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Results for “Nuclear Localization Signals”

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PubMed2004

Multiple domains control the subcellular localization and activity of ETR-3, a regulator of nuclear and cytoplasmic RNA processing events.

Embryonic lethal abnormal vision (ELAV) type RNA binding protein 3 (ETR-3; also called NAPOR, CUGBP2, or BRUNOL3) has been implicated in the regulation of nuclear and cytoplasmic RNA processing events, including alternative splicing, RNA editing, stability and translation. Here, we report that the ETR-3 protein contains multiple regions that control its subcellular localization and are important for its activity as a splicing regulator. We cloned ETR-3 from chicken heart and fused it to the C terminus of green fluorescent protein (GFPcETR3vL). GFPcETR3vL is found predominantly in the nucleus and is an active regulator of alternative splicing in cotransfection assays with a cardiac troponin T minigene. ETR-3 contains two N-terminal RNA recognition motifs (RRMs), a 210-amino acid divergent domain, and a C-terminal RRM. We demonstrate that the C terminus contains a strong nuclear localization signal overlapping the third RRM, which can confer nuclear localization on a normally cytoplasmic pyruvate kinase chimera. Additional deletions revealed nuclear localization and export activities in the divergent domain of ETR-3, as well as regions within the first two RRMs that are important for cytoplasmic localization. The nuclear export activity of the divergent domain is sensitive to leptomycin B, indicating that export to the cytoplasm is mediated via a CRM1-dependent pathway. The C terminus and a region within the divergent domain were also shown to be important for splicing activity of ETR-3. This is the first characterization of protein domains involved in mediating the subcellular localization and splicing activity of a member of the CELF family of RNA processing regulators.

Alternative Splicing↗

PubMed2006

Subcellular localization of CIAPIN1.

Cytokine-induced apoptosis inhibitor 1 (CIAPIN1) is a newly identified anti-apoptotic molecule. Our previous studies have demonstrated that CIAPIN1 is ubiquitously expressed in normal fetal and adult human tissues and confers multidrug resistance in gastric cancer cells, possibly by upregulating the expression of multidrug resistance gene 1 and multidrug resistance-related protein 1. However, fundamental biological functions of CIAPIN1 have not been elucidated. In this study, we first predicted the subcellular localization of CIAPIN1 with bioinformatic approaches and then characterized the intracellular localization of CIAPIN1 in both human and mouse cells by a combination of techniques including (a)immunohistochemistry and immunofluorescence, (b) His-tagged CIAPIN1 expression, and (c)subcellular fractionation and analysis of CIAPIN1 in the fractions by Western blotting. All methods produced consistent results; CIAPIN1 was localized in both the cytoplasm and the nucleus and was accumulated in the nucleolus. Bioinformatic prediction disclosed a putative nuclear localization signal and a putative nuclear export signal within both human and mouse CIAPIN1. These findings suggest that CIAPIN1 may undergo a cytoplasm-nucleus-nucleolus translocation.

Animals↗

PubMed2006

The diverging roles of calmodulin and PKC in the regulation of p21 intracellular localization.

Intracellular localization plays an important role in the functional regulation of the cyclin-dependent kinase inhibitor p21. While nuclear functions have been linked to the tumor suppressor activity of p21, cytoplasmatic functions are oncogenic. We have recently shown that Ser153 phosphorylation of p21 by PKC contributes to its cytoplasmatic accumulation, and that this phosphorylation is inhibited by Ca(2+)-dependent calmodulin binding to the C-terminal region of p21. Consequently, PKC and calmodulin/Ca(2+) play diverging roles in the regulation of p21 intracellular localization. Other kinases such as AKT and MIRK/dyrk1B also phosphorylate p21 near the nuclear localization signal, thus inhibiting its nuclear accumulation. We discuss here the effects of such phosphorylations on p21 functionality, as well as its relevance to cell cycle progression and differentiation.

Animals↗

PubMed2002

Subcellular localization of MURA and MURB proteins encoded by the maize MuDR transposon.

MuDR controls transposition of the Mu transposable element family in Zea mays L. It produces two major transcripts: mudrA and mudrB, mudrA encodes the MURA transposase, but no specific function has been ascribed to mudrB, which lacks strong homology to known genes. Using transient expression assays in onion epidermal cells, we defined three monopartite nuclear localization signals (NLSs) of MURA; each was functionally sufficient for nuclear targeting of MURA:GUS fusion proteins. Interestingly, one NLS (NLS-A3) is produced by the splicing of the third intron. In contrast, there were no clear NLS in MURB, and the major form of MURB aggregated in the cytoplasm. Self-interaction of MURA and of MURB was also shown in a yeast two-hybrid assay. To test whether interactions of MURA and MURB can occur at the level of protein translocation into the nucleus, a cytoplasmically localized MURB:GFP was co-expressed with MURA or with the GUS fusion proteins. Co-expression did not change the localization pattern of either MURA or MURB; MURA and MURB do not detectably interact in a yeast two-hybrid assay. These results suggest that MURA and MURB do not mutually affect their localization, at least in the forms examined here.

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↗

PubMed2002

Nuclear translocation of extracellular superoxide dismutase.

Histochemical examination of mouse tissues showed nuclear staining of extracellular superoxide dismutase (EC-SOD), and the nuclear translocation of EC-SOD was also confirmed in cultured cells that had been transfected with its gene, as shown by immunohistochemistry and Western blot analysis. The EC-SOD which was secreted into the medium was incorporated into 3T3-L1 cells and a significant fraction of the material taken up was localized in the nucleus. Site-directed mutagenesis indicated that the heparin-binding domain of EC-SOD functions as the nuclear localization signal. These results suggest that the mechanism of the nuclear transport of EC-SOD involves a series of N-terminal signal peptide- and C-terminal heparin-binding domain-dependent processes of secretion, re-uptake and the subsequent nuclear translocation. The findings herein provide support for the view that the role of EC-SOD is to protect the genome DNA from damage by reactive oxygen species and/or the transcriptional regulation of redox-sensitive gene expression.

3T3 Cells↗

PubMed2003

Arabidopsis transportin1 is the nuclear import receptor for the circadian clock-regulated RNA-binding protein AtGRP7.

We characterized the Arabidopsis orthologue of the human nuclear import receptor transportin1 (TRN1). Like the human receptor, Arabidopsis TRN1 recognizes nuclear import signals on proteins that are different from the classical basic nuclear localization signals. The M9 domain of human heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is the prototype of such signals. We show that AtTRN1 binds to similar domains in hnRNP-like proteins from plants. AtTRN1 also interacts with human hnRNP A1 and with yeast Nab2p, two classical import cargo proteins of transportin in these organisms. Like all nuclear transport receptors of the importin-beta family, AtTRN1 binds to the regulatory GTPase Ran from Arabidopsis. We demonstrated that the amino terminus of AtTRN1 is necessary for this interaction. Recombinant AtTRN1 conferred nuclear import of fluorescently labelled BSA-M9 peptide conjugates in permeabilized HeLa cells, functionally replacing human TRN1 in these in vitro nuclear import assays. We identified three plant substrate proteins that interact with AtTRN1 and contain M9-like domains: a novel Arabidopsis hnRNP that shows high similarity to human hnRNP A1 and two small RNA-binding proteins from Arabidopsis, AtGRP7 and AtGRP8. Nuclear import activity of the M9-like domains of these plant proteins was demonstrated in vivo by their ability to confer partial nuclear re-localisation of a GFP fusion protein containing a nuclear export signal. In addition, fluorescently labelled AtGRP7 was specifically imported into nuclei of permeabilized HeLa cells by Arabidopsis AtTRN1 and human TRN1. These results suggest that the transportin-mediated nuclear import pathway is highly conserved between man, yeast and plants.

Amino Acid Sequence↗

PubMed2003

Interaction of the parathyroid hormone receptor with the 14-3-3 protein.

The receptor for parathyroid hormone (PTH) and PTH-related protein (PTHrP) regulates calcium homeostasis, bone remodeling and skeletal development. 14-3-3 proteins bind to signaling proteins and act as molecular scaffolds and regulators of subcellular localization. We show that the parathyroid hormone receptor (PTHR) interacts with 14-3-3 and the proteins colocalize within the cell. 14-3-3 interacts with the C-terminal tail of the receptor containing a consensus 14-3-3 binding motif, but additional binding sites are also used. Protein kinase-A treatment of the receptor and especially the C-terminal tail reduces 14-3-3 binding. The expressed C-terminal tail is primarily localized in the nucleus, supporting the function of a putative nuclear localization signal that could be involved in the previously described nuclear localization of PTHR. The observed interaction between PTHR and the 14-3-3 protein implies that 14-3-3 could contribute to regulation of PTHR signaling.

14-3-3 Proteins↗

PubMed2001

The C-terminal half of Hsp90 is responsible for its cytoplasmic localization.

With some exceptions, research so far has shown heat shock protein (Hsp) 90 to be a cytoplasmic protein. Here, we studied the sequence determinants which dictate the subcellular localization of Hsp90. By constructing hybrid molecules between a nuclear protein, progesterone receptor (PR), and parts of Hsp90, we demonstrated that the C-terminal but not the N-terminal half of Hsp90 can prevent nuclear translocation of the PR. Studies with an antibody raised against a region which contains the major nuclear localization signal (NLS) of the PR suggest that the inhibition of nuclear localization is not due to steric hindrance of the NLS of the PR by Hsp90 sequences in hybrid molecules. In order to characterize further the cytoplasmic anchoring of Hsp90 we constructed four chimeric molecules between the C-terminal half of Hsp90 and estrogen receptor (ER) with different numbers of nuclear localization protosignals (proto-NLS). When the C-terminal half of Hsp90 was fused with ER containing no or one proto-NLS, the hybrid molecule was located exclusively in the cytoplasm. When the nuclear translocation signal was strengthened by adding two or three protosignals, the hybrid molecule was exclusively nuclear. These results suggest that the C-terminal half of Hsp90 contains a sequence which is responsible for the cytoplasmic localization of the protein. Further deletions of the molecule suggested that the cytoplasmic anchoring signal is located between amino acids 333 and 664.

Amino Acid Sequence↗

PubMed2003

Alterations in the post-translational modification and intracellular trafficking of clusterin in MCF-7 cells during apoptosis.

Clusterin is a heterodimeric, disulfide-linked 70-80 kDa glycoprotein that is induced during regression of most, if not all, hormone-dependent epithelial tissues. These studies describe the biogenesis and intracellular trafficking of clusterin in MCF-7 cells before and after the initiation of apoptosis with antiestrogens and TNF alpha. Under physiological conditions, clusterin is modified in the endoplasmic reticulum (ER), and proteolytically cleaved in the Golgi to generate discrete alpha and beta chains prior to secretion. Treatment with TNFalpha or the antiestrogen, ICI 182,780, induces apoptosis in MCF-7 cells and leads to substantial changes in the activity of Golgi-resident enzymes, significantly altering the biogenesis of clusterin. This leads to the appearance of a 50-53 kDa uncleaved, nonglycosylated, disulfide-linked isoform of clusterin that accumulates in the nucleus. While clusterin contains a cryptic SV-40-like nuclear localization signal, mutation of this sequence does not affect the nuclear accumulation of the disulfide-linked nuclear isoform. Confocal microscopy demonstrates that the nuclear accumulation of clusterin is coincident with DNA fragmentation. These data suggest that, at least in secretory epithelial cells, retrograde transport from the Golgi to the ER of a nonglycosylated, uncleaved isoform and the subsequent translocation of clusterin to the nucleus occur in dying cells.

Amino Acid Sequence↗

PubMed2004

Multiple elements regulate nuclear/cytoplasmic shuttling of FOXO1: characterization of phosphorylation- and 14-3-3-dependent and -independent mechanisms.

FOXO1, a Forkhead transcription factor, is an important target of insulin and growth factor action. Phosphorylation of Thr-24, Ser-256 and Ser-319 promotes nuclear exclusion of FOXO1, yet the mechanisms regulating nuclear/cytoplasmic shuttling of FOXO1 are poorly understood. Previous studies have identified an NLS (nuclear localization signal) in the C-terminal basic region of the DBD (DNA-binding domain), and a leucine-rich, leptomycin-B sensitive NES (nuclear export signal) located further downstream. Here, we find that other elements in the DBD also contribute to nuclear localization, and that multiple mechanisms contribute to nuclear exclusion of FOXO1. Phosphorylation of Ser-319 and a cluster of nearby residues (Ser-322, Ser-325 and Ser-329) functions co-operatively with the nearby NES to promote nuclear exclusion. The N-terminal region of FOXO1 (amino acids 1-149) also is sufficient to promote nuclear exclusion, and does so through multiple mechanisms. Amino acids 1-50 are sufficient to promote nuclear exclusion of green fluorescent protein fusion proteins, and the phosphorylation of Thr-24 is required for this effect. A leucine-rich, leptomycin B-sensitive export signal is also present nearby. Phosphorylated FOXO1 binds 14-3-3 proteins, and co-precipitation studies with tagged proteins indicate that 14-3-3 binding involves co-operative interactions with both Thr-24 and Ser-256. Ser-256 is located in the C-terminal region of the DBD, where 14-3-3 proteins may interfere both with DNA-binding and with nuclear-localization functions. Together, these studies demonstrate that multiple elements contribute to nuclear/cytoplasmic shuttling of FOXO1, and that phosphorylation and 14-3-3 binding regulate the cellular distribution and function of FOXO1 through multiple mechanisms. The presence of these redundant mechanisms supports the concept that the regulation of FOXO1 function plays a critical role in insulin and growth factor action.

14-3-3 Proteins↗

PubMed2004

Neurofibromin is actively transported to the nucleus.

Mutations in the neurofibromatosis type 1 (NF1) tumor suppressor gene predispose individuals to a variety of benign and malignant tumors. Many tumor suppressors 'shuttle' between the nucleus and the cytoplasm, thus regulating their function. By expressing different NF1 constructs in COS-7 cells (encompassing exons 28-49 and fused to the green fluorescent protein), we identified a functional nuclear localization signal (NLS) in exon 43. Mutation of the NLS completely abolishes the nuclear entry of the NF1-derivative fusion protein. A highly expressed splice variant that lacks this NLS controls the localization and hence the function of neurofibromin. The localization of neurofibromin in the nucleus may provide novel clues to unknown functions for NF1.

Active Transport, Cell Nucleus↗

PubMed2006

A multifunctional PEI-based cationic polyplex for enhanced systemic p53-mediated gene therapy.

We recently reported a novel coupling strategy involving salicylhydroxamic acid and phenyl(di)boronic acid molecules to attach the CNGRC peptide to PEI/DNA for CD13 targeting in tumors. Here, we doubly coupled Simian Virus (SV) 40 peptide-(nuclear localization signal)) and oligonucleotide-based (DNA nuclear targeting signal) nuclear signals to the same vector using peptide nucleic acid chemistry. This vector, CNGRC/PEG/PEI/DNA-betagal/NLS/DNTS, was predominantly localized in the cell nucleus, yielding about 200-fold higher betagal gene expression in vitro, more than 20-fold increase in tumor-specific gene delivery, and a robust betagal gene expression as demonstrated in stained tumor sections. For gene therapy purposes, we further engineered a similar targeting polyplex, CNGRC/PEG/PEI/DNA-p53/NLS/DNTS, with EBV-based episomal vector for sustained p53 gene expression. A distribution of vector DNA and apoptosis in p53-containing tumors was observed, yielding a significant tumor regression and 95% animal survival after 60 days. This multicomponent vector also co-targeted tumor and tumor-associated endothelial cells but not normal cells, and had more efficient therapeutic index than each vector administered as a single modality. The use of an efficient coupling strategy without compromising the vector's integrity for DNA condensation and endosomal escape; nuclear import; tumor-specific and persistent p53 gene expression clearly provides a basis for developing a single combinatorial approach for non-viral gene therapy.

Animals↗

PubMed2002

Differential intracellular localization of the human and mouse endonuclease III homologs and analysis of the sorting signals.

The mammalian endonuclease III homolog NTH1 is a DNA glycosylase/AP lyase that recognizes oxidized pyrimidine bases. Here, we compared the intracellular localization of human and mouse NTH1 and analyzed their sorting signals by examining expression of enhanced green fluorescent protein (EGFP)-tagged NTH1 protein. Full-length hNTH1 was sorted exclusively into nuclei. Deletion analysis showed that two basic amino acid clusters, which constitute the nuclear localization signal (NLS), are essential for nuclear sorting. Moreover, disruption of the NLS by deletion or substitution of arginine residue(s) altered the localization of the protein to mitochondria. In contrast, most mNTH1 molecules were sorted into mitochondria, with a relatively small amount localized in nuclei. Deletion analysis indicated that the mitochondrial targeting sequence of mNTH1 is contained within the N-terminal 38 amino acids. Alignment of the N-terminal sequence of human and mouse NTH1 showed that mNTH1 lacks a basic amino acid cluster corresponding to one of the NLS sequences found in hNTH1. Nuclear localization of mNTH1 was increased when this NLS sequence was added to mNTH1 through the addition of appropriate amino acids. The fact that transcription of the hNTH1 gene is initiated at multiple sites indicated that three isoforms of hNTH1 protein are translated using different initiation codons. However, no difference in intracellular localization was observed among three isoforms of hNTH1 with different N-terminal sequences.

Amino Acid Sequence↗

PubMed2000

Nuclear and subnuclear targeting sequences of the protein phosphatase-1 regulator NIPP1.

NIPP1 is a nuclear subunit of protein phosphatase-1 (PP1) that colocalizes with pre-mRNA splicing factors in speckles. We report here that the nuclear and subnuclear targeting of NIPP1, when expressed in HeLa cells or COS-1 cells as a fusion protein with the enhanced-green-fluorescent protein (EGFP), are mediated by distinct sequences. While NIPP1-EGFP can cross the nuclear membrane passively, the active transport to the nucleus is mediated by two independent nuclear localization signals in the central domain of NIPP1, which partially overlap with binding site(s) for PP1. Furthermore, the concentration of NIPP1-EGFP in the nuclear speckles requires the 'ForkHead-Associated' domain in the N terminus. This domain is also required for the nuclear retention of NIPP1 when active transport is blocked. Our data imply that the nuclear and subnuclear targeting of NIPP1 are controlled independently.

Amino Acid Sequence↗

PubMed2006

Human cytomegalovirus DNA polymerase catalytic subunit pUL54 possesses independently acting nuclear localization and ppUL44 binding motifs.

The catalytic subunit of human cytomegalovirus (HCMV) DNA polymerase pUL54 is a 1242-amino-acid protein, whose function, stimulated by the processivity factor, phosphoprotein UL44 (ppUL44), is essential for viral replication. The C-terminal residues (amino acids 1220-1242) of pUL54 have been reported to be sufficient for ppUL44 binding in vitro. Although believed to be important for functioning in the nuclei of infected cells, no data are available on either the interaction of pUL54 with ppUL44 in living mammalian cells or the mechanism of pUL54 nuclear transport and its relationship with that of ppUL44. The present study examines for the first time the nuclear import pathway of pUL54 and its interaction with ppUL44 using dual color, quantitative confocal laser scanning microscopy on live transfected cells and quantitative gel mobility shift assays. We showed that of two nuclear localization signals (NLSs) located at amino acids 1153-1159 (NLSA) and 1222-1227 (NLSB), NLSA is sufficient to confer nuclear localization on green fluorescent protein (GFP) by mediating interaction with importin alpha/beta. We also showed that pUL54 residues 1213-1242 are sufficient to confer ppUL44 binding abilities on GFP and that pUL54 and ppUL44 can be transported to the nucleus as a complex. Our work thus identified distinct sites within the HCMV DNA polymerase, which represent potential therapeutic targets and establishes the molecular basis of UL54 nuclear import.

Amino Acid Motifs↗

PubMed2004

NLS-dependent nuclear localization of p120ctn is necessary to relieve Kaiso-mediated transcriptional repression.

The Armadillo catenin p120(ctn) regulates cadherin adhesive strength at the plasma membrane and interacts with the novel BTB/POZ transcriptional repressor Kaiso in the nucleus. The dual localization of p120(ctn) at cell-cell junctions and in the nucleus suggests that its nucleocytoplasmic trafficking is tightly regulated. Here we report on the identification of a specific and highly basic nuclear localization signal (NLS) in p120(ctn). The functionality of the NLS was validated by its ability to direct the nuclear localization of a heterologous beta-galactosidase-GFP fusion protein. Mutating two key positively charged lysines to neutral alanines in the NLS of full-length p120(ctn) inhibited both p120(ctn) nuclear localization as well as the characteristic p120(ctn)-induced branching phenotype that correlates with increased cell migration. However, while these findings and others suggested that nuclear localization of p120(ctn) was crucial for the p120(ctn)-induced branching phenotype, we found that forced nuclear localization of both wild-type and NLS-mutated p120(ctn) did not induce branching. Recently, we also found that one role of p120(ctn) was to regulate Kaiso-mediated transcriptional repression. However, it remained unclear whether p120(ctn) sequestered Kaiso in the cytosol or directly inhibited Kaiso transcriptional activity in the nucleus. Using minimal promoter assays, we show here that the regulatory effect of p120(ctn) on Kaiso transcriptional activity requires the nuclear translocation of p120(ctn). Therefore, an intact NLS in p120(ctn) is requisite for its first identified regulatory role of the transcriptional repressor Kaiso.

Alanine↗

PubMed2005

Huntingtin interacting protein 1 modulates the transcriptional activity of nuclear hormone receptors.

Internalization of activated receptors regulates signaling, and endocytic adaptor proteins are well-characterized in clathrin-mediated uptake. One of these adaptor proteins, huntingtin interacting protein 1 (HIP1), induces cellular transformation and is overexpressed in some prostate cancers. We have discovered that HIP1 associates with the androgen receptor through a central coiled coil domain and is recruited to DNA response elements upon androgen stimulation. HIP1 is a novel androgen receptor regulator, significantly repressing transcription when knocked down using a silencing RNA approach and activating transcription when overexpressed. We have also identified a functional nuclear localization signal at the COOH terminus of HIP1, which contributes to the nuclear translocation of the protein. In conclusion, we have discovered that HIP1 is a nucleocytoplasmic protein capable of associating with membranes and DNA response elements and regulating transcription.

Amino Acid Sequence↗