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Results for “Nuclear Localization Signals”

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PubMed2004

Chicken anemia virus induced apoptosis: underlying molecular mechanisms.

In 1990, the chicken anemia virus (CAV) genome was cloned by us and proven to be representative for CAV isolates worldwide. This genome contains unique promoter/enhancer replication elements and genes. Upon infection of its target cells, CAV replicates via a double-stranded (ds) DNA intermediate. From this ds CAV molecule, a single mRNA is transcribed, which encodes for three distinct proteins VP1, VP2, and VP3 or apoptin. Its capsid contains only the VP1 protein. However, for the production of the neutralizing epitope, co-synthesis of VP1 and VP2 are needed. CAV genomes with mutations in the 12 bp insert of the promoter/enhancer region were shown to produce immunogenic functional CAV particles. Mutations in these and other regulatory elements of CAV might also decrease its virus load resulting in a reduced pathogenic effect. CAV causes fatal cytopathogenic effects in e.g. chicken thymocytes via apoptosis. Under in vitro conditions, CAV replicates only in transformed chicken cell lines, which indicates that at least a part of the CAV life-cycle requires transformed-like cellular events. In these transformed cell lines, the synthesis of the apoptin protein alone mimics the CAV-induced apoptosis, whereas the VP2 protein also harbors some apoptotic activity. Extensive studies on apoptin resulted in the characterization of domains essential for its apoptotic activity and nuclear localization, which seems to be related with its ability to induce apoptosis. Therefore, both VP2 and apoptin are of interest in reducing the pathogenicity of CAV infections. A series of biomedical studies on apoptin have been carried out in human cell systems, which are informative about the mechanism of CAV-induced apoptosis in chicken (transformed) cells. Synthesis of apoptin alone induces apoptosis in various human transformed and/or tumorigenic cell lines, but not in normal human diploid cells. A striking difference in the cellular localization of apoptin was observed in human normal diploid cells versus tumor cells. In all tumor cells, apoptin is located mainly in the heterochromatic regions of the nucleus, whereas in normal cells it is present in peri-nuclear structures. Apoptin contains a bipartite nuclear localization signal, and one domain that resemble a nuclear export signal. Elucidation of parts of the apoptin-induced apoptotic pathway revealed unique characteristics: apoptin-induced apoptosis is independent of the tumor suppressor p53. The anti-apoptotic protein Bcl-2 does not inhibit but even accelerates apoptin-induced apoptosis in tumor cells, whereas over expression of Bcl-2 in normal cells has no effect on the apoptin activity. Upstream caspases are not involved, whereas downstream caspase 3 is, but seems not to be essential. A number of novel proteins were shown to interact with apoptin in transformed cells. Future studies of apoptin, VP2 and related cellular proteins in chicken cells will unravel the regulatory aspects of CAV-induced apoptosis.

Animals↗

PubMed2003

The E7 oncoprotein of high-risk human papillomavirus type 16 enters the nucleus via a nonclassical Ran-dependent pathway.

E7, the major transforming protein of high-risk human papillomavirus (HPV), type 16, binds and inactivates the retinoblastoma protein (pRb), and the Rb-related proteins p107 and p130. HPV16 E7 is a nuclear protein lacking a classical basic nuclear localization signal. In this study we investigated the nuclear import of HPV16 E7 oncoprotein in digitonin-permeabilized HeLa cells. HPV16 E7 nuclear import was independent of pRb, as an E7(DeltaDLYC) variant defective in pRb binding was imported into the nuclei of digitonin-permeabilized cells as efficiently as wild-type E7 in the presence of exogenous cytosol. Interestingly, we discovered that HPV16 E7 is imported into the nuclei via a novel pathway different from those mediated by Kap alpha2beta1 heterodimers, Kap beta1, or Kap beta2. Nuclear accumulation of E7 required Ran and was not inhibited by the RanG19V-GTP variant, an inhibitor of Kap beta mediated import pathways. Together the data suggest that HPV16 E7 translocates through the nuclear pores via a nonclassical Ran-dependent pathway, independent of the main cytosolic Kap beta import receptors.

Active Transport, Cell Nucleus↗

PubMed2003

Cell cycle dependent intracellular distribution of two spliced isoforms of TCP/ILF3 proteins.

TCP80 is an approximately 80kDa mammalian cytoplasmic protein that binds to a set of mRNAs and inhibits their translation in vitro and ex vivo. This protein has high sequence similarity to interleukin-2 enhancer-binding factors (NF90/ILF3) and the M-phase phosphoprotein (MPP4)/DRBP76. A 110kDa immunologic isoform of TCP80/NF90/MPP4/DRBP76, termed TCP110, also is present in cytoplasm and nuclei of many types of cells. A cDNA sequence coding for TCP110 was derived by 5(')RACE. The TCP110 sequence is identical to ILF3. The gene coding for TCP110/ILF3 mapped to human chromosome 19 and the gene organization was analyzed using TCP80 and TCP110/ILF3 cDNA sequences. The TCP/ILF3 gene spans >34.8kb and contains 21 exons. At least one alternatively spliced product involving exons 19-21 exists and predicts the formation of either TCP80 or TCP110/ILF3. However, the functional relationships of TCP80 and TCP110/ILF3 required elucidation. The metabolic turnover rates and subcellular distribution of TCP80 and TCP110/ILF3 during the cell cycle showed TCP80 to be relatively stable (t(1/2)=5 days) in the cytoplasmic compartment. In comparison, TCP110/ILF3 migrated between the cytoplasmic and nuclear compartments during the cell cycle. The TCP110 C-terminal segment contains an additional nuclear localizing signal that plays a role in its nuclear translocation. This study indicates that the multiple cellular functions, i.e., translation control, interleukin-2 enhancer binding, or cell division, of TCP/ILF3 are fulfilled by alternatively spliced isoforms.

Alternative Splicing↗

PubMed2001

Disruption of the FG nucleoporin NUP98 causes selective changes in nuclear pore complex stoichiometry and function.

The NUP98 gene encodes precursor proteins that generate two nucleoplasmically oriented nucleoporins, NUP98 and NUP96. By using gene targeting, we have selectively disrupted the murine NUP98 protein, leaving intact the expression and localization of NUP96. We show that NUP98 is essential for mouse gastrulation, a developmental stage that is associated with rapid cell proliferation, but dispensable for basal cell growth. NUP98-/- cells had an intact nuclear envelope with a normal number of embedded nuclear pore complexes. Typically, NUP98-deficient cells contained on average approximately 5-fold more cytoplasmic annulate lamellae than control cells. We found that a set of cytoplasmically oriented nucleoporins, including NUP358, NUP214, NUP88, and p62, assembled inefficiently into nuclear pores of NUP98-/- cells. Instead, these nucleoporins were prominently associated with the annulate lamellae. By contrast, a group of nucleoplasmically oriented nucleoporins, including NUP153, NUP50, NUP96, and NUP93, had no affinity for annulate lamellae and assembled normally into nuclear pores. Mutant pores were significantly impaired in transport receptor-mediated docking of proteins with a nuclear localization signal or M9 import signal and showed weak nuclear import of such substrates. In contrast, the ability of mutant pores to import ribosomal protein L23a and spliceosome protein U1A appeared intact. These observations show that NUP98 disruption selectively impairs discrete protein import pathways and support the idea that transport of distinct import complexes through the nuclear pore complex is mediated by specific subsets of nucleoporins.

Active Transport, Cell Nucleus↗

PubMed2001

The human decatenation checkpoint.

Chromatid catenation is actively monitored in human cells, with progression from G(2) to mitosis being inhibited when chromatids are insufficiently decatenated. Mitotic delay was quantified in normal and checkpoint-deficient human cells during treatment with ICRF-193, a topoisomerase II catalytic inhibitor that prevents chromatid decatenation without producing topoisomerase-associated DNA strand breaks. Ataxia telangiectasia (A-T) cells, defective in DNA damage checkpoints, showed normal mitotic delay when treated with ICRF-193. The mitotic delay in response to ICRF-193 was ablated in human fibroblasts expressing an ataxia telangiectasia mutated- and rad3-related (ATR) kinase-inactive ATR allele (ATR(ki)). BRCA1-mutant HCC1937 cells also displayed a defect in ICRF-193-induced mitotic delay, which was corrected by expression of wild-type BRCA1. Phosphorylations of hCds1 or Chk1 and inhibition of Cdk1 kinase activity, which are elements of checkpoints associated with DNA damage or replication, did not occur during ICRF-193-induced mitotic delay. Over-expression of cyclin B1 containing a dominant nuclear localization signal, and inhibition of Crm1-mediated nuclear export, reversed ICRF-193-induced mitotic delay. In combination, these results imply that ATR and BRCA1 enforce the decatenation G(2) checkpoint, which may act to exclude cyclin B1/Cdk1 complexes from the nucleus. Moreover, induction of ATR(ki) produced a 10-fold increase in chromosomal aberrations, further emphasizing the vital role for ATR in genetic stability.

Ataxia Telangiectasia↗

PubMed1999

Degradation of the basic helix-loop-helix/Per-ARNT-Sim homology domain dioxin receptor via the ubiquitin/proteasome pathway.

The basic helix-loop-helix/Per-ARNT-Sim homology domain dioxin receptor (DR) translocates to the nucleus upon binding of aromatic hydrocarbon ligands typified by dioxin, whereupon it partners the Ah receptor nuclear translocator and initiates transcription. Concurrently, ligand binding down-regulates receptor levels via an unknown mechanism. In this study we show that receptor levels are dependent upon cellular compartmentalization, with entry into the nucleus leading to the rapid destruction of the DR. Ligand-induced DR translocation was bypassed by adding a heterologous nuclear localization signal to the DR, creating a constitutively nuclear form of the dioxin receptor (DRNLS). The DRNLS protein was shown to be unstable with a half-life of </=1 h whether partnering ARNT or HSP90. Thus, the structural changes induced by ligand binding have no inherent effect on DR stability but are critical in transporting the receptor prior to degradation. The proteolytic pathway that degrades the nuclear receptor is suggested to involve ubiquitination as it was inhibited by the proteasome inhibitor MG132 or co-expression of DRNLS with the ubiquitin mutant UbK48R. Incubation of cells expressing DRNLS with the phosphatase inhibitor calyculin resulted in the rapid phosphorylation and ubiquitination of DRNLS, suggesting that a nuclear kinase is required to trigger receptor proteolysis. Overall, this study demonstrates a novel mechanism of proteolysis whereby the simple relocation of a transcription factor from cytoplasm to nucleus initiates its rapid destruction.

Animals↗

PubMed2001

Nuclear import of the yeast AP-1-like transcription factor Yap1p is mediated by transport receptor Pse1p, and this import step is not affected by oxidative stress.

The yeast AP-1-like transcription factor, Yap1p, is essential for the oxidative stress response in budding yeast. Yap1p is located predominantly in the cytoplasm; however, upon imposition of oxidative stress, Yap1p concentrates in the nucleus and activates target genes. Yap1p is constitutively transported in and out of the nucleus. Oxidative stress inhibits the Crm1p/Xpo1p-dependent nuclear export step, resulting in nuclear accumulation of Yap1p. In this study, we examined the mechanism for Yap1p nuclear import, and determined whether the import step is affected by oxidative stress. The nuclear accumulation of Yap1p required the activity of the small GTPase, Ran/Gsp1p. Under conditions in pse1-1 cells carrying a temperature-sensitive mutation of the importin beta family member PSE1/KAP121, nuclear translocation of Yap1p was inhibited dramatically. In an in vitro assay, we showed that Yap1p could directly bind to Pse1p and that this interaction was dissociated by Ran-GTP. These results indicate that Pse1p is the nuclear import receptor for Yap1p. In addition to Pse1p, we suggest that Kap123p, which is homologous to Pse1p, has a minor effect on the nuclear import of Yap1p. Furthermore, we identified the nuclear localization signal of Yap1p and demonstrated that the nuclear import of Yap1p was not affected by oxidative stress.

Cell Nucleus↗

PubMed1999

Txk, a nonreceptor tyrosine kinase of the Tec family, is expressed in T helper type 1 cells and regulates interferon gamma production in human T lymphocytes.

Differentiation of human T cells into T helper (Th)1 and Th2 cells is vital for the development of cell-mediated and humoral immunity, respectively. However, the precise mechanism responsible for the Th1 cell differentiation is not fully clarified. We have studied the expression and function of Txk, a member of the Tec family of nonreceptor tyrosine kinases. We found that Txk expression is restricted to Th1/Th0 cells with IFN-gamma producing potential. Txk transfection of Jurkat T cells resulted in a several-fold increase of IFN-gamma mRNA expression and protein production; interleukin (IL)-2 and IL-4 production were unaffected. Antisense oligodeoxynucleotide of Txk specifically inhibited IFN-gamma production of normal peripheral blood lymphocytes, antigen-specific Th1 clones, and Th0 clones; IL-2 and IL-4 production by the T cells was unaffected. Txk cotransfection led to the enhanced luciferase activity of plasmid (p)IFN-gamma promoter/enhancer (pIFN-gamma[-538])-luciferase-transfected Jurkat cells upon mitogen activation. Txk transfection did not affect IL-2 and IL-4 promoter activities. Thus, Txk specifically upregulates IFN-gamma gene transcription. In fact, Txk translocated from cytoplasm into nuclei upon activation and transfection with a mutant Txk expression plasmid that lacked a nuclear localization signal sequence did not enhance IFN-gamma production by the cells, indicating that nuclear localization of Txk is obligatory for the enhanced IFN-gamma production. In addition, IL-12 treatment of peripheral blood CD4(+) T cells enhanced the Txk expression, whereas IL-4 treatment completely inhibited it. These results indicate that Txk expression is intimately associated with development of Th1/Th0 cells and is significantly involved in the IFN-gamma production by the cells through Th1 cell-specific positive transcriptional regulation of the IFN-gamma gene.

Cell Differentiation↗

PubMed1996

GLE2, a Saccharomyces cerevisiae homologue of the Schizosaccharomyces pombe export factor RAE1, is required for nuclear pore complex structure and function.

To identify and characterize novel factors required for nuclear transport, a genetic screen was conducted in the yeast Saccharomyces cerevisiae. Mutations that were lethal in combination with a null allele of the gene encoding the nucleoporin Nup100p were isolated using a colony-sectoring assay. Three complementation groups of gle (for GLFG lethal) mutants were identified. In this report, the characterization of GLE2 is detailed. GLE2 encodes a 40.5-kDa polypeptide with striking similarity to that of Schizosaccharomyces pombe RAE1. In indirect immunofluorescence and nuclear pore complex fractionation experiments, Gle2p was associated with nuclear pore complexes. Mutated alleles of GLE2 displayed blockage of polyadenylated RNA export; however, nuclear protein import was not apparently diminished. Immunofluorescence and thin-section electron microscopic analysis revealed that the nuclear pore complex and nuclear envelope structure was grossly perturbed in gle2 mutants. Because the clusters of herniated pore complexes appeared subsequent to the export block, the structural perturbations were likely indirect consequences of the export phenotype. Interestingly, a two-hybrid interaction was detected between Gle2p and Srp1p, the nuclear localization signal receptor, as well as Rip1p, a nuclear export signal-interacting protein. We propose that Gle2p has a novel role in mediating nuclear transport.

Amino Acid Sequence↗

PubMed1992

Isolation and characterization of a novel gene with differential expression in benign and malignant human breast tumours.

We report the identification of a novel cDNA representing an mRNA showing significantly higher levels of expression in benign breast lesions than in carcinomas. This cDNA was identified by differential screening of a cDNA library generated from a breast carcinoma, and shows consistently higher expression in fibroadenomas than in carcinomas. The expression in both benign and malignant tissues is highest in epithelial cells as determined by in situ hybridization to tissue sections. The nucleotide sequence of the full-length cDNA has been determined, and the deduced protein is highly basic with no signal or transmembrane sequence, but two potential nuclear localization signals. Neither the DNA nor the protein sequence show any significant homology to sequences in current databases. The cDNA hybridizes to multiple sequences within both human and other mammalian genomes, but to single genomic sequences in Drosophila, Physarum and Schizosaccharomyces pombe. This cDNA therefore represents a highly conserved gene sequence. We have identified only one major transcript in human cells, and it seems likely that there are several pseudogenes within the human genome.

Adenofibroma↗

PubMed2000

Nucleocytoplasmic shuttling of the aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that acts in concert with the AhR nuclear translocator (ARNT), and alters gene expression in response to environmental contaminants such as 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD). We have previously shown that AhR contains both a nuclear localization signal (NLS), AhR(13-39), and a nuclear export signal (NES), AhR(55-75), in its NH(2)-terminal region. In this study, we obtained direct evidence for the nucleocytoplasmic shuttling of AhR and show the biological significance of the shuttling in terms of the transcriptional activation of its target gene, CYP1A1. When AhR(13-75) fused with glutathione S-transferase (GST)-green fluorescent protein (GFP) was microinjected into the nucleus of a polykaryotic of BHK21 cell, the GST-AhR(13-75)-GFP migrated from one nucleus to the other. This event, nucleocytoplasmic shuttling, was completely inhibited in the presence of leptomycin B (LMB). The interaction between chromosome region maintenance 1 (CRM1) and endogenous AhR was shown by immunoprecipitation with antibodies to AhR followed by immunoblot analysis with antibodies to CRM1. The inhibition of the nuclear export of AhR by LMB repressed the transcriptional activation of the CYP1A1 gene. The findings suggest that nuclear-cytoplasmic shuttling of AhR is essential for the inducible expression of the CYP1A1 protein.

Animals↗

PubMed1999

Analysis of the phosphorylation sites of herpes simplex virus type 1 regulatory protein ICP27.

The herpes simplex virus type 1 (HSV-1) regulatory protein ICP27 is a 63-kDa phosphoprotein required for viral replication. ICP27 has been shown to contain both stable phosphate groups and phosphate groups that cycle on and off during infection (K. W. Wilcox, A. Kohn, E. Sklyanskaya, and B. Roizman, J. Virol. 33:167-182, 1980). Despite extensive genetic analysis of the ICP27 gene, there is no information available about the sites of the ICP27 molecule that are phosphorylated during viral infection. In this study, we mapped several of the phosphorylation sites of ICP27 following in vivo radiolabeling. Phosphoamino acid analysis showed that serine is the only amino acid that is phosphorylated during infection. Two-dimensional phosphopeptide mapping showed a complex tryptic phosphopeptide pattern with at least four major peptides and several minor peptides. In addition, ICP27 purified from transfected cells yielded a similar phosphopeptide pattern, suggesting that cellular kinases phosphorylate ICP27 during viral infection. In vitro labeling showed that protein kinase A (PKA), PKC, and casein kinase II (CKII) were able to differentially phosphorylate ICP27, resulting in distinct phosphopeptide patterns. The major phosphorylation sites of ICP27 appeared to cluster in the N-terminal portion of the protein, such that a frameshift mutant that encodes amino acids 1 to 163 yielded a phosphopeptide pattern very similar to that seen with the wild-type protein. Further, using small deletion and point mutations in kinase consensus sites, we have elucidated individual serine residues that are phosphorylated in vivo. Specifically, the serine at residue 114 was highly phosphorylated by PKA and the serine residues at positions 16 and 18 serve as targets for CKII phosphorylation in vivo. These kinase consensus site mutants were still capable of complementing the growth of an ICP27-null mutant virus. Interestingly, phosphorylation of the serine at residue 114, which lies within the major nuclear localization signal, appeared to modulate the efficiency of nuclear import of ICP27.

Herpesvirus 1, Human↗

PubMed2000

Evidence for separable functions of Srp1p, the yeast homolog of importin alpha (Karyopherin alpha): role for Srp1p and Sts1p in protein degradation.

Srp1p (importin alpha) functions as the nuclear localization signal (NLS) receptor in Saccharomyces cerevisiae. The srp1-31 mutant is defective in this nuclear localization function, whereas an srp1-49 mutant exhibits defects that are unrelated to this localization function, as was confirmed by intragenic complementation between the two mutants. RPN11 and STS1 (DBF8) were identified as high-dosage suppressors of the srp1-49 mutation but not of the srp1-31 mutation. We found that Sts1p interacts directly with Srp1p in vitro and also in vivo, as judged by coimmunoprecipitation and two-hybrid analyses. Mutants of Sts1p that cannot interact with Srp1p are incapable of suppressing srp1-49 defects, strongly suggesting that Sts1p functions in a complex with Srp1p. STS1 also interacted with the second suppressor, RPN11, a subunit of the 26S proteasome, in the two-hybrid system. Further, degradation of Ub-Pro-beta-galactosidase, a test substrate for the ubiquitin-proteasome system, was defective in srp1-49 but not in srp1-31. This defect in protein degradation was alleviated by overexpression of either RPN11 or STS1 in srp1-49. These results suggest a role for Srp1p in regulation of protein degradation separate from its well-established role as the NLS receptor.

ATP-Binding Cassette Transporters↗

PubMed2004

Nucleocytoplasmic shuttling of JAZ, a new cargo protein for exportin-5.

Exportin-5 is a nuclear export receptor for certain classes of double-stranded RNA (dsRNA), including pre-micro-RNAs, viral hairpin RNAs, and some tRNAs. It can also export the RNA binding proteins ILF3 and elongation factor EF1A. However, the rules that determine which RNA binding proteins are exportin-5 cargoes remain unclear. JAZ possesses an unusual dsRNA binding domain consisting of multiple C2H2 zinc fingers. We found that JAZ binds to exportin-5 in a Ran-GTP- and dsRNA-dependent manner. Exportin-5 stimulates JAZ shuttling, and gene silencing of exportin-5 reduces shuttling. Recombinant exportin-5 also stimulates nuclear export of JAZ in permeabilized cells. JAZ also binds to ILF3, and surprisingly, this interaction is RNA independent, even though it requires the dsRNA binding domains of ILF3. Exportin-5, JAZ, and ILF3 can form a heteromeric complex with Ran-GTP and dsRNA, and JAZ increases ILF3 binding to exportin-5. JAZ does not contain a classical nuclear localization signal, and in digitonin-permeabilized cells, nuclear accumulation of JAZ does not require energy or cytosol. Nonetheless, low temperatures prevent JAZ import, suggesting that nuclear entry does not occur via simple diffusion. Together, these data suggest that JAZ is exported by exportin-5 but translocates back into nuclei by a facilitated diffusion mechanism.

Active Transport, Cell Nucleus↗

PubMed2006

Adenovirus core protein pVII is translocated into the nucleus by multiple import receptor pathways.

Adenoviruses are nonenveloped viruses with an approximately 36-kb double-stranded DNA genome that replicate in the nucleus. Protein VII, an abundant structural component of the adenovirus core that is strongly associated with adenovirus DNA, is imported into the nucleus contemporaneously with the adenovirus genome shortly after virus infection and may promote DNA import. In this study, we evaluated whether protein VII uses specific receptor-mediated mechanisms for import into the nucleus. We found that it contains potent nuclear localization signal (NLS) activity by transfection of cultured cells with protein VII fusion constructs and by microinjection of cells with recombinant protein VII fusions. We identified three NLS-containing regions in protein VII by deletion mapping and determined important NLS residues by site-specific mutagenesis. We found that recombinant protein VII and its NLS-containing domains strongly and specifically bind to importin alpha, importin beta, importin 7, and transportin, which are among the most abundant cellular nuclear import receptors. Moreover, these receptors can mediate the nuclear import of protein VII fusions in vitro in permeabilized cells. Considered together, these data support the hypothesis that protein VII is a major NLS-containing adaptor for receptor-mediated import of adenovirus DNA and that multiple import pathways are utilized to promote efficient nuclear entry of the viral genome.

Active Transport, Cell Nucleus↗

PubMed1999

Truncated BRCA2 is cytoplasmic: implications for cancer-linked mutations.

BRCA2 mutations predispose carriers mainly to breast cancer. The vast majority of BRCA2 mutations are predicted to result in a truncated protein product. The smallest known cancer-associated deletion removes from the C terminus only 224 of the 3,418 residues constituting BRCA2, suggesting that these terminal amino acids are crucial for BRCA2 function. A series of green fluorescent protein (GFP)-tagged BRCA2 deletion mutants revealed that nuclear localization depends on two nuclear localization signals that reside within the final 156 residues of BRCA2. Consistent with this observation, an endogenous truncated BRCA2 mutant (6174delT) was found to be cytoplasmic. Together, these studies provide a simple explanation for why the vast majority of BRCA2 mutants are nonfunctional: they do not translocate into the nucleus.

BRCA2 Protein↗

PubMed2006

Delivery of condensed DNA by liposomal non-viral gene delivery system into nucleus of dendritic cells.

In this study, we developed novel double-membranous non-viral gene delivery system modified with SV-40 T antigen-derived nuclear localization signal (NLS-DMEND) for delivery of luciferase plasmid DNA to nucleus of non-dividing mouse bone marrow-derived dendritic cells (BMDC). Intracellular trafficking and gene expression of NLS-DMEND in the BMDC were evaluated. Condensed DNA was observed in the nucleus by confocal laser scanning microscopy, and the NLS-DMEND induced significant luciferase activity in the BMDC. It was suggested that the condensed DNA particle transferred into nucleus via energy dependent manner, since the nuclear transfer was inhibited by metabolic inhibitors. In conclusion, condensed plasmid DNA was delivered into the nucleus of non-dividing BMDC by NLS-DMEND.

Animals↗

PubMed2005

XBP1 forms a regulatory loop with BMP-4 and suppresses mesodermal and neural differentiation in Xenopus embryos.

The active form of the Xenopus X-box binding protein 1 (xXBP1) partially synergizes and partially antagonizes with BMP-4 signaling. xXBP1 overexpression inhibits mesoderm differentiation and formation of neural tissues. A functional knockdown promotes differentiation of lateral and dorsal mesoderm but not of ventral mesoderm and of neuroectoderm. We show that the active form of xXBP1 in gastrula and early neurula stage embryos is generated by removal of exon 4 and not by an endoribonuclease activity in the endoplasmic reticulum. The N-terminal region of xXBP1 which contains the basic leucine-zipper also contains a nuclear localization signal and both, the N-terminal as well as the C-terminal regions are required for xXBP1 function. The effects of xXBP1 are in part correlated to a regulatory loop between xXBP1 and BMP-4. xXBP1 and BMP-4 stimulate mutually the transcription of each other, but xXBP1 inhibits the BMP-4 target gene, Xvent-2. Both, in vitro and in vivo assays demonstrate that xXBP1 interacts with BMP-4 and Xvent-2B promoters. GST-pulldown assays reveal that xXBP1 can interact with c-Jun, the transcriptional co-activator p300 and with the BMP-4 responsive Smad1. On the other hand, xXBP1 also binds to the inhibitory Smads, Smad6 and Smad7, that can act as transcriptional co-repressors. Based on these data, we conclude that xXBP1 might function as an inhibitor of mesodermal and neural tissue formation by acting either as transcriptional activator or as repressor. This dual activity depends upon binding of co-factors being involved in the formation of distinct transcription complexes.

Alternative Splicing↗