©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Independent Signaling of grp78 Gene Transcription and Phosphorylation of Eukaryotic Initiation Factor 2 by the Stressed Endoplasmic Reticulum (*)

(Received for publication, October 17, 1994; and in revised form, December 12, 1994)

Margaret A. Brostrom (§) C. Robert Prostko Debra Gmitter Charles O. Brostrom

From the Department of Pharmacology, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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-2alpha phosphorylation and grp78 mRNA induction were measured in GH(3) pituitary cells subjected to varied degrees of ER stress to ascertain whether activation of an eIF-2alpha kinase is involved in both events. grp78 mRNA was induced at low concentrations of ionomycin and dithiothreitol that did not provoke eIF-2alpha phosphorylation or inhibition of amino acid incorporation. Mobilization of the bulk of cell-associated Ca and the induction of grp78 mRNA occurred at comparable low concentrations of ionomycin, whereas phosphorylation of eIF-2alpha and inhibition of protein synthesis required higher ionophore concentrations. Pretreatment for 1 h with cycloheximide suppressed grp78 mRNA induction and eIF-2alpha phosphorylation in response to either stressor. Prolonged (17 h) cycloheximide blockade increased eIF-2alpha phosphorylation without inducing grp78 mRNA. Upon release from the blockade, grp78 mRNA was induced and eIF-2alpha was dephosphorylated. Translational tolerance to ionomycin or dithiothreitol, accompanied by dephosphorylation of eIF-2alpha, was observed whenever grp78 mRNA was induced. Induction of grp78 mRNA preceded significant eIF-2alpha phosphorylation during treatment with brefeldin A. It is concluded that signaling of grp78 gene transcription can occur independently of eIF-2alpha phosphorylation or translational repression and that greater degrees of ER stress are required for eIF-2alpha phosphorylation than for grp78 mRNA induction.


INTRODUCTION

Alterations in the functional status of the endoplasmic reticulum (ER) (^1)signal the increased synthesis of ER resident protein chaperones(1, 2) . Signaling is evoked by such conditions as depletion of ER Ca stores, introduction of a reducing environment, suppression of protein glycosylation, or overexpression of secretory proteins and is regulated at the level of gene transcription. Grp78/BiP, the chaperone induced most prominently by ER stress, has been hypothesized to function in the correct folding and assembly of proteins during early protein processing(3) , in the retention of improperly folded proteins that accumulate within the ER lumen when processing is distressed(4) , and in the translocation of proteins from the cytosol to the ER for processing(5, 6) . The gene for Grp78 possesses a highly conserved promoter region that confers ER stress inducibility and binds specific transcription factors(7, 8, 9) . Although the mechanism whereby the stressed ER of mammalian cells signals the activation of gene transcription is unclear, an ER transmembrane kinase appears key to such signaling in yeast(10, 11) . Termed IRE1 or ERN1, this kinase is structurally similar to growth factor receptor kinases with its N terminus on the lumenal side and cytoplasmic C terminus carrying the kinase domain. By analogy, IRE1 may phosphorylate as yet unidentified substrate proteins on serine or threonine residues.

ER perturbants, including Ca-mobilizing and thiol-reducing agents, also signal the rapid suppression of mRNA translation at initiation within min(12, 13) . This inhibition occurs in conjunction with the phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF-2) (14, 15) which is known to constrain eIF-2B activity and thereby reduce formation of the 43 S preinitiation complex(16) . Several hours exposure of cells to either Ca-mobilizing or thiol-reducing drugs results in the prominent induction of grp78, the dephosphorylation of eIF-2alpha, and the partial (40-80%) recovery of rates of translational initiation and amino acid incorporation(17, 18) . All of these events are blocked by actinomycin D. Translational recovery is associated with the development of cross-tolerance to Ca-mobilizing or reducing stressors and is decreased by antisense oligonucleotides directed against grp78 mRNA.

HCR and PKR, mammalian eIF-2alpha kinases that phosphorylate the alpha-subunit at serine residue 51, are strongly implicated in rapid translational repression under conditions wherein continued protein synthesis might prove injurious(16, 19, 20) . HCR, the heme-regulated eIF-2alpha kinase of erythroid cells, is inactive when complexed with certain cytosolic chaperones (21, 22) and may function to regulate gene transcription(23) . PKR is interferon-inducible, activated by double-stranded RNA produced during the replicative cycle of certain viruses, localized to ribosomes of the rough ER, and may function importantly in controlling growth and differentiation(16, 19, 20) . PKR has recently been demonstrated to activate gene transcription through regulation of NF-kappaB(24) . The possibility that activation of an eIF-2alpha kinase during ER stress is required for signaling of grp78 gene transcription has not been explored. The following study was undertaken to ascertain whether phosphorylation of eIF-2alpha and induction of grp78 mRNA occur under identical degrees of ER stress and whether the phosphorylation is obligatory for grp78 gene transcription.


EXPERIMENTAL PROCEDURES

Materials

Plasmids carrying cDNAs used in this study were kindly provided by Drs. A. S. Lee, University of Southern California Medical School (hamster grp78) and D. Cleveland, Johns Hopkins University (beta-actin). Monoclonal antibody to eIF-2alpha and highly purified eIF-2 were the generous gifts of Drs. E. Henshaw, University of Rochester, and J. Dholakia, University of Louisville School of Medicine, respectively. Unless indicated otherwise, chemicals and reagents were purchased from the Sigma.

General Procedures

GH(3) cells were propagated and maintained as described previously(25) . Harvested cells (2 times 10^6/ml) were equilibrated for 30 min in serum-free Ham's F-10 medium adjusted to contain 0.2 mM Ca prior to use. Amino acid incorporation was measured as described (26) for incubations conducted in triplicate, and results are presented as the average ± range of values obtained. Cell-associated Ca determinations were conducted as described previously (17) , and the average ± range of values obtained for incubations conducted in quadruplicate is provided.

Preparation of DNA Probes and Extraction and Analysis of mRNA

CsCl-purified recombinant plasmids p3C5 (grp78) and pA1 (beta-actin) were linearized by digestion with an appropriate restriction enzyme, extracted with phenol/chloroform, and precipitated with ethanol. Total cellular RNA was isolated using the acid guanidinium isothiocyanate method(27) , except for inclusion of one additional phenol/chloroform extraction. RNA concentrations were estimated by absorbance at 260 nm, and measurement of various mRNAs by dot blot analysis using labeled cDNA probes was performed as described previously (18) with the exception that radioactivity was quantified by a PhosphorImager (Molecular Dynamics). Results are expressed as the average ± range of values obtained for triplicate mRNA determinations.

Determination of the Phosphorylation State of the alpha-Subunit of eIF-2

Cells (2 times 10^6) pelleted by centrifugation were dissolved by the addition of sample buffer (250 µl, 40 °C) containing 3.5% ampholines (4 parts pH 4-8 and 1 part pH 3.5-10), 1% beta-mercaptoethanol, 0.2% Tween 20, and 12.5 M urea. The preparations were then subjected to slab gel isoelectric focusing over a pI range of 5-7 (BDH Resolyte ampholines) in the presence of 9.5 M urea (Boehringer Mannheim) to separate the phosphorylated and unphosphorylated forms of eIF-2alpha (28) . Gels were incubated for 30 min in 0.75 M Tris-Cl, pH 8.8, 0.2% SDS, and transferred to Immobilon-P membranes for 2.5 h at 250 mA in 12.5 mM Tris, 100 mM glycine, 20% methanol, and 0.02% SDS. After treatment with monoclonal anti-eIF-2alpha as described(14) , the blots were blocked with 5% milk protein in phosphate-buffered saline at pH 7.4 containing 0.3 mg/ml BSA, incubated with horseradish peroxidase-conjugated goat anti-mouse IgG, and the immunoreactive peptides detected using an ECL kit (Amersham Corp.). Films were scanned with the Imagestar scanning program. Analysis of the relative amounts of phosphorylated and unphosphorylated subunit, which differ by approximately 0.1 pI unit, was performed on a Macintosh Quadra 700 computer using the public domain National Institutes of Health Image program (written by Wayne Rasband at the United States National Institutes of Health and available from the Internet by anonymous ftp from zippy.nimh.nih.gov or on floppy disk from NTIS, 5285 Port Royal Rd., Springfield, VA 22161, part number PB93-504868).

Measurement of Translational Tolerance to Ionomycin

Preparations were incubated with or without ER stressors as described in the text. Ionomycin-treated preparations were adjusted with fatty acid-free BSA (2 mg/ml) to bind ionophore (29) and incubations continued for 15 min. Variously treated preparations were washed twice by centrifugation and resuspension in fresh medium without stressor or BSA. Following resuspension to 2 times 10^6 cells/ml of fresh medium lacking stressor, washed preparations were equilibrated for 15 min. Ionomycin (1 µM) was then added to portions of each preparation, and leucine incorporation was determined for triplicate incubation samples after 15 min of treatment. Results are expressed as the percent inhibition of incorporation observed upon challenge with 1 µM ionomycin.


RESULTS

Ionomycin and Dithiothreitol Concentration Dependencies for eIF-2 Phosphorylation and grp78 mRNA Induction

Exposure of cultured cells to Ca-mobilizing or thiol-reducing agents results in the inhibition of amino acid incorporation, the accumulation of monosomes and ribosomal subunits, and the reduction of the cellular content of 43 S preinitiation complex(12, 13) . GH(3) cells, which exhibit these responses especially rapidly, concurrently display an average 10-20-fold increase in the amount of phosphorylated eIF-2alpha and a 50% reduction in eIF-2B activity at 15% fractional phosphorylation of eIF-2alpha(14, 15) . GH(3) cells also induce grp78 mRNA rapidly (1-3 h) in response to these agents but require cAMP elevation and/or a phorbol ester for optimal inductions(18) . Increased synthesis of Grp78 in GH(3) cells is invariably accompanied by development of translational cross-tolerance to the perturbants and by the dephosphorylation of eIF-2alpha(14, 15, 17, 18) . It was unclear from previous studies with these cells, however, that comparable degrees of ER perturbation signal each of these various events.

The ionophore concentration dependencies for inhibition of amino acid incorporation, increased eIF-2alpha phosphorylation, Ca mobilization, induction of grp78 mRNA, and development of translational tolerance were compared within the same preparation of GH(3) cells ( Fig. 1and Fig. 2). Cells suspended in medium containing phorbol ester and cholera toxin were incubated for 15 min or 3 h with increasing concentrations of ionomycin prior to various analyses. At 15 min of incubation, leucine incorporation into protein was suppressed by ionomycin at concentrations exceeding 30 nM, with 0.1 and 1 µM drug providing 48 and 88% inhibitions, respectively (Fig. 1A). eIF-2alpha phosphorylation increased in parallel with inhibition of incorporation, with approximately 20% of the factor being phosphorylated at 1 µM ionomycin (Fig. 1A and Fig. 2, upper panel). Following 3 h of exposure, however, these parameters became resistant to suppression by the ionophore. Some residual eIF-2alpha phosphorylation and inhibition of leucine incorporation was manifested at the higher ionophore concentrations (Fig. 1B and Fig. 2, lower panel).


Figure 1: Phosphorylation of eIF-2alpha, induction of grp78 mRNA, expression of translational tolerance, and mobilization of cell-associated Ca at varying concentrations of ionomycin. GH(3) cells in medium containing 0.6 µM PMA and 50 ng/ml cholera toxin were treated for 15 min or 3 h with the indicated concentrations of ionomycin. A, incubations were conducted for 15 min. [^3H]Leucine was then added to portions of each preparation, and pulse incorporation into protein was determined after 15 min (circle). Additional samples were taken to quantitate the percentage of eIF-2alpha in the phosphorylated form after immunoblotting as described under ``Experimental Procedures'' (bullet). B, incubations were conducted for 3 h. [^3H]Leucine incorporation (circle) and the percentage of eIF-2alpha in the phosphorylated form (bullet) were then determined. C, incubations were conducted for 3 h. Total RNA was extracted and subjected to dot blot analysis using radiolabeled cDNA probes for cellular mRNAs encoding grp78 (bullet) or beta-actin (). The content of each mRNA in untreated cells at time 0 was assigned a value of 1.0, and the results are expressed as the relative concentration after 3-h incubation. Additional preparations were subjected to a washing procedure to remove ionomycin and were subsequently rechallenged with ionomycin (1 µM) or solvent (Me(2)SO, 0.5%) for 15 min. [^3H]Leucine incorporation into protein was then determined. The percent inhibition of incorporation provided by ionomycin is plotted inversely on the ordinate as a measure of translational tolerance to the ionophore (circle). D, cells preloaded with CaCl(2) were challenged as above with the indicated concentrations of ionomycin, and cell-associated Ca was determined after 15 min () or 3 h (box) of incubation.




Figure 2: Ionomycin concentration dependence of eIF-2alpha phosphorylation during brief and extended incubations. GH(3) cells were treated with the indicated concentrations of ionomycin under conditions described in the legend to Fig. 1. Upper panel, 15-min incubation; lower panel, 3-h incubation. Preparations were denatured, subjected to slab gel isoelectric focussing, and immunoblotted for eIF-2alpha as indicated under ``Experimental Procedures.'' The position of the phosphorylated form of eIF-2alpha is indicated by the arrow.



The induction of grp78 mRNA occurred at low concentrations of ionophore that did not significantly affect eIF-2alpha phosphorylation or rates of amino acid incorporation. grp78 mRNA, measured after 3 h of incubation (Fig. 1C), was induced to increasing degrees between 5 and 50 nM ionomycin. Translational tolerance, measurable in washed preparations as an increased resistance of incorporation to inhibition by 1 µM ionomycin, developed concomitantly with the induction of grp78 mRNA (Fig. 1C). Cell-associated Ca was largely mobilized over the range of 5 and 100 nM ionomycin at either incubation time (Fig. 1D). A similar experiment was conducted to determine the concentrations of dithiothreitol required for inhibition of amino acid incorporation, eIF-2alpha phosphorylation, induction of grp78 mRNA, and development of translational tolerance (Fig. 3). Amino acid incorporation was inhibited and eIF-2alpha was phosphorylated within 15 min at concentrations of reducing agent in excess of 60 µM (Fig. 3A). Effects were half-maximal at approximately 150 µM drug. After 3 h good recoveries of incorporation and dephosphorylation of eIF-2alpha were observed for cells exposed to the previously inhibitory concentrations of dithiothreitol (Fig. 3B). Both the induction of grp78 mRNA and the development of translational tolerance to ionomycin occurred at low concentrations of dithiothreitol (30-50 µM) that were not inhibitory to amino acid incorporation or productive of eIF-2alpha phosphorylation (Fig. 3C). Actin mRNA concentrations did not increase during longer incubations with either ionomycin or dithiothreitol (Fig. 1C and 3C).


Figure 3: Phosphorylation of eIF-2alpha, induction of grp78 mRNA, and expression of translational tolerance at varying concentrations of dithiothreitol. GH(3) cells in medium containing 0.6 µM PMA and 50 ng/ml cholera toxin were treated for 15 min or 3 h with the indicated concentrations of dithiothreitol. A, incubations were conducted for 15 min. [^3H]Leucine incorporation into protein (circle) and the percentage of eIF-2alpha in the phosphorylated form (bullet) were then determined. B, incubations were conducted for 3 h. [^3H]Leucine incorporation (circle) and the percentage of eIF-2alpha in the phosphorylated form (bullet) were then determined. C, incubations were conducted for 3 h. Samples were taken for extraction of total cellular RNA, and mRNAs for grp78 (bullet) and beta-actin () were quantitated by dot blot analysis. As in Fig. 1, results shown are normalized to the basal concentration of mRNA in control cells at time 0. Additional preparations were washed to remove dithiothreitol and were rechallenged with ionomycin (1 µM) or solvent for 15 min. [^3H]leucine incorporation into protein was determined, and the percent inhibition provided by ionomycin was plotted inversely on the ordinate as a measure of translational tolerance (circle).



eIF-2 Phosphorylation and grp78 mRNA Induction in the Presence of Cycloheximide

Induction of grp78 mRNA by ionophore A23187 (30) or mercaptoethanol (31) in hamster fibroblasts is significantly reduced by cycloheximide or puromycin pretreatments. To ascertain whether continuous protein synthesis is required for grp78 mRNA induction in GH(3) cells, preparations were treated with cycloheximide at various times before and after challenge with ionophore (Table 1). Almost complete inhibition of amino acid incorporation was obtained with the elongation blocker. A 10-fold induction of grp78 mRNA was observed for cells incubated with ionophore for 3 h without cycloheximide. In contrast message content increased only 2-fold when cycloheximide was added 10 min before ionophore. As the time period between the two additions was extended the induction of grp78 mRNA increased. For example, grp78 mRNA was induced almost 6-fold if cycloheximide was added 90 min after ionophore. Actin mRNA was unaffected by treatments with cycloheximide and/or ionophore ( Table 1and Table 2).





The ability of cycloheximide to counteract the phosphorylation of eIF-2alpha and the induction of grp78 mRNA in cells responding to either ionomycin or dithiothreitol was examined in additional incubations (Table 2). Pretreatment for 1 h with the elongation blocker resulted in almost complete suppression of grp78 mRNA induction in response to either stressor. eIF-2alpha phosphorylation occurring in response to the stressors was also diminished in the presence of cycloheximide. Phosphorylation after 15-min treatment with 0.6 mM dithiothreitol was abolished, whereas phosphorylation in response to 15 min of treatment with 1 µM ionomycin was reduced approximately 60% by the inhibitor. The degree to which cycloheximide suppressed eIF-2alpha phosphorylation depended on the concentration of stressor (Fig. 4). Phosphorylation in response to 0.2 mM dithiothreitol or 0.2 µM ionophore was eliminated by cycloheximide whereas that in response to 1.5 mM dithiothreitol or 2 µM ionophore was partially suppressed. The dephosphorylation that routinely accompanies recovery of amino acid incorporation in cells exposed for 3 h to ionophore did not occur in incubations containing cycloheximide (Table 2). Rather, the residual phosphorylation observed upon 15-min challenge with ionophore in the presence of cycloheximide was preserved during the extended incubation.


Figure 4: Effect of cycloheximide on eIF-2alpha phosphorylation following treatment with two different concentrations of dithiothreitol or ionomycin. GH(3) cells were pretreated in the absence (upper panel) or presence (lower panel) of cycloheximide (50 µM) for 1 h. Dithiothreitol (0.2 or 1.5 mM) or ionomycin (0.2 or 2 µM) was added as indicated. After 15 min preparations were denatured, subjected to slab gel isoelectric focussing, and immunoblotted for eIF-2alpha. The arrow indicates the position of the phosphorylated form of eIF-2alpha.



grp78 mRNA Induction during Recovery from Prolonged Cycloheximide Treatment

A strategy was developed whereby grp78 mRNA induction could be produced without the use of either Ca-mobilizing or reducing agents and a clear dissociation from eIF-2alpha phosphorylation could be observed. GH(3) cells were subjected to a prolonged (17 h) incubation with cycloheximide to deplete substrates for ER protein processing. The cells were then washed twice by centrifugation and resuspension in Ham's F-10 medium to remove the inhibitor and were incubated in fresh medium to rapidly reintroduce substrates for ER protein processing. Aliquots were incubated in the absence or presence of actinomycin D for 30 min or 3.5 h. Samples were then taken for measurements of leucine incorporation, grp78 mRNA and actin mRNA concentrations, and eIF-2alpha phosphorylation (Table 3). Cycloheximide-pretreated preparations quickly regained the ability to incorporate leucine, with incorporation values approaching those of nonpretreated control preparations at 3.5 h. grp78 mRNA in cycloheximidepretreated preparations, which did not differ from that of nonpretreated controls at time 0, was found to rise during the incubation, with 3-fold and 6-fold inductions attained after 30 min and 3.5 h, respectively. These inductions were blocked by actinomycin D. Actin mRNA concentrations did not change during the incubations. As observed previously(15) , eIF-2alpha phosphorylation increased significantly during prolonged cycloheximide treatment. Dephosphorylation of the factor, which was complete after 3.5 h recovery in the absence of cycloheximide, was prevented by actinomycin D. During comparable incubations of nonpretreated controls, grp78 mRNA was not increased and the phosphorylation state of eIF-2alpha was not affected.



Translational tolerance to ionomycin was expressed after 3 h, but not after 15 min, of recovery from cycloheximide pretreatment (Table 4). Development of the accommodation was prevented when cells were allowed to recover in the presence of actinomycin D.



eIF-2 Phosphorylation and grp78 mRNA Induction by Brefeldin A

The fungal metabolite brefeldin A, which causes resorption of the Golgi apparatus into the ER and inhibits protein secretion, induces grp78 gene expression in cultured fibroblasts and hepatoma cells(32, 33) . grp78 mRNA inductions by the metabolite and by Ca-mobilizing drugs required comparable incubation times. Brefeldin A was recently reported to inhibit protein synthesis in GH(3) cells through phosphorylation of eIF-2alpha(34) . More than 90 min were required for expression of the inhibition, suggesting that grp78 mRNA induction and eIF-2alpha phosphorylation are concurrent, rather than interdependent, signals. grp78 mRNA induction and eIF-2alpha phosphorylation were determined for the same preparation of GH(3) cells challenged with brefeldin A (Table 5; Fig. 5). No significant inhibition of leucine incorporation or increase in eIF-2alpha phosphorylation was observed after 15-min or 1-h treatments. After 4 h with 5 or 10 µg/ml brefeldin A wherein grp78 mRNA was significantly (11-14-fold) induced, incorporation was reduced modestly (24-37%) and 5-7% of eIF-2alpha was phosphorylated. As expected, profound suppression of incorporation and prominent eIF-2alpha phosphorylation occurred when these cells were exposed to ionomycin for 15 min. Translational accommodation, dephosphorylation of eIF-2, and induction of grp78 mRNA occurred after 4 h with the ionophore.




Figure 5: Time dependence of eIF-2alpha phosphorylation upon treatment with brefeldin A or ionomycin. Conditions are as described in the legend to Table 5. GH(3) cells were challenged with either 5 or 10 µg/ml brefeldin A (BFA) or with 1 µM ionomycin as indicated. After incubation for 15 min, 1 h, or 4 h, preparations were denatured, subjected to slab gel isoelectric focussing, and immunoblotted for eIF-2alpha. The arrow indicates the position of the phosphorylated form of eIF-2alpha.




DISCUSSION

The event most commonly invoked in the signaling of increased grp78 gene transcription is the accumulation of unfolded proteins in the lumen of the ER. The ER perturbants used in this study to induce grp78 mRNA either cause the lumenal accumulation of unprocessed intermediates or introduce nonresident proteins into the lumen(29, 32, 33, 34, 35, 36, 37, 38) . It is less clear how release from prolonged elongation blockade, which also provoked considerable grp78 mRNA induction, could cause unfolded or alien proteins to accumulate in the ER and/or protein processing to be impaired. In this regard Dorner et al.(39) observed that expression of normal secretory proteins at higher than normal concentrations retarded processing of these proteins and stimulated grp78 gene transcription. It is conceivable, therefore, that prolonged incubation in the absence of substrates for protein processing causes down-regulation of the ER processing system and that flooding the ER with new substrates after such down-regulation causes underprocessed intermediates to accumulate.

It is clear from the findings described in this report that neither an increase in the phosphorylation of eIF-2alpha nor a decrease in the rate of mRNA translation is required for signaling of grp78 gene transcription by ER perturbants. In agreement with earlier studies(30, 31) , grp78 gene transcription in response to ER perturbants was actually repressed when full translational blockade was imposed. These findings are entirely consistent with the proposal (40) that Grp78 suppresses IRE1 activity when complexed with the kinase and that grp78 gene transcription is signaled when unfolded processing intermediates rob the kinase of the chaperone. Cycloheximide would be expected to block synthesis of precursors of such unfolded intermediates that would otherwise accumulate in the lumen of the stressed ER. An alternative explanation for the findings described in Table 1is that continuous synthesis of a cytosolic factor possessing a rapid turnover rate under nonstressed conditions is obligatory for signaling of grp78 gene transcription during ER stress. Turnover would necessarily be retarded under conditions that perturb the ER and could conceivably involve rapid catabolism or removal from the cytosol, either by export to the extracellular fluid or translocation into an organelle such as the ER. An arrest of protein translocation into the ER during stress, resulting in the accumulation of an IRE1-phosphorylatable gene regulatory factor in the cytosol, remains compatible with available information. Phosphorylation of eIF-2alpha by Ca ionophore or dithiothreitol was also reduced when protein synthesis was blocked. Cycloheximide inhibitions were only partial at higher doses of the perturbants, however, consistent with the proposal that eIF-2alpha phosphorylation is signaled under conditions wherein Grp78 is insufficient for management of stress within the ER.

The mammalian counterpart of the protein kinase IRE1, which is required for signaling of grp78 gene transcription in yeast(10, 11) , has not yet been characterized. It is improbable, however, that this enzyme signals both grp78 gene transcription and eIF-2alpha phosphorylation during ER stress. To function as an eIF-2alpha kinase, mammalian IRE1 would have to display different substrate specificities at different degrees of ER stress. Such an enzyme would be unique.

Studies are currently in progress in our laboratory to describe the mechanism whereby the highly stressed ER signals eIF-2alpha phosphorylation. We have recently identified an eIF-2alpha kinase activity that is stimulated in intact cells in response to ER perturbants and is detectible in cell lysates. (^2)The properties of this kinase appear identical with those of PKR, the interferon-inducible, double-stranded RNA-activated protein kinase implicated in control of growth and differentiation. Further investigations will be required to understand how the ER prompts the activation of a protein kinase residing outside the organelle.

The phosphorylation state of eIF-2alpha was found to correlate closely with the rate of amino acid incorporation during increasing degrees of ER stress (Fig. 1Fig. 2Fig. 3). Equally notable was the close correlation between degree of expression of translational tolerance to ionomycin and extent of grp78 mRNA induction. It was also clear that dephosphorylation of eIF-2alpha, which always accompanied translational recovery during extended incubations at high (1 µM) ionophore concentrations (e.g.(14) and (15) ; Fig. 2and 5), did not occur when grp78 mRNA induction was suppressed by cycloheximide (Table 2). These findings, as well as those reported previously(17, 18) , favor a role for new Grp78 in expression of the accommodation. How the chaperone acts to suppress eIF-2alpha phosphorylation and thereby maintain translational activity remains unclear. Grp78 has been proposed to function as part of a translocation system for protein entry into the ER(5, 6) ; this system may conceivably bind to and inhibit an eIF-2alpha kinase. The binding of cytosolic chaperones to HCR suppresses the activity of this eIF-2alpha kinase in rabbit reticulocyte lysates(21, 22) . Addition of denatured proteins to such lysates signals activation of the kinase, presumably as a consequence of dissociation of chaperone-enzyme complexes(41) . It is therefore attractive to speculate that Grp78 regulates an eIF-2alpha kinase in intact non-erythroid cells during stresses that promote protein unfolding in the ER.

In many respects both eIF-2 phosphorylation and grp78 induction behave as though they are subject to inhibition by a catalytic ER pool of Grp78. Any condition involving the depletion of this pool, including the accumulation of very early ER protein folding intermediates, overproduction of high amounts of specific proteins, or interdiction of ER to Golgi vesicular traffic, would ultimately trigger the two responses. The induction of grp78 is clearly the more sensitive of the two. Under such conditions protein synthesis would become dependent on a continuing synthesis of Grp78 as is, indeed, observed.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant DK 35393, National Science Foundation Grant IBN 90-21616, and a grant from the Foundation of Medicine and Dentistry of New Jersey. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed. Tel.: 908-235-4086; Fax: 908-235-4073.

(^1)
The abbreviations used are: ER, endoplasmic reticulum; Grp78/BiP, glucose-regulated stress protein 78 or immunoglobulin heavy chain binding protein; eIF, eukaryotic initiation factor; BSA, bovine serum albumin; PMA, phorbol 12-myristate 13-acetate; HCR, heme-regulated eIF-2alpha kinase; PKR, interferon-inducible, double-stranded RNAactivated eIF-2alpha kinase.

(^2)
C. R. Prostko, J. N. Dholakia, M. A. Brostrom, and C. O. Brostrom, manuscript in preparation.


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