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At least 955 records · Page 53Linked to original sources
PubMed2001

Nucleocytoplasmic RNA transport in retroviral replication.

Retroviral replication is highly dependent on post-transcriptional regulation because a single primary transcript directs synthesis of many viral proteins. The identification and characterization of two post-transcriptional regulatory systems (Rev/RRE and CTE) revealed the efficient use of cellular transport pathways by retroviruses to achieve production of infectious progeny virus. The Rev/RRE system of HIV-1 consists of the viral Rev protein which binds to its target sequence on incompletely spliced RNAs and channels these into the CRM1-dependent export pathway, which is normally used for export of cellular proteins and RNAs (U snRNAs and 5 S rRNA). The CTE, on the other hand, directly recruits the cellular mRNA export receptor TAP to the viral RNA. Both systems have in common that they recruit a key player of a specific cellular export pathway and this recruitment appears to out-compete the respective cellular target molecules. The fact that CTE can functionally substitute for Rev/RRE, yielding a replication-competent virus, indicates that very short sequence elements are sufficient for post-transcriptional control. The presence of short dominant export signals could relieve the selective pressure on the remainder of the genome to maintain a sequence that is easily exported. The resultant increase in permitted sequence space may increase the potential for immune escape, thereby providing a selective advantage for the virus. Replication of the CTE-dependent HIV-1 variant is significantly impaired compared with the wild-type virus. Considering that post-transcriptional control in the case of HIV is also used to provide a temporal switch from the early phase of regulatory protein expression to the late phase of virion production, one may suggest that the CRM1 export pathway is advantageous for the rapid delivery of large amounts of cargo (i.e. HIV RNA). This would be in accordance with its normal function because CRM1 has been shown to direct the nuclear export of cellular regulatory proteins which must be accomplished rapidly as well. In summary, retroviruses have evolved fascinating ways to deal with their cellular environment and to make use of cellular transport pathways, allowing nuclear export of intron-containing RNAs which are normally restricted to the nucleus. Specific signals on the viral RNAs recruit key factors of cellular export, thus bypassing these restrictions and ensuring efficient viral replication.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2003

Nuclear export of the glucocorticoid receptor is accelerated by cell fusion-dependent release of calreticulin.

Nucleocytoplasmic exchange of nuclear hormone receptors is hypothesized to allow for rapid and direct interactions with cytoplasmic signaling factors. In addition to recycling between a naïve, chaperone-associated cytoplasmic complex and a liganded chaperone-free nuclear form, the glucocorticoid receptor (GR) has been observed to shuttle between nucleus and cytoplasm. Nuclear export of GR and other nuclear receptors has been proposed to depend on direct interactions with calreticulin, which is predominantly localized to the lumen of the endoplasmic reticulum. We show that rapid calreticulin-mediated nuclear export of GR is a specific response to transient disruption of the endoplasmic reticulum that occurs during polyethylene glycol-mediated cell fusion. Using live and digitonin-permeabilized cells we demonstrate that, in the absence of cell fusion, GR nuclear export occurs slowly over a period of many hours independent of direct interaction with calreticulin. Our findings temper expectations that nuclear receptors respond rapidly and directly to cytoplasmic signals in the absence of additional regulatory control. These results highlight the importance of verifying findings of nucleocytoplasmic trafficking using techniques in addition to heterokaryon cell fusion.

Active Transport, Cell Nucleus↗
PubMed2001

Control of p53 ubiquitination and nuclear export by MDM2 and ARF.

p53 and ARF-INK4a are the two most frequently altered loci in human tumors. The activity of p53 protein is inhibited during normal cell growth by the proto-oncoprotein MDM2 through either repression of p53-mediated transcription in the nucleus or proteasomal degradation of p53 protein in the cytoplasm. Responding to oncogenic signal-activated cell hyperproliferation, ARF-mediated antagonism of MDM2 inhibition results in p53 becoming active and its protein levels rising. The biochemical mechanisms of ubiquitination and nuclear export that underlie the functions of ARF and MDM2 in p53 control continue to emerge.

ADP-Ribosylation Factor 1↗
PubMed2001

Investigation of nucleo-cytoplasmic transport using UV-guided microinjection.

Active nucleo-cytoplasmic transport is mediated by dynamic signal-mediated pathways. We investigated the effects of transcription inhibitors or fluorescent lectins on nuclear import mediated by nuclear localization signals (NLSs). Therefore, a novel experimental approach that allows the controlled sequential introduction of fluorescent substances into living cells was established. A microinjection system equipped with an UV-source enabled us to identify fluorescent-labeled cells for the subsequent introduction of additional fluorescent compounds, in order to study their interactions in vivo. Cells were initially labeled either by expression of autofluorescent proteins or by microinjection of fluorescent substances. Transcription inhibitors did not affect nuclear transport mediated by classical NLSs but inhibited import mediated by the M9-domain of hnRNPA1. Comparison of a mono- and bipartite NLS revealed that the bipartite signal was more active in import. Sequential injection of differentially labeled nuclear import and export substrates allowed monitoring of import and export simultaneously in the same living cell. The introduced experimental approach will also be useful to analyze a variety of biological processes in living mammalian cells.

Amino Acid Sequence↗
PubMed1991

Nuclear mRNA export.

Nuclear mRNA export through the nuclear pore complex has been proposed to be a unidirectional, signal-mediated and energy-dependent process. Evidence exists that this process can be influenced by many factors including other steps in the pathway of cytoplasmic mRNA formation, sequences of the RNA substrate that are either transcribed or added co- or post-transcriptionally, and extracellular effectors.

Biological Transport, Active↗
PubMed2005

Cytoplasmic targeting of mutant poly(A)-binding protein nuclear 1 suppresses protein aggregation and toxicity in oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by progressive eyelid drooping, swallowing difficulties and proximal limb weakness. The autosomal dominant form of this disease is caused by a polyalanine expansion from 10 to 12-17 residues, located at the N-terminus of the poly(A)-binding protein nuclear 1 (PABPN1). A distinct pathological hallmark of OPMD is the presence of filamentous intranuclear aggregates in patients' skeletal muscle cells. Wildtype PABPN1 protein is expressed ubiquitously and was shown to be mostly concentrated in discrete nuclear domains called 'speckles'. Using an established cell- culture model, we show that most mutant PABPN1- positive (alanine expanded form) intranuclear aggregates are structures distinct from intranuclear speckles. In contrast, the promyelocytic leukaemia protein, a major component of nuclear bodies, strongly colocalized to intranuclear aggregates of mutant PABPN1. Wildtype PABPN1 can freely shuttle between the nucleus and cytoplasm. We determined whether the nuclear environment is necessary for mutant PABPN1 inclusion formation and cellular toxicity. This was achieved by inactivating the mutant PABPN1 nuclear localization signal and by generating full-length mutant PABPN1 fused to a strong nuclear export sequence. A green fluorescence protein tag inserted at the N-terminus of both wildtype PABPN1 (ala10) and mutant PABPN1 (ala17) proteins allowed us to visualize their subcellular localization. Targeting mutant PABPN1 to the cytoplasm resulted in a significant suppression of both intranuclear aggregates formation and cellular toxicity, two histological consequences of OPMD. Our results indicate that the nuclear localization of mutant PABPN1 is crucial to OPMD pathogenesis.

Amino Acid Sequence↗
PubMed2004

RXRalpha acts as a carrier for TR3 nuclear export in a 9-cis retinoic acid-dependent manner in gastric cancer cells.

Retinoid X receptor (RXR) plays a crucial role in the cross talk between retinoid receptors and other hormone receptors including the orphan receptor TR3, forming different heterodimers that transduce diverse steroid/thyroid hormone signaling. Here we show that RXRalpha exhibits nucleocytoplasmic shuttling in MGC80-3 gastric cancer cells and that RXRalpha shuttling is energy-dependent through a nuclear pore complex (NPC)-mediated pathway for its import and an intact DNA binding domain-mediated pathway for its export. In the presence of its ligand 9-cis retinoic acid, RXRalpha was almost exclusively located in the cytoplasm. More importantly, we also show that RXRalpha acts as a carrier to assist translocation of TR3, which plays an important role in apoptosis. Both RXRalpha and TR3 colocalized in the nucleus; however, upon stimulation by 9-cis retinoic acid they cotranslocated to the cytoplasm and then localized in the mitochondria. TR3 export depends on RXRalpha, as in living cells GFP-TR3 alone did not result in export from the nucleus even in the presence of 9-cis retinoic acid, whereas GFP-TR3 cotransfected with RXRalpha was exported out of the nucleus in response to 9-cis retinoic acid. Moreover, specific reduction of RXRalpha levels caused by anti-sense RXRalpha abolished TR3 nuclear export. In contrast, specific knockdown of TR3 by antisense-TR3 or TR3-siRNA did not affect RXRalpha shuttling. These results indicate that RXRalpha is responsible for TR3 nucleocytoplasmic translocation, which is facilitated by the RXRalpha ligand 9-cis retinoic acid. In addition, mitochondrial TR3, but not RXRalpha, was critical for apoptosis, as TR3 mutants that were distributed in the mitochondria induced apoptosis in the presence or absence of 9-cis retinoic acid. These data reveal a novel aspect of RXRalpha function, in which it acts as a carrier for nucleocytoplasmic translocation of orphan receptors.

3T3 Cells↗
PubMed2002

Activated glycogen synthase-3 beta suppresses cardiac hypertrophy in vivo.

The adult myocardium responds to a variety of pathologic stimuli by hypertrophic growth that frequently progresses to heart failure. The calcium/calmodulin-dependent protein phosphatase calcineurin is a potent transducer of hypertrophic stimuli. Calcineurin dephosphorylates members of the nuclear factor of activated T cell (NFAT) family of transcription factors, which results in their translocation to the nucleus and activation of calcium-dependent genes. Glycogen synthase kinase-3 (GSK-3) phosphorylates NFAT proteins and antagonizes the actions of calcineurin by stimulating NFAT nuclear export. To determine whether activated GSK-3 can act as an antagonist of hypertrophic signaling in the adult heart in vivo, we generated transgenic mice that express a constitutively active form of GSK-3 beta under control of a cardiac-specific promoter. These mice were physiologically normal under nonstressed conditions, but their ability to mount a hypertrophic response to calcineurin activation was severely impaired. Similarly, cardiac-specific expression of activated GSK-3 beta diminished hypertrophy in response to chronic beta-adrenergic stimulation and pressure overload. These findings reveal a role for GSK-3 beta as an inhibitor of hypertrophic signaling in the intact myocardium and suggest that elevation of cardiac GSK-3 beta activity may provide clinical benefit in the treatment of pathologic hypertrophy and heart failure.

Animals↗
PubMed1999

Translocation of cyclin B1 to the nucleus at prophase requires a phosphorylation-dependent nuclear import signal.

BACKGROUND: At M phase, cyclin B1 is phosphorylated in the cytoplasmic retention sequence (CRS), which is required for nuclear export. During interphase, cyclin B1 shuttles between the nucleus and the cytoplasm because constitutive nuclear import is counteracted by rapid nuclear export. In M phase, cyclin B moves rapidly into the nucleus coincident with its phosphorylation, an overall movement that might be caused simply by a decrease in its nuclear export. However, the questions of whether CRS phosphorylation is required for cyclin B1 translocation in mitosis and whether a reduction in nuclear export is sufficient to explain its rapid relocalisation have not been addressed. RESULTS: We have used two forms of green fluorescent protein to analyse simultaneously the translocation of wild-type cyclin B1 and a phosphorylation mutant of cyclin B1 in mitosis, and correlated this with an in vitro nuclear import assay. We show that cyclin B1 rapidly translocates into the nucleus approximately 10 minutes before breakdown of the nuclear envelope, and that this movement requires the CRS phosphorylation sites. A cyclin B1 mutant that cannot be phosphorylated enters the nucleus after the wild-type protein. Phosphorylation of the CRS creates a nuclear import signal that enhances cyclin B1 import in vitro and in vivo, in a manner distinct from the previously described import of cyclin B1 mediated by importin beta. CONCLUSIONS: We show that phosphorylation of human cyclin B1 is required for its rapid translocation to the nucleus towards the end of prophase. Phosphorylation enhances cyclin B1 nuclear import by creating a nuclear import signal. The phosphorylation of the CRS is therefore a critical step in the control of mitosis.

Amino Acid Sequence↗
PubMed2006

PPM1A functions as a Smad phosphatase to terminate TGFbeta signaling.

TGFbeta signaling controls diverse normal developmental processes and pathogenesis of diseases including cancer and autoimmune and fibrotic diseases. TGFbeta responses are generally mediated through transcriptional functions of Smads. A key step in TGFbeta signaling is ligand-induced phosphorylation of receptor-activated Smads (R-Smads) catalyzed by the TGFbeta type I receptor kinase. However, the potential of Smad dephosphorylation as a regulatory mechanism of TGFbeta signaling and the identity of Smad-specific phosphatases remain elusive. Using a functional genomic approach, we have identified PPM1A/PP2Calpha as a bona fide Smad phosphatase. PPM1A dephosphorylates and promotes nuclear export of TGFbeta-activated Smad2/3. Ectopic expression of PPM1A abolishes TGFbeta-induced antiproliferative and transcriptional responses, whereas depletion of PPM1A enhances TGFbeta signaling in mammalian cells. Smad-antagonizing activity of PPM1A is also observed during Nodal-dependent early embryogenesis in zebrafish. This work demonstrates that PPM1A/PP2Calpha, through dephosphorylation of Smad2/3, plays a critical role in terminating TGFbeta signaling.

Active Transport, Cell Nucleus↗
PubMed1999

In or out? Regulating nuclear transport.

The compartmentalization of proteins within the nucleus or cytoplasm of a eukaryotic cell offers opportunity for regulation of cell cycle progression and signalling pathways. Nuclear localization of proteins is determined by their ability to interact with specific nuclear import and export factors. In the past year, substrate phosphorylation has emerged as a common mechanism for controlling this interaction.

Biological Transport↗
PubMed1999

Genetic evidence for interactions between yeast importin alpha (Srp1p) and its nuclear export receptor, Cse1p.

The yeast Srp1p protein functions as an import receptor for proteins bearing basic nuclear localization signals. Cse1p, the yeast homolog of mammalian CAS, recycles Srp1p back to the cytoplasm after import substrates have been released into the nucleoplasm. In this report we describe genetic interactions between SRP1 and CSE1. Results from genetic suppression and synthetic lethality studies demonstrate that these gene products interact to ensure accurate chromosome segregation. We also describe new mutant alleles of CSE1 and analyze a new temperature-sensitive allele of CSE1, cse1-2. This allele causes high levels of chromosome missegregation and cell cycle arrest during mitosis at the nonpermissive temperature.

Amino Acid Sequence↗
PubMed2006

CaM kinase II selectively signals to histone deacetylase 4 during cardiomyocyte hypertrophy.

Class IIa histone deacetylases (HDACs) regulate a variety of cellular processes, including cardiac growth, bone development, and specification of skeletal muscle fiber type. Multiple serine/threonine kinases control the subcellular localization of these HDACs by phosphorylation of common serine residues, but whether certain class IIa HDACs respond selectively to specific kinases has not been determined. Here we show that calcium/calmodulin-dependent kinase II (CaMKII) signals specifically to HDAC4 by binding to a unique docking site that is absent in other class IIa HDACs. Phosphorylation of HDAC4 by CaMKII promotes nuclear export and prevents nuclear import of HDAC4, with consequent derepression of HDAC target genes. In cardiomyocytes, CaMKII phosphorylation of HDAC4 results in hypertrophic growth, which can be blocked by a signal-resistant HDAC4 mutant. These findings reveal a central role for HDAC4 in CaMKII signaling pathways and have implications for the control of gene expression by calcium signaling in a variety of cell types.

14-3-3 Proteins↗
PubMed2001

Substrate-induced nuclear export and peripheral compartmentalization of hepatic glucokinase correlates with glycogen deposition.

Hepatic glucokinase (GK) is acutely regulated by binding to its nuclear-anchored regulatory protein (GKRP). Although GK release by GKRP is tightly coupled to the rate of glycogen synthesis, the nature of this association is obscure. To gain insight into this coupling mechanism under physiological stimulating conditions in primary rat hepatocytes, we analyzed the subcellular distribution of GK and GKRP with immunofluorescence, and glycogen deposition with glycogen cytochemical fluorescence, using confocal microscopy and quantitative image analysis. Following stimulation, a fraction of the GK signal translocated from the nucleus to the cytoplasm. The reduction in the nuclear to cytoplasmic ratio of GK, an index of nuclear export, correlated with a >50% increase in glycogen cytochemical fluorescence over a 60 min stimulation period. Furthermore, glycogen accumulation was initially deposited in a peripheral pattern in hepatocytes similar to that of GK. These data suggest that a compartmentalization exists of both active GK and the initial sites of glycogen deposition at the hepatocyte surface.

Active Transport, Cell Nucleus↗
PubMed2003

Proteolytic release of the carboxy-terminal fragment of proHB-EGF causes nuclear export of PLZF.

Cleavage of membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF) via metalloprotease activation yields amino- and carboxy-terminal regions (HB-EGF and HB-EGF-C, respectively), with HB-EGF widely recognized as a key element of epidermal growth factor receptor transactivation in G protein-coupled receptor signaling. Here, we show a biological role of HB-EGF-C in cells. Subsequent to proteolytic cleavage of proHB-EGF, HB-EGF-C translocated from the plasma membrane into the nucleus. This translocation triggered nuclear export of the transcriptional repressor, promyelocytic leukemia zinc finger (PLZF), which we identify as an HB-EGF-C binding protein. Suppression of cyclin A and delayed entry of S-phase in cells expressing PLZF were reversed by the production of HB-EGF-C. These results indicate that released HB-EGF-C functions as an intracellular signal and coordinates cell cycle progression with HB-EGF.

Active Transport, Cell Nucleus↗
PubMed2006

Impaired nuclear import of mammalian Dlx4 proteins as a consequence of rapid sequence divergence.

Dlx genes encode a developmentally important family of transcription factors with a variety of functions and sites of action during vertebrate embryogenesis. The murine Dlx4 gene is an enigmatic member of the family; little is known about the normal developmental function(s) of Dlx4. Here, we show that Dlx4 is expressed in the murine placenta and in a trophoblast cell line where the protein localizes to both the nucleus and cytoplasm. Despite the presence of several leucine/valine-rich motifs that match known nuclear export sequences, cytoplasmic Dlx4 is not due to CRM-1-mediated nuclear export. Rather, nuclear import of Dlx4 is compromised by specific residues that flank the nuclear localization signal. One of these residues represents a novel conserved feature of the Dlx4 protein in placental mammals, and the second represents novel variation within mouse Dlx4 isoforms. Comparison of orthologous protein sequences reveals a particularly high rate of non-synonymous change in the coding regions of mammalian Dlx4 genes. Since impaired nuclear localization is unlikely to enhance the function of a nuclear transcription factor, these data point to reduced selection pressure as the basis for the rapid divergence of the Dlx4 gene within the mammalian clade.

Active Transport, Cell Nucleus↗
PubMed1999

Nuclear transport and transcriptional regulation.

Studies over the past 10 years have provided major insights into the molecular mechanisms responsible for active transport of macromolecules in and out of the nucleus. Nucleocytoplasmic transport pathways correspond to active and signal-mediated processes that involve substrates, adaptors and receptors. Regulation of both nuclear import and nuclear export is mainly exerted at the level of transport complex formation and has emerged as one of the most efficient mechanisms to adapt gene expression to the cell environment by restricting the access of transcriptional regulators to their target genes.

Animals↗
PubMed2002

Hepatitis delta virus ribonucleoproteins shuttle between the nucleus and the cytoplasm.

Hepatitis delta virus (HDV) infection of individuals infected with hepatitis B virus (HBV) is associated with more severe liver damage and an increased risk of fulminant disease. HDV is a single-stranded RNA virus that encodes a single protein, the delta antigen, which is expressed in two forms, small (S-HDAg) and large (L-HDAg). Here we show that although HDV ribonucleoproteins are mainly detected in the nucleus, they are also present in the cytoplasm of cells infected with HDV or transfected with HDV cDNA. Making use of an heterokaryon assay, we demonstrate that HDV ribonucleoproteins shuttle continuously between the nucleus and the cytoplasm. In the absence of HDV RNA, both forms of the delta antigen are retained in the nucleus, whereas in the absence of the delta antigen, HDV RNA is predominantly detected in the cytoplasm. Coexpression of HDV RNA and S-HDAg (which binds to the viral RNA and contains a nuclear localization signal) results in nuclear accumulation of the viral RNA. This suggests that HDV RNA mediates export of viral particles to the cytoplasm whereas the delta antigen triggers their reimport into the nucleus.

3T3 Cells↗