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Results for “Nuclear Localization Signals”

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PubMed2003

Proteins that mediate the nuclear entry of the goat uterine estrogen receptor activation factor (E-RAF): identification of a molecular basis for the inhibitory effect of progesterone on estrogen action.

A 66 kDa transport protein, tp66, has been identified as the protein that mediates the nuclear transport of the estrogen receptor activation factor (E-RAF). Indirect evidence shows that tp66 influences the transport of E-RAF mainly by recognizing the nuclear localization signals (NLS) on the latter. A 38 kDa nuclear pore complex protein (npcp38) has been identified to which tp66-E-RAF complex gets 'docked' prior to the nuclear entry of E-RAF. Progesterone binding to E-RAF serves to dissociate E-RAF from the tp66 thereby inhibiting the nuclear entry of E-RAF. The demonstration of the high affinity progesterone binding property of E-RAF adds credibility to the above findings. A change in conformation of E-RAF being brought about by progesterone binding is evident from the results of the circular dichroism (CD) analysis. This appears to be the fundamental reason behind the dissociation of the tp66-E-RAF complex under progesterone influence and provides a molecular basis for the estrogen 'antagonistic' action of progesterone. A nuclear run-on transcription assay clearly demonstrates the transcription-activation function of E-RAF II, also reaffirming the functional role of tp66 in the nuclear entry of E-RAF.

Active Transport, Cell Nucleus↗

PubMed1991

Nuclear and nuclear envelope binding proteins of the glucocorticoid receptor nuclear localization peptide identified by crosslinking.

The molecular mechanisms underlying the nuclear entry of steroid receptors and possible regulation of steroid hormone action during receptor passage across the nuclear envelope have not been elucidated. A nuclear localization signal has been identified in the hinge region of the glucocorticoid receptor. A synthetic peptide corresponding to this sequence was radio-iodinated and incubated with high salt- and detergent-extracted rat liver nuclei or nuclear envelope in the presence of crosslinker. After SDS-PAGE, two nuclear polypeptides of 60 and 76 kDa which had been specifically crosslinked were identified by autoradiography. A 60 kDa polypeptide was also crosslinked in the nuclear envelope fraction. ATP and elevated temperatures enhanced the crosslinking of both nuclear peptides. Finally, we showed that the pattern of crosslinking of the simian virus 40 large tumour antigen nuclear localization signal was identical to that of the glucocorticoid receptor signal to the nuclear polypeptides. The crosslinked peptides are good candidates for nuclear importers of the glucocorticoid receptor. In addition, the data suggest that these binding sites may be part of a general mechanism for nuclear entry of proteins.

Adenosine Triphosphate↗

PubMed2006

Nucleocytoplasmic shuttling of HMGB1 is regulated by phosphorylation that redirects it toward secretion.

The high mobility group box 1 (HMGB1) protein can be secreted by activated monocytes and macrophages and functions as a late mediator of sepsis. HMGB1 contains two nuclear localization signals (NLSs) for controlled nuclear transport, and acetylation of both NLSs of HMGB1 is involved in nuclear transport toward secretion. However, phosphorylation of HMGB1 and its relation to nuclear transport have not been shown. We show here that HMGB1 is phosphorylated and dynamically shuttled between cytoplasmic and nuclear compartments according to its phosphorylation state. Phosphorylation of HMGB1 was detected by metabolic labeling and Western blot analysis after treatments with TNF-alpha and okadaic acid, a phosphatase inhibitor. Hyperphosphorylated HMGB1 in RAW 264.7 and human monocytes was relocated to the cytoplasm. In a nuclear import assay, phosphorylated HMGB1 in the cytoplasm did not enter the nucleus. We mutated serine residues of either or both NLSs of HMGB1 to glutamic acid to simulate a phosphorylated state and examined the binding of HMGB1 to karyopherin-alpha1, which was identified as the nuclear import protein for HMGB1 in this study. Substitution to glutamic acid in either NLSs decreased the binding with karyopherin-alpha1 by approximately 50%; however, substitution of both NLSs showed no binding, and HMGB1 was relocated to the cytoplasm and subsequently secreted. These data support the hypothesis that HMGB1 could be phosphorylated and that the direction of transport is regulated by phosphorylation of both NLS regions.

Animals↗

PubMed2006

Nucleocytoplasmic traffic disorder induced by cardioviruses.

Some picornaviruses, for example, poliovirus, increase bidirectional permeability of the nuclear envelope and suppress active nucleocytoplasmic transport. These activities require the viral protease 2A(pro). Here, we studied nucleocytoplasmic traffic in cells infected with encephalomyocarditis virus (EMCV; a cardiovirus), which lacks the poliovirus 2A(pro)-related protein. EMCV similarly enhanced bidirectional nucleocytoplasmic traffic. By using the fluorescent "Timer" protein, which contains a nuclear localization signal, we showed that the cytoplasmic accumulation of nuclear proteins in infected cells was largely due to the nuclear efflux of "old" proteins rather than impaired active nuclear import of newly synthesized molecules. The nuclear envelope of digitonin-treated EMCV-infected cells permitted rapid efflux of a nuclear marker protein. Inhibitors of poliovirus 2A(pro) did not prevent the EMCV-induced efflux. Extracts from EMCV-infected cells and products of in vitro translation of viral RNAs contained an activity increasing permeability of the nuclear envelope of uninfected cells. This activity depended on the expression of the viral leader protein. Mutations disrupting the zinc finger motif of this protein abolished its efflux-inducing ability. Inactivation of the L protein phosphorylation site (Thr47-->Ala) resulted in a delayed efflux, while a phosphorylation-mimicking (Thr47-->Asp) replacement did not significantly impair the efflux-inducing ability. Such activity of extracts from EMCV-infected cells was suppressed by the protein kinase inhibitor staurosporine. As evidenced by electron microscopy, cardiovirus infection resulted in alteration of the nuclear pores, but it did not trigger degradation of the nucleoporins known to be degraded in the poliovirus-infected cells. Thus, two groups of picornaviruses, enteroviruses and cardioviruses, similarly alter the nucleocytoplasmic traffic but achieve this by strikingly different mechanisms.

Active Transport, Cell Nucleus↗

PubMed2001

Nuclear import of Spo12p, a protein essential for meiosis.

In Saccharomyces cerevisiae, Spo12p is involved in mitosis and is essential for meiosis. We found that Spo12p is imported into the nucleus by the karyopherin Kap121p. A complex containing Spo12p and Kap121p was isolated from cytosol and was also reconstituted with recombinant proteins, indicating that this interaction is direct. Spo12p was mislocalized to the cytosol in pse1-1, a temperature-sensitive strain harboring a mutation of Kap121p, at the permissive temperature, confirming an essential role for Kap121p in Spo12p import. Spo12p was also mislocalized in a pse1-1/pse1-1 homozygous strain, suggesting it is imported via the same pathway in diploid cells. Furthermore, we found that pse1-1/pse1-1 shows a sporulation defect similar to that of spo12Delta/spo12Delta. In addition, we have characterized the Spo12p nuclear localization signal, mapped it to residues 76-130, and identified residues within this region that are important for nuclear localization signal function.

Amino Acid Sequence↗

PubMed1990

Expression and targeting of intracellular antibodies in mammalian cells.

Genes encoding the heavy and light chains of a hapten-specific IgM antibody were modified by site-directed mutagenesis to destroy the hydrophobic leader sequences and allow expression in the cytoplasm of non-lymphoid cells. The in situ assembly of the mutant heavy and light chains was tested in transfected cell lines by immunofluorescence using anti-idiotypic antibodies. A positive diffuse cytoplasmic staining was observed. This demonstrated that the antibody polypeptide chains could assemble in the cell cytoplasm and led us to ask whether antibodies could be further targeted to the nucleus. Mutations were therefore made in which the leader sequence of the light chain was replaced by the nuclear localization signal of the SV40 large T antigen. Transfectants in which the heavy chain lacking the hydrophobic leader was expressed together with a light chain carrying the nuclear localization signal were selected and a nuclear distribution of the assembled antibody was found. Thus, it should prove possible to target a specific antibody to the cell nucleus with the aim of interfering with the function of a nuclear antigen.

Amino Acid Sequence↗

PubMed2001

DNA binding domains in diverse nuclear receptors function as nuclear export signals.

BACKGROUND: The nuclear receptor superfamily of transcription factors directs gene expression through DNA sequence-specific interactions with target genes. Nuclear import of these receptors involves recognition of a nuclear localization signal (NLS) by importins, which mediate translocation into the nucleus. Nuclear receptors lack a leucine-rich nuclear export signal (NES), and export is insensitive to leptomycin B, indicating that nuclear export is not mediated by Crm1. RESULTS: We set out to define the NES in the glucocorticoid receptor (GR) and to characterize the export pathway. We found that the 69 amino acid DNA binding domain (DBD) of GR, which is unrelated to any known NES, is necessary and sufficient for export. Mutational analysis revealed that a 15 amino acid sequence between the two zinc binding loops in the GR-DBD confers nuclear export to a GFP reporter protein, and alanine-scanning mutagenesis was used to identify the residues within this sequence that are critical for export. The DBD is highly related (41%-88% identity) in steroid, nonsteroid, and orphan nuclear receptors, and we found that the DBDs from ten different nuclear receptors all function as export signals. DBD-dependent nuclear export is saturable, and prolonged nuclear localization of the GR increases its transcriptional activity. CONCLUSIONS: Multiple members of the nuclear receptor superfamily use a common pathway to exit the nucleus. We propose that NLS-mediated import and DBD-mediated export define a shuttling cycle that integrates the compartmentalization and activity of nuclear receptors.

Active Transport, Cell Nucleus↗

PubMedDate not supplied

Receptor-like properties of the 26 kDa transmembrane form of TNF.

Most members of the TNF family of proteins exist as transmembrane proteins with relatively long intracellular domains, and a number of them are involved in the ill-defined phenomenon of "reverse signaling". We have identified a putative nuclear localization signal in the cytoplasmic domain of TNF which proved to be functional in two assays. Western analysis identified an approximately 10 kDa peptide corresponding to the transmembrane and cytoplasmic domains of TNF after the proteolytic liberation of the 17 kDa, soluble form of TNF. This 10 kDa peptide was enriched in internal membranes and nuclear fractions of disrupted cells. Immune electron-microscopic studies proved its localization in transport vesicles and the nucleus. The nuclear transport of the intracellular segment of TNF resembles the signaling process through the Notch-type of receptors. Indeed, the presence of the 10 kDa peptide seems to influence the expression of another inflammatory cytokine, interleukin-1 beta. These findings suggest that the transmembrane form of TNF has receptor-like properties and its interaction with the receptors initiates a bidirectional signaling.

Active Transport, Cell Nucleus↗

PubMed2004

Aprataxin, the causative protein for EAOH is a nuclear protein with a potential role as a DNA repair protein.

Early-onset ataxia with ocular motor apraxia and hypoalbuminemia (EAOH) is an autosomal recessive neurodegenerative disorder characterized by early-onset ataxia, ocular motor apraxia, and hypoalbuminemia. Recently, the causative gene for EAOH, APTX, has been identified. Of the two splicing variants of APTX mRNA, the short and the long forms, long-form APTX mRNA was found to be the major isoform. Aprataxin is mainly located in the nucleus, and, furthermore, the first nuclear localization signal located near the amino terminus of the long-form aprataxin is essential for its nuclear localization. We found, based on the yeast two-hybrid and coimmunoprecipitation experiments, that the long-form but not the short-form aprataxin interacts with XRCC1 (x-ray repair cross-complementing group 1). Interestingly the amino terminus of the long-form aprataxin is homologous with polynucleotidekinase-3'-phosphatase, which has been demonstrated to be involved in base excision repair, a subtype of single-strand DNA break repair, through interaction with XRCC1, DNA polymerase beta, and DNA ligase III. These results strongly support the possibility that aprataxin and XRCC1 constitute a multiprotein complex and are involved in single-strand DNA break repair, and furthermore, that accumulation of unrepaired damaged DNA underlies the pathophysiological mechanisms of EAOH.

Alternative Splicing↗

PubMed2001

An RNA recognition motif (RRM) is required for the localization of PTB-associated splicing factor (PSF) to subnuclear speckles.

Using fusions with green fluorescent protein (GFP), we have identified sequences in the polypyrimidine tract binding protein-associated splicing factor (PSF) that are involved in nuclear and subnuclear localization. Like other splicing factors, PSF localizes to the nucleus, is absent from nucleoli, and accumulates in punctate structures within the nucleus referred to as speckles. However, PSF lacks the known speckle localization domains that have been identified in other proteins. Instead, the localization of PSF to speckles is dependent on an RNA recognition motif (RRM). PSF comprises an N-terminal proline- and glutamine-rich domain, two RRMs (RRM1 and RRM2), and a C-terminal region that contains two nuclear localization signals, both of which are required for complete nuclear localization. Deletion of RRM2 led to a complete loss of speckle localization and resulted in diffuse accumulation of PSF in the nucleus, indicating that RRM2 is required for subnuclear localization. Thus, PSF appears to localize to speckles through a novel pathway that is dependent on its second RRM. Consistent with the use of a novel subnuclear targeting pathway, PSF redistributes to perinucleolar clusters upon the addition of a transcription inhibitor whereas other splicing factors display increased localization to speckles in the absence of transcription. A yeast two-hybrid screen identified four-and-a-half LIM-only protein 2 (FHL2) as a potential RRM2 interaction partner, indicating a possible role for zinc-finger or LIM domains in the localization of splicing factors to subnuclear speckles.

Active Transport, Cell Nucleus↗

PubMed2003

Ggamma subunit-selective G protein beta 5 mutant defines regulators of G protein signaling protein binding requirement for nuclear localization.

The signal transducing function of Gbeta(5) in brain is unknown. When studied in vitro Gbeta(5) is the only heterotrimeric Gbeta subunit known to interact with both Ggamma subunits and regulators of G protein signaling (RGS) proteins. When tested with Ggamma, Gbeta(5) interacts with other classical components of heterotrimeric G protein signaling pathways such as Galpha and phospholipase C-beta. We recently demonstrated nuclear expression of Gbeta(5) in neurons and brain (Zhang, J. H., Barr, V. A., Mo, Y., Rojkova, A. M., Liu, S., and Simonds, W. F. (2001) J. Biol. Chem. 276, 10284-10289). To gain further insight into the mechanism of Gbeta(5) nuclear localization, we generated a Gbeta(5) mutant deficient in its ability to interact with RGS7 while retaining its ability to bind Ggamma, and we compared its properties to the wild-type Gbeta(5). In HEK-293 cells co-transfection of RGS7 but not Ggamma(2) supported expression in the nuclear fraction of transfected wild-type Gbeta(5). In contrast the Ggamma-preferring Gbeta(5) mutant was not expressed in the HEK-293 cell nuclear fraction with either co-transfectant. The Ggamma-selective Gbeta(5) mutant was also excluded from the cell nucleus of transfected PC12 cells analyzed by laser confocal microscopy. These results define a requirement for RGS protein binding for Gbeta(5) nuclear expression.

Active Transport, Cell Nucleus↗

PubMed2004

Expression of plant protein phosphatase 2C interferes with nuclear import of the Agrobacterium T-complex protein VirD2.

Agrobacterium tumefaciens transfers DNA to plant cells as a single-stranded DNA molecule (the T-strand) covalently linked to VirD2 protein. VirD2 contains nuclear localization signal sequences that presumably help direct the T-strand to the plant nucleus. We identified a tomato cDNA clone, DIG3, that encodes a protein that interacts with the C-terminal region of VirD2. DIG3 encodes an enzymatically active type 2C serine/threonine protein phosphatase. Overexpression of DIG3 in tobacco BY-2 protoplasts inhibited nuclear import of a beta-glucuronidase-VirD2 nuclear localization signal fusion protein. Thus, DIG3 may be involved in nuclear import of the VirD2 protein and, consequently, the VirD2/transferred DNA complex.

Agrobacterium tumefaciens↗

PubMed2004

Regulation of nucleocytoplasmic localizatioin of the atDjC6 chaperone protein.

The sequence of the atDjC6 chaperone protein includes three potential nuclear localization signal (NLS) sequences (A-C) and three potential nuclear export signal (NES) sequences (X-Z). The subcellular localization of atDjC6 was studied by scanning laser confocal microscopy of chimera with the green-fluorescent protein (GFP) transiently expressed in tobacco BY-2 cells. The localization of the atDjC6::GFP chimera was coincident with that of the nuclear stain propidium iodide. Site-directed mutagenesis was used to verify the predicted NLS sequences. Each was individually fused to GFP and tested for protein localization. The individual NLS sequences were sufficient to direct partial nuclear localization of GFP, although the targeting information within NLS-B is apparently conformation sensitive. Site-directed mutagenesis of the NES sequences increased the amount of each chimera that was nuclearly localized, indicating a decrease in nuclear export. When any pair of NLS sequences were appended to GFP, the chimera were entirely nuclearly localized. Quantitative two-hybrid analysis was used to verify that the decoding of NLS sequence information involves interaction with the NLS-receptor protein importin-alpha. Each of the NLS sequences is flanked by a site of potential Ser phosphorylation, and recombinant atDjC6 could be phosphorylated in vitro. Mutagenesis of Ser residues to the P-Ser mimic Asp interfered with nuclear targeting, apparently by preventing recognition or binding by importin-alpha. Our results are consistent with a regulated nucleocytoplasmic localization of the atDjC6 chaperone protein.

Algorithms↗

PubMed1992

NIP1, a gene required for nuclear transport in yeast.

Cytochrome c with a nuclear localization signal added at the N terminus was mistargeted to the nucleus, resulting in a yeast strain deficient in mitochondrial cytochrome c. Reversion of this strain allowed the isolation of temperature-conditional mutants defective in nuclear transport, as demonstrated with one of these mutants, nip1-1, that was shown to be defective in nuclear accumulation of a LacZ protein containing a nuclear localization signal of the yeast ribosomal protein L29. The NIP1+ gene was cloned and shown to encode a 93,143-Da protein. Furthermore, an epitope-labeled NIP1 protein migrated in SDS/polyacrylamide gels with a mass of approximately 100,000 Da and was shown by immunofluorescence to localize mainly in the cytoplasm. NIP1+ was shown to be an essential gene by gene disruption experiments. Intriguingly, NIP1 has a serine-rich acidic N-terminal region that is similar in this regard to the N-terminal region of a previously described nuclear localization signal-binding protein, NSR1.

Alleles↗

PubMed2004

Nuclear localization of Tob is important for regulation of its antiproliferative activity.

TOB: is a member of an antiproliferative gene family that includes btg1, pc3/tis21/btg2, pc3b, ana/btg3, and tob2. Exogenous overexpression of the family proteins suppresses cell proliferation. These proteins participate in transcriptional regulation of several genes. Here, we show that Tob is a nuclear protein that is imported into the nucleus through a nuclear localization signal (NLS)-mediated mechanism. Mutation in the NLS sequence of Tob affects its nuclear localization and impairs antiproliferative activity. Additionally, Tob contains a nuclear export signal (NES). In oncogenic ErbB2-transformed cells, nuclear export of Tob is facilitated by NES-mediated mechanism, resulting in decrease of its antiproliferative activity. These results indicate that regulation of nuclear localization of Tob is important for its antiproliferative activity.

Animals↗

PubMed1995

Identification of nuclear and nucleolar localization signals in the herpes simplex virus regulatory protein ICP27.

Previous work has shown that the herpes simplex virus type 1 (HSV-1) regulatory protein ICP27 localizes to the cell nucleus and that certain mutant ICP27 polypeptides localize preferentially in nucleoli. To map the signals in ICP27 which mediate its nuclear localization, we identified the portions of ICP27 which can direct a cytoplasmic protein, pyruvate kinase (PK), to nuclei. Our results demonstrate that ICP27 contains multiple nuclear localization signals (NLSs) that function with differing efficiencies. First, ICP27 possesses a strong NLS, mapping to residues 110 to 137, which bears similarity to the bipartite NLSs found in Xenopus laevis nucleoplasmin and other proteins. Second, ICP27 possesses one or more weak NLSs which map to a carboxyl-terminal portion of the protein between residues 140 and 512. Our PK-targeting experiments also demonstrate that ICP27 contains a relatively short sequence, mapping to residues 110 to 152, that can function as a nucleolar localization signal (NuLS). This signal includes ICP27's strong NLS as well as 15 contiguous residues which consist entirely of arginine and glycine. This latter sequence is very similar to an RGG box, a putative RNA-binding motif found in a number of cellular proteins which are involved in nuclear RNA processing. To confirm the results of the PK-targeting experiments, we mutated the ICP27 gene by deleting sequences encoding either the strong NLS or the RGG box. Deletion of the strong NLS (residues 109 to 138) resulted in an ICP27 molecule that was only partially defective for nuclear localization, while deletion of the RGG box (residues 139 to 153) resulted in a molecule that was nuclear localized but excluded from nucleoli. Recombinant HSV-1s bearing either of these deletions were unable to replicate efficiently in Vero cells, suggesting that ICP27's strong NLS and RGG box carry out important in vivo functions.

Amino Acid Sequence↗

PubMed1994

Nuclear localization and cell cycle regulation of a murine protein tyrosine phosphatase.

MPTP is a murine homolog of the human T-cell protein tyrosine phosphatase (PTPase) and the rat PTP-S enzyme. Enzymatic activity of this ubiquitously expressed protein was demonstrated in immunoprecipitates from NIH 3T3 cells and in recombinant protein overexpressed in bacteria. Expression of beta-galactosidase-MPTP MPTP chimeric proteins in COS1 cells identified a nuclear localization signal at the carboxyl terminus of the MPTP that was sufficient to direct beta-galactosidase as well as a tagged version of the MPTP to the nucleus. Deletion analysis of amino acids within the nuclear targeting signal showed that this sequence does not conform to the bipartite type of nuclear localization signals. Furthermore, it was shown that the steady-state levels of MPTP RNA fluctuate in a cell cycle-specific manner. On the basis of these experiments, we discuss the possible function of MPTP in the cell cycle and other nuclear processes.

Amino Acid Sequence↗

PubMed1995

Stimulation of nuclear import by simian virus 40-transformed cell extracts is dependent on protein kinase activity.

We previously reported that both the nuclear import rate of large karyophilic gold particles and the functional size of the pores are significantly greater in simian virus 40-transformed fibroblasts (the SV-T2 cell line) than in nontransformed BALB/c 3T3 cells. In this study, we found that cytosolic fractions obtained from SV-T2 cultures can increase nuclear transport capacity (both import rate and pore size) when microinjected into BALB/c 3T3 cells. The transport-enhancing function of the extracts can be abolished by the protein kinase inhibitors staurosporine and K252a as well as 5'-p-fluorosulfonylbenzoyladenosine and protein phosphatase 2A, which, although less specific, also interfere with kinase activity. Increases in transport capacity of the same magnitude as that produced by the SV-T2 extracts were obtained by microinjecting protein kinase A or C or recombinant mitogen-activated protein kinase. These data provide further support for the interpretation that the enhancer is a protein kinase. From experiments performed with specific kinase inhibitor peptides, it appears likely that protein kinase C is the active factor in the SV-T2 cytosolic fractions; however, this will require further verification. It was also determined, by using gold particles coated with bovine serum albumin conjugated to synthetic nuclear localization signal peptides that lacked phosphorylation sites, that the enhancer affects the transport machinery rather than the activity of the nuclear localization signals.

3T3 Cells↗