Regulated expression of GRP78 during vasopressin-induced hypertrophy of heart-derived myocytes.

Although the development of cellular hypertrophy is widely believed to involve Ca(2+) signaling, potential supporting roles for sequestered Ca(2+) in this process have not been explored. H9c2 cardiomyocytes respond to arginine vasopressin with an initial mobilization of Ca(2+) stores and reduced rates of mRNA translation followed by repletion of Ca(2+) stores, up-regulation of translation beyond initial rates, and the development of hypertrophy. Rates of synthesis of the endoplasmic reticulum (ER) chaperones, GRP78 and GRP94, were found to increase preferentially at early times of vasopressin treatment. Total GRP78 content increased 2- to 3-fold within 8 h after which the chaperone was subject to post-translational modification. Preferential synthesis of GRP78 and the increase in chaperone content both occurred at pM vasopressin concentrations and were abolished at supraphysiologic Ca(2+) concentrations. Co-treatment with phorbol myristate acetate decreased vasopressin-dependent Ca(2+) mobilization and slowed appearance of new GRP78 molecules in response to the hormone, whereas 24 h pretreatment with phorbol ester prolonged vasopressin-dependent Ca(2+) mobilization and further increased rates of GRP78 synthesis in response to the hormone. Findings did not support a role for newly synthesized GRP78 in translational up-regulation by vasopressin. However up-regulation, which does not depend on Ca(2+) sequestration, appeared to expedite chaperone expression. This report provides the first evidence that a Ca(2+)-mobilizing hormone at physiologic concentrations signals increased expression of GRP78. Translational tolerance to depletion of ER Ca(2+) stores, typifying a robust ER stress response, did not accompany vasopressin-induced hypertrophy.

Vasopressin-induced hypertrophy in H9c2 heart-derived myocytes.

Protein synthesis in H9c2 heart-derived myocytes responds biphasically to arginine vasopressin (1 microM). An initial 50% inhibition attributable to Ca(2+) mobilization from the sarcoplasmic/endoplasmic reticulum is followed by a recovery that subsequently converts to a 1.5-fold stimulation. This study was undertaken to ascertain whether vasopressin programs H9c2 cells to undergo hypertrophy or to proliferate and whether early translational inhibition is required for programming. Translational suppression was observed only at vasopressin concentrations (>1 nM) causing extensive (>50%) depletion of Ca(2+) stores and was diminished at supraphysiologic extracellular Ca(2+) concentrations. Stimulation of protein synthesis, by contrast, was unaffected by changes in extracellular Ca(2+), depended on gene transcription, was suppressed by a protein kinase C pseudosubstrate sequence (peptide 19-27), and was observed at pM vasopressin concentrations. Activation of MAP kinases, phosphoinositide 3-kinase, calcineurin, S6 kinase, or eIF4 could not be implicated in the stimulation, which persisted for 24 h. Vasopressin-treated H9c2 cells underwent hypertrophy by standard criteria. Cellular protein accumulation occurred at pM hormone concentrations, was blocked by peptide 19-27, was observed regardless of retinoic acid pretreatment to prevent myogenic transdifferentiation, and preceded full repletion of Ca(2+) stores. It is proposed that H9c2 cells, which possess all basic features of V1-vasopressin receptor signaling, provide a convenient model for investigating vasopressin-induced myocyte hypertrophy. Early translational suppression is not needed for vasopressin-induced H9c2 myocyte hypertrophy whereas activation of protein kinase C appears essential.

Endoplasmic reticulum (ER)-associated degradation of misfolded N-linked glycoproteins is suppressed upon inhibition of ER mannosidase I.

To examine the role of early carbohydrate recognition/trimming reactions in targeting endoplasmic reticulum (ER)-retained, misfolded glycoproteins for ER-associated degradation (ERAD), we have stably expressed the cog thyroglobulin (Tg) mutant cDNA in Chinese hamster ovary cells. We found that inhibitors of ER mannosidase I (but not other glycosidases) acutely suppressed Cog Tg degradation and also perturbed the ERAD process for Tg reduced with dithiothreitol as well as for gamma-carboxylation-deficient protein C expressed in warfarin-treated baby hamster kidney cells. Kifunensine inhibition of ER mannosidase I also suppressed ERAD in castanospermine-treated cells; thus, suppression of ERAD does not require lectin-like binding of ER chaperones calnexin and calreticulin to monoglucosylated oligosaccharides. Notably, the undegraded protein fraction remained completely microsome-associated. In pulse-chase studies, kifunensine-sensitive degradation was still inhibitable even 1 h after Tg synthesis. Intriguingly, chronic treatment with kifunensine caused a 3-fold accumulation of Cog Tg in Chinese hamster ovary cells and did not lead to significant induction of the ER unfolded protein response. We hypothesize that, in a manner not requiring lectin-like activity of calnexin/calreticulin, the recognition or processing of a specific branched N-linked mannose structure enhances the efficiency of glycoprotein retrotranslocation from the ER lumen.

Longitudinal quantification of human immunodeficiency virus type 1 DNA and RNA in long-term nonprogressors.

Twenty patients with human immunodeficiency virus type 1 (HIV-1) infection for >7 years, no HIV-1-related symptoms, no treatment, and CD4+ cell counts >500/microL were included in a prospective study in 1993. Four years later, 12 patients had progressed (SPs), while 8 had not (long-term nonprogressors [LTNPs]). At inclusion, HIV-1 RNA, but not DNA, levels were higher in SPs. During follow-up, a consistent increase in HIV-1 RNA was seen in only 1 LTNP. In 2 LTNPs, plasma viremia was persistently undetectable or <110 copies/mL. Infectious virus was isolated from only 1 LTNP and from 11 SPs. In 4 LTNPs, HIV-1 DNA levels decreased spontaneously with time. The restricted viral replication and the declining HIV-1 DNA levels suggest that the HIV-1 infection can be controlled efficiently in a few LTNPs, leading to a decrease in the total virus burden with time.

The dynamic role of GRP78/BiP in the coordination of mRNA translation with protein processing.

The role of GRP78/BiP in coordinating endoplasmic reticular (ER) protein processing with mRNA translation was examined in GH3 pituitary cells. ADP-ribosylation of GRP78 and eukaryotic initiation factor (eIF)-2alpha phosphorylation were assessed, respectively, as indices of chaperone inactivation and the inhibition of translational initiation. Inhibition of protein processing by ER stress (ionomycin and dithiothreitol) resulted in GRP78 deribosylation and eIF-2 phosphorylation. Suppression of translation relative to ER protein processing (cycloheximide) produced approximately 50% ADP-ribosylation of GRP78 within 90 min without eIF-2 phosphorylation. ADP-ribosylation was reversed in 90 min by cycloheximide removal in a manner accelerated by ER stressors. Cycloheximide sharply reduced eIF-2 phosphorylation in response to ER stressors for about 30 min; sensitivity returned as GRP78 became increasingly ADP-ribosylated. Reduced sensitivity of eIF-2 to phosphorylation appeared to derive from the accumulation of free, unmodified chaperone as proteins completed processing without replacements. Prolonged (24 h) incubations with cycloheximide resulted in the selective loss of the ADP-ribosylated form of GRP78 and increased sensitivity of eIF-2 phosphorylation in response to ER stressors. Brefeldin A decreased ADP-ribosylation of GRP78 in parallel with increased eIF-2 phosphorylation. The cytoplasmic stressor, arsenite, which inhibits translational initiation through eIF-2 phosphorylation without affecting the ER, also produced ADP-ribosylation of GRP78.

Low relative frequencies of CD26(+) CD4(+) cells in long-term nonprogressing human immunodeficiency virus type 1-infected subjects.

A broad antibody panel was used for immunophenotyping of human immunodeficiency virus type 1 (HIV-1)-infected patients who were long-term nonprogressors (LTNP). The LTNP were compared with patients in the early phase of infection and patients who had progressed to advanced immunodeficiency. Changes in CD8(+) subset distribution were observed mainly at acquisition of HIV-1 infection, whereas CD4(+) subset changes appeared during progression of HIV-1 infection. The decreasing levels of CD4(+) cells were characterized by an increasing frequency of cells expressing the activation markers HLA-Dr and CD45RO but not the CD28 surface antigen. The LTNP exhibited significant changes compared to HIV-negative patients in almost all markers. Compared to patients in the early phase of infection, the only difference was a relatively lower frequency of CD4(+) cells expressing CD26 among the LTNP. The results show that HIV-1-infected persons who have no signs of immunodeficiency despite many years of infection have an immunophenotypic pattern that is substantially different from that of noninfected persons. Despite the long duration of infection, the LTNP exhibit a pattern similar to that of newly infected persons, with the exception of lower expression of CD26 on CD4(+) cells.

Human immunodeficiency virus type 1 disease progression, CCR5 genotype, and specific immune responses.

The correlation among the presence of a 32-bp deletion in the CC-chemokine receptor 5 (CCR5) gene, disease progression, and human immunodeficiency virus type 1 (HIV-1)-specific immune responses was analyzed for a cohort of 79 Caucasian HIV-1-infected patients. The CCR5 genotype (CCR5/CCR5 = wild type/wild type or delta32CCR5/CCR5 = 32-bp deletion/wild type) in peripheral blood mononuclear cells was determined by PCR, followed by sequencing of both wild-type and delta32CCR5 gene fragments. HIV-1-specific humoral responses to gp41 and V3MN peptides were determined by enzyme immunoassays. The prevalence of the delta32CCR5 allele was lower among 37 patients with rapid progression (progression to AIDS or to a CD4 cell count of <200 x 10(6)/liter in less than 9 years; P < 0.01) compared to that for 42 patients with slow progression (no AIDS and CD4 cell count of >200 x 10(6)/liter after at least 9 years from infection) or to that for 25 non-HIV-1-infected Swedish blood donors (P < 0.05). No differences were observed in the wild-type CCR5 sequences between the different groups of patients. For three analyzed patients, the 32-bp delta32CCR5 gene deletions were identical. The antibody titers against gp41 and a V3MN peptide in patients with the delta32CCR5/CCR5 genotype were not significantly different from those in pair-matched CCR5/CCR5 controls. However, in 13 analyzed patients, a stronger serum neutralizing activity was associated with the delta32CCR5/CCR5 genotype. Thus, a CCR5/CCR5 genotype correlates with a shortened AIDS-free HIV-1 infection period and possibly with a worse neutralizing activity, without an evident influence on the antibody response to two major antigenic regions of HIV-1 envelope.

An examination of the role of increased cytosolic free Ca2+ concentrations in the inhibition of mRNA translation.

Mobilization of Ca2+ sequestered by the endoplasmic reticulum (ER) produces the phosphorylation of initiation factor (eIF) 2, whereas an increase in cytosolic free Ca2+ ([Ca2+]i) due to plasmalemmal Ca2+ influx increases the phosphorylation of elongation factor (eEF) 2. In nucleated mammalian cells, depletion of ER Ca2+ stores has been demonstrated to inhibit translational initiation, but evidence that increased [Ca2+]i per se causes slowing of peptide chain elongation is lacking. L-type Ca2+ channel activity of GH3 pituitary cells, which are enriched in calmodulin-dependent eEF-2 kinase, was manipulated such that the impact of [Ca2+]i on eEF-2 phosphorylation and translational rate could be examined for up to 10 min without inhibiting initiation. At 1 mM extracellular Ca2+, resting [Ca2+]i values were high (154-255 nM) and eEF-2 was phosphorylated. The Ca2+ channel antagonist, nisoldipine, lowered [Ca2+]i and reduced eEF-2 phosphorylation by half but had no effect on amino acid incorporation. The Ca2+ channel agonist, Bay K 8644, produced sustained elevations of [Ca2+]i that were associated with 25-50% increases in eEF-2 phosphorylation, but no changes in protein synthetic rates occurred. Larger Ca2+ influxes were achievable with either 25 mM KCl or KCl plus Bay K 8644. These treatments further increased eEF-2 phosphorylation (50-100% above control) and inhibited leucine incorporation by 20-70% but ATP content was reduced by 25-50% and total cell-associated Ca2+ contents rose by 3- to 13-fold. eIF-2alpha was not phosphorylated during these treatments. Addition of low concentrations of ionomycin, which do not lower ATP content, was associated with complex changes in [Ca2+]i that resembled alterations in eEF-2 phosphorylation. The inhibition of leucine incorporation in response to ionomycin, however, coincided only with the phosphorylation of eIF-2alpha, not eEF-2. It is concluded that changes in [Ca2+]i occurring in the absence of ATP depletion alter the phosphorylation state of eEF-2 but are not regulatory for mRNA translation.

Regulation of protein synthesis in ventricular myocytes by vasopressin. The role of sarcoplasmic/endoplasmic reticulum Ca2+ stores.

Protein synthesis in H9c2 ventricular myocytes was subject to rapid inhibition by agents that release Ca2+ from the sarcoplasmic/endoplasmic reticulum, including thapsigargin, ionomycin, caffeine, and arginine vasopressin. Inhibitions were attributable to the suppression of translational initiation and were coupled to the mobilization of cell-associated Ca2+ and the phosphorylation of eIF2alpha. Ionomycin and thapsigargin produced relatively stringent degrees of Ca2+ mobilization that produced an endoplasmic reticulum (ER) stress response. Translational recovery was associated with the induction of ER chaperones and resistance to translational inhibition by Ca2+-mobilizing agents. Vasopressin at physiologic concentrations mobilized 60% of cell-associated Ca2+ and decreased protein synthesis by 50% within 20-30 min. The inhibition of protein synthesis was exerted through an interaction at the V1 vascular receptor, was imposed at physiologic extracellular Ca2+ concentrations, and became refractory to hormonal washout within 10 min of treatment. Inhibition was found to attenuate after 30 min, with full recovery occurring in 2 h. Translational recovery did not involve an ER stress response but rather was derived from the partial repletion of intracellular Ca2+ stores. Longer exposures to vasopressin were invariably accompanied by increased rates of protein synthesis. Translational inhibition by vasopressin, but not by Ca2+-mobilizing drugs, was both preventable and reversible by treatment with phorbol ester, which reduced the extent of Ca2+ mobilization occurring in response to the hormone. Larger and more prolonged translational inhibitions occurred after down-regulation of protein kinase C. This report provides the first compelling evidence that hormonally induced mobilization of sarcoplasmic/endoplasmic reticulum Ca2+ stores is regulatory upon mRNA translation.

Regulation of translational initiation during cellular responses to stress.

Chemicals and conditions that damage proteins, promote protein misfolding, or inhibit protein processing trigger the onset of protective homeostatic mechanisms resulting in "stress responses" in mammalian cells. Included in these responses are an acute inhibition of mRNA translation at the initiation step, a subsequent induction of various protein chaperones, and the recovery of mRNA translation. Separate, but closely related, stress response systems exist for the endoplasmic reticulum (ER), relating to the induction of specific "glucose-regulated proteins" (GRPs), and for the cytoplasm, pertaining to the induction of the "heat shock proteins" (HSPs). Activators of the ER stress response system, including Ca(2+)-mobilizing and thiol-reducing agents, are discussed and compared to activators of the cytoplasmic stress system, such as arsenite, heavy metal cations, and oxidants. An emerging integrative literature is reviewed that relates protein chaperones associated with cellular stress response systems to the coordinate regulation of translational initiation and protein processing. Background information is presented describing the roles of protein chaperones in the ER and cytoplasmic stress response systems and the relationships of chaperones and protein processing to the regulation of mRNA translation. The role of chaperones in regulating eIF-2 alpha kinase activities, eIF-2 cycling, and ribosomal loading on mRNA is emphasized. The putative role of GRP78 in coupling rates of translation to processing is modeled, and functional relationships between the HSP and GRP chaperone systems are discussed.

Apoptosis of CD4+ and CD19+ cells during human immunodeficiency virus type 1 infection--correlation with clinical progression, viral load, and loss of humoral immunity.

Enhanced rates of programmed cell death (apoptosis) have been detected in T cells and B cells from human immunodeficiency virus type 1 (HIV-1)-infected individuals. To evaluate the possible relevance of this event to HIV pathogenesis and disease progression, apoptosis in CD4+ T lymphocytes and CD19+ B lymphocytes, viral load, and neutralizing antibody titers were assayed in HIV-1-infected slow progressors and progressors. A correlation was found between progressive disease and apoptosis of CD4+ T cells. The extent of apoptosis in CD4+ cells was similar in slow progressors and seronegative control subjects. By contrast, we found elevated levels of B-cell apoptosis in all HIV-1-infected individuals compared with seronegative control subjects, with a tendency toward increased levels of apoptosis with progressive disease. Apoptosis in CD4+ T cells and CD19+ B cells correlated with viral RNA levels in plasma. Furthermore, higher rates of B-cell apoptosis were observed in individuals with poor neutralizing activity against a panel of six clinical HIV-1 isolates. From these results we conclude that the extent of apoptosis in cultured CD4+ cells and CD19+ cells appears to parallel the decline in CD4 cell counts in infected individuals. The finding of a relation between apoptosis in B cells and poor neutralizing capacity suggests that apoptosis may be related to loss of immune function. A role for apoptosis in the pathogenesis of AIDS is also supported by the strong correlation between viral load and rates of apoptosis in CD4+ T cells.

Analysis of the endoplasmic reticular Ca2+ requirement for alpha1-antitrypsin processing and transport competence.

Depletion of Ca2+ sequestered within the endoplasmic reticulum (ER) of HepG2 hepatoma cells results in the luminal accumulation of immature alpha1-antitrypsin possessing Man8-9 GlcNAc2 oligosaccharide side chains. This study explores the basis for this arrest and describes consequent alterations in the size and rate of secretion of the complex endoglycosidase H-resistant form of the protein. Inhibition of glucosidase I and II with castanospermine or alpha-1,2-mannosidase with 1-deoxymannojirimycin produced altered ER processing intermediates that were rapidly secreted. Subsequent mobilization of ER Ca2+ stores resulted in the appearance and retention of slightly larger related forms of these intermediates. Retention of glycosylated intermediates was not ascribable to an association with alpha1,2-mannosidase or lectin-like chaperones, the intermediates were not degraded and all evidence of ER retention or size alterations produced by Ca2+ depletion was quickly reversed by Ca2+ restoration. Cells that were Ca2+ depleted for 2 h slowly secreted an abnormal slightly smaller complex oligosaccharide form of alpha1-antitrypsin at approximately the same rate as the non-glycosylated protein generated by treatment with tunicamycin. The hypothesis that Ca2+ affects the folding and ER transport competence of glycosylated forms of alpha1-antitrypsin is discussed.

Site-directed serology of HIV-1 subtype B infection: relation between virus specific antibody levels and disease progression.

Activated T helper (Th) cell-dependent (TD) antibody responses were determined over an 8-10 year period in 28 patients infected with HIV-1 subtype B. Twelve patients remain asymptomatic with normal CD4+ cell counts for 101-114 months. These individuals were defined as long-term asymptomatic (LTA). Sixteen patients progressed to severe immunodeficiency within 58-120 months. In samples derived close to the diagnosis of HIV-1, CD4+ cell counts were higher among the LTAs (P < 0.01). Antibody production driven by activated Th cells was determined using peptides corresponding to HIV-1 V3US/Eur, gp41, and the hepatitis C virus (HCV) core proteins. The less Th cell-dependent B cell antibody response was represented by measles virus immunity. Close to HIV-1 diagnosis, variable third (V3), gp41, HCV core, and measles antibody titres were at similar levels among the LTAs and the progressors. With time the LTAs displayed unchanged levels of V3 and gp41 antibodies, and slightly decreasing levels of HCV core antibodies (P < 0.05). In contrast, the progressors showed a decrease in all these antibody responses (P < 0.05, for all). In both groups, the levels of measles antibody remained stable. Our data show that no significant change of the antibody responses of LTAs is seen, even after 101-114 months of known HIV-1 infection. Furthermore, the marked decrease of TD antibody production in the progressors suggests that activated Th cells may be excellent targets for HIV-1 infection.

Inhibition of translational initiation by activators of the glucose-regulated stress protein and heat shock protein stress response systems. Role of the interferon-inducible double-stranded RNA-activated eukaryotic initiation factor 2alpha kinase.

Depletion of endoplasmic reticulum (ER) Ca2+ perturbs protein folding and processing within the organelle while inhibiting translational initiation through activation of the double-stranded RNA-activated eukaryotic initiation factor (eIF)-2alpha kinase (PKR) (Prostko, C. R., Dholakia, J. N., Brostrom, M. A., and Brostrom, C. O. (1995) J. Biol. Chem. 270, 6211-6215). The glucose-regulated stress protein (GRP) chaperones are subsequently induced. We now report that sodium arsenite, a prototype for stressors fostering cytoplasmic protein misfolding, also inhibits translational initiation through activation of PKR while subsequently inducing the heat shock protein (HSP) chaperones. Arsenite neither mobilized ER-associated Ca2+ nor slowed peptide chain elongation. Various HSP-inducing chemicals caused rapid phosphorylation of eIF-2alpha. When incubated with double-stranded RNA, extracts derived from arsenite-treated cells displayed greater degrees of phosphorylation of PKR and eIF-2alpha than did control extracts. Cells overexpressing a dominant negative PKR mutation resisted translational inhibition and eIF-2alpha phosphorylation in response to ER or cytoplasmic stressors. Induction of either the HSP or GRP chaperones was accompanied by development of translational tolerance to either Ca2+-mobilizing agents or arsenite. Following induction of the HSPs by arsenite, cells remained susceptible to induction of the GRPs by Ca2+-mobilizing agents. Conversely, cells possessing induced GRP contents in response to Ca2+-mobilizing agents readily induced the HSPs in response to arsenite. It is concluded that the two chaperone systems function independently except for their mutual suppression of PKR.

Translational suppression by Ca2+ ionophores: reversibility and roles of Ca2+ mobilization, Ca2+ influx, and nucleotide depletion.

The divalent cation selective ionophores A23187 and ionomycin were compared for their effects on the Ca2+ contents, nucleotide contents, and protein synthetic rates of several types of cultured cells. Both ionophores reduced amino acid incorporation by approximately 85% at low concentrations (50-300 nmol/L) in cultured mammalian cells without reducing ATP or GTP contents. At these concentrations A23187 and ionomycin each promoted substantial Ca2+ efflux, whereas at higher concentrations a large influx of the cation was observed. Ca2+ influx occurred at lower ionophore concentrations and to greater extents in C6 glioma and P3X63Ag8 myeloma than in GH3 pituitary cells. The ATP and GTP contents of the cells and their ability to adhere to growth surfaces declined sharply at ionophore concentrations producing increased Ca2+ influx. Prominent reductions of nucleotide contents occurred in EGTA-containing media that were further accentuated by extracellular Ca2+. Ionomycin produced more Ca2+ influx and nucleotide decline than comparable concentrations of A23187. The inhibition of amino acid incorporation and mobilization of cell-associated Ca2+ by ionomycin were readily reversed in GH3 cells by fatty acid-free bovine serum albumin, whereas the effects of A23187 were only partially reversed. Amino acid incorporation was further suppressed by ionophore concentrations depleting nucleotide contents. Mitochondrial uncouplers potentiated Ca2+ accumulation in response to both ionophores. At cytotoxic concentrations Lubrol PX abolished protein synthesis but did not cause Ca2+ influx. Nucleotide depletion at high ionophore concentrations is proposed to result from increased plasmalemmal Ca2+-ATPase activity and dissipation of mitochondrial proton gradients and to cause intracellular Ca2+ accumulation. Increased Ca2+ contents in response to Ca2+ ionophores are proposed as an indicator of ionophore-induced cytotoxicity.

Biological and molecular characterization of subtype D, G, and A/D recombinant HIV-1 transmissions in Sweden.

HIV-1 can be subdivided into at least nine genetic subtypes (A through H and O), but in Europe and the United States there is an almost complete dominance of subtype B. In this study three Swedish HIV-1 transmission chains of subtypes other than subtype B have been biologically and molecularly characterized. The three index cases were African men. The p17 gag and env V3 regions of the HIV-1 genome were directly sequenced from uncultured lymphocytes. Phylogenetic analyses showed that the HIV-1 variants with each transmission group were genetically closely related, supporting the epidemiological information. The individuals in transmission groups I (n = 3) and II (n = 2) carried subtype G and D virus, respectively. Interestingly, all four individuals in transmission group III displayed a recombinant genotype with subtype D p17 gag sequence and subtype A V3 sequence. The biological phenotype of virus isolates (rapid/high, syncytium-inducing; or slow/low, non-syncytium-inducing) correlated with the clinical stage of the infected individual. The study also suggested that the correlation between biological phenotype and V3 genotype that has been established for subtype B HIV-1 variants may be valid also for other subtypes. This study demonstrates that HIV-1 variants of subtypes other than B, including a subtype A/D recombinant, are being transmitted in Europe.

Activation of the double-stranded RNA-regulated protein kinase by depletion of endoplasmic reticular calcium stores.

Perturbants of the endoplasmic reticulum (ER), including Ca(2+)-mobilizing agents, provoke a rapid suppression of translational initiation in conjunction with an increased phosphorylation of the alpha-subunit of eukaryotic initiation factor (eIF)-2. Depletion of ER Ca2+ stores was found to signal the activation of a specific eIF-2 alpha kinase. Analysis of extracts derived from cultured cells that had been pretreated with Ca2+ ionophore A23187 or thapsigargin revealed a 2-3-fold increase in eIF-2 alpha kinase activity without detectable changes in eIF-2 alpha phosphatase activity. A peptide of 65-68 kDa, which was phosphorylated concurrently with eIF-2 alpha in extracts of pretreated cells, was identified as the interferon-inducible, double-stranded RNA (dsRNA)-regulated protein kinase (PKR). Depletion of ER Ca2+ stores did not alter the PKR contents of extracts. When incubated with reovirus dsRNA, extracts derived from cells with depleted ER Ca2+ stores displayed greater degrees of phosphorylation of PKR and of eIF-2 alpha than did control extracts. The enhanced dsRNA-dependent phosphorylation of PKR was observed regardless of prior induction of the kinase with interferon. Lower concentrations of dsRNA were required for maximal phosphorylation of PKR in extracts of treated as compared to control preparations. These findings suggest that PKR mediates the translational suppression occurring in response to perturbation of ER Ca2+ homeostasis.

Independent signaling of grp78 gene transcription and phosphorylation of eukaryotic initiator factor 2 alpha by the stressed endoplasmic reticulum.

Perturbation of endoplasmic reticular (ER) function signals increased expression of the gene encoding the ER resident chaperone Grp78/BiP and rapid suppression of translational initiation accompanied by phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF-2). eIF-2 alpha phosphorylation and grp78 mRNA induction were measured in GH3 pituitary cells subjected to varied degrees of ER stress to ascertain whether activation of an eIF-2 alpha kinase is involved in both events. grp78 mRNA was induced at low concentrations of ionomycin and dithiothreitol that did not provoke eIF-2 alpha phosphorylation or inhibition of amino acid incorporation. Mobilization of the bulk of cell-associated Ca2+ and the induction of grp78 mRNA occurred at comparable low concentrations of ionomycin, whereas phosphorylation of eIF-2 alpha and inhibition of protein synthesis required higher ionophore concentrations. Pretreatment for 1 h with cycloheximide suppressed grp78 mRNA induction and eIF-2 alpha phosphorylation in response to either stressor. Prolonged (17 h) cycloheximide blockade increased eIF-2 alpha phosphorylation without inducing grp78 mRNA. Upon release from the blockade, grp78 mRNA was induced and eIF-2 alpha was dephosphorylated. Translational tolerance to ionomycin or dithiothreitol, accompanied by dephosphorylation of eIF-2 alpha, was observed whenever grp78 mRNA was induced. Induction of grp78 mRNA preceded significant eIF-2 alpha phosphorylation during treatment with brefeldin A. It is concluded that signaling of grp78 gene transcription can occur independently of eIF-2 alpha phosphorylation or translational repression and that greater degrees of ER stress are required for eIF-2 alpha phosphorylation than for grp78 mRNA induction.