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Results for “Nuclear Export Signals”

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At least 217 records · Page 12Linked to original sources

PubMed2005

Growth suppression of Leydig TM3 cells mediated by aryl hydrocarbon receptor.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin induces developmental toxicity in reproductive organs. To elucidate the function of AhR, we generated stable transformants of TM3 cells overexpressing wild-type aryl hydrocarbon receptor (AhR) or its mutants which carried mutations in nuclear localization signal or nuclear export signal. In the presence of 3-methylcholanthrene (MC), proliferation of the cells transfected with wild-type AhR was completely suppressed, whereas cells expressing AhR mutants proliferated in a manner equivalent to control TM3 cells, suggesting AhR-dependent growth inhibition. The suppression was associated with up-regulation of cyclin-dependent kinase inhibitor p21Cip1, which was abolished by pretreatment with actinomycin D. A p38 MAPK specific inhibitor, SB203580, blocked the increase of p21Cip1 mRNA in response to MC. Treatment with indigo, another AhR ligand, failed to increase of p21Cip1 mRNA, although up-regulation of mRNA for CYP1A1 was observed. These data suggest AhR in Leydig cells mediates growth inhibition by inducing p21Cip1.

Animals↗

PubMed2005

Controlled intracellular localization and enhanced antisense effect of oligonucleotides by chemical conjugation.

Oligonucleotides can be covalently linked to peptides composed of any sequence of amino acids by solid phase fragment condensation. The peptides incorporated into the conjugates include nuclear localizing signals (NLS), nuclear export signals (NES), membrane fusion domain of some viral proteins and some designed peptides with amphipathic character. Evaluation of biological properties of DNA-peptide conjugates indicated that (a) the conjugates could bind to target RNA and dsDNA with increased affinity, (b) the conjugates were more resistant to cellular nuclease degradation, (c) the conjugate-RNA hybrids could activate RNase H as effectively as native oligonucleotides, (d) the conjugates with fusion peptides showed largely enhanced cellular uptake, (e) the conjugates with NLS could be predominantly delivered into the cell nucleus, (f) the conjugates with NES could be localized in the cytoplasm. As a result, antisense oligonucleotides conjugated with NLS could inhibit human telomerase in human leukemia cells much more strongly than phosphorothioate oligonucleotides.

Amino Acid Sequence↗

PubMed2006

Spatio-temporal regulation of mitogen-activated protein kinase (MAPK) signalling by protein phosphatases.

ERK (extracellular-signal-regulated kinase) is a MAPK (mitogen-activated protein kinase), which regulates diverse physiological functions including cell proliferation, differentiation, transformation and survival. It is now clear that in addition to the duration and magnitude of signalling through this MAPK pathway, the spatial restriction of MAPK activity plays a key role in determining the physiological outcome of signalling. Recent work has led to the discovery of MAPK-binding proteins, which contain either nuclear localization signals or nuclear export signals. These include MAPK activators and specific protein phosphatases, which may act to both regulate MAPK activity and the subcellular localization of their substrate. This represents a mechanism by which signalling in response to extracellular stimuli may be modulated in terms of both magnitude/duration and spatial restriction thus allowing differential access of the activated MAPK to target proteins and the interpretation of this information by cells to determine an appropriate physiological response.

Animals↗

PubMed2002

Determinants of nuclear and cytoplasmic ubiquitin-mediated degradation of MyoD.

The ubiquitin-proteasome system is responsible for the regulation and turnover of many short-lived proteins both in the cytoplasm and in the nucleus. Degradation can occur via two distinct pathways, an N terminus-dependent pathway and a lysine-dependent pathway. The pathways are characterized by the site of initial ubiquitination of the protein, the N terminus or an internal lysine, respectively. MyoD, a basic helix-loop-helix transcription factor, is a substrate for the ubiquitin-proteasome pathway and is degraded in the nucleus. It is preferentially tagged for degradation on the N terminus and thus is degraded by the N terminus-dependent pathway. Addition of a 6x Myc tag to the N terminus of MyoD can force degradation through the lysine-dependent pathway by preventing ubiquitination at the N-terminal site. Modifications of the nuclear localization signal and nuclear export signal of MyoD restrict ubiquitination and degradation to the cytoplasm or the nucleus. Using these mutants, we determined which degradation pathway is dominant in the cytoplasm and the nucleus. Our results suggest that the lysine-dependent pathway is the more active pathway within the cytoplasm, whereas in the nucleus the two pathways are both active in protein degradation.

Cell Nucleus↗

PubMed2005

Phosphorylation of Ser-446 determines stability of MKP-7.

MAPK cascades can be negatively regulated by members of the MAPK phosphatase (MKP) family. However, how MKP activity is regulated is not well characterized. MKP-7, a JNK-specific phosphatase, possesses a unique COOH-terminal stretch (CTS) in addition to domains conserved among MKP family members. The CTS contains several motifs such as a nuclear localization signal, a nuclear export signal, PEST sequences, and a serine residue (Ser-446) that can be phosphorylated by activated ERK, suggesting an important regulatory role(s).(35)S-pulse labeling experiments indicate that the half-life of MKP-7 is 1.5 h, a period significantly elongated by deleting the CTS. We also show that overexpressed MKP-7 is polyubiquitinated when co-expressed with ubiquitin and that proteasome inhibitors markedly inhibit MKP-7 degradation. We also determined that MKP-7 phosphorylated at Ser-446 has a longer half-life than unphosphorylated form of the wild type protein, as does a phospho-mimic mutant of MKP-7. These results indicate that activation of the ERK pathway strongly blocks JNK activation through stabilization of MKP-7 mediated by phosphorylation.

Amino Acid Motifs↗

PubMed2000

Rev protein of human immunodeficiency virus type 1 and cellular exportin 1 protein relocalize each other to a subnucleolar structure.

The exportin 1/crml protein associates with leucine-rich nuclear export signals (NESs) and mediates nuclear export in various experimental systems. We show here that exportin 1 and the NES-containing human immunodeficiency virus (HIV) type 1 Rev protein relocalize each other to a characteristic dotlike structure within the nucleoli of human cells. On treatment with actinomycin D, Rev remains in these dots longer than in the rest of the nucleoli, arguing that the nucleolar dots do not represent sites of high transport turnover. Transient expression of exportin 1 strongly reduces the expression of a reporter that depends on the export of HIV RNA. When export of hepatitis B virus RNA and simple retrovirus RNA, as well as spliced mRNA, was assayed in this way, exportin 1 inhibited reporter expression to a lesser extent. Thus, an excess of exportin 1 may downregulate Rev-mediated RNA export by sequestering Rev to a subnucleolar structure.

Base Sequence↗

PubMed2003

Antisense effects of DNA-peptide conjugates.

Antisense conjugate DNA covalently bound to functional amines, sugars or peptides such as nuclear localization signals (NLS), nuclear export signals (NES) and artificial amphiphilic alpha-helical and beta-sheet peptides were synthesized by solid phase fragment condensation (SPFC). Inhibitory effects on human telomerase of synthesized antisense conjugate DNAs were evaluated by TRAP assay. Antisense conjugate DNAs showed several times higher inhibitory effects on telomerase activity compared to native antisense DNA in cell lysis solutions. Antisense conjugate DNAs showed comparable or higher as same as inhibitory activity compared with antisense phosphorothioate DNA (S-DNA). Interestingly, antisense conjugate DNA showed much higher inhibition against telomerase activity with native antisense DNA or antisense phosphorothioate DNA in cellular system. We observed enhanced cellular uptake and positively controlled intracellular localization of conjugate DNAs by use of confocal fluorescent microscope.

Amino Acid Sequence↗

PubMed2005

Highly sensitive inhibition of hTERT mRNA expression and telomerase activity by DNA-signal-peptide conjugates.

In the present study, we investigated the antisense properties of conjugate oligonucleotides (ODNs) inhibiting human telomerase activity. Conjugate oligonucleotides assembled with signal peptides, artificially designed peptides, amines and sugars were synthesized by solid phase fragment condensation (SPFC) in sufficient yields. Conjugate ODNs showed a high resistance to nuclease degradation and sufficient binding affinity to target RNA, comparatively rapid and sufficient intracellular delivery and specific localization controlled by signal peptides (nuclear localization signals, NLS; nuclear export signals, NES). ODN-NLS conjugates demonstrated high antisense inhibitory effects against human telomerase activity into the nucleus (e. g, phosphorothioate conjugate inhibited the telomerase activity over 95%), whereas ODN-NES conjugates inhibited target mRNA expression into the cytoplasm.

Biological Transport↗

PubMed2006

Immunohistochemistry predicts nucleophosmin (NPM) mutations in acute myeloid leukemia.

Nucleophosmin (NPM) exon-12 mutations occur in 50% to 60% of adult acute myeloid leukemia (AML) with normal karyotype and are predictors of favorable prognosis. We evaluated bone marrow or peripheral blood samples from 450 adult patients with AML of the GIMEMA (Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto)/AML12 EORTC (European Organization for Research and Treatment of Cancer) trial to (1) search for new exon-12 NPM mutations; (2) determine whether NPM immunostaining on paraffin-embedded biopsies predicts NPM mutations; and (3) investigate altered nucleocytoplasmic NPM traffic in primary AML cells. Fourteen NPM mutations, including 8 new variants, were identified. All 200 AML cases expressing cytoplasmic NPM (NPMc(+) AML) carried NPM mutations. None of the 250 cases with nucleus-restricted NPM (NPMc(-) AML) was mutated. At the C-terminus, NPM leukemic mutants carried mutations of only tryptophan 290 or of both tryptophans 288 and 290 and a new nuclear export signal (NES) motif, which appear to underlie their nuclear export. The specific Crm1/exportin-1 inhibitor leptomycin-B relocated NPM mutants from cytoplasm to nucleus of primary NPMc(+) AML cells, demonstrating that nuclear export is NES dependent. NPM mutants bound and recruited wild-type NPM into leukemic cell cytoplasm. Because alterations at C-terminus of leukemic NPM mutants are similar, immunohistochemistry detects all exon-12 NPM mutations and is a valuable, inexpensive tool in the diagnostic-prognostic work-up of patients with AML with normal karyotype.

Active Transport, Cell Nucleus↗

PubMed1999

Nuclear import and export pathways.

Macromolecules enter or leave the nucleus by using nuclear localization signals (NLS), or nuclear export signals (NES), respectively. Different types of NLS and NES are recognized directly or indirectly via adapters, by transport receptors. All transport receptors identified thus far are members of the same family and share an ability to shuttle between the nucleus and the cytoplasm, and to interact with the small GTPase Ran and with nucleoporins at the nuclear pore complex (NPC). The GTPase Ran regulates the interaction of transport receptors with either cargoes, or adapters, or nucleoporins and is crucial in providing directionality to nuclear import and export. Surprisingly, GTP hydrolysis by Ran is not required for translocation of some receptor/cargo complexes through the NPC. One of the challenges for the future will be to establish the mechanisms of translocation through the NPC of different transport receptors together with their cargoes. J. Cell. Biochem. Suppls. 32/33:76-83, 1999.

Animals↗

PubMed2006

Nuclear translocation of the pro-apoptotic Bcl-2 family member Bok induces apoptosis.

The anti-apoptotic members of the Bcl-2 family, such as Bcl-2 and Bcl-XL, play a central role in preventing the induction of apoptosis via the intrinsic apoptotic pathway. It has been previously shown that induction of apoptosis by the pro-apoptotic Bcl-2 family member Bok is not antagonized by either Bcl-2 or Bcl-xL, suggesting that Bok might have a unique role in the apoptotic cascade. We showed here that human Bok is the only member of the Bcl-2 family to have a leucine-rich sequence indicative of a nuclear export signal within its BH3 domain. Western blot analysis of nuclear and cytoplasmic fractions identified Bok in both the nucleus and the cytoplasm of HEK 293T cells, HeLa cells, and breast cancer cells, and its nuclear concentration increased after treatment of those cells with leptomycin B, an inhibitor of the exportin Crm1. Immunocytochemistry of flag-tagged Bok confirmed its nuclear localization. Mutating the nuclear export signal of Bok by site-directed mutagenesis resulted in an increase in its nuclear localization and apoptotic activity. We also found that Crm1 interacted with wild-type Bok but not with the mutated form. These results suggest that nuclear export of Bok is a regulated process mediated by Crm1, and constitutes the first report of a link between the apoptotic activity and nuclear localization of a pro-apoptotic member of the Bcl-2 family.

Active Transport, Cell Nucleus↗

PubMed2001

Identification of three functions of the adenovirus e4orf6 protein that mediate p53 degradation by the E4orf6-E1B55K complex.

Complexes containing adenovirus E4orf6 and E1B55K proteins play critical roles in productive infection. Both proteins interact directly with the cellular tumor suppressor p53, and in combination they promote its rapid degradation. To examine the mechanism of this process, degradation of exogenously expressed p53 was analyzed in p53-null human cells infected with adenovirus vectors encoding E4orf6 and/or E1B55K. Coexpression of E4orf6 and E1B55K greatly reduced both the level and the half-life of wild-type p53. No effect was observed with the p53-related p73 proteins, which did not appear to interact with E4orf6 or E1B55K. Mutant forms of p53 were not degraded if they could not efficiently bind E1B55K, suggesting that direct interaction between p53 and E1B55K may be required. Degradation of p53 was independent of both MDM2 and p19ARF, regulators of p53 stability in mammalian cells, but required an extended region of E4orf6 from residues 44 to 274, which appeared to possess three separate biological functions. First, residues 39 to 107 were necessary to interact with E1B55K. Second, an overlapping region from about residues 44 to 218 corresponded to the ability of E4orf6 to form complexes with cellular proteins of 19 and 14 kDa. Third, the nuclear retention signal/amphipathic arginine-rich alpha-helical region from residues 239 to 253 was required. Interestingly, neither the E4orf6 nuclear localization signal nor the nuclear export signal was essential. These results suggested that if nuclear-cytoplasmic shuttling is involved in this process, it must involve another export signal. Degradation was significantly blocked by the 26S proteasome inhibitor MG132, but unlike the HPV E6 protein, E4orf6 and E1B55K were unable to induce p53 degradation in vitro in reticulocyte lysates. Thus, this study implies that the E4orf6-E1B55K complex may direct p53 for degradation by a novel mechanism.

Adenoviridae↗

PubMed2006

BMAL1 shuttling controls transactivation and degradation of the CLOCK/BMAL1 heterodimer.

CLOCK and BMAL1 are bHLH-PAS-containing transcription factors that bind to E-box elements and are indispensable for expression of core circadian clock components such as the Per and Cry genes. A key step in expression is the heterodimerization of CLOCK and BMAL1 and their accumulation in the nucleus with an approximately 24-h periodicity. We show here that nucleocytoplasmic shuttling of BMAL1 is essential for transactivation and for degradation of the CLOCK/BMAL1 heterodimer. Using serial deletions and point mutants, we identified a functional nuclear localization signal and Crm1-dependent nuclear export signals in BMAL1. Transient-transfection experiments revealed that heterodimerization of CLOCK and BMAL1 accelerates their turnover, as well as E-box-dependent clock gene transcription. Moreover, in embryonic mouse fibroblasts, robust transcription of Per2 is tightly associated with massive degradation of the CLOCK/BMAL1 heterodimer. CRY proteins suppressed this process during the transcription-negative phase and led to nuclear accumulation of the CLOCK/BMAL1 heterodimer. Thus, these findings suggest that the decrease of BMAL1 abundance during the circadian cycle reflects robust transcriptional activation of clock genes rather than inhibition of BMAL1 synthesis.

ARNTL Transcription Factors↗

PubMed2005

Nuclear import and export signals in control of Nrf2.

Nrf2 binds to the antioxidant response element and regulates expression and antioxidant induction of a battery of chemopreventive genes. In this study, we have identified nuclear import and export signals of Nrf2 and show that the nuclear import and export of Nrf2 is regulated by antioxidants. We demonstrate that Nrf2 contains a bipartite nuclear localization signal (NLS) and a leucine-rich nuclear export signal, which regulate Nrf2 shuttling in and out of the nucleus. Immunofluorescence and immunoblot analysis revealed that Nrf2 accumulates in the nucleus within 15 min of antioxidant treatment and is exported out of nucleus by 8 h after treatment. Nrf2 mutant lacking the NLS failed to enter the nucleus and displayed diminished expression and induction of the downstream NAD(P)H:quinone oxidoreductase 1 gene. The Nrf2 NLS sequence, when fused to green fluorescence protein, resulted in the nuclear accumulation of green fluorescence protein, indicating that this signal sequence was sufficient to direct nuclear localization of Nrf2. A nuclear export signal (NES) was characterized in the C terminus of Nrf2, the deletion of which caused Nrf2 to accumulate predominantly in the nucleus. The Nrf2 NES was sensitive to leptomycin B and could function as an independent export signal when fused to a heterologous protein. Further studies demonstrate that NES-mediated nuclear export of Nrf2 is required for degradation of Nrf2 in the cytosol. These results led to the conclusion that Nrf2 localization between cytosol and nucleus is controlled by both nuclear import and export of Nrf2, and the overall distribution of Nrf2 is probably the result from a balance between these two processes. Antioxidants change this balance in favor of nuclear accumulation of Nrf2, leading to activation of chemopreventive proteins. Once this is achieved, Nrf2 exits the nucleus for binding to INrf2 and degradation.

Active Transport, Cell Nucleus↗

PubMed2002

Members of the tristetraprolin family of tandem CCCH zinc finger proteins exhibit CRM1-dependent nucleocytoplasmic shuttling.

Members of the tristetraprolin (TTP) family of CCCH tandem zinc finger proteins can bind directly to certain types of AU-rich elements (AREs) in mRNA. Experiments in TTP-deficient mice have shown that TTP is involved in the physiological destabilization of at least two cytokine mRNAs, those encoding tumor necrosis factor alpha and granulocyte-macrophage colony-stimulating factor. The two other known mammalian members of the TTP family, CMG1 and TIS11D, also contain ARE-binding CCCH tandem zinc finger domains and can also destabilize ARE-containing mRNAs. To investigate the effects of primary sequence on the subcellular localization of these proteins, we constructed green fluorescent protein fusions with TTP, CMG1, and TIS11D; these were predominantly cytoplasmic when expressed in 293 or HeLa cells. Deletion and mutation analyses revealed functional nuclear export signals in the amino terminus of TTP and in the carboxyl termini of CMG1 and TIS11D. This type of leucine-rich nuclear export signal interacts with the nuclear export receptor CRM1; abrogation of CRM1 activity resulted in nuclear accumulation of TTP, CMG1, and TIS11D. These proteins are thus nucleocytoplasmic shuttling proteins and rely on CRM1 for their export from the nucleus. Although TTP, CMG1, and TIS11D lack known nuclear import sequences, mapping experiments revealed that their nuclear accumulation required an intact tandem zinc finger domain but did not require RNA binding ability. These findings suggest possible roles for nuclear import and export in the regulation of cellular TTP, CMG1, and TIS11D activity.

Amino Acid Sequence↗

PubMed2004

DNA damage induces p53-dependent BRCA1 nuclear export.

The tumor suppressor gene BRCA1 plays an important role in the response to DNA damage. BRCA1 function is regulated by a variety of mechanisms including transcriptional control, phosphorylation, and protein-protein interactions. Recent studies have shown that BRCA1 is a nuclear-cytoplasmic shuttle protein. Its subcellular localization is controlled by a nuclear localization signal-mediated nuclear import via the importin receptor pathway and a nuclear export signal-facilitated nuclear export through a CRM1-dependent pathway. Using the human breast cancer cell line, MCF7, the subcellular distribution of BRCA1 was assessed by immunohistochemical staining and Western blotting analyses of fractionated subcellullar extracts. Ionizing radiation stimulated BRCA1 nuclear export in a dose-dependent manner. This DNA damage-induced BRCA1 nuclear export utilized a CRM1-dependent mechanism and also required wild-type p53, whose function was abrogated by the E6 protein in MCF7 cells. In addition, the dependence on p53 was confirmed using a second cell type operating a tetracycline-inducible system. The effect of ionizing radiation on BRCA1 export was observed in every phase of the cell cycle, although BRCA1 localization did vary between the G(1), S, and G(2)/M phases. These results imply that, in addition to ATM-, ATR-, and Chk2-dependent phosphorylations, cytoplasmic relocalization of BRCA1 protein is a mechanism whereby BRCA1 function is regulated in response to DNA damage.

Active Transport, Cell Nucleus↗

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↗

PubMed2005

Identification of nuclear import and export signals within Fli-1: roles of the nuclear import signals in Fli-1-dependent activation of megakaryocyte-specific promoters.

The Ets factor Friend leukemia integration 1 (Fli-1) is an important regulator of megakaryocytic (Mk) differentiation. Here, we demonstrate two novel nuclear localization signals (NLSs) within Fli-1: one (NLS1) is located at the N terminus, and another (NLS2) is within the Ets domain. Nuclear accumulation of Fli-1 reflected the combined functional effects of the two discrete NLSs. Each NLS can independently direct nuclear transport of a carrier protein, with mutations within the NLSs affecting nuclear accumulation. NLS1 has a bipartite motif, whereas the NLS2 region contains a nonclassical NLS. Both NLSs bind importin alpha (IMPalpha) and IMPbeta, with NLS1 and NLS2 being predominantly recognized by IMPalpha and IMPbeta, respectively. Fli-1 also contains one nuclear export signal. Leptomycin B abolished its cytoplasmic accumulation, showing CRM1 dependency. We demonstrate that Ets domain binding to specific target DNA effectively blocks IMP binding, indicating that the targeted DNA binding plays a role in localizing Fli-1 to its destination and releasing IMPs for recycling back to the cytoplasm. Finally, by analyzing full-length Fli-1 carrying NLS1, NLS2, and combined NLS1-NLS2 mutations, we conclude that two functional NLSs exist in Fli-1 and that each NLS is sufficient to target Fli-1 to the nucleus for activation of Mk-specific genes.

Active Transport, Cell Nucleus↗