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PubMed2004

Isoform-specific differences in rapid nucleocytoplasmic shuttling cause distinct subcellular distributions of 14-3-3 sigma and 14-3-3 zeta.

Nucleocytoplasmic transport of proteins plays an important role in the regulation of many cellular processes. Differences in nucleocytoplasmic shuttling can provide a basis for isoform-specific biological functions for members of multigene families, like the 14-3-3 protein family. Many organisms contain multiple 14-3-3 isoforms, which play a role in numerous processes, including signalling, cell cycle control and apoptosis. It is still unclear whether these isoforms have specialised biological functions and whether this specialisation is based on isoform-specific ligand binding, expression regulation or specific localisation. Therefore, we studied the subcellular distribution of 14-3-3 sigma and 14-3-3 zeta in vivo in various mammalian cell types using yellow fluorescent protein fusions and isoform-specific antibodies. 14-3-3 sigma was mainly localised in the cytoplasm and only low levels were present in the nucleus, whereas 14-3-3 zeta was found at relatively higher levels in the nucleus. Fluorescence recovery after photobleaching (FRAP) experiments indicated that the 14-3-3 proteins rapidly shuttle in and out of the nucleus through active transport and that the distinct subcellular distributions of 14-3-3 sigma and 14-3-3 zeta are caused by differences in nuclear export. 14-3-3 sigma had a 1.7x higher nuclear export rate constant than 14-3-3 zeta, while import rate constants were equal. The 14-3-3 proteins are exported from the nucleus at least in part by a Crm1-dependent, leptomycin B-sensitive mechanism. The differences in subcellular distribution of 14-3-3 that we found in this study are likely to reflect a molecular basis for isoform-specific biological specialisation.

14-3-3 Proteins↗
PubMed2002

Smurf1 regulates the inhibitory activity of Smad7 by targeting Smad7 to the plasma membrane.

Smad ubiquitin regulatory factor 1 (Smurf1), a HECT-type E3 ubiquitin ligase, interacts with inhibitory Smad7 and induces cytoplasmic localization of Smad7. Smurf1 then associates with transforming growth factor-beta type I receptor (TbetaR-I) and enhances the turnover of this receptor. However, the mechanisms of the nuclear export and plasma membrane localization of the Smurf1.Smad7 complex have not been elucidated. We show here that Smurf1 targets Smad7 to the plasma membrane through its N-terminal conserved 2 (C2) domain. Both wild-type Smurf1 (Smurf1(WT)) and Smurf1 lacking the C2 domain (Smurf1(deltaC2)) bound to Smad7 and translocated nuclear Smad7 to the cytoplasm. However, unlike Smurf1(WT), Smurf1(deltaC2) did not move to the plasma membrane and failed to recruit Smad7 to the cell surface TbetaR-II.TbetaR-I complex. Moreover, although Smurf1(deltaC2) induced ubiquitination of Smad7, it failed to induce the ubiquitination and degradation of TbetaR-I and did not enhance the inhibitory activity of Smad7. Thus, these results suggest that the plasma membrane localization of Smad7 by Smurf1 requires the C2 domain of Smurf1 and is essential for the inhibitory effect of Smad7 in the transforming growth factor-beta signaling pathway.

Activin Receptors, Type I↗
PubMed2006

The fragile X mental retardation protein interacts with a distinct mRNA nuclear export factor NXF2.

Loss of fragile X mental retardation protein, FMRP, causes the fragile X syndrome. Highly expressed in the brain and testis, FMRP has been implicated in the transport and translation of specific mRNAs. Here we show that FMRP and the mRNA nuclear export factor NXF2 co-express in the mouse male germ cells and hippocampal neurons and that FMRP associates with NXF2 but not with its close relative NXF1. We thus hypothesize that FMRP and NXF2 may act in concert to promote the nucleocytoplasmic transport of specific mRNAs in male germ cells and neurons.

Active Transport, Cell Nucleus↗
PubMed2003

Route of steroid-activated macromolecules through nuclear pores imaged with atomic force microscopy.

In eukaryotic cells, two concentric membranes, the nuclear envelope (NE), separate the nucleus from the cytoplasm. The NE is punctured by nuclear pore complexes (NPCs; molecular mass 120 MDa) that serve as regulated pathways for macromolecules entering and leaving the nuclear compartment. Transport across NPCs occurs through central channels. Such import and export of macromolecules through individual NPCs can be elicited in the Xenopus laevis oocyte by injecting the mineralocorticoid aldosterone and can be visualized with atomic force microscopy. The electrical NE resistance in intact cell nuclei can be measured in parallel. Resistance increases when macromolecules are engaged with the NPC. This article describe six observations made from these experiments and the conclusions that can be drawn from them. (i) A homogeneous population of macromolecules (approx. 100 kDa) attaches to the cytoplasmic face of the NPC 2 min after aldosterone injection. They are most likely to be aldosterone receptors. After a few minutes, they have disappeared. (ii) Large plugs (approx. molecular mass 1 MDa) appear in the central channels 20 min after hormone injection. They are most likely to be ribonucleoproteins exiting the nucleus. (iii) Electrical resistance measurements in isolated nuclei reveal transient electrical NE resistance peaks: an early (2 min) peak and a late (20 min) peak. Electrical peaks reflect macromolecule interaction with the NPC. (iv) Spironolactone blocks both the early and late peaks. This indicates that classic aldosterone receptors are involved in the pregenomic (early) and post-genomic (late) responses. (v) Actinomycin D and, independently, RNase A block the late electrical peak, confirming that plugs are genomic in nature. (vi) Intracellular calcium chelation blocks both early and late electrical peaks. Thus, the release of calcium from internal stores, which is known to be the first intracellular signal in response to aldosterone, is a prerequisite for the late genomic response.

Aldosterone↗
PubMed2005

Trafficking of nuclear heparin-binding epidermal growth factor-like growth factor into an epidermal growth factor receptor-dependent autocrine loop in response to oxidative stress.

Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) accumulates in the nucleus in aggressive transitional cell carcinoma (TCC) cells and this histologic feature is a marker of poor prognosis in human bladder cancer tissues. Here we report that HB-EGF can be exported from the nucleus during stimulated processing and secretion of the growth factor. Production of reactive oxygen species (ROS) resulted in mobilization of the HB-EGF precursor, proHB-EGF, from the nucleus of TCCSUP bladder cancer cells to a detergent-resistant membrane compartment, where the growth factor was cleaved by a metalloproteinase-mediated mechanism and shed into the extracellular space. Inhibition of nuclear export suppressed HB-EGF shedding. Production of ROS resulted in EGF receptor (EGFR) and Akt1 phosphorylation in HB-EGF-expressing cells. HB-EGF also stimulated cell proliferation and conferred cytoprotection when cells were challenged with cisplatin. These findings show that the nucleus can serve as an intracellular reservoir for a secreted EGFR ligand and, thus, can contribute to an autocrine loop leading to cell proliferation and protection from apoptotic stimuli.

Active Transport, Cell Nucleus↗
PubMed2001

Identification of Sam68 arginine glycine-rich sequences capable of conferring nonspecific RNA binding to the GSG domain.

Sam68 is an RNA-binding protein that contains a heterogeneous nuclear ribonucleoprotein K homology domain embedded in a larger RNA binding domain called the GSG (GRP33, Sam68, GLD-1) domain. This family of proteins is often referred to as the STAR (signal transduction and activators of RNA metabolism) proteins. It is not known whether Sam68 is a general nonspecific RNA-binding protein or whether it recognizes specific response elements in mRNAs with high affinity. Sam68 has been shown to bind homopolymeric RNA and a synthetic RNA sequence called G8-5 that has a core UAAA motif. Here we performed a structure function analysis of Sam68 and identified two arginine glycine (RG)-rich regions that confer nonspecific RNA binding to the Sam68 GSG domain. In addition, by using chimeric proteins between Sam68 and QKI-7, we demonstrated that one of the Sam68 RG-rich sequences of 26 amino acids was sufficient to confer homopolymeric RNA binding to the GSG domain of QKI-7, another STAR protein. Furthermore, that minimal sequence can also give QKI-7 the ability (as Sam68) to functionally substitute for HIV-1 REV to facilitate the nuclear export of RNAs. Our studies suggest that neighboring RG-rich sequences may impose nonspecific RNA binding to GSG domains. Because the Sam68 RNA binding activity is negatively regulated by tyrosine phosphorylation, our data lead us to propose that Sam68 might be a specific RNA-binding protein when tyrosine phosphorylated.

Amino Acid Sequence↗
PubMed2000

Deficient gene expression in protein kinase inhibitor alpha Null mutant mice.

Protein kinase inhibitor (PKI) is a potent endogenous inhibitor of the cyclic AMP (cAMP)-dependent protein kinase (PKA). It functions by binding the free catalytic (C) subunit with a high affinity and is also known to export nuclear C subunit to the cytoplasm. The significance of these actions with respect to PKI's physiological role is not well understood. To address this, we have generated by homologous recombination mutant mice that are deficient in PKIalpha, one of the three isoforms of PKI. The mice completely lack PKI activity in skeletal muscle and, surprisingly, show decreased basal and isoproterenol-induced gene expression in muscle. Further examination revealed reduced levels of the phosphorylated (active) form of the transcription factor CREB (cAMP response element binding protein) in the knockouts. This phenomenon stems, at least in part, from lower basal PKA activity levels in the mutants, arising from a compensatory increase in the level of the RIalpha subunit of PKA. The deficit in gene induction, however, is not easily explained by current models of PKI function and suggests that PKI may play an as yet undescribed role in PKA signaling.

Animals↗
PubMed2002

Receptor (MT(1)) mediated influence of melatonin on cAMP concentration and insulin secretion of rat insulinoma cells INS-1.

Recent functional, autoradiographic, and molecular investigations have shown that the pineal secretory product melatonin reduces the forskolin-stimulated insulin secretion from isolated pancreatic islets of neonate rats. Autoradiographic and binding studies as well as reverse transcriptase-polymerase chain reaction (RT-PCR) experiments proved that these effects are mediated through specific, high-affinity pertussis-toxin-sensitive Gi-protein-coupled MT(1) receptors and subsequent inhibition of the adenylyl cyclase/cyclic adenosine monophosphate (cAMP) system. This hypothesis was proved by blocking the intracellular signal transduction pathway using the non-hydrolyzable guanosine triphosphate analog guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) or the competitive melatonin receptor antagonist luzindole. Both GTPgammaS and luzindole diminished the melatonin effect. We have published these prior results elsewhere. So far, however, no information is available on both whether the MT1 receptors are located on the beta-cells and whether the consecutive functional reactions are based on a direct influence of melatonin on the insulin producing beta-cells. In order to examine this question, we used a glucose responsive insulin producing insulinoma cell line INS-1 isolated from rats. Comparable with the results of islets the competitive receptor antagonist luzindole diminished the insulin-decreasing effect of melatonin. In addition, our RT-PCR experiments, using specific primers for the rat melatonin receptor MT(1) showed that this melatonin receptor mRNA is also expressed in the INS-1 cells. Furthermore we radioimmunologically analyzed the forskolin-stimulated cAMP concentration in the superfusate. Similar to insulin secretion, the cAMP concentration was significantly reduced by melatonin. Following the hypothesis that cAMP is actively secreted from INS-1 cells by an energy-dependent mechanism based on either a OAT1/ROAT1 like anion exchanger or MDR-like transport systems, we used probenecid (p-[dipropylsulfamoyl] benzoic acid), a known inhibitor of cAMP extrusion. Probenecid blocks the export of cAMP by acting on transport mechanisms which are as yet not completely understood. Consistently, insulin secretion was increased and cAMP concentration diminished. The application of the phosphodiesterase inhibitor IBMX (3-isobutyl-1-methylxanthine) caused a marked rise of insulin secretion as well as cAMP concentration in the perifusate. From these data we conclude that the MT1 receptor is located on the INS-1 cell and therefore in general on pancreatic beta-cells.

1-Methyl-3-isobutylxanthine↗
PubMed2002

Nuclear degradation of p53 occurs during down-regulation of the p53 response after DNA damage.

The principal regulator of p53 stability is HDM2, an E3 ligase that mediates p53 degradation via the ubiquitin-26S proteasome pathway. The current model holds that p53 degradation occurs exclusively on cytoplasmic proteasomes and hence has an absolute requirement for nuclear export of p53 via the CRM-1 pathway. However, proteasomes are abundant in both cytosol and nucleus, and no studies have been done to determine under what physiological circumstances p53 degradation might occur in the nucleus. We analyzed HDM2-mediated degradation of endogenous p53 in the presence of various nuclear export inhibitors of CRM-1, including leptomycin B (LMB), a noncompetitive, specific, and fast-acting inhibitor; and HTLV1-Rex protein, a potent competitive inhibitor. We found that significant HDM2-mediated p53 degradation took place in the presence of LMB or HTLV1-Rex, indicating that endogenous p53 degradation occurs locally in the nucleus, in parallel to cytoplasmic degradation. Moreover, p53 null cells that coexpressed export-defective mutants of p53 and HDM2 retained partial competence for p53 degradation. It is important that nuclear degradation of p53 occurred during the poststress recovery phase of a p53 response, after DNA damage ceased. We propose that the capability of local p53 degradation within the nucleus provides a tighter and faster control during the down-regulatory phase, when an active p53 program needs to be turned off quickly.

Active Transport, Cell Nucleus↗
PubMed2005

Role of FXR in regulating bile acid homeostasis and relevance for human diseases.

Recent studies reveal that bile acids are signalling molecules that activate several nuclear receptors and regulate many physiological pathways and processes to maintain bile acid and cholesterol homeostasis. Analysis of orphan receptor expression patterns in enterohepatic tissues identified bile acids as ligands for farnesoid X receptor (FXR). The primary bile acid chenodeoxycholic acid (CDCA) was shown to be the most potent FXR ligand in vitro at an EC50 of 10-50 microM. FXR can also be activated by the secondary bile acids lithocholic acid (LCA) and deoxycholic acid (DCA). Upon activation FXR heterodimerises with 9-cis retinoic X receptor (RXR) and regulates a cohort of genes involved in cholesterol catabolism and bile acids biosynthesis. Thus bile acid-activated FXR directly induces expression of Small Heterodimer Partner (SHP), a nuclear receptor that suppresses bile acid biosynthesis down-regulates the Na+ taurocholate cotransport peptide (NTCP), a pump depicted to transport bile acids from the lumen into hepatocyte, and induces expression of bile salt export pump (BSEP), the principal bile acid efflux transporter in the liver. As demonstrated by the Fxr null mice, FXR defends the liver against cholestasis. The 6-ethyl derivative of CDCA (6-ECDCA) is approximately 100 fold more potent than CDCA in activating FXR in vitro. In vivo administration of 6-ECDCA protects against cholestasis induced by estrogen and LCA in rats providing evidence that development of potent FXR agonists might represent a new approach for the treatment of cholestastic disorders.

Animals↗
PubMed2007

Role of Ca2+ activation and bilobal structure of calmodulin in nuclear and nucleolar localization.

Ca2+ signalling to the nucleus is thought to occur by calmodulin entry into the nucleus where calmodulin has many functions. In the present study we have investigated the role of Ca2+ and the N- and C-terminal lobes of calmodulin in its subnuclear targeting by using fluorescently labelled calmodulin and its mutants and confocal microscopy. Our data show, first, that Ca2+ stimulation induces a reorganization of subnuclear structures to which apo-calmodulin can bind. Secondly, Ca2+-independent association of the C-terminal lobe is seen with subnuclear structures such as chromatin, the nuclear envelope and the nucleoli. Thirdly, Ca2+-dependent accumulation of both calmodulin and the C-terminal calmodulin lobe occurs in the nucleoli. The N-terminal lobe of calmodulin does not show significant binding to subnuclear structures although, similarly to the C-terminal lobe, it accumulates in the nucleoplasm of wheat germ agglutinin-blocked nuclei suggesting that a facilitated nuclear export mechanism exists for calmodulin.

Active Transport, Cell Nucleus↗
PubMed2002

The human homologue of yeast ArgRIII protein is an inositol phosphate multikinase with predominantly nuclear localization.

The function of the transcription regulator ArgRIII in the expression of several genes involved in the metabolism of arginine in yeast has been well studied. It was previously reported that it is also an inositol phosphate multikinase and an important factor of the mRNA export pathway [reviewed by Shears (2000) Bioessays 22, 786-789]. In the present study we report the cloning of a full-length 1248-bp cDNA encoding a human inositol phosphate multikinase (IPMK). This protein has a calculated molecular mass of 47.219 kDa. Functionally important motifs [inositol phosphate-binding site, ATP-binding site, catalytically important SSLL (Ser-Ser-Leu-Leu) domain] are conserved between the human IPMK and yeast ArgRIII. Bacterially expressed protein demonstrated an inositol phosphate multikinase activity similar to that of yeast ArgRIII. Ins(1,4,5)P3 is phosphorylated at positions 3 and 6 up to Ins(1,3,4,5,6)P5. The human IPMK fused with a fluorescent protein tag is localized predominantly in the nucleus when transiently expressed in mammalian cells. A basic cluster in the protein's C-terminus is positively involved in nuclear targeting. These findings are consistent with the concept of a nuclear inositol phosphate signalling and phosphorylation pathway in mammalian cells.

Amino Acid Sequence↗
PubMed2006

A Salmonella type III secretion effector interacts with the mammalian serine/threonine protein kinase PKN1.

Essential to salmonellae pathogenesis is an export device called the type III secretion system (TTSS), which mediates the transfer of bacterial effector proteins from the bacterial cell into the host cell cytoplasm. Once inside the host cell, these effectors are then capable of altering a variety of host cellular functions in order to promote bacterial survival and colonization. SspH1 is a Salmonella enterica serovar Typhimurium TTSS effector that localizes to the mammalian nucleus and down-modulates production of proinflammatory cytokines by inhibiting nuclear factor (NF)-kappaB-dependent gene expression. To identify mammalian binding partners of SspH1 a yeast two-hybrid screen against a human spleen cDNA library was performed. It yielded a serine/threonine protein kinase called protein kinase N 1 (PKN1). The leucine-rich repeat domain of SspH1 was demonstrated to mediate this interaction and also inhibition of NF-kappaB-dependent gene expression. This suggested that PKN1 may play a role in modulation of the NF-kappaB signalling pathway. Indeed, we found that expression of constitutively active PKN1 in mammalian cells results in a decrease, while depletion of PKN1 by RNA interference causes an increase in NF-kappaB-dependent reporter gene expression. These data indicate that SspH1 may inhibit the host's inflammatory response by interacting with PKN1.

Animals↗
PubMed1993

Association with terminal exons in pre-mRNAs: a new role for the U1 snRNP?

Psoralen cross-linking experiments in HeLa cell nuclear extracts have revealed the binding of U1 snRNA to substrates containing the SV40 late and adenovirus L3 polyadenylation signals. The sites of U1 cross-linking to the substrates map different distances upstream of the AAUAAA sequence to regions with limited complementarity to the 5' end of U1 snRNA. U1 cross-linking to the same site in the SV40 late pre-mRNA is enhanced by the addition of an upstream 3' splice site, which also enhances polyadenylation. Examination of different nuclear extracts reveals a correlation between U1 cross-linking and the coupling of splicing and polyadenylation, suggesting that the U1 snRNP participates in the coordination of these two RNA-processing events. Mutational analyses demonstrate that U1/substrate association cannot be too strong for coupling to occur and suggest that the U1 snRNP plays a similar role in recognition of internal and 3' terminal exons. Possible mechanisms for communication between the splicing and polyadenylation machineries are discussed, as well as how interaction of the U1 snRNP with 3' terminal exons might contribute to mRNA export.

Adenoviridae↗
PubMed2006

Oncogenic conversion of the thyroid hormone receptor by altered nuclear transport.

Nuclear receptors (NRs) are transcription factors whose activity is modulated by ligand binding. These receptors are at the core of complex signaling pathways and act as integrators of many cellular signals. In the last decade our understanding of NRs has greatly evolved. In particular, regulation of NR subcellular dynamics has emerged as central to their activity. Research on the subcellular distribution of the thyroid hormone receptor (TR) has revealed new dimensions in the complexity of NR regulation, and points to the possibility that NR mislocalization plays a key role in oncogenesis. For many years, TR was thought to reside exclusively in the nucleus. It is now known that TR is a dynamic protein that shuttles between the nucleus and cytoplasm. TR is localized to the nucleus in a phosphorylated form, suggesting that compartment-specific phosphorylation mediates cross-talk between multiple cell signaling pathways. The oncoprotein v-ErbA, a viral-derived dominant negative variant of TR is actively exported to the cytoplasm by the CRM1 export receptor. Strikingly, the oncoprotein causes mislocalization of cellular TR and some of its coactivators by direct interaction. Here, we offer some perspectives on the role of subcellular trafficking in the oncogenic conversion of TR, and propose a new model for oncoprotein dominant negative activity.

Journal Article↗
PubMed2002

Nuclear translocation of cell adhesion kinase beta/proline-rich tyrosine kinase 2.

Cell adhesion kinase beta (CAKbeta/PYK2) is a protein-tyrosine kinase of the focal adhesion kinase (FAK) family. Whereas FAK predominantly localizes at focal adhesions, CAK beta localizes at the perinuclear region in fibroblasts. Here we expressed in cultured cells two point mutants of CAKbeta, P717A and P859A, each of which had lost one of its two PXXP motifs, the ligand sequence for SH3 domains, found at the CAKbeta C-terminal region. We observed a remarkable change in the subcellular distribution of the P859A mutant; while that of the P717A mutant was the same as the wild type. The P859A mutant localized exclusively in the cell nucleus in all cell lines examined. Wild-type CAKbeta also accumulated in the nucleus when cells were treated with an inhibitor of the nuclear export of proteins. These results indicate that CAK beta shuttles between the cytoplasm and the nucleus. On nuclear accumulation of P859A-CAKbeta, a CAKbeta-binding protein, Hic-5, also accumulated in the nucleus. P859A-CAKbeta and co-expressed Hic-5 formed nuclear speckles, in which one other CAK beta-binding protein, p130(Cas), was also concentrated. These findings on nuclear translocation of CAK beta imply that CAKbeta may regulate nuclear processes such as transcription, particularly because Hic-5 was recently shown to be a coactivator of nuclear receptors.

Active Transport, Cell Nucleus↗
PubMed2004

ZAP is a CRM1-dependent nucleocytoplasmic shuttling protein.

The zinc finger antiviral protein (ZAP) is a recently isolated host antiviral factor. It specifically inhibits the replication of Moloney murine leukemia virus (MMLV) and Sindbis virus (SIN) by preventing the accumulation of viral RNA in the cytoplasm. In this report, we demonstrate that ZAP is predominantly localized in the cytoplasm at steady state but shuttles between the nucleus and the cytoplasm in a CRM1-dependent manner. Two nuclear localization sequences (NLS) and one nuclear export sequence (NES) were identified. One NLS was mapped to amino acids 68-RARVCRRK-75 and the other mapped to a region including amino acids K405 and K406. The NES was mapped to amino acids 284-LEDVSVDV-291. These findings help to understand why ZAP specifically prevents the accumulation of viral RNA in the cytoplasm. These findings also suggest possible functions of ZAP in the nucleus.

Active Transport, Cell Nucleus↗
PubMed1999

The eps15 homology (EH) domain-based interaction between eps15 and hrb connects the molecular machinery of endocytosis to that of nucleocytosolic transport.

The Eps15 homology (EH) module is a protein-protein interaction domain that establishes a network of connections involved in various aspects of endocytosis and sorting. The finding that EH-containing proteins bind to Hrb (a cellular cofactor of the Rev protein) and to the related protein Hrbl raised the possibility that the EH network might also influence the so-called Rev export pathway, which mediates nucleocytoplasmic transfer of proteins and RNAs. In this study, we demonstrate that Eps15 and Eps15R, two EH-containing proteins, synergize with Hrb and Hrbl to enhance the function of Rev in the export pathway. In addition, the EH-mediated association between Eps15 and Hrb is required for the synergistic effect. The interaction between Eps15 and Hrb occurs in the cytoplasm, thus pointing to an unexpected site of action of Hrb, and to a possible role of the Eps15-Hrb complex in regulating the stability of Rev.

Adaptor Proteins, Signal Transducing↗