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Results for “Nuclear Localization Signals”

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PubMed2006

Human cytomegalovirus UL84 protein contains two nuclear export signals and shuttles between the nucleus and the cytoplasm.

Previous studies defined pUL84 of human cytomegalovirus as an essential regulatory protein with nuclear localization that was proposed to act during initiation of viral-DNA synthesis. Recently, we demonstrated that a complex domain of 282 amino acids within pUL84 functions as a nonconventional nuclear localization signal. Sequence inspection of this domain revealed the presence of motifs with homology to leucine-rich nuclear export signals. Here, we report the identification of two functional, autonomous nuclear export signals and show that pUL84 acts as a CRM-1-dependent nucleocytoplasmic shuttling protein. This suggests an unexpected cytoplasmic role for this essential viral regulatory protein.

Amino Acid Motifs↗

PubMed1999

Control of NF-kappa B transcriptional activation by signal induced proteolysis of I kappa B alpha.

In unstimulated cells the transcription factor NF-kappa B is held in the cytoplasm in an inactive state by I kappa B inhibitor proteins. Ultimately activation of NF-kappa B is achieved by ubiquitination and proteasome-mediated degradation of I kappa B alpha and we have therefore investigated factors which control this proteolysis. Signal-induced degradation of I kappa B alpha exposes the nuclear localization signal of NF-kappa B, thus allowing it to translocate into the nucleus and activate transcription from responsive genes. An autoregulatory loop is established when NF-kappa B induces expression of the I kappa B alpha gene and newly synthesized I kappa B alpha accumulates in the nucleus where it negatively regulates NF-kappa B-dependent transcription. As part of this post-induction repression, the nuclear export signal on I kappa B alpha mediates transport of NF-kappa B-I kappa B alpha complexes from the nucleus to the cytoplasm. As nuclear export of I kappa B alpha is blocked by leptomycin B this drug was used to examine the effect of cellular location on susceptibility of I kappa B alpha to signal-induced degradation. In the presence of leptomycin B, I kappa B alpha is accumulated in the nucleus and in this compartment is resistant to signal-induced degradation. Thus signal-induced degradation of I kappa B alpha is mainly, if not exclusively a cytoplasmic process. An efficient nuclear export of I kappa B alpha is therefore essential for maintaining a low level of I kappa B alpha in the nucleus and allowing NF-kappa B to be transcriptionally active upon cell stimulation. We have detected a modified form of I kappa B alpha, conjugated to the small ubiquitin-like protein SUMO-1, which is resistant to signal-induced degradation. SUMO-1 modified I kappa B alpha remains associated with NF-kappa B and thus overexpression of SUMO-1 inhibits the signal-induced activation of NF-kappa B-dependent transcription. Reconstitution of the conjugation reaction with highly purified proteins demonstrated that in the presence of a novel E1 SUMO-1 activating enzyme, Ubch9 directly conjugated SUMO-1 to I kappa B alpha on residues K21 and K22, which are also used for ubiquitin modification. Thus, while ubiquitination targets proteins for rapid degradation, SUMO-1 modification acts antagonistically to generate proteins resistant to degradation.

Animals↗

PubMed1999

The net repressor is regulated by nuclear export in response to anisomycin, UV, and heat shock.

The ternary complex factors (TCFs) are targets for Ras/mitogen-activated protein kinase signalling pathways. They integrate the transcriptional response at the level of serum response elements in early-response genes, such as the c-fos proto-oncogene. An important aim is to understand the individual roles played by the three TCFs, Net, Elk1, and Sap1a. Net, in contrast to Elk1 and Sap1a, is a strong repressor of transcription. We now show that Net is regulated by nuclear-cytoplasmic shuttling in response to specific signalling pathways. Net is mainly nuclear under both normal and basal serum conditions. Net contains two nuclear localization signals (NLSs); one is located in the Ets domain, and the other corresponds to the D box. Net also has a nuclear export signal (NES) in the conserved Ets DNA binding domain. Net is apparently unique among Ets proteins in that a particular leucine in helix 1, a structural element, generates a NES. Anisomycin, UV, and heat shock induce active nuclear exclusion of Net through a pathway that involves c-Jun N-terminal kinase kinase and is inhibited by leptomycin B. Nuclear exclusion relieves transcriptional repression by Net. The specific induction of nuclear exclusion of Net by particular signalling pathways shows that nuclear-cytoplasmic transport of transcription factors can add to the specificity of the response to signalling cascades.

Amino Acid Motifs↗

PubMed1999

Human hepatitis B virus X protein is detectable in nuclei of transfected cells, and is active for transactivation.

Subcellular localization and transactivation of human hepatitis B virus X protein (HBx), a plausible causative factor for hepatocellular carcinogenesis, were studied in transiently transfected cells. The transactivation was detected not only by the cis-element driven chloramphenicol acetyltransferase (CAT) assay but also by immunostaining of CAT protein cotransfected into human hepatoma cell line HepG2. Scanning fluorescence microscopy showed the majority of immunological signals of HBx to be at the perinuclear region of transfected cytoplasm. HBx was also clearly detectable in the nucleus, though less intensely expressed. This was confirmed by Western analysis and coimmunoprecipitation of HBx with transcription factor IIB (TFIIB) in subcellular fractionations. The percentage of HBx-positive cells coincided with that of CAT-positive cells, and confocal laser microscopy revealed the coexistence of CAT signals in GFP-HBx positive cells. The SV40 large T antigen nuclear localization signal (NLS) appended HBx, regardless of whether NLS was added to the N- or C-terminus, transactivated all the examined X-responsive elements (XRE) similarly as did wild-type HBx. Similar results were obtained in p53 negative Saos-2 cells. The detected nuclear HBx may be involved in modulating the transcription at the promoter level whereas the HBx in cytoplasm may be working through signal transduction pathways.

Cell Nucleus↗

PubMed2001

Nuclear localization of the plant protein Ser/Thr phosphatase PP7.

Recently we identified novel plant Ser/Thr phosphatases, termed PP7, which belong to the PPP family and have no known close homologs in other kingdoms. We now addressed the intracellular location of Arabidopsis thaliana PP7 using GFP fusions and confocal laser scanning microscopy. PP7. GFP fusion was expressed transiently or stably in Nicotiana benthamiana. PP7. GFP was found to be a predominantly nuclear protein. Effects of cytoskeleton-disrupting drugs indicate that cytoskeleton may be required for efficient PP7. GFP delivery to the nucleus. Deletion of a potential nuclear localization signal in the first insert in the catalytic domain, as well as exposure to the dark, cold, high salinity and abscisic acid failed to prevent nuclear localization of PP7. GFP. Deletion of the 44 C-terminal amino acids resulted in a fusion protein located exclusively in the cytoplasm. The results suggest a possible similarity of the nuclear targeting signals in PP7 and the PP5/PPT subfamily.

Abscisic Acid↗

PubMed2001

Identification of hepatocyte growth factor activator inhibitor type 2 (HAI-2)-related small peptide (H2RSP): its nuclear localization and generation of chimeric mRNA transcribed from both HAI-2 and H2RSP genes.

A novel small gene, designated hepatocyte growth factor activator inhibitor type 2 (HAI-2)-related small peptide (H2RSP) was cloned and characterized in the process of the search for splicing variant forms of HAI-2 by 3'-rapid amplification of cDNA ends (RACE). The gene consisted of 4 exons spanning approximately 1 kb and was located in 11 kb downstream of HAI-2 gene (19q.13.11). The novel transcript identified by 3'-RACE was thought to be chimerically transcribed from both HAI-2 (exons 1-7) and H2RSP (exons 2-4) genes. Wild-type H2RSP mRNA (0.5 kb) was detected abundantly in various tissues including the gastrointestinal tract, whereas chimeric mRNA (1.5 kb) was found mainly in the kidney, prostate, and placenta by Northern blot analysis. The predicted amino acid sequence of H2RSP contained two unique domains, namely the serine-rich region (exon 3) and the lysine-rich region (exon 4). Transfection of deleted series of H2RSP cDNAs fused to enhanced green fluorescent protein (EGFP) into HeLa cells revealed that H2RSP has nuclear localization signal in the lysine-rich region. Immunohistochemical study using anti-H2RSP polyclonal antibody indeed revealed the nuclear localization of this peptide in vivo. These results suggest that H2RSP and H2RSP/HAI-2 chimeric peptides might function as a transcriptional regulatory peptide at the nucleus.

Amino Acid Sequence↗

PubMed1995

A hybrid baculovirus-bacteriophage T7 transient expression system.

A hybrid recombinant baculovirus-bacteriophage T7 expression system was developed for transient expression in insect cells of plasmids with foreign genes provided with a T7 promoter. The coding sequence for T7 RNA polymerase, with or without a nuclear localization signal, was inserted into the genome of Autographa californica nuclear polyhedrosis virus. Recombinant viruses stably expressed T7 RNA polymerase in insect cells. Upon transfection of infected insect cells with plasmids containing the genes for chloramphenicol acetyltransferase (CAT), the hepatitis B virus precore-, core- or e- antigens under control of the T7 promoter, transient expression of these genes was detected by ELISA. The results obtained indicate that this baculovirus/T7 system provides a simple and widely applicable tool for transient gene expression studies.

Animals↗

PubMed2002

Identification of a novel transcription factor, ELYS, expressed predominantly in mouse foetal haematopoietic tissues.

BACKGROUND: The precise mechanism governing the generation of haematopoietic stem cells still remains to be understood, partly because the molecules required for early haematopoiesis have not fully been identified. RESULTS: We have identified a novel gene expressed in embryonic haematopoietic tissues, designated ELYS (for embryonic large molecule derived from yolk sac), which has no significant homology with any other known molecules. Based on the cDNA sequence, mouse ELYS protein is composed of 2243 amino acid residues and contains an AT-hook DNA-binding domain, eight nuclear localization signals (NLSs) at the C-terminal region, three nuclear export signals (NESs) and two WD repeats at the N-terminal region. ELYS has a potential to shuttle between the cytoplasm and nucleus. When in the nucleus, ELYS is present in the nuclear matrix. Fusions of the yeast GAL4 DNA-binding domain and various ELYS mutants reveal the presence of transcriptional activation and inhibitory domains. The ELYS gene is predominantly expressed in embryonic haematopoietic tissues, i.e. foetal liver, spleen, and thymus, whereas the expression is down-regulated in the adult. In the aorta-gonad-mesonephros (AGM) region of an 11.5 dpc mouse embryo, ELYS is expressed in the endothelium lining the dorsal aorta. In the adult bone marrow, ELYS is notably expressed in the Lin-/c-kit+/Sca-1+ population. CONCLUSIONS: We have reported the isolation and characterization of a novel molecule, ELYS. ELYS seems to be a nuclear transcription factor associated with both early and mature haematopoietic events.

Amino Acid Sequence↗

PubMed2002

RAP80, a novel nuclear protein that interacts with the retinoid-related testis-associated receptor.

In this study, we describe the characterization of a novel nuclear protein, referred to as RAP80. The RAP80 cDNA was cloned from a human testis cDNA library and encodes a 719-amino acid protein containing two potential CX(2)CX(11)HX(3)C-type zinc finger motifs at its carboxyl-terminal region. Analysis of its genomic structure revealed that the RAP80 gene covers more than 90 kb and consists of 15 exons and 14 introns. Fluorescence in situ hybridization mapped the RAP80 gene to human chromosome 5q35. RAP80 mRNA is expressed in many human tissues, but its expression is particularly high in testis. In situ hybridization showed that RAP80 is highly expressed in germ cells of mouse testis but is not differentially regulated during spermatogenesis. Confocal microscopy showed that RAP80 is localized to the nucleus, where it is distributed in a speckled pattern. Deletion analysis showed that a bipartite nuclear localization signal at the amino terminus is important in mediating nuclear transport of RAP80. Monohybrid analysis showed that RAP80 might function as an active repressor of transcription. Mammalian two-hybrid analysis demonstrated that RAP80 was able to interact with the retinoid-related testis-associated receptor (RTR), an orphan receptor that has been implicated in the control of embryonic development and spermatogenesis. Pull-down analysis showed that RAP80 and RTR physically interact in vitro. Deletion and point mutation analyses revealed that part of the hinge domain of RTR is required for this interaction. RAP80 is able to inhibit the interaction of RTR with the co-repressor N-CoR likely by competing with N-CoR for RTR binding. Our results suggest that RAP80 may be functioning as a modulator of RTR signaling.

Alternative Splicing↗

PubMed2000

Suppression of post-transcriptional gene silencing by a plant viral protein localized in the nucleus.

Post-transcriptional gene silencing (PTGS) is a homology-dependent RNA degradation process that may target RNA exclusively in the cytoplasm. In plants, PTGS functions as a natural defense mechanism against viruses. We reported previously that the 2b protein encoded by cucumber mosaic cucumovirus (CMV) is a virulence determinant and a suppressor of PTGS initiation in transgenic Nicotiana benthamiana. By fusion with the green fluorescent protein, we now show that the CMV 2b protein localizes to the nuclei of tobacco suspension cells and whole plants via an arginine-rich nuclear localization signal, (22)KRRRRR(27). We further demonstrate that the nuclear targeting of the 2b protein is required for the efficient suppression of PTGS, indicating that PTGS may be blocked in the nucleus. In addition, our data indicate that the PTGS suppressor activity is important, but not sufficient, for virulence determination by the 2b protein.

Amino Acid Sequence↗

PubMed2003

Transcriptional activation of the NF-kappaB p65 subunit by mitogen- and stress-activated protein kinase-1 (MSK1).

Nuclear factor kappaB (NF-kappaB) is one of the key regulators of transcription of a variety of genes involved in immune and inflammatory responses. NF-kappaB activity has long been thought to be regulated mainly by IkappaB family members, which keep the transcription factor complex in an inactive form in the cytoplasm by masking the nuclear localization signal. Nowadays, the importance of additional mechanisms controlling the nuclear transcription potential of NF-kappaB is generally accepted. We show that the mitogen-activated protein kinase inhibitors SB203580 and PD98059 or U0126, as well as a potent mitogen- and stress- activated protein kinase-1 (MSK1) inhibitor H89, counteract tumor necrosis factor (TNF)-mediated stimulation of p65 transactivation capacity. Mutational analysis of p65 revealed Ser276 as a target for phosphorylation and transactivation in response to TNF. Moreover, we identified MSK1 as a nuclear kinase for p65, since MSK1 associates with p65 in a stimulus-dependent way and phosphorylates p65 at Ser276. This effect represents, together with phosphorylation of nucleosome components such as histone H3, an essential step leading to selective transcriptional activation of NF-kappaB-dependent gene expression.

Animals↗

PubMed1996

Early transcription of Agrobacterium T-DNA genes in tobacco and maize.

We developed a sensitive procedure to investigate the kinetics of transcription of an Agrobacterium tumefaciens transferred (T)-DNA-encoded beta-glucuronidase gusA (uidA) gene soon after infection of plant suspension culture cells. The procedure uses a reverse transcriptase-polymerase chain reaction and enables detection of gusA transcripts within 18 to 24 hr after cocultivation of the bacteria with either tobacco or maize cells. Detection of gusA transcripts depended absolutely on the intact virulence (vir) genes virB, virD1/virD2, and virD4 within the bacterium. Mutations in virC and virE resulted in delayed and highly attenuated expression of the gusA gene. A nonpolar transposon insertion into the C-terminal coding region of virD2 resulted in only slightly decreased production of gusA mRNA, although this insertion resulted in the loss of the nuclear localization sequence and the important omega region from VirD2 protein and rendered the bacterium avirulent. However, expression of gusA transcripts in tobacco infected by this virD2 mutant was more transient than in cells infected by a wild-type strain. Infection of tobacco cells with an Agrobacterium strain harboring a mutant virD2 allele from which the omega region had been deleted resulted in similar transient expression of gusA mRNA. These data indicate that the C-terminal nuclear localization signal of the VirD2 protein is not essential for nuclear uptake of T-DNA and further suggest that the omega domain of VirD2 may be required for efficient integration of T-DNA into the plant genome. The finding that the initial kinetics of gusA gene expression in maize cells are similar to those shown in infected tobacco cells but that the presence of gusA mRNA in maize is highly transient suggests that the block to maize transformation involves T-DNA integration and not T-DNA entry into the cell or nuclear targeting.

Bacterial Proteins↗

PubMed1996

Nuclear pore-targeting complex and its role on nuclear protein transport.

The process of selective nuclear protein transport is divided into at least two steps: 1) ATP-independent, nuclear localization signal (NLS)-dependent binding to the cytoplasmic face of nuclear pores and 2) ATP-dependent translocation through the nuclear pores. Using a digitonin-permeabilized cell-free transport assay, it was found that a karyophile forms a stable complex with a cytoplasmic fraction to target the nuclear pores. Since this complex shows nuclear pore-binding activity, we have referred to it as the nuclear Pore-Targeting Complex (PTAC). The complex contains two essential proteins. The 58 kDa component of PTAC (PTAC 58; importin alpha; karyopherin alpha) was found to bind directly to NLS. The 97 kDa component of PTAC (PTAC 97; importin beta; karyopherin beta) associates with PTAC 58, but not karyophile. A complex of PTAC 58 and PTAC 97 targets nuclear pores, depending on the presence of a karyophile. The data suggest that the initial step in nuclear protein transport occurs as a result of complex formation of a karyophile with PTAC 58 which is, in turn, bound to PTAC 97.

Animals↗

PubMed2000

Nucleocytoplasmic shuttling of heterodimeric splicing factor U2AF.

The U2 small nuclear ribonucleoprotein auxiliary factor (U2AF) is a heterodimeric splicing factor composed of 65-kDa (U2AF(65)) and 35-kDa (U2AF(35)) subunits. The large subunit of U2AF recognizes the intronic polypyrimidine tract, a sequence located adjacent to the 3' splice site that serves as an important signal for both constitutive and regulated pre-mRNA splicing. The small subunit U2AF(35) interacts with the 3' splice site dinucleotide AG and is essential for regulated splicing. Like several other proteins involved in constitutive and regulated splicing, both U2AF(65) and U2AF(35) contain an arginine/serine-rich (RS) domain. In the present study we determined the role of RS domains in the subcellular localization of U2AF. Both U2AF(65) and U2AF(35) are shown to shuttle continuously between the nucleus and the cytoplasm by a mechanism that involves carrier receptors and is independent from binding to mRNA. The RS domain on either U2AF(65) or U2AF(35) acts as a nuclear localization signal and is sufficient to target a heterologous protein to the nuclear speckles. Furthermore, the results suggest that the presence of an RS domain in either U2AF subunit is sufficient to trigger the nucleocytoplasmic import of the heterodimeric complex. Shuttling of U2AF between nucleus and cytoplasm possibly represents a means to control the availability of this factor to initiate spliceosome assembly and therefore contribute to regulate splicing.

3T3 Cells↗

PubMed2001

Nuclear translocation of the N-terminal prodomain of interleukin-16.

Interleukin-16 (IL-16) is a pleiotropic cytokine that functions as a chemoattractant factor, a modulator of T cell activation, and an inhibitor of human immunodeficiency virus (HIV) replication. These diverse functions are exclusively attributed to the secreted C-terminal peptide of 121 amino acids (mature IL-16), which is cleaved from the precursor protein (pro-IL-16) by caspase-3. Human pro-IL-16 is comprised of 631 amino acids with three PDZ domains, one of which is present in secreted mature IL-16. No cellular localization or biologic functions have been ascribed to the unusually large and highly conserved N-terminal prodomain formed as a result of proteolytic release of the third PDZ domain of pro-IL-16. Here we show that the N-terminal prodomain of pro-IL-16 translocates into the nucleus following cleavage of the C-terminal segment. The nuclear localization signal of pro-IL-16 consists of a classical bipartite nuclear targeting motif. We also show that the nuclear targeting of the IL-16 prodomain induces a G(0)/G(1) arrest in the cell cycle. Taken together, the high degree of conservation of the prodomain among species, the presence of two PDZ motifs, and the nuclear localization and subsequent inhibitory effect on cell cycle progression suggest that pro-IL-16 is cleaved into two functional proteins, a C-terminal-secreted cytokine and an N-terminal product, which affects the cell cycle.

Animals↗

PubMed2005

S-nitrosylated GAPDH initiates apoptotic cell death by nuclear translocation following Siah1 binding.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) influences cytotoxicity, translocating to the nucleus during apoptosis. Here we report a signalling pathway in which nitric oxide (NO) generation that follows apoptotic stimulation elicits S-nitrosylation of GAPDH, which triggers binding to Siah1 (an E3 ubiquitin ligase), nuclear translocation and apoptosis. S-nitrosylation of GAPDH augments its binding to Siah1, whose nuclear localization signal mediates translocation of GAPDH. GAPDH stabilizes Siah1, facilitating its degradation of nuclear proteins. Activation of macrophages by endotoxin and of neurons by glutamate elicits GAPDH-Siah1 binding, nuclear translocation and apoptosis, which are prevented by NO deletion. The NO-S-nitrosylation-GAPDH-Siah1 cascade may represent an important molecular mechanism of cytotoxicity.

Animals↗

PubMed2006

Identification and characterization of the nuclear localization/retention signal in the EWS proto-oncoprotein.

Ewing sarcoma (EWS) protein, a member of a large family of RNA-binding proteins, contains an N-terminal transcriptional activation domain (EAD) and a C-terminal RNA-binding domain (RBD). Due to its multifunctional properties EWS protein is involved in processes such as gene expression, RNA processing and transport, and cell signaling. Chimeric EWS proteins generated by chromosomal translocations cause malignant tumors. EWS protein is located predominantly in the nucleus, but was found also in the cytosol and associated with the cell membrane. The determinants responsible for the nuclear localization of the protein were as yet unknown. We identified the nuclear localization signal of EWS protein at its C terminus (C-NLS), which is required for the nuclear import and retention of the protein. The C-NLS sequence is conserved in related proto-oncoproteins suggesting an NLS function also in these proteins. Two arginine residues, due to their positive charge, a proline residue and a tyrosine residue are essential for C-NLS function. The nuclear localization of EWS protein is independent of the regions in RBD containing numerous arginine methylation sites, RNA-recognition and zinc finger motifs. Regions in EAD guide the subnuclear partition of EWS protein and contain another but different NLS that allows nucleocytoplasmic shuttling of the N-terminal domain.

Amino Acid Motifs↗

PubMed2005

Ran GTPase regulates Mad2 localization to the nuclear pore complex.

In yeast and mammalian cells, the spindle assembly checkpoint proteins Mad1p and Mad2p localize to the nuclear pore complex (NPC) during interphase. Deletion of MAD1 or MAD2 did not affect steady-state nucleocytoplasmic distribution of a classical nuclear localization signal-containing reporter, a nuclear export signal-containing reporter, or Ran localization. We utilized cells with conditional mutations in the yeast Ran GTPase pathway to examine the relationship between Ran and targeting of checkpoint regulators to the NPC. Mutations that disrupt the concentration of Ran in the nucleus displaced Mad2p but not Mad1p from the NPC. The displacement of Mad2p in M-phase cells was correlated with activation of the spindle checkpoint. Our observations demonstrate that Mad2p localization at NPCs is sensitive to nuclear levels of Ran and suggest that release of Mad2p from NPCs is closely linked with spindle assembly checkpoint activation in yeast. This is the first evidence indicating that Ran affects the localization of Mad2p to the NPC.

Animals↗