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Results for “Nuclear Export Signals”

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At least 703 records · Page 39Linked to original sources
PubMed2004

Phosphorylation by the beta-catenin/MAPK complex promotes 14-3-3-mediated nuclear export of TCF/POP-1 in signal-responsive cells in C. elegans.

In C. elegans embryos, a Wnt/MAPK signaling pathway downregulates the TCF/LEF transcription factor POP-1, resulting in a lower nuclear level in signal-responsive cells compared to their sisters. Although the beta-catenin WRM-1 is required for POP-1 downregulation, a direct interaction between these two proteins does not seem to be required, as the beta-catenin-interacting domain of POP-1 is dispensable for both POP-1 downregulation and function in early embryos. We show here that WRM-1 downregulates POP-1 by promoting its phosphorylation by the MAP kinase LIT-1 and subsequent nuclear export via a 14-3-3 protein, PAR-5. In signal-responsive cells, we also detect a concurrent upregulation of nuclear LIT-1 that is dependent on Wnt/MAPK signaling. Our results suggest a model whereby Wnt/MAPK signaling downregulates POP-1 levels in responsive cells, in part by increasing nuclear LIT-1 levels, thereby increasing POP-1 phosphorylation and PAR-5-mediated nuclear export.

14-3-3 Proteins↗
PubMed2005

Stress kinase signaling regulates androgen receptor phosphorylation, transcription, and localization.

Activation of signal transduction kinase cascades is known to alter androgen receptor (AR) activity, but the molecular mechanisms are still poorly defined. Here we show that stress kinase signaling regulates Ser 650 phosphorylation and AR nuclear export. In LNCaP prostate cancer cells, activation of either MAPK kinase (MKK) 4:c-Jun N-terminal kinase (JNK) or MKK6:p38 signaling pathways increased Ser 650 phosphorylation, whereas pharmacologic inhibition of JNK or p38 signaling led to a reduction of AR Ser 650 phosphorylation. Both p38alpha and JNK1 phosphorylated Ser 650 in vitro. Small interfering RNA-mediated knockdown of either MKK4 or MKK6 increased endogenous prostate-specific antigen (PSA) transcript levels, and this increase was blocked by either bicalutamide or AR small interfering RNA. Stress kinase inhibition of PSA transcription is, therefore, dependent on the AR. Similar experiments involving either activation or inhibition of MAPK/ERK kinase:ERK signaling had little effect on Ser 650 phosphorylation or PSA mRNA levels. Ser 650 is proximal to the DNA binding domain that contains a nuclear export signal. Mutation of Ser 650 to alanine reduced nuclear export of the AR, whereas mutation of Ser 650 to the phosphomimetic amino acid aspartate restored AR nuclear export. Pharmacologic inhibition of stress kinase signaling reduced wild-type AR nuclear export equivalent to the S650A mutant without affecting nuclear export of the S650D mutant. Our data suggest that stress kinase signaling and nuclear export regulate AR transcriptional activity.

Active Transport, Cell Nucleus↗
PubMed1999

The novel human HUEL (C4orf1) gene maps to chromosome 4p12-p13 and encodes a nuclear protein containing the nuclear receptor interaction motif.

A 3250-bp novel human cDNA sequence was isolated from the MRC-5 human embryonic lung cell line by the rapid amplification of cDNA ends technique. This gene was designated HUEL and given the symbol C4orf1 by the HUGO Nomenclature Committee. Within HUEL was identified a continuous ORF of 1704 bp encoding a predicted hydrophilic protein of 568 amino acids with a calculated molecular mass of 63,410 Da. The putative protein contains the LXXLL signature motif considered necessary and sufficient for binding of certain coactivators to liganded nuclear receptors, as well as nuclear localization signals, a nuclear export-like signal, a zinc finger-like motif, an acidic region, and two leucine zipper-like domains. Northern blot analysis of human fetal tissues revealed 3. 4-kb transcripts, while RT-PCR demonstrated HUEL expression in a wide range of human adult tissues and cancer cell lines. In the SiHa, HT-1080, and G-401 cancer lines was detected an alternative transcript in which a 166-bp segment was excluded by exon skipping, which is predicted to culminate in a protein with a modified and truncated C-terminus. HUEL was localized to chromosome region 4p12-p13 by fluorescence in situ hybridization. In Western blots, affinity-purified antibodies raised against a HUEL-specific synthetic peptide could recognize a distinct protein band of approximately 70 kDa. Immunoblotting of subcellular fractions and indirect immunofluorescence of human embryonic lung cells demonstrated the distribution of HUEL predominantly in the cytoplasm, with an apparently cytoskeletal association. However, in smaller or dividing PLC/PRF/5 and TONG liver carcinoma cells, there was a translocation of HUEL from the cytoplasm to the nucleus. Taken together, these data suggest that HUEL plays a role in transcriptional regulation.

Adult↗
PubMed2005

Nuclear targeting of transforming growth factor-beta-activated Smad complexes.

Upon stimulation by the transforming growth factor beta (TGF-beta), Smad2 and Smad3 are phosphorylated at their C termini and assemble into stable heteromeric complexes with Smad4. These complexes are the functional entities that translocate into the nucleus and regulate the expression of TGF-beta target genes. Here we report that the TGF-beta-activated phospho-Smad3/Smad4 complex utilizes an importin-independent mechanism for nuclear import and engages different nucleoporins for nuclear import compared with the monomeric Smad4. Within the heteromeric complex, phospho-Smad3 appears to dominate over Smad4 in the nuclear import process and guides the complex to its nuclear destination. We also demonstrate that the binding of phospho-Smad3 to Smad4 prevents Smad4 from interacting with the nuclear export receptor chromosome region maintenance 1. In this way, TGF-beta signaling suppresses nuclear export of Smad4 by chromosome region maintenance 1 and thereby targets Smad4 into the nucleus. Indeed tumorigenic mutations in Smad4 that affect its interaction with Smad2 or Smad3 impair nuclear accumulation of Smad4 in response to TGF-beta.

Active Transport, Cell Nucleus↗
PubMed2006

Nuclear export of retinoid X receptor alpha in response to interleukin-1beta-mediated cell signaling: roles for JNK and SER260.

As the obligate heterodimer partner to class II nuclear receptors, the retinoid X receptor alpha (RXRalpha) plays a vital physiological role in the regulation of multiple hepatic functions, including bile formation, intermediary metabolism, and endobiotic/xenobiotic detoxification. Many RXRalpha-regulated genes are themselves suppressed in inflamed liver via unknown mechanisms, which constitute a substantial component of the negative hepatic acute phase response. In this study we show that RXRalpha, generally considered a stable nuclear resident protein, undergoes rapid nuclear export in response to signals initiated by the pro-inflammatory cytokine interleukin-1beta (IL-1beta), a central activator of the acute phase response. Within 30 min of exposure to IL-1beta, nuclear levels of RXRalpha are markedly suppressed in human liver-derived HepG2 cells, temporally coinciding with its appearance in the cytoplasm. The nuclear residence of RXRalpha is maintained by inhibiting c-jun N-terminal kinase (JNK, curcumin or SP600125) or CRM-1-mediated nuclear export (Leptomycin B). Pretreatment with the proteasome inhibitor MG132 blocks IL-1beta-mediated reductions in nuclear RXRalpha levels while increasing accumulation in the cytoplasm. Mutational studies identify one residue, serine 260, a JNK phosphoacceptor site whose phosphorylation status had an unknown role in RXRalpha function, as critical for IL-1beta-mediated nuclear export of transfected human RXRalpha-green fluorescent fusion constructs. These findings indicate that inflammation-mediated cell signaling leads to rapid and profound reductions in nuclear RXRalpha levels, via a multistep, JNK-dependent mechanism involving Ser260, nuclear export, and proteasomal degradation. Thus, inflammation-meditated cell signaling targets RXRalpha for nuclear export and degradation; a potential mechanism that explains the broad suppression of RXRalpha-dependent gene expression in the inflamed liver.

Active Transport, Cell Nucleus↗
PubMed2005

Insights into a CRM1-mediated RNA-nuclear export pathway in Trypanosoma cruzi.

Nuclear export and import of proteins and RNAs is a regulated process that permits the control of protein expression during cell development and differentiation. In all eukaryotic organisms transport of proteins to specific cellular compartments requires specific signaling sequences. Proteins that shuttle between nucleus and cytoplasm bear nuclear localization signals (NLS) and/or nuclear export signals (NES) and some of them can carry mRNAs, as part of shuttling ribonucleoprotein complexes. In this work we describe in the protozoan parasite Trypanosoma cruzi, a CRM1/exportin1 nuclear export factor named TcCRM1. This protein contains the conserved central region (CCR) that interacts with NES sequences present within cargo molecules, and the Cys residue involved in covalent binding to the Streptomyces metabolite leptomycin B (LMB). By subcellular fractionation we show that TcCRM1, a protein of about 117 kDa, has nuclear localization. We also demonstrate that LMB inhibits the replication of T. cruzi in a dose-dependent manner. In situ hybridization experiments performed with a Texas red-coupled oligo(dT) probe revealed that LMB produced a partial short-term accumulation of a poly(A)+RNA subset in the nucleus. Some mRNAs such as HSP70, TcUBP2/1 and TcPABP1 are reduced or disappeared from the cytoplasm of LMB treated cells. In sharp contrast with metazoans, no effect was observed on two U snRNAs subcellular localization, implying that a different export route might exist for these RNAs in trypanosomes.

Active Transport, Cell Nucleus↗
PubMed2025

Multiplexed Dual-Color Fluorescence-Based Distinction Between Nuclear Trapping and Translocation of FOXO3.

FOXO3 is a transcription factor that mainly exerts its functions in the cell nucleus. The amino acid sequence of FOXO3 contains a nuclear localization sequence (NLS) and a nuclear export sequence (NES) allowing for nuclear/cytoplasmic shuttling that plays an important role in regulating FOXO3 activity. Nuclear accumulation of FOXO3 proteins can be the result of translocation to the nucleus triggered by upstream regulatory input or trapping of FOXO3 within the nucleus through the inhibition of its nuclear export via the receptor CRM1. In order to distinguish these two modes of FOXO3 activation, we have generated a multiplexed assay. The development of this platform includes a reporter cell line that monitors CRM1 activity by using RFP-labeled HIV-1 Rev. protein with a strong heterologous NES. Simultaneously, the intracellular localization of FOXO3 can be monitored by a second cell line stably expressing GFP-FOXO3. Here we describe a detailed protocol on how to co-culture these reporter cell lines and use them to interrogate compound-induced FOXO3 activation in order to understand the mode of action.

Forkhead Box Protein O3↗
PubMed1996

Exogenously injected nuclear import factor p10/NTF2 inhibits signal-mediated nuclear import and export of proteins in living cells.

p10/NTF2 is a cytosolic factor which is required for the translocation step in nuclear protein import in an in vitro assay with digitonin-permeabilized cells. To study the functional roles of p10/NTF2 on protein transport between the nucleus and cytoplasm in living cells, recombinant p10/NTF2 was micro-injected into cultured mammalian cells. Cytoplasmically injected p10/NTF2 strongly inhibited the nuclear import of co-injected NLS-containing substrates in a dose-dependent manner but had no effect on the diffusive import of small non-nuclear proteins. Moreover, when injected into the cell nucleus, p10/NTF2 inhibited the nuclear export of NES-containing substrates. The results suggest that the nuclear import factor p10/NTF2 may also be involved in the nuclear export of proteins and that the protein transport efficiency between the nucleus and cytoplasm may be regulated by the intracellular level of p10/NTF2.

Animals↗
PubMed2000

SAC3 may link nuclear protein export to cell cycle progression.

Selective movement of proteins between the nucleus and the cytoplasm is a regulatory mechanism exploited extensively by the eukaryotic cell. We have identified the evolutionarily conserved Sac3 protein, which was implicated previously in the regulation of mitosis [Bauer, A. & Kölling, R. (1996) J. Cell Sci. 109, 1575-1583] as a novel mediator of nuclear protein export. We show that Sac3p is localized to the nuclear pore, where it interacts with nucleoporins. Loss of SAC3 function results in a block in nuclear export of a nuclear export signal-containing reporter protein. Our results also demonstrate that SAC3 interacts genetically with the nuclear protein export factors Crm1p/Xpo1p and Yrb2p. Taken together, these data indicate a link between nuclear protein export and transition through the cell cycle.

Amino Acid Sequence↗
PubMed2001

Regulation of p53 localization.

Despite intensive study of p53, the regulation of p53 cellular localization is still poorly understood. This is an overview of the elements and molecules involved in p53 nucleocytoplasmic transportation. These include the nuclear import and export signals of p53, inhibition of p53 nuclear import and export by oligomerization, MDM2-mediated p53 nuclear export, and possible roles of p53 phosphorylation in regulating p53 cellular localization. Finally, questions regarding p53 cellular trafficking will also be discussed.

Amino Acid Sequence↗
PubMed2006

Nuclear assembly of UGA decoding complexes on selenoprotein mRNAs: a mechanism for eluding nonsense-mediated decay?

Recoding of UGA from a stop codon to selenocysteine poses a dilemma for the protein translation machinery. In eukaryotes, two factors that are crucial to this recoding process are the mRNA binding protein of the Sec insertion sequence, SBP2, and the specialized elongation factor, EFsec. We sought to determine the subcellular localization of these selenoprotein synthesis factors in mammalian cells and thus gain insight into how selenoprotein mRNAs might circumvent nonsense-mediated decay. Intriguingly, both EFsec and SBP2 localization differed depending on the cell line but significant colocalization of the two proteins was observed in cells where SBP2 levels were detectable. We identify functional nuclear localization and export signals in both proteins, demonstrate that SBP2 undergoes nucleocytoplasmic shuttling, and provide evidence that SBP2 levels and localization may influence EFsec localization. Our results suggest a mechanism for the nuclear assembly of the selenocysteine incorporation machinery that could allow selenoprotein mRNAs to circumvent nonsense-mediated decay, thus providing new insights into the mechanism of selenoprotein translation.

Active Transport, Cell Nucleus↗
PubMed1997

A role for the M9 transport signal of hnRNP A1 in mRNA nuclear export.

Among the nuclear proteins associated with mRNAs before their export to the cytoplasm are the abundant heterogeneous nuclear (hn) RNPs. Several of these contain the M9 signal that, in the case of hnRNP A1, has been shown to be sufficient to signal both nuclear export and nuclear import in cultured somatic cells. Kinetic competition experiments are used here to demonstrate that M9-directed nuclear import in Xenopus oocytes is a saturable process. Saturating levels of M9 have, however, no effect on the import of either U snRNPs or proteins carrying a classical basic NLS. Previous work demonstrated the existence of nuclear export factors specific for particular classes of RNA. Injection of hnRNP A1 but not of a mutant protein lacking the M9 domain inhibited export of mRNA but not of other classes of RNA. This suggests that hnRNP A1 or other proteins containing an M9 domain play a role in mRNA export from the nucleus. However, the requirement for M9 function in mRNA export is not identical to that in hnRNP A1 protein transport.

Animals↗
PubMed1997

The location of the transport gate in the nuclear pore complex.

Signal-mediated nuclear transport is a gated process that occurs through a central transporter element located within the pore complex. The purpose of this investigation was to identify the region of the transporter that functions as the gate; i.e. the region that restricts passive diffusion of macromolecules through the pores. To accomplish this, small gold particles coated with polyethylene glycol (PEG; total particle diameter 40-70 A) or large PEG-particles (total diameter 110-270 A) were microinjected into the cytoplasm or nucleoplasm of Xenopus oocytes. Since PEG does not contain either nuclear import or export signals, it is assumed that the particles distribute by simple diffusion. The cells were fixed after 5 or 30 minutes and subsequently examined using TEM. The distribution of the particles located adjacent to and within the pore complexes was then mapped. The results obtained at both 5 and 30 minutes after cytoplasmic injections of small gold were basically the same. The particles readily entered the transporter but, on the average, were approximately 11 times more concentrated in the cytoplasmic half of this structure. The opposite distribution was observed following nuclear injections, i.e. the particles that were located in the transporter were approximately 7 times more numerous in the nuclear half. Our data indicate that there is a single transport gate located in the central domain of the transporter that restricts passive diffusion. The large particles that were injected into the cytoplasm migrated to the surface of the pore complex, but entered the transporter less frequently than small gold. Interestingly, the diffusion of large PEG-particles to the surface of the pores following nuclear injection was greatly restricted; however, this was not the case for similar size particles that were coated with protein containing nuclear export signals (NES). The latter results suggest that the NES is not only required for translocation, but also for migration within the nucleoplasm.

Amino Acid Sequence↗
PubMed2005

Cytoplasmic tethering of a RING protein RBCK1 by its splice variant lacking the RING domain.

RBCC protein interacting with PKC 1 (RBCK1) is a transcription factor belonging to the RING-IBR protein family and has been shown to shuttle between the nucleus and cytoplasm, possessing both the nuclear export and localization signals within its amino acid sequence. RBCK2, lacking the C-terminal half of RBCK1 including the RING-IBR domain, has also been identified as an alternative splice variant of RBCK1. RBCK2 shows no transcriptional activity and instead it represses the transcriptional activity of RBCK1. Here, we show that RBCK2 is present usually in the cytoplasm containing two Leu-rich regions that presumably serve as a nuclear export signal (NES). Moreover, an NES-disrupted RBCK1 that is mostly localized within the nucleus is translocated to the cytoplasm when coexpressed with RBCK2, suggesting that RBCK2 serves as a cytoplasmic tethering protein for RBCK1. We propose a novel and general function of RING-lacking splice variants of RING proteins to control the intracellular localization and functions of the parental RING proteins by forming a hetero-oligomeric complex.

Alternative Splicing↗
PubMed2002

Tpr is localized within the nuclear basket of the pore complex and has a role in nuclear protein export.

Tpr is a coiled-coil protein found near the nucleoplasmic side of the pore complex. Since neither the precise localization of Tpr nor its functions are well defined, we generated antibodies to three regions of Tpr to clarify these issues. Using light and EM immunolocalization, we determined that mammalian Tpr is concentrated within the nuclear basket of the pore complex in a distribution similar to Nup153 and Nup98. Antibody localization together with imaging of GFP-Tpr in living cells revealed that Tpr is in discrete foci inside the nucleus similar to several other nucleoporins but is not present in intranuclear filamentous networks (Zimowska et al., 1997) or in long filaments extending from the pore complex (Cordes et al., 1997) as proposed. Injection of anti-Tpr antibodies into mitotic cells resulted in depletion of Tpr from the nuclear envelope without loss of other pore complex basket proteins. Whereas nuclear import mediated by a basic amino acid signal was unaffected, nuclear export mediated by a leucine-rich signal was retarded significantly. Nuclear injection of anti-Tpr antibodies in interphase cells similarly yielded inhibition of protein export but not import. These results indicate that Tpr is a nucleoporin of the nuclear basket with a role in nuclear protein export.

Active Transport, Cell Nucleus↗
PubMed2003

Nucleocytoplasmic transport signals affect the age at onset of abnormalities in knock-in mice expressing polyglutamine within an ectopic protein context.

In order to better understand the role of nuclear localization of polyglutamine in the human CAG repeat disorders, gene targeting was used to add either nuclear localization (NLS) or nuclear export (NES) signals to versions of the mouse Hprt protein containing expanded polyglutamine (HprtQ150). The NLS increased levels of nuclear HprtQ150 protein in the mouse brain and hastened both the presentation of neuronal intranuclear inclusions (NIIs) and the onset of behavioral abnormalities. The NES reduced levels of nuclear HprtQ150 protein in mouse brain and delayed both the presentation of NIIs and the onset of behavioral abnormalities. Together these results indicate the nucleus is the primary site of toxicity in HprtQ150 mice. Furthermore, the signals did not alter the relative regional distribution of NIIs, suggesting that factors other than nuclear access dictate the regional specificity of NII formation in this mouse model.

Active Transport, Cell Nucleus↗
PubMed2005

The highly structured encapsidation signal of MuLV RNA is involved in the nuclear export of its unspliced RNA.

The encapsidation signal (Psi) of retroviruses is located in the 5' UTR of the viral genomic unspliced transcript and is highly structured. In the Psi of murine leukaemia virus (MuLV), four stem-loops, called A, B, C and D, promote dimerization and encapsidation of the MuLV unspliced RNA into virions. Through analysis of Psi-deleted transcripts, we found that the AB and CD motifs independently enhanced the cytoplasmic accumulation of RNAs. Furthermore, we showed that over-expression of the Psi sequence in the infected cells led to a competition with the nuclear export of unspliced MuLV transcripts, revealing a new function for these stem-loops in the transport of viral intron-containing RNAs from the nucleus to the cytoplasm.

5' Untranslated Regions↗