Cloning of a gene (RIG-G) associated with retinoic acid-induced differentiation of acute promyelocytic leukemia cells and representing a new member of a family of interferon-stimulated genes

In a cell line (NB4) derived from a patient with acute promyelocytic leukemia, all-trans-retinoic acid (ATRA) and interferon (IFN) induce the expression of a novel gene we call RIG-G (for retinoic acid-induced gene G). This gene codes for a 58-kDa protein containing 490 amino acids with several potential sites for post-translational modification. In untreated NB4 cells, the expression of RIG-G is undetectable. ATRA treatment induces the transcriptional expression of RIG-G relatively late (12–24 hr) in a protein synthesis-dependent manner, whereas IFN-α induces its expression early (30 min to 3 hr). Database search has revealed a high-level homology between RIG-G and several IFN-stimulated genes in human (ISG54K, ISG56K, and IFN-inducible and retinoic acid-inducible 58K gene) and some other species, defining a well conserved gene family. The gene is composed of two exons and has been mapped by fluorescence in situ hybridization to chromosome 10q24, where two other human IFN-stimulated gene members are localized. A synergistic induction of RIG-G expression in NB4 cells by combined treatment with ATRA and IFNs suggests that a collaboration exists between their respective signaling pathways.

Nuclear translocation of NF-κB is increased in dopaminergic neurons of patients with Parkinson disease

Evidence from postmortem studies suggest an involvement of oxidative stress in the degeneration of dopaminergic neurons in Parkinson disease (PD) that have recently been shown to die by apoptosis, but the relationship between oxidative stress and apoptosis has not yet been elucidated. Activation of the transcription factor NF-κB is associated with oxidative stress-induced apoptosis in several nonneuronal in vitro models. To investigate whether it may play a role in PD, we looked for the translocation of NF-κB from the cytoplasm to the nucleus, evidence of its activation, in melanized neurons in the mesencephalon of postmortem human brain from five patients with idiopathic PD and seven matched control subjects. In PD patients, the proportion of dopaminergic neurons with immunoreactive NF-κB in their nuclei was more than 70-fold that in control subjects. A possible relationship between the nuclear localization of NF-κB in mesencephalic neurons of PD patients and oxidative stress in such neurons has been shown in vitro with primary cultures of rat mesencephalon, where translocation of NF-κB is preceded by a transient production of free radicals during apoptosis induced by activation of the sphingomyelin-dependent signaling pathway with C2-ceramide. The data suggest that this oxidant-mediated apoptogenic transduction pathway may play a role in the mechanism of neuronal death in PD.

Deregulated expression of c-Myc in a translocation-negative plasmacytoma on extrachromosomal elements that carry IgH and myc genes

The induced expression of c-Myc in plasmacytomas in BALB/c mice is regularly associated with nonrandom chromosomal translocations that juxtapose the c-myc gene to one of the Ig loci on chromosome 12 (IgH), 6 (IgK), or 16 (IgL). The DCPC21 plasmacytoma belongs to a small group of plasmacytomas that are unusual in that they appear to be translocation-negative. In this paper, we show the absence of any c-myc-activating chromosomal translocation for the DCPC21 by using fluorescent in situ hybridization, chromosome painting, and spectral karyotyping. We find that DCPC21 harbors c-myc and IgH genes on extrachromosomal elements (EEs) from which c-myc is transcribed, as shown by c-myc mRNA tracks and extrachromosomal gene transfer experiments. The transcriptional activity of these EEs is supported further by the presence of the transcription-associated phosphorylation of histone H3 (H3P) on the EEs. Thus, our data suggest that in this plasmacytoma, c-Myc expression is achieved by an alternative mechanism. The expression of the c-Myc oncoprotein is initiated outside the chromosomal locations of the c-myc gene, i.e., from EEs, which can be considered functional genetic units. Our data also imply that other “translocation-negative” experimental and human tumors with fusion transcripts or oncogenic activation may indeed carry translocation(s), however, in an extrachromosomal form.

Withdrawal of IL-7 induces Bax translocation from cytosol to mitochondria through a rise in intracellular pH

IL-7 functions as a trophic factor during T lymphocyte development by a mechanism that is partly based on the induction of Bcl-2, which protects cells from apoptosis. Here we report a mechanism by which cytokine withdrawal activates the prodeath protein Bax. On loss of IL-7 in a dependent cell line, Bax protein translocated from the cytosol to the mitochondria, where it integrated into the mitochondrial membrane. This translocation was attributable to a conformational change in the Bax protein itself. We show that a rise in intracellular pH preceded mitochondrial translocation and triggered the change in Bax conformation. Intracellular pH in the IL-7-dependent cells rose steadily to peak over pH 7.8 by 6 hr after cytokine withdrawal, paralleling the time point of Bax translocation (a similar alkalinization and Bax translocation was also observed after IL-3 withdrawal from a dependent cell line). The conformation of Bax was directly altered by pH of 7.8 or higher and was demonstrated by increased protease sensitivity, exposure of N terminus epitopes, and exposure of a hydrophobic domain in the C terminus. Eliminating charged amino acids at the C or N termini of Bax induced a conformational change similar to that induced by raising pH, implicating these residues in the pH effect. Therefore, we have shown that by either cytokine withdrawal, experimental manipulation of pH, or site-directed mutagenesis, Bax protein changes conformation, exposing membrane-seeking domains, thereby inducing mitochondrial translocation and initiating the cascade of events leading to apoptotic death.

Metabotropic glutamate receptor-initiated translocation of protein kinase p90rsk to polyribosomes: A possible factor regulating synaptic protein synthesis

Maintenance of lasting synaptic efficacy changes requires protein synthesis. We report here a mechanism that might influence translation control at the level of the single synapse. Stimulation of metabotropic glutamate receptors in hippocampal slices induces a rapid protein kinase C-dependent translocation of multifunction kinase p90rsk to polyribosomes; concomitantly, there is enhanced phosphorylation of at least six polyribosome binding proteins. Among the polyribosome bound proteins are the p90rsk-activating kinase ERK-2 and a known p90rsk substrate, glycogen synthase kinase 3β, which regulates translation efficiency via eukaryotic initiation factor 2B. Thus metabotropic glutamate receptor stimulation could induce synaptic activity-dependent translation via translocation of p90rsk to ribosomes.

The t(8;21) chromosomal translocation in acute myelogenous leukemia modifies intranuclear targeting of the AML1/CBFα2 transcription factor

Targeting of gene regulatory factors to specific intranuclear sites may be critical for the accurate control of gene expression. The acute myelogenous leukemia 8;21 (AML1/ETO) fusion protein is encoded by a rearranged gene created by the ETO chromosomal translocation. This protein lacks the nuclear matrix-targeting signal that directs the AML1 protein to appropriate gene regulatory sites within the nucleus. Here we report that substitution of the chromosome 8-derived ETO protein for the multifunctional C terminus of AML1 precludes targeting of the factor to AML1 subnuclear domains. Instead, the AML1/ETO fusion protein is redirected by the ETO component to alternate nuclear matrix-associated foci. Our results link the ETO chromosomal translocation in AML with modifications in the intranuclear trafficking of the key hematopoietic regulatory factor, AML1. We conclude that misrouting of gene regulatory factors as a consequence of chromosomal translocations is an important characteristic of acute leukemias.

Roles of SecG in ATP- and SecA-dependent protein translocation

SecA, the translocation ATPase in Escherichia coli, undergoes cycles of conformational changes (insertion/deinsertion) in response to ATP and a preprotein. The membrane-embedded portion of protein translocase, SecYEG, has crucial roles in the SecA-driven preprotein translocation reaction. We previously identified a secY mutation (secY205) that did not allow an ATP- and preprotein-dependent (productive) insertion of SecA as well as secA mutations that suppressed the secY205 translocation defect. One of the suppressor mutations, secA36, also suppressed the cold-sensitive phenotype of the secG deletion mutant. In vitro experiments at 20°C showed that inverted membrane vesicles lacking SecG were almost inactive in combination with the wild-type SecA protein in translocation of proOmpA as well as in the accompanying ATP hydrolysis. In contrast, the SecA36 mutant protein was found to be able to execute the translocation activity fully at this temperature, even in the absence of SecG. A SecG requirement and its alleviation by the SecA36 alteration also were shown for the SecA insertion reaction. The finding that the SecA36 protein no longer requires assistance from SecG in its insertion and in its catalysis of protein translocation agrees with the idea that SecG normally assists in the functioning of SecA. In agreement with this notion, when the intrinsic SecA function was compromised by a lowered ATP concentration, SecG became essential even at 37°C and even for the SecA36 protein. We propose that in the normal translocase, SecG cooperates with SecA to facilitate efficient movement of preprotein in each catalytic cycle of SecA.

ExoY, an adenylate cyclase secreted by the Pseudomonas aeruginosa type III system

The exoenzyme S regulon is a set of coordinately regulated virulence genes of Pseudomonas aeruginosa. Proteins encoded by the regulon include a type III secretion and translocation apparatus, regulators of gene expression, and effector proteins. The effector proteins include two enzymes with ADP-ribosyltransferase activity (ExoS and ExoT) and an acute cytotoxin (ExoU). In this study, we identified ExoY as a fourth effector protein of the regulon. ExoY is homologous to the extracellular adenylate cyclases of Bordetella pertussis (CyaA) and Bacillus anthracis (EF). The homology among the three adenylate cyclases is limited to two short regions, one of which possesses an ATP-binding motif. In assays for adenylate cyclase activity, recombinant ExoY (rExoY) catalyzed the formation of cAMP with a specific activity similar to the basal activity of CyaA. In contrast to CyaA and EF, rExoY activity was not stimulated or activated by calmodulin. A 500-fold stimulation of activity was detected following the addition of a cytosolic extract from Chinese hamster ovary (CHO) cells. These results indicate that a eukaryotic factor, distinct from calmodulin, enhances rExoY catalysis. Site-directed mutagenesis of residues within the putative active site of ExoY abolished adenylate cyclase activity. Infection of CHO cells with ExoY-producing strains of P. aeruginosa resulted in the intracellular accumulation of cAMP. cAMP accumulation within CHO cells depended on an intact type III translocation apparatus, demonstrating that ExoY is directly translocated into the eukaryotic cytosol.

Yersinia enterocolitica induces apoptosis in macrophages by a process requiring functional type III secretion and translocation mechanisms and involving YopP, presumably acting as an effector protein

Yersiniae, causative agents of plague and gastrointestinal diseases, secrete and translocate Yop effector proteins into the cytosol of macrophages, leading to disruption of host defense mechanisms. It is shown in this report that Yersinia enterocolitica induces apoptosis in macrophages and that this effect depends on YopP. Functional secretion and translocation mechanisms are required for YopP to act, strongly suggesting that this protein exerts its effect intracellularly, after translocation into the macrophages. YopP shows a high level of sequence similarity with AvrRxv, an avirulence protein from Xanthomonas campestris, a plant pathogen that induces programmed cell death in plant cells. This indicates possible similarities between the strategies used by pathogenic bacteria to elicit programmed cell death in both plant and animal hosts.

Detection of Transient In Vivo Interactions between Substrate and Transporter during Protein Translocation into the Endoplasmic Reticulum

The split-ubiquitin technique was used to detect transient protein interactions in living cells. Nub, the N-terminal half of ubiquitin (Ub), was fused to Sec62p, a component of the protein translocation machinery in the endoplasmic reticulum of Saccharomyces cerevisiae. Cub, the C-terminal half of Ub, was fused to the C terminus of a signal sequence. The reconstitution of a quasi-native Ub structure from the two halves of Ub, and the resulting cleavage by Ub-specific proteases at the C terminus of Cub, serve as a gauge of proximity between the two test proteins linked to Nub and Cub. Using this assay, we show that Sec62p is spatially close to the signal sequence of the prepro-α-factor in vivo. This proximity is confined to the nascent polypeptide chain immediately following the signal sequence. In addition, the extent of proximity depends on the nature of the signal sequence. Cub fusions that bore the signal sequence of invertase resulted in a much lower Ub reconstitution with Nub-Sec62p than otherwise identical test proteins bearing the signal sequence of prepro-α-factor. An inactive derivative of Sec62p failed to interact with signal sequences in this assay. These in vivo findings are consistent with Sec62p being part of a signal sequence-binding complex.

Involvement of ATP-dependent Pseudomonas Exotoxin Translocation from a Late Recycling Compartment in Lymphocyte Intoxication Procedure

Pseudomonas exotoxin (PE) is a cytotoxin which, after endocytosis, is delivered to the cytosol where it inactivates protein synthesis. Using diaminobenzidine cytochemistry, we found over 94% of internalized PE in transferrin (Tf) -positive endosomes of lymphocytes. When PE translocation was examined in a cell-free assay using purified endocytic vesicles, more than 40% of endosomal 125I-labeled PE was transported after 2 h at 37°C, whereas a toxin inactivated by point mutation in its translocation domain was not translocated. Sorting of endosomes did not allow cell-free PE translocation, whereas active PE transmembrane transport was observed after > 10 min of endocytosis when PE and fluorescent-Tf were localized by confocal immunofluorescence microscopy within a rab5-positive and rab4- and rab7-negative recycling compartment in the pericentriolar region of the cell. Accordingly, when PE delivery to this structure was inhibited using a 20°C endocytosis temperature, subsequent translocation from purified endosomes was impaired. Translocation was also inhibited when endosomes were obtained from cells labeled with PE in the presence of brefeldin A, which caused fusion of translocation-competent recycling endosomes with translocation-incompetent sorting elements. No PE processing was observed in lymphocyte endosomes, the full-sized toxin was translocated and recovered in an enzymatically active form. ATP hydrolysis was found to directly provide the energy required for PE translocation. Inhibitors of endosome acidification (weak bases, protonophores, or bafilomycin A1) when added to the assay did not significantly affect 125I-labeled PE translocation, demonstrating that this transport is independent of the endosome-cytosol pH gradient. Nevertheless, when 125I-labeled PE endocytosis was performed in the presence of one of these molecules, translocation from endosomes was strongly inhibited, indicating that exposure to acidic pH is a prerequisite for PE membrane traversal. When applied during endocytosis, treatments that protect cells against PE intoxication (low temperatures, inhibitors of endosome acidification, and brefeldin A) impaired 125I-labeled PE translocation from purified endosomes. We conclude that PE translocation from a late receptor recycling compartment is implicated in the lymphocyte intoxication procedure.

Transduction of Basolateral-to-Apical Signals across Epithelial Cells: Ligand-stimulated Transcytosis of the Polymeric Immunoglobulin Receptor Requires Two Signals

Transcytosis of the polymeric immunoglobulin receptor (pIgR) is stimulated by binding of its ligand, dimeric IgA (dIgA). During this process, dIgA binding at the basolateral surface of the epithelial cell transmits a signal to the apical region of the cell, which in turn stimulates the transport of dIgA–pIgR complex from a postmicrotubule compartment to the apical surface. We have previously reported that the signal of stimulation was controlled by a protein-tyrosine kinase (PTK) activated upon dIgA binding. We now show that this signal of stimulation moves across the cell independently of pIgR movement or microtubules and acts through the tyrosine kinase activity by releasing Ca++ from inositol trisphosphate–sensitive intracellular stores. Surprisingly we have found that a second independent signal is required to achieve dIgA-stimulated transcytosis of pIgR. This second signal depends on dIgA binding to the pIgR solely at the basolateral surface and the ability of pIgR to dimerize. This enables pIgR molecules that have bound dIgA at the basolateral surface to respond to the signal of stimulation once they reach the postmicrotubule compartment. We propose that the use of two signals may be a general mechanism by which signaling receptors maintain specificity along their signaling and trafficking pathways.

Folding of Active β-Lactamase in the Yeast Cytoplasm before Translocation into the Endoplasmic Reticulum

Polypeptides targeted to the yeast endoplasmic reticulum (ER) posttranslationally are thought to be kept in the cytoplasm in an unfolded state by Hsp70 chaperones before translocation. We show here that Escherichia coli β-lactamase associated with Hsp70, but adopted a native-like conformation before translocation in living Saccharomyces cerevisiae cells. β-Lactamase is a globular trypsin-resistant molecule in authentic form. For these studies, it was linked to the C terminus of a yeast polypeptide Hsp150Δ, which conferred posttranslational translocation and provided sites for O-glycosylation. We devised conditions to retard translocation of Hsp150Δ-β-lactamase. This enabled us to show by protease protection assays that an unglycosylated precursor was associated with the cytoplasmic surface of isolated microsomes, whereas a glycosylated form resided inside the vesicles. Both proteins were trypsin resistant and had similar β-lactamase activity and Km values for nitrocefin. The enzymatically active cytoplasmic intermediate could be chased into the ER, followed by secretion of the activity to the medium. Productive folding in the cytoplasm occurred in the absence of disulfide formation, whereas in the ER lumen, proper folding required oxidation of the sulfhydryls. This suggests that the polypeptide was refolded in the ER and consequently, at least partially unfolded for translocation.

The Formation of an Insulin-responsive Vesicular Cargo Compartment Is an Early Event in 3T3-L1 Adipocyte Differentiation

Differentiating 3T3-L1 cells exhibit a dramatic increase in the rate of insulin-stimulated glucose transport during their conversion from proliferating fibroblasts to nonproliferating adipocytes. On day 3 of 3T3-L1 cell differentiation, basal glucose transport and cell surface transferrin binding are markedly diminished. This occurs concomitant with the formation of a distinct insulin-responsive vesicular pool of intracellular glucose transporter 1 (GLUT1) and transferrin receptors as assessed by sucrose velocity gradients. The intracellular distribution of the insulin-responsive aminopeptidase is first readily detectable on day 3, and its gradient profile and response to insulin at this time are identical to that of GLUT1. With further time of differentiation, GLUT4 is expressed and targeted to the same insulin-responsive vesicles as the other three proteins. Our data are consistent with the notion that a distinct insulin-sensitive vesicular cargo compartment forms early during fat call differentiation and its formation precedes GLUT4 expression. The development of this compartment may result from the differentiation-dependent inhibition of constitutive GLUT1 and transferrin receptor trafficking such that there is a large increase in, or the new formation of, a population of postendosomal, insulin-responsive vesicles.

Role of Tyrosine Phosphorylation in Ligand-induced Regulation of Transcytosis of the Polymeric Ig Receptor

The polymeric Ig receptor (pIgR) transcytoses its ligand, dimeric IgA (dIgA), from the basolateral to the apical surface of epithelial cells. Although the pIgR is constitutively transcytosed in the absence of ligand, binding of dIgA stimulates transcytosis of the pIgR. We recently reported that dIgA binding to the pIgR induces translocation of protein kinase C, production of inositol triphosphate, and elevation of intracellular free calcium. We now report that dIgA binding causes rapid, transient tyrosine phosphorylation of several proteins, including phosphatidyl inositol-specific phospholipase C-γl. Protein tyrosine kinase inhibitors or deletion of the last 30 amino acids of pIgR cytoplasmic tail prevents IgA-stimulated protein tyrosine kinase activation, tyrosine phosphorylation of phospholipase C-γl, production of inositol triphosphate, and the stimulation of transcytosis by dIgA. Analysis of pIgR deletion mutants reveals that the same discrete portion of the cytoplasmic domain, residues 727–736 (but not the Tyr734), controls both the ability of pIgR to cause dIgA-induced tyrosine phosphorylation of the phospholipase C-γl and to undergo dIgA-stimulated transcytosis. In addition, dIgA transcytosis can be strongly stimulated by mimicking phospholipase C-γl activation. In combination with our previous results, we conclude that the protein tyrosine kinase(s) and phospholipase C-γl that are activated upon dIgA binding to the pIgR control dIgA-stimulated pIgR transcytosis.