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• W2110374497 abstract "Calcium depletion from the endoplasmic reticulum inhibits protein synthesis and correlates with increased phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF-2α) by a mechanism that does not require ongoing protein synthesis. To elucidate whether protein synthesis inhibition requires eIF-2α phosphorylation and whether eIF-2α 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-2α, a mutant eIF-2α (S51A) that is resistant to phosphorylation, or a dominant negative mutant PKR (K296P in catalytic subdomain II). Expression of either mutant eIF-2α 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-2α 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-2α upon A23187 treatment. In addition, overexpression of the HIV TAR RNA binding protein also inhibited eIF-2α phosphorylation upon A23187 treatment. Taken together, our data show that calcium depletion activates PKR to phosphorylate eIF-2α, and this activation is likely mediated through the PKR RNA binding domain. Calcium depletion from the endoplasmic reticulum inhibits protein synthesis and correlates with increased phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF-2α) by a mechanism that does not require ongoing protein synthesis. To elucidate whether protein synthesis inhibition requires eIF-2α phosphorylation and whether eIF-2α 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-2α, a mutant eIF-2α (S51A) that is resistant to phosphorylation, or a dominant negative mutant PKR (K296P in catalytic subdomain II). Expression of either mutant eIF-2α 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-2α 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-2α upon A23187 treatment. In addition, overexpression of the HIV TAR RNA binding protein also inhibited eIF-2α phosphorylation upon A23187 treatment. Taken together, our data show that calcium depletion activates PKR to phosphorylate eIF-2α, and this activation is likely mediated through the PKR RNA binding domain. Utilization of eukaryotic initiation factor-2 (eIF-2)1 1The abbreviations used are: eIF-2eukaryotic translation initiation factor 2PKRdouble-stranded RNA-activated protein kinaseERendoplasmic reticulumPAGEpolyacrylamide gel electrophoresiswtwild typeTRBPTAR RNA binding proteindsdouble stranded. 1The abbreviations used are: eIF-2eukaryotic translation initiation factor 2PKRdouble-stranded RNA-activated protein kinaseERendoplasmic reticulumPAGEpolyacrylamide gel electrophoresiswtwild typeTRBPTAR RNA binding proteindsdouble stranded. is an important control point in the regulation of protein synthesis initiation(1Jackson R.J. Trachsel H. Translations in Eukaryotes. CRC Press, Inc., Boca Raton, FL1990: 193-229Google Scholar, 2Hershey J.W.B. Annu. Rev. Biochem. 1991; 60: 717-755Crossref PubMed Scopus (840) Google Scholar). eIF-2 is a heterotrimer composed of α, β, and γ subunits and forms a ternary complex with initiator methionine tRNAi 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(3Amesz H. Boumans H. Haubrich-Morre T. Voorma H.O. Benne R. Eur. J. Biochem. 1979; 98: 513-520Crossref PubMed Scopus (53) Google Scholar, 4Konieczny A. Safer B. J. Biol. Chem. 1983; 258: 3402-3408Abstract Full Text PDF PubMed Google Scholar). The phosphorylation of serine residue 51 in eIF-2α increases the affinity of eIF-2 for GDP and thereby prevents GTP exchange and inhibits further initiation events(5Rowlands A.G. Panniers R. Henshaw E.C. J. Biol. Chem. 1988; 263: 5526-5533Abstract Full Text PDF PubMed Google Scholar). In mammalian cells, protein synthesis is immediately controlled through environmental changes. For example, protein synthesis is rapidly inhibited in response to heat shock(6Craig E. CRC Crit. Rev. Biochem. 1985; 18: 239-280Crossref PubMed Scopus (569) Google Scholar, 7Duncan R. Hershey J.W.B. J. Biol. Chem. 1985; 260: 5493-5497Abstract Full Text PDF PubMed Google Scholar), amino acid deprivation, glucose deprivation, serum starvation(8Duncan R. Hershey J.W.B. J. Biol. Chem. 1984; 259: 11882-11889Abstract Full Text PDF PubMed Google Scholar), or viral infection(9Katze M.B. Semin. Virol. 1993; 4: 259-268Crossref Scopus (47) Google Scholar, 10Schneider R.J. Shenk T. Annu. Rev. Biochem. 1987; 56: 317-332Crossref PubMed Scopus (134) Google Scholar). Under these conditions, phosphorylation of eIF-2α correlates with inhibition of protein synthesis, although the specific kinases and/or phosphatases that regulate eIF-2α phosphorylation are poorly characterized(11Hershey J.W.B. Semin. Virol. 1993; 4: 201-207Crossref Scopus (10) Google Scholar).In mammalian cells, two related serine/threonine protein kinases that phosphorylate eIF-2α are the hemin-regulated inhibitor and the double-stranded RNA-activated inhibitor (PKR)(12Samuel C.E. J. Biol. Chem. 1993; 268: 7603-7606Abstract Full Text PDF PubMed Google Scholar). 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(12Samuel C.E. J. Biol. Chem. 1993; 268: 7603-7606Abstract Full Text PDF PubMed Google Scholar, 13Meurs E. Chong K. Galabru J. Thomas N.S. Kerr I.M. Williams B.R. Hovanessian A.G. Cell. 1990; 62: 379-390Abstract Full Text PDF PubMed Scopus (812) Google Scholar, 14Manche L. Green S.R. Schmedt C. Mathews M.B. Mol. Cell. Biol. 1992; 12: 5238-5248Crossref PubMed Scopus (414) Google Scholar). After activation, PKR displays two known distinct substrate specificities: autophosphorylation (15Galabru J. Katze M.G. Robert N. Hovanessian A.G. Eur. J. Biochem. 1989; 178: 581-589Crossref PubMed Scopus (118) Google Scholar, 16Thomis D.C. Samuel C.E. J. Virol. 1993; 67: 7695-7700Crossref PubMed Google Scholar) and phosphorylation of eIF-2α(2Hershey J.W.B. Annu. Rev. Biochem. 1991; 60: 717-755Crossref PubMed Scopus (840) Google Scholar). Recent studies also suggest that PKR activates the nuclear transcription factor NFκB through phosphorylation of its inhibitor IκB(17Kumar A. Haque J. Lacoste J. Hiscott J. Williams B.R.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6288-6292Crossref PubMed Scopus (508) Google Scholar). PKR-mediated phosphorylation of eIF-2α and subsequent inhibition of protein synthesis is implicated in the antiviral and antiproliferative effect of interferons(18Hovanessian A.G. Semin. Virol. 1993; 4: 237-245Crossref Scopus (49) Google Scholar, 19Mathews M.B. Semin. Virol. 1993; 4: 247-257Crossref Scopus (64) Google Scholar). Interferon-resistant viruses encode specific gene products that circumvent the translation inhibition imposed by PKR activation(9Katze M.B. Semin. Virol. 1993; 4: 259-268Crossref Scopus (47) Google Scholar, 10Schneider R.J. Shenk T. Annu. Rev. Biochem. 1987; 56: 317-332Crossref PubMed Scopus (134) Google Scholar). In addition to viral gene products, cellular proteins such as the TAR RNA binding protein (TRBP) (20Park H. Davies M.V. Langland J.O. Chang H-W. Nam Y.S. Tartaglia J. Paoletti E. Jacobs B.L. Kaufman R.J. Venkatesan S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4713-4717Crossref PubMed Scopus (185) Google Scholar) and p58 (21Lee T.G. Tang N. Thompson S. Miller J. Katze M.G. Mol. Cell. Biol. 1994; 14: 2331-2342Crossref PubMed Google Scholar) have been implicated in regulating PKR activation and may regulate eIF-2α 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 (22Koromilas A.E. Roy S. Barber G.N. Katze M.G. Sonenberg N. Science. 1992; 257: 1685-1689Crossref PubMed Scopus (494) Google Scholar, 23Meurs E.F. Galabru J. Barber G.N. Katze M.G. Hovanessian A.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 232-236Crossref PubMed Scopus (414) Google Scholar) and suggests that regulation of eIF-2α 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(24Berridge M.J. Annu. Rev. Biochem. 1987; 56: 159-193Crossref PubMed Scopus (2440) Google Scholar, 25Sambrook J.F. Cell. 1990; 61: 197-199Abstract Full Text PDF PubMed Scopus (237) Google Scholar). In response to hormonally generated inositol triphosphate, calcium is released to the cytoplasm as part of the signal transduction cascade(24Berridge M.J. Annu. Rev. Biochem. 1987; 56: 159-193Crossref PubMed Scopus (2440) Google Scholar). Sequestered calcium can be released experimentally by treating cells with calcium ionophores (26Weissmann G. Anderson P. Serhan C. Samuelsson E. Goodman E. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 1506-1510Crossref PubMed Scopus (52) Google Scholar) or inhibitors of the microsomal calcium-dependent ATPase such as thapsigargin(27Thastrup O. Cullen P.J. Drobak B.K. Hanley M.R. Dawson A.P. Drbak B.K. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2466-2470Crossref PubMed Scopus (2986) Google Scholar). Depletion of calcium from the endoplasmic reticulum correlates with inhibition of protein synthesis and increased eIF-2α phosphorylation (28Preston S.F. Berlin R.D. Cell Calcium. 1992; 13: 303-312Crossref PubMed Scopus (30) Google Scholar, 29Prostko C.R. Brostrom M.A. Malara E.M. Brostrom C.O. J. Biol. Chem. 1992; 267: 16751-16754Abstract Full Text PDF PubMed Google Scholar), although the serine/threonine kinase(s) involved is unknown. We have studied the effect of overexpression of wild-type and mutants of both eIF-2α and PKR on eIF-2α phosphorylation and translation upon calcium depletion. Our data suggest that PKR mediates eIF-2α phosphorylation and inhibits protein synthesis.EXPERIMENTAL PROCEDURESVector ConstructionThe eIF-2α wild-type and S51A mutant (30Pathak V.K. Schindler D. Hershey J.W.B. Mol. Cell. Biol. 1988; 8: 993-995Crossref PubMed Scopus (67) Google Scholar) cDNAs encoded on 1.6-kilobase EcoRI fragments were subcloned into the unique EcoRI site of the pEDmtxrVA¯ expression vector, a derivative of pED-mtxr(31Kaufman R.J. Davies M.V. Wasley L.C. Michnick D. Nucleic Acids Res. 1991; 19: 4485-4490Crossref PubMed Scopus (231) Google Scholar) lacking the adenovirus VAI RNA gene. An XbaI-SalI fragment from pBS-8.4 (encoding wild-type PKR cDNA (13Meurs E. Chong K. Galabru J. Thomas N.S. Kerr I.M. Williams B.R. Hovanessian A.G. Cell. 1990; 62: 379-390Abstract Full Text PDF PubMed Scopus (812) Google Scholar) and kindly provided by Dr. B. Williams (Cleveland Clinic, OH)) was subcloned into the expression vector pMT2 (32Kaufman R.J. Davies M.V. Pathak V.K. Hershey J.W.B. Mol. Cell. Biol. 1989; 9: 946-958Crossref PubMed Scopus (332) Google Scholar). 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-mtxrVA¯. Site-directed mutagenesis (32Kaufman R.J. Davies M.V. Pathak V.K. Hershey J.W.B. Mol. Cell. Biol. 1989; 9: 946-958Crossref PubMed Scopus (332) Google Scholar) 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(33Davies M.V. Elroy-Stein O. Jagus R. Moss B. Kaufman R.J. J. Virol. 1992; 66: 1943-1950Crossref PubMed Google Scholar). 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¯ (33Davies M.V. Elroy-Stein O. Jagus R. Moss B. Kaufman R.J. J. Virol. 1992; 66: 1943-1950Crossref PubMed Google Scholar). 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 LineseIF-2α wild-type, mutant S51A, or PKR K296P contained within the pEDmtxrVA¯ vector were cotransfected with pSV2Neo (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-mtxrVA¯ PKR and pSV2Neo (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 [35S]methionine and [35S]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(34Dorner A.J. Kaufman R.J. Methods Enzymol. 1990; 185: 577-596Crossref PubMed Scopus (56) Google Scholar). Protein concentrations were determined(35Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213377) Google Scholar), 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-2α were measured by Western blot analysis using polyclonal anti-PKR antibody or polyclonal anti-eIF-2α antibody (kindly provided by Drs. Hovanessian and Hershey).COS-1 Cell DNA Transfection and AnalysisCOS-1 cells were transfected by the DEAE-dextran procedure(36Kaufman R.J. Methods Enzymol. 1990; 185: 487-511Crossref PubMed Scopus (148) Google Scholar). At 60 h post-transfection, cells were treated with calcium ionophore A23187 for 15 min at 37°C and then labeled with [35S]methionine and [35S]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-2α was immunoprecipitated from cell extracts using monoclonal anti-eIF-2α antibody (kindly provided by the late Dr. Henshaw) and protein A-Sepharose as the immunoadsorbent. Steady state levels of eIF-2α were measured by SDS-PAGE and Western immunoblotting procedures using anti-eIF-2α polyclonal antibody(37Davies M.V. Furtado M. Hershey J.W.B. Thimmappaya B. Kaufman R.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9163-9167Crossref PubMed Scopus (45) Google Scholar). 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-2α expressed in COS-1 cells at 60 h post-transfection or in NIH3T3 cells was monitored by labeling cells with 2 ml of [32P]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-2α 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.RESULTSNIH3T3 cells were derived that overexpress either eIF-2α wt or a mutant eIF-2α S51A in which the site of PKR-mediated phosphorylation was mutated to alanine. Western blot analysis demonstrated that the steady state level of eIF-2α was significantly elevated in both wild-type (6.4-fold, Fig. 1A, lane 2) and mutant eIF-2α S51A (8.8-fold, Fig. 1A, lane 3) transfected cell lines compared to a cell line transfected with the original vector alone (Fig. 1A, lane 1). The overexpression of eIF-2α 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, lanes 4 and 5). Overexpression of PKR K296P increased the growth rate and increased saturation density similar to previous observations(22Koromilas A.E. Roy S. Barber G.N. Katze M.G. Sonenberg N. Science. 1992; 257: 1685-1689Crossref PubMed Scopus (494) Google Scholar, 23Meurs E.F. Galabru J. Barber G.N. Katze M.G. Hovanessian A.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 232-236Crossref PubMed Scopus (414) Google Scholar).The effect of wild-type eIF-2α, eIF-2α 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-2α wt. In contrast, A23187 treatment reduced protein synthesis to 31% in cells that overexpress mutant eIF-2α S51A and to 29% in cells that overexpress PKR mutant K296P. Cells overexpressing either mutant eIF-2α 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-2α wt (Fig. 1B). Overexpression of wild-type eIF-2α, mutant eIF-2α S51A, or mutant PKR K296P did not detectably alter the specificity of polypeptides synthesized compared to control cells (Fig. 1C, lanes 1, 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-2α 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-2α in the control and transfected cells was measured by immunoprecipitation of equal amounts of protein from the cell extracts with anti-eIF-2α antibody and analysis by SDS-PAGE. The synthesis of eIF-2α was increased in cells transfected with eIF-2α wt or eIF-2α S51A (Fig. 1D, lanes 3 and 5) compared to control NIH3T3 or cells that overexpress PKR K296P (Fig. 1D, lanes 1 and 7). eIF-2α synthesis was reduced approximately 5-fold upon A23187 treatment in control NIH3T3 cells (Fig. 1D, lanes 1 and 2) and in cells that overexpress eIF-2α wt (Fig. 1D, lanes 3 and 4). In contrast, eIF-2α synthesis was not significantly reduced upon A23187 treatment in the cells that overexpress mutant eIF-2α S51A (Fig. 1D, lanes 5 and 6) or mutant PKR K296P (Fig. 1D, lanes 7 and 8). The effects of A23187 treatment on eIF-2α synthesis paralleled the effects on global protein synthesis in the different cell lines.The effect of calcium depletion on phosphorylation of eIF-2α was measured in NIH3T3 cells that overexpress eIF-2α wt. Immunoprecipitation of eIF-2α from [32P]phosphate-labeled NIH3T3 cells that overexpress eIF-2α wt detected [32P]phosphate incorporation into eIF-2α (Fig. 2, lane 1). Treatment with either A23187 or thapsigargin increased the [32P]phosphate incorporation by 3-fold (Fig. 2, lanes 3 and 5). Whereas treatment with the translation elongation inhibitor cycloheximide (10 μg/ml, a concentration that inhibited protein synthesis greater than 90%) slightly reduced [32P]phosphate incorporation in NIH3T3 cells that overexpress eIF-2α wt, it did not significantly inhibit the increase in phosphorylation observed upon treatment with A23187 or thapsigargin (Fig. 2, lanes 4 and 6). These results show that calcium depletion mediated by either A23187 or thapsigargin increases eIF-2α phosphorylation by a mechanism that does not require ongoing protein synthesis.Figure 2:Increased eIF-2α phosphorylation by calcium depletion does not require ongoing protein synthesis. NIH3T3 cells that overexpress wild-type eIF-2α were labeled with [32P]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-2α monoclonal antibody and analyzed by SDS-PAGE.View Large Image Figure ViewerDownload Hi-res image Download (PPT)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(38Lodish H.F. Kong N. J. Biol. Chem. 1990; 265: 10893-10899Abstract Full Text PDF PubMed Google Scholar). 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, lanes 4 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, lanes 5 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. Panel A, 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.” Panel B, cells were pulse labeled with [35S]methionine and [35S]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.”View Large Image Figure ViewerDownload Hi-res image Download (PPT)The role of RNA binding in the ability for PKR mutant K296P to inhibit eIF2α 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(33Davies M.V. Elroy-Stein O. Jagus R. Moss B. Kaufman R.J. J. Virol. 1992; 66: 1943-1950Crossref PubMed Google Scholar). 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, lane 2), 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, lanes 3 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, lanes 3 and 4) and were specifically immunoprecipitated with anti-T7 antibody (Fig. 4A, lanes 6 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(20Park H. Davies M.V. Langland J.O. Chang H-W. Nam Y.S. Tartaglia J. Paoletti E. Jacobs B.L. Kaufman R.J. Venkatesan S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4713-4717Crossref PubMed Scopus (185) Google Scholar).Figure 4:PKR mutant K296P, truncated PKR 1-243, and TRBP inhibit A23187-induced eIF-2α 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. Panel A, The T7-tagged constructs were cotransfected into COS-1 cells with the adenosine deaminase expression vector p9A (33Davies M.V. Elroy-Stein O. Jagus R. Moss B. Kaufman R.J. J. Virol. 1992; 66: 1943-1950Crossref PubMed Google Scholar). At 60 h post-transfection, cells were pulse labeled with [35S]methionine and [35S]cysteine, and cell extracts were prepared for analysis by SDS-PAGE before (lanes 1-4) and after (lanes 5-7) immunoprecipitation with anti-T7 monoclonal antibody. Panels B and C, COS-1 cells were cotransfected with the wild-type eIF-2α cDNA in the expression plasmid pED-mtxrVA¯ and the indicated T7-tagged expression vectors and analyzed for eIF-2α phosphorylation as described under “Experimental Procedures.”View Large Image Figure ViewerDownload Hi-res image Download (PPT)The effect of PKR mutant K296P, truncated 1-243 PKR, and TRBP on phosphorylation of eIF-2α in response to A23187 treatment was studied by cotransfection of these expression vectors with an eIF-2α 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-2α overexpressed from a second plasmid (pED-2α wt) can be monitored. At 60 h post-transfection, the effect of A23187 treatment on the phosphorylation status of the overexpressed eIF-2α was determined by measuring the amount of [32P]phosphate incorporation into eIF-2α and comparing it to the steady state eIF-2α level measured by Western blot analysis. Western blot analysis showed that cells cotransfected with pED-2α wt had increased levels of eIF-2α (Fig. 4, B and C (bottom), lanes 10-16 and 19-22) compared to cells that did not receive DNA (Fig. 4, B and C (bottom), lanes 8, 9, 17, and 18). In the presence of wild-type PKR cotransfection, the level of eIF-2α was slightly reduced (Fig. 4B (bottom), lane 16), consistent with inhibition of protein synthesis mediated by wild-type PKR. In" @default.
• W2110374497 created "2016-06-24" @default.
• W2110374497 creator A5015378855 @default.
• W2110374497 creator A5066475880 @default.
• W2110374497 creator A5088836753 @default.
• W2110374497 date "1995-07-01" @default.
• W2110374497 modified "2023-09-29" @default.
• W2110374497 title "Calcium Depletion from the Endoplasmic Reticulum Activates the Double-stranded RNA-dependent Protein Kinase (PKR) to Inhibit Protein Synthesis" @default.
• W2110374497 cites W1488906799 @default.
• W2110374497 cites W1493558178 @default.
• W2110374497 cites W1506260722 @default.
• W2110374497 cites W1529554110 @default.
• W2110374497 cites W1549105044 @default.
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• W2110374497 cites W159356339 @default.
• W2110374497 cites W1608735237 @default.
• W2110374497 cites W1639005451 @default.
• W2110374497 cites W179070074 @default.
• W2110374497 cites W1807534989 @default.
• W2110374497 cites W1878018656 @default.
• W2110374497 cites W1968987342 @default.
• W2110374497 cites W1970812281 @default.
• W2110374497 cites W1986422175 @default.
• W2110374497 cites W1989136432 @default.
• W2110374497 cites W1992155962 @default.
• W2110374497 cites W1998365327 @default.
• W2110374497 cites W2002725367 @default.
• W2110374497 cites W2012690458 @default.
• W2110374497 cites W2021174259 @default.
• W2110374497 cites W2036853011 @default.
• W2110374497 cites W2037044371 @default.
• W2110374497 cites W2040353939 @default.
• W2110374497 cites W2044915637 @default.
• W2110374497 cites W2045493915 @default.
• W2110374497 cites W2045765561 @default.
• W2110374497 cites W2063535351 @default.
• W2110374497 cites W2067128865 @default.
• W2110374497 cites W2072808030 @default.
• W2110374497 cites W2080133175 @default.
• W2110374497 cites W2086155529 @default.
• W2110374497 cites W2089033636 @default.
• W2110374497 cites W2118635464 @default.
• W2110374497 cites W2130684922 @default.
• W2110374497 cites W2136189765 @default.
• W2110374497 cites W2155564065 @default.
• W2110374497 cites W2156638980 @default.
• W2110374497 cites W2165055107 @default.
• W2110374497 cites W2175307231 @default.
• W2110374497 cites W2199020419 @default.
• W2110374497 cites W2215417236 @default.
• W2110374497 cites W4293247451 @default.
• W2110374497 cites W72118176 @default.
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