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Results for “Nuclear Localization Signals”

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PubMed1999

Structure-function analysis of the protein-binding domains of Mac1p, a copper-dependent transcriptional activator of copper uptake in Saccharomyces cerevisiae.

The Mac1 protein in Saccharomyces cerevisiae is essential for the expression of yeast high affinity copper uptake. A positive transcription factor, Mac1p binds via its N-terminal domain to GCTC elements in the promoters of CTR1 and FRE1, encoding a copper permease and metal reductase, respectively. Mac1p-dependent transcriptional activation is negatively regulated by copper. We have mapped the domains in Mac1p responsible for its nuclear localization and for the protein-protein interactions that underlie its transcriptional activity. Immunofluorescence studies indicate that Mac1p contains two nuclear localization signals, one each in the N- and C-terminal halves of the protein. Yeast one-hybrid analysis demonstrates that the copper-dependent transcriptional activity in Mac1p resides primarily in a cysteine-rich element encompassing residues 264-279. Two-hybrid analysis indicates that a copper-independent Mac1p-Mac1p interaction linked to DNA binding is due primarily to a predicted helix in the C-terminal region of the protein encompassing residues 388-406. Point mutations within this putative helix abrogate the Mac1-Mac1 interaction in vivo and formation of a ternary (Mac1p)(2).DNA complex in vitro. When produced in normal abundance, Mac1pI396D and Mac1pF400D helix mutants do not support transcriptional activation in vivo consistent with an essential Mac1p dimerization in transcriptional activation. Lastly, the one- and two-hybrid data indicate that an intramolecular interaction between the DNA-binding and transactivation domains negatively modulates Mac1p activity.

Binding Sites↗

PubMed2005

Nucleocytoplasmic distribution of opioid growth factor and its receptor in tongue epithelium.

The subcellular distributions of the opioid growth factor (OGF), [Met(5)]-enkephalin, and opioid growth factor receptor (OGFr) in the epithelium of the rat tongue were determined in order to reveal structure-function relationships. Laser scanning confocal microscopic analysis showed that both OGF and OGFr were colocalized in the paranuclear cytoplasm and in the nuclei of keratinocytes in the stratum basale. Using immunoelectron microscopy and postembedding techniques, double labeling experiments disclosed that complexes of OGF-OGFr were colocalized on the outer nuclear envelope, in the paranuclear cytoplasm, perpendicular to the nuclear envelope in a putative nuclear pore complex, and in the nucleus adjacent to heterochromatin. Anti-OGF IgG alone was detected in the cytoplasm, and anti-OGFr IgG alone was associated with the outer nuclear envelope. Study of chronic treatment with the opioid antagonist, naltrexone (NTX), which blocks opioid-receptor binding, revealed the presence of OGFr immunoreactivity alone in the cytoplasm and the nucleus; some OGF-OGFr complexes were also observed. Colocalization of OGFr and karyopherin (importin) beta was recorded in the cytoplasm and nucleus. These results in tongue epithelium are the first to suggest that OGFr resides on the outer nuclear envelope, where OGF interacts with OGFr; that the OGF-OGFr complex translocates between cytoplasm and nucleus at the nuclear pore; and that the nuclear localization signal of OGFr interacts with karyopherin beta for nuclear transport. These novel data also indicate that signal transduction for cell proliferation appears to involve an OGF-OGFr complex that interfaces with chromatin in the nucleus. Moreover, the unique finding that OGFr was found in the cytoplasm and nucleus in NTX-treated specimens may suggest that NTX-OGFr complexes have the same pathway as OGF-OGFr.

Animals↗

PubMed2000

Carboxy terminal region of the Fanconi anemia protein, FANCG/XRCC9, is required for functional activity.

Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with eight complementation groups. Four of the FA genes have been cloned, and at least three of the encoded proteins, FANCA, FANCC, and FANCG/XRCC9, interact in a nuclear complex, required for the maintenance of normal chromosome stability. In the current study, mutant forms of the FANCA and FANCG proteins have been generated and analyzed with respect to protein complex formation, nuclear translocation, and functional activity. The results demonstrate that the amino terminal two-thirds of FANCG (FANCG amino acids 1-428) binds to the amino terminal nuclear localization signal (NLS) of the FANCA protein. On the basis of 2-hybrid analysis, the FANCA/FANCG binding is a direct protein-protein interaction. Interestingly, a truncated mutant form of the FANCG protein, lacking the carboxy terminus, binds in a complex with FANCA and translocates to the nucleus; however, this mutant protein fails to bind to FANCC and fails to correct the mitomycin C sensitivity of an FA-G cell line. Taken together, these results demonstrate that binding of FANCG to the amino terminal FANCA NLS sequence is necessary but not sufficient for the functional activity of FANCG. Additional amino acid sequences at the carboxy terminus of FANCG are required for the binding of FANCC in the complex. (Blood. 2000;96:1625-1632)

Animals↗

PubMed2001

Identification of a tissue-specific putative transcription factor in breast tissue by serological screening of a breast cancer library.

Application of SEREX (serological analysis of recombinant tumor cDNA expression libraries) to different tumor types has led to the identification of several categories of human tumor antigens. In this study, the analysis of a breast cancer library with autologous patient serum led to the isolation of seven genes, designated NY-BR-1 through NY-BR-7. NY-BR-1, representing 6 of 14 clones isolated, showed tissue-restricted mRNA expression in breast and testis but not in 13 other normal tissues tested. Among tumor specimens, NY-BR-1 mRNA expression was found in 21 of 25 breast cancers but in only 2 of 82 nonmammary tumors. Structural analysis of NY-BR-1 cDNA and the corresponding genomic sequences in the recently released working draft of human genome indicated that NY-BR-1 is composed of 37 exons and has an open reading frame of 4.0-4.2 kb, encoding a peptide of Mr 150,000-160,000. A bipartite nuclear localization signal motif indicates a nuclear site for NY-BR-1, and the presence of a bZIP site (DNA-binding site followed by leucine zipper motif) suggests that NY-BR-1 is a transcription factor. Additional structural features include five tandem ankyrin repeats, implying a role for NY-BR-1 in protein-protein interactions. NY-BR-1 thus represents a breast tissue-specific putative transcription factor with autoimmunogenicity in breast cancer patients. In addition to NY-BR-1, a homologous gene, NY-BR-1.1, was identified in this study. NY-BR-1.1 shares 54% amino acid homology with NY-BR-1 and also shows tissue-restricted mRNA expression. However, unlike NY-BR-1, NY-BR-1.1 mRNA is expressed in brain, in addition to breast and testis. The exon structure of NY-BR-1.1 remains to be defined. Using human genome database, NY-BR-1 was localized to chromosome 10p11-p12, and NY-BR-1.1 was tentatively localized to chromosome 9.

Alternative Splicing↗

PubMed2003

The DFNA15 deafness mutation affects POU4F3 protein stability, localization, and transcriptional activity.

A mutation in the POU4F3 gene (BRN-3.1, BRN3C) is responsible for DFNA15 (MIM 602459), autosomal-dominant nonsyndromic hearing loss. POU4F3 is a member of the POU family of transcription factors and is essential for inner-ear hair cell maintenance. To test the potential effects of the human POU4F3 mutation, we performed a series of experiments in cell culture to mimic the human mutation. Mutant POU4F3 loses most of its transcriptional activity and most of its ability to bind to DNA and does not function in a dominant-negative manner. Moreover, whereas wild-type POU4F3 is found exclusively in the nucleus, our studies demonstrate that the mutant protein is localized both to the nucleus and the cytoplasm. Two nuclear localization signals were identified; both are essential for proper nuclear entry of POU4F3 protein. We found that the mutant protein half-life is longer than that of the wild type. We propose that the combination of defects caused by the mutation on the function of the POU4F3 transcription factor eventually leads to hair cell morbidity in affected family H members.

Amino Acid Sequence↗

PubMed2004

The brain-specific double-stranded RNA-binding protein Staufen2: nucleolar accumulation and isoform-specific exportin-5-dependent export.

The mammalian double-stranded RNA-binding proteins Staufen (Stau1 and Stau2) are involved in RNA localization in polarized neurons. In contrast to the more ubiquitously expressed Stau1, Stau2 is mainly expressed in the nervous system. In Drosophila, the third double-stranded RNA-binding domain (RBD3) of Staufen is essential for RNA interaction. When conserved amino acids within the RBD3 of Stau2 were mutated to render Stau2 defective for RNA binding, the mutant Stau2 proteins accumulate predominantly in the nucleolus. This is in contrast to wild type Stau2 that mostly localizes in the cytosol. The nuclear import is dependent on a nuclear localization signal in close proximity to the RBD3. The nuclear export of Stau2 is not dependent on CRM1 but rather on Exportin-5. We show that Exportin-5 interacts with the RBD3 of wild type Stau2 in an RNA-dependent manner in vitro but not with mutant Stau2. When Exportin-5 is down-regulated by RNA interference, only the largest isoform of Stau2 (Stau2(62)) preferentially accumulates in the nucleolus. It is tempting to speculate that Stau2(62) binds RNA in the nucleus and assembles into ribonucleoparticles, which are then exported via the Exportin-5 pathway to their final destination.

Animals↗

PubMed2005

Identification of domains of ataxia-telangiectasia mutated required for nuclear localization and chromatin association.

Ataxia-telangiectasia mutated (ATM) is essential for rapid induction of cellular responses to DNA double strand breaks (DSBs). In this study, we mapped a nuclear localization signal (NLS), 385KRKK388, within the amino terminus of ATM and demonstrate its recognition by the conventional nuclear import receptor, the importin alpha1/beta1 heterodimer. Although mutation of this NLS resulted in green fluorescent protein (GFP) x ATM(NLSm) localizing predominantly within the cytoplasm, small amounts of nuclear GFP x ATM(NLSm) were still sufficient to elicit a DNA damage response. Insertion of an heterologous nuclear export signal between GFP and ATM(NLSm) resulted in complete cytoplasmic localization of ATM, concomitantly reducing the level of substrate phosphorylation and increasing radiosensitivity, which indicates a functional requirement for ATM nuclear localization. Interestingly, the carboxyl-terminal half of ATM, containing the kinase domain, which localizes to the cytoplasm, could not autophosphorylate itself or phosphorylate substrates, nor could it correct radiosensitivity in response to DSBs even when targeted to the nucleus by insertion of an exogenous NLS, demonstrating that the ATM amino terminus is required for optimal ATM function. Moreover, we have shown that the recruitment/retention of ATM at DSBs requires its kinase activity because a kinase-dead mutant of GFP x ATM failed to form damage-induced foci. Using deletion mutation analysis we mapped a domain in ATM (amino acids 5-224) required for its association with chromatin, which may target ATM to sites of DNA damage. Combined, these data indicate that the amino terminus of ATM is crucial not only for nuclear localization but also for chromatin association, thereby facilitating the kinase activity of ATM in vivo.

Animals↗

PubMed2006

The l2 minor capsid protein of low-risk human papillomavirus type 11 interacts with host nuclear import receptors and viral DNA.

Analysis of the interactions of low-risk human papillomavirus type 11 (HPV11) L2 with karyopherin beta (Kap beta) nuclear import receptors revealed that L2 interacted with Kap beta 1, Kap beta 2, and Kap beta 3 and formed a complex with the Kap alpha 2 beta 1 heterodimer. HPV11 L2 contains two nuclear localization signals (NLSs)-in the N terminus and the C terminus-that could mediate its nuclear import via a classical pathway. Each NLS was functional in vivo, and deletion of both of them abolished L2 nuclear localization. Both NLSs interacted with the viral DNA. Thus, HPV11 L2 can interact with several karyopherins and the viral DNA and may enter the nucleus via multiple pathways.

Active Transport, Cell Nucleus↗

PubMed2001

A role for Hsc70 in regulating nucleocytoplasmic transport of a temperature-sensitive p53 (p53Val-135).

Mouse temperature-sensitive p53(Val-135) accumulates in the nucleus and acts as a "wild-type" at 32 degrees C while it is sequestered in the cytoplasm at 37 degrees C. The cytoplasmic p53(Val-135) relocalized into the nucleus upon inhibition of the nuclear export at 37 degrees C, whereas a mutation in a major bipartite nuclear localization signal (NLS) caused constitutive cytoplasmic localization, indicating that it shuttled between the cytoplasm and the nucleus by its own nuclear export signal and NLS rather than tethered to cytoplasmic structures. Although the full-length p53(Val-135) did not bind the import receptor at 37 degrees C, a C-terminally truncated p53(Val-135) lacking residues 326-390 did bind it. Molecular chaperones such as Hsc70 were associated with p53(Val-135) at 37 degrees C but not at 32 degrees C. When the nuclear export was blocked by leptomycin B, only a fraction lacking Hsc70 was specifically accumulated in the nucleus. Immunodepletion of Hsc70 from the reticulocyte lysate caused p53(Val-135) to bind the import receptor. This binding was blocked by supplying the cell extract containing Hsc70 but not by the addition of recombinant Hsc70 alone. We suggest that the association with the Hsc70-containing complex prevents the NLS from the access of the import receptor through the C-terminal region of p53(Val-135) at 37 degrees C, whereas its dissociation at 32 degrees C allows rapid nuclear import.

Amino Acid Sequence↗

PubMed1996

PO149, a new member of pollen pectate lyase-like gene family from alfalfa.

PO149 is a low-copy-number gene expressed in the late stages of pollen development. The promoter region contains no similarities in DNA sequence to those of other pollen-specific genes, except for a tobacco sequence (AAATGA), which occurs four times in this alfalfa gene and much further upstream than in tobacco. Four distinct TATA boxes were detected in the promoter with the distal and proximal TATA boxes being separated by a spacer of 269 nucleotides. Hairpin loop structures were found in the 5'- and 3'-untranslated regions of PO149 mRNA. The coding region of PO149 is interrupted by two introns and encodes a putative prepeptide of 450 amino acids with homology to pollen pectate lyase-like proteins and pollen allergens. The coding region also contains sequences characteristic of both a signal peptide and a nuclear localization signal.

Allergens↗

PubMed1994

Cytoplasmic-nuclear trafficking of progesterone receptor. In vivo study of the mechanism of action of antiprogestins.

The signal responsible for the nuclear localization of the progesterone receptor has been characterized. The study of the mechanism of this nuclear localization has revealed that the receptor continuously shuttles between the nucleus and the cytoplasm. The receptor diffuses into the cytoplasm and is constantly and actively transported back into the nucleus. Preliminary evidence suggests that the same mechanism exists for estradiol and glucocorticoid receptors. Experiments designed to study the traffic of steroid hormone receptors have been applied to the determination of the molecular mechanism of action of antisteroids. Using these techniques, we have shown that two major antiprogestins, RU486 and ZK98299, act at the same point in the cell as the hormone.

Animals↗

PubMed1994

Fibroblast growth factor-2 (FGF-2) in the nucleus: translocation process and targets.

FGF-2 (basic fibroblast growth factor) was recently detected in the nucleus of a variety of cell types. The large isoforms contain a functional nuclear localization signal that allows their nuclear accumulation in producing cells, while a small amount of FGF-2 added exogenously to target cells is translocated to the nucleus in phase G1 of the cell cycle according to an unknown process. We report here using Chinese hamster ovary cell mutants bearing deficiency in heparan sulfate proteoglycans (HSPGs) synthesis that HSPGs are required for transport of exogenous FGF-2 to the nucleus. Furthermore a co-transport was suggested since an active complex containing FGF-2 and HSPGs was isolated from nuclei of treated cells. Several FGF-2 nuclear targets were described. In vivo as in vitro, it activates rDNA transcription and it binds to a specific DNA sequence that is present in the non-transcribed spacer of ribosomal genes. In vitro, FGF-2 has a strong affinity for histone H1 and it activates the protein kinase CKII. In the nucleus FGF-2 could regulate gene expression through modulation of chromatin structure.

Animals↗

PubMed2006

Nek1 shares structural and functional similarities with NIMA kinase.

The Aspergillus NIMA serine/threonine kinase plays a pivotal role in controlling entrance into mitosis. A major function attributed to NIMA is the induction of chromatin condensation. We show here that the founder murine NIMA-related kinase, Nek1, is larger than previously reported, and that the full-length protein conserves the structural hallmarks of NIMA. Even though Nek1 bears two classical nuclear localization signals (NLS), the endogenous protein localizes to the cytoplasm. Ectopic overexpression of various Nek1 constructs suggests that the C-terminus of Nek1 bears cytoplasmic localization signal(s). Overexpression of nuclear constructs of Nek1 resulted in abnormal chromatin condensation, with the DNA mainly confined to the periphery of the nucleus. Advanced condensation phenotype was associated with nuclear pore complex dispersal. The condensation was not accompanied by up-regulation of mitotic or apoptotic markers. A similar phenotype has been described following NIMA overexpression, strengthening the notion that the mammalian Nek1 kinase has functional similarity to NIMA.

Active Transport, Cell Nucleus↗

PubMed2001

Characterization of the nuclear import pathway for HIV-1 integrase.

The karyophilic properties of the human immunodeficiency virus, type I (HIV-1) pre-integration complex (PIC) allow the virus to infect non-dividing cells. To better understand the mechanisms responsible for nuclear translocation of the PIC, we investigated nuclear import of HIV-1 integrase (IN), a PIC-associated viral enzyme involved in the integration of the viral genome in the host cell DNA. Accumulation of HIV-1 IN into nuclei of digitonin-permeabilized cells does not result from passive diffusion but rather from an active transport that occurs through the nuclear pore complexes. HIV-1 IN is imported by a saturable mechanism, implying that a limiting cellular factor is responsible for this process. Although IN has been previously proposed to contain classical basic nuclear localization signals, we found that nuclear accumulation of IN does not involve karyopherins alpha, beta1, and beta2-mediated pathways. Neither the non-hydrolyzable GTP analog, guanosine 5'-O-(thiotriphosphate), nor the GTP hydrolysis-deficient Ran mutant, RanQ69L, significantly affects nuclear import of IN, which depends instead on ATP hydrolysis. Therefore these results support the idea that IN import is not mediated by members of the karyopherin beta family. More generally, in vitro nuclear import of IN does not require addition of cytosolic factors, suggesting that cellular factor(s) involved in this active but atypical pathway process probably remain associated with the nuclear compartment or the nuclear pore complexes from permeabilized cells.

Biological Transport↗

PubMed2001

The transcription factor SOX9 regulates cell cycle and differentiation genes in chondrocytic CFK2 cells.

SOX9 is a transcription factor that is essential for chondrocyte differentiation and cartilage formation. We stably overexpressed SOX9 cDNA in the rat chondrocytic cell line CFK2. Compared with the vector control, a greater proportion of SOX9-transfected cells accumulated in the G0/G1 phase. This was associated with an increase in mRNA and protein expression of p21(cip1), an inhibitor of cyclin-dependent kinase activity. SOX9 enhanced p21(cip1) promoter activity in a luciferase reporter assay. CFK2 cells overexpressing SOX9 became more elongated and adhesive and demonstrated a shift in cytoplasmic F-actin distribution. N-cadherin mRNA levels were elevated in the SOX9-transfected cells, and SOX9 enhanced N-cadherin promoter activity. By electrophoretic mobility shift assay, nuclear extracts of SOX9-transfected CFK2 cells specifically bound an oligonucleotide comprising an N-cadherin promoter region containing a consensus SOX9-binding motif. The transcriptional activity of SOX9 depended upon nuclear localization signals required for SOX9 nuclear entry. Differentiation of transfected CFK2 cells was accelerated as evidenced by more rapid accumulation of alkaline phosphatase activity, increased production of proteoglycans, and increased calcium accumulation, and this was associated with decreased ERK1 expression. These studies demonstrate that SOX9 alters the rate of cell cycle progression of chondrocytes and their differentiation by enhancing or inhibiting the expression of selected genes, including p21(cip1) and ERK1, and that N-cadherin is an additional direct target of this transcriptional regulator.

Animals↗

PubMed2005

B23 regulates GADD45a nuclear translocation and contributes to GADD45a-induced cell cycle G2-M arrest.

Gadd45a is an important player in cell cycle G2-M arrest in response to genotoxic stress. However, the underlying mechanism(s) by which Gadd45a exerts its role in the control of cell cycle progression remains to be further defined. Gadd45a interacts with Cdc2, dissociates the Cdc2-cyclin B1 complex, alters cyclin B1 nuclear localization, and thus inhibits the activity of Cdc2/cyclin B1 kinase. These observations indicate that Gadd45a nuclear translocation is closely associated with its role in cell cycle G2-M arrest. Gadd45a has been characterized as a nuclear protein, but it does not contain a classical nuclear localization signal, suggesting that Gadd45a nuclear translocation might be mediated through different nuclear import machinery. Here we show that Gadd45a associates directly with B23 (nucleophosmin), and the B23-interacting domain is mapped at the central region (61-100 amino acids) of the Gadd45a protein using a series of Myc tag-Gadd45a deletion mutants. Deletion of this central region disrupts Gadd45a association with B23 and abolishes Gadd45a nuclear translocation. Suppression of endogenous B23 through a short interfering RNA approach disrupts Gadd45a nuclear translocation and results in impaired Gadd45a-induced cell cycle G2-M arrest. These findings demonstrate a novel association of B23 and Gadd45a and implicate B23 as an important regulator in Gadd45a nuclear import.

Active Transport, Cell Nucleus↗

PubMed2003

A docking site determining specificity of Pbs2 MAPKK for Ssk2/Ssk22 MAPKKKs in the yeast HOG pathway.

Mitogen-activated protein kinase (MAPK) cascades are conserved signaling modules composed of three sequentially activated kinases (MAPKKK, MAPKK and MAPK). Because individual cells contain multiple MAPK cascades, mechanisms are required to ensure the fidelity of signal transmission. In yeast, external high osmolarity activates the HOG (high osmolarity glycerol) MAPK pathway, which consists of two upstream branches (SHO1 and SLN1) and common downstream elements including the Pbs2 MAPKK and the Hog1 MAPK. The Ssk2/Ssk22 MAPKKKs in the SLN1 branch, when activated, exclusively phosphorylate the Pbs2 MAPKK. We found that this was due to an Ssk2/Ssk22-specific docking site in the Pbs2 N-terminal region. The Pbs2 docking site constitutively bound the Ssk2/Ssk22 kinase domain. Docking site mutations drastically reduced the Pbs2-Ssk2/Ssk22 interaction and hampered Hog1 activation by the SLN1 branch. Fusion of the Pbs2 docking site to a different MAPKK, Ste7, allowed phosphorylation of Ste7 by Ssk2/Ssk22. Thus, the docking site contributes to both the efficiency and specificity of signaling. During these analyses, we also found a nuclear export signal and a possible nuclear localization signal in Pbs2.

Amino Acid Sequence↗

PubMed2003

PEP: Predictions for Entire Proteomes.

PEP is a database of Predictions for Entire Proteomes. The database contains summaries of analyses of protein sequences from a range of organisms representing all three major kingdoms of life: eukaryotes, prokaryotes and archaea. All proteins publicly available for organisms were aligned against SWISS-PROT, TrEMBL and PDB. Additionally, the following annotations are provided: secondary structure, transmembrane helices, coiled coils, regions of low complexity, signal peptides, PROSITE motifs, nuclear localization signals and classes of cellular function. Proteins that contain long regions without regular secondary structure are also identified. We have produced a related database of structural domain-like fragments derived from PEP and clusters based on homology between all fragments. The PEP database, fragments and clusters are distributed freely as a set of flat files and have been integrated into SRS. The PEP group of databases can be accessed from: http://cubic.bioc.columbia.edu/pep.

Animals↗