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At least 883 records · Page 49Linked to original sources
PubMed2003

Neuronal activity-dependent nucleocytoplasmic shuttling of HDAC4 and HDAC5.

The class II histone deacetylases, HDAC4 and HDAC5, directly bind to and repress myogenic transcription factors of the myocyte enhancer factor-2 (MEF-2) family thereby inhibiting skeletal myogenesis. During muscle differentiation, repression of gene transcription by MEF-2/HDAC complexes is relieved due to calcium/calmodulin-dependent (CaM) kinase-induced translocation of HDAC4 and HDAC5 to the cytoplasm. MEF-2 proteins and HDACs are also highly expressed in the nervous system and have been implicated in neuronal survival and differentiation. Here we investigated the possibility that the subcellular localization of HDACs, and thus their ability to repress target genes, is controlled by synaptic activity in neurones. We found that, in cultured hippocampal neurones, the localization of HDAC4 and HDAC5 is dynamic and signal-regulated. Spontaneous electrical activity was sufficient for nuclear export of HDAC4 but not of HDAC5. HDAC5 translocation to the cytoplasm was induced following stimulation of calcium flux through synaptic NMDA receptors or L-type calcium channels; glutamate bath application (stimulating synaptic and extrasynaptic NMDA receptors) antagonized nuclear export. Activity-induced nucleocytoplasmic shuttling of both HDACs was partially blocked by the CaM kinase inhibitor KN-62 with HDAC5 nuclear export being more sensitive to CaM kinase inhibition than that of HDAC4. Thus, the subcellular localization of HDACs in neurones is specified by neuronal activity; differences in the activation thresholds for HDAC4 and HDAC5 nuclear export provides a mechanism for input-specific gene expression.

Active Transport, Cell Nucleus↗
PubMed2003

N-Acetyl-seryl-aspartyl-lysyl-proline inhibits TGF-beta-mediated plasminogen activator inhibitor-1 expression via inhibition of Smad pathway in human mesangial cells.

Recent large clinical trials indicate that angiotensin-converting enzyme inhibitors (ACE-I) attenuate the detrimental outcome of progressive renal disease. The hemoregulatory tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP, AcSDKP) is hydrolyzed by ACE, and plasma Ac-SDKP level is increased by fivefold after treatment with ACE-I. Ac-SDKP was found to ameliorate cardiac and renal fibrosis in hypertensive animal models. However, the molecular mechanisms by which Ac-SDKP mediates anti-fibrotic effects remain unclear. This study is an examination of the interaction between Ac-SDKP and transforming growth factor-beta (TGF-beta), one of the key cytokines in the progression of renal disease, in human mesangial cells. Ac-SDKP inhibited TGF-beta1-induced plasminogen activator inhibitor-1 (PAI-1) and alpha2 (I) collagen mRNA. Ac-SDKP suppressed not only TGF-beta1-induced Smad2 phosphorylation at Ser-465/467 in a dose-dependent manner, but also the nuclear accumulation of receptor-regulated Smads (R-Smad), Smad2 and Smad3. As expected, Ac-SDKP inhibited TGF-beta-responsive Smad-dependent luciferase reporters, 3TP-luc and 4xSBE-luc. Immunofluorescence analysis revealed that the inhibitory Smad, Smad7, was exported to the cytoplasm from the nucleus by the treatment with Ac-SDKP. These findings provide novel evidence that Ac-SDKP inhibits TGF-beta signal transduction through the suppression of R-Smad activation via nuclear export of Smad7, highlighting an alternative mechanism involved in the reno-protective efficacy of ACE-I.

Cell Culture Techniques↗
PubMed2000

Corf, the Rev/Rex homologue of HTDV/HERV-K, encodes an arginine-rich nuclear localization signal that exerts a trans-dominant phenotype when mutated.

Regulation of nucleo-cytoplasmic export of viral transcripts by a viral protein (Rev/Rex) is a characteristic feature in the replication cycle of complex retroviruses. We recently reported that the endogenous retrovirus family HTDV/HERV-K encodes a protein, Corf, that is a cellular Counterpart of Rev/Rex function and thus a new component of nucleo-cytoplasmic pathways. In HTDV/HERV-K-expressing cells, Corf is localized within the nucleoli. Here we describe the nuclear localization signal (NLS) of the Corf protein. Mutations in the NLS lead to cytoplasmic accumulation of the mutated protein and abrogate Corf function in a trans-dominant way.

Amino Acid Sequence↗
PubMed2002

Apoptotic regulation by the Crk adapter protein mediated by interactions with Wee1 and Crm1/exportin.

The adapter protein Crk contains an SH2 domain and two SH3 domains. Through binding of particular ligands to the SH2 domain and the N-terminal SH3 domain, Crk has been implicated in a number of signaling processes, including regulation of cell growth, cell motility, and apoptosis. We report here that the C-terminal SH3 domain, never shown to bind any specific signaling molecules, contains a binding site for the nuclear export factor Crm1. We find that a mutant Crk protein, deficient in Crm1 binding, promotes apoptosis. Moreover, this nuclear export sequence mutant [NES(-) Crk] interacts strongly, through its SH2 domain, with the nuclear tyrosine kinase, Wee1. Collectively, these data suggest that a nuclear population of Crk bound to Wee1 promotes apoptotic death of mammalian cells.

Active Transport, Cell Nucleus↗
PubMed2003

CRM1-mediated nuclear export and regulated activity of the Receptor Tyrosine Kinase antagonist YAN require specific interactions with MAE.

ETS family transcription factors serve as downstream effectors of signal transduction pathways, mediating cellular proliferation, differentiation and, when misregulated, tumorigenesis. The transcriptional repressor YAN prevents inappropriate responses to Receptor Tyrosine Kinase signaling by outcompeting POINTED for access to target gene promoters. We demonstrate that the molecular mechanism underlying downregulation of YAN involves CRM1-mediated nuclear export and define a novel role in this context for MAE, a co-factor previously implicated in facilitating MAPK phosphorylation of YAN. In addition to promoting YAN downregulation, MAE also participates in an inhibitory feedback loop that attenuates POINTED-P2 activation. Thus, we propose that MAE plays multiple independent roles in fine-tuning the levels of POINTED and YAN activity in accordance with changing RTK signaling conditions.

Active Transport, Cell Nucleus↗
PubMed2000

Exportin 4: a mediator of a novel nuclear export pathway in higher eukaryotes.

Transport receptors of the importin beta superfamily account for many of the nuclear import and export events in eukaryotic cells. They mediate translocation through nuclear pore complexes, shuttle between nucleus and cytoplasm and co-operate with the RanGTPase system to regulate their interactions with cargo molecules in a compartment-specific manner. We used affinity chromatography on immobilized RanGTP to isolate further candidate nuclear transport receptors and thereby identified exportin 4 as the most distant member of the importin beta family so far. Exportin 4 appears to be conserved amongst higher eukaryotes, but lacks obvious orthologues in yeast. It mediates nuclear export of eIF-5A (eukaryotic translation initiation factor 5A) and possibly that of other cargoes. The export signal in eIF-5A appears to be complex and to involve the hypusine modification that is unique to eIF-5A. We discuss possible cellular roles for nuclear export of eIF-5A.

Amino Acid Sequence↗
PubMed2026

TNFα-dependent modulation of WT1-MMP9 regulatory axis links developmental and inflammatory pathways in glaucoma.

Glaucomas are heterogeneous optic neuropathies associated with extracellular matrix dysregulation, abnormal ocular morphogenesis, and inflammatory signaling. Targeted deep sequencing of 586 primary congenital glaucoma (PCG) cases and 1,757 controls identified rare pathogenic variants in multiple genes, including WT1 and MMP9. Notably, WT1 variants clustered within the nuclear export sequence. Further, functional analyses showed that combined wt1-pax6 suppression in zebrafish disrupted ocular morphogenesis, highlighting developmental interdependence. In human trabecular meshwork cells, WT1 acted as a transcriptional repressor of MMP9, while TNF-α signaling triggered nitric oxide-dependent nuclear export of WT1, resulting in delayed MMP9 upregulation. This effect was reversible by inhibiting nuclear export or nitric oxide synthase. A patient-derived mutation in the nuclear-export region of WT1, disrupted this regulatory switch, causing abnormal MMP9 expression. These findings position WT1 as an important regulator linking developmental and inflammatory mechanisms in glaucoma pathogenesis.

anterior segment dysgenesis↗
PubMed2002

Nuclear export of NF90 is required for interleukin-2 mRNA stabilization.

In response to T cell activation signals, the half-life of interleukin-2 (IL-2) mRNA is greatly extended. The cis elements mediating IL-2 mRNA stabilization are located in its 5' and 3' untranslated regions (UTR). The 3'UTR also contains AU-rich elements (AREs) that mediate rapid IL-2 mRNA degradation in the cytoplasm of nonstimulated T cells. NF90, a previously described RNA binding protein, binds to a subregion of the 3'UTR that contains several AREs and slows down the degradation of IL-2 mRNA. In nonstimulated cells, NF90 is mostly nuclear, but T cell activation results in its accumulation in the cytoplasm. The nuclear export of NF90 is required for IL-2 mRNA stabilization.

3' Untranslated Regions↗
PubMed2003

The LIM-only protein FHL2 is a serum-inducible transcriptional coactivator of AP-1.

Proteins with LIM domains have been implicated in transcriptional regulation. The four and half LIM domain (FHL) group of LIM-only proteins is composed of five members, some of which have been shown to have intrinsic activation function. Here we show that FHL2 is the only member of the family whose expression is inducible upon serum stimulation in cultured cells. Induction of FHL2 is coordinated in time with the increased levels of two early-response products, the oncoproteins Fos and Jun. FHL2 associates with both Jun and Fos, in vitro and in vivo. The FHL2-Jun interaction requires the Ser-63-Ser-73 JNK phosphoacceptor sites in c-Jun, but not their phosphorylation. FHL2 powerfully stimulates Fos- and Jun-dependent transcription, thereby acting as an inducible coactivator of AP-1 function. Moreover, we show that intracellular localization of FHL2 is controlled by signaling events and a Crm1-dependent active nuclear export mechanism. Thus, FHL2, as an inducible coactivator of AP-1, coordinately participates with Fos and Jun in the early transcriptional response to serum factors.

Animals↗
PubMed2000

Gene activation by Varicella-zoster virus IE4 protein requires its dimerization and involves both the arginine-rich sequence, the central part, and the carboxyl-terminal cysteine-rich region.

Varicella-zoster virus (VZV) open reading frame 4-encoded protein (IE4) possesses transactivating properties for VZV genes as well as for those of heterologous viruses. Since most transcription factors act as dimers, IE4 dimerization was studied using the mammalian two-hybrid system. Introduction of mutations in the IE4 open reading frame demonstrated that both the central region and the carboxyl-terminal cysteine-rich domain were important for efficient dimerization. Within the carboxyl-terminal domain, substitution of amino acids encompassing residues 443-447 totally abolished dimerization. Gene activation by IE4 was studied by transient transfection with an IE4 expression plasmid and a reporter gene under the control of either the human immunodeficiency virus, type 1, long terminal repeat or the VZV thymidine kinase promoter. Regions of IE4 important for dimerization were also shown to be crucial for transactivation. In addition, the arginine-rich domains Rb and Rc of the amino-terminal region were also demonstrated to be important for transactivation, whereas the Ra domain as well as an acidic and bZIP-containing regions were shown to be dispensable for gene transactivation. A nucleocytoplasmic shuttling of IE4 has also been characterized, involving a nuclear localization signal identified within the Rb domain and a nuclear export mechanism partially depending on Crm-1.

Amino Acid Sequence↗
PubMed2005

Evaluations of HIV type 1 rev gene diversity and functional domains following perinatal transmission.

The human immunodeficiency virus type 1 (HIV-1) rev exons 1 and 2 sequences were analyzed from six mother-infant pairs following perinatal transmission. The rev open reading frame was maintained with a frequency of 93.96% in six mother-infant pairs' sequences. There was a low degree of viral heterogeneity and estimates of genetic diversity in mother-infant pairs' rev sequences. However, the distances of rev sequences between epidemiologically unlinked individuals were greater than in epidemiologically linked mother-infant pairs. Furthermore, phylogenetic parameters revealed that the epidemiologically linked mother-infant pairs were closer evolutionarily to each other as compared with epidemiologically unlinked mother-infant pairs. Both mothers and infants were under positive selection pressure as determined by the ratios of nonsynonymous to synonymous substitutions. The functional domains required for Rev activity, including nuclear export of RNA, RNA binding domain, and nuclear import signals, were conserved in all mother-infant pairs' sequences. The conservation of functional domains of rev and a low degree of heterogeneity following vertical transmission are consistent with an indispensable role of rev in the HIV-1 life cycle.

Adult↗
PubMed2005

Requirement of an additional Sam68 domain for inhibition of human immunodeficiency virus type 1 replication by Sam68 dominant negative mutants lacking the nuclear localization signal.

Human immunodeficiency virus type 1 (HIV-1) replication requires active nuclear export of unspliced and incompletely spliced HIV-1 RNA transcripts. This process is evolutionally made possible by expression of HIV-1 Rev, one of the three HIV-1 proteins encoded by completely spliced HIV-1 RNAs. Evidence has accumulated to suggest that Sam68 plays an important role in HIV-1 replication through HIV-1 Rev protein. In the present study, we further examined the structure-function relationship of Sam68 protein in relation to HIV-1 replication. We identified a Sam68 domain located between aa269 and aa321 to be involved in the HIV-inhibitory effects of Sam68 dominant negative mutants lacking the nuclear localization signal (NLS). Deletion of this domain abrogated inhibition of HIV-1 replication by these mutants. HIV-1 Rev protein appeared to mediate the HIV-inhibitory effects of these mutants and by this domain, as assessed by Rev-dependent chloramphenicol acetyltransferase reporter gene assay, in trans rev-defective HIV-1 complementation assay, and RNase protection assay. The HIV-inhibitory mutants containing this domain were further found to have diminished binding affinity to the wild-type Sam68 and to be associated with cytoplasmic retention of exclusively nuclear localized wild type Sam68. Taken together, these results further ascertain the important role of Sam68 in HIV-1 Rev function and viral replication, and suggest that the HIV-inhibitory effects of Sam68 dominant negative mutants directly result from their binding to endogenous Sam68 and their interference with nuclear localization of endogenous Sam68.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Efficient nuclear export of p65-IkappaBalpha complexes requires 14-3-3 proteins.

IkappaB are responsible for maintaining p65 in the cytoplasm under non-stimulating conditions and promoting the active export of p65 from the nucleus following NFkappaB activation to terminate the signal. We now show that 14-3-3 proteins regulate the NFkappaB signaling pathway by physically interacting with p65 and IkappaBalpha proteins. We identify two functional 14-3-3 binding domains in the p65 protein involving residues 38-44 and 278-283, and map the interaction region of IkappaBalpha in residues 60-65. Mutation of these 14-3-3 binding domains in p65 or IkappaBalpha results in a predominantly nuclear distribution of both proteins. TNFalpha treatment promotes recruitment of 14-3-3 and IkappaBalpha to NFkappaB-dependent promoters and enhances the binding of 14-3-3 to p65. Disrupting 14-3-3 activity by transfection with a dominant-negative 14-3-3 leads to the accumulation of nuclear p65-IkappaBalpha complexes and the constitutive association of p65 with the chromatin. In this situation, NFkappaB-dependent genes become unresponsive to TNFalpha stimulation. Together our results indicate that 14-3-3 proteins facilitate the nuclear export of IkappaBalpha-p65 complexes and are required for the appropriate regulation of NFkappaB signaling.

14-3-3 Proteins↗
PubMed1998

Nuclear export of the stress-activated protein kinase p38 mediated by its substrate MAPKAP kinase-2.

BACKGROUND: Mitogen-activated protein (MAP) kinases (or extracellular signal regulated kinases; Erks) and stress-activated protein (SAP) kinases mediate cellular responses to a wide variety of signals. In the Erk MAP kinase pathway, activation of MAP kinases takes place in the cytoplasm and the activated enzyme moves to the nucleus. This translocation to the nucleus is essential to MAP kinase signalling because it enables the kinase to phosphorylate transcription factors. Whether components of the pathway mediated by the SAP kinase p38 change their cellular location on activation is not clear; we have therefore studied the cellular localisation of components of this pathway before and after stimulation. RESULTS: The p38 SAP kinase substrate MAP-kinase-activated protein kinase-2 (MAPKAP kinase-2) contains a putative nuclear localisation signal which we show is functional and required for activation by a variety of stimuli. Following phosphorylation of MAPKAP kinase-2, nuclear p38 was exported to the cytoplasm in a complex with MAPKAP kinase-2. Export of MAPKAP kinase-2 required phosphorylation by p38 but did not appear to require the kinase activity of MAPKAP kinase-2. The p38 activators MKK3 and MKK6 were present in both the nucleus and the cytoplasm, consistent with a role in activating p38 in the nucleus. CONCLUSIONS: In the p38 SAP kinase pathway, MAPKAP kinase-2 serves both as an effector of p38 by phosphorylating substrates and as a determinant of cellular localisation of p38. Nuclear export of p38 and MAPKAP kinase-2 may permit them to phosphorylate substrates in the cytoplasm.

Amino Acid Sequence↗
PubMed2004

Transportin2 functions as importin and mediates nuclear import of HuR.

The RanGTP-binding nuclear transport receptors transportin1 (TRN1) and transportin2 (TRN2) are highly similar in sequence but are reported to function in nuclear import and export, respectively. Here we show that TRN2 possesses properties of a nuclear import receptor. TRN1/2 both interacted with a similar set of RNA-binding proteins in a RanGTP-sensitive manner. TRN2 bound RanGTP with high affinity, a feature of nuclear import receptors. As expected of an import complex, RanGTP also disrupted the interaction between TRN2 and HuR, an RNA-binding protein previously described as a TRN2 export substrate. The HuR nucleocytoplasmic shuttling signal, a sequence resembling the M9 nuclear import signal of hnRNP A1, was necessary and sufficient for TRN-mediated nuclear import of HuR in vitro. Finally, crosscompetition experiments demonstrated that HuR nucleocytoplasmic shuttling signal and M9 are imported along redundant pathways involving TRN1/2, substantiating the function of TRN2 in nuclear import.

Amino Acid Sequence↗
PubMed2001

Nuclear export of human beta-catenin can occur independent of CRM1 and the adenomatous polyposis coli tumor suppressor.

beta-Catenin is a mediator of the Wnt-signaling pathway. In many cancers, beta-catenin is stabilized and accumulates in the nucleus where it associates with lymphoid-enhancing factor 1/ T-cell transcription factors to activate genes involved in cell transformation. Previously, we showed that adenomatous polyposis coli (APC) protein can regulate beta-catenin localization by nuclear export. In this study, we used in vitro transport assays to test whether cellular beta-catenin can exit the nucleus independent of APC and the CRM1 export receptor. In digitonin-permeabilized SW480 (APC(mut/mut)) tumor cells, nuclear beta-catenin decreased >60% in export reactions in the absence of exogenous factors. Under similar conditions, nuclear c-ABL was only exported after the addition of cytosolic extract, and the export was blocked by the CRM1-specific inhibitor, leptomycin B. The nuclear export of beta-catenin was not blocked by leptomycin B treatment, revealing a CRM1- and APC-independent pathway. The export of beta-catenin was sensitive to lower temperatures and the removal of ATP, indicating an active process. Ectopically expressed yellow fluorescent protein-beta-catenin also displayed CRM1-independent export. Conversely, the overexpression of the CRM1 transporter moderately stimulated export of nuclear beta-catenin, confirming that beta-catenin exits the nucleus by at least two distinct pathways. The shuttling ability of tumor cell beta-catenin has implications for its regulation and its role in transferring signals between the nucleus and plasma membrane.

Adenomatous Polyposis Coli Protein↗
PubMed2003

Nuclear export of ERK3 by a CRM1-dependent mechanism regulates its inhibitory action on cell cycle progression.

Extracellular signal-regulated kinase 3 (ERK3) is an atypical member of the mitogen-activated protein kinase family of serine/threonine kinases. Little is known on the regulation of ERK3 function. Here, we report that ERK3 is constitutively localized in the cytoplasmic and nuclear compartments. In contrast to other mitogen-activated protein kinases, the cellular distribution of ERK3 remains unchanged in response to common mitogenic or stress stimuli and is independent of the enzymatic activity or phosphorylation of the kinase. The cytoplasmic localization of ERK3 is directed by a CRM1-dependent nuclear export mechanism. Treatment of cells with leptomycin B causes the nuclear accumulation of ERK3 in a high percentage of cells. Moreover, ectopic expression of CRM1 promotes the cytoplasmic relocalization of ERK3, whereas overexpression of snurportin 1, which binds CRM1 with high affinity, inhibits the nuclear export of ERK3. We also show that CRM1 binds to ERK3 in vitro. Importantly, we show that enforced localization of ERK3 in the nucleus or cytoplasm markedly attenuates the ability of the kinase to induce cell cycle arrest in fibroblasts. Our results suggest that nucleocytoplasmic shuttling of ERK3 is required for its negative regulatory effect on cell cycle progression.

Active Transport, Cell Nucleus↗
PubMed2000

Yeast nucleoporins involved in passive nuclear envelope permeability.

The vertebrate nuclear pore complex (NPC) harbors an approximately 10-nm diameter diffusion channel that is large enough to admit 50-kD polypeptides. We have analyzed the permeability properties of the Saccharomyces cerevisiae nuclear envelope (NE) using import (NLS) and export (NES) signal-containing green fluorescent protein (GFP) reporters. Compared with wild-type, passive export rates of a classical karyopherin/importin (Kap) Kap60p/Kap95p-targeted NLS-GFP reporter (cNLS-GFP) were significantly faster in nup188-Delta and nup170-Delta cells. Similar results were obtained using two other NLS-GFP reporters, containing either the Kap104p-targeted Nab2p NLS (rgNLS) or the Kap121p-targeted Pho4p NLS (pNLS). Elevated levels of Hsp70 stimulated cNLS-GFP import, but had no effect on the import of rgNLS-GFP. Thus, the role of Hsp70 in NLS-directed import may be NLS- or targeting pathway-specific. Equilibrium sieving limits for the diffusion channel were assessed in vivo using NES-GFP reporters of 36-126 kD and were found to be greater than wild-type in nup188-Delta and nup170-Delta cells. We propose that Nup170p and Nup188p are involved in establishing the functional resting diameter of the NPC's central transport channel.

Biological Transport↗