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Results for “Nuclear Localization Signals”

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At least 721 records · Page 40Linked to original sources

PubMed2000

Subcellular localization of prostaglandin H synthase-2 in a human amnion cell line: implications for nuclear localized prostaglandin signaling pathways.

We have determined that prostaglandin H synthase-2 localises strongly to the nuclear membrane as well as being found in the endoplasmic reticulum in human amnion-derived WISH cells which have been stimulated with interleukin 1beta and phorbol ester. This is consistent with findings in cells of non-reproductive origin. There is strong evidence that prostaglandin J2 derivatives, which in other tissues exhibit tumour suppressing, antiproliferative and/or differentiation promoting activities, act through binding of intracellular receptors which then enter the nucleus. In addition, some arachidonic acid derivatives are clearly generated by enzymes at the nuclear envelope and localise to sites in nuclei or bind sites in nuclei. The WISH cell line will make an excellent system for studying these perinuclear intracellular prostanoid signaling mechanisms.

Amnion↗

PubMed1998

Signal transduction in hypoxic cells: inducible nuclear translocation and recruitment of the CBP/p300 coactivator by the hypoxia-inducible factor-1alpha.

In response to decreased cellular oxygen concentrations the basic helix-loop-helix (bHLH)/PAS (Per, Arnt, Sim) hypoxia-inducible transcription factor, HIF-1alpha, mediates activation of networks of target genes involved in angiogenesis, erythropoiesis and glycolysis. Here we demonstrate that the mechanism of activation of HIF-1alpha is a multi-step process which includes hypoxia-dependent nuclear import and activation (derepression) of the transactivation domain, resulting in recruitment of the CREB-binding protein (CBP)/p300 coactivator. Inducible nuclear accumulation was shown to be dependent on a nuclear localization signal (NLS) within the C-terminal end of HIF-1alpha which also harbors the hypoxia-inducible transactivation domain. Nuclear import of HIF-1alpha was inhibited by either deletion or a single amino acid substitution within the NLS sequence motif and, within the context of the full-length protein, these mutations also resulted in inhibition of the transactivation activity of HIF-1alpha and recruitment of CBP. However, nuclear localization per se was not sufficient for transcriptional activation, since fusion of HIF-1alpha to the heterologous GAL4 DNA-binding domain generated a protein which showed constitutive nuclear localization but required hypoxic stimuli for function as a CBP-dependent transcription factor. Thus, hypoxia-inducible nuclear import and transactivation by recruitment of CBP can be functionally separated from one another and play critical roles in signal transduction by HIF-1alpha.

Amino Acid Substitution↗

PubMed2005

Endosomal transport of ErbB-2: mechanism for nuclear entry of the cell surface receptor.

The cell membrane receptor ErbB-2 migrates to the nucleus. However, the mechanism of its nuclear translocation is unclear. Here, we report a novel mechanism of its nuclear localization that involves interaction with the transport receptor importin beta1, nuclear pore protein Nup358, and a host of players in endocytic internalization. Knocking down importin beta1 using small interfering RNA oligonucleotides or inactivation of small GTPase Ran by RanQ69L, a dominant-negative mutant of Ran, causes a nuclear transport defect of ErbB-2. Mutation of a putative nuclear localization signal in ErbB-2 destroys its interaction with importin beta1 and arrests nuclear translocation, while inactivation of nuclear export receptor piles up ErbB-2 within the nucleus. Additionally, blocking of internalization by a dominant-negative mutant of dynamin halts its nuclear localization. Thus, the cell membrane-embedded ErbB-2, through endocytosis using the endocytic vesicle as a vehicle, importin beta1 as a driver and Nup358 as a traffic light, migrates from the cell surface to the nucleus. This novel mechanism explains how a receptor tyrosine kinase on the cell surface can be translocated into the nucleus. This pathway may serve as a general mechanism to allow direct communication between cell surface receptors and the nucleus, and our findings thus open a new era in understanding direct trafficking between the cell membrane and nucleus.

Active Transport, Cell Nucleus↗

PubMed2000

Genetic alterations disrupting the nuclear localization of the retinoblastoma-related gene RB2/p130 in human tumor cell lines and primary tumors.

The prototypic tumor suppressor gene, the retinoblastoma gene (RB/ p105), is mutated in a variety of human tumors. However, to date, mutational data on retinoblastoma family members p107 and RB2/p130 in tumors is lacking. We studied the expression of pRb2/p130 by immunocytochemistry and Western blot analysis in a panel of human osteosarcoma and lymphoid cell lines. Only the lymphoid cell lines showed an abnormal cytoplasmic localization of pRb2/p130, suggesting possible alterations within the region of nuclear localization signaling. We screened these cell lines for genetic alterations of the RB2/p130 gene in the region of the putative bipartite nuclear localization signal (NLS). This region is highly homologous with that of the RB/p105 gene. In addition, we screened four primary Burkitt's lymphomas for genetic alterations in the RB2/p130 gene. Naturally occurring mutations, which disrupt the putative bipartite NLS, were found in lymphoma cell lines and primary tumors, but not in the osteosarcoma cell lines, where normal nuclear localization of the protein was detectable. Site-directed mutagenesis and transfection assay using NLS mutants displayed markedly reduced biological activity as measured by flow cytometric analysis. This study clearly describes RB2/ p130 as an important target for mutations and subsequent inactivation in lymphoma pathogenesis, thus validating that RB2/p130 is a classical tumor suppressor gene.

Amino Acid Substitution↗

PubMed1998

Determination of the functional domain organization of the importin alpha nuclear import factor.

Although importin alpha (Imp alpha) has been shown to act as the receptor for basic nuclear localization signals (NLSs) and to mediate their recruitment to the importin beta nuclear import factor, little is known about the functional domains present in Imp alpha, with the exception that importin beta binding is known to map close to the Imp alpha NH2 terminus. Here, we demonstrate that sequences essential for binding to the CAS nuclear export factor are located near the Imp alpha COOH terminus and include a critical acidic motif. Although point mutations introduced into this acidic motif inactivated both CAS binding and Imp alpha nuclear export, a putative leucine-rich nuclear export signal proved to be neither necessary nor sufficient for Imp alpha nuclear export. Analysis of sequences within Imp alpha that bind to the SV-40 T antigen NLS or to the similar LEF-1 NLS revealed that both NLSs interact with a subset of the eight degenerate armadillo (Arm) repeats that form the central part of Imp alpha. However, these two NLS-binding sites showed only minimal overlap, thus suggesting that the degeneracy of the Arm repeat region of Imp alpha may serve to facilitate binding to similar but nonidentical basic NLSs. Importantly, the SV-40 T NLS proved able to specifically inhibit the interaction of Imp alpha with CAS in vitro, thus explaining why the SV-40 T NLS is unable to also function as a nuclear export signal.

Amino Acid Sequence↗

PubMed2003

Karyopherin-mediated nuclear import of the homing endonuclease VMA1-derived endonuclease is required for self-propagation of the coding region.

VMA1-derived endonuclease (VDE), a site-specific endonuclease in Saccharomyces cerevisiae, enters the nucleus to generate a double-strand break in the VDE-negative allelic locus, mediating the self-propagating gene conversion called homing. Although VDE is excluded from the nucleus in mitotic cells, it relocalizes at premeiosis, becoming localized in both the nucleus and the cytoplasm in meiosis. The nuclear localization of VDE is induced by inactivation of TOR kinases, which constitute central regulators of cell differentiation in S. cerevisiae, and by nutrient depletion. A functional genomic approach revealed that at least two karyopherins, Srp1p and Kap142p, are required for the nuclear localization pattern. Genetic and physical interactions between Srp1p and VDE imply direct involvement of karyopherin-mediated nuclear transport in this process. Inactivation of TOR signaling or acquisition of an extra nuclear localization signal in the VDE coding region leads to artificial nuclear localization of VDE and thereby induces homing even during mitosis. These results serve as evidence that VDE utilizes the host systems of nutrient signal transduction and nucleocytoplasmic transport to ensure the propagation of its coding region.

Active Transport, Cell Nucleus↗

PubMed2005

Identification and characterization of two novel splicing isoforms of human estrogen-related receptor beta.

CONTEXT: Estrogen-related receptor beta (ERRbeta) was one of the first two orphan nuclear receptors reported and is believed to play important roles in estrogen-regulated pathways. Embryo lethality of ERRbeta-null mice indicated that ERRbeta is essential for embryo development. OBJECTIVE: Two novel splicing isoforms of human (h) ERRbeta, hERRbeta2-Delta10 and short-form hERRbeta, were identified during the cloning of previously reported hERRbeta-hERRbeta2. We aim to investigate the functional differences of these three human ERRbeta-splicing isoforms. RESULTS AND CONCLUSIONS: A genomic sequence comparison within and flanking the ERRbeta genes of eight species demonstrated that short-form hERRbeta lacks an F domain and is the matched homolog of mouse and rat ERRbeta proteins in humans. However, hERRbeta2-Delta10 and the previously reported hERRbeta2 isoforms are primate specific. RT-PCR analysis showed that short-form hERRbeta has a wide distribution in the 24 of 27 human tissues and cell lines tested, whereas hERRbeta2 and hERRbeta2-Delta10 were only expressed in testis and kidney. The three human ERRbeta-splicing isoforms have different transcriptional activities when measured on an estrogen response element-driven luciferase reporter in transfection assays. The localization of a nuclear localization signal of short-form hERRbeta was also determined. Interestingly, the F domain of hERRbeta2 alters the function of the nuclear localization signal. Therefore, the ERRbeta isoforms are likely to have diverse biological functions in vivo, and characterizing the three isoforms of ERRbeta will lead to an understanding of the multiple levels of gene regulation involved in steroid receptor-signaling pathways in humans and may provide novel therapeutic targets for human diseases.

Alternative Splicing↗

PubMed2002

Identification of two nuclear import signals in the alpha-gene product ICP22 of herpes simplex virus 1.

The herpes simplex virus 1 (HSV-1) infected cell protein 22 (ICP22) is a multifunctional viral regulator that localizes in the nucleus of infected cells. ICP22 is required for optimal virus replication in certain cell types and is subject to extensive posttranslational modification. To map the signals in ICP22 which mediate its efficient nuclear localization, we investigated the nuclear import of fusion proteins comprising various fragments of ICP22 fused to green fluorescent protein (GFP) or beta-galactosidase (beta-Gal). These data demonstrated that ICP22 contains two independent regions with nuclear localization signal (NLS) activity. NLS1 maps to ICP22 amino acid position 16-31 and closely resembles the classical bipartite NLS of the type originally identified in nucleoplasmin. In contrast, NLS2 maps to ICP22 amino acid position 118-131 and contains multiple critical basic residues. Furthermore, fusion of both NLSs to chimeric glutathione-S-transferase (GST)-GFP protein and subsequent cytoplasmic microinjection of the respective transport substrates allowed us to monitor nuclear import in real-time. These data demonstrated that both ICP22-derived NLSs mediated efficient nuclear import with identical kinetics, resulting in complete nuclear accumulation of the chimeric transport cargoes at approximately 30 min postinjection. Finally, our data provide new insights into the domain structure of the multifunctional alpha-gene product ICP22 of HSV-1.

Active Transport, Cell Nucleus↗

PubMed2005

The cell cycle independence of HIV infections is not determined by known karyophilic viral elements.

Human immunodeficiency virus and other lentiviruses infect cells independent of cell cycle progression, but gammaretroviruses, such as the murine leukemia virus (MLV) require passage of cells through mitosis. This property is thought to be important for the ability of HIV to infect resting CD4+ T cells and terminally differentiated macrophages. Multiple and independent redundant nuclear localization signals encoded by HIV have been hypothesized to facilitate migration of viral genomes into the nucleus. The integrase (IN) protein of HIV is one of the HIV elements that targets to the nucleus; however, its role in nuclear entry of virus genomes has been difficult to describe because mutations in IN are pleiotropic. To investigate the importance of the HIV IN protein for infection of non-dividing cells, and to investigate whether or not IN was redundant with other viral signals for cell cycle-independent nuclear entry, we constructed an HIV-based chimeric virus in which the entire IN protein of HIV was replaced by that of MLV. This chimeric virus with a heterologous IN was infectious at a low level, and was able to integrate in an IN-dependent manner. Furthermore, this virus infected non-dividing cells as well as it infected dividing cells. Moreover, we used the chimeric HIV with MLV IN to further eliminate all of the other described nuclear localization signals from an HIV genome--matrix, IN, Viral Protein R, and the central polypurine tract--and show that no combination of the virally encoded NLS is essential for the ability of HIV to infect non-dividing cells.

CD4-Positive T-Lymphocytes↗

PubMed1997

Mediators of nuclear protein import target karyophilic proteins to pore complexes of cytoplasmic annulate lamellae.

Nuclear pore complexes are constitutive structures of the nuclear envelope in eukaryotic cells and represent the sites where transport of molecules between nucleus and cytoplasm takes place. However, pore complexes of similar structure, but with largely unknown functional properties, are long known to occur also in certain cytoplasmic cisternae that have been termed annulate lamellae (AL). To analyze the capability of the AL pore complex to interact with the soluble mediators of nuclear protein import and their karyophilic protein substrates, we have performed a microinjection study in stage VI oocytes of Xenopus laevis. In these cells AL are especially abundant and can easily be identified by light and electron microscopy. Following injection into the cytoplasm, fluorochrome-labeled mediators of two different nuclear import pathways, importin beta and transportin, not only associate with the nuclear envelope but also with AL. Likewise, nuclear localization signals (NLS) of the basic and M9 type, but not nuclear export signals, confer targeting and transient binding of fluorochrome-labeled proteins to cytoplasmic AL. Mutation or deletion of the NLS signals prevents these interactions. Furthermore, binding to AL is abolished by dominant negative inhibitors of nuclear protein import. Microinjections of gold-coupled NLS-bearing proteins reveal specific gold decoration at distinct sites within the AL pore complex. These include such at the peripheral pore complex-attached fibrils and at the central "transporter" and closely resemble those of "transport intermediates" found in electron microscopic studies of the nuclear pore complex (NPC). These data demonstrate that AL can represent distinct sites within the cytoplasm of transient accumulation of nuclear proteins and that the AL pore complex shares functional binding properties with the NPC.

Animals↗

PubMed1998

Cse1p is required for export of Srp1p/importin-alpha from the nucleus in Saccharomyces cerevisiae.

In metazoan cells, the CAS protein has been shown to function as a recycling factor for the importin-alpha subunit of the classical nuclear localization signal receptor, exporting importin-alpha from the nucleus to allow its participation in multiple rounds of nuclear import. CAS is a member of a family of proteins that bear homology to the larger subunit of the nuclear localization signal receptor, importin-beta, and that are found in all eukaryotes from yeast to humans. Sequence similarity identifies the product of the Saccharomyces cerevisiae CSE1 gene as a potential CAS homologue. Here we present evidence that Cse1p is the functional homologue of CAS: Cse1p is required to prevent accumulation of Srp1p/importin-alpha in the nucleus, it localizes to the nuclear envelope in a pattern typical of nuclear transport receptors, and it associates in vivo with Srp1p in a nucleotide-specific manner. We show further that mutations in CSE1 and SRP1 have specific effects on their association and on the intracellular localization of Cse1p.

Biological Transport↗

PubMed1994

Nuclear import of Agrobacterium VirD2 and VirE2 proteins in maize and tobacco.

Previously, we have shown that Agrobacterium-plant cell transferred DNA (T-DNA) transport into the host cell nucleus is likely mediated by two specific bacterial proteins, VirD2 and VirE2. Here, we used these proteins to study molecular pathways of nuclear import. First, the role of VirE2 nuclear localization signals (NLSs) in the T-DNA transport pathway was examined by using tobacco plants transgenic for deletion mutants of VirE2. In these plants, the virulence of wild-type Agrobacterium was reduced possibly by competition for the cellular nuclear import machinery. Second, we analyzed the nuclear localization of VirE2 and VirD2 in the nonhost monocot maize. Part of the known recalcitrance of monocots to transformation by Agrobacterium could be due to a potential selectivity in nuclear import pathways in monocotyledonous and dicotyledonous plants. Nuclear transport of VirD2 and VirE2 in maize leaves and roots was compared to that in tobacco protoplasts and roots. Both proteins accumulated in maize leaf and tobacco protoplast nuclei as well as in nuclei of immature root cells. In contrast, VirD2 and VirE2 expressed in mature roots of maize and tobacco remained cytoplasmic. Point mutations of VirE2 nuclear localization signals, NSE 1 and NSE 2, also revealed that, in maize, the NSE 1 signal was mainly responsible for nuclear import; in contrast, both signals functioned independently in tobacco protoplasts.

Amino Acid Sequence↗

PubMed2005

Defective calmodulin-mediated nuclear transport of the sex-determining region of the Y chromosome (SRY) in XY sex reversal.

The sex-determining region of the Y chromosome (SRY) plays a key role in human sex determination, as mutations in SRY can cause XY sex reversal. Although some SRY missense mutations affect DNA binding and bending activities, it is unclear how others contribute to disease. The high mobility group domain of SRY has two nuclear localization signals (NLS). Sex-reversing mutations in the NLSs affect nuclear import in some patients, associated with defective importin-beta binding to the C-terminal NLS (c-NLS), whereas in others, importin-beta recognition is normal, suggesting the existence of an importin-beta-independent nuclear import pathway. The SRY N-terminal NLS (n-NLS) binds calmodulin (CaM) in vitro, and here we show that this protein interaction is reduced in vivo by calmidazolium, a CaM antagonist. In calmidazolium-treated cells, the dramatic reduction in nuclear entry of SRY and an SRY-c-NLS mutant was not observed for two SRY-n-NLS mutants. Fluorescence spectroscopy studies reveal an unusual conformation of SRY.CaM complexes formed by the two n-NLS mutants. Thus, CaM may be involved directly in SRY nuclear import during gonadal development, and disruption of SRY.CaM recognition could underlie XY sex reversal. Given that the CaM-binding region of SRY is well-conserved among high mobility group box proteins, CaM-dependent nuclear import may underlie additional disease states.

Active Transport, Cell Nucleus↗

PubMed2004

The MB2 gene family of Plasmodium species has a unique combination of S1 and GTP-binding domains.

BACKGROUND: Identification and characterization of novel Plasmodium gene families is necessary for developing new anti-malarial therapeutics. The products of the Plasmodium falciparum gene, MB2, were shown previously to have a stage-specific pattern of subcellular localization and proteolytic processing. RESULTS: Genes homologous to MB2 were identified in five additional parasite species, P. knowlesi, P. gallinaceum, P. berghei, P. yoelii, and P. chabaudi. Sequence comparisons among the MB2 gene products reveal amino acid conservation of structural features, including putative S1 and GTP-binding domains, and putative signal peptides and nuclear localization signals. CONCLUSIONS: The combination of domains is unique to this gene family and indicates that MB2 genes comprise a novel family and therefore may be a good target for drug development.

Amino Acid Sequence↗

PubMed2006

An evolutionarily conserved translation initiation mechanism regulates nuclear or mitochondrial targeting of DNA ligase 1 in Arabidopsis thaliana.

The Arabidopsis DNA ligase 1 gene (AtLIG1) is indispensable for cell viability. AtLIG1 expresses one major and two minor mRNA transcripts differing only in the length of the 5' untranslated leader sequences preceding a common ORF. Control of AtLIG1 isoform production and intracellular targeting depends upon mechanisms controlling the choice of translation initiation site within the AtLIG1 ORF. Confocal laser scanning microscopy of green fluorescent protein-tagged AtLIG1 isoforms expressed in Arabidopsis revealed that translation of AtLIG1 mRNA transcripts from the first in-frame start codon produces an AtLIG1 isoform that is targeted exclusively to the mitochondria. Translation initiation from the second in-frame start codon produces an AtLIG1 isoform targeted only to the nucleus. There is no evidence for AtLIG1-GFP being targeted to chloroplasts. The mitochondrial AtLIG1 isoform possesses both an N-terminal mitochondrial-targeting signal and an internal bipartite nuclear localization signal (NLS) yet is targeted only to mitochondria, demonstrating a hierarchical dominance of the mitochondrial presequence over the NLS. The length of the 5'-UTR and more significantly the nucleotide context around alternative start codons in the AtLIG1 transcripts affect translation initiation to ensure a balanced synthesis of both nuclear and mitochondrial AtLIG1 isoforms, probably via a context-dependent leaky ribosome scanning mechanism.

Arabidopsis↗

PubMed2002

Molecular characterization of nucleus-localized RNA-binding proteins from higher plants.

We report the isolation and characterization of genes from the higher plants Arabidopsis, spinach and tobacco which code for nucleus-localized RNA-binding proteins. Common features of these polypeptides are glycine/arginine-rich regions with several RGG repeats at their N- and C-termini, which are sufficient for RNA binding in northwestern assays. All polypeptides analysed contain two basic bipartite nuclear localization signals and translational fusions harbouring these regions with the beta-glucuronidase gene direct the fusion proteins into the nucleus. Nuclear localization was confirmed by cellular fractionation with a polyclonal antiserum raised against the over-expressed tobacco protein NtRGG1p. Two or three copies of related RGG genes appear to be present in the analysed organisms and the expression of some of them is regulated: a tobacco gene is light-regulated and a spinach gene is preferentially expressed in roots. Possible biological functions of this class of RNA-binding proteins as well as structure/function relationships related to the modular structure are discussed.

Amino Acid Sequence↗

PubMed2001

Nuclear export of mammalian PERIOD proteins.

The timing of mammalian circadian rhythm is determined by interlocking negative and positive transcriptional feedback loops that govern the cyclic expression of both clock regulators and output genes. In mammals, nuclear localization of the circadian regulators PER1-3 is controlled by multiple mechanisms, including multimerization with PER and CRY proteins. In addition, nuclear entry of mammalian PER1 (mPER1) can be regulated by a phosphorylation-dependent masking of its nuclear localization signal. Here we present evidence suggesting that nuclear localization of PER proteins is a dynamic process determined by both nuclear import and previously unrecognized nuclear export pathways. Examination of the subcellular localization of a series of truncated mPER1 proteins demonstrated that cytoplasmic localization is mediated by an 11-amino acid region with homology to leucine-rich nuclear export signals (NESs). Similar sequences were identified in mPER2 and mPER3 as well as in several insect PER proteins. The putative NESs from mPER1 and mPER2 were able to direct cytoplasmic accumulation when fused to a heterologous protein. Mutations in conserved NES residues and the nuclear export inhibitor leptomycin B each blocked the function of the NES. Full-length mPER1 was also exported from microinjected Xenopus laevis oocyte nuclei in an NES-dependent manner. The presence of a functional NES in mPER1 and mPER2 as well as related sequences in a variety of other PER proteins suggests that nuclear export may be a conserved and important feature of circadian regulators.

Amino Acid Sequence↗

PubMed2003

Cloning and molecular characterization of a novel gene strongly induced by the adenovirus E1A gene in rat thyroid cells.

Expression of the adenovirus E1A gene in the rat thyroid differentiated cell line PC Cl 3 induces thyrotropin-independent cell growth and impairs differentiation. However, the malignant phenotype is achieved only when the PC E1A cells are infected with other murine retroviruses carrying the v-abl, v-raf or polyoma middle-T genes. To determine through which genes E1A affects thyroid cells, we differentially screened PC Cl 3 and PC E1A cells. Here we report a new gene, named CL2, that is upregulated in PC E1A cells. The CL2 transcript is 4.4 kb long and encodes a 949 amino-acid protein. Conceptual translation of the open reading frame showed one product with a signal peptide, multiple nuclear localization signals and three newly described domains. Furthermore, in vivo, this protein was located juxtanuclear, which is suggestive of Golgian localization, and also in cytoplasm and nucleus/nucleolus. Finally, CL2 gene expression was drastically downregulated in human thyroid neoplastic cell lines and tissues.

Adenovirus E1A Proteins↗