuclear export of proteins.

3T3 Cells↗

PubMed1999

Membrane-tethered Drosophila Armadillo cannot transduce Wingless signal on its own.

Drosophila Armadillo and its vertebrate homolog beta-catenin are key effectors of Wingless/Wnt signaling. In the current model, Wingless/Wnt signal stabilizes Armadillo/beta-catenin, which then accumulates in nuclei and binds TCF/LEF family proteins, forming bipartite transcription factors which activate transcription of Wingless/Wnt responsive genes. This model was recently challenged. Overexpression in Xenopus of membrane-tethered beta-catenin or its paralog plakoglobin activates Wnt signaling, suggesting that nuclear localization of Armadillo/beta-catenin is not essential for signaling. Tethered plakoglobin or beta-catenin might signal on their own or might act indirectly by elevating levels of endogenous beta-catenin. We tested these hypotheses in Drosophila by removing endogenous Armadillo. We generated a series of mutant Armadillo proteins with altered intracellular localizations, and expressed these in wild-type and armadillo mutant backgrounds. We found that membrane-tethered Armadillo cannot signal on its own; however it can function in adherens junctions. We also created mutant forms of Armadillo carrying heterologous nuclear localization or nuclear export signals. Although these signals alter the subcellular localization of Arm when overexpressed in Xenopus, in Drosophila they have little effect on localization and only subtle effects on signaling. This supports a model in which Armadillo's nuclear localization is key for signaling, but in which Armadillo intracellular localization is controlled by the availability and affinity of its binding partners.

Animals↗

PubMed1998

Distinct domains of IkappaBalpha regulate c-Rel in the cytoplasm and in the nucleus.

IkappaBalpha is a critical regulator of Rel/NF-KB-mediated gene activation. It controls the induction of NF-KB factors by retaining them in the cytoplasm and also functions in the nucleus to terminate the induction process. In this study, we show that IkappaBalpha regulates the transcriptional activity of c-Rel in the nuclear compartment. We also demonstrate that discrete functional domains of IkappaBalpha are responsible for the cytoplasmic and nuclear regulation of c-Rel. We show that the determinants for the cytoplasmic regulation of c-Rel reside in the N-terminal and central ankyrin regions of IkappaBalpha and that the N-terminal domain of IkappaBalpha is required to mask the c-Rel nuclear localization signal. Importantly, IkappaBalpha sequences necessary to regulate c-Rel in the nucleus map to its central ankyrin domain and to a few negatively charged amino acids that immediately follow in the C-terminal IkappaBalpha PEST domain. The mapping of the IkappaBalpha determinants that control the cytoplasmic and nuclear activities of c-Rel to specific regions of the molecule suggests that IkappaBalpha inhibitors could be designed to antagonize Rel/NF-kappaB activity in different subcellular compartments or at defined stages of activation.

Animals↗

PubMed2002

Nuclear import of factors involved in signaling is inhibited in C3H/10T1/2 cells treated with tetradecylthioacetic acid.

The non-beta-oxidisable tetradecylthioacetic acid (TTA) is incorporated into cellular membranes when C3H/10T1/2 cells are cultured in TTA-containing medium. We here demonstrate that this alteration in cellular membranes affect the nuclear translocation of proteins involved in signal transduction. Analysis of cellular fatty acid composition shows that TTA and TTA:1n-8 constitute approximately 40 mol% of total fatty acids in cellular/nuclear membranes. Activation of c-fos expression is significantly inhibited in TTA-treated cells but the enzymatic activation of mitogen activated protein kinase (ERK) is not affected. Immunofluorescence and confocal microscopy studies demonstrate that in mitogene-stimulated TTA-treated cells, the translocation of phosphorylated ERK1/2, protein kinase C alpha (PKC alpha), and PKC beta(1) from the cytoplasm into the nucleus is considerably decreased and delayed. Concomitant with a decreased nuclear import, ERK1/2 dephosphorylation is decreased in TTA-treated cells. There is no TTA-induced inhibition of nuclear import of proteins with a classical nuclear localization signal (NLS), as seen by in vitro nuclear import experiments of BSA fused to the NLS from SV40 large T, or in vivo studies of hnRNP A1 nuclear import. The expression levels of Importin alpha, Importin beta, Importin 7, and NTF2 are not altered in the TTA-treated cells. Taken together, our data indicate that TTA treatment causes changes in cellular fatty acid composition that negatively affect NLS-independent mechanisms of protein translocation through the nuclear pore complex.

Active Transport, Cell Nucleus↗

PubMed2001

Apoptosis induced by human Fas-associated factor 1, hFAF1, requires its ubiquitin homologous domain, but not the Fas-binding domain.

FAF1 is a Fas-binding protein without typical death domain. Instead, FAF1 has several domains found in proteins of ubiquitination pathway. Transient overexpression of hFAF1 in BOSC23 cells caused membrane blebbing and cell body condensation which were characteristics of apoptosis. Subsequent analysis revealed that overexpression of hFAF1 induced nuclear condensation, appearance of phosphatidylserines in the outer leaflet of the cellular membrane, and caspase 3 activation. The apoptotic potential of hFAF1 required downstream ubiquitin homologous domain (UB2) and adjacent nuclear localization signal but not the Fas-binding domain. Our data showed that mere intrinsic overexpression of hFAF1 initiated apoptosis in the absence of any extrinsic death signal in BOSC23 cells.

Active Transport, Cell Nucleus↗

PubMed2006

Characterization of calcineurin-dependent response element binding protein and its involvement in copper-metallothionein gene expression in Neurospora.

In continuation of our recent observations indicating the presence of a lone calcineurin-dependent response element (CDRE) in the -3730bp upstream region of copper-induced metallothionein (CuMT) gene of Neurospora [K.S. Kumar, S. Dayananda, C. Subramanyam, Copper alone, but not oxidative stress, induces copper-metallothionein gene in Neurospora crassa, FEMS Microbiol. Lett. 242 (2005) 45-50], we isolated and characterized the CDRE-binding protein. The cloned upstream region of CuMT gene was used as the template to specifically amplify CDRE element, which was immobilized on CNBr-activated Sepharose 4B for use as the affinity matrix to purify the CDRE binding protein from nuclear extracts obtained from Neurospora cultures grown in presence of copper. Two-dimensional gel electrophoresis of the affinity purified protein revealed the presence of a single 17kDa protein, which was identified and characterized by MALDI-TOF. Peptide mass finger printing of tryptic digests and analysis of the 17kDa protein matched with the regulatory beta-subunit of calcineurin (Ca(2+)-calmodulin dependent protein phosphatase). Parallel identification of nuclear localization signals in this protein by in silico analysis suggests a putative role for calcineurin in the regulation of CuMT gene expression.

Amino Acid Sequence↗

PubMed2004

Analysis of multiple Invs transcripts in mouse and MDCK cells.

Infantile nephronophthisis is associated with cystic kidneys, situs inversus, and INVS mutations. The function of the INVS product, inversin, is unknown but evidence suggests there are multiple inversin isoforms with differing molecular weights, cellular localization patterns, and binding partners. We used Northern blots, RT-PCR, and sequence analysis to identify alternative INVS transcripts. Northern blots probed with Invs cDNA detected four bands in normal mouse kidney. RT-PCR of mouse kidney RNA revealed Invs transcripts with skipping of exon 5, 11, or 13. We sequenced canine (MDCK-II cells) INVS and determined that the corresponding full-length protein shares identity with mouse (74%) and human (84%) inversin. Canine INVS produces a transcript that skips exon 12. Exon skips cause loss of inversin protein motifs, including ankyrin repeats, IQ domains, destruction boxes, and nuclear localization signals. Identification of INVS splice variants will help us determine which inversin protein motifs contribute to left-right asymmetry and kidney development.

Alternative Splicing↗