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PubMed2003

The M-phase-promoting factor modulates the sensitivity of the Ca2+ stores to inositol 1,4,5-trisphosphate via the actin cytoskeleton.

The resumption of the meiotic cycle (maturation) induced by 1-methyladenine in prophase-arrested starfish oocytes is indicated by the breakdown of the germinal vesicle and is characterized by the increased sensitivity of the Ca2+ stores to inositol 1,4,5-trisphosphate (InsP3) to InsP3 starting at the animal hemisphere (where the germinal vesicle was originally located) and propagating along the animal/vegetal axis of the oocyte. This initiates Ca2+ signals around the germinal vesicle before nuclear envelope breakdown. Previous studies have suggested that the final activation of the maturation-promoting factor (MPF), a cyclin-dependent kinase, which is the major element controlling the entry of eukaryotic cells into the M phase, occurs in the nucleus. MPF is then exported to the cytoplasm where its activity is autocatalytically amplified following a similar animal/vegetal spatial pattern. We have investigated whether activated MPF was involved in the increased sensitivity of the Ca2+ response to InsP3. We have found that the development of increased sensitivity of the Ca2+ stores to InsP3 receptors together with the Ca2+ signals in the perinuclear region was blocked in oocytes treated with the specific MPF inhibitor roscovitine. That the nuclear MPF activation is indeed required for changes of the InsP3 receptors sensitivity was shown by enucleating or by dissecting oocytes into vegetal and animal hemispheres prior to the addition of 1-MA. MPF activity 50 min after 1-methyladenine addition was much lower in the enucleated oocytes and in the vegetal hemisphere, which did not contain the germinal vesicle, as compared with the animal hemisphere, which did contain it. The Ca2+ increase induced by InsP3 under these experimental conditions correlated with the changes in actin cytoskeleton induced by MPF.

Actins↗
PubMed2004

Increased importin-beta-dependent nuclear import of the actin modulating protein CapG promotes cell invasion.

CapG (gCap39) is a ubiquitous gelsolin-family actin modulating protein involved in cell signalling, receptor-mediated membrane ruffling, phagocytosis and motility. CapG is the only gelsolin-related actin binding protein that localizes constitutively to both nucleus and cytoplasm. Structurally related proteins like severin and fragmin are cytoplasmic because they contain a nuclear export sequence that is absent in CapG. Increased CapG expression has been reported in some cancers but a causal role for CapG in tumour development, including invasion and metastasis, has not been explored. We show that moderate expression of green fluorescent protein-tagged CapG (CapG-EGFP) in epithelial cells induces invasion into collagen type I and precultured chick heart fragments. Nuclear export sequence-tagged CapG-EGFP fails to induce invasion, whereas point mutations in the nuclear export sequence permitting nuclear re-entry restore cellular invasion. Nuclear import of CapG is energy-dependent and requires the cytosolic receptor importin beta but not importin alpha. Nuclear CapG does not possess intrinsic transactivation activity but suppresses VP16 transactivation of a luciferase reporter gene in a dose-dependent manner. Furthermore, invasion requires signalling through the Ras-phosphoinositide 3-kinase pathway and Cdc42 or RhoA, but not Rac1. We show for the first time active nuclear import of an actin binding protein, and our findings point to a role for nuclear CapG in eliciting invasion, possibly through interfering with the cellular transcription machinery.

Actins↗
PubMed2003

Parathyroid hormone-related protein (PTHrP): a nucleocytoplasmic shuttling protein with distinct paracrine and intracrine roles.

Parathyroid hormone-related protein (PTHrP) was first discovered as a circulating factor secreted by certain cancers responsible for the syndrome of humoral hypercalcemia of malignancy. PTHrP possesses distinct paracrine and intracrine signaling roles. The similarity of its N-terminus to that of parathyroid hormone (PTH) enables it to share PTH's paracrine signaling properties, whereas the rest of the molecule possesses other functions, largely relating to an intracrine signaling role in the nucleus/nucleolus in regulating apoptosis and cell proliferation. Recent advances have shown that intracellularly expressed PTHrP is able to shuttle in cell-cycle- and signal-dependent fashion between nucleus and cytoplasm through the action of the distinct intracellular transport receptors importin beta 1 and exportin 1 (Crm1) mediating nuclear import and export of PTHrP, respectively. Together, the import and export pathways constitute an integrated system for PTHrP subcellular localization. Intriguingly, PTHrP nuclear/nucleolar import is dependent on microtubule integrity, transport to the nucleus appearing to occur in vectorial fashion along microtubules, mediated in part by the action of importin beta 1. PTHrP has recently been shown to be able to bind to RNA, meaning that PTHrP's nucleocytoplasmic shuttling ability may relate to a specific role within the nucleus/nucleolus to regulate RNA synthesis and/or transport.

Amino Acid Sequence↗
PubMed2006

Leptomycin B, a metabolite of Streptomyces, inhibits the expression of inducible nitric oxide synthase in BV2 microglial cells.

Overexpression of inducible nitric oxide synthase (iNOS) and the resultant overproduction of NO has been implicated in neuronal inflammatory diseases. Leptomycin B (LMB), a metabolite of Streptomyces, has been identified as a specific inhibitor of CRM1 nuclear export receptor. In this study, we evaluated the effect of LMB on lipopolysaccharide (LPS)-induced iNOS expression in BV2 cells, a murine microglial cells and the associated mechanisms. LMB strongly inhibited LPS-induced iNOS protein and mRNA expressions in BV2 cells in which 10 ng/ml of LMB (18 nM) was sufficient to greatly down-regulate iNOS by LPS, suggesting the potency of LMB to inhibit iNOS. The data of iNOS promoter-driven luciferase assay further suggested that the LMB inhibitory effect was in part due to inhibition of iNOS transcription. However, LPS-induced activation of various intracellular signaling proteins, such as nuclear factor-kappaB (NF-kappaB), extracellular signal-regulated kinases (ERKs), p38s, and c-Jun N-terminal kinases (JNKs), whose activations are known to be important for iNOS expression by LPS in BV2 cells, were not affected in the presence of LMB. Together, these results suggest that LMB inhibits iNOS expression in response to LPS in BV2 microglia, and the inhibition seems to be associated with blockage of CRM1-mediated iNOS mRNA nuclear export and also in part transcriptional down-regulation of iNOS, but not through modulation of NF-kappaB and the mitogen-activated protein kinase signaling pathways.

Active Transport, Cell Nucleus↗
PubMed2006

Integration of morphological and cytophysiological studies on estrogen receptor.

Ultrastructural and immunocytochemical studies of an intra-nuclear particle, the perichromatin granule (PCG), demonstrated the presence of processed mRNA in this structure. Ovariectomy caused an increase in the number of PCGs in uterine cells and administration of estradiol drastically reduced the nuclear pool of PCGs in 15 min. In vitro studies demonstrated that this depletion was accompanied by an increase of the export of previously synthesized RNA. Similar quantitative changes of the abundance of PCG and of the rate of the export of RNA were found in ventral prostate after orchiectomy and testosterone restitution, as well as in the target cells of FSH, LH, TSH, and ACTH. These results taken together led us to conclude that PCGs constitute an intra-nuclear compartment of a few processed mRNA in equilibrium with transcription and export. This mRNA is rapidly transferred to the cytoplasm by specific hormone signals.

Animals↗
PubMed2003

In vitro measurement of nuclear permeability changes in apoptosis.

In the eukaryotic cell, exchange of biomolecules between nucleus and cytoplasm is a highly regulated process which responds sensitively to changes of the environment. One well-known cellular response to environmental challenges is cell death by apoptosis. In fact, apoptosis has been shown to affect the nucleocytoplasmic transport machinery, in particular the nuclear pore, by modulating its size exclusion limit for passive diffusion. The underlying molecular factors are still unknown, mainly because of the lack of a suitable system to detect and quantitate the apoptotic effects on the nuclear pore. Here we present an assay that was designed to measure alterations of the permeability of the nuclear envelope under apoptotic conditions. The assay is based on the well-established technique of selective permeabilization of the plasma membrane with digitonin and allows assessment of permeability changes in nonfixed samples. It comprises a computer program, called Nuclear Permeability Assay, for the quantitation of the nuclear fluorescence signal, which may be generally employed for the evaluation of in vitro transport systems using semipermeabilized cells, such as assays for nuclear import and export.

Active Transport, Cell Nucleus↗
PubMed1999

Nucleocytoplasmic transport: Inside out regulation.

Cells can respond to extracellular signals by redistributing transcription regulators between the nucleus and cytosol. Recent findings in budding yeast indicate that the nuclear transport receptor Msn5p mediates the nuclear export of several transcription regulators after their phosphorylation in the nucleus.

Biological Transport↗
PubMed2000

Phosphorylation of the nuclear transport machinery down-regulates nuclear protein import in vitro.

We have examined whether signal-mediated nucleocytoplasmic transport can be regulated by phosphorylation of the nuclear transport machinery. Using digitonin-permeabilized cell assays to measure nuclear import and export, we found that the phosphatase inhibitors okadaic acid and microcystin inhibit transport mediated by the import receptors importin beta and transportin, but not by the export receptor CRM1. Several lines of evidence, including the finding that transport inhibition is partially reversed by the broad specificity protein kinase inhibitor staurosporine, indicate that transport inhibition is due to elevated phosphorylation of a component of the nuclear transport machinery. The kinases and phosphatases involved in this regulation are present in the permeabilized cells. A phosphorylation-sensitive component of the nuclear transport machinery also is present in permeabilized cells and is most likely a component of the nuclear pore complex. Substrate binding by the importin alpha.beta complex and the association of the complex with the nucleoporins Nup358/RanBP2 and Nup153 are not affected by phosphatase inhibitors, suggesting that transport inhibition by protein phosphorylation does not involve these steps. These results suggest that cells have mechanisms to negatively regulate entire nuclear transport pathways, thus providing a means to globally control cellular activity through effects on nucleocytoplasmic trafficking.

Adenosine Triphosphate↗
PubMed1990

Regulation of the expression of the regulatory subunit of cAMP-dependent protein kinase II beta in Friend erythroleukemic cells. Evidence for posttranscriptional control and a central role for the C subunit.

Friend erythroleukemic cells provide a system for studying the regulation of the expression of regulatory (R) and catalytic (C) subunit isoforms of cAMP-dependent protein kinases. Friend cells contain RI alpha, two RII subunits previously designated RII-52 and RII-54, and C alpha. When the cells are treated with 0.2 mM methylisobutylxanthine (MIX) and either 20 microM forskolin or 0.5 mM 8-Br-cAMP, RI alpha content declines 50-75% because of a large decrease in the t1/2 value for the dissociated RI alpha subunit; RII-54 expression is invariant, but the amount and rate of synthesis of RII-52 increases 10-15-fold (Schwartz, D. A., and Rubin, C. S. (1985) J. Biol. Chem. 260, 6296-6303). We now demonstrate that RII-52 and RII-54 correspond to RII beta and RII alpha, respectively. When cAMP levels are elevated in Friend cells the abundance of the 3.3-kilobase RII beta mRNA increases 25-30-fold in parallel with the rate of RII beta subunit synthesis indicating that pretranslational control is operative. Other R and C mRNAs are not markedly induced. Surprisingly, the rate of transcriptional initiation of the RII beta gene and the stability of RII beta mRNA are not altered during RII beta induction. Rather, the induction of RII beta mRNA is associated with the accumulation of major (3.4 kilobases) and minor (4 kilobases) RII beta pre-mRNAs in the nucleus. It appears that the cAMP signal-transduction system alters a nuclear protein(s) such that either the proportion of RII beta pre-mRNAs that are processed to mature mRNAs and are exported to the cytoplasm is greatly increased or the nuclear precursors are stabilized. Thus, regulation is exerted at a posttranscriptional level. In order to establish directly a causal role for C in RII beta induction and to rule out artifacts introduced by the use of drugs such as forskolin, MIX, and cAMP analogs we stably transfected Friend cells with a vector containing C alpha cDNA under the regulation of the zinc-activated metallothionein I promoter. The addition of 0.15 mM ZnSO4 caused the accumulation of dissociated C subunits and the selective induction of RII beta.

1-Methyl-3-isobutylxanthine↗
PubMed2001

Deoxycholic acid suppresses p53 by stimulating proteasome-mediated p53 protein degradation.

Bile acids, principally deoxycholic acid (DCA), have been implicated in the promotion of colon tumorigenesis in both animals and humans. Increasing evidence suggests that bile acids may exert their tumor promoting activity by modulating intracellular signaling and altering gene expression. In this study we have investigated the effect of bile acids on the tumor suppressor p53 using the human colon tumor cell line HCT116, which retains the wild-type p53 gene and functional p53 signaling in response to DNA damage. We found that exposure of the cells to elevated concentrations of DCA suppressed accumulation of p53 protein as well as p53 transactivation and impaired the p53 response of the cells to DNA damaging agents, such as ionizing radiation. Neither ursodeoxycholic acid, a putative chemopreventive agent, nor cholic acid, which is biologically inert, had any effect on p53 protein level and transactivation activity. Further examination revealed that instead of inhibition, DCA induced p53 mRNA in a dose-dependent manner, indicating that the inhibitory effect of DCA on p53 protein is mediated by a post-transcriptional mechanism. Both lactacystin, a specific inhibitor of the 26S proteasome, and leptomycin B, a specific inhibitor of the nuclear export protein CRM1, could block the effect that DCA had on p53 protein levels, suggesting that DCA suppressed p53 by stimulating the process of proteasome-mediated degradation of p53. Significantly, blocking extracellular signal-regulated kinase (ERK) signaling, but not protein kinase C (PKC), blunted suppression by DCA of p53 protein levels and transactivation activity, suggesting that DCA suppressed p53, in part, by stimulating the ERK signaling pathway. Both ERK and PKC signaling have been previously demonstrated to be stimulated by DCA. These results suggest a novel signaling mechanism of bile acids that may play an important role in colon tumor promotion mediated by bile acids.

Carcinogens↗
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↗
PubMed1999

Nuclear shuttling of yeast scaffold Ste5 is required for its recruitment to the plasma membrane and activation of the mating MAPK cascade.

Localization of Ste5 to GP at the plasma membrane is essential for transmission of the pheromone signal to associated MAP kinase cascade enzymes. Here, we show that this crucial localization requires prior shuttling of Ste5 through the nucleus. Ste5 shuttles through the nucleus constitutively during vegetative growth. Pheromone enhances nuclear export of Ste5, and this pool translocates vectorially to the cell periphery. Remarkably, Ste5 that cannot transit the nucleus is unable to localize at the periphery and activate the pathway, while Ste5 with enhanced transit through the nucleus has enhanced ability to localize to the periphery and activate the pathway. This novel regulatory scheme may ensure that cytoplasmic Ste5 does not activate downstream kinases in the absence of pheromone and could be applicable to other membrane-recruited signaling proteins.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Class II histone deacetylases confer signal responsiveness to the ankyrin-repeat proteins ANKRA2 and RFXANK.

Class II histone deacetylases (HDACs) contain unique amino-terminal extensions that mediate interactions with members of the myocyte enhancer factor-2 (MEF2) family of transcription factors and responsiveness to kinases, including Ca2+/calmodulin-dependent kinase (CaMK). Despite intense investigation of class II HDACs, little is known of MEF2-independent mechanisms for transcriptional repression by these chromatin-modifying enzymes. Here, we demonstrate that class II HDACs 4 and 5 physically associate with ankyrin-repeat proteins ANKRA2 and RFXANK (RFX-B/Tvl-1/ANKRA1). ANKRA2 is a megalin- and BKCa potassium channel-interacting factor, whereas RFXANK is a positive regulator of major histocompatibility complex II (MHC II) gene expression. HDAC4 and HDAC5 interact with the ankyrin repeats of ANKRA2 and RFXANK and, through association with RFXANK, repress MHC II promoter activation. HDACs 4 and 5 also repress endogenous HLA-DRA gene expression induced by CIITA. Phosphorylation of class II HDACs by CaMK results in CRM1-dependent nuclear export of HDAC/RFXANK complexes. These results define a novel transcriptional pathway under the control of class II HDACs and suggest a role for these transcriptional repressors as signal-responsive regulators of antigen presentation.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Z-DNA, a new in situ marker for transcription.

Z-DNA forms transiently behind the active RNA polymerases, because of the mechanical torsional stress produced during transcription. In this paper, we explore the possibility that the distribution of Z-DNA stretches signals the sites related to nuclear transcription. To localize transcription, the in situ assay for active RNA polymerases, that allows the elongation of the already initiated transcripts but no initiation of new ones (run-on experiments), was carried out in isolated nuclei of Allium cepa L. root meristems. Both nucleolar and non-nucleolar sites appeared labelled. Nucleoli were most active in transcription than the multiple non-nucleolar foci altogether. In situ immunodetection of Z-DNA provided images that were comparable to those obtained after the run-on assay, with one exception: while Z-DNA and transcription sites were scattered throughout the whole nucleus, Z-DNA also accumulated in the nuclear periphery, where no transcription foci were detected in run-on assays. The peripheral Z-conformation signals might correspond to dsRNA segments present in the pre-mRNA in the process of their export to cytoplasm. The Z-containing structures nearly disappeared when non-nucleolar RNA polymerase II-dependent transcription had been previously abolished by the adenosine analogue DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole). This inhibition selectively decreased the amount of all nucleoplasmic Z structures. On the other hand, the inhibition of the nucleolar RNA polymerase I by cordycepin (3'-deoxyadenosine) prevented the presence of Z-DNA in nucleoli. We propose to use the in situ immunodetection of Z-DNA as a marker of the transcription level in both nucleolus and non-peripheral nucleoplasmic regions of nuclei. Co-detection of Z-DNA and of intermediate filament (IF) proteins, the major components of the nuclear matrix, was also carried out. The IFA antibody recognizes a conserved epitope essential for dimerization of the multiple IF proteins. They co-localized with most nucleolar Z-DNA, but not with the nucleoplasmic ones. In the nuclear periphery, the Z-positive signals were adjacent to the IF proteins constituting the lamina, though both signals did not often co-localize.

Allium↗
PubMed1992

Movement of macromolecules between the cytoplasm and the nucleus in yeast.

Since the first description of signals for nuclear protein localization, studies with yeast have played an important role in our understanding of nuclear protein import. Very recent experiments suggest that new insights into the poorly understood process of RNA export will also emerge from analyses of yeast. Recent advances have facilitated our understanding of protein and RNA exchange between the nucleus and cytoplasm.

Amino Acid Sequence↗
PubMed2026

Nuclear export inhibition activates TP53 pathways and is a potent therapeutic strategy in atypical teratoid rhabdoid tumors.

BACKGROUND: Atypical teratoid/rhabdoid tumor (ATRT) is an aggressive central nervous system tumor mostly affecting young children. Improved and less toxic therapies for children with ATRT are imperative due to the toxicities associated with current treatments. Furthermore, existing therapies do not address the underlying genetic drivers of ATRT. In this study, we sought to determine whether exportin-1 (XPO1) is a genetic dependency and therapeutic target in ATRT. METHODS: We utilized an integrative approach harnessing patient-derived ATRT cell lines, functional genomics, pharmacologic assays, transcriptomics, and in vivo intracranial xenograft models to systematically test the hypothesis that XPO1 is a novel dependency in ATRT. RESULTS: Analysis of RNA-sequencing datasets revealed high XPO1 expression in ATRT cells compared to other pediatric brain tumor cell lines. Both CRISPR/Cas9 genetic knockdown and pharmacologic inhibition of XPO1 using 6 selective inhibitors of nuclear export (SINEs) in patient-derived atypical teratoid/rhabdoid tumor (ATRT) cells led to significant reduction in cell viability and proliferation. Furthermore, we observed increased apoptosis, G0 phase cell cycle arrest, and upregulation of TP53 signaling pathways in cells treated with the SINE selinexor. Consistently, our transcriptomic data revealed the upregulation of apoptosis and TP53 signaling pathways and concomitant depletion of cell cycle gene sets. In vivo, selinexor in combination with radiation and cyclophosphamide led to significant reduction in tumor volume and increased animal survival in intracranial ATRT xenograft models. CONCLUSIONS: Our data reveals XPO1 as a novel genetic dependency and potent therapeutic target in ATRT.

atypical teratoid rhabdoid tumor↗
PubMed2000

Identification of a functional nuclear export sequence in BRCA1.

Germ-line mutations in the tumor suppressor gene Brca1 confer increased susceptibility to breast and ovarian cancers. BRCA1 is a 1863-amino acid protein with roles in transcriptional regulation and the cellular responses to DNA damage. Given its function in these nuclear processes, the subcellular localization of BRCA1 is an important issue and has been the object of recent controversy. BRCA1 contains two nuclear localization signals and is most frequently detected in the cell nucleus by immunofluorescence microscopy. In this study, we show that BRCA1 is a nuclear-cytoplasmic shuttling protein, capable of both entering and exiting the nucleus. We identified a functional Rev-type nuclear export sequence ((81)QLVEELLKIICAFQLDTGL) near the amino terminus of BRCA1 that facilitates export via the CRM1/exportin pathway. Mutational inactivation of this nuclear export sequence, or treatment of cells with the CRM1-specific export inhibitor leptomycin B, induced nuclear accumulation of ectopic full-length BRCA1. Moreover, overexpression of the CRM1 export receptor resulted in decreased nuclear localization of endogenous BRCA1. The unexpected ability of BRCA1 to shuttle between nucleus and cytoplasm may have implications for the regulation and function of this tumor suppressor.

3T3 Cells↗
PubMed1991

Pathways for the nuclear transport of proteins and RNAs.

The nuclear pore complex catalyses the import and export of both proteins and RNAs. The molecular mechanisms of RNA and protein translocation through the nuclear pore are likely to be similar; however, their signals and targeting apparatus may differ. Recent insights into RNA transport have come from studies of kinetic control mechanisms and the preconditions for translocation that include processing, RNP assembly, and a targeting function for 5' caps.

Journal Article↗