©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Activation of the Double-stranded RNA-regulated Protein Kinase by Depletion of Endoplasmic Reticular Calcium Stores (*)

(Received for publication, January 9, 1995)

C. Robert Prostko (1) Jaydev N. Dholakia (2) Margaret A. Brostrom (1) Charles O. Brostrom (1)(§)

From the  (1)Department of Pharmacology, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854 and the (2)Department of Biochemistry, University of Louisville School of Medicine, Louisville, Kentucky 40292

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Perturbants of the endoplasmic reticulum (ER), including Ca-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 Ca stores was found to signal the activation of a specific eIF-2alpha kinase. Analysis of extracts derived from cultured cells that had been pretreated with Ca ionophore A23187 or thapsigargin revealed a 2-3-fold increase in eIF-2alpha kinase activity without detectable changes in eIF-2alpha phosphatase activity. A peptide of 65-68 kDa, which was phosphorylated concurrently with eIF-2alpha in extracts of pretreated cells, was identified as the interferon-inducible, double-stranded RNA (dsRNA)-regulated protein kinase (PKR). Depletion of ER Ca stores did not alter the PKR contents of extracts. When incubated with reovirus dsRNA, extracts derived from cells with depleted ER Ca stores displayed greater degrees of phosphorylation of PKR and of eIF-2alpha 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 Ca homeostasis.


INTRODUCTION

PKR (^1)(dsRNA-dependent/regulated protein kinase) is a ubiquitously expressed enzyme induced by interferon alpha or beta and activated during viral infection in various cell types (reviewed in (1, 2, 3) ). The activated enzyme catalyzes the phosphorylation of the alpha-subunit of eukaryotic initiation factor (eIF)-2, resulting in the arrest of translation at initiation, and is thought to function in both the anti-proliferative and antiviral actions of interferon. The possibility that this enzyme possesses multiple substrates and/or serves more broadly in cellular control mechanisms is supported by various recent findings. For example, PKR activates gene transcription through regulation of NF-kappaB (4) and is strongly implicated in the control of differentiation and growth (5, 6, 7, 8, 9, 10) .

The endoplasmic reticulum (ER) functions critically in the early processing of newly synthesized secretory, lysosomal and integral membrane proteins. Metabolic perturbation of the ER with Ca-mobilizing or thiol-reducing agents interrupts the processing and folding of newly synthesized polypeptides within the ER lumen(11) . These disruptions of ER function signal the immediate suppression of translational initiation coincident with the phosphorylation of eIF-2alpha(12, 13) . These perturbants also provoke a subsequent stress response that is characterized by the induction of certain ER resident protein chaperones termed the glucose-regulated proteins(14) . Greater degrees of ER stress are required for signaling of eIF-2alpha phosphorylation than for induction of the glucose-regulated proteins(15) , an observation consistent with the existence of a pathway to slow synthesis of precursors when protein processing is seriously impaired. The mechanism whereby perturbation of the ER signals increased phosphorylation of eIF-2alpha is currently undefined. Decreased phosphatase activity has been suggested as responsible for phosphorylation of eIF-2alpha in perfused liver deprived of a single essential amino acid(16) , whereas activation of eIF-2alpha kinases has been proposed to regulate phosphorylation of the factor under other conditions(1) . The present report provides evidence that depletion of ER Ca stores signals the activation of an eIF-2alpha kinase and that the activated enzyme is PKR.


EXPERIMENTAL PROCEDURES

Materials

Acrylamide, horseradish peroxidase-conjugated goat anti-rabbit IgG, and protein molecular weight standards were from Bio-Rad. Enhanced chemiluminescence (ECL) kits and adenosine 5`-[-P]triphosphate (3000 Ci/mmol) were purchased from Amersham Corp. Protein A-Sepharose and ultrapure urea were obtained from Boehringer Mannheim. [^3H]Leucine was from ICN. The following researchers graciously provided reagents used in this study: Dr. E. Henshaw, University of Rochester (monoclonal anti-eIF-2alpha); Drs. M. Katze and G. Barber, University of Washington (rabbit anti-human PKR); Dr. J.-J. Chen, Harvard-MIT (rabbit anti-mouse PKR); Dr. S. Pestka, Robert Wood Johnson Medical School (interferon alpha A/D Bgl); and Dr. A. Shatkin, Rutgers Center for Advanced Biotechnology and Medicine (reovirus dsRNA). Other chemicals and reagents were from Sigma unless otherwise indicated.

General Methodology

Rat GH(3) pituitary cells were cultured in Ham's F-10, human HeLa cells in Ham's F-12, and mouse NIH-3T3 cells in Dulbecco's modified Eagle's medium. Media for propagating cell lines contained 10% fetal bovine serum. Prior to use cells were washed once with sterile saline and placed in defined, serum-free modified Ham's F-10 containing 0.2 mM Ca, 100 µM leucine, and 35 µM methionine. Methods for measurement of amino acid incorporation(17) , isoelectric focusing and immunoblotting of eIF-2alpha(13, 14, 16) , and purification of homogeneous eIF-2 (18) have been described previously. Protein concentrations were determined as described (19) with albumin as standard. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described(20) .

Measurement of eIF-2alpha Kinase Activity

After treatments, cells were harvested by centrifugation and homogenized in 20 mM Hepes, pH 7.4, 50 mM KCl, 1.5 mM Mg(2)OAc, and 0.4% Triton X-100. Preparations were then centrifuged at 12,000 times g for 10 min at 4 °C. Supernatant fractions (extracts) were adjusted to equal protein concentrations and were either used immediately or quick-frozen and stored at -80 °C. Phosphorylation of eIF-2alpha was measured in vitro in 20-30 µl of 20 mM Hepes, pH 7.4, 40 mM KCl, 1 mM Mg(2)OAc, and 0.5 mM dithiothreitol (with <0.1% Triton X-100). Cellular protein, ATP, and eIF-2 concentrations are indicated in the legends. Reactions were conducted at 32 °C for 2 min (GH(3)) or 30 °C for 15 min (HeLa and NIH-3T3) and terminated by addition of sample buffer for SDS-PAGE(20) . Immunoprecipitation of PKR was accomplished by incubation at 4 °C for 2 h with 2-3 µl of PKR antiserum, followed by addition of protein A-Sepharose (10 µl, 1 h). Immune complexes were washed four or five times with 10 mM Hepes, pH 7.4, 25 mM KCl, 0.1% Triton X-100 before denaturation by boiling in sample buffer.


RESULTS

Increased eIF-2alpha Kinase Activity in Extracts Derived from Cells with Depleted Ca Stores

GH(3) cells exposed to agents that mobilize Ca from the ER, such as Ca ionophores, EGTA, or thapsigargin, exhibit a rapid phosphorylation of the alpha-subunit of eIF-2(12, 13, 15) . The possibility that ER Ca depletion signals the activation of an eIF-2alpha kinase that is preserved during cell lysis was explored. Extracts prepared from A23187-treated and control preparations were incubated with [-P]ATP and increasing concentrations of highly purified eIF-2. The phosphoproteins derived from the incubations were then separated by SDS-PAGE and analyzed by autoradiography (Fig. 1A). Greater degrees of eIF-2alpha phosphorylation occurred in extracts derived from the A23187-treated cells at each concentration of eIF-2 as compared to those prepared from untreated cells. The direct addition of A23187, EGTA, or thapsigargin to the extracts did not affect eIF-2alpha phosphorylation (not shown). The phosphorylation of eIF-2alpha was attributed to an eIF-2 kinase, since no inhibition of eIF-2alpha phosphatase activity in extracts of ionophore-treated cells was observed. Phosphate was removed from P-labeled eIF-2alpha, prepared by incubation of purified eIF-2 with [-P]ATP and the heme-regulated eIF-2alpha kinase of red cells(16) , at slow but identical rates in extracts of control and treated preparations (not shown). Based on the quantitation of incorporation of radioactive phosphate into the alpha-subunit, eIF-2alpha kinase activity was increased 2-3-fold relative to extracts derived from untreated cells (Fig. 1B).


Figure 1: eIF-2alpha kinase activity of extracts derived from GH(3) cells pretreated with or without Ca ionophore. Cells in serum-free medium were incubated for 7.5 min with or without the addition of 1 µM A23187. Extracts (10 µg of protein) derived from each preparation were incubated for 2 min with [-P]ATP (0.5 µM) and the indicated amounts of highly purified eIF-2. Mixtures were then subjected to SDS-PAGE followed by autoradiography (panel A). Incorporation of radioactive phosphate into the alpha-subunit of eIF-2 was quantitated for each experimental condition with an AMBIS radioanalytic imaging system (panelB). The circles and triangles indicate pretreatments with and without A23187, respectively.



Properties of the eIF-2alpha Kinase and Identification as PKR

Two mammalian eIF-2alpha kinases, each of which is strongly implicated in translational regulation, have been identified and characterized(1, 2, 3) . HCR, the heme-regulated eIF-2alpha kinase, is typically present at high concentrations in erythroid cells. PKR, the dsRNA-regulated, interferon-inducible eIF-2alpha kinase, is thought to be more broadly distributed. To determine whether the kinase signaled by depletion of ER Ca stores is interferon-inducible and regulated by dsRNA, cell lines other than GH(3) were employed. HeLa and NIH-3T3 cells were selected because these lines induce PKR in response to interferon (5, 6, 7, 21) and are suitable for studies with antibodies against human (22) and murine PKR(23) .

Pretreatment of HeLa cells with interferon alpha modestly reduced leucine incorporation (Table 1) and slightly increased eIF-2alpha phosphorylation (Table 1; Fig. 2, lanes1 and 3). Upon challenge of the cells with either A23187 or thapsigargin, however, leucine incorporation was inhibited approximately 80-90% and approximately 40-50% of eIF-2alpha molecules became phosphorylated regardless of interferon pretreatment (Table 1; Fig. 2, lanes2, 3, 5, and 6). Identical findings were obtained with NIH-3T3 cells (not shown).




Figure 2: Effects of Ca-mobilizing drugs on eIF-2alpha phosphorylation in HeLa cells cultured in the absence or presence of interferon. Cells were cultured for 18 h with or without interferon alpha (IFN, 1000 units/ml). Cultures were washed and suspended in defined medium without further additions, with A23187 (1 µM in combination with 1 mM EGTA to hasten depletion of HeLa Ca stores(31) , or with thapsigargin (Tg, 10 µM) as indicated. Incubations without drug or with thapsigargin were conducted for 45 min. The incubation with A23187/EGTA was conducted for 20 min. After centrifugation at 1000 times g, cells were lysed and subjected to slab gel isoelectric focusing over a pI range of 4-8. Immunoblotting for eIF-2alpha was performed as described(13, 14, 16) . The arrows indicate the migration positions of the phosphorylated (eIF-2alpha(P)) and non-phosphorylated (eIF-2alpha) subunits.



PKR when activated by dsRNA is subject to autophosphorylation(1, 2, 3) . HeLa cells were pretreated with interferon alpha to increase PKR content and were challenged with A23187 or thapsigargin to mobilize ER sequestered Ca. A 2-3-fold increase in eIF-2alpha kinase activity was observed in extracts of challenged, as compared to control, preparations (Fig. 3A, lanes1, 3, and 5). A 68-kDa polypeptide was observed to phosphorylate simultaneously with eIF-2alpha in these extracts. Addition of low concentrations of reovirus dsRNA to incubations caused a marked increase in the phosphorylations of this polypeptide and of eIF-2alpha (lanes1 and 2), with greater increases observed in extracts of A23187 or thapsigargin-treated preparations (lanes2, 4, and 6). Pretreatment with interferon caused a 5-fold induction of the 68-kDa polypeptide, which was identified as PKR by immunoblotting (Fig. 3B, lanes1 and 2). Challenge with A23187 (lane3) or thapsigargin (lane4) did not affect the amount of immunoreactive PKR detected in extracts of interferon-pretreated cells.


Figure 3: dsRNA-dependent phosphorylation of eIF-2alpha and PKR and the PKR contents of extracts derived from HeLa cells treated with or without Ca-mobilizing agents. Cells were cultured for 18 h with interferon alpha (IFN, 1000 units/ml) and treated with A23187/EGTA or thapsigargin (Tg) as described in the legend to Fig. 2. Extracts (20 µg of protein) of each preparation were incubated for 15 min with [-P]ATP (0.5 µM) and purified eIF-2 (75 ng) with or without reovirus dsRNA (0.1 µg/ml) as indicated. Mixtures were subjected to SDS-PAGE, and autoradiography was performed (panelA). Migration positions of eIF-2alpha and human PKR are indicated. Additional aliquots (75 µl) of these extracts and of a lysate of cells cultured without interferon were subjected to immunoblotting for measurement of relative PKR concentrations (panelB). 1, no interferon (IFN); 2, with interferon; 3, A23187-treated; 4, thapsigargin-treated.



Extracts of HeLa cells that had not been pretreated with interferon did not contain detectable eIF-2alpha kinase activity regardless of whether dsRNA was added (not shown). Therefore, experiments were conducted to establish whether the increased dsRNA-dependent phosphorylations of eIF-2alpha and PKR in extracts of cells with depleted ER Ca stores were dependent on interferon pretreatment. NIH-3T3 cells and an antibody established to efficiently immunoprecipitate PKR from extracts of murine cells were employed for this purpose. Cells cultured with and without interferon alpha were either treated with A23187 or thapsigargin or carried as untreated controls. Cell extracts from each preparation were incubated with or without the addition of dsRNA, and PKR was subsequently isolated by immunoprecipitation. Phosphorylation of PKR was determined after SDS-PAGE and autoradiography (Fig. 4A). Phosphorylation of the kinase in extracts of untreated controls was detectable only if incubations were conducted with added dsRNA (lanes1 and 2). By contrast, PKR phosphorylation was distinguishable in extracts of A23187 or thapsigargin-treated preparations incubated without dsRNA (lanes3 and 5); addition of dsRNA to these extracts caused the kinase to become highly phosphorylated (lanes4 and 6). When interferon-pretreated preparations were subjected to these protocols, PKR phosphorylation was further amplified for extracts prepared from cells treated with ER Ca perturbants and for incubations conducted with added dsRNA (lanes 7-12).


Figure 4: dsRNA-dependent phosphorylation of PKR in extracts of NIH-3T3 cells pretreated with or without interferon and Ca-mobilizing drugs: correlation with activation of eIF-2alpha kinase. Cells were cultured for 18 h with or without interferon alpha (IFN, 1000 units/ml). Cells were then incubated in serum-free medium for 7.5 min without addition, with A23187 (1 µM), or with thapsigargin (1 µM). Extracts (20 µg protein) derived from each preparation were incubated for 15 min with [-P]ATP (0.5 µM) and purified eIF-2 (0.5 µg) with or without the addition of reovirus dsRNA (0.1 µg/ml) as indicated. PKR was then immunoprecipitated from each reaction mixture, immunoprecipitates were subjected to electrophoresis, and autoradiography was performed (panelA). Supernatant fractions remaining after immunoprecipitation of samples corresponding to lanes7, 9, and 11 in panelA were subjected to SDS-PAGE and analyzed for eIF-2alpha phosphorylation by autoradiography (panelB).



The increase in PKR phosphorylation attributable to depletion of the Ca stores of interferon-treated NIH-3T3 cells was associated with markedly increased eIF-2alpha kinase activity. Supernatant fractions remaining after immunoprecipitation of PKR from extracts corresponding to lanes7, 9, and 11 of Fig. 4A were examined for extent of eIF-2alpha phosphorylation (Fig. 4B). Extracts of cells challenged with A23187 (lane2) or with thapsigargin (lane3) actively incorporated phosphate into eIF-2alpha without addition of dsRNA, whereas extracts of unchallenged controls did not (lane1).

Experiments were also performed to ascertain whether Ca store depletion affected the dsRNA concentration dependence of PKR phosphorylation in vitro. Extracts derived from A23187-treated and untreated NIH-3T3 cells were incubated with 10 µM [-P]ATP and increasing concentrations of reovirus dsRNA. PKR was then isolated by immunoprecipitation, electrophoresis and autoradiography were performed, and PKR phosphorylation was quantitated by densitometry. The characteristic biphasic response to changes in dsRNA concentration (2, 3) was observed (Fig. 5). Low concentrations of dsRNA enhanced PKR phosphorylation, whereas concentrations in excess of 0.1 µg/ml were inhibitory. Phosphorylation of PKR in extracts of treated preparations was significantly greater than that in extracts of control preparations regardless of dsRNA concentration, with a 11-fold differential observed at 0.03 µg/ml. Somewhat lower concentrations of dsRNA were required for maximal PKR phosphorylation in extracts of A23187-treated, as compared with control, preparations.


Figure 5: dsRNA concentration dependence of PKR phosphorylation in extracts of NIH-3T3 cells pretreated with or without A23187. Cells were cultured for 18 h with interferon alpha (1000 units/ml) and then incubated in serum-free medium for 7.5 min with or without A23187 (1 µM). Extracts were incubated as described in the legend to Fig. 4but with 10 µM [-P]ATP and the indicated concentrations of reovirus dsRNA. PKR was immunoprecipitated from each reaction mixture, and electrophoresis and autoradiography were performed. Incorporation of radioactive phosphate into PKR was quantitated by densitometry. Results are expressed as percent of the maximum phosphorylation observed. The circles and triangles indicate pretreatments with and without A23187, respectively.




DISCUSSION

By several criteria the phosphorylation of eIF-2alpha and suppression of translational initiation occurring in eukaryotic cells in response to ER Ca-mobilizing agents involve the activation of the well known, interferon-inducible, dsRNA-activated eIF-2alpha kinase, PKR. Addition of dsRNA to extracts of NIH-3T3 cells with depleted ER Ca stores resulted in the phosphorylation of immunoprecipitable PKR that did not occur in extracts derived from untreated cells. Interferon pretreatment of the cells broadened this differential. Increased phosphorylation of PKR was also detectable in incubations without dsRNA for extracts derived from cells treated with Ca-mobilizing agents. Phosphorylation of PKR in vitro invariably correlated with the phosphorylation of eIF-2alpha.

While the PKR activity of the extracts appears to be stable for some weeks when stored at -80 °C, the eIF-2alpha kinase activity of extracts was clearly inefficient compared to that of intact cells. We found that the kinase activity of crude extracts was optimally manifested during brief incubations conducted with highly purified eIF-2alpha and [-P]ATP of high specific activity. Sufficient expression of eIF-2alpha kinase by the cells from which extracts were derived was critical. For example, eIF-2alpha was not noticeably phosphorylated in extracts of HeLa or NIH-3T3 cells unless the cells were pretreated with interferon to induce PKR. It should be noted that, although interferon pretreatment increased PKR concentrations, the induced enzyme was not expressed in an activated form. Neither the phosphorylation state of eIF-2alpha nor the rate of leucine incorporation was prominently affected by interferon treatment. Similarly, the percentage of total eIF-2alpha phosphorylated in response to Ca-mobilizing agents did not change as a function of interferon pretreatment. Extracts derived from interferon-pretreated cells that were challenged with Ca-mobilizing drugs, however, exhibited significantly (HeLa) or markedly (NIH-3T3) increased eIF-2alpha kinase activity as compared to extracts of pretreated, non-challenged controls. Treatment with interferon, therefore, improved our detection in vitro of the eIF-2alpha kinase activation occurring in vivo. While all of the eIF-2 kinase of our extracts was apparently PKR, we could not exclude the possibility that other eIF-2 kinase activities were activated in vivo that were inactivated by lysis.

PKR is reported to be modestly phosphorylated in vivo and to phosphorylate in vitro in the absence of dsRNA(5) . Autophosphorylation is widely viewed as essential to the mechanism whereby dsRNA activates PKR both in vivo and in vitro. Binding of dsRNA to the amino terminus of the latent enzyme prompts the autophosphorylation(24) , resulting in a conformational change that is believed necessary for expression of eIF-2alpha kinase activity. Current evidence favors the hypotheses that latent PKR exists as a non-phosphorylated, ribosome-associated monomer and as a cytosolic dimer of partly phosphorylated subunits (25) and that cross-phosphorylation promotes dimerization(26, 27) . Whether mobilization of sequestered ER Ca stores causes PKR to autophosphorylate and/or to dimerize in vivo remains to be established. Activation of this cytosolic protein kinase by perturbation of the ER may represent a unique signaling mechanism. Depletion of ER Ca stores did not cause the enzyme to be compartmentalized, as has been reported for eIF-2alpha kinase activity after heat shock(28) . Increased cytoplasmic free Ca could not be implicated in the activation since identical findings were obtained when cells were exposed to extracellular EGTA to deplete the cytoplasmic cation pool. It is clear, however, that activation of PKR by this pathway involves a modification that is not destroyed during cell lysis, that favors autophosphorylation in vitro, and that renders the enzyme more susceptible to activation by low concentrations of dsRNA in vitro. Further investigations will be needed to ascertain whether an endogenous dsRNA activator, such as has been described in 3T3-F442A cells(29) , is generated or whether an endogenous inhibitor, such as the 58-kDa tetratricopeptide repeat protein(30) , dissociates from the enzyme in cells with depleted ER Ca stores.

The data of this report indicate that PKR is subject to activation by Ca mobilizing agents that are known to disrupt ER protein processing and to inhibit translational initiation in intact cells. This information, in conjunction with a lack of other candidates for the eIF-2alpha kinase activity, indicates that PKR probably functions in capacities extending beyond combating viral infections. Inhibition of translational initiation characterized by phosphorylation of eIF-2alpha occurs in eukaryotic cells experiencing various hormonal, nutritional, or environmental stressors(1, 2, 3) . While the exact components of the signaling system emanating from the ER that trigger PKR activation remain to be defined, the initiating events appear to involve the disruption of protein processing. It is clear that conditions or agents that either mobilize sequestered Ca from or disrupt the oxidizing environment of the organelle prevent the proper folding of newly synthesized proteins. Substantial evidence suggests that ER chaperones such as GRP78 that function in protein processing become limiting under such circumstances but are induced within several hours. This induction is accompanied by the dephosphorylation of eIF-2alpha and a limited resumption (approximately 50%) of translation(12, 13, 15) . It therefore appears that the activation of eIF-2 kinase is inversely proportional to the availability of GRP78.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant DK 35393, National Science Foundation Grant BNS 9021616, and a grant from the Foundation of the University 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 should be addressed: Dept. of Pharmacology, UMDNJ-Robert Wood Johnson Medical School, 675 Hoes Ln., Piscataway, NJ 08854. Tel.: 908-235-5240; Fax: 908-235-4073.

(^1)
The abbreviations used are: PKR, dsRNA-dependent/regulated protein kinase; ER, endoplasmic reticulum; eIF, eukaryotic initiation factor; dsRNA, double-stranded RNA; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Drs. Prakash Srivasatava and Randy Kaufman for helpful discussions concerning this work.


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