©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Calcium Depletion from the Endoplasmic Reticulum Activates the Double-stranded RNA-dependent Protein Kinase (PKR) to Inhibit Protein Synthesis (*)

Sri Prakash Srivastava (1), Monique V. Davies (3), Randal J. Kaufman (1) (2)(§)

From the (1)Department of Biological Chemistry and the (2)Howard Hughes Medical Institute, University of Michigan Medical Center, Ann Arbor, Michigan 48105 and the (3)Genetics Institute, Cambridge, Massachusetts 02140

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Calcium depletion from the endoplasmic reticulum inhibits protein synthesis and correlates with increased phosphorylation of the subunit of eukaryotic initiation factor 2 (eIF-2) by a mechanism that does not require ongoing protein synthesis. To elucidate whether protein synthesis inhibition requires eIF-2 phosphorylation and whether eIF-2 phosphorylation is mediated by the double-stranded RNA-dependent protein kinase (PKR), we studied protein synthesis in response to calcium depletion mediated by calcium ionophore A23187 in cell lines overexpressing wild-type eIF-2, a mutant eIF-2 (S51A) that is resistant to phosphorylation, or a dominant negative mutant PKR (K296P in catalytic subdomain II). Expression of either mutant eIF-2 or mutant PKR partially protected NIH3T3 cells from inhibition of protein synthesis upon A23187 treatment. In contrast, overexpression of wild-type PKR increased sensitivity to protein synthesis inhibition mediated by A23187 treatment. In a COS-1 monkey cell transient transfection system, increased eIF-2 phosphorylation in response to A23187 treatment was inhibited by expression of the dominant negative PKR mutant. Overexpression of the PKR regulatory RNA binding domain, independent of the PKR catalytic domain, was sufficient to inhibit increased phosphorylation of eIF-2 upon A23187 treatment. In addition, overexpression of the HIV TAR RNA binding protein also inhibited eIF-2 phosphorylation upon A23187 treatment. Taken together, our data show that calcium depletion activates PKR to phosphorylate eIF-2, and this activation is likely mediated through the PKR RNA binding domain.


INTRODUCTION

Utilization of eukaryotic initiation factor-2 (eIF-2)()is an important control point in the regulation of protein synthesis initiation(1, 2) . eIF-2 is a heterotrimer composed of , , and subunits and forms a ternary complex with initiator methionine tRNA and GTP. This ternary complex binds to the 40 S ribosomal subunit and to mRNA to form a 48 S species. Upon 60 S ribosomal subunit joining to the 48 S species, eIF-2-bound GTP is hydrolyzed to GDP. For eIF-2 to promote another round of initiation, GTP must exchange for GDP, a reaction catalyzed by the guanine nucleotide exchange factor or eIF-2B(3, 4) . The phosphorylation of serine residue 51 in eIF-2 increases the affinity of eIF-2 for GDP and thereby prevents GTP exchange and inhibits further initiation events(5) . In mammalian cells, protein synthesis is immediately controlled through environmental changes. For example, protein synthesis is rapidly inhibited in response to heat shock(6, 7) , amino acid deprivation, glucose deprivation, serum starvation(8) , or viral infection(9, 10) . Under these conditions, phosphorylation of eIF-2 correlates with inhibition of protein synthesis, although the specific kinases and/or phosphatases that regulate eIF-2 phosphorylation are poorly characterized(11) .

In mammalian cells, two related serine/threonine protein kinases that phosphorylate eIF-2 are the hemin-regulated inhibitor and the double-stranded RNA-activated inhibitor (PKR)(12) . Hemin-regulated protein kinase is primarily expressed in erythroid cells and is activated by reduced levels of hemin. PKR is found in most cells, its synthesis is induced by interferon, and its activity is dependent upon dsRNA. PKR has a conserved serine/threonine protein kinase catalytic domain in its carboxyl terminus and two RNA binding motifs in its amino terminus(12, 13, 14) . After activation, PKR displays two known distinct substrate specificities: autophosphorylation (15, 16) and phosphorylation of eIF-2(2) . Recent studies also suggest that PKR activates the nuclear transcription factor NFB through phosphorylation of its inhibitor IB(17) . PKR-mediated phosphorylation of eIF-2 and subsequent inhibition of protein synthesis is implicated in the antiviral and antiproliferative effect of interferons(18, 19) . Interferon-resistant viruses encode specific gene products that circumvent the translation inhibition imposed by PKR activation(9, 10) . In addition to viral gene products, cellular proteins such as the TAR RNA binding protein (TRBP) (20) and p58 (21) have been implicated in regulating PKR activation and may regulate eIF-2 phosphorylation to affect global protein synthesis and cell growth in response to a variety of different stimuli. Expression of a dominant negative mutant of PKR transforms NIH3T3 cells (22, 23) and suggests that regulation of eIF-2 phosphorylation may represent a significant mechanism for growth control.

The endoplasmic reticulum (ER) is the site where initial folding and processing of newly synthesized secretory proteins occurs and is also the major site of calcium storage in the cell(24, 25) . In response to hormonally generated inositol triphosphate, calcium is released to the cytoplasm as part of the signal transduction cascade(24) . Sequestered calcium can be released experimentally by treating cells with calcium ionophores (26) or inhibitors of the microsomal calcium-dependent ATPase such as thapsigargin(27) . Depletion of calcium from the endoplasmic reticulum correlates with inhibition of protein synthesis and increased eIF-2 phosphorylation (28, 29), although the serine/threonine kinase(s) involved is unknown. We have studied the effect of overexpression of wild-type and mutants of both eIF-2 and PKR on eIF-2 phosphorylation and translation upon calcium depletion. Our data suggest that PKR mediates eIF-2 phosphorylation and inhibits protein synthesis.


EXPERIMENTAL PROCEDURES

Vector Construction

The eIF-2 wild-type and S51A mutant (30) cDNAs encoded on 1.6-kilobase EcoRI fragments were subcloned into the unique EcoRI site of the pEDmtxVA expression vector, a derivative of pED-mtx(31) lacking the adenovirus VAI RNA gene. An XbaI-SalI fragment from pBS-8.4 (encoding wild-type PKR cDNA (13) and kindly provided by Dr. B. Williams (Cleveland Clinic, OH)) was subcloned into the expression vector pMT2 (32). Subsequently, a 2-kilobase PstI fragment (5`-PstI from the pMT2 polylinker and 3`-PstI in the 3`-untranslated region of PKR) was subcloned into the PstI cloning site of pED-mtxVA. Site-directed mutagenesis (32) introduced a K296P (codon AAA to CCA) mutation into the wild-type PKR expression vector. The EcoRI-PstI fragment encoding PKR mutant K296P was subcloned into the expression vector pETF VA to yield pK296P ETF(33) . T7 epitope-tagged versions of wild-type PKR, K296P, and a truncated PKR comprising amino acid residues 1-243 encoding the RNA binding domain were constructed by polymerase chain reaction amplification using specific primers to introduce a T7 epitope tag at the carboxyl-terminal end of PKR or after residue 243. The 5`-primer was 5`-AACTGCAGCCACCATGGCTGGTGATCTTTCA-3`. The 3`-primer for full-length PKR was 5`-ACGCGTCGACCTAACCCATTTGCTGTCCACC-AGTCATGCTAGCCATACATGTGTGTCGTTCATT-3`. The 3`-primer for truncated PKR was 5`-ACGCGTCGACCTAACCCATTTGCTGTCCACCAGTCATGCTAGCCATTGCCAAAGATCTTTTTGC-3`. Amplified fragments were subcloned into the TA cloning vector (Invitrogen). PstI/SalI fragments were isolated from the TA cloning vector and subcloned into pETF VA (33). DNA sequence analysis confirmed the correct sequence at the 5`- and 3`-ends of the polymerase chain reaction amplified and cloned fragments.

Derivation and Characterization of Cell Lines

eIF-2 wild-type, mutant S51A, or PKR K296P contained within the pEDmtxVA vector were cotransfected with pSVNeo (ratio of 10:1) into NIH3T3 cells (provided by Dr. S. Aaronson), and transformants were selected for growth in G418 (1 mg/ml, Life Technologies, Inc.). NCTC clone 929 cells (ATCC CCL 1) were cotransfected with pED-mtxVA PKR and pSVNeo (ratio of 10:1), selected for growth in G418 (1.0 mg/ml), and subsequently selected for gene amplification by growth in sequentially increasing concentrations of methotrexate up to 0.3 µM. All cell lines used in this study were cloned from selected pools by limiting dilution.

Exponentially growing cells were treated with calcium ionophore A23187 (Sigma) in Dulbecco's modified essential medium containing 10% heat-inactivated fetal bovine serum for 15 min at 37 °C. Cells were rinsed with methionine-free and cysteine-free medium and then pulse labeled in methionine-free and cysteine-free Dulbecco's modified essential medium containing 50 µCi/ml [S]methionine and [S]cysteine (DuPont NEN) for 15 min in the presence of drug. Cells were rinsed in cold phosphate-buffered saline and harvested in NP4O lysis buffer as described(34) . Protein concentrations were determined(35) , and equal amounts of protein were analyzed by reducing SDS-polyacrylamide gel electrophoresis (PAGE). Autoradiography was performed after treatment with EN/HANCE (DuPont NEN). Expression of PKR and eIF-2 were measured by Western blot analysis using polyclonal anti-PKR antibody or polyclonal anti-eIF-2 antibody (kindly provided by Drs. Hovanessian and Hershey).

COS-1 Cell DNA Transfection and Analysis

COS-1 cells were transfected by the DEAE-dextran procedure(36) . At 60 h post-transfection, cells were treated with calcium ionophore A23187 for 15 min at 37 °C and then labeled with [S]methionine and [S]cysteine in methionine and cysteine-free minimal essential media (Life Technologies, Inc.) for an additional 15 min. Cell extracts were harvested in Nonidet P-40 lysis buffer in the presence of the protease inhibitors soybean trypsin inhibitor (1.0 mg/ml), aprotinin (1.0 mg/ml), and phenylmethylsulfonyl fluoride (1.0 mM). eIF-2 was immunoprecipitated from cell extracts using monoclonal anti-eIF-2 antibody (kindly provided by the late Dr. Henshaw) and protein A-Sepharose as the immunoadsorbent. Steady state levels of eIF-2 were measured by SDS-PAGE and Western immunoblotting procedures using anti-eIF-2 polyclonal antibody(37) . T7 epitope-tagged proteins were detected by immunoprecipitation from cell extracts using anti-T7 antibody (Novagen, Madison, WI) and protein A-Sepharose as the immunoadsorbent.

The phosphorylation status of eIF-2 expressed in COS-1 cells at 60 h post-transfection or in NIH3T3 cells was monitored by labeling cells with 2 ml of [P]phosphate (400 µCi of orthophosphoric acid, DuPont NEN) for 4 h. Thapsigargin (300 nM, Sigma) or A23187 were added during the last 15 min of the labeling. Cycloheximide (10 µg/ml, Sigma) was added 15 min prior to the drug treatments and was maintained through the course of the labeling. Cell extracts were prepared and immunoprecipitated with monoclonal anti-eIF-2 antibody using protein A-Sepharose as the immunoadsorbent. Samples were analyzed by SDS-PAGE and autoradiography. All band intensities were quantified using NIH Image 1.55b program.


RESULTS

NIH3T3 cells were derived that overexpress either eIF-2 wt or a mutant eIF-2 S51A in which the site of PKR-mediated phosphorylation was mutated to alanine. Western blot analysis demonstrated that the steady state level of eIF-2 was significantly elevated in both wild-type (6.4-fold, Fig. 1A, lane2) and mutant eIF-2 S51A (8.8-fold, Fig. 1A, lane3) transfected cell lines compared to a cell line transfected with the original vector alone (Fig. 1A, lane1). The overexpression of eIF-2 wild-type or S51A mutant did not significantly alter the cell growth rate or saturation density of the cells (data not shown). NIH3T3 cells were derived that overexpress a PKR mutant K296P in catalytic subdomain II. Western blot analysis using an anti-PKR polyclonal antibody detected expression of PKR K296P migrating at 72 kDa under conditions where the endogenous PKR was not detected in nontransfected cells (Fig. 1A, lanes4 and 5). Overexpression of PKR K296P increased the growth rate and increased saturation density similar to previous observations(22, 23) .


Figure 1: Expression of eIF-2 S51A or PKR K296P protects cells from A23187-mediated inhibition of protein synthesis. PanelA, cell extracts were prepared for Western immunoblot analysis using anti-eIF-2 polyclonal antibody (lanes1-3) or anti-PKR polyclonal antibody (lanes4 and 5) as described under ``Experimental Procedures.'' Clones shown were obtained from transfection with pED-mtxVA vector alone (lanes1 and 4), eIF-2 wild-type vector (wt 5, lane2), eIF-2 S51A (51(2), lane3), and mutant PKR K296P (KP3A, lane5). PanelB, cells were treated with increasing concentrations of A23187, and [S]methionine and [S]cysteine incorporation into trichloroacetic acid-precipitable protein was measured as described under ``Experimental Procedures.'' The cpm/µg protein are represented as the percent of control obtained from a parallel dish without A23187 treatment. PanelC, SDS-PAGE analysis of extracts from cells pulse labeled with (+) or without (-) A23187 treatment was as described under ``Experimental Procedures.'' The figure shows an autoradiogram of the dried gel. Cell lines analyzed are indicated at the top. Molecular weight markers are indicated at the right. PanelD, cell extracts were immunoprecipitated with anti-eIF-2 monoclonal antibody and analyzed by SDS-PAGE.



The effect of wild-type eIF-2, eIF-2 mutant S51A, or PKR mutant K296P overexpression on translation inhibition in response to calcium depletion was monitored by treating exponentially growing NIH3T3 cells with the calcium ionophore A23187 for 15 min and metabolic pulse labeling. Compared to untreated cells, A23187 treatment reduced protein synthesis to 13% in the original NIH3T3 cells and to 14% in cells that overexpress eIF-2 wt. In contrast, A23187 treatment reduced protein synthesis to 31% in cells that overexpress mutant eIF-2 S51A and to 29% in cells that overexpress PKR mutant K296P. Cells overexpressing either mutant eIF-2 S51A or PKR mutant K296P were more resistant to inhibition of protein synthesis over a range of A23187 concentrations compared to control cells that overexpress eIF-2 wt (Fig. 1B). Overexpression of wild-type eIF-2, mutant eIF-2 S51A, or mutant PKR K296P did not detectably alter the specificity of polypeptides synthesized compared to control cells (Fig. 1C, lanes1, 3, 5, and 7). In addition, A23187 treatment did not significantly alter the spectrum of polypeptide synthesized in these cell lines. These results suggest that expression of either mutant eIF-2 S51A or mutant PKR K296P protected the cells from inhibition of global protein synthesis upon A23187 treatment and did not protect translation of selective mRNAs.

The synthesis of eIF-2 in the control and transfected cells was measured by immunoprecipitation of equal amounts of protein from the cell extracts with anti-eIF-2 antibody and analysis by SDS-PAGE. The synthesis of eIF-2 was increased in cells transfected with eIF-2 wt or eIF-2 S51A (Fig. 1D, lanes3 and 5) compared to control NIH3T3 or cells that overexpress PKR K296P (Fig. 1D, lanes1 and 7). eIF-2 synthesis was reduced approximately 5-fold upon A23187 treatment in control NIH3T3 cells (Fig. 1D, lanes1 and 2) and in cells that overexpress eIF-2 wt (Fig. 1D, lanes3 and 4). In contrast, eIF-2 synthesis was not significantly reduced upon A23187 treatment in the cells that overexpress mutant eIF-2 S51A (Fig. 1D, lanes5 and 6) or mutant PKR K296P (Fig. 1D, lanes7 and 8). The effects of A23187 treatment on eIF-2 synthesis paralleled the effects on global protein synthesis in the different cell lines.

The effect of calcium depletion on phosphorylation of eIF-2 was measured in NIH3T3 cells that overexpress eIF-2 wt. Immunoprecipitation of eIF-2 from [P]phosphate-labeled NIH3T3 cells that overexpress eIF-2 wt detected [P]phosphate incorporation into eIF-2 (Fig. 2, lane1). Treatment with either A23187 or thapsigargin increased the [P]phosphate incorporation by 3-fold (Fig. 2, lanes3 and 5). Whereas treatment with the translation elongation inhibitor cycloheximide (10 µg/ml, a concentration that inhibited protein synthesis greater than 90%) slightly reduced [P]phosphate incorporation in NIH3T3 cells that overexpress eIF-2 wt, it did not significantly inhibit the increase in phosphorylation observed upon treatment with A23187 or thapsigargin (Fig. 2, lanes4 and 6). These results show that calcium depletion mediated by either A23187 or thapsigargin increases eIF-2 phosphorylation by a mechanism that does not require ongoing protein synthesis.


Figure 2: Increased eIF-2 phosphorylation by calcium depletion does not require ongoing protein synthesis. NIH3T3 cells that overexpress wild-type eIF-2 were labeled with [P]phosphate for 4 h and then treated with A23187 or thapsigargin in the presence (+) and absence (-) of cycloheximide (CHX) as described under ``Experimental Procedures.'' Cell extracts were prepared and immunoprecipitated with anti-eIF-2 monoclonal antibody and analyzed by SDS-PAGE.



To further characterize the role of PKR in response to calcium depletion, we studied the effect of wild-type PKR overexpression on protein synthesis upon A23187 treatment. To date, we have not been able to obtain wild-type PKR overexpression in stably transfected NIH3T3 cells, possibly due to its negative effect on cell growth(38) . However, we were able to establish mouse fibroblast L929 cell lines that overexpress wild-type PKR. Western blot analysis of two independent cell lines transfected with wild-type PKR detected PKR protein under conditions where the endogenous PKR was not detected in nontransfected cells (Fig. 3A, data not shown). Treatment with 1 µM A23187 reduced protein synthesis to 40% in control L929 cells and to 17% in cells that overexpress wild-type PKR (Fig. 3B, lanes4 and 7). Treatment with 5 µM A23187 completely inhibited protein synthesis in cells that overexpress wild-type PKR, whereas residual protein synthesis was detected in control L929 cells (Fig. 3B, lanes5 and 8). Similar results were derived with an independently derived clone of L929 cells that overexpresses wild-type PKR (data not shown). These results demonstrate that overexpression of wild-type PKR sensitizes the cell to inhibition of protein synthesis mediated by calcium depletion.


Figure 3: Overexpression of wild-type PKR sensitizes L929 cells to A23187. PanelA, cell lysates from the original L292 cells and from clone 2 were prepared and analyzed by Western blot analysis using anti-PKR polyclonal antibody as described under ``Experimental Procedures.'' PanelB, cells were pulse labeled with [S]methionine and [S]cysteine in the presence (1, 5) or absence (-) of A23187 treatment (1 or 5 µM as indicated), and cell extracts were prepared for SDS-PAGE as described under ``Experimental Procedures.''



The role of RNA binding in the ability for PKR mutant K296P to inhibit eIF2 phosphorylation in response to A23187 was studied by transient overexpression of either the PKR RNA binding domain (amino acid residues 1-243) expressed independently or of another RNA binding protein, the HIV TRBP in COS-1 cells. The PKR mutant K296P and the amino-terminal 1-243 amino acids of PKR were engineered with a bacteriophage T7 epitope tag in the carboxyl terminus. The ability of these engineered molecules to inhibit activation of endogenous PKR was tested by a cotransfection assay to measure the rescue of translation of a reporter mRNA encoding adenosine deaminase that is not translated due to PKR activation(33) . Each construct was cotransfected with the adenosine deaminase expression vector p9A, and total protein synthesis was measured by SDS-PAGE analysis of cell extracts. Upon cotransfection with the T7-tagged wild-type PKR expression vector, adenosine deaminase translation was not detected (Fig. 4A, lane2), whereas cotransfection with either the T7-tagged RNA binding domain(1-243) or the T7-tagged PKR mutant K296P increased adenosine deaminase translation (Fig. 4A, lanes3 and 4). In addition, a 35-kDa polypeptide and a 72-kDa polypeptide, the expected size for the T7-tagged proteins, were detected upon analysis of the total cell extracts (Fig. 4A, lanes3 and 4) and were specifically immunoprecipitated with anti-T7 antibody (Fig. 4A, lanes6 and 7). These results show that addition of the T7 tag to the carboxyl terminus of either PKR K296P or the RNA binding domain did not interfere with the ability of these proteins to inhibit endogenous PKR and rescue adenosine deaminase translation. Previous studies demonstrated inhibition of endogenous PKR activity by expression of TRBP in COS-1 cells(20) .


Figure 4: PKR mutant K296P, truncated PKR 1-243, and TRBP inhibit A23187-induced eIF-2 phosphorylation. Human cDNAs encoding PKR wild-type, mutant K296P, or truncated 1-243 were engineered with a T7 epitope tag in the pETF VA vector. PanelA, The T7-tagged constructs were cotransfected into COS-1 cells with the adenosine deaminase expression vector p9A (33). At 60 h post-transfection, cells were pulse labeled with [S]methionine and [S]cysteine, and cell extracts were prepared for analysis by SDS-PAGE before (lanes1-4) and after (lanes5-7) immunoprecipitation with anti-T7 monoclonal antibody. PanelsB and C, COS-1 cells were cotransfected with the wild-type eIF-2 cDNA in the expression plasmid pED-mtxVA and the indicated T7-tagged expression vectors and analyzed for eIF-2 phosphorylation as described under ``Experimental Procedures.''



The effect of PKR mutant K296P, truncated 1-243 PKR, and TRBP on phosphorylation of eIF-2 in response to A23187 treatment was studied by cotransfection of these expression vectors with an eIF-2 expression vector. Since the subpopulation of transfected cells express both plasmid DNA molecules, the effect of protein expression from one plasmid on the phosphorylation status of eIF-2 overexpressed from a second plasmid (pED-2 wt) can be monitored. At 60 h post-transfection, the effect of A23187 treatment on the phosphorylation status of the overexpressed eIF-2 was determined by measuring the amount of [P]phosphate incorporation into eIF-2 and comparing it to the steady state eIF-2 level measured by Western blot analysis. Western blot analysis showed that cells cotransfected with pED-2 wt had increased levels of eIF-2 (Fig. 4, B and C (bottom), lanes10-16 and 19-22) compared to cells that did not receive DNA (Fig. 4, B and C (bottom), lanes8, 9, 17, and 18). In the presence of wild-type PKR cotransfection, the level of eIF-2 was slightly reduced (Fig. 4B (bottom), lane16), consistent with inhibition of protein synthesis mediated by wild-type PKR. In addition, A23187 treatment did not alter the steady state level of eIF-2 (Fig. 4, B and C (bottom), lanes11, 13, 15, 20, 22). Analysis of immunoprecipitated eIF-2 by SDS-PAGE and autoradiography showed significant [P]phosphate incorporation into eIF-2 in cells transfected with pED-2 wt alone (Fig. 4B, lane10). A23187 treatment increased phosphate incorporation into eIF-2 by 5-fold (Fig. 4B, lane11). In the presence of wild-type PKR cotransfection, eIF-2 was significantly phosphorylated (Fig. 4B, lane16) to a degree similar to that observed by treatment with A23187 (Fig. 4B, lane11). In cells cotransfected with PKR mutant K296P (Fig. 4B, lane12) or the RNA binding domain alone (Fig. 4B, lane14), phosphorylation of eIF-2 was reduced and, most significantly, was not increased upon A23187 treatment (Fig. 4B, lanes13 and 15). Similarly, cotransfection with the TRBP expression vector with pED-2 wt showed that TRBP expression inhibited eIF-2 phosphorylation in the absence or presence of A23187 treatment (Fig. 4C, lanes21 and 22) compared to the absence of TRBP (Fig. 4C, lanes19 and 20). Thus, expression of either the PKR RNA binding domain or TRBP, another dsRNA binding protein, can inhibit eIF-2 phosphorylation mediated by calcium depletion.


DISCUSSION

The data presented here demonstrate that PKR mediates inhibition of protein synthesis in response to calcium depletion by phosphorylation of eIF-2. The endoplasmic reticulum is the principal cellular storage site for calcium and is also the site where initial folding of newly synthesized secretory proteins occurs. Intralumenal calcium is required for the folding and secretion of selective proteins (39). Depletion of intralumenal calcium immediately disrupts normal protein folding, inhibits further translation initiation events, and increases phosphorylation of eIF-2(40) . Agents that disrupt protein folding in the ER also induce eIF-2 phosphorylation and inhibit protein synthesis(41) . The prolonged presence of unfolded proteins in the ER activates transcription of the glucose-regulated genes through a mechanism that requires ongoing protein synthesis(42, 43, 44) . In contrast, the immediate increase in eIF-2 phosphorylation in response to calcium depletion studied here did not require ongoing protein synthesis. Thus, it appears these two events are mediated through different signaling pathways. It is presently unknown whether the proximal signal to induce eIF-2 phosphorylation is calcium depletion or the presence of misfolded proteins in the ER.

Overexpression of eIF-2 mutant S51A, the site of phosphorylation mediated by eIF-2 kinases PKR and hemin-regulated protein kinase (12), partially protected the cells from protein synthesis inhibition mediated by calcium ionophore A23187 treatment. These findings suggest that calcium depletion can inhibit protein synthesis through an eIF-2 kinase that phosphorylates serine residue 51. Expression of either the PKR dominant negative mutant K296P or eIF-2 mutant S51A protected cells to the same degree from inhibition of protein synthesis mediated by calcium depletion. In addition, overexpression of wild-type PKR sensitized cells to ionophore A23187 treatment, further implicating PKR in mediating the response. This latter finding is consistent with observations that interferon treatment, known to induce PKR expression, sensitizes cells to the antiviral effects of A23187 treatment(45) . It is unlikely that altered calcium storage resulting from transformation mediated by PKR mutant K296P expression (22, 23) elicits protection from the A23187 insult because similar protection was observed by transient expression of the PKR K296P in COS-1 cells, and increased A23187 sensitivity was observed upon stable overexpression of wild-type PKR in L929 cells. The sum of these results strongly implicates PKR in mediating eIF-2 phosphorylation in response to calcium depletion.

PKR is activated by binding to dsRNA(14, 46, 47) . The shortest RNA-RNA duplexes capable of binding PKR are approximately 30 base pairs; however, the binding efficiency increases with increasing length up to 85 base pairs(14) . In addition, some single-stranded RNAs by virtue of their stem-loop structures can bind and either promote or prevent activation of PKR(19) . Following stable dsRNA-PKR complex formation, a conformational change likely occurs with subsequent autophosphorylation and activation(19) . PKR can also be activated by polyanion molecules (48), although the mechanism is poorly understood. A transient expression system was used to characterize the inducer of PKR-mediated eIF-2 phosphorylation in response to calcium depletion. Our results demonstrated that expression of a functionally defective PKR mutant K296P, the RNA binding domain alone, or the dsRNA binding protein TRBP all inhibited the A23187-induced phosphorylation of eIF-2 and lead us to propose two mechanisms by which these proteins inhibit the calcium-mediated activation of PKR. First, the PKR mutant K296P, the PKR RNA binding domain, and TRBP may form dimers with endogenous wild-type PKR and subsequently prevent its trans-autophosphorylation and activation upon calcium depletion. In support of this possibility, we have recently demonstrated by coimmunoprecipitation that intact PKR interacts with both the PKR RNA binding domain (residues 1-243) as well as TRBP when coexpressed in COS-1 cells.()Alternatively, since the K296P mutant PKR, the RNA binding domain, and TRBP all have in common the ability to bind dsRNA, it is possible that calcium depletion induces a unique dsRNA PKR activator. Increased cytosolic calcium may alter the secondary structure of some RNA molecules to become good PKR activators. Although there are several known cellular gene products that inhibit PKR activity(20, 49, 50, 51) , the finding that overexpression of mutant PKR K296P, the PKR RNA binding domain, and TRBP all prevent PKR-mediated phosphorylation of eIF-2 in response to calcium depletion suggests it is unlikely that calcium depletion directly inhibits the activity of a cellular PKR inhibitor.

Recent results suggest that PKR may phosphorylate IB to activate the transcription factor NFB and induce expression from NFB-dependent promoters(17) . In addition, calcium mobilization is a primary mechanism in signal transduction and is implicated in the activation of specific gene transcription upon cell activation or differentiation(52) . The question arises as to why calcium depletion from the ER would be associated with both transcriptional activation as well as translational inhibition? We propose that change in a cell program involves a coupling between disassembly of polysomes mediated by PKR activation and recruitment of free ribosomes to newly transcribed mRNAs. It is possible that this coordinated response at both the level of mRNA generation and at the level of mRNA utilization is required to establish specific changes in gene expression.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants HL5777 and HL5273 (to R. J. K.). 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: Tel.: 313-763-9037; Fax: 313-763-9323.

The abbreviations used are: eIF-2, eukaryotic translation initiation factor 2; PKR, double-stranded RNA-activated protein kinase; ER, endoplasmic reticulum; PAGE, polyacrylamide gel electrophoresis; wt, wild type; TRBP, TAR RNA binding protein; ds, double stranded.

S. Wu and R. J. Kaufman, unpublished results.


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