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PubMed2005

Calmodulin-dependent protein kinase IV regulates nuclear export of Cabin1 during T-cell activation.

Calcium signaling is critical for activation of T lymphocytes and has been proposed to be transduced through multiple calmodulin target proteins. Whereas the calcineurin-NFAT signaling module is critical for all mammalian T cells, the role of calmodulin-dependent kinase IV (CaMKIV) in mouse naïve CD4+ T-cell activation remains enigmatic. We have applied lentivius-mediated RNA interference of CaMKIV to human T cells and found that knockdown of CaMKIV abrogates T-cell receptor-mediated transcription of the IL-2 gene. We demonstrate that CaMKIV directly phosphorylates Cabin1, a transcriptional corepressor for myocyte enhancer factor 2, creating a docking site for 14-3-3, which causes its nuclear export. CaMKIV-mediated nuclear export of Cabin1 is likely to account for a significant part of the requirement of CaMKIV during human T-cell activation.

14-3-3 Proteins↗
PubMed2002

Calpain as an effector of the Gq signaling pathway for inhibition of Wnt/beta -catenin-regulated cell proliferation.

Signaling pathways interact to integrate and regulate information flow in evoking complex cellular responses. We have studied the mechanisms and consequences of interactions between the Gq and Wnt/beta-catenin pathways. In human colon carcinoma SW480 cells, activation of the Gq pathway inhibits beta-catenin signaling as determined by transcriptional reporter and cell proliferation assays. Ca(2+) release from internal stores results in nuclear export and calpain-mediated degradation of beta-catenin in the cytoplasm. Galphaq does not inhibit the effects of constitutively activated DeltaN-XTCF3-VP16 chimera in SW480 cells. Similarly, in HEK293 cells the Gq pathway suppresses beta-catenin-T cell factor/lymphocyte enhancer factor-1 transcriptional activity induced by Wnt/Frizzled interaction or glycogen synthase kinase-3beta-resistant beta-catenin, but not DeltaN-XTCF3-VP16. We conclude that Gq signaling promotes nuclear export and calpain-mediated degradation of beta-catenin, which therefore contributes to the inhibition of Wnt/beta-catenin pathway.

Calcium↗
PubMed1992

Distinct molecular signals for nuclear import of the nucleolar snRNA, U3.

Export to the cytoplasm of U3 RNA transcribed from a rat U3 gene injected into the nucleus of Xenopus oocytes indicates that the biogenesis of U3 RNA, like that of the previously studied Sm-precipitable nucleoplasmic snRNAs (U1, U2, U4, and U5), includes a cytoplasmic phase. The regulation of import of the U3 snRNA into the nucleus has been analyzed by injection of synthetic human U3 transcripts into the cytoplasm of Xenopus oocytes. Binding of the major autoantigenic protein of the U3 snRNP, fibrillarin, and cap trimethylation can occur in the cytoplasm, but neither are required for import. The 3'-terminal 13 nucleotides are required for optimal import and cap trimethylation and participate in a phylogenetically conserved U3 structural element, a short 3'-terminal stem. An artificial construct containing the 3'-terminal 13 nucleotides, including the 3'-terminal stem, but only 56 nucleotides of the 217 nucleotides in U3, appears to be sufficient for import. The presence of the 3'-terminal stem in all snRNAs known to be imported suggests that it might be a universal element required for nuclear import.

Animals↗
PubMed2000

Identification of an export control sequence and a requirement for the KH domains in ICP27 from herpes simplex virus type 1.

The herpes simplex virus type 1 (HSV-1) immediate-early protein ICP27 is an RNA-binding protein that performs multiple functions required for the expression of HSV-1 genes during a productive infection. One essential function involves shuttling between the nucleus and the cytoplasm. Some of the domains identified in ICP27 include a leucine-rich nuclear export sequence (NES), a nuclear localization signal, three KH-like RNA-binding domains, and an RGG-box type RNA-binding motif. To study the contribution of two of the essential domains in ICP27 to HSV gene expression, we generated recombinant herpesviruses carrying deleterious mutations in the NES and KH domains of ICP27. To accomplish this, we fused the green fluorescent protein (GFP) to ICP27 and utilized fluorescence as a marker to isolate recombinant herpesviruses. Fusion of GFP to wild-type ICP27 did not disturb its localization or function or significantly reduce virus yield. Analysis of HSV gene expression in cells infected with a recombinant virus carrying a point mutation in the first KH-like RNA-binding domain revealed that nuclear export of ICP27 was not blocked, and the expression of only a subset of ICP27-dependent late genes was affected. These findings suggest that individual KH-like RNA-binding motifs in ICP27 may be involved in binding distinct RNAs. Analysis of recombinant viruses carrying a lethal mutation in the NES of ICP27 was not accomplished because this mutation results in a strong dominant-negative phenotype. Finally, we demonstrate that shuttling by ICP27 is regulated by an export control sequence adjacent to its NES that functions like the inhibitory sequence element found adjacent to the NES of NS1 from influenza virus.

Amino Acid Sequence↗
PubMed2003

A novel splicing regulator shares a nuclear import pathway with SR proteins.

Alternative splicing of precursor mRNA is often regulated by serine/arginine-rich proteins (SR proteins) and hnRNPs, and varying their concentration in the nucleus can be a mechanism for controlling splice site selection. To understand the nucleocytoplasmic transport mechanism of splicing regulators is of key importance. SR proteins are delivered to the nucleus by transportin-SRs (TRN-SRs), importin beta-like nuclear transporters. Here we identify and characterize a non-SR protein, RNA-binding motif protein 4 (RBM4), as a novel substrate of TRN-SR2. TRN-SR2 interacts specifically with RBM4 in a Ran-sensitive manner. TRN-SR2 indeed mediates the nuclear import of a recombinant protein containing the RBM4 C-terminal domain. This domain serves as a signal for both nuclear import and export, and for nuclear speckle targeting. Finally, both in vivo and in vitro splicing analyses demonstrate that RBM4 not only modulates alternative pre-mRNA splicing but also acts antagonistically to authentic SR proteins in splice site and exon selection. Thus, a novel splicing regulator with opposite activities to SR proteins shares an identical import pathway with SR proteins to the nucleus.

Active Transport, Cell Nucleus↗
PubMed2006

Analysis of the subcellular localization, function, and proteolytic control of the Arabidopsis cyclin-dependent kinase inhibitor ICK1/KRP1.

Recent studies have shown that cyclin-dependent kinase (CDK) inhibitors can have a tremendous impact on cell cycle progression in plants. In animals, CDK inhibitors are tightly regulated, especially by posttranslational mechanisms of which control of nuclear access and regulation of protein turnover are particularly important. Here we address the posttranslational regulation of INHIBITOR/INTERACTOR OF CDK 1 (ICK1)/KIP RELATED PROTEIN 1 (KRP1), an Arabidopsis (Arabidopsis thaliana) CDK inhibitor. We show that ICK1/KRP1 exerts its function in the nucleus and its presence in the nucleus is controlled by multiple nuclear localization signals as well as by nuclear export. In addition, we show that ICK1/KRP1 localizes to different subnuclear domains, i.e. in the nucleoplasm and to the chromocenters, hinting at specific actions within the nuclear compartment. Localization to the chromocenters is mediated by an N-terminal domain, in addition we find that this domain may be involved in cyclin binding. Further we demonstrate that ICK1/KRP1 is an unstable protein and degraded by the 26S proteasome in the nucleus. This degradation is mediated by at least two domains indicating the presence of at least two different pathways impinging on ICK1/KRP1 protein stability.

Active Transport, Cell Nucleus↗
PubMed2001

RNA export mediated by tap involves NXT1-dependent interactions with the nuclear pore complex.

Nuclear export of ribonucleoprotein complexes requires cis-acting signals and recognition by receptors that mediate translocation through the nuclear pore complex. Translocation is likely to involve a series of physical interactions between the ribonucleoprotein complex and nucleoporins within the nuclear pore complex. Here, we have characterized the function of NXT1 in the context of the Tap-dependent RNA export pathway. Tap has been implicated in the nuclear export of RNA transcripts derived from Mason-Pfizer monkey virus that contain the constitutive transport element. We demonstrate that NXT1 stimulates binding of a Tap-RNA complex to nucleoporins in vitro, and we provide mutational analysis that shows these interactions are necessary for nuclear export of an intron-containing viral mRNA in vivo. Tap contains separate domains for binding to nucleoporins and NXT1, both of which are critical for its export function. RNA export is mediated by a heterodimer of Tap and NXT1, and the function of NXT1 on this pathway is to regulate the affinity of the Tap-RNA complex for nucleoporins within the nuclear pore complex. We propose that NXT1-dependent binding of the Tap-RNA complex to the nucleoporin p62, which we have reconstituted in vitro using recombinant proteins, represents a single step of the translocation reaction.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2003

Nuclear export and plasma membrane recruitment of the Ste5 scaffold are coordinated with oligomerization and association with signal transduction components.

The Ste5 scaffold activates an associated mitogen-activated protein kinase cascade by binding through its RING-H2 domain to a Gbetagamma dimer (Ste4/Ste18) at the plasma membrane in a recruitment event that requires prior nuclear shuttling of Ste5. Genetic evidence suggests that Ste5 must oligomerize to function, but its impact on Ste5 function and localization is unknown. Herein, we show that oligomerization affects Ste5 activity and localization. The majority of Ste5 is monomeric, suggesting that oligomerization is tightly regulated. Increasing the pool of Ste5 oligomers increases association with Ste11. Remarkably, Ste5 oligomers are also more efficiently exported from the nucleus, retained in the cytoplasm by Ste11 and better recruited to the plasma membrane, resulting in constitutive activation of the mating mitogen-activated protein kinase cascade. Coprecipitation tests show that the RING-H2 domain is the key determinant of oligomerization. Mutational analysis suggests that the leucine-rich domain limits the accessibility of the RING-H2 domain and inhibits export and recruitment in addition to promoting Ste11 association and activation. Our results suggest that the major form of Ste5 is an inactive monomer with an inaccessible RING-H2 domain and Ste11 binding site, whereas the active form is an oligomer that is more efficiently exported and recruited and has a more accessible RING-H2 domain and Ste11 binding site.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Actin dynamics control SRF activity by regulation of its coactivator MAL.

Rho GTPases regulate the transcription factor SRF via their ability to induce actin polymerization. SRF activity responds to G actin, but the mechanism of this has remained unclear. We show that Rho-actin signaling regulates the subcellular localization of the myocardin-related SRF coactivator MAL, rearranged in t(1;22)(p13;q13) AML. The MAL-SRF interaction displays the predicted properties of a Rho-regulated SRF cofactor. MAL is predominantly cytoplasmic in serum-starved cells, but accumulates in the nucleus following serum stimulation. Activation of the Rho-actin signaling pathway is necessary and sufficient to promote MAL nuclear accumulation. MAL N-terminal sequences, including two RPEL motifs, are required for the response to signaling, while other regions mediate its nuclear export (or cytoplasmic retention) and nuclear import. MAL associates with unpolymerized actin through its RPEL motifs. Constitutively cytoplasmic MAL derivatives interfere with MAL redistribution and Rho-actin signaling to SRF. MAL associates with several SRF target promoters regulated via the Rho-actin pathway.

3T3 Cells↗
PubMed2003

Cadmium induces nuclear export of Bach1, a transcriptional repressor of heme oxygenase-1 gene.

The export of certain nuclear proteins is involved in the regulation of various nuclear functions, including transcription. In some cases, the export of target proteins is induced upon environmental or cellular cues, resulting in conditional gene expression. The small Maf proteins appear to be critical regulators of heme oxygenase (HO)-1, an anti-oxidant defense enzyme that degrades heme into iron, carbon monoxide, and biliverdin. Although ho-1 is repressed by Bach1/small Maf heterodimers, it is activated by Nrf2/small Maf heterodimers, indicating that Bach1 and Nrf2 compete with each other. We anticipated that the nuclear concentration of Bach1 might be regulated to ensure that the entire system effectively responds to various stimuli. We carried out detailed domain analysis of Bach1 in an effort to understand how various inducers of HO-1 inactivate Bach1. We show here that cadmium, a strong inducer of HO-1, activates the nuclear export of Bach1. This cadmium-induced export of Bach1 was mediated in trans by its C-terminal region that is conserved between Bach1 and Bach2. The nuclear export of Bach2 was also induced by cadmium, indicating that the cadmium responsibility is shared between Bach1 and Bach2. The nuclear export of Bach1 was dependent on Crm1/Exportin-1 as well as the extracellular signal-regulated kinase-1/2 (ERK1/2) activity. These results indicate that the nuclear export of Bach1 constitutes an important regulatory mechanism to relieve the Bach1-mediated repression of genes such as ho-1.

Base Sequence↗
PubMed2004

PEA-15 is inhibited by adenovirus E1A and plays a role in ERK nuclear export and Ras-induced senescence.

Oncogenic ras activates multiple signaling pathways to enforce cell proliferation in tumor cells. The ERK1/2 mitogen-activated protein kinase pathway is required for the transforming effects of ras, and its activation is often sufficient to convey mitogenic stimulation. However, in some settings oncogenic ras triggers a permanent cell cycle arrest with features of cellular senescence. How the Ras/ERK1/2 pathway activates different cellular programs is not well understood. Here we show that ERK1/2 localize predominantly in the cytoplasm during ras-induced senescence. This cytoplasmic localization seems to be dependent on an active nuclear export mechanism and can be rescued by the viral oncoprotein E1A. Consistent with this hypothesis, we showed that E1A dramatically down-regulated the expression of the ERK1/2 nuclear export factor PEA-15. Also, RNA interference against PEA-15 restored the nuclear localization of phospho-ERK1/2 in Ras-expressing primary murine embryo fibroblasts and stimulated their escape from senescence. Because senescence prevents the transforming effect of oncogenic ras, our results suggest a tumor suppressor function for PEA-15 that operates by means of controlling the localization of phospho-ERK1/2.

Active Transport, Cell Nucleus↗
PubMed2003

Regulation of cell survival and proliferation by the FOXO (Forkhead box, class O) subfamily of Forkhead transcription factors.

Recently, the FOXO (Forkhead box, class O) subfamily of Forkhead transcription factors has been identified as direct targets of phosphoinositide 3-kinase-mediated signal transduction. The AFX (acute-lymphocytic-leukaemia-1 fused gene from chromosome X), FKHR (Forkhead in rhabdomyosarcoma) and FKHR-L1 (FKHR-like 1) transcription factors are directly phosphorylated by protein kinase B, resulting in nuclear export and inhibition of transcription. This signalling pathway was first identified in the nematode worm Caenorhabditis elegans, where it has a role in regulation of the life span of the organism. Studies have shown that this evolutionarily conserved signalling module has a role in regulation of both cell-cycle progression and cell survival in higher eukaryotes. These effects are co-ordinated by FOXO-mediated induction of a variety of specific target genes that are only now beginning to be identified. Interestingly, FOXO transcription factors appear to be able to regulate transcription through both DNA-binding-dependent and -independent mechanisms. Our understanding of the regulation of FOXO activity, and defining specific transcriptional targets, may provide clues to the molecular mechanisms controlling cell fate decisions to divide, differentiate or die.

Animals↗
PubMed2005

Inhibition of albumin synthesis in chronic diseases: molecular mechanisms.

The albumin gene is expressed specifically in the liver after birth, and this expression is regulated predominantly at the transcriptional level. Regulatory proteins occupy specific DNA sequences within the promoter and enhancer of the albumin gene. The interaction between the CCAAT/enhancer binding protein (C/EBP)-beta and the albumin DNA is critical for albumin synthesis. Cachexia-induced hypoalbuminemia is mediated by tumor necrosis factor (TNF)-alpha. In turn, TNF-alpha stimulates oxidative stress, NO synthesis, and phosphorylation of C/EBP-beta within its nuclear localization signal (NLS). Consequently, C/EBP-beta is exported from the nucleus, preventing it to act as a transcriptional factor on the albumin gene. Antioxidants, NOS inhibitors. and dominant negative, nonphosphorylatable C/EBP-beta peptides block phosphorylation of C/EBP-beta within the NLS and its nuclear export as well as rescue the abnormal albumin gene expression, suggesting potential therapeutic interventions.

Albumins↗
PubMed2006

The long and winding road to gut homeostasis.

PURPOSE OF REVIEW: The open ecosystem of the alimentary tract, harboring approximately 1 kg of bacteria, exhibits a rapid, but tightly controlled turnover. Impaired nuclear receptor function can give rise to perturbation in the gut, leading to inflammation and possibly neoplasia. Intriguingly, bacteria-dependent signaling pathways can modulate, and in turn be modulated by, a subset of nuclear receptors. This review attempts to highlight how microbes and nuclear receptors could jointly regulate gut homeostasis. RECENT FINDINGS: Commensal bacteria can utilize peroxisome proliferator activated receptor-gamma-dependent nuclear export of RelA as a novel mechanism to attenuate inflammatory signals triggered by a pathogen. Other nuclear receptors, such as liver X receptor, vitamin D receptor and farnesoid X receptor were also recently shown to interact with bacteria-induced mammalian inflammatory pathways. Although details of this interplay are still being unraveled, a role for these and other nuclear receptors in gastrointestinal inflammation and possibly neoplasia is beyond dispute. SUMMARY: The commensal microflora is being accorded due importance in regulating homeostasis of the gastrointestinal tract. Recent data suggest that the molecular messengers used by these bacteria include nuclear receptors. Exploiting mechanisms of nuclear receptor activity as drug targets, together with a detailed knowledge of the microbiota, could improve our understanding of gut-related ailments, and aid in mitigating their symptoms.

Animals↗
PubMed2001

E2F4 is exported from the nucleus in a CRM1-dependent manner.

E2F is a family of transcription factors required for normal cell cycle control and for cell cycle arrest in G1. E2F4 is the most abundant E2F protein in many cell types. In quiescent cells, it is localized to the nucleus, where it is bound to the retinoblastoma-related protein p130. During entry into the cell cycle, the protein disappears from the nucleus and appears in the cytoplasm. The mechanism by which this change occurs has, in the past, been unclear. We have found that E2F4 is actively exported from the nucleus and that leptomycin B, a specific inhibitor of nuclear export, inhibits this process. E2F4 export is mediated by two hydrophobic export sequences, mutations in either of which result in export failure. Individual export mutants of E2F4, but not a mutant with inactivation of both export signals, can be efficiently excluded from the nucleus by forced coexpression of the nuclear export receptor CRM1. Similarly, CRM1 overexpression can prevent cell cycle arrest induced by the cyclin kinase inhibitor p16(INK4a), an E2F4-dependent process. Taken together, these data suggest that nuclear export contributes to the regulation of E2F4 function, including its ability to regulate exit from G1 in association with a suitable pocket protein.

Amino Acid Sequence↗
PubMed2002

A constitutively nuclear form of NFATx shows efficient transactivation activity and induces differentiation of CD4(+)CD8(+) T cells.

The Ca(2+) signal facilitates nuclear translocation of NFAT through the dephosphorylation of clustered serine residues in the calcium regulatory domain by the Ca(2+)/calmodulin-dependent phosphatase calcineurin. The conformation of dephosphorylated NFAT exposes the nuclear localization signal for translocation into the nucleus and masks the nuclear export sequence to keep the protein in the nucleus. It has been reported that deletion of some serine-rich motifs masking the nuclear localization signal results in the translocation of NFAT into the nucleus, but that the nuclear export sequence located at the N terminus also needs to be deleted for NFATx (NFAT4/NFATc3) to exert efficient transactivation function. Here, we report that deletion of the critical serine-rich motifs of NFATx leads to a conformation that efficiently exposes the nuclear localization signal and that has stronger transcription activity compared with the fully activated wild-type protein in the presence of the nuclear export sequence. This also suggests that the regulation of the transactivation domain by phosphorylation observed in NFAT1 may not contribute significantly to the transcription activity of NFATx. The expression of this constitutively nuclear form of NFATx in the CD4(+)CD8(+) T cell line facilitates differentiation into the CD4 single-positive stage upon stimulation with phorbol ester. Our data suggest that NFATx is involved in the regulation of co-receptor expression during differentiation into the CD4 single-positive stage.

Amino Acid Sequence↗
PubMed1997

Dominant-negative mutants of importin-beta block multiple pathways of import and export through the nuclear pore complex.

Nuclear protein import proceeds through the nuclear pore complex (NPC). Importin-beta mediates translocation via direct interaction with NPC components and carries importin-alpha with the NLS substrate from the cytoplasm into the nucleus. The import reaction is terminated by the direct binding of nuclear RanGTP to importin-beta which dissociates the importin heterodimer. Here, we analyse the sites of interaction on importin-beta for its multiple partners. Ran and importin-alpha respectively require residues 1-364 and 331-876 of importin-beta for binding. Thus, RanGTP-mediated release of importin-alpha from importin-beta is likely to be an active displacement rather than due to simple competition between Ran and importin-alpha for a common binding site. Importin-beta has at least two non-overlapping sites of interaction with the NPC, which could potentially be used sequentially during translocation. Our data also suggest that termination of import involves a transient release of importin-beta from the NPC. Importin-beta fragments which bind to the NPC, but not to Ran, resist this release mechanism. As would be predicted from this, these importin-beta mutants are very efficient inhibitors of NLS-dependent protein import. Surprisingly, however, they also inhibit M9 signal-mediated nuclear import as well as nuclear export of mRNA, U snRNA, and the NES-containing Rev protein. This suggests that mediators of these various transport events share binding sites on the NPC and/or that mechanisms exist to coordinate translocation through the NPC via different nucleocytoplasmic transport pathways.

Animals↗