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Results for “Nuclear Export Signals”

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PubMed2000

Ubiquitin-mediated proteolysis of a short-lived regulatory protein depends on its cellular localization.

In this study we demonstrate that the Deg1 degradation signal of the transcriptional repressor Matalpha2 confers compartment-specific turnover to a reporter protein. Rapid degradation of a Deg1-containing fusion protein is observed only when the reporter is efficiently imported into the nucleus. In contrast, a reporter that is constantly exported from the nucleus exhibits an extended half-life. Furthermore, nuclear import functions are crucial for both Deg1-induced degradation as well as for the turnover of the endogenous Matalpha2 protein. The conjugation of ubiquitin to a Deg1-containing reporter protein is abrogated in mutants affected in nuclear import. Obviously, the Deg1 signal initiates rapid proteolysis within the nucleoplasm, whereas in the cytosol it mediates turnover via a slower pathway. In both pathways the ubiquitin-conjugating enzymes Ubc6p/Ubc7p play a pivotal role. These observations imply that both the cellular targeting of a substrate and the compartment-specific activity of components of the ubiquitin-proteasome system define the half-life of naturally short-lived proteins.

Carrier Proteins↗
PubMed2005

Cell cycle-dependent nucleocytoplasmic shuttling of the neurofibromatosis 2 tumour suppressor merlin.

The neurofibromatosis 2 tumour suppressor merlin/schwannomin is structurally related to the ezrin-radixin-moesin family of proteins, which anchor actin cytoskeleton to specific membrane proteins and participate in cell signalling. Merlin inhibits cell growth with a yet unknown mechanism. As most tumour suppressors are linked to cell cycle control, we investigated merlin's behaviour during cell cycle. In glioma and osteosarcoma cells, endogenous merlin was targeted to the nucleus in a cell cycle-specific manner. Merlin accumulated perinuclearly at the G2/M phase, and shifted to the nucleus at early G1. During mitosis, merlin localized to mitotic spindles and at the contractile ring. Nuclear merlin was strongly reduced in confluent cells. Blocking of the CRM1/exportin nuclear export pathway led to accumulation of merlin in the nucleus. Activation of the p21-activated kinase or protein kinase A, which result in phosphorylation of merlin, did not affect its nuclear localization. Merlin regulates the activity of extracellular signal-regulated kinase 2 (ERK2) and nuclear localization of both proteins was induced by cell adhesion. Unlike ERK2, nuclear localization of merlin was not, however, dependent on intact actin cytoskeleton. These results link merlin to events related to cell cycle control and may help to resolve its tumour suppressor function.

Active Transport, Cell Nucleus↗
PubMed1997

Regulated nuclear translocation of the Mig1 glucose repressor.

Glucose represses the transcription of many genes in bakers yeast (Saccharomyces cerevisiae). Mig1 is a Cys2-His2 zinc finger protein that mediates glucose repression of several genes by binding to their promoters and recruiting the general repression complex Ssn6-Tup1. We have found that the subcellular localization of Mig1 is regulated by glucose. Mig1 is imported into the nucleus within minutes after the addition of glucose and is just as rapidly transported back to the cytoplasm when glucose is removed. This regulated nuclear localization requires components of the glucose repression signal transduction pathway. An internal region of the protein separate from the DNA binding and repression domains is necessary and sufficient for glucose-regulated nuclear import and export. Changes in the phosphorylation status of Mig1 are coincident with the changes in its localization, suggesting a possible regulatory role for phosphorylation. Our results suggest that a glucose-regulated nuclear import and/or export mechanism controls the activity of Mig1.

DNA-Binding Proteins↗
PubMedDate not supplied

The role of nuclear envelope calcium in modifying nuclear pore complex structure.

Some of the most important trafficking processes in cells involve transport across the nuclear envelope. Whether it is the import of transcription factors or the export of RNA, the only known portal across the double lipid bilayer that forms the nuclear envelope are the macromolecular pores known as nuclear pore complexes (NPCs). Understanding how signals influence the conformation of the NPC is important for testing models of, and perhaps modifying, transport across the nuclear envelope. Here we summarize high-resolution atomic force microscopy studies of NPC structure following manipulation of nuclear envelope calcium stores of nuclei from Xenopus laevis oocytes. The results show that the release of calcium from these stores through the specific activation of inositol 1,4,5-trisphosphate receptors leads to changes in NPC structure observable from both sides of the nuclear envelope. The diameter of the NPC is also sensitive to these calcium stores and increases upon calcium release. Western blot analysis reveals the presence of ryanodine receptors in the nuclear envelope of X. laevis oocytes, although in low abundance. Activation of these calcium channels also leads to the displacement of the central mass and changes in NPC diameter. This change in structure may involve a displacement of the cytoplasmic and nuclear rings of the NPC towards each other, leading to the apparent emergence of the central mass from both sides of the NPC. The changes in conformation and diameter of the NPC may alter cargo access and binding to phenylalanine-glycine repeats lining the pore, thus altering transport.

Adenosine↗
PubMed2006

Staufen1 is imported into the nucleolus via a bipartite nuclear localization signal and several modulatory determinants.

Mammalian Stau1 (Staufen1), a modular protein composed of several dsRBDs (double-stranded RNA-binding domains), is probably involved in mRNA localization. Although Stau1 is mostly described in association with the rough endoplasmic reticulum and ribosomes in the cytoplasm, recent studies suggest that it may transit through the nucleus/nucleolus. Using a sensitive yeast import assay, we show that Stau1 is actively imported into the nucleus through a newly identified bipartite nuclear localization signal. As in yeast, the bipartite nuclear localization signal is necessary for Stau1 nuclear import in mammalian cells. It is also required for Stau1 nucleolar trafficking. However, Stau1 nuclear transit seems to be regulated by mechanisms that involve cytoplasmic retention and/or facilitated nuclear export. Cytoplasmic retention is mainly achieved through the action of dsRBD3, with dsRBD2 playing a supporting role in this function. Similarly, dsRBD3, but not its RNA-binding activity, is critical for Stau1 nucleolar trafficking. The function of dsRBD3 is strengthened or stabilized by the presence of dsRBD4 but prevented by the interdomain between dsRBD2 and dsRBD3. Altogether, these results suggest that Stau1 nuclear trafficking is a highly regulated process involving several determinants. The presence of Stau1 in the nucleus/nucleolus suggests that it may be involved in ribonucleoprotein formation in the nucleus and/or in other nuclear functions not necessarily related to mRNA transport.

Amino Acid Sequence↗
PubMed1996

A suboptimal 5' splice site is a cis-acting determinant of nuclear export of polyomavirus late mRNAs.

Mouse polyomavirus has been used as a model system to study nucleocytoplasmic transport of mRNA. Three late mRNAs encoding the viral capsid proteins are generated by alternative splicing from common pre-mRNA molecules. mRNAs encoding the virion protein VP2 (mVP2) harbor an unused 5' splice site, and more than half of them remain fully unspliced yet are able to enter the cytoplasm for translation. Examination of the intracellular distribution of late viral mRNAs revealed, however, that mVP2 molecules are exported less efficiently than are mVP1 and mVP3, in which the 5' splice site has been removed by splicing. Point mutations and deletion analyses demonstrated that the efficiency of mVP2 export is inversely correlated with the strength of the 5' splice site and that unused 3' splice sites present in the mRNA have little or no effect on export. These results suggest that the unused 5' splice site is a key player in mVP2 export. Interestingly, mRNAs carrying large deletions but retaining the 5' splice site exhibited a wild-type mVP2 export phenotype, suggesting that there are no other constitutive cis-acting sequences involved in mVP2 export. RNA stability measurements confirmed that the subcellular distribution differences between these mRNAs were not due to differential half-lives between the two cellular compartments. We therefore conclude that the nuclear export of mVP2 is strongly influenced by a suboptimal 5' splice site. Furthermore, results comparing spliced and unspliced forms of mVP2 molecules indicated that the process of splicing does not enhance nuclear export. Since mVP2 and some of its mutant forms can accumulate in the cytoplasm in the absence of splicing, we propose that splicing is not a prerequisite for mRNA export in the polyomavirus system; rather, removal of splicing machinery from mRNAs may be required. The possibility that export of other viral mRNAs can be influenced by suboptimal splicing signals is also discussed.

3T3 Cells↗
PubMed2003

Acetylation regulates subcellular localization of the Wnt signaling nuclear effector POP-1.

Lymphoid enhancer factor/T-cell factor (LEF/TCF) are transcription factors that mediate the Wnt signaling pathway, and have crucial roles during embryonic development in various organisms. Here we report that acetylation enhances nuclear retention of POP-1, the Caenorhabditis elegans LEF/TCF homolog, through increasing nuclear import and blocking nuclear export. We identify three lysines that are acetylated in vivo, and demonstrate their essential requirement for proper nuclear localization and biological activity of POP-1 during C. elegans embryogenesis. The conservation of these lysines among other LEF/TCF family members suggests that acetylation may be an important, evolutionarily conserved mechanism regulating subcellular distribution of LEF/TCF factors.

Acetylation↗
PubMed2000

Akt-dependent cytokine production in mast cells.

Cross-linking of FcepsilonRI induces the activation of three protein tyrosine kinases, Lyn, Syk, and Bruton's tyrosine kinase (Btk), leading to the secretion of a panel of proinflammatory mediators from mast cells. This study showed phosphorylation at Ser-473 and enzymatic activation of Akt/protein kinase B, the crucial survival kinase, upon FcepsilonRI stimulation in mouse mast cells. Phosphorylation of Akt is regulated positively by Btk and Syk and negatively by Lyn. Akt in turn can regulate positively the transcriptional activity of interleukin (IL)-2 and tumor necrosis factor (TNF)-alpha promoters. Transcription from the nuclear factor kappaB (NF-kappaB), nuclear factor of activated T cells (NF-AT), and activator protein 1 (AP-1) sites within these promoters is under the control of Akt activity. Accordingly, the signaling pathway involving IkappaB-alpha, a cytoplasmic protein that binds NF-kappaB and inhibits its nuclear translocation, appears to be regulated by Akt in mast cells. Catalytic activity of glycogen synthase kinase (GSK)-3beta, a serine/threonine kinase that phosphorylates NF-AT and promotes its nuclear export, seems to be inhibited by Akt. Importantly, Akt regulates the production and secretion of IL-2 and TNF-alpha in FcepsilonRI-stimulated mast cells. Altogether, these results revealed a novel function of Akt in transcriptional activation of cytokine genes via NF-kappaB, NF-AT, and AP-1 that contributes to the production of cytokines.

Animals↗
PubMed2004

Asr1p, a novel yeast ring/PHD finger protein, signals alcohol stress to the nucleus.

During fermentation, yeast cells are exposed to increasing amounts of alcohol, which is stressful and affects both growth and viability. On the molecular level, numerous aspects of alcohol stress signaling remain unresolved. We have identified a novel yeast Ring/PHD finger protein that constitutively shuttles between nucleus and cytoplasm but accumulates in the nucleus upon exposure to ethanol, 2-propanol, or 1-butanol. Subcellular localization of this protein is not altered by osmotic, oxidative, or heat stress or during nitrogen or glucose starvation. Because of its exclusive sensitivity to environmental alcohol, the protein was called Asr1p for Alcohol Sensitive Ring/PHD finger 1 protein. Nuclear accumulation of Asr1p is rapid, reversible, and requires a functional Ran/Gsp1p gradient. Asr1p contains two N terminally located leucine-rich nuclear export sequences (NES) required for nuclear export. Consistently, it accumulates in the nucleus of xpo1-1 cells at restrictive temperature and forms a trimeric complex with the exportin Xpo1p and Ran-GTP. Deletion of ASR1 leads to sensitivity in growth on medium containing alcohol or detergent, consistent with a function of Asr1p in alcohol-related signaling. Asr1p is the first reported protein that changes its subcellular localization specifically upon exposure to alcohol and therefore represents a key element in the analysis of alcohol-responsive signaling.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Failure of a medulloblastoma-derived mutant of SUFU to suppress WNT signaling.

Germline mutations of APC in patients with Turcot syndrome (colon cancer and medulloblastoma), was well as somatic mutations of APC, beta-catenin, and Axin in sporadic medulloblastomas (MBs) have shown the importance of WNT signaling in the pathogenesis of MB. A subset of children with MB have germline mutations of SUFU, a known inhibitor of Hedgehog signal transduction. A recent report suggested that murine Sufu can bind beta-catenin, export it from the nucleus, and thereby repress beta-catenin/T-cell factor (Tcf)-mediated transcription. We show that an MB-derived mutant of SUFU has lost the ability to decrease nuclear levels of beta-catenin, and cannot inhibit beta-catenin/Tcf-mediated transcription as compared to wild type SUFU. Our results suggest that loss of function of SUFU results in overactivity of both the Sonic Hedgehog, and the WNT signaling pathways, leading to excessive proliferation and failure to differentiate resulting in MB.

Carrier Proteins↗
PubMed2004

Activation of cAMP response element-mediated gene expression by regulated nuclear transport of TORC proteins.

The CREB family of proteins are critical mediators of gene expression in response to extracellular signals and are essential regulators of adaptive behavior and long-term memory formation. The TORC proteins were recently described as potent CREB coactivators, but their role in regulation of CREB activity remained unknown. TORC proteins were found to be exported from the nucleus in a CRM1-dependent fashion. A high-throughput microscopy-based screen was developed to identify genes and pathways capable of inducing nuclear TORC accumulation. Expression of the catalytic subunit of PKA and the calcium channel TRPV6 relocalized TORC1 to the nucleus. Nuclear accumulation of the three human TORC proteins was induced by increasing intracellular cAMP or calcium levels. TORC1 and TORC2 translocation in response to calcium, but not cAMP, was mediated by calcineurin, and TORC1 was shown to be directly dephosphorylated by calcineurin. TORC function was shown to be essential for CRE-mediated gene expression induced by cAMP, calcium, or GPCR activation, and nuclear transport of TORC1 was sufficient to activate CRE-dependent transcription. Drosophila TORC was also shown to translocate in response to calcineurin activation in vivo. Thus, TORC nuclear translocation is an essential, conserved step in activation of cAMP-responsive genes.

Active Transport, Cell Nucleus↗
PubMed2000

The fragile X-related proteins FXR1P and FXR2P contain a functional nucleolar-targeting signal equivalent to the HIV-1 regulatory proteins.

Fragile X syndrome is caused by the absence of the fragile X mental-retardation protein (FMRP). FMRP and the fragile X-related proteins 1 and 2 (FXR1P and FXR2P) form a gene family with functional similarities, such as RNA binding, polyribosomal association and nucleocytoplasmic shuttling. In a previous study, we found that FMRP and FXR1P shuttle between cytoplasm and nucleoplasm, while FXR2P shuttles between cytoplasm and nucleolus. The nuclear and nucleolar-targeting properties of these proteins were investigated further. Here, we show that FXR2P contains in its C-terminal part, a stretch of basic amino acids 'RPQRRNRSRRRRFR' that resemble the nucleolar-targeting signal (NoS) of the viral protein Rev. This particular sequence is also present within exon 15 of the FXR1 gene. This exon undergoes alternative splicing and is therefore only present in some of the FXR1P isoforms. We investigated the intracellular distribution of various FXR1P isoforms with (iso-e and iso-f) and without (iso-d) the potential NoS in transfected COS cells treated with the nuclear export inhibitor leptomycin-B. Both iso-e and iso-f showed a nucleolar localization, as observed for FXR2P; iso-d was detected in the nucleo-plasm outside the nucleoli. Further, when a labelled 16-residue synthetic peptide corresponding to the NoS of FXR1P was added to human fibroblast cultures a clear nucleolar signal was observed. Based on these data we argue that the intranuclear distribution of FXR2P and FXR1P isoforms is very likely to be mediated by a similar NoS localized in their C-terminal region. This domain is absent in some FXR1P isoforms as well as in all FMRP isoforms, suggesting functional differences for this family of proteins, possibly related to RNA metabolism in different tissues.

Amino Acid Sequence↗
PubMed2005

Influenza a viruses with mutations in the m1 helix six domain display a wide variety of morphological phenotypes.

Several functions required for the replication of influenza A viruses have been attributed to the viral matrix protein (M1), and a number of studies have focused on a region of the M1 protein designated "helix six." This region contains an exposed positively charged stretch of amino acids, including the motif 101-RKLKR-105, which has been identified as a nuclear localization signal, but several studies suggest that this domain is also involved in functions such as binding to the ribonucleoprotein genome segments (RNPs), membrane association, interaction with the viral nuclear export protein, and virus assembly. In order to define M1 functions in more detail, a series of mutants containing alanine substitutions in the helix six region were generated in A/WSN/33 virus. These were analyzed for RNP-binding function, their capacity to incorporate into infectious viruses by using reverse genetics, the replication properties of rescued viruses, and the morphological phenotypes of the mutant virus particles. The most notable effect that was identified concerned single amino acid substitution mutants that caused significant alterations to the morphology of budded viruses. Whereas A/WSN/33 virus generally forms particles that are predominantly spherical, observations made by negative stain electron microscopy showed that several of the mutant virions, such as K95A, K98A, R101A, and K102A, display a wide range of shapes and sizes that varied in a temperature-dependent manner. The K102A mutant is particularly interesting in that it can form extended filamentous particles. These results support the proposition that the helix six domain is involved in the process of virus assembly.

Amino Acid Sequence↗
PubMed2001

Inhibition of nuclear import by protein kinase B (Akt) regulates the subcellular distribution and activity of the forkhead transcription factor AFX.

AFX belongs to a subfamily of Forkhead transcription factors that are phosphorylated by protein kinase B (PKB), also known as Akt. Phosphorylation inhibits the transcriptional activity of AFX and changes the steady-state localization of the protein from the nucleus to the cytoplasm. Our goal was threefold: to identify the cellular compartment in which PKB phosphorylates AFX, to determine whether the nuclear localization of AFX plays a role in regulating its transcriptional activity, and to elucidate the mechanism by which phosphorylation alters the localization of AFX. We show that phosphorylation of AFX by PKB occurs in the nucleus. In addition, nuclear export mediated by the export receptor, Crm1, is required for the inhibition of AFX transcriptional activity. Both phosphorylated and unphosphorylated AFX, however, bind Crm1 and can be exported from the nucleus. These results suggest that export is unregulated and that phosphorylation by PKB is not required for the nuclear export of AFX. We show that AFX enters the nucleus by an active, Ran-dependent mechanism. Amino acids 180 to 221 of AFX comprise a nonclassical nuclear localization signal (NLS). S193, contained within this atypical NLS, is a PKB-dependent phosphorylation site on AFX. Addition of a negative charge at S193 by mutating the residue to glutamate reduces nuclear accumulation. PKB-mediated phosphorylation of AFX, therefore, attenuates the import of the transcription factor, which shifts the localization of the protein from the nucleus to the cytoplasm and results in the inhibition of AFX transcriptional activity.

3T3 Cells↗
PubMed2004

Negative regulation of transforming growth factor-beta (TGF-beta) signaling by WW domain-containing protein 1 (WWP1).

Smad7 negatively regulates transforming growth factor (TGF)-beta superfamily signaling by binding to activated type I receptors, thereby preventing the phosphorylation of receptor-regulated Smads (R-Smads), as well as by recruiting HECT-type E3 ubiquitin ligases to degrade type I receptors through a ubiquitin-dependent mechanism. To elucidate the regulatory mechanisms of TGF-beta signaling, we searched for novel members of proteins that interact with Smad7 using a yeast two-hybrid system. One of the proteins identified was the WW domain-containing protein 1 (WWP1) that is structurally related to Smad ubiquitin regulatory factors (Smurfs), E3 ubiquitin ligases for Smads and TGF-beta superfamily receptors. Using a TGF-beta-responsive reporter in mammalian cells, we found that WWP1 inhibited transcriptional activities induced by TGF-beta. Similar to Smurfs, WWP1 associated with Smad7 and induced its nuclear export, and enhanced binding of Smad7 to TGF-beta type I receptor to cause ubiquitination and degradation of the receptor. Consistent with these results, WWP1 inhibited phosphorylation of Smad2 induced by TGF-beta. WWP1 thus negatively regulates TGF-beta signaling in cooperation with Smad7. However, unlike Smurfs, WWP1 failed to ubiquitinate R-Smads and SnoN. Importantly, WWP1 and Smurfs were expressed in distinct patterns in human tissues and carcinoma cell lines, suggesting unique pathophysiological roles of WWP1 and Smurfs.

Active Transport, Cell Nucleus↗
PubMed2003

Constitutively elevated nuclear export activity opposes Ca2+-dependent NFATc3 nuclear accumulation in vascular smooth muscle: role of JNK2 and Crm-1.

The transcription factor NFAT (nuclear factor of activated T-cells) is a cytosolic phosphoprotein that accumulates in the nucleus following dephosphorylation by the calcium (Ca2+)/calmodulin-dependent phosphatase, calcineurin. A defining feature of stimuli that induce NFAT nuclear accumulation/activation is a sustained increase in global intracellular Ca2+. Contrary to expectations, we have found that a sustained elevation of intracellular Ca2+, induced by membrane potential depolarization and mediated by voltage-dependent Ca2+ channels, does not result in nuclear localization of the NFATc3 isoform in smooth muscle. However, vasoconstrictors (e.g. uridine triphosphate (UTP)) and growth factors, which elevate intracellular Ca2+ and engage multiple intracellular signaling pathways, induce a robust increase in smooth muscle nuclear NFATc3. Here we show that depolarizing stimuli that normally fail to induce NFATc3 nuclear accumulation in arterial smooth muscle effectively induce nuclear accumulation under conditions in which Crm-1-dependent or JNK2-mediated nuclear export processes are disrupted. Consistent with an important regulatory role for JNK, UTP exerts a suppressive effect on JNK activity in smooth muscle. Export of nuclear NFATc3 following UTP-induced nuclear accumulation is dramatically slowed in cerebral arteries from JNK2-/- animals. These data indicate that in smooth muscle, stimulation of Ca2+-dependent, calcineurin-mediated nuclear import and suppression of Crm-1/JNK-dependent nuclear export are both required for induction of NFATc3 nuclear accumulation. These results highlight the dynamic interplay between influences that promote and oppose NFAT nuclear accumulation and suggest that in arterial smooth muscle suppression of constitutive nuclear export activity is an important property of NFAT-activating stimuli.

Active Transport, Cell Nucleus↗
PubMed2006

MAPK signalling: ERK5 versus ERK1/2.

Extracellular-signal-regulated kinase 5 (ERK5) is a member of the mitogen-activated protein kinase (MAPK) family and, similar to ERK1/2, has the Thr-Glu-Tyr (TEY) activation motif. Both ERK5 and ERK1/2 are activated by growth factors and have an important role in the regulation of cell proliferation and cell differentiation. Moreover, both the ERK5 and the ERK1/2 pathways are sensitive to PD98059 and U0126, which are two well-known inhibitors of the ERK pathway. Despite these similarities, recent studies have revealed distinctive features of the ERK5 pathway: ERK5 has a key role in cardiovascular development and neural differentiation; ERK5 nuclear translocation is controlled by its own nuclear localizing and nuclear export activities; and the carboxy-terminal half of ERK5, which follows its kinase catalytic domain, has a unique function.

Animals↗