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PubMed1999

Biology of the fragile X mental retardation protein, an RNA-binding protein.

The fragile X syndrome, an X-linked disease, is the most frequent cause of inherited mental retardation. The syndrome results from the absence of expression of the FMR1 gene (fragile mental retardation 1) owing to the expansion of a CGG trinucleotide repeat located in the 5' untranslated region of the gene and the subsequent methylation of its CpG island. The FMR1 gene product (FMRP) is a cytoplasmic protein that contains two KH domains and one RGG box, characteristics of RNA-binding proteins. FMRP is associated with mRNP complexes containing poly(A)+mRNA within actively translating polyribosomes and contains nuclear localization and export signals making it a putative transporter (chaperone) of mRNA from the nucleus to the cytoplasm. FMRP is the archetype of a novel family of cytoplasmic RNA-binding proteins that includes FXR1P and FXR2P. Both of these proteins are very similar in overall structure to FMRP and are also associated with cytoplasmic mRNPs. Members of the FMR family are widely expressed in mouse and human tissues, albeit at various levels, and seem to play a subtle choreography of expression. FMRP is most abundant in neurons and is absent in muscle. FXR1P is strongly expressed in muscle and low levels are detected in neurons. The complex expression patterns of the FMR1 gene family in different cells and tissues suggest that independent, however similar, functions for each of the three FMR-related proteins might be expected in the selection and metabolism of tissue-specific classes of mRNA. The molecular mechanisms altered in cells lacking FMRP still remain to be elucidated as well as the putative role(s) of FXR1P and FXR2P as compensatory molecules.

Animals↗
PubMed1998

The Fanconi anemia proteins FAA and FAC function in different cellular compartments to protect against cross-linking agent cytotoxicity.

Fanconi anemia (FA) is an autosomal recessive disease characterized by chromosomal instability, bone marrow failure, and a high risk of developing malignancies. Although the disorder is genetically heterogeneous, all FA cells are defined by their sensitivity to the apoptosis-inducing effect of cross-linking agents, such as mitomycin C (MMC). The cloned FA disease genes, FAC and FAA, encode proteins with no homology to each other or to any known protein. We generated a highly specific antibody against FAA and found the protein in both the cytoplasm and nucleus of mammalian cells. By subcellular fractionation, FAA is also associated with intracellular membranes. To identify the subcellular compartment that is relevant for FAA activity, we appended nuclear export and nuclear localization signals to the carboxy terminus of FAA and enriched its localization in either the cytoplasm or the nucleus. Nuclear localization of FAA was both necessary and sufficient to correct MMC sensitivity in FA-A cells. In addition, we found no evidence for an interaction between FAA and FAC either in vivo or in vitro. Together with a previous finding that FAC is active in the cytoplasm but not in the nucleus, our results indicate that FAA and FAC function in separate subcellular compartments. Thus, FAA and FAC, if functionally linked, are more likely to be in a linear pathway rather than form a macromolecular complex to protect against cross-linker cytotoxicity.

Antibodies, Monoclonal↗
PubMed1997

Nucleocytoplasmic transport of 5S ribosomal RNA.

Nucleocytoplasmic transport of 5S ribosomal RNA in Xenopus oocytes occurs in the context of small, non-ribosomal RNPs. The complex with the zinc finger protein TFIIIA (7S RNP) is exported from the nucleus and stored in the cytoplasm, whereas the complex with the ribosomal protein L5 (5S RNP) shuttles between the nucleus and the cytoplasm. Nuclear import- and export-signals appear to reside within the protein moiety of these RNPs. Import of TFIIIA is inhibited by RNA binding, whereas nuclear transfer of L5 is not influenced by RNA binding. We propose that the export capacity of both, TFIIIA and L5, is regulated by the interaction with 5S ribosomal RNA.

Journal Article↗
PubMed2002

Inserting a nuclear targeting signal into a replication-competent Moloney murine leukemia virus affects viral export and is not sufficient for cell cycle-independent infection.

The effects of inserting reported nuclear localization signals (NLSs) into the Moloney murine leukemia virus (Mo-MuLV) integrase (IN) protein, within a replication-competent viral construct, were studied. In contrast to the virus harboring IN fused to the simian virus 40 (SV40) large T antigen NLS (SV40 NLS) (J. A. Seamon, M. Adams, S. Sengupta, and M. J. Roth, Virology 274:412-419, 2000), a codon-modified SV40 NLS was stably expressed during viral propagation. Incorporation of the codon-modified SV40 NLS into IN, however, altered the packaging of the Gag-Pol precursor in the virus; viral particles contained decreased levels of reverse transcriptase (RT) and IN. In addition, the virus showed delayed kinetics of viral DNA synthesis upon infection. A panel of infectious MuLVs containing alternative IN-NLS fusions was generated and assayed for cell cycle-independent infection. Viral infection with the NLS-tagged proteins, however, remained dependent on passage of the cells through mitosis. This finding has direct implications for engineering murine-based retroviral vectors for gene therapy.

Amino Acid Sequence↗
PubMed1999

Being at the right place at the right time: the role of nuclear transport in dynamic transcriptional regulation in yeast.

As a consequence of compartmentalization in eukaryotic cells, the transcriptional response to extracellular stimuli requires signals to be transmitted from the cytoplasm to the nucleus. Thus, nuclear import and export of signal transduction and transcription factors provide an important level of transcriptional control in eukaryotes. Recent investigations have illustrated the importance of this control for dynamic gene expression, as will be discussed on transcriptional activators and repressors (Mac1p, Mig1p, Msn2p/4p, Swi5p) and protein kinases (Hog1p, Sty1/Spc1, protein kinase A).

Animals↗
PubMed2006

Platelet-derived growth factor BB induces nuclear export and proteasomal degradation of CREB via phosphatidylinositol 3-kinase/Akt signaling in pulmonary artery smooth muscle cells.

Cyclic AMP response element binding protein (CREB) content is diminished in smooth muscle cells (SMCs) in remodeled pulmonary arteries from animals with pulmonary hypertension and in the SMC layers of atherogenic systemic arteries and cardiomyocytes from hypertensive individuals. Loss of CREB can be induced in cultured SMCs by chronic exposure to hypoxia or platelet-derived growth factor BB (PDGF-BB). Here we investigated the signaling pathways and mechanisms by which PDGF elicits depletion of SMC CREB. Chronic PDGF treatment increased CREB ubiquitination in SMCs, while treatment of SMCs with the proteasome inhibitor lactacystin prevented decreases in CREB content. The nuclear export inhibitor leptomycin B also prevented depletion of SMC CREB alone or in combination with lactacystin. Subsequent studies showed that PDGF activated extracellular signal-regulated kinase, Jun N-terminal protein kinase, and phosphatidylinositol 3 (PI3)-kinase pathways in SMCs. Inhibition of these pathways blocked SMC proliferation in response to PDGF, but only inhibition of PI3-kinase or its effector, Akt, blocked PDGF-induced CREB loss. Finally, chimeric proteins containing enhanced cyan fluorescent protein linked to wild-type CREB or CREB molecules with mutations in several recognized phosphorylation sites were introduced into SMCs. PDGF treatment reduced the levels of each of these chimeric proteins except for one containing mutations in adjacent serine residues (serines 103 and 107), suggesting that CREB loss was dependent on CREB phosphorylation at these sites. We conclude that PDGF stimulates nuclear export and proteasomal degradation of CREB in SMCs via PI3-kinase/Akt signaling. These results indicate that in addition to direct phosphorylation, proteolysis and intracellular localization are key mechanisms regulating CREB content and activity in SMCs.

Acetylcysteine↗
PubMedDate not supplied

Nuclear import and export of proteins: the molecular basis for intracellular signaling.

A family of latent cytoplasmic transcription factors termed Stats are activated by a variety of cytokines, and are then translocated into the nucleus where they activate transcription. Recent advances in nuclear protein import have shown that the extracellular signal-dependent nuclear import of Stat1 is mediated via complex formation with NPI-1 (a member of the alpha subunit family) and the beta subunit of the nuclear pore-targeting complex, and a small GTPase, Ran. The unique transport pathway of Stat1, which is different from that of the SV40T-antigen, indicates that a complex divergence exists in the function of transport factors and transport pathways.

Animals↗
PubMed1998

Macromolecular exchanges between the nucleus and cytoplasm.

The control of transcription and translation is of fundamental importance in cell biology. In this regard, the nuclear envelope is in a unique position to contribute to the regulation of these events, by directing macromolecular exchanges between the nucleus and cytoplasm. Such exchanges occur through the nuclear pore complexes, mainly by signal-mediated processes. Different signals are required for import and export. Specific cytoplasmic or nuclear receptors initially bind the signal-containing substrate, and the complex subsequently interacts with the pores. Additional factors then assist in translocation across the envelope. Current research is focused mainly on further characterization of transport receptors, translocation factors, as well as components of the nuclear pore complex, i.e., the nucleoporins. The ultimate goal is to understand the molecular interactions that occur among the different components of the transport apparatus, the energy sources for transport, and how variations in transport capacity are generated.

Animals↗
PubMed2005

Antagonistic regulation of Yan nuclear export by Mae and Crm1 may increase the stringency of the Ras response.

Phosphorylation of Yan, a major target of Ras signaling, leads to Crm1-dependent Yan nuclear export, a response that is regulated by Yan polymerization. Yan SAM (sterile alpha motif) domain mutations preventing polymerization result in Ras-independent, but Crm1-dependent Yan nuclear export, suggesting that polymerization prevents Yan export. Mae, which depolymerizes Yan, competes with Crm1 for binding to Yan. Phosphorylation of Yan favors Crm1 in this competition and counteracts inhibition of nuclear export by Mae. These findings suggest that, prior to Ras activation, the Mae/Yan interaction blocks premature nuclear export of Yan monomers. After activation, transcriptional up-regulation of Mae apparently leads to complete depolymerization and export of Yan.

Animals↗
PubMed1999

Identification of novel import and export signals of human TAP, the protein that binds to the constitutive transport element of the type D retrovirus mRNAs.

The nuclear export of the unspliced type D retrovirus mRNA depends on the cis-acting constitutive transport RNA element (CTE) that has been shown to interact with the human TAP (hTAP) protein promoting the export of the CTE-containing mRNAs. We report here that hTAP is a 619-amino-acid protein extending the previously identified protein by another 60 residues at the N terminus and that hTAP shares high homology with the predicted rat and mouse TAP proteins. We found that hTAP is a nuclear protein that accumulates in the nuclear rim and the nucleoplasm. We further demonstrated that hTAP is able to shuttle between the nucleus and the cytoplasm. Identification of the signals responsible for nuclear import (NLS) and export (NES) revealed that they are distinct but partially overlapping. NLS and NES of hTAP are active transferable signals that do not share similarities with known elements. The C-terminal portion contributes further to hTAP's nuclear retention and contains a signal(s) for nuclear rim association. Taken together, our data show that hTAP is a dynamic protein capable of bidirectional trafficking across the nuclear envelope. These data further support hTAP's role as an export factor of the CTE-containing mRNAs.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2006

Hdm2 nuclear export, regulated by insulin-like growth factor-I/MAPK/p90Rsk signaling, mediates the transformation of human cells.

Insulin-like growth factor (IGF)-I receptor activation leads to enhanced proliferation and cell survival via the MAP kinase and phosphatidylinositol 3-kinase-signaling pathways. Upon stimulation by IGF-I, the Hdm2 oncoprotein is phosphorylated by AKT, leading to its rapid nuclear translocation and subsequent inhibition of p53. We now show that IGF-I stimulation regulates the nuclear export of Hdm2 and p53 via the MAP kinase pathway. Inhibition of p38 MAPK or MEK via pharmacological means or expression of dominant negative proteins inhibited the cytoplasmic accumulation of Hdm2 and increased Hdm2 and p53 protein levels, whereas constitutively active p90Rsk promoted the nuclear export of Hdm2. Expression of constitutively active p90Rsk with E1A, oncogenic H-Ras, and hTERT resulted in the anchorage-independent growth of normal human fibroblasts. Our findings link p90Rsk-mediated modulation of Hdm2 nuclear to cytoplasmic shuttling with the diminished ability of p53 to regulate cell cycle checkpoints that ultimately leads to transformation.

Active Transport, Cell Nucleus↗
PubMed2005

Nuclear export determines the cytokine sensitivity of STAT transcription factors.

Cytokine-dependent gene activation critically depends upon the tyrosine phosphorylation (activation) of STAT transcription factors at membrane-bound cytokine receptors. The extent of STAT activation and hence the specificity of signaling is primarily determined by structural complementarity between the SH2 domain of the STATs and the tyrosine-phosphorylated receptor chains. Here, we identified constitutive nucleocytoplasmic shuttling as another mechanism that controls the differential activation of STAT transcription factors. Our analysis of nucleocytoplasmic cycling of STAT1 revealed that the expression of the alternatively spliced transactivation domain and its signal-dependent serine phosphorylation maximized the rate of nuclear export. Export modulation occurred independently of retention factors or the export receptor CRM1, and was observed both before and during stimulation of cells with cytokines. Our data indicated a dual role for the transactivation domain. It enhanced the nuclear retention of activated STAT1, but had the opposite effect on inactivated molecules. Accordingly, and despite their identical receptor recognition, the STAT1 splice variants differed strongly in the amplitude of tyrosine phosphorylation and in the duration of the cytokine signal. Thus, regulated nuclear export determined the cytokine sensitivity of the shuttling STAT1 transcription factors by controlling their availability at the receptor kinase complex.

Active Transport, Cell Nucleus↗
PubMed2006

Phosphorylation of tyrosine 568 controls nuclear export of Nrf2.

Nuclear factor Nrf 2, under normal conditions, is retained in the cytosol by INrf 2. Antioxidants and oxidants antagonize this interaction, resulting in the release of Nrf 2. Nrf 2 translocates to the nucleus binds to ARE and activates a battery of chemopreventive genes. Once this is achieved, Nrf 2 is exported out of the nucleus, binds with INrf 2, and degrades. Nrf 2 contains well defined signals that control nuclear import and export of Nrf 2. The present studies demonstrate that phosphorylation of tyrosine 568 is required for Crm1-mediated nuclear export and degradation of Nrf 2. Mutation of tyrosine 568 to alanine and phenylalanine resulted in the loss of interaction with Crm1 and abrogation of nuclear export of Nrf 2. Nrf 2Y568A is deficient in nuclear export and displays delayed degradation compared with wild-type Nrf 2. In addition, Src inhibitor PP2 caused nuclear accumulation of Nrf 2 in normal and hydrogen peroxide-treated cells but had no effect on localization of mutant Nrf 2Y568A. Further experiments with small interfering RNA revealed that Fyn phosphorylated Nrf 2Y568 leading to nuclear export and degradation of Nrf 2.

Active Transport, Cell Nucleus↗
PubMed2004

Cell density regulates intracellular localization of aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that plays a role as an intracellular mediator of the xenobiotic signaling pathway. AhR contains signals for both nuclear localization and nuclear export (NES). The objective of this study was to demonstrate how AhR intracellular distribution was regulated physiologically in cells. We found that cell density, but not the cell cycle, influenced the subcellular distribution of AhR in a keratinocyte cell line, HaCaT: AhR was predominantly nuclear at sparse cell densities, both nuclear and cytoplasmic at subconfluence, and predominantly cytoplasmic at confluence. Stable transfectants of HaCaT carrying a reporter gene fused with xenobiotic responsive element showed an association between xenobiotic responsive element-mediated transcription and AhR relocalization. Leptomycin B promoted nuclear accumulation of AhR irrespective of cell density, suggesting that this alteration may be because of a change of the regulation of the nuclear export of AhR. We found that Ser-68 in the NES of AhR was phosphorylated after nuclear accumulation of activated AhR and the nuclear export of a chimeric GST-AhR-GFP fusion protein was suppressed by substitution of a serine residue (Ser-68) to aspartic acid, which mimics the negative charge of phosphorylation. This novel cell density-dependent AhR relocalization was affected by exposure to SB203580, okadaic acid, and low Ca(2+) concentrations. These findings strongly suggest that cell density regulates the intracellular localization and function of AhR, because of modulation of nuclear export activity. The p38 MAPK-mediated phosphorylation of the NES and its dephosphorylation, regulated by cell-cell contact signals, may have pivotal roles in the novel AhR relocalization.

Active Transport, Cell Nucleus↗
PubMed2001

Nuclear export of phosphorylated C/EBPbeta mediates the inhibition of albumin expression by TNF-alpha.

Decreased albumin expression is a frequent feature of cachexia patients afflicted with chronic diseases, including cancer, and a major contributor to their morbidity. Here we show that tumor necrosis-alpha (TNF-alpha) treatment of primary mouse hepatocytes or TNF-alpha overexpression in a mouse model of cachexia induces oxidative stress, nitric oxide synthase (NOS) expression and phosphorylation of C/EBPbeta on Ser239, within the nuclear localization signal, thus inducing its nuclear export, which inhibits transcription from the albumin gene. SIN-1, a NO donor, duplicated the TNF-alpha effects on hepatocytes. We found similar molecular abnormalities in the liver of patients with cancer-cachexia. The cytoplasmic localization and association of C/EBPbeta-PSer239 with CRM1 (exportin-1) in TNF-alpha-treated hepatocytes was inhibited by leptomycin B, a blocker of CRM1 activity. Hepatic cells expressing the non-phosphorylatable C/EBPbeta alanine mutant were refractory to the inhibitory effects of TNF-alpha on albumin transcription since the mutant remained localized to the nucleus. Treatment of TNF-alpha mice with antioxidants or NOS inhibitors prevented phosphorylation of C/EBPbeta on Ser239 and its nuclear export, and rescued the abnormal albumin gene expression.

Active Transport, Cell Nucleus↗
PubMed2004

Nuclear export of the nonenveloped parvovirus virion is directed by an unordered protein signal exposed on the capsid surface.

It is uncertain whether nonenveloped karyophilic virus particles may actively traffic from the nucleus outward. The unordered amino-terminal domain of the VP2 major structural protein (2Nt) of the icosahedral parvovirus minute virus of mice (MVM) is internal in empty capsids, but it is exposed outside of the shell through the fivefold axis of symmetry in virions with an encapsidated single-stranded DNA genome, as well as in empty capsids subjected to a heat-induced structural transition. In productive infections of transformed and normal fibroblasts, mature MVM virions were found to efficiently exit from the nucleus prior to cell lysis, in contrast to the extended nuclear accumulation of empty capsids. Newly formed mutant viruses lacking the three phosphorylated serine residues of 2Nt were hampered in their exit from the human transformed NB324K nucleus, in correspondence with the capacity of 2Nt to drive microinjected phosphorylated heated capsids out of the nucleus. However, in normal mouse A9 fibroblasts, in which the MVM capsid was phosphorylated at similar sites but with a much lower rate, the nuclear exit of virions and microinjected capsids harboring exposed 2Nt required the infection process and was highly sensitive to inhibition of the exportin CRM1 in the absence of a demonstrable interaction. Thus, the MVM virion exits the nucleus by accessing nonconventional export pathways relying on cell physiology that can be intensified by infection but in which the exposure of 2Nt remains essential for transport. The flexible 2Nt nuclear transport signal may illustrate a common structural solution used by nonenveloped spherical viruses to propagate in undamaged host tissues.

Active Transport, Cell Nucleus↗
PubMed2006

Nucleolar trafficking is essential for nuclear export of intronless herpesvirus mRNA.

The nucleolus is the largest subnuclear structure and is plurifunctional in nature. Here, we demonstrate that nucleolar localization of a key herpesvirus regulatory protein is essential for its role in virus mRNA nuclear export. The herpesvirus saimiri ORF57 protein is a nucleocytoplasmic shuttle protein that is conserved in all herpesviruses and orchestrates the nuclear export of viral intronless mRNAs. We demonstrate that expression of the ORF57 protein induces nucleolar redistribution of human TREX (transcription/export) proteins that are involved in mRNA nuclear export. Moreover, we describe a previously unidentified nucleolar localization signal within ORF57 that is composed of two distinct nuclear localization signals. Intriguingly, point mutations that ablate ORF57 nucleolar localization lead to a failure of ORF57-mediated viral mRNA nuclear export. Furthermore, nucleolar retargeting of the ORF57 mutant was achieved by the incorporation of the HIV-1 Rev nucleolar localization signal, and analysis demonstrated that this modification was sufficient to restore viral mRNA nuclear export. This finding represents a unique and fundamental role for the nucleolus in nuclear export of viral mRNA.

Active Transport, Cell Nucleus↗
PubMed2005

Crosstalk between ERK2 and RXR regulates nuclear import of transcription factor NGFI-B.

Transcription factor NGFI-B initiates apoptosis when allowed to translocate to mitochondria. Retinoid-X receptor (RXR), another member of the nuclear receptor family, regulates NGFI-B signaling through heterodimerization and nuclear export. Growth factor EGF activates ERK2, which phosphorylates NGFI-B and determines if NGFI-B is allowed to translocate to mitochondria. In the present study, EGF treatment resulted in an increased nuclear import of NGFI-B. Likewise, active ERK2 resulted in a preferential nuclear localization of NGFI-B. When coexpressed with RXR the nuclear import and nuclear localization induced by active ERK2 were strongly reduced. In the presence of its ligand 9-cis-retinoic acid, RXR no longer inhibited ERK2-induced nuclear import. Thus, RXR serves a permissive role for ERK2-mediated nuclear accumulation of NGFI-B. This finding represents a novel crosstalk between ERK2 and RXR signaling pathways, and explains how two independent inhibitors of apoptosis (EGF and 9-cis-retinoic acid) may cooperate to regulate nuclear targeting of apoptosis inducer NGFI-B.

Active Transport, Cell Nucleus↗