Export of mRNA from microinjected nuclei of Xenopus laevis oocytes.

Export of mRNA from the nucleus to the cytoplasm was studied in mature Xenopus laevis oocytes. In vitro transcribed, capped 32P-labeled mRNA was microinjected into nuclei, and its appearance in the cytoplasm measured by counting radioactivity or by RNA extraction and gel electrophoresis. Both for a 5.0-kb transferrin receptor mRNA and a 2.0-kb 4F2 antigen heavy chain mRNA we found saturable transport with an apparent Km of 3.6 x 10(8) molecules per oocyte nucleus. Under non-saturating conditions the half-time for mRNA export from the nucleus was approximately 2 min at 20 degrees C. At higher concentrations of injected mRNA this half-time was prolonged, and the maximal transport rate was reached at approximately 1.6 x 10(8) molecules/min. mRNA transport showed properties of an energy-dependent mechanism, since it was inhibited at 4 degrees C or by ATP depletion. Co-injection of the cap dinucleotide m7GpppG blocked the export effectively, suggesting a role for the cap in this process. The export was also inhibited by the pre-injection of wheat germ agglutinin. The effect of the lectin was specific and abolished by co-injection of N-acetylglucosamine. Finally, we found significant competitive inhibition in mRNA export by the presence of tRNA. Our results suggest that mRNA transport is a facilitated process which may share common steps with tRNA transport. Preliminary gel retardation experiments show that injected mRNA associates with endogenous nuclear proteins and suggest an exchange of some of the bound components during the transport to the cytoplasm.

NXT1 is necessary for the terminal step of Crm1-mediated nuclear export.

Soluble factors are required to mediate nuclear export of protein and RNA through the nuclear pore complex (NPC). These soluble factors include receptors that bind directly to the transport substrate and regulators that determine the assembly state of receptor-substrate complexes. We recently reported the identification of NXT1, an NTF2-related export factor that stimulates nuclear protein export in permeabilized cells and undergoes nucleocytoplasmic shuttling in vivo (Black, B.E., L. Lévesque, J.M. Holaska, T.C. Wood, and B.M. Paschal. 1999. Mol. Cell. Biol. 19:8616-8624). Here, we describe the molecular characterization of NXT1 in the context of the Crm1-dependent export pathway. We find that NXT1 binds directly to Crm1, and that the interaction is sensitive to the presence of Ran-GTP. Moreover, mutations in NXT1 that reduce binding to Crm1 inhibit the activity of NXT1 in nuclear export assays. We show that recombinant Crm1 and Ran are sufficient to reconstitute nuclear translocation of a Rev reporter protein from the nucleolus to an antibody accessible site on the cytoplasmic side of the NPC. Further progress on the export pathway, including the terminal step of Crm1 and Rev reporter protein release, requires NXT1. We propose that NXT1 engages with the export complex in the nucleoplasm, and that it facilitates delivery of the export complex to a site on the cytoplasmic side of NPC where the receptor and substrate are released into the cytoplasm.

Evidence for a role of CRM1 in signal-mediated nuclear protein export.

Chromosome maintenance region 1 (CRM1), a protein that shares sequence similarities with the karyopherin beta family of proteins involved in nuclear import pathway, was shown to form a complex with the leucine-rich nuclear export signal (NES). This interaction was inhibited by leptomycin B, a drug that prevents the function of the CRM1 protein in yeast. To analyze the role of the CRM1-NES interaction in nuclear export, a transport assay based on semipermeabilized cells was developed. In this system, which reconstituted NES-, cytosol-, and energy-dependent nuclear export, leptomycin B specifically blocked export of NES-containing proteins. Thus, the CRM1 protein could act as a NES receptor involved in nuclear protein export.

Impairment of mycobacterial but not viral immunity by a germline human STAT1 mutation.

Interferons (IFN) alpha/beta and gamma induce the formation of two transcriptional activators: gamma-activating factor (GAF) and interferon-stimulated gamma factor 3 (ISGF3). We report a natural heterozygous germline STAT1 mutation associated with susceptibility to mycobacterial but not viral disease. This mutation causes a loss of GAF and ISGF3 activation but is dominant for one cellular phenotype and recessive for the other. It impairs the nuclear accumulation of GAF but not of ISGF3 in heterozygous cells stimulated by IFNs. Thus, the antimycobacterial, but not the antiviral, effects of human IFNs are principally mediated by GAF.

Thymotaxin, a chemotactic protein, is identical to beta 2-microglobulin.

Thymotaxin, an 11-kilodalton protein chemotactic for rat bone marrow hematopoietic precursors, was purified from media conditioned by a rat thymic epithelial cell line. The NH2-terminal sequence of thymotaxin was identical to that of rat beta 2-microglobulin (beta 2m). Antibodies to beta 2m removed thymotaxin activity from the fraction containing the 11-kilodalton protein. Chemotactic activity was observed with rat plasma beta 2m, human beta 2m, and mouse recombinant beta 2m, further supporting the identity of thymotaxin with beta 2m. The directional migration, as opposed to random movement, of the cells was also confirmed. The only rat bone marrow cells that migrated toward beta 2m were Thy1+ immature lymphoid cells devoid of T cell, B cell, and myeloid cell differentiation markers.

Interaction between hnRNPA1 and IkappaBalpha is required for maximal activation of NF-kappaB-dependent transcription.

Transcriptional activation of NF-kappaB is mediated by signal-induced phosphorylation and degradation of its inhibitor, IkappaBalpha. NF-kappaB activation induces a rapid resynthesis of IkappaBalpha which is responsible for postinduction repression of transcription. Following resynthesis, IkappaBalpha translocates to the nucleus, removes template bound NF-kappaB, and exports NF-kappaB to the cytoplasm in a transcriptionally inactive form. Here we demonstrate that IkappaBalpha interacts directly with another nucleocytoplasmic shuttling protein, hnRNPA1, both in vivo and in vitro. This interaction requires one of the N-terminal RNA binding domains of hnRNPA1 and the C-terminal region of IkappaBalpha. Cells lacking hnRNPA1 are defective in NF-kappaB-dependent transcriptional activation, but the defect in these cells is complemented by ectopic expression of hnRNPA1. hnRNPA1 expression in these cells increased the amount of IkappaBalpha degradation, compared to that of the control cells, in response to activation by Epstein-Barr virus latent membrane protein 1. Thus in addition to regulating mRNA processing and transport, hnRNPA1 also contributes to the control of NF-kappaB-dependent transcription.

RanGTP-binding protein NXT1 facilitates nuclear export of different classes of RNA in vitro.

To better characterize the mechanisms responsible for RNA export from the nucleus, we developed an in vitro assay based on the use of permeabilized HeLa cells. This new assay supports nuclear export of U1 snRNA, tRNA, and mRNA in an energy- and Xenopus extract-dependent manner. U1 snRNA export requires a 5' monomethylated cap structure, the nuclear export signal receptor CRM1, and the small GTPase Ran. In contrast, mRNA export does not require the participation of CRM1. We show here that NXT1, an NTF2-related protein that binds directly to RanGTP, strongly stimulates export of U1 snRNA, tRNA, and mRNA. The ability of NXT1 to promote export is dependent on its capacity to bind RanGTP. These results support the emerging view that NXT1 is a general export factor, functioning on both CRM1-dependent and CRM1-independent pathways of RNA export.

Direct association and nuclear import of the hepatitis B virus X protein with the NF-kappaB inhibitor IkappaBalpha.

The X protein of hepatitis B virus (HBV) is a transcriptional activator which is required for infection and may play an important role in HBV-associated hepatocarcinogenesis. It has been suggested that X acts as a nuclear coactivator or stimulates several signal transduction pathways by acting in the cytoplasm. One of these pathways leads to the nuclear translocation of NF-kappaB. A recent report indicates that X activates NF-kappaB by acting on two cytoplasmic inhibitors of this family of transcription factors: IkappaBalpha and the precursor/inhibitor p105. We demonstrate here that X directly interacts with IkappaBalpha, which is able to transport it to the nucleus by a piggyback mechanism. This transport requires a region of IkappaBalpha (the second ankyrin repeat) which has been demonstrated to be involved in its nuclear import following NF-kappaB activation. Using deletion mutants, we showed that amino acids 249 to 253 of IkappaBalpha (located in the C-terminal part of the sixth ankyrin repeat) play a critical role in the interaction with X. This small region overlaps one of the domains of IkappaBalpha mediating the interaction with the p50 and p65 subunits of NF-kappaB and is also close to the nuclear export sequence of IkappaBalpha, therefore providing a potential explanation for the nuclear accumulation of IkappaBalpha with X. This association can also be observed upon the induction of endogenous IkappaBalpha by tumor necrosis factor alpha (TNF-alpha) treatment of Chang cells expressing X. In accordance with this observation, band shift analysis indicates that X induces a sustained NF-kappaB activation following TNF-alpha treatment, probably by preventing the reassociation of newly synthesized nuclear IkappaBalpha with DNA-bound NF-kappaB complexes.

p95vav associates with the nuclear protein Ku-70.

The proto-oncogene vav is expressed solely in hematopoietic cells and plays an important role in cell signaling, although little is known about the proteins involved in these pathways. To gain further information, the Src homology 2 (SH2) and 3 (SH3) domains of Vav were used to screen a lymphoid cell cDNA library by the yeast two-hybrid system. Among the positive clones, we detected a nuclear protein, Ku-70, which is the DNA-binding element of the DNA-dependent protein kinase. In Jurkat and UT7 cells, Vav is partially localized in the nuclei, as judged from immunofluorescence and confocal microscopy studies. By using glutathione S-transferase fusion proteins derived from Ku-70 and coimmunoprecipitation experiments with lysates prepared from human thymocytes and Jurkat and UT7 cells, we show that Vav associates with Ku-70. The interaction of Vav with Ku-70 requires only the 150-residue carboxy-terminal portion of Ku-70, which binds to the 25 carboxy-terminal residues of the carboxy SH3 domain of Vav. A proline-to-leucine mutation in the carboxy SH3 of Vav that blocks interaction with proline-rich sequences does not modify the binding of Ku-70, which lacks this motif. Therefore, the interaction of Vav with Ku-70 may be a novel form of protein-protein interaction. The potential role of Vav/Ku-70 complexes is discussed.

Taxol affects nuclear lamina and pore complex organization and inhibits import of karyophilic proteins into the cell nucleus.

Treatment of human carcinoma cells with Taxol induces focal unraveling of the nuclear lamina and extensive clustering or ectopic localization of the nuclear pore complexes. These striking aberrations develop when the cells are transferred to drug-free medium and are allowed to complete mitosis. As could be confirmed by terminal deoxynucleotidyl transferase-mediated nick end labeling assays, 4,6-diamidino-2-phenylindole staining, 5-bromo-2-deoxyuridine incorporation, and examination of the nuclear lamins by Western blotting, the malformation of the nuclear envelope is not a consequence of apoptosis or G1 arrest. In fact, Taxol-treated cells possessing a defective nuclear envelope remain alive and replication-competent for at least 24 h, undergoing programmed death 72 h after removal of the drug. While still in the nonapoptotic state, these cells lose the ability to import karyophilic proteins into the nucleus. Diminished nucleocytoplasmic transport through the nuclear pore complex can be readily demonstrated by in vitro assays involving digitonin-permeabilized cells or in vivo monitoring of nuclear factor-kappaB translocation upon stimulation with tumor necrosis factor-alpha. These observations reveal novel cellular targets of antimicrotubule drugs and may pave the way for improved schemes of anticancer treatment.

Expression of IkappaBalpha in the nucleus of human peripheral blood T lymphocytes.

According to current models the inhibitory capacity of I(kappa)B(alpha) would be mediated through the retention of Rel/NF-kappaB proteins in the cytosol. However, I(kappa)B(alpha) has also been detected in the nucleus of cell lines and when overexpressed by transient transfection. To gain better insight into the potential role of nuclear I(kappa)B(alpha) in a physiological context we have analysed its presence in the nucleus of human peripheral blood T lymphocytes (PBL). We demonstrate the nuclear localization of I(kappa)B(alpha) in PBL by different techniques: Western blot, indirect immunofluorescence and electron microscopy. Low levels of nuclear I(kappa)B(alpha) were detected in resting cells whereas a superinduction was obtained after PMA activation. The nuclear pool of I(kappa)B(alpha) showed a higher stability than cytosolic I(kappa)B(alpha) and was partially independent of the resynthesis of the protein. Unexpectedly, the presence of nuclear I(kappa)B(alpha) did not inhibit NF-kappaB binding to DNA and this phenomenon was not due to the presence of IkappaBbeta at the nuclear level. Immunoprecipitation experiments failed to demonstrate an association between nuclear I(kappa)B(alpha) and NF-kappaB proteins. Our results demonstrate that in resting and PMA-activated human PBL, I(kappa)B(alpha) is present in the nucleus in an apparently inactive form unable to disrupt NF-kappaB binding from DNA.

Nuclear transport and transcriptional regulation.

Studies over the past 10 years have provided major insights into the molecular mechanisms responsible for active transport of macromolecules in and out of the nucleus. Nucleocytoplasmic transport pathways correspond to active and signal-mediated processes that involve substrates, adaptors and receptors. Regulation of both nuclear import and nuclear export is mainly exerted at the level of transport complex formation and has emerged as one of the most efficient mechanisms to adapt gene expression to the cell environment by restricting the access of transcriptional regulators to their target genes.

Activation of NF-kappaB by Akt upregulates Snail expression and induces epithelium mesenchyme transition.

Carcinoma progression is associated with the loss of epithelial features, and the acquisition of mesenchymal characteristics and invasive properties by tumour cells. The loss of cell-cell contacts may be the first step of the epithelium mesenchyme transition (EMT) and involves the functional inactivation of the cell-cell adhesion molecule E-cadherin. Repression of E-cadherin expression by the transcription factor Snail is a central event during the loss of epithelial phenotype. Akt kinase activation is frequent in human carcinomas, and Akt regulates various cellular mechanisms including EMT. Here, we show that Snail activation and consequent repression of E-cadherin may depend on AKT-mediated nuclear factor-kappaB (NF-kappaB) activation, and that NF-kappaB induces Snail expression. Expression of the NF-kappaB subunit p65 is sufficient for EMT induction, validating this signalling module during EMT. NF-kappaB pathway activation is associated with tumour progression and metastasis of several human tumour types; E-cadherin acts as a metastasis suppressor protein. Thus, this signalling and transcriptional network linking AKT, NF-kappaB, Snail and E-cadherin during EMT is a potential target for antimetastatic therapeutics.

Domains of Crm1 involved in the formation of the Crm1, RanGTP, and leucine-rich nuclear export sequences trimeric complex.

Nuclear export of proteins containing a leucine-rich nuclear export sequence (NES) is mediated by a specific NES receptor known as Crm1. This protein, which is related to the karyopherin beta family, interacts directly with NES in a RanGTP-dependent manner. To characterize the domains of Crm1 involved in formation of the trimeric Crm1-NES-RanGTP complex, N- and C-terminal deletion mutants of Crm1 were generated and their ability to bind NES and RanGTP in vitro was analyzed. Our results indicate that two regions of Crm1 are required for the formation of the trimeric Crm1-NES-RanGTP complex, the N-terminal domain of Crm1 and the central domain of the receptor, starting after residue 160 with an essential region between 566 and 720. The N-terminal domain is homologous to the RanGTP-binding domain of karyopherin beta and therefore is likely involved in the interaction with RanGTP. Consequently, the central domain likely corresponds to the NES-binding site of Crm1.

Characterization of Ca2+ fluxes in rat liver plasma-membrane vesicles.

Inside-out plasma-membrane vesicles isolated from rat liver [Prpic, Green, Blackmore & Exton (1984) J. Biol. Chem. 259, 1382-1385] accumulated a substantial amount of 45Ca2+ when they were incubated in a medium whose ionic composition and pH mimicked those of cytosol and which contained MgATP. The Vmax of the initial 45Ca2+ uptake rate was 2.9 +/- 0.6 nmol/min per mg and the Km for Ca2+ was 0.50 +/- 0.08 microM. The ATP-dependent 45Ca2+ uptake by inside-out plasma-membrane vesicles was about 20 times more sensitive to saponin than was the ATP-dependent uptake by a microsomal preparation. The 45Ca2+ efflux from the inside-out vesicles, which is equivalent to the Ca2+ influx in intact cells, was increased when the free Ca2+ concentration in the medium was decreased. The Ca2+ antagonists La3+ and Co2+ inhibited the 45Ca2+ efflux from the vesicles. Neomycin stimulated the Ca2+ efflux in the presence of either a high or a low free Ca2+ concentration. These results confirm that polyvalent cations regulate Ca2+ fluxes through the plasma membrane.

SUMO-1 conjugation in vivo requires both a consensus modification motif and nuclear targeting.

SUMO-1 is a small ubiquitin-related modifier that is covalently linked to many cellular protein targets. Proteins modified by SUMO-1 and the SUMO-1-activating and -conjugating enzymes are located predominantly in the nucleus. Here we define a transferable sequence containing the PsiKXE motif, where Psi represents a large hydrophobic amino acid, that confers the ability to be SUMO-1-modified on proteins to which it is linked. Whereas addition of short sequences from p53 and IkappaBalpha, containing the PsiKXE motif, to a carrier protein is sufficient for modification in vitro, modification in vivo requires the additional presence of a nuclear localization signal. Thus, protein substrates must be targeted to the nucleus to undergo SUMO-1 conjugation.

Protein export from the nucleus.

The evolution of the nucleus imposed on eukaryotic cells the necessity to strictly control exchange of molecules between the nucleus and the remainder of the cell, not only to protect and correctly transmit genetic information, but also to coordinate nuclear and cytoplasmic functions. Studies over the past 10 years have provided major insights into the molecular mechanisms responsible for transport of molecules between the nucleus and the cytoplasm. In addition, regulation of the nucleocytoplasmic distribution of diverse cellular factors has emerged as one of the most efficient mechanism to adapt gene expression to the cell environment, for example by controlling the access of transcriptional regulators to their target genes. In this review, we focus on the molecular basis of protein nuclear export that relies on interactions between targeting sequences present on the cargoes, specific export receptors or exportins and nuclear pore proteins, with special emphasis on the role of the Ran GTPase and associated proteins in this process.

IkappaBalpha and IkappaBalpha /NF-kappa B complexes are retained in the cytoplasm through interaction with a novel partner, RasGAP SH3-binding protein 2.

IkappaBalpha inhibits the transcriptional activity of NF-kappaB both in the cytoplasm by preventing the nuclear translocation of NF-kappaB and in the nucleus where it dissociates NF-kappaB from DNA and transports it back to the cytoplasm. Cytoplasmic localization of inactive NF-kappaB/IkappaBalpha complexes is controlled by mutual masking of nuclear import sequences of NF-kappaB p65 and IkappaBalpha and active CRM1-mediated nuclear export. Here, we describe an additional mechanism accounting for the cytoplasmic anchoring of IkappaBalpha or NF-kappaB/IkappaBalpha complexes. The N-terminal domain of IkappaBalpha contains a sequence responsible for the cytoplasmic retention of IkappaBalpha that is specifically recognized by G3BP2, a cytoplasmic protein that interacts with both IkappaBalpha and IkappaBalpha/NF-kappaB complexes. G3BP2 is composed of an N-terminal domain homologous to the NTF2 protein, followed by an acidic domain sufficient for the interaction with the IkappaBalpha cytoplasmic retention sequence, a region containing five PXXP motifs and a C-terminal domain containing RNA-binding motifs. Overexpression of G3BP2 directly promotes retention of IkappaBalpha in the cytoplasm, indicating that subcellular distribution of IkappaBalpha and NF-kappaB/IkappaBalpha complexes likely results from a equilibrium between nuclear import, nuclear export, and cytoplasmic retention. The molecular organization of G3BP2 suggests that this putative scaffold protein might connect the NF-kappaB signal transduction cascade with cellular functions such as nuclear transport or RNA metabolism.