The nuclear hormone receptor coactivator SRC-1 is a specific target of p300.

p300 and its family member, CREB-binding protein (CBP), function as key transcriptional coactivators by virtue of their interaction with the activated forms of certain transcription factors. In a search for additional cellular targets of p300/CBP, a protein-protein cloning strategy, surprisingly identified SRC-1, a coactivator involved in nuclear hormone receptor transcriptional activity, as a p300/CBP interactive protein. p300 and SRC-1 interact, specifically, in vitro and they also form complexes in vivo. Moreover, we show that SRC-1 encodes a new member of the basic helix-loop-helix-PAS domain family and that it physically interacts with the retinoic acid receptor in response to hormone binding. Together, these results implicate p300 as a component of the retinoic acid signaling pathway, operating, in part, through specific interaction with a nuclear hormone receptor coactivator, SRC-1.

Activation of an interleukin 1 converting enzyme-dependent apoptosis pathway by granzyme B.

Cytotoxic T lymphocytes (CTL) can induce apoptosis through a granzyme B-based killing mechanism. Here we show that in cells undergoing apoptosis by granzyme B, both p45 pro-interleukin 1 beta converting enzyme (ICE) and pro-CPP32 are processed. Using ICE deficient (ICE -/-) mice, embryonic fibroblasts exhibit high levels of resistance to apoptosis by granzyme B or granzyme 3, while B lymphoblasts are granzyme B-resistant, thus identifying an ICE-dependent apoptotic pathway that is activated by CTL granzymes. In contrast, an alternative ICE-independent pathway must also be activated as ICE -/- thymocytes remain susceptible to apoptosis by both granzymes. In ICE -/- B cells or HeLa cells transfected with mutant inactive ICE or Ich-1S that exhibit resistance to granzyme B, CPP32 is processed to p17 and poly(ADP-ribose) polymerase is cleaved indicating that this protease although activated was not associated with an apoptotic nuclear phenotype. Using the peptide inhibitor Ac-DEVD-CHO, apoptosis as well as p45 ICE hydrolysis are suppressed in HeLa cells, suggesting that a CPP32-like protease is upstream of ICE. In contrast, p34cdc2 kinase, which is required for granzyme B-induced apoptosis, remains inactive in ICE -/- B cells indicating it is downstream of ICE. We conclude that granzyme B activates an ICE-dependent cell death pathway in some cell types and requires a CPP32-like Ac-DEVD-CHO inhibitable protease acting upstream to initiate apoptosis.

Differential expression of the ToxR regulon in classical and E1 Tor biotypes of Vibrio cholerae is due to biotype-specific control over toxT expression.

The two major disease-causing biotypes of Vibrio cholerae, classical and El Tor, exhibit differences in their epidemic nature. Their behavior in the laboratory also differs in that El Tor strains produce two major virulence factors, cholera toxin (CT) and the toxin coregulated pilus (TCP), only under very restricted growth conditions, whereas classical strains do so in standard laboratory medium. Expression of toxin and TCP is controlled by two activator proteins, ToxR and ToxT, that operate in cascade fashion with ToxR controlling the synthesis of ToxT. Both biotypes express equivalent levels of ToxR, but only classical strains appear to express ToxT when grown in standard medium. In this report we show that restrictive expression of CT and TCP can be overcome in El Tor strains by expressing ToxT independently of ToxR. An El Tor strain lacking functional ToxT does not express CT or TCP, ruling out existence of a cryptic pathway for virulence regulation in this biotype. These results may have implications for understanding the evolution of El Tor strains toward reduced virulence with respect to classical strains.

Apoptosis of nur77/N10-transgenic thymocytes involves the Fas/Fas ligand pathway.

The orphan nuclear receptor Nur77/N10 has recently been demonstrated to be involved in apoptosis of T cell hybridomas. We report here that chronic expression of Nur77/N10 in thymocytes of transgenic mice results in a dramatic reduction of CD4+CD8+ double-positive as well as CD4+CD8- and CD4-CD8+ single-positive cell populations due to an early onset of apoptosis. CD4-CD8- double-negative and CD25+ precursor cells, however, are unaffected. Moreover, nur77/N10-transgenic thymocytes show increased expression of Fas ligand (FasL), while the levels of the Fas receptor (Fas) are not increased. The mouse spontaneous mutant gld (generalized lymphoproliferative disease) carries a point mutation in the extracellular domain of the FasL gene that abolishes the ability of FasL to bind to Fas. Thymuses from nur77/N10-transgenic mice on a gld/gld background have increased cellularity and an almost normal profile of thymocyte subpopulations. Our results demonstrate that one pathway of apoptosis triggered by Nur77/N10 in double-positive thymocytes occurs through the upregulation of FasL expression resulting in increased signaling through Fas.

Inhibitors of the proteasome pathway interfere with induction of nitric oxide synthase in macrophages by blocking activation of transcription factor NF-kappa B.

The objective of this study was to elucidate the role of the proteasome pathway or multicatalytic proteinase complex in the induction of immunologic nitric oxide (NO) synthase (iNOS) in rat alveolar macrophages activated by lipopolysaccharide. Macrophages were incubated in the presence of lipopolysaccharide plus test agent for up to 24 hr. Culture media were analyzed for accumulation of stable oxidation products of NO (NO2- + N03-, designated as NOX-), cellular RNA was extracted for determination of iNOS mRNA levels by Northern blot analysis, and nuclear extracts were prepared for determination of NF-kappa B by electrophoretic mobility-shift assay. Inhibitors of calpain (alpha-N-acetyl-Leu-Leu-norleucinal; N-benzyloxycarbonyl-Leu-leucinal) and the proteasome (N-benzyloxycarbonyl-Ile-Glu-(O-t-Bu)-Ala-leucinal) markedly inhibited or abolished the induction of iNOS in macrophages. The proteinase inhibitors interfered with lipopolysaccharide-induced NOX- production by macrophages, and this effect was accompanied by comparable interference with the appearance of both iNOS mRNA and NF-kappa B. Calpain inhibitors elicited effects at concentrations of 1-100 microM, whereas the proteasome inhibitor was 1000-fold more potent, producing significant inhibitory effects at 1 nM. The present findings indicate that the proteasome pathway is essential for lipopolysaccharide-induced expression of the iNOS gene in rat alveolar macrophages. Furthermore, the data support the view that the proteasome pathway is directly involved in promoting the activation of NF-kappa B and that the induction of iNOS by lipopolysaccharide involves the transcriptional action of NF-kappaB.

Characterization of a second secreted IgE isoform and identification of an asymmetric pathway of IgE assembly.

A number of alternatively spliced epsilon transcripts have been detected in IgE-producing B cells, in addition to the mRNAs encoding the classical membrane and secreted IgE heavy (H) chains. In a recent study, we examined the protein products of three of these alternatively spliced isoforms and found that they are intracellularly retained and degraded because of their inability to assemble into complete IgE molecules. We have now similarly examined a more recently described epsilon mRNA species that is generated by splicing between a donor splice site immediately upstream of the stop codon in the H-chain constant region exon 4 (CH4) and an acceptor site located in the 3' part of the second membrane exon. We show that this isoform is efficiently secreted by both plasma cells and B lymphocytes and therefore represents a second secreted IgE isoform (epsilon S2). The epsilon S2 H chain is only six amino acids longer than the classical secreted Ig H chain (epsilon S1) and contains a C-terminal cysteine, which is a characteristic sequence feature of mu and alpha H chains. However, unlike IgM and IgA, the epsilon S2 C-terminal cysteine (Cys-554) does not induce polymerization of H2L2 molecules (where L is light chain), but rather creates a disulfide bond between the two H chains that increases the rate of association into covalently bound H2L2 monomers. This C-terminal cysteine also does not function as an intracellular retention element because the epsilon S2 isoform was secreted in amounts equal to that of the epsilon S1, both in B lymphocytes and in plasma cells. The epsilon S2 H chains secreted by B lymphocytes differed from the epsilon S1 H chains in the extent of glycosylation. Interestingly, a difference in glycosylation between B-lymphocytes and plasma cells was also noted for both isoforms. The presence of the Cys-554 also allowed the identification of a distinctive asymmetric pathway of IgE assembly, common to both types of epsilon H chains.

Nuclear/cytoplasmic localization of the von Hippel-Lindau tumor suppressor gene product is determined by cell density.

The product of the von Hippel-Lindau (VHL) tumor suppressor gene, the gene inactivated in VHL disease and in sporadic clear-cell renal carcinomas, has recently been shown to have as a functional target the transcription elongation complex, elongin (also called SIII). Here it is shown that there is a tightly regulated, cell-density-dependent transport of VHL into and/or out of the nucleus. In densely grown cells, the VHL protein is predominantly in the cytoplasm, whereas in sparse cultures, most of the protein can be detected in the nucleus. We have identified a putative nuclear localization signal in the first 60 and first 28 amino acids of the human and rat VHL protein, respectively. Sequences in the C-terminal region of the VHL protein may also be required for localization to the cytosol. These findings provide the initial indication of a novel cell density-dependent pathway that is responsible for the regulation of VHL cellular localization.

Stimulation-dependent I kappa B alpha phosphorylation marks the NF-kappa B inhibitor for degradation via the ubiquitin-proteasome pathway.

The nuclear translocation of NF-kappa B follows the degradation of its inhibitor, I kappa B alpha, an event coupled with stimulation-dependent inhibitor phosphorylation. Prevention of the stimulation-dependent phosphorylation of I kappa B alpha, either by treating cells with various reagents or by mutagenesis of certain putative I kappa B alpha phosphorylation sites, abolishes the inducible degradation of I kappa B alpha. Yet, the mechanism coupling the stimulation-induced phosphorylation with the degradation has not been resolved. Recent reports suggest a role for the proteasome in I kappa B alpha degradation, but the mode of substrate recognition and the involvement of ubiquitin conjugation as a targeting signal have not been addressed. We show that of the two forms of I kappa B alpha recovered from stimulated cells in a complex with RelA and p50, only the newly phosphorylated form, pI kappa B alpha, is a substrate for an in vitro reconstituted ubiquitin-proteasome system. Proteolysis requires ATP, ubiquitin, a specific ubiquitin-conjugating enzyme, and other ubiquitin-proteasome components. In vivo, inducible I kappa B alpha degradation requires a functional ubiquitin-activating enzyme and is associated with the appearance of high molecular weight adducts of I kappa B alpha. Ubiquitin-mediated protein degradation may, therefore, constitute an integral step of a signal transduction process.

Recruitment of hepatocyte nuclear factor 4 into specific intranuclear compartments depends on tyrosine phosphorylation that affects its DNA-binding and transactivation potential.

Hepatocyte nuclear factor 4 (HNF-4) is a prominent member of the family of liver-enriched transcription factors, playing a role in the expression of a large number of liver-specific genes. We report here that HNF-4 is a phosphoprotein and that phosphorylation at tyrosine residue(s) is important for its DNA-binding activity and, consequently, for its transactivation potential both in cell-free systems and in cultured cells. Tyrosine phosphorylation did not affect the transport of HNF-4 from the cytoplasm to the nucleus but had a dramatic effect on its subnuclear localization. HNF-4 was concentrated in distinct nuclear compartments, as evidenced by in situ immunofluorescence and electron microscopy. This compartmentalization disappeared when tyrosine phosphorylation was inhibited by genistein. The correlation between the intranuclear distribution of HNF-4 and its ability to activate endogenous target genes demonstrates a phosphorylation signal-dependent pathway in the regulation of transcription factor activity.

Mammalian karyopherin alpha 1 beta and alpha 2 beta heterodimers: alpha 1 or alpha 2 subunit binds nuclear localization signal and beta subunit interacts with peptide repeat-containing nucleoporins.

Although only 44% identical to human karyopherin alpha 1, human karyopherin alpha 2 (Rch1 protein) substituted for human karyopherin alpha 1 (hSRP-1/NPI-1) in recognizing a standard nuclear localization sequence and karyopherin beta-dependent targeting to the nuclear envelope of digitonin-permeabilized cells. By immunofluorescence microscopy of methanol-fixed cells, karyopherin beta was localized to the cytoplasm and the nuclear envelope and was absent from the nuclear interior. Digitonin permeabilization of buffalo rat liver cells depleted their endogenous karyopherin beta. Recombinant karyopherin beta can bind directly to the nuclear envelope of digitonin-permeabilized cells at 0 degree C (docking reaction). In contrast, recombinant karyopherin alpha 1 or alpha 2 did not bind unless karyopherin beta was present. Likewise, in an import reaction (at 20 degrees C) with all recombinant transport factors (karyopherin alpha 1 or alpha 2, karyopherin beta, Ran, and p10) import depended on karyopherin beta. Localization of the exogenously added transport factors after a 30-min import reaction showed karyopherin beta at the nuclear envelope and karyopherin alpha 1 or alpha 2, Ran, and p10 in the nuclear interior. In an overlay assay with SDS/PAGE-resolved and nitrocellulose-transferred proteins of the nuclear envelope, 35S-labeled karyopherin beta bound to at least four peptide repeat-containing nucleoporins--Nup358, Nup214, Nup153, and Nup98.

Transposon tagging of tobacco mosaic virus resistance gene N: its possible role in the TMV-N-mediated signal transduction pathway.

Plants can recognize and resist invading pathogens by signaling the induction of rapid defense responses. Often these responses are mediated by single dominant resistance genes (R genes). The products of R genes have been postulated to recognize the pathogen and trigger rapid host defense responses. Here we describe isolation of the classical resistance gene N of tobacco that mediates resistance to the well-characterized pathogen tobacco mosaic virus (TMV). The N gene was isolated by transposon tagging using the maize Activator (Ac) transposon. We confirmed isolation of the N gene by complementation of the TMV-sensitive phenotype with a genomic DNA fragment. Sequence analysis of the N gene shows that it encodes a protein with an amino-terminal domain similar to that of the cytoplasmic domains of the Drosophila Toll protein and the interleukin 1 receptor in mammals, a putative nucleotide-binding site and 14 imperfect leucine-rich repeats. The presence of these functional domains in the predicted N gene product is consistent with the hypothesis that the N resistance gene functions in a signal transduction pathway. Similarities of N to Toll and the interleukin 1 receptor suggest a similar signaling mechanism leading to rapid gene induction and TMV resistance.

Anterior axis duplication in Xenopus induced by the over-expression of the cadherin-binding protein plakoglobin.

Plakoglobin interacts with both classical and desmosomal cadherins. It is closely related to Drosophila aramadillo (arm) gene product; arm acts in the wingless (wg)-signaling pathway to establish segment polarity. In Xenopus, homologs of wg--i.e., wnts, can produce anterior axis duplications by inducing dorsal mesoderm. Studies in Drosophila suggest that wnt acts by increasing the level of cytoplasmic armadillo protein (arm). To test whether simply increasing the level of plakoglobin mimics the effects of exogenous wnts in Xenopus, we injected fertilized eggs with RNA encoding an epitope-tagged form of plakoglobin; this induced both early radial gastrulation and anterior axis duplication. Exogenous plakoglobin accumulates in the nuclei of embryonic cells. Plakoglobin binds to the tail domain of the desmosomal cadherin desmoglein 1. When RNA encoding the tail domain of desmoglein was coinjected with plakoglobin RNA, both the dorsalizing effect and nuclear accumulation of plakoglobin were suppressed. Mutational analysis indicates that the central arm repeat region of plakoglobin is sufficient to induce axis duplication and that this polypeptide accumulates in the nuclei of embryonic cells. These data show that increased plakoglobin levels can, by themselves, generate the intracellular signals involved in the specification of dorsal mesoderm.

The nuclear safety framework in the European Union after Fukushima. Egmont Paper No. 73, December 2014

Summary. On 11 March 2011, a devastating earthquake struck Japan and caused a major nuclear accident at the Fukushima Daiichi nuclear plant. The disaster confirmed that nuclear reactors must be protected even against accidents that have been assessed as highly unlikely. It also revealed a well-known catalogue of problems: faulty design, insufficient back-up systems, human error, inadequate contingency plans, and poor communications. The catastrophe triggered the rapid launch of a major re-examination of nuclear reactor security in Europe. It also stopped in its tracks what had appeared to be a ‘nuclear renaissance’, both in Europe and globally, especially in the emerging countries. Under the accumulated pressure of rising demand and climate warming, many new nuclear projects had been proposed. Since 2011 there has been more ambivalence, especially in Europe. Some Member States have even decided to abandon the nuclear sector altogether. This Egmont Paper aims to examine the reactions of the EU regarding nuclear safety since 2011. Firstly, a general description of the nuclear sector in Europe is provided. The nuclear production of electricity currently employs around 500,000 people, including those working in the supply chain. It generates approximately €70 billion per year. It provides roughly 30% of the electricity consumed in the EU. At the end of 2013, there were 131 nuclear power reactors active in the EU, located in 14 countries. Four new reactors are under construction in France, Slovakia and Finland. Secondly, this paper will present the Euratom legal framework regarding nuclear safety. The European Atomic Energy Community (EAEC or Euratom) Treaty was signed in 1957, and somewhat obscured by the European Economic Community (EEC) Treaty. It was a more classical treaty, establishing institutions with limited powers. Its development remained relatively modest until the Chernobyl catastrophe, which provoked many initiatives. The most important was the final adoption of the Nuclear Safety Directive 2009/71. Thirdly, the general symbiosis between Euratom and the International Atomic Energy Agency (IAEA) will be explained. Fourthly, the paper analyses the initiatives taken by the EU in the wake of the Fukushima catastrophe. These initiatives are centred around the famous ‘stress tests’. Fifthly, the most important legal change brought about by this event was the revision of Directive 2009/71. Directive 2014/87 has been adopted quite rapidly, and has deepened in various ways the role of the EU in nuclear safety. It has reinforced the role and effective independence of the national regulatory authorities. It has enhanced transparency on nuclear safety matters. It has strengthened principles, and introduced new general nuclear safety objectives and requirements, addressing specific technical issues across the entire life cycle of nuclear installations, and in particular, nuclear power plants. It has extended monitoring and the exchange of experiences by establishing a European system of peer reviews. Finally, it has established a mechanism for developing EU-wide harmonized nuclear safety guidelines. In spite of these various improvements, Directive 2014/87 Euratom still reflects the ambiguity of the Euratom system in general, and especially in the field of nuclear safety. The use of nuclear energy remains controversial among Member States. Some of them remain adamantly in favour, others against or ambivalent. The intervention of the EAEC institutions remains sensitive. The use of the traditional Community method remains limited. The peer review method remains a very peculiar mechanism that deserves more attention.

APPL proteins link Rab5 to nuclear signal transduction via an endosomal compartment

Signals generated in response to extracellular stimuli at the plasma membrane are transmitted through cytoplasmic transduction cascades to the nucleus. We report the identification of a pathway directly linking the small GTPase Rab5, a key regulator of endocytosis, to signal transduction and mitogenesis. This pathway operates via APPL1 and APPL2, two Rab5 effectors, which reside on a subpopulation of endosomes. In response to extracellular stimuli such as EGF and oxidative stress, APPL1 translocates from the membranes to the nucleus where it interacts with the nucleosome remodeling and histone deacetylase multiprotein complex NuRD/MeCP1, an established regulator of chromatin structure and gene expression. Both APPL1 and APPL2 are essential for cell proliferation and their function requires Rab5 binding. Our findings identify an endosomal compartment bearing Rab5 and APPL proteins as an intermediate in signaling between the plasma membrane and the nucleus.

Regulation of endocytosis, nuclear translocation, and signaling of fibroblast growth factor receptor 1 by E-cadherin

Fibroblast growth factor (FGF) receptors (FGFRs) signal to modulate diverse cellular functions, including epithelial cell morphogenesis. In epithelial cells, E-cadherin plays a key role in cell-cell adhesion, and its function can be regulated through endocytic trafficking. In this study, we investigated the location, trafficking, and function of FGFR1 and E-cadherin and report a novel mechanism, based on endocytic trafficking, for the coregulation of E-cadherin and signaling from FGFR1. FGF induces the internalization of surface FGFR1 and surface E-cadherin, followed by nuclear translocation of FGFR1. The internalization of both proteins is regulated by common endocytic machinery, resulting in cointernalization of FGFR1 and E-cadherin into early endosomes. By blocking endocytosis, we show that this is a requisite, initial step for the nuclear translocation of FGFR1. Overexpression of E-cadherin blocks both the coendocytosis of E-cadherin and FGFR1, the nuclear translocation of FGFR1 and FGF-induced signaling to the mitogen-activated protein kinase pathway. Furthermore, stabilization of surface adhesive E-cadherin, by overexpressing p120(ctn), also blocks internalization and nuclear translocation of FGFR1. These data reveal that conjoint endocytosis and trafficking is a novel mechanism for the coregulation of E-cadherin and FGFR1 during cell signaling and morphogenesis.