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Results for “Nuclear Localization Signals”

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PubMed2000

Identification of novel residues involved in nuclear localization of a baculovirus polyhedrin protein.

A baculovirus polyhedrin protein has proven to possess a nuclear localization signal (NLS) sequence and a domain required for supramolecular assembly. Here we investigated five Bombyx mori nucleopolyhedrovirus (BmNPV) mutants that did not produce polyhedra. Two of five mutants were generated during routine baculoviral expression vector screening, and three were isolated by treatment with the mutagen 5-bromo-2'-deoxyuridine (BrdU). Marker rescue mapping and nucleotide sequence analysis showed that mutations in the polyhedrin gene caused the altered phenotype of these mutants. Biochemical fractionation indicated that cells infected with these mutants exhibited polyhedrin protein in both the nucleus and the cytoplasm. Electron microscopic observation revealed that polyhedrin produced by these mutants ocurred in both the nucleus and the cytoplasm, but did not form a crystalline lattice. Despite the incompleteness of polyhedrin nuclear localization, the NLSs of the five mutants were unchanged, although some of the mutations occurred within residues just outside of the domain reported to be required for polyhedron assembly (4). This result suggests that (a) the polyhedrin NLS directs polyhedrin to the nucleus, but the efficiency of this localization is regulated by regions other than the NLS (probably, polyhedrin conformation and its association with the nucleus are also involved), and (b) formation of a crystalline lattice may also be determined by several domains within polyhedrin.

Animals↗

PubMed2002

Identification and characterization of SRSP1, a rat gene differentially expressed during spermatogenesis and coding for a serine stretch-containing protein.

By means of mRNA differential display and cDNA library screening we have characterized a novel gene of the rat that is differentially expressed during spermatogenesis. Northern blot analyses showed that its mRNA reaches the highest level during the first meiotic prophase. The transcript appears to be testis-specific as it was not detectable in any of the nine other tissues tested. The full length ORF encodes a putative phosphoprotein containing a serine stretch and a bipartite nuclear localization signal, which we call Srsp1 ("serin-rich spermatogenic protein 1"). Comparison of the cDNA from SRSP1 with genomic sequences in databases allowed us to determine the number of introns; some putative regulatory sequences are proposed. This is the first report of a gene differentially expressed during spermatogenesis that codes for a protein containing a serine stretch.

Amino Acid Sequence↗

PubMed1998

Localization and functions of steroid hormone receptors.

This review focuses on the subcellular localization of steroid hormone receptors (SHRs), taking into account the technical problems of immunohistochemistry and the characteristics of nuclear localization signals (NLSs) of each receptor, on the interaction between SHRs and cellular components, and on the possible roles of sex SHRs in the reproductive organs. It is concluded that SHRs are basically localized in the nucleus, regardless of hormonal status, and that considerable amounts of unliganded SHRs may be present in the cytoplasm of target cells in exceptional cases. Most immunohistochemical results that demonstrate nuclear translocation of liganded SHRs seem to be responsible for insufficient fixation. Immunoelectron microscopy shows that SHRs associate with the chromatin in absence or presence of hormones and that intranuclear translocation of liganded SHRs from the condensed chromatin to euchromatin which observed in some cell types, may be a passive process caused by consequence of conformational changes in the chromatin binding receptors. Histochemical data suggest that the nuclear matrix (NM) is not a main binding site of liganded SHRs in the nucleus. The artificial formation of intermolecular disulfide bonds during NM preparation presumably causes the entrapment of liganded SHRs into the fraction. It seems that heat shock protein 90 (hsp90) does not form stable complexes with unliganded receptors in vivo, and it interacts with SHRs transiently cooperating with other heat shock proteins as a chaperone that helps folding of newly synthesized and refolding of denatured receptors. Estrogens transiently induce a number of nuclear protooncogenes, such as c-fos and c-jun family proteins, which act as transcription factors through estrogen receptor (ER) system in the endometrial epithelium of mature and immature rodents. Therefore, it is suggested that the changes in concentrations of these gene products trigger the proliferation and differentiation of uterine epithelium. In addition, ER system, not only in stroma cells but in the epithelial cells appears to participate in the growth response and abnormalities of epithelium elicited by the exogenous estrogen treatment at the neonatal period.

Animals↗

PubMed2001

Repression of TFII-I-dependent transcription by nuclear exclusion.

TFII-I is an unusual transcription factor possessing both basal and signal-induced transcriptional functions. Here we report the characterization of a TFII-I-related factor (MusTRD1/BEN) that regulates transcriptional functions of TFII-I by controlling its nuclear residency. MusTRD1/BEN has five or six direct repeats, each containing helix--loop--helix motifs, and, thus, belongs to the TFII-I family of transcription factors. TFII-I and MusTRD1/BEN, when expressed individually, show predominant nuclear localization. However, when the two proteins are coexpressed ectopically, MusTRD1/BEN locates almost exclusively to the nucleus, whereas TFII-I is largely excluded from the nucleus, resulting in a loss of TFII-I-dependent transcriptional activation of the c-fos promoter. Mutation of a consensus nuclear localization signal in MusTRD1/BEN results in a reversal of nuclear residency of the two proteins and a concomitant gain of c-fos promoter activity. These data suggest a means of transcriptional repression by competition at the level of nuclear occupancy.

Animals↗

PubMed2003

Nuclear localization of the Hermes transposase depends on basic amino acid residues at the N-terminus of the protein.

For the Hermes transposable element to be mobilized in its eukaryotic host, the transposase, encoded by the element, must make contact with its DNA. After synthesis in the cytoplasm, the transposase has to be actively imported into the nucleus because its size of 70.1 kDa prevents passive diffusion through the nuclear pore. Studies in vitro using transient expression of a Hermes-EGFP fusion protein in Drosophila melanogaster Schneider 2 cells showed the transposase was located predominantly in the nucleus. In silico sequence analysis, however, did not reveal any nuclear localization signal (NLS). To identify the sequence(s) responsible for localization of Hermes transposase in the nucleus, truncated or mutated forms of the transposase were examined for their influence on sub-cellular localization of marker proteins fused to the transposase. Using the same expression system and a GFP-GUS fusion double marker, residues 1-110 were recognized as sufficient, and residues 1-32 as necessary, for nuclear localization. Amino acid K25 greatly facilitated nuclear localization, indicating that at least this basic amino acid plays a significant role in this process. This sequence overlaps the proposed DNA binding region of the Hermes transposase and is not necessarily conserved in all members of the hAT transposable element family.

Amino Acid Sequence↗

PubMed2006

Regulated activating Thr172 phosphorylation of cyclin-dependent kinase 4(CDK4): its relationship with cyclins and CDK "inhibitors".

Cyclin-dependent kinase 4 (CDK4) is a master integrator of mitogenic and antimitogenic extracellular signals. It is also crucial for many oncogenic transformation processes. Various molecular features of CDK4 activation remain poorly known or debated, including the regulation of its association with D-type cyclins, its activating Thr172 phosphorylation, and the roles of Cip/Kip CDK "inhibitors" in these processes. Thr172 phosphorylation of CDK4 was reinvestigated using two-dimensional gel electrophoresis in various experimental systems, including human fibroblasts, canine thyroid epithelial cells stimulated by thyrotropin, and transfected mammalian and insect cells. Thr172 phosphorylation of CDK4 depended on prior D-type cyclin binding, but Thr172 phosphorylation was also found in p16-bound CDK4. Opposite effects of p27 on cyclin D3-CDK4 activity observed in different systems depended on its stoichiometry in this complex. Thr172-phosphorylated CDK4 was enriched in complexes containing p21 or p27, even at inhibitory levels of p27 that precluded CDK4 activity. Deletion of the p27 nuclear localization signal sequence relocalized cyclin D3-CDK4 in the cytoplasm but did not affect CDK4 phosphorylation. Within cyclin D3 complexes, T-loop phosphorylation of CDK4, but not of CDK6, was directly regulated, identifying it as a determining target for cell cycle control by extracellular factors. Collectively, these unexpected observations indicate that CDK4-activating kinase(s) should be reconsidered.

Animals↗

PubMed2001

Import of Agrobacterium T-DNA into plant nuclei: two distinct functions of VirD2 and VirE2 proteins.

To study the mechanism of nuclear import of T-DNA, complexes consisting of the virulence proteins VirD2 and VirE2 as well as single-stranded DNA (ssDNA) were tested for import into plant nuclei in vitro. Import of these complexes was fast and efficient and could be inhibited by a competitor, a nuclear localization signal (NLS) coupled to BSA. For import of short ssDNA, VirD2 was sufficient, whereas import of long ssDNA additionally required VirE2. A VirD2 mutant lacking its C-terminal NLS was unable to mediate import of the T-DNA complexes into nuclei. Although free VirE2 molecules were imported into nuclei, once bound to ssDNA they were not imported, implying that when complexed to DNA, the NLSs of VirE2 are not exposed and thus do not function. RecA, another ssDNA binding protein, could substitute for VirE2 in the nuclear import of T-DNA but not in earlier events of T-DNA transfer to plant cells. We propose that VirD2 directs the T-DNA complex to the nuclear pore, whereas both proteins mediate its passage through the pore. Therefore, by binding to ssDNA, VirE2 may shape the T-DNA complex such that it is accepted for translocation into the nucleus.

Active Transport, Cell Nucleus↗

PubMed2003

Functional domains required for the Saccharomyces cerevisiae Mus81-Mms4 endonuclease complex formation and nuclear localization.

The Saccharomyces cerevisiae Mms4 and Mus81 proteins form a specific complex, which functions as an endonuclease specific for branched DNA molecules and protects cells from killing by DNA alkylation damage, but not damage induced by ionizing radiations. In an effort to further understand the structure and functions of the Mus81-Mms4 complex, we attempted to define domains required for complex formation and nuclear localization through deletion and mutagenesis analyses. Combined yeast two-hybrid and co-immunoprecipitation experiments indicate that the C-terminal 100 amino acids of both Mus81 and Mms4 are required and sufficient for heterodimer formation. However, a single amino acid substitution in Mms4 in the N-terminal region is able to abolish the interaction, which suggests that the three-dimensional structure is also important for Mms4 to interact with Mus81. By fusion to green fluorescent protein and in vivo subcellular localization studies, we demonstrate that Mms4 and Mus81 are nuclear proteins and can be localized to the nucleus independently. Deletion analyses indicate that one of two putative nuclear localization signals (residues 244-263) in Mms4 is required for localization, whereas the N-terminal half of Mus81 is necessary and sufficient for its localization to the nucleus.

Cell Nucleus↗

PubMed2003

Elevated activity of the large form of ADAR1 in vivo: very efficient RNA editing occurs in the cytoplasm.

Mammalian cells express small and large forms of the RNA editing enzyme ADAR1, referred to as ADAR1-S and ADAR1-L, respectively. Here we observed that ADAR1-L was >70-fold more active than was ADAR1-S when assayed with a substrate that could be edited in either the nucleus or cytoplasm, and was also much more active when assayed with a substrate that was generated in the cytoplasm during viral replication. In contrast, when a substrate that could only be edited within the nucleus was assayed, the activity of ADAR1-S was found to be somewhat higher than that of ADAR1-L. We show here not only that editing could occur in the cytoplasm but also that the process was extremely efficient, occurred rapidly, and could occur in the absence of translation. Consistent with the observation that editing in the cytoplasm can be very efficient, deletion of the nuclear localization signal from ADAR2 resulted in a protein with 15-fold higher activity when tested with a substrate that contained an editing site in the mature message. In addition to its potential role in an antiviral response, we propose that ADAR1-L is the form primarily responsible for editing mRNAs in which the editing site is retained after processing.

Adenosine Deaminase↗

PubMed2003

JUMONJI, a critical factor for cardiac development, functions as a transcriptional repressor.

JUMONJI (JMJ) is a nuclear factor that is critical for normal cardiovascular development, evidenced by the analysis of jmj homozygous mutant mice. However, the molecular function of JMJ remains to be elucidated. In the present study, we investigated whether JMJ is a transcriptional modulator. Reporter gene assays using the GAL4-DNA binding domain fused to JMJ and a reporter gene consisting of the GAL4 binding sites upstream of a luciferase reporter gene indicated that JMJ functions as a powerful transcriptional repressor. The DNA binding motif of JMJ was determined using CASTing experiments by incubating a random oligonucleotide library with the GST-JMJ fusion protein coupled to agarose beads. Among the selected binding oligonucleotides, the high affinity DNA binding sequences were identified by gel retardation assays. JMJ repressed expression of the reporter genes containing the high affinity JMJ binding sequences, indicating that JMJ is a DNA-binding transcriptional repressor. The domains for transcriptional repression, DNA binding, and nuclear localization signal were mapped by mutational analyses using reporter gene assays, gel retardation assays, and immunostaining experiments, respectively. The present data demonstrate for the first time that JMJ functions as a DNA-binding transcriptional repressor. Therefore, JMJ may play a critical role in transcription factor cascade to regulate expression of heart-specific genes and normal cardiac development.

Animals↗

PubMed1999

Noninvasive intracellular delivery of functional peptides and proteins.

In order to probe intracellular signaling based on interactions of thousands of proteins expressed in the living cell, new methods of noninvasive delivery of functional peptides and proteins to cells have been developed. These include cellular import of peptides and proteins based on the cell-membrane-permeable properties of the hydrophobic region of a signal peptide sequence. The prototypical cell-permeable SN50 peptide, which contains the nuclear localization signal sequence of NK-kappaB p50, has been applied in multiple cell types to block nuclear import of this and other transcription factors. Further developments, including site-specific ligation of bipartite import peptides and production of import-competent recombinant proteins, provide the means for easy and rapid delivery of peptides and proteins to a wide spectrum of cells in order to regulate intracellular pathways involved in adhesion, signaling and trafficking to the nucleus.

Animals↗

PubMed2000

Differential effects of C-terminal molecular tagged integrase on replication competent moloney murine leukemia virus.

Moloney murine leukemia virus (M-MuLV) proviruses carrying integrase (IN) protein tagged either with a simian virus 40 (SV40) nuclear localization signal (NLS) or various antigenic epitopes were generated. Hexahistidine (His(6)), hemagluttinin (HA), or two consecutive HA sequences (2XHA) were fused to the C-terminus of IN as antigenic markers. These epitope-tagged IN proteins were stably expressed through multiple rounds of infection. The IN-His(6), IN-HA, and IN-2XHA proteins, purified from virus, could be immunoprecipitated with antibodies against His(6) and HA, respectively. An M-MuLV provirus encoding the SV40 large T antigen NLS fused to IN at the same position as the epitope tags was also passaged through cells. In contrast to the stability of the epitope tags, the SV40 NLS sequence was rapidly mutated by a frameshift mutation that introduced negatively charged amino acids into the basic NLS. The instability of the NLS suggests that the strong nuclear localization of the IN-SV40 NLS may have detrimental effects on virus assembly. These observations have implications for studying nuclear transport properties of M-MuLV and for engineering a murine-based retroviral vector for gene therapy.

Amino Acid Sequence↗

PubMed2003

Primary structure and compartmentalization of Drosophila melanogaster host cell factor.

Human host cell factor-1 (HCF-1) is a large, 2035-residue nuclear protein that interacts with cellular and viral transcription factors. It contains an N-terminal kelch domain, C-terminal fibronectin type III (FnIII) domain, and a central region including tandem repeats which act as cleavage sites. A second human HCF-1 related gene encodes a protein with a high degree of homology in both the N-terminal kelch domain and C-terminal FnIII domain, but lacks the central portion and as a result is considerably smaller at 792 residues. A unique HCF orthologue has been found in Caenorhabditis elegans which is structurally more related to HCF-2 than HCF-1. Here we report the cloning and expression of the single Drosophila melanogaster host cell factor orthologue (dHCF). The dHCF is 1500 residues in size, intermediate between HCF-1 and HCF-2 and contains an N-terminal kelch domain, and C-terminal FnIII domain both of which show a very high degree of identity, and a central region of some 700 residues with more limited homology. Despite containing a central region no repeat-related motifs were apparent. The dHCF is expressed as a single unprocessed polypeptide consistent with the lack of the internal HCF-1 processing sites, and exhibits a predominantly nuclear localization. We show that this nuclear localization is dependent on a bipartite nuclear localization signal at the C-terminus of the protein, which contains a long spacer of 20 amino acids between two basic clusters. Finally, we also show that dHCF is unable to rescue the tsBN67 cell cycle arrest phenotype. These results indicate that dHCF is an orthologue of HCF-1, although both proteins might not be functionally exchangeable.

Amino Acid Sequence↗

PubMed2004

A human immunodeficiency virus type 1 Tat-like arginine-rich RNA-binding domain is essential for HEXIM1 to inhibit RNA polymerase II transcription through 7SK snRNA-mediated inactivation of P-TEFb.

The HEXIM1 protein inhibits the kinase activity of P-TEFb (CDK9/cyclin T) to suppress RNA polymerase II transcriptional elongation in a process that specifically requires the 7SK snRNA, which mediates the interaction of HEXIM1 with P-TEFb. In an attempt to define the sequence requirements for HEXIM1 to interact with 7SK and inactivate P-TEFb, we have identified the first 18 amino acids within the previously described nuclear localization signal (NLS) of HEXIM1 as both necessary and sufficient for binding to 7SK in vivo and in vitro. This 7SK-binding motif was essential for HEXIM1's inhibitory action, as the HEXIM1 mutants with this motif replaced with a foreign NLS failed to interact with 7SK and P-TEFb and hence were unable to inactivate P-TEFb. The 7SK-binding motif alone, however, was not sufficient to inhibit P-TEFb. A region C-terminal to this motif was also required for HEXIM1 to associate with P-TEFb and suppress P-TEFb's kinase and transcriptional activities. The 7SK-binding motif in HEXIM1 contains clusters of positively charged residues reminiscent of the arginine-rich RNA-binding motif found in a wide variety of proteins. Part of it is highly homologous to the TAR RNA-binding motif in the human immunodeficiency virus type 1 (HIV-1) Tat protein, which was able to restore the 7SK-binding ability of a HEXIM1 NLS substitution mutant. We propose that a similar RNA-protein recognition mechanism may exist to regulate the formation of both the Tat-TAR-P-TEFb and the HEXIM1-7SK-P-TEFb ternary complexes, which may help convert the inactive HEXIM1/7SK-bound P-TEFb into an active one for Tat-activated and TAR-dependent HIV-1 transcription.

Amino Acid Sequence↗

PubMed2006

Inhibitory effects of homeodomain-interacting protein kinase 2 on the aorta-gonad-mesonephros hematopoiesis.

Definitive hematopoiesis starts in the aorta-gonad-mesonephros (AGM) region of the mouse embryo. Our previous studies revealed that STAT3, a gp130 downstream transcription factor, is required for AGM hematopoiesis and that homeodomain-interacting protein kinase 2 (HIPK2) phosphorylates serine-727 of STAT3. HIPK2 is a serine/threonine kinase known to be involved in transcriptional repression and apoptosis. In the present study, we examined the role of HIPK2 in hematopoiesis in mouse embryo. HIPK2 transcripts were found in fetal hematopoietic tissues such as the mouse AGM region and fetal liver. In cultured AGM cells, HIPK2 protein was detected in adherent cells. Functional analyses of HIPK2 were carried out by introducing wild-type and mutant HIPK2 constructs into AGM cultures. Production of CD45(+) hematopoietic cells was suppressed by forced expression of HIPK2 in AGM cultures. This suppression required the kinase domain and nuclear localization signals of HIPK2, but the kinase activity was dispensable. HIPK2-overexpressing AGM-derived nonadherent cells did not form cobblestone-like colonies in cultures with stromal cells. Furthermore, overexpression of HIPK2 in AGM cultures impeded the expansion of CD45(low)c-Kit(+) cells, which exhibit the immature hematopoietic progenitor phenotype. These data indicate that HIPK2 plays a negative regulatory role in AGM hematopoiesis in the mouse embryo.

Animals↗

PubMed2002

Arabidopsis thaliana atrab28: a nuclear targeted protein related to germination and toxic cation tolerance.

The Arabidopsis gene Atrab28 has been shown to be expressed during late embryogenesis. The pattern of expression of Atrab28 mRNA and protein during embryo development is largely restricted to provascular tissues of mature embryos, and in contrast to the maize Rab28 homologue it cannot be induced by ABA and dehydration in vegetative tissues. Here, we have studied the subcellular location of Atrab28 protein and the effect of its over-expression in transgenic Arabidopsis plants. The Atrab28 protein was mainly detected in the nucleus and nucleolus of cells from mature embryos. In frame fusion of Atrab28 to the reporter green fluorescent protein (GFP) directed the GFP to the nucleus in transgenic Arabidopsis and in transiently transformed onion cells. Analysis of chimeric constructs identified an N-terminal region of 60 amino acids containing a five amino acid motif QPKRP that was necessary for targeting GFP to the nucleus. These results indicate that Atrab28 protein is targeted to the nuclear compartments by a new nuclear localization signal (NLS). Transgenic Arabidopsis plants, with gain of Atrab28 function, showed faster germination rates under either standard or salt and osmotic stress conditions. Moreover, improved cation toxicity tolerance was also observed not only during germination but also in seedlings. These results suggest a role of Atrab28 in the ion cell balance during late embryogenesis and germination.

Adaptation, Physiological↗

PubMed2006

Novel splice variants of ING4 and their possible roles in the regulation of cell growth and motility.

The ING4 gene is a candidate tumor suppressor gene that functions in cell proliferation, contact inhibition, and angiogenesis. We identified three novel splice variants of ING4 with differing activities in controlling cell proliferation, cell spreading, and cell migration. ING4_v1 (the longest splice variant), originally identified as ING4, encodes an intact nuclear localization signal (NLS), whereas the other three splice variants (ING4_v2, ING4_v3, and ING4_v4) lack the full NLS, resulting in increased cytoplasmic localization of these proteins. We found that one of the three ING4 variants, ING4_v2, is expressed at the same level as the original ING4 (ING4_v1), suggesting that ING4 variants may have significant biological functions. Growth suppressive effects of the variants that have a partial NLS (ING4_v2 and ING4_v4) were attenuated by a weaker effect of the variants on p21(WAF1) promoter activation. ING4_v4 lost cell spreading and migration suppressive effects; on the other hand, ING4_v2 retained a cell migration suppressive effect but lost a cell spreading suppressive effect. Therefore, ING4_v2, which localized primarily into cytoplasm, might have an important role in the regulation of cell migration. We also found that ING4_v4 played dominant-negative roles in the induction of p21(WAF1) promoter activation and in the suppression of cell motility by ING4_v1. In addition, ING4 variants had different binding affinities to two cytoplasmic proteins, protein-tyrosine phosphatase, receptor type, f polypeptide (PTPRF), interacting protein (liprin), alpha1, and G3BP2a. Understanding the functions of the four splice variants may aid in defining their roles in human carcinogenesis.

Adaptor Proteins, Signal Transducing↗

PubMed2005

PTEN enters the nucleus by diffusion.

Despite much evidence for phosphatidylinositol phosphate (PIP)-triggered signaling pathways in the nucleus, there is little understanding of how the levels and activities of these proteins are regulated. As a first step to elucidating this problem, we determined whether phosphatase and tensin homolog deleted on chromosome 10 (PTEN) enters the nucleus by passive diffusion or active transport. We expressed various PTEN fusion proteins in tsBN2, HeLa, LNCaP, and U87MG cells and determined that the largest PTEN fusion proteins showed little or no nuclear localization. Because diffusion through nuclear pores is limited to proteins of 60,000 Da or less, this suggests that nuclear translocation of PTEN occurs via diffusion. We examined PTEN mutants, seeking to identify a nuclear localization signal (NLS) for PTEN. Mutation of K13 and R14 decreased nuclear localization, but these amino acids do not appear to be part of an NLS. We used fluorescence recovery after photobleaching (FRAP) to demonstrate that GFP-PTEN can passively pass through nuclear pores. Diffusion in the cytoplasm is retarded for the PTEN mutants that show reduced nuclear localization. We conclude that PTEN enters the nucleus by diffusion. In addition, sequestration of PTEN in the cytoplasm likely limits PTEN nuclear translocation.

Cell Line, Tumor↗