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Results for “Nuclear Localization Signals”

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PubMed1995

A polymorphic bipartite motif signals nuclear targeting of early auxin-inducible proteins related to PS-IAA4 from pea (Pisum sativum).

The plant hormone, indoleacetic acid (IAA), transcriptionally activates two early genes in pea, PS-IAA4/5 and PS-IAA6 that encode short-lived nuclear proteins. The identification of the nuclear localization signals (NLS) in PS-IAA4 and PS-IAA6 using progressive deletion analysis and site-directed mutagenesis is reported. A C-terminal SV40-type NLS is sufficient to direct the beta-glucuronidase reporter to the nucleus of transiently transformed tobacco protoplasts, but is dispensible for nuclear localization of both proteins. The dominant and essential NLS in PS-IAA4 and PS-IAA6 overlap with a bipartite basic motif which is polymorphic and conserved in related proteins from other plant species, having the consensus sequence (KKNEK)KR-X(24-71)-(RSXRK)/(RK/RK). Both basic elements of this motif in PS-IAA4, (KR-X41-RSYRK), function interdependently as a bipartite NLS. However, in PS-IAA6 (KKNEKKR-X36-RKK) the upstream element of the corresponding motif contains additional basic residues which allow its autonomous function as an SV40-type monopartite NLS. The spacer-length polymorphism, X(24-70), in respective bipartite NLS peptides of several PS-IAA4-like proteins from Arabidopsis thaliana does not affect nuclear targeting function. The structural and functional variation of the bipartite basic motif in PS-IAA4-like proteins supports the proposed integrated consensus of NLS.

Amino Acid Sequence↗

PubMed1992

The interaction of amphiregulin with nuclei and putative nuclear localization sequence binding proteins.

Amphiregulin (AR) is a 23 kDa, bifunctional growth modulating glycoprotein belonging to the epidermal growth factor (EGF) family of polypeptide growth regulators. AR possesses two putative nuclear localization sequences (NLS), binds to DNA sepharose, and localizes to the nucleoli of human ovarian surface epithelial carcinoma cells suggesting that AR has a direct nuclear role. We have found that 125I-labeled AR, when exogenously applied to several carcinoma cell lines, associated with nuclei in a time, temperature, and concentration dependent fashion. The control peptide, EGF, also associated with these fractions but at approximately 20% of the efficiency of AR. Cross-linking experiments with 125I-labeled AR and nuclear fractions derived from various carcinoma and normal cell lines demonstrated that AR binds two proteins of molecular mass 205 and 120 kDa. AR binding to these nuclear fraction proteins was specific and saturable as shown by competition experiments utilizing both SV-40 large T antigen NLS and an AR derived peptide encompassing both putative AR NLS. The combined results suggest that nuclear interactions may play a significant role in AR induced growth responses.

Amino Acid Sequence↗

PubMed1996

Nuclear import of protein kinases and cyclins.

Karyophilic and acidic clusters were found in most nonmembrane serine/threonine protein kinases whose primary structure was examined. These karyophilic clusters might mediate the anchoring of the kinase molecules to transporter proteins for their regulated nuclear import and might constitute the nuclear localization signals (NLS) of the kinase molecules. In contrast to protein transcription factors that are exclusively nuclear possessing strong karyophilic peptides composed of at least four arginines (R) and lysines (K) within an hexapeptide flanked by proline and glycine helix-breakers, protein kinases often contain one histidine and three K+R residues; this is proposed to specify a weak NLS structure resulting in the nuclear import of a fraction of the total cytoplasmic kinase molecules as well as in their weak retention in the different ionic strength nuclear environment. Putative NLS peptides in protein kinases may also contain hydrophobic or bulky aromatic amino acids proposed to further diminish their capacity to act as strong NLS. Most kinases lacking karyophilic clusters (c-Mos, v-Mos, sea star MAP, and yeast KIN28, SRA1, SRA3, TPK1, TPK2) also lack acidic clusters, which is in contrast to most kinases containing both acidic and karyophilic peptides; this and the presence of R/K clusters in the transporter proteins supports a role of acidic clusters on kinases in nuclear import. Cyclins B lack karyophilic signals and are proposed to be imported into nuclei via their association with Cdc2.

Amino Acid Sequence↗

PubMed1995

The two isoforms of mouse terminal deoxynucleotidyl transferase differ in both the ability to add N regions and subcellular localization.

Two alternatively spliced terminal deoxynucleotidyl transferase transcripts, TdTS and TdTL which code respectively for proteins of 509 and 529 amino acids have been previously identified in the mouse thymus. Here we show that the same two transcripts are also present in B lineage cells from bone marrow. In addition we demonstrate that the corresponding 20 amino acid insertion found near the carboxy-terminal end of TdTL significantly alters the function of the enzyme. In contrast to TdTS, TdTL does not catalyse N region insertions at the recombination junction of a V(D)J site-specific recombination substrate. In an attempt to explain the lack of N region insertions we have characterized the different parameters which distinguish the two isoforms of TdT. Examination of transfected cell extracts revealed a reduced capacity of TdTL to add nucleotides to the 3' end of DNA, consistent with a lower terminal transferase activity. Furthermore, the half-life of the TdTL protein in these cells is 2-fold shorter than that of TdTS. Finally, despite the fact that TdTL has the same nuclear localization signal as TdTS, the cellular localization of the two isoforms was strikingly different. In contrast to nuclear TdTS, TdTL was found exclusively in the cytoplasm. All these characteristics could contribute to the functional difference between the two isoforms of TdT. However, the subcellular localization of TdTL on its own can account for its inability to add N regions.

Alternative Splicing↗

PubMed1998

[Nuclear transcription factor kappa B (NF-kappa B)].

INTRODUCTION: The transcription factor NF-kappa B is a key regulator of genes involved in responses to infection, inflammation and stress. It was first identified as a protein with ADN-binding activity specific for the 10-base-pair kappa B site in the immunoglobulin k light-chain enhancer of B lymphocytes. CURRENT KNOWLEDGE AND KEY POINTS: NF-kappa B is normally present in the cell cytoplasm bound to an inhibitory I kappa B protein. The nuclear localization signal (NLS) in NF-kappa B is a short amino acid signal sequence involved in nuclear transport. Inducers of NF-kappa activation trigger dissociation and degradation of I kappa B from the NF-kappa B complex. This allows NF-kappa B to translocate to the nucleus and bind to kappa B DNA sites. Repression of NF-kappa B dependent gene expression is one of the major elements of immunosuppression by glucocorticoids. FUTURES PROSPECTS AND PROJECTS: Endothelial cells at sites of inflammatory responses express a variety of genes that are under the control of nuclear factor NF-kappa B. This transcriptional factor with its inhibitors may be linked in an autoregulatory system that can be activated by multiple signals relevant to vascular pathophysiology. NF-kappa B is therefore an obvious target for new types of anti-inflammatory treatment.

Endothelium↗

PubMed2001

PHF3-specific antibody responses in over 60% of patients with glioblastoma multiforme.

Glioblastoma multiforme (GBM), a malignant astrocytic tumour, represents the most frequent tumour of the human brain. Nevertheless, its molecular pathology is not well understood. We utilized the immune system, which contributes to cancer protection, to help identify new GBM-related genes. By screening a human GBM cDNA library with autologous patient serum (SEREX-approach), we isolated a gene termed PHF3 (PHD finger protein 3). The gene product of PHF3 is immunogenic in GBM as tested in an allogenic patient serum screening demonstrating antibodies in 24 of 39 (61.53%) sera, whereas none of the 14 healthy persons had antibodies against PHF3. While previous SEREX studies revealed allogenic antibody responses up to 40%, our results for PHF3 represent the highest reported rate for a specific antibody response. We show that GBM patients with an antibody response against PHF3 show significant better survival than patients without PHF3-specific antibodies. Because the amino acid sequence of PHF3 contains a PHD finger (also termed LAP motif), a TFIIS homology, a proline rich region and nuclear localization signals, it supposedly functions as a transcription factor. A polyclonal antibody generated against PHF3 shows nuclear expression in most investigated formalin-fixed, paraffin embedded tissues. In GBM, PHF3 expression is concentrated in cells surrounding necroses.

Adult↗

PubMed1996

Identification of functional domains and evolution of Tc1-like transposable elements.

Tc1-like transposable elements from teleost fish have been phylogenetically examined to determine the mechanisms involved in their evolution and conserved domains of function. We identified two new functional domains in these elements. The first is a bipartite nuclear localization signal, indicating that transposons can take advantage of the transport machinery of host cells for nuclear uptake of their transposases. The second is a novel combination of a paired domain-related protein motif juxtaposed to a leucine zipper-like domain located in the putative DNA-binding regions of the transposases. This domain coexists with a special inverted repeat structure in certain transposons in such phylogenetically distant hosts as fish and insects. Our data indicate that reassortment of functional domains and horizontal transmission between species are involved in the formation and spread of new types of transposable elements.

Amino Acid Sequence↗

PubMed1993

Phosphorylation on protein kinase C sites inhibits nuclear import of lamin B2.

The nuclear lamina is a karyoskeletal structure at the nucleoplasmic surface of the inner nuclear membrane. Its assembly state is regulated by phosphorylation of the intermediate filament type lamin proteins. Strong evidence has been obtained for a causal link between phosphorylation of lamins by the p34cdc2 protein kinase and disassembly of the nuclear lamina during mitosis. In contrast, no information is currently available on the role of lamin phosphorylation during interphase of the cell cycle. Here, we have identified four protein kinase C phosphorylation sites in purified chicken lamin B2 as serines 400, 404, 410, and 411. In vivo, the tryptic peptide containing serines 400 and 404 is phosphorylated throughout interphase, whereas serines 410 and 411 become phosphorylated specifically in response to activation of protein kinase C by phorbol ester. Prompted by the close proximity of serines 410/411 to the nuclear localization signal of lamin B2, we have studied the influence of phosphorylation of these residues on nuclear transport. Using an in vitro assay, we show that phosphorylation of lamin B2 by protein kinase C strongly inhibits transport to the nucleus. Moreover, phorbol ester treatment of intact cells leads to a substantial reduction of the rate of nuclear import of newly synthesized lamin B2 in vivo. These findings have implications for the dynamic structure of the nuclear lamina, and they suggest that the modulation of nuclear transport rates by cytoplasmic phosphorylation may represent a general mechanism for regulating nuclear activities.

Amino Acid Sequence↗

PubMed1996

The dmf1/mid1 gene is essential for correct positioning of the division septum in fission yeast.

Little is known about the mechanisms that establish the position of the division plane in eukaryotic cells. Wild-type fission yeast cells divide by forming a septum in the middle of the cell at the end of mitosis. Dmf1 mutants complete mitosis and initiate septum formation, but the septa that form are positioned at random locations and angles in the cell, rather than in the middle. We have cloned the dmf1 gene as a suppressor of the cdc7-24 mutant. The dmf1 mutant is allelic with mid1. The gene encodes a novel protein containing a putative nuclear localization signal, and a carboxy-terminal PH domain. In wild-type cells, Dmf1p is nuclear during interphase, and relocates to form a medial ring at the cell cortex coincident with the onset of mitosis. This relocalization occurs before formation of the actin ring and is associated with increased phosphorylation of Dmf1p. The Dmf1p ring can be formed in the absence of an actin ring, but depends on some of the genes required for actin ring formation. When the septum is completed and the cells separate, Dmf1p staining is once again nuclear. These data implicate Dmf1p as an important element in assuring correct placement of the division septum in Schizosaccharomyces pombe cells.

Amino Acid Sequence↗

PubMed2006

AKT can be activated in the nucleus.

To investigate issues about AKT/PKB nuclear localization in cells, we examined endogenous or transiently transfected AKT localization in cancer cell lines by immunofluorescence. We found that AKT can be detected in both the nucleus and cytoplasm of HEK 293, HeLa and MCF7E cells. It was found that an active process mediates AKT nuclear translocation as shown by fusing AKT with GFP3 protein. The cellular distribution pattern of serial deletion mutants from GFP3-HA-AKT revealed that more than one segment of AKT is required for AKT nuclear translocation, while the individual segment does not have any apparent nuclear transport activity. These results implied that the signal mediating AKT nuclear translocation is conformation dependent, or more likely, is dependent upon association with other proteins. It was also found that AKT does not contain any apparent nuclear export signal. Furthermore, we found that nuclear AKT was activated in MCF7E cells upon stimulation. The possibility that nuclear activated AKT was translocated from the cytoplasm was excluded through the generation of a chimeric AKT protein, in which a strong nuclear localization signal was fused to the C-terminal of AKT.

Active Transport, Cell Nucleus↗

PubMed2006

Fusion of the BCL9 HD2 domain to E1A increases the cytopathic effect of an oncolytic adenovirus that targets colon cancer cells.

BACKGROUND: The Wnt signaling pathway is activated by mutations in the APC and beta-catenin genes in many types of human cancer. beta-catenin is stabilized by these mutations and activates transcription in part by acting as a bridge between Tcf/LEF proteins and the HD2 domain of the BCL9 coactivator. We have previously described oncolytic adenoviruses with binding sites for Tcf/LEF transcription factors inserted into the early viral promoters. These viruses replicate selectively in cells with activation of the Wnt pathway. To increase the activity of these viruses we have fused the viral transactivator E1A to the BCL9 HD2 domain. METHODS: Luciferase assays, co-immunoprecipitation and Western blotting, immunofluorescent cell staining and cytopathic effect assays were used to characterize the E1A-HD2 fusion protein and virus in vitro. Growth curves of subcutaneous SW620 colon cancer xenografts were used to characterize the virus in vivo. RESULTS: The E1A-HD2 fusion protein binds to beta-catenin in vivo and activates a Tcf-regulated luciferase reporter better than wild-type E1A in cells with activated Wnt signaling. Expression of the E1A-HD2 protein promotes nuclear import of beta-catenin, mediated by the strong nuclear localization signal in E1A. Tcf-regulated viruses expressing the fusion protein show increased expression of viral proteins and a five-fold increase in cytopathic effect (CPE) in colorectal cancer cell lines. There was no change in viral protein expression or CPE in HeLa cells, indicating that E1A-HD2 viruses retain selectivity for cells with activation of the Wnt signaling pathway. Despite increasing the cytopathic effect of the virus in vitro, fusion of the HD2 domain to E1A did not increase the burst size of the virus in vitro or the anti-tumor effect of the virus in an SW620 xenograft model in vivo. CONCLUSION: Despite an increase in the nuclear pool of beta-catenin, the effects on viral activity in colon cancer cells were small, suggesting that factors acting downstream of beta-catenin are limiting for viral replication and toxicity in these cells. The approach of fusing E1A to a protein domain implicated in oncogenic signaling could be used to selectively increase the activity of oncolytic viruses targeting several other pathways defective in cancer.

Adenovirus E1A Proteins↗

PubMed1999

DIO-1 is a gene involved in onset of apoptosis in vitro, whose misexpression disrupts limb development.

The DIO-1 (death inducer-obliterator-1) gene, identified by differential display PCR in pre-B WOL-1 cells undergoing apoptosis, encodes a putative transcription factor whose protein has two Zn finger motifs, nuclear localization signals, and transcriptional activation domains, expressed in the limb interdigitating webs during development. When overexpressed, DIO-1 translocates to the nucleus and activates apoptosis in vitro. Nuclear translocation as well as induction of apoptosis are lost after deletion of the nuclear localization sequences. DIO-1 apoptotic induction is prevented by caspase inhibitors and Bcl-2 overexpression. The in vivo role of DIO-1 was studied by misexpressing DIO-1 during chicken limb development. The most frequently observed phenotype was an arrest in limb outgrowth, an effect that correlates with the inhibition of mesodermal and ectodermal genes involved in this process. Our data demonstrate the ability of DIO-1 to trigger apoptotic processes in vitro and suggest a role for this gene in cell death during development.

Amino Acid Sequence↗

PubMed1995

Signals and structural features involved in integral membrane protein targeting to the inner nuclear membrane.

We have examined transfected cells by immunofluorescence microscopy to determine the signals and structural features required for the targeting of integral membrane proteins to the inner nuclear membrane. Lamin B receptor (LBR) is a resident protein of the nuclear envelope inner membrane that has a nucleoplasmic, amino-terminal domain and a carboxyl-terminal domain with eight putative transmembrane segments. The amino-terminal domain of LBR can target both a cytosolic protein to the nucleus and a type II integral protein to the inner nuclear membrane. Neither a nuclear localization signal (NLS) of a soluble protein, nor full-length histone H1, can target an integral protein to the inner nuclear membrane although they can target cytosolic proteins to the nucleus. The addition of an NLS to a protein normally located in the inner nuclear membrane, however, does not inhibit its targeting. When the amino-terminal domain of LBR is increased in size from approximately 22.5 to approximately 70 kD, the chimeric protein cannot reach the inner nuclear membrane. The carboxyl-terminal domain of LBR, separated from the amino-terminal domain, also concentrates in the inner nuclear membrane, demonstrating two nonoverlapping targeting signals in this protein. Signals and structural features required for the inner nuclear membrane targeting of proteins are distinct from those involved in targeting soluble polypeptides to the nucleoplasm. The structure of the nucleocytoplasmic domain of an inner nuclear membrane protein also influences targeting, possibly because of size constraints dictated by the lateral channels of the nuclear pore complexes.

Animals↗

PubMed2000

Molecular cloning and expression analysis of a putative nuclear protein, SR-25.

We cloned a full-length mouse cDNA and its human homologue encoding a novel protein designated as "SR-25." In Northern blot analysis, SR-25 mRNA was expressed in all organs tested, and relatively abundant in testis and thymus. Deduced amino acid sequences of mouse SR-25 and human SR-25 showed 77.7% identity. SR-25 has a serine-arginine repeat (SR repeat) and two types of amino acid clusters: a serine cluster and a highly basic cluster. Based on the presence of many nuclear localizing signals and a similarity to RNA splicing proteins, SR-25 is strongly suggested to be a nuclear protein and may contribute to RNA splicing.

Amino Acid Sequence↗

PubMed2000

Isolation, genomic organization, and expression analysis of the mouse and rat homologs of MEFV, the gene for familial mediterranean fever.

Familial Mediterranean fever (FMF) is a recessive disorder characterized by episodes of fever with serositis or synovitis. Recently the FMF gene (MEFV) was cloned; the protein product, pyrin/marenostrin, is thought to regulate inflammation in myeloid cells. In this manuscript we report the mouse and rat homologs of MEFV. The murine gene contains ten exons with a coding sequence of 2304 bp, while the rat homolog has nine exons with a coding sequence of 2253 bp. A considerable amino acid sequence homology was observed between the mouse and human (47.6% identity and 65.5% similarity) and between the mouse and rat genes (73.5% identity and 82.1% similarity). The predicted rodent proteins have several important domains and signals found in human pyrin, including a B-box zinc finger domain, Robbins-Dingwall nuclear localization signal, and coiled-coil domain. However, perhaps because of an ancient frame-shift mutation, neither the mouse nor the rat protein has an intact C-terminal B30.2 domain, in which most FMF-associated mutations have been found in human MEFV. Nevertheless, like the human gene, mouse Mefv is expressed in peripheral blood granulocytes but not lymphocytes. Consistent with its expression in granulocytes, Mefv was detected at high levels in the primary follicles and marginal zones of the splenic white pulp. Mefv is localized on mouse Chromosome (Chr) 16, region A3-B1, extending a region of synteny with human Chr 16p13.3. Development of knockout and knockin mouse models may provide further insights into the functional evolution of this gene.

Amino Acid Sequence↗

PubMed2006

14-3-3 proteins integrate E2F activity with the DNA damage response.

The E2F family is composed of at least eight E2F and two DP subunits, which in cells exist as E2F/DP heterodimers that bind to and regulate E2F target genes. While DP-1 is an essential and widespread component of E2F, much less is known about the DP-3 subunit, which exists as a number of distinct protein isoforms that differ in several respects including the presence of a nuclear localisation signal (NLS). We show here that the NLS region of DP-3 harbours a binding site for 14-3-3epsilon, and that binding of 14-3-3epsilon alters the cell cycle and apoptotic properties of E2F. DP-3 responds to DNA damage, and the interaction between DP-3 and 14-3-3epsilon is under DNA damage-responsive control. Further, 14-3-3epsilon is present in the promoter region of certain E2F target genes, and reducing 14-3-3epsilon levels induces apoptosis. These results identify a new level of control on E2F activity and, at a more general level, suggest that 14-3-3 proteins integrate E2F activity with the DNA damage response.

14-3-3 Proteins↗

PubMed2003

Interactions between plant RING-H2 and plant-specific NAC (NAM/ATAF1/2/CUC2) proteins: RING-H2 molecular specificity and cellular localization.

Numerous, highly conserved RING-H2 domains are found in the model plant Arabidopsis thaliana (thale cress). To characterize potential RING-H2 protein interactions, the small RING-H2 protein RHA2a was used as bait in a yeast two-hybrid screen. RHA2a interacted with one of the plant-specific NAC [NAM ('no apical meristem'), ATAF1/2, CUC2 ('cup-shaped cotyledons 2')] transcription factors, here named ANAC (abscisic acid-responsive NAC). The core RING-H2 domain was sufficient for the interaction. The ability of 11 structurally diverse RING-H2 domains to interact with ANAC was then examined. Robust interaction was detected for three of the domains, suggesting multi-specificity for the interaction. The domains that interacted with ANAC contain a glutamic acid residue in a position corresponding to a proline in many RING-H2 domains. Conversion of this glutamic acid residue into proline in RHA2a decreased its ability to interact with ANAC, most likely by changing the interaction surface. This suggested that a short, divergent region in RING-H2 domains modulate interaction specificity. ANAC contains a degenerate bipartite nuclear localization signal (NLS), while RHG1a, also identified as an ANAC interaction partner, contains a basic NLS. Both signals localized beta-glucuronidase reporter fusions to the nucleus. N-terminally truncated RHA2a also directed nuclear localization, apparently dependent on basic amino acids in the RING-H2 domain. Nuclear co-localization of the RING-H2 proteins and ANAC may enable their interaction in vivo to regulate the activity of the ANAC transcription factor.

Amino Acid Sequence↗

PubMed2002

Cut1/separase C-terminus affects spindle pole body positioning in interphase of fission yeast: pointed nuclear formation.

BACKGROUND: The separase-securin complex is required for anaphase. Separase activated by securin destruction cleaves the cohesin subunit Scc1/Rad21 enriched in kinetochores. Fission yeast Cut1/separase resides in interphase cytoplasm and mobilizes to the spindle and the spindle pole bodies (SPBs) in mitosis, while Cut2/securin remains in the nucleus from interphase to metaphase, and temporarily locates at the short spindle. RESULTS: We here report a novel SPB-led dynamic nuclear movement in fission yeast, when the Cut1 C-terminal fragment is over-expressed. The tip of the pointed nucleus contained both SPB and centromeric DNA, and rapidly moved along the bundled cytoplasmic microtubules. The same pointed nucleus was produced when the human separase C-fragment was over-expressed. The pointed nuclear formation did not require the protease site of separase, but required the conserved C-terminus and a microtubule- and kinetochore-binding protein Mtc1/Alp14, a homologue of frog XMAP215 and budding yeast Stu2. The movement-inducing C-fragment should be cytoplasmic, as the pointed nucleus was abolished when the fragment contained the NLS (nuclear localization signal). CONCLUSIONS: Overproduced separase C-fragment abolishes correct SPB-positioning in interphase. Resulting pointed nuclear formation (alternatively called 'pigtail movement') requires cytoplasmic microtubules and Mtc1/Alp14.

Catalytic Domain↗