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Results for “Nuclear Localization Signals”

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PubMed1989

A karyophilic signal sequence in adenovirus type 5 E1A is functional in Xenopus oocytes but not in somatic cells.

The carboxy-terminal five amino acids of the adenovirus type 5 E1A gene product are necessary and sufficient for this protein to become localized in the nuclei of somatic cells. In this report, we demonstrate that E1A contains a second nuclear localization signal, which resides within residues 140 to 185 and which functions in Xenopus oocytes but not Xenopus or mammalian somatic cells. These data demonstrate the cell-type-specific utilization of a nuclear localization signal.

Adenovirus Early Proteins↗

PubMed2002

Control of intracellular dynamics of mammalian period proteins by casein kinase I epsilon (CKIepsilon) and CKIdelta in cultured cells.

Recent studies have shown that casein kinase I epsilon (CKIepsilon) is an essential regulator of the mammalian circadian clock. However, the detailed mechanisms by which CKIepsilon regulates each component of the circadian negative-feedback loop have not been fully defined. We show here that mPer proteins, negative limbs of the autoregulatory loop, are specific substrates for CKIepsilon and CKIdelta. The CKI phosphorylation of mPer1 and mPer3 proteins results in their rapid degradation, which is dependent on the ubiquitin-proteasome pathway. Moreover, CKIepsilon and CKIdelta are able to induce nuclear translocation of mPer3, which requires its nuclear localization signal. The mutation in potential phosphorylation sites on mPer3 decreased the extent of both nuclear translocation and degradation of mPer3 that are stimulated by CKIepsilon. CKIepsilon and CKIdelta affected the inhibitory effect of mPer proteins on the transcriptional activity of BMAL1-CLOCK, but the inhibitory effect of mCry proteins on the activity of BMAL1-CLOCK was unaffected. These results suggest that CKIepsilon and CKIdelta regulate the mammalian circadian autoregulatory loop by controlling both protein turnover and subcellular localization of mPer proteins.

ARNTL Transcription Factors↗

PubMed1998

Cloning, characterization, and chromosomal localization of human superillin (SVIL).

Supervillin is a 205-kDa F-actin binding protein originally isolated from bovine neutrophils. This protein is tightly associated with both actin filaments and plasma membranes, suggesting that it forms a high-affinity link between the actin cytoskeleton and the membrane. Human supervillin cDNAs cloned from normal human kidney and from the cervical carcinoma HeLa S3 predict a bipartite structure with three potential nuclear localization signals in the NH2-terminus and three potential actin-binding sequences in the COOH-terminus. In fact, throughout its length, the COOH-terminal half of supervillin is similar to segments 2-6 plus the COOH-terminal "headpiece" of villin, an actin-binding protein in intestinal microvilli. A comparison of the bovine and human sequences indicates that supervillin is highly conserved at the amino acid level, with 79.2% identity of the NH2-terminus and conservation of three of the four nuclear localization signals found in bovine supervillin. The COOH-terminus is even more conserved, with 95.1% amino acid identity overall and 100% conservation of the villin-like headpiece. Supervillin mRNAs are expressed in all human tissue tested, bu are most abundant in muscle, bone marrow, thyroid gland, and salivary gland; comparatively little message is found in brain. Human supervillin mRNA is approximately 7.5 kb; this message is especially abundant in HeLa S3 cervical carcinoma, SW480 adenocarcinoma, and A549 lung carcinoma cell lines. The human supervillin gene (SVIL) is localized to a single chromosomal locus at 10p11.2, a region that is deleted in some prostate tumors.

Amino Acid Sequence↗

PubMed2006

PINCH-1 expression during early avian embryogenesis: implications for neural crest and heart development.

The invasion of the cardiac neural crest (CNC) into the outflow tract (OFT) and subsequent OFT septation are critical events during vertebrate heart development. We previously had performed four modified differential display (DD) screens in the chick embryo to identify genes that may be involved in CNC and heart development. Full-length sequence of one of the DD clones has been obtained and identified as chick PINCH-1. This particularly interesting new cysteine-histidine-rich protein contains five protein-binding LIM domains (five double zinc fingers), a nuclear localization signal, and a nuclear export signal, allowing it to participate in integrin and growth factor signaling and possibly act as a transcription factor. We show here for the first time that chick PINCH-1 is expressed in neural crest cells, both in the neural fold and cardiac OFT, and is also expressed in mesoderm derived-structures, including the myocardium, during avian embryogenesis. The normal expression pattern and overexpression in neural crest cell explants suggest that PINCH-1 may be a regulator of neural crest cell adhesion and migration.

Amino Acid Sequence↗

PubMed1994

In vitro nuclear import of snRNPs: cytosolic factors mediate m3G-cap dependence of U1 and U2 snRNP transport.

We have established an in vitro snRNP nuclear import system using digitonin permeabilized somatic cells supplemented with cytosolic extracts. As model karyophiles we used digoxygenin labelled U1 snRNPs or fluorescein labelled U2 snRNPs. In vitro nuclear import of snRNPs is inhibited by anti-pore component antibodies, consistent with transport occurring through nuclear pores. This import requires ATP, cytosolic factors and a nuclear localization signal (NLS). SnRNP nuclear accumulation is saturable and distinct from protein transport. Nuclear import of snRNPs, in permeabilized NRK cells supplemented with somatic cell cytosol, requires the same NLS structures as those identified in micro-injected mammalian cells. In contrast to the situation in Xenopus oocytes, the m3G-cap is not required for in vitro nuclear import of U1 and U2 snRNPs in somatic cells. Instead, assembly of the Sm-core domain is both necessary and sufficient to mediate snRNP nuclear targeting. Interestingly, when the in vitro system was provided with cytosol from Xenopus oocytes instead of somatic cells, U1 and U2 snRNP nuclear import was provided with cytosol from Xenopus oocytes instead of somatic cells, U1 and U2 snRNP nuclear import was m3G-cap dependent. These results indicate that soluble cytosolic factors mediate the differential m3G-cap dependence of U1 and U2 snRNP nuclear import in somatic cells and oocytes. We also demonstrate the existence of a soluble cytosolic factor whose interaction with the U2 snRNP m3G-cap is both saturable and essential for U2 snRNP nuclear import in Xenopus oocytes.

Amino Acid Sequence↗

PubMed1994

p40MO15 associates with a p36 subunit and requires both nuclear translocation and Thr176 phosphorylation to generate cdk-activating kinase activity in Xenopus oocytes.

p40MO15, a cdc2-related protein, is the catalytic subunit of the kinase (CAK, cdk-activating kinase) responsible for Thr161/Thr160 phosphorylation and activation of cdk1/cdk2. We have found that strong overexpression of p40MO15 only moderately increases CAK activity in Xenopus oocytes, indicating that a regulatory CAK subunit (possibly a cyclin-like protein) limits the ability to generate CAK activity in p40MO15 overexpressing oocytes. This 36 kDa subunit was microsequenced after extensive purification of CAK activity. Production of Xenopus CAK activity was strongly reduced in enucleated oocytes overexpressing p40MO15 and p40MO15 shown to contain a nuclear localization signal required for nuclear translocation and generation of CAK activity. p40MO15 was found to be phosphorylated on Ser170 and Thr176 by proteolytic degradation, radiosequencing of tryptic peptides and mutagenesis. Thr176 phosphorylation is required and Ser170 phosphorylation is dispensable for p40MO15 to generate CAK activity upon association with the 36 kDa regulatory subunit. Finally, Thr176 and Ser170 phosphorylations are not intramolecular autophosphorylation reactions. Taken together, the above results identify protein-protein interactions, nuclear translocation and phosphorylation (by an unidentified kinase) as features of p40MO15 that are required for the generation of active CAK.

Amino Acid Sequence↗

PubMed1999

The ins and outs of HIV Rev.

The Rev protein of the human immunodeficiency virus mediates the nuclear export of the intron-containing viral messages. This export is a consequence of the continuous shuttling of HIV Rev between the nucleus and cytoplasm. This shuttling is mediated by a nuclear localization signal and a nuclear export signal contained within Rev. Recently, several factors which are required for the movement of Rev through the nuclear pore have been identified. This review will focus on these factors and their role the nucleocytoplasmic shuttling of HIV Rev.

Cell Nucleus↗

PubMed1998

Induction of a regular nuclear lattice by overexpression of NuMA.

Transient overexpression of nuclear mitotic apparatus protein (NuMA) in HeLa cells results in ordered lattices which can fill the nucleus and which are stable to detergent extraction. Electron microscopy reveals a quasi-hexagonal organization with an average spacing between the vertices of approximately 170 nm and short 6-nm-diameter rods connecting the vertices. Overexpression of a NuMA construct with an in-frame addition in the coiled-coil domain shows hexagons with the spacing increased by 42% while constructs with deletions in the coiled-coil domain yield hexagons with the spacing decreased by 40 and 19%. NuMA constructs truncated at residue 2005 or 2030 in the tail domain cause a drastic reorganization of nuclear components with relocation of the DNA, histone H1, and nucleoli to the nuclear rim. A construct lacking the head and much of the coiled-coil region also affects nuclear organization. In contrast, NuMA constructs truncated at residue 1950 or 1935 which lack the nuclear localization signal display normal nuclear structure but form cytoplasmic aggregates which also display hexagonal organization. Immunoelectron microscopy confirms that the nuclear lattices are built from NuMA. We discuss the importance of the different domains of NuMA for building the ordered in vivo lattices and whether NuMA could play a structural role in the architecture of the normal interphase nucleus.

Antigens, Nuclear↗

PubMed2003

Nuclear localization and transcriptional repression are confined to separable domains in the circadian protein CRYPTOCHROME.

Circadian rhythms are driven by molecular clocks composed of interlocking transcription/translation feedback loops. CRYPTOCHROME (CRY) proteins are critical components of these clocks and repress the activity of the transcription factor heterodimer CLOCK/BMAL1. Unlike the homologous DNA repair enzyme 6-4 PHOTOLYASE, CRYs have extended carboxyl-terminal tails and cannot repair DNA damage (reviewed in ). Unlike mammals, Xenopus laevis contains both CRYs (xCRYs) and 6-4 PHOTOLYASE (xPHOTOLYASE), providing an excellent comparative tool to study CRY repressive function. We can extend findings to CRYs in general because xCRYs share high sequence homology with mammalian CRYs. We show here that deletion of xCRYs' C-terminal domain produces proteins that are, like xPHOTOLYASE, unable to suppress CLOCK/BMAL1 activation. However, these truncations also cause the proteins to be cytoplasmically localized. A heterologous nuclear localization signal (NLS) restores the truncation mutants' nuclear localization and repressive activity. Our results demonstrate that the CRYs' C termini are essential for nuclear localization but not necessary for the suppression of CLOCK/BMAL1 activation; this finding indicates that these two functions reside in separable domains. Furthermore, the functional differences between CRYs and PHOTOLYASE can be attributed to the few amino acid changes in the conserved portions of these proteins.

Amino Acid Sequence↗

PubMed2006

The nuclear accumulation of the Fanconi anemia protein FANCE depends on FANCC.

The Fanconi anemia (FA) protein FANCE is an essential component of the nuclear FA core complex, which is required for monoubiquitination of the downstream target FANCD2, an important step in the FA pathway of DNA cross-link repair. FANCE is predominantly localized in the nucleus and acts as a molecular bridge between the FA core complex and FANCD2, through direct binding of both FANCC and FANCD2. At present, it is poorly understood how the nuclear accumulation of FANCE is regulated and therefore we investigated the nuclear localization of this FA protein. We found that FANCE has a strong tendency to localize in the nucleus, since the addition of a nuclear export signal does not interfere with the nuclear localization of FANCE. We also demonstrate that the nuclear accumulation of FANCE does not rely solely on its nuclear localization signal motifs, but also on FANCC. The other FA proteins are not involved in the nuclear accumulation of FANCE, indicating a tight relationship between FANCC and FANCE, as suggested from their direct interaction. Finally, we show that the region of FANCE interacting with FANCC appears to be different from the region involved in binding FANCD2. This strengthens the idea that FANCE recruits FANCD2 to the core complex, without interfering with the binding of FANCC.

Active Transport, Cell Nucleus↗

PubMed1998

Molecular cloning and characterization of a human brain-specific gene implicated in neuronal differentiation.

A 2.5 kb human cDNA clone containing a CAG trinucleotide repeat, designated HB20, was isolated from a human fetal brain library. Northern analysis on multi-tissue blots and various cell lines confirmed that HB20 is specifically expressed in the brain. Its expression is low in two glioma cells, moderate in a neuron precursor cell, NT2, but absent in lymphoma, cervical carcinoma, or colonic carcinoma cells. Significant increase of HB20 mRNA was shown along with retinoic acid-induced terminal differentiation of NT2 cells into neuron cells, hNT. Homology comparison of the predicted HB20 amino acid sequence with the current database revealed that it belongs to a newly recognized protein family composed of nucleosome assembly proteins and SET proto-oncogene, which has been shown to interact specifically with B-type cyclins involved in the control of cell proliferation. Together with the detection of nuclear localization signals and apparent nuclear distribution of expressed protein, HB20 is likely to be a brain-specific nuclear protein, functioning in the process of neuronal differentiation.

Amino Acid Sequence↗

PubMed2002

Genomic organization and chromosomal localization of the importin alpha1 gene in the mouse.

Importin alpha1 (also referred to as NPI1 or importin alphaS1) gene encodes a member of the cytosolic receptor protein superfamily that recognizes classical monopartite and bipartite basic type nuclear localization signals and mediates nuclear protein import via an importin beta-dependent pathway. Here we report on the organization of the importin alpha1 locus in the mouse genome. The gene is approximately 40 kb in length from the translation initiation codon to the poly(A) additional site. The translated region of the gene is comprised of 13 coding exons and the exon-intron boundaries conform to the GT/AG rule. Importin alpha1 was mapped to a middle region of mouse chromosome 16 by fluorescence in situ hybridization analysis. Moreover, it was found by reverse transcriptase polymerase chain reaction analysis that importin alpha1 is widely expressed in various tissues in adult mice and at various stages during embyogenesis. This study is the first example that provides detailed genomic information on nuclear transport factors such as importins and exportins and provides a basis for further studies such as the generation of mutants in mice for purposes of investigating the role of importin alpha1 in development and differentiation.

Animals↗

PubMed2002

Hepatitis delta virus ribonucleoproteins shuttle between the nucleus and the cytoplasm.

Hepatitis delta virus (HDV) infection of individuals infected with hepatitis B virus (HBV) is associated with more severe liver damage and an increased risk of fulminant disease. HDV is a single-stranded RNA virus that encodes a single protein, the delta antigen, which is expressed in two forms, small (S-HDAg) and large (L-HDAg). Here we show that although HDV ribonucleoproteins are mainly detected in the nucleus, they are also present in the cytoplasm of cells infected with HDV or transfected with HDV cDNA. Making use of an heterokaryon assay, we demonstrate that HDV ribonucleoproteins shuttle continuously between the nucleus and the cytoplasm. In the absence of HDV RNA, both forms of the delta antigen are retained in the nucleus, whereas in the absence of the delta antigen, HDV RNA is predominantly detected in the cytoplasm. Coexpression of HDV RNA and S-HDAg (which binds to the viral RNA and contains a nuclear localization signal) results in nuclear accumulation of the viral RNA. This suggests that HDV RNA mediates export of viral particles to the cytoplasm whereas the delta antigen triggers their reimport into the nucleus.

3T3 Cells↗

PubMed2006

Identification of nuclear import mechanisms for the neuronal Cdk5 activator.

The activation of Cdk5 by p35 plays a pivotal role in a multitude of nervous system activities ranging from neuronal differentiation to degeneration. A fraction of Cdk5 and p35 localizes in the nucleus where Cdk5-p35 exerts its functions via protein phosphorylation, and p35 displays a dynamic localization between the cytoplasm and the nucleus. Here, we examined the nuclear import properties of p35. In nuclear import assays, p35 was actively transported into the nuclei of digitonin-permeabilized HeLa cells and cortical neurons by cytoplasmic carrier-mediated mechanisms. Importin-beta, importin-5, and importin-7 were identified to import p35 into the nuclei via a direct interaction with it. An N-terminal region of p35 was defined to interact with the above importins, serving as a nuclear localization signal. Finally, we show that the nuclear localization of p35 does not require the association of Cdk5. Furthermore, Cdk5 and importin-beta/5/7 are mutually exclusive in binding to p35. These results suggest that p35 employs pathways distinct from that used by Cdk5 for transport to the nucleus.

Active Transport, Cell Nucleus↗

PubMed2006

Nucleocytoplasmic shuttling of the NAD+-dependent histone deacetylase SIRT1.

Sir2 (silent information regulator 2) is an NAD(+)-dependent histone deacetylase that contributes to longevity in yeast. SIRT1, a mammalian Sir2 ortholog, deacetylates histones and various transcription factors, including p53, FOXO proteins, and peroxisome proliferator-activated receptor-gamma. We found that its subcellular localization varied in different tissues of the adult mouse. Some subsets of neurons predominantly expressed SIRT1 in the cytoplasm, but ependymal cells expressed it in both the nucleus and cytoplasm. On the other hand, spermatocytes expressed SIRT1 only in the nucleus. Cardiomyocytes in the day 12.5 mouse embryo expressed SIRT1 exclusively in the nucleus, but in the adult heart, they expressed it in both the cytoplasm and nucleus. C2C12 myoblast cells expressed SIRT1 in the nucleus, but it localized to the cytoplasm after differentiation. LY294002, an inhibitor of phosphoinositide 3-hydroxykinase, strongly inhibited the nuclear localization of SIRT1 in undifferentiated C2C12 cells. In a heterokaryon assay, SIRT1 shuttled between the nucleus and cytoplasm, and leptomycin B, an inhibitor of CRM1-mediated nuclear exportation, inhibited this shuttling. Two nuclear localization signals and two nuclear export signals were identified by deletion and site-directed mutation analyses. Overexpressed nuclear (but not cytoplasmic or dominant-negative) SIRT1 enhanced the deacetylation of histone H3 in C2C12 cells. Moreover, only the nuclear form suppressed the apoptosis of C2C12 cells induced by antimycin A, an oxidative stressor. These findings indicate that nucleocytoplasmic shuttling is a novel regulatory mechanism of SIRT1, which may participate in differentiation and in inhibition of cell death.

Active Transport, Cell Nucleus↗

PubMed2000

APC-mediated downregulation of beta-catenin activity involves nuclear sequestration and nuclear export.

Mutational inactivation of adenomatous polyposis coli (APC) initiates most colon carcinomas. APC functions include targeting cytoplasmic beta-catenin, a Wnt pathway mediator, for proteolysis. Although APC shuttles between cytoplasm and nucleus, the role of nuclear APC protein, particularly with respect to nuclear beta-catenin levels and activity, remains unclear. Here, we demonstrate that APC lacking functional nuclear localization signals (NLSs) or nuclear export signals (NESs) does not effectively downregulate nuclear beta-catenin levels; neither does wild-type APC when nuclear export is blocked. While APC bearing mutated NLSs could not downregulate beta-catenin-mediated transcriptional activation, APC lacking NESs remained active. Consistent with the hypothesis that nuclear APC lacking NESs can inhibit beta-catenin function by sequestration, we show that endogenous APC and beta-catenin proteins interact within the nucleus. These data demonstrate that nuclear APC binding to beta-catenin, and then inducing its nuclear export, plays a critical role in the control of nuclear beta-catenin levels and activity.

Adenomatous Polyposis Coli↗

PubMed1997

An alternative pathway for gene regulation by Myc.

The c-Myc protein activates transcription as part of a heteromeric complex with Max. However, Myc-transformed cells are characterized by loss of expression of several genes, suggesting that Myc may also repress gene expression. Two-hybrid cloning identifies a novel POZ domain Zn finger protein (Miz-1; Myc-interacting Zn finger protein-1) that specifically interacts with Myc, but not with Max or USF. Miz-1 binds to start sites of the adenovirus major late and cyclin D1 promoters and activates transcription from both promoters. Miz-1 has a potent growth arrest function. Binding of Myc to Miz-1 requires the helix-loop-helix domain of Myc and a short amphipathic helix located in the carboxy-terminus of Miz-1. Expression of Myc inhibits transactivation, overcomes Miz-1-induced growth arrest and renders Miz-1 insoluble in vivo. These processes depend on Myc and Miz-1 association and on the integrity of the POZ domain of Miz-1, suggesting that Myc binding activates a latent inhibitory function of this domain. Fusion of a nuclear localization signal induces efficient nuclear transport of Miz-1 and impairs the ability of Myc to overcome transcriptional activation and growth arrest by Miz-1. Our data suggest a model for how gene repression by Myc may occur in vivo.

3T3 Cells↗

PubMed2003

Ehp53, an Entamoeba histolytica protein, ancestor of the mammalian tumour suppressor p53.

This paper reports the identification of Ehp53, a p53-like Entamoeba histolytica protein, which binds to the human p53 DNA consensus sequence (oli-p53). Monoclonal antibodies against p53 (Ab-1 and Ab-2) recognized a single 53 kDa spot in two-dimensional gels and inhibited the formation of complexes produced by E. histolytica nuclear extracts and oli-p53. Additionally, E. histolytica gene promoter sequences with high homology to oli-p53 formed complexes with nuclear proteins that were abolished by oli-p53. Ehp53 protein levels increased in UV-irradiated trophozoites. This protein was also detected in Entamoeba moshkovskii and Entamoeba invadens. By confocal microscopy, Ehp53 was located in the nuclei, EhkO organelles and cytoplasm. The Ehp53-encoding gene was cloned and its predicted amino acid sequence showed 30-54 % and 50-57 % homology with important domains of the human and the Drosophila melanogaster p53 proteins, respectively. This homology included the tetramerization domain, the nuclear export signal and a nuclear localization signal. Ehp53 also contains seven of the eight DNA-binding residues and two of the four Zn(2+)-binding sites described for p53. A recombinant Ehp53 was recognized by Ab-2. Ehp53 is believed to be the first p53-like protein found in protozoa and may be the evolutionary ancestor of the mammalian p53.

Amino Acid Sequence↗