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• W2011994162 abstract "Initiation of translation from most cellular mRNAs occurs via scanning; the 40 S ribosomal subunit binds to the m7G-cap and then moves along the mRNA until an initiation codon is encountered. Some cellular mRNAs contain internal ribosome entry sequences (IRESs) within their 5′-untranslated regions, which allow initiation independently of the 5′-cap. This study investigated the ability of cellular stress to regulate the activity of IRESs in cellular mRNAs. Three stresses were studied that cause the phosphorylation of the translation initiation factor, eIF2α, by activating specific kinases: (i) amino acid starvation, which activates GCN2; (ii) endoplasmic reticulum (ER) stress, which activates PKR-like ER kinase, PERK kinase; and (iii) double-stranded RNA, which activates double-stranded RNA-dependent protein kinase (PKR) by mimicking viral infection. Amino acid starvation and ER stress caused transient phosphorylation of eIF2α during the first hour of treatment, whereas double-stranded RNA caused a sustained phosphorylation of eIF2α after 2 h. The effects of these treatments on IRES-mediated initiation were investigated using bicistronic mRNA expression vectors. No effect was seen for the IRESs from the mRNAs for the chaperone BiP and the protein kinase Pim-1. In contrast, translation mediated by the IRESs from the cationic amino acid transporter, cat-1, and of the cricket paralysis virus intergenic region, were stimulated 3- to 10-fold by all three treatments. eIF2α phosphorylation was required for the response because inactivation of phosphorylation prevented the stimulation. It is concluded that cellular stress can stimulate translation from some cellular IRESs via a mechanism that requires the phosphorylation of eIF2α. Moreover, there are distinct regulatory patterns for different cellular mRNAs that contain IRESs within their 5′-untranslated regions. Initiation of translation from most cellular mRNAs occurs via scanning; the 40 S ribosomal subunit binds to the m7G-cap and then moves along the mRNA until an initiation codon is encountered. Some cellular mRNAs contain internal ribosome entry sequences (IRESs) within their 5′-untranslated regions, which allow initiation independently of the 5′-cap. This study investigated the ability of cellular stress to regulate the activity of IRESs in cellular mRNAs. Three stresses were studied that cause the phosphorylation of the translation initiation factor, eIF2α, by activating specific kinases: (i) amino acid starvation, which activates GCN2; (ii) endoplasmic reticulum (ER) stress, which activates PKR-like ER kinase, PERK kinase; and (iii) double-stranded RNA, which activates double-stranded RNA-dependent protein kinase (PKR) by mimicking viral infection. Amino acid starvation and ER stress caused transient phosphorylation of eIF2α during the first hour of treatment, whereas double-stranded RNA caused a sustained phosphorylation of eIF2α after 2 h. The effects of these treatments on IRES-mediated initiation were investigated using bicistronic mRNA expression vectors. No effect was seen for the IRESs from the mRNAs for the chaperone BiP and the protein kinase Pim-1. In contrast, translation mediated by the IRESs from the cationic amino acid transporter, cat-1, and of the cricket paralysis virus intergenic region, were stimulated 3- to 10-fold by all three treatments. eIF2α phosphorylation was required for the response because inactivation of phosphorylation prevented the stimulation. It is concluded that cellular stress can stimulate translation from some cellular IRESs via a mechanism that requires the phosphorylation of eIF2α. Moreover, there are distinct regulatory patterns for different cellular mRNAs that contain IRESs within their 5′-untranslated regions. The vast majority of eukaryotic mRNAs is translated via the scanning mechanism (1Hellen C.U. Sarnow P. Genes Dev. 2001; 15: 1593-1612Crossref PubMed Scopus (806) Google Scholar, 2Pestova T.V. Kolupaeva V.G. Lomakin I.B. Pilipenko E.V. Shatsky I.N. Agol V.I. Hellen C.U. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7029-7036Crossref PubMed Scopus (603) Google Scholar). This mechanism involves the recognition of the 5′-end of the mRNA and its m7G-cap structure by the translation initiator factor eIF4F, which is composed of eIF4A, eIF4G, and eIF4E. This is followed by binding of the 40 S ribosomal subunit/eIF2·GTP·Met-tRNAi ternary complex and scanning downstream to the initiation codon (1Hellen C.U. Sarnow P. Genes Dev. 2001; 15: 1593-1612Crossref PubMed Scopus (806) Google Scholar, 2Pestova T.V. Kolupaeva V.G. Lomakin I.B. Pilipenko E.V. Shatsky I.N. Agol V.I. Hellen C.U. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7029-7036Crossref PubMed Scopus (603) Google Scholar). Following GTP hydrolysis, the 60 S ribosomal subunit joins the complex to form the 80 S ribosome (3Hershey J. Merrick W. Sonenberg N. Hershey J.W.B. Mathews M.B Translational Control of Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2000: 33-88Google Scholar). Recently, it has been shown that translation of some mRNAs is initiated by cap-independent mechanisms (1Hellen C.U. Sarnow P. Genes Dev. 2001; 15: 1593-1612Crossref PubMed Scopus (806) Google Scholar, 4Jackson R.J. Kaminski A. RNA (N. Y.). 1995; 1: 985-1000PubMed Google Scholar). Elements within the 5′-untranslated region (UTR) 1The abbreviations used are: UTRuntranslated regionIGRintergenic regionIRESinternal ribosome entry siteICSintercistronic spacerORFopen reading frameCATchloramphenicol acetyltransferasecat-1cationic amino acid transporter-1CrPVcricket paralysis virusdsRNAdouble-stranded RNAPKRdouble-stranded RNA-dependent protein kinasePERKPKR-like ER kinaseERendoplasmic reticulumLUCluciferaseFLUCfirefly LUCRLUCrenilla LUCpoly(IC)poly(I)·poly(C)DMEMDulbecco's modified Eagle's medium of the mRNAs known as internal ribosome entry sequences (IRESs) can direct ribosome binding without the need for the eIF4F complex (1Hellen C.U. Sarnow P. Genes Dev. 2001; 15: 1593-1612Crossref PubMed Scopus (806) Google Scholar, 5Johannes G. Sarnow P. RNA (N. Y.). 1998; 4: 1500-1513Crossref PubMed Scopus (223) Google Scholar). This mode of initiation has mainly been described for viral RNAs, which are translated in infected cells when cap-dependent translation is inhibited (6Johannes G. Carter M.S. Eisen M.B. Brown P.O. Sarnow P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13118-13123Crossref PubMed Scopus (324) Google Scholar). Some cellular mRNAs also contain IRESs in their 5′-UTRs (7Martinez-Salas E. Ramos R. Lafuente E. Lopez de Quinto S. J. Gen. Virol. 2001; 82: 973-984Crossref PubMed Scopus (110) Google Scholar). It has been shown that translation of some of these IRESs is regulated by the cell cycle (8Pyronnet S. Pradayrol L. Sonenberg N. Mol. Cell. 2000; 5: 607-616Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar), developmental stage (9Creancier L. Morello D. Mercier P. Prats A.C. J. Cell Biol. 2000; 150: 275-281Crossref PubMed Scopus (119) Google Scholar), apoptosis (10Clemens M.J. Bommer U.A. Int. J. Biochem. Cell Biol. 1999; 31: 1-23Crossref PubMed Scopus (206) Google Scholar, 11Clemens M.J. Bushell M. Jeffrey I.W. Pain V.M. Morley S.J. Cell Death Differ. 2000; 7: 603-615Crossref PubMed Scopus (207) Google Scholar), and cellular stress (12Fernandez J. Yaman I. Mishra R. Merrick W.C. Snider M.D. Lamers W.H. Hatzoglou M. J. Biol. Chem. 2001; 276: 12285-12291Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 13Fernandez J.M. Bode B. Koromilas A. Diehl J.A. Krukovets I. Snider M.D. Hatzoglou M. J. Biol. Chem. 2002; 7: 7Google Scholar, 14Fernandez J. Yaman I. Merrick W.C. Koromilas A. Wek R.C. Sood R. Hensold J. Hatzoglou M. J. Biol. Chem. 2002; 277: 2050-2058Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Many important features of how IRESs in cellular mRNAs mediate translation initiation are poorly understood. It is not known how many different types of IRESs are found in cellular mRNAs. In addition, the mechanisms by which IRESs are regulated and the number of different control mechanisms are poorly understood. untranslated region intergenic region internal ribosome entry site intercistronic spacer open reading frame chloramphenicol acetyltransferase cationic amino acid transporter-1 cricket paralysis virus double-stranded RNA double-stranded RNA-dependent protein kinase PKR-like ER kinase endoplasmic reticulum luciferase firefly LUC renilla LUC poly(I)·poly(C) Dulbecco's modified Eagle's medium We have recently shown that the mRNA for the Arg/Lys transporter, cat-1, contains an IRES sequence (12Fernandez J. Yaman I. Mishra R. Merrick W.C. Snider M.D. Lamers W.H. Hatzoglou M. J. Biol. Chem. 2001; 276: 12285-12291Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). This IRES is located in the 5′-UTR, which also contains a 48-residue open reading frame (14Fernandez J. Yaman I. Merrick W.C. Koromilas A. Wek R.C. Sood R. Hensold J. Hatzoglou M. J. Biol. Chem. 2002; 277: 2050-2058Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Translation initiation from this IRES is increased during amino acid starvation when global and cap-dependent protein synthesis is decreased (15Kimball S. Jefferson L. Sonenberg N. Hershey J.W.B. Mathews M.B Translational Control of Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2000: 561-580Google Scholar), allowing cat-1 protein expression when amino acids are limiting (12Fernandez J. Yaman I. Mishra R. Merrick W.C. Snider M.D. Lamers W.H. Hatzoglou M. J. Biol. Chem. 2001; 276: 12285-12291Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). The phosphorylation of translation initiation factors plays a key role in this regulation (14Fernandez J. Yaman I. Merrick W.C. Koromilas A. Wek R.C. Sood R. Hensold J. Hatzoglou M. J. Biol. Chem. 2002; 277: 2050-2058Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). During amino acid starvation, phosphorylation of eIF2α increases, which decreases its activity, causing reduced levels of ternary complexes (16Pain V.M. Biochimie (Paris). 1994; 76: 718-728Crossref PubMed Scopus (50) Google Scholar). In addition, eIF4F activity is decreased due to dephosphorylation of eIF4E and the eIF4E-binding protein 4E-BP-1 (16Pain V.M. Biochimie (Paris). 1994; 76: 718-728Crossref PubMed Scopus (50) Google Scholar). It was shown previously that phosphorylation of eIF2α by the kinase GCN2, whose activity is stimulated by uncharged tRNAs, is required for enhanced cat-1 IRES activity during amino acid deprivation (14Fernandez J. Yaman I. Merrick W.C. Koromilas A. Wek R.C. Sood R. Hensold J. Hatzoglou M. J. Biol. Chem. 2002; 277: 2050-2058Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). In this report, we expand these studies of the regulation of IRES activity by eIF2α phosphorylation. It is shown that two other types of cellular stress that increase eIF2α phosphorylation also stimulate translation mediated by the cat-1 IRES. Agents that cause the accumulation of unfolded proteins within the endoplasmic reticulum (ER) trigger the unfolded protein response by activating the eIF2α kinase, PERK, in the ER membrane (17Kaufman R.J. Genes Dev. 1999; 13: 1211-1233Crossref PubMed Scopus (1944) Google Scholar). We show that thapsigargin, which mobilizes sequestered Ca2+ from the ER, and tunicamycin, which disrupts protein glycosylation, increase cat-1 IRES-mediated translation by the activation of PERK. We also show that double-stranded RNA (dsRNA), which mimics viral infection (18Proud C.G. Trends Biochem. Sci. 1995; 20: 241-246Abstract Full Text PDF PubMed Scopus (200) Google Scholar), stimulates translation mediated by the cat-1 IRES by activating the eIF2α kinase, PKR. These results demonstrate that this IRES can be regulated by a variety of cellular stresses that stimulate eIF2α phosphorylation. We also tested whether other cellular IRESs can be regulated by these cellular stresses. The IRESs from the BiP and Pim-1 mRNAs were studied. BiP is a chaperone protein that assists in protein folding within the ER (19Sarnow P. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5795-5799Crossref PubMed Scopus (87) Google Scholar). Transcription of the BiP gene is induced as part of the unfolded protein response (20Lee A.S. Trends Biochem. Sci. 2001; 26: 504-510Abstract Full Text Full Text PDF PubMed Scopus (925) Google Scholar). The BiP mRNA is translated under these conditions via an IRES element found within its 5′-UTR (21Macejak D.G. Sarnow P. Nature. 1991; 353: 90-94Crossref PubMed Scopus (423) Google Scholar). Pim-1 is a serine-threonine kinase that functions with c-Myc in cellular transformation (22Saris C.J. Domen J. Berns A. EMBO J. 1991; 10: 655-664Crossref PubMed Scopus (265) Google Scholar). This mRNA is translated in poliovirus-infected cells via an IRES element within its 5′-UTR (23Wilson J.E. Powell M.J. Hoover S.E. Sarnow P. Mol. Cell. Biol. 2000; 20: 4990-4999Crossref PubMed Scopus (251) Google Scholar). We show here that the activity of these IRESs is maintained but not increased by the cellular stresses that increase eIF2α phosphorylation. These results demonstrate that IRESs from cellular mRNAs are a diverse group because they are not all regulated by the same mechanism. The following bicistronic mRNA expression vectors have been described previously: pSVCAT/cat1-5′-UTRf/LUC, which encodes an mRNA containing CAT as the 5′-cistron and LUC as the 3′-cistron (14Fernandez J. Yaman I. Merrick W.C. Koromilas A. Wek R.C. Sood R. Hensold J. Hatzoglou M. J. Biol. Chem. 2002; 277: 2050-2058Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). The intercistronic spacer (ICS) is the 270-bp 5′-UTR of the cat-1 mRNA (14Fernandez J. Yaman I. Merrick W.C. Koromilas A. Wek R.C. Sood R. Hensold J. Hatzoglou M. J. Biol. Chem. 2002; 277: 2050-2058Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). pSVCAT/BiP/LUC encodes an mRNA with the 5′-UTR of the BiP mRNA as the ICS (21Macejak D.G. Sarnow P. Nature. 1991; 353: 90-94Crossref PubMed Scopus (423) Google Scholar). Three bicistronic plasmids encoding renilla luciferase (RLUC) as the 5′-cistron and firefly luciferase (FLUC) as the 3′-cistron were used (23Wilson J.E. Powell M.J. Hoover S.E. Sarnow P. Mol. Cell. Biol. 2000; 20: 4990-4999Crossref PubMed Scopus (251) Google Scholar). The IGR construct (SV40/T7ΔEMCV/Fluc-IGR/CrPVORF2) contains 207 bp from the intergenic region (IGR) of cricket paralysis virus (CrPV) in the ICS (23Wilson J.E. Powell M.J. Hoover S.E. Sarnow P. Mol. Cell. Biol. 2000; 20: 4990-4999Crossref PubMed Scopus (251) Google Scholar, 24Wilson J.E. Pestova T.V. Hellen C.U. Sarnow P. Cell. 2000; 102: 511-520Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar). The IGRmut construct contains the CrPV IGR with the CC residues corresponding to bases 6214 and 6215 of the CrPV sequence mutated to GG (23Wilson J.E. Powell M.J. Hoover S.E. Sarnow P. Mol. Cell. Biol. 2000; 20: 4990-4999Crossref PubMed Scopus (251) Google Scholar, 24Wilson J.E. Pestova T.V. Hellen C.U. Sarnow P. Cell. 2000; 102: 511-520Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar). The Pim-1 construct contains the 5′-UTR of the human Pim-1 mRNA in the ICS in place of the CrPV sequence. Expression vectors for PERK, PERK-mut, GCN2, GCN2-mut, and eIF2α S-A, were kindly provided by D. Ron (New York University School of Medicine). The cDNAs in all vectors were inserted at the XbaI/Hin dIII site of pCDNA3. In these vectors, transcription is directed by the cytomegalovirus promoter (25Brewer J.W. Diehl J.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12625-12630Crossref PubMed Scopus (364) Google Scholar). All cells were maintained in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS). Plasmid DNAs were transfected into C6 rat glioma cells (5 × 105/35-mm dish) using the calcium phosphate technique (26Hyatt S.L. Aulak K.S. Malandro M. Kilberg M.S. Hatzoglou M. J. Biol. Chem. 1997; 272: 19951-19957Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Cotransfections were performed with equimolar amounts of plasmid DNAs. Cells were cultured for 48 h in growth medium followed by incubation under test conditions for the indicated times. Control cells were incubated in DMEM/F12 supplemented with FBS dialyzed against phosphate-buffered saline (26Hyatt S.L. Aulak K.S. Malandro M. Kilberg M.S. Hatzoglou M. J. Biol. Chem. 1997; 272: 19951-19957Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Cells were starved for amino acids by incubating in Krebs-Ringer buffer supplemented with dialyzed FBS (26Hyatt S.L. Aulak K.S. Malandro M. Kilberg M.S. Hatzoglou M. J. Biol. Chem. 1997; 272: 19951-19957Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). No difference in the regulation of the cat-1 gene by amino acid starvation was observed when Krebs-Ringer buffer containing all amino acids was used in place of DMEM/F12 medium (26Hyatt S.L. Aulak K.S. Malandro M. Kilberg M.S. Hatzoglou M. J. Biol. Chem. 1997; 272: 19951-19957Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Cells were also incubated with 2.5 μg/ml tunicamycin, 400 nm thapsigargin, or 100 μg/ml poly(I)·poly(C) (poly(IC)) for the appropriate times. To address the role of PKR kinase, wild-type mouse embryo fibroblasts (PKR+/+) or fibroblasts with the kinase inactivated by homologous recombination (PKR−/−) were used (27Yang Y.L. Reis L.F. Pavlovic J. Aguzzi A. Schafer R. Kumar A. Williams B.R. Aguet M. Weissmann C. EMBO J. 1995; 14: 6095-6106Crossref PubMed Scopus (568) Google Scholar). Cell extracts were prepared and analyzed for LUC (Tropix Luciferase Assay Kit) and CAT activities as described previously (28Leahy P. Crawford D.R. Grossman G. Gronostajski R.M. Hanson R.W. J. Biol. Chem. 1999; 274: 8813-8822Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The activities were normalized to the protein content of the cell extracts, which was measured using the Bio-Rad DC assay. Cells transfected with dual luciferase plasmids were lysed, and RLUC and FLUC were measured using the Promega Dual Luciferase Analysis Kit. The expression of eIF2α and eIF4E was analyzed by Western blotting. The expression of phospho-eIF2α and phospho-eIF4E was analyzed using antibodies specific for the phosphorylated forms of these proteins, all as described previously (13Fernandez J.M. Bode B. Koromilas A. Diehl J.A. Krukovets I. Snider M.D. Hatzoglou M. J. Biol. Chem. 2002; 7: 7Google Scholar). Our previous studies have shown that translation from the cat-1 IRES is stimulated by amino acid starvation via a mechanism that requires phosphorylation of eIF2α by GCN2 kinase (14Fernandez J. Yaman I. Merrick W.C. Koromilas A. Wek R.C. Sood R. Hensold J. Hatzoglou M. J. Biol. Chem. 2002; 277: 2050-2058Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). It is known that several other cellular stresses induce phosphorylation of eIF2α, including ER stress and the presence of dsRNA (18Proud C.G. Trends Biochem. Sci. 1995; 20: 241-246Abstract Full Text PDF PubMed Scopus (200) Google Scholar), which occurs during viral infection. Consequently, we investigated whether these stresses also stimulate translation from the cat-1 IRES. Studying IRESs in the 5′-UTR of cellular mRNAs is difficult. It is believed that these mRNAs are translated by both cap-dependent and independent mechanisms under normal conditions. IRES-mediated translation may be activated under stress conditions when cap-dependent translation decreases (1Hellen C.U. Sarnow P. Genes Dev. 2001; 15: 1593-1612Crossref PubMed Scopus (806) Google Scholar). However, it is difficult to know how translation is initiated in vivo because cap-dependent and independent initiation cannot be readily distinguished. To study IRES-mediated translation exclusively, we used the bicistronic expression vector, CAT/cat1-5′-UTRf/LUC, employed in our previous studies (14Fernandez J. Yaman I. Merrick W.C. Koromilas A. Wek R.C. Sood R. Hensold J. Hatzoglou M. J. Biol. Chem. 2002; 277: 2050-2058Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). The mRNA synthesized from this vector contains open reading frames for the CAT and LUC enzymes (Fig. 1 A). The first cistron encodes CAT, which is translated by a cap-dependent mechanism. The second cistron encodes LUC, which is translated only if initiation occurs in the intercistronic spacer, which contains the entire 270-bp 5′-UTR of the cat-1 mRNA. To test the effect of ER stress on translation from the cat-1 IRES, C6 glioma cells transiently transfected with CAT/cat1-5′-UTRf/LUC were treated with tunicamycin or thapsigargin (Fig. 1 B). Tunicamycin interferes with protein folding in the ER by blocking the glycosylation of Asn residues of newly made proteins (25Brewer J.W. Diehl J.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12625-12630Crossref PubMed Scopus (364) Google Scholar). Thapsigargin induces ER stress by depleting ER Ca2+ stores (29Harding H.P. Novoa I.I. Zhang Y. Zeng H. Wek R. Schapira M. Ron D. Mol. Cell. 2000; 6: 1099-1108Abstract Full Text Full Text PDF PubMed Scopus (2430) Google Scholar). Tunicamycin and thapsigargin decreased CAT activity after 1.5 h, consistent with the inhibition of cap-dependent translation by these agents. In contrast, both treatments caused slow increases in LUC activity. Increases were only seen after 3 h of treatment, and activity then increased throughout the 12-h course of the experiment. Changes in both CAT and LUC activities are reflected in the LUC/CAT ratio, which increased by 3 h of treatment and was 16 times the control level by 12 h (Fig. 1 B). These results demonstrate that translation from the cat-1 IRES is increased by treatments that induce ER stress. Moreover, the long lag and slow increase in translation are similar to the kinetics of increased translation during amino acid starvation (12Fernandez J. Yaman I. Mishra R. Merrick W.C. Snider M.D. Lamers W.H. Hatzoglou M. J. Biol. Chem. 2001; 276: 12285-12291Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). To test the effects of dsRNA, C6 cells transiently transfected with the CAT/cat1-5′-UTRf/LUC vector were treated with poly(IC). This treatment had effects similar to tunicamycin and thapsigargin (Fig. 1 B). LUC activity increased, but only after a lag. There was also a decrease in CAT activity. These changes are reflected in an increase in the LUC/CAT ratio. An increase was first seen after 6 h of treatment, and the highest activity was observed after 12 h, although dsRNA caused a smaller increase (7-fold) than the other treatments. These results indicate that dsRNA increases translation mediated by the cat-1 IRES with a long lag period and a persistent increase in activity. We have shown previously that amino acid starvation increases translation from the cat-1 IRES via a mechanism that involves phosphorylation of the translation initiation factor, eIF2α, by GCN2 kinase. GCN2, which is active when uncharged tRNAs are present, is one of at least four kinases known to phosphorylate eIF2α, regulating the activity of this factor in response to distinct upstream signals (27Yang Y.L. Reis L.F. Pavlovic J. Aguzzi A. Schafer R. Kumar A. Williams B.R. Aguet M. Weissmann C. EMBO J. 1995; 14: 6095-6106Crossref PubMed Scopus (568) Google Scholar). eIF2α is also phosphorylated by PERK kinase, which is stimulated during ER stress, and by PKR kinase, which is activated by dsRNA (18Proud C.G. Trends Biochem. Sci. 1995; 20: 241-246Abstract Full Text PDF PubMed Scopus (200) Google Scholar). To determine whether the effects of ER stress and dsRNA on translation from the cat-1 IRES are mediated by PERK and PKR, the effects of overexpressing dominant-negative kinase mutants were studied (25Brewer J.W. Diehl J.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12625-12630Crossref PubMed Scopus (364) Google Scholar). To examine the involvement of PERK, the effects of amino acid starvation and thapsigargin were studied in C6 cells cotransfected with CAT/cat1-5′-UTRf/LUC and an expression plasmid encoding either wild-type or dominant-negative PERK (Fig. 2 A). The stimulation of cat-1 IRES-mediated translation by amino acid starvation or thapsigargin was not affected by overexpression of wild-type PERK (Fig. 2 A). The 15-fold increase in LUC/CAT ratio is similar to that observed in cells with no PERK overexpression (Fig. 1 B and Ref. 13Fernandez J.M. Bode B. Koromilas A. Diehl J.A. Krukovets I. Snider M.D. Hatzoglou M. J. Biol. Chem. 2002; 7: 7Google Scholar). Expression of the dominant-negative PERK mutant caused a decrease in LUC expression mediated by the cat-1 IRES, suggesting that basal PERK activity regulates IRES activity (Fig. 2 A, compare control values). Amino acid starvation of these cells increased translation mediated by the IRES. In fact, the LUC/CAT ratio increased by 15-fold as compared with untreated cells expressing mutant PERK, the same increase seen in cells overexpressing wild-type PERK (Fig. 2 A). In contrast, thapsigargin treatment only caused a 4-fold increase in the LUC/CAT ratio in cells expressing mutant PERK. This experiment demonstrates that PERK is required for the control of IRES activity by ER stress but not by amino acid starvation. To further support this finding, the effect of a dominant-negative mutant of GCN2 on the response of the cat-1 IRES to ER stress was studied. We have shown previously that overexpression of this mutant in C6 cells blocks the increase in cat-1 IRES activity by amino acid starvation (14Fernandez J. Yaman I. Merrick W.C. Koromilas A. Wek R.C. Sood R. Hensold J. Hatzoglou M. J. Biol. Chem. 2002; 277: 2050-2058Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). In contrast, thapsigargin caused a large increase in LUC expression in C6 cells overexpressing mutant GCN2 (Fig. 2 B). These data support the idea that ER stress stimulates cat-1 IRES-mediated translation via PERK, whereas amino acid starvation stimulates translation via GCN2. A similar experiment was performed to examine the importance of PKR kinase. These experiments took advantage of a well characterized mouse embryo fibroblast cell line in which PKR has been inactivated by homologous recombination (27Yang Y.L. Reis L.F. Pavlovic J. Aguzzi A. Schafer R. Kumar A. Williams B.R. Aguet M. Weissmann C. EMBO J. 1995; 14: 6095-6106Crossref PubMed Scopus (568) Google Scholar). In wild-type cells (PKR+/+), all three cellular stresses caused increased translation from the cat-1 IRES (Fig. 2 C). The only difference between the results from these cells and C6 cells was the level of stimulation by dsRNA. In PKR+/+ cells, all three stresses increased the LUC/CAT ratio by the same extent, whereas the increase caused by dsRNA in C6 cells was only half that caused by the other two stresses. In PKR−/− cells, translation from the cat-1 IRES was induced by both amino acid starvation and thapsigargin, consistent with the idea that PKR kinase does not mediate the effects of these cellular stresses (Fig. 2 C). In contrast, stimulation of IRES-mediated translation by dsRNA was abolished in these mutant cells. Taken together, these results support our hypothesis that translation mediated by the cat-1 IRES is regulated by eIF2α phosphorylation. Moreover, they support the idea that this regulation involves several independent signaling pathways: GCN2 kinase mediates the effects of amino acid starvation, PERK mediates the effects of ER stress, and PKR mediates the effects of dsRNA. Our results suggest that phosphorylation of eIF2α by specific kinases is important in the regulation of the cat-1 IRES in response to cellular stress. To support this conclusion, we examined the effect of these cellular stresses on the phosphorylation of eIF2α. This was accomplished by Western blot analysis using antibodies specific for either total eIF2α or the phosphorylated form of this protein. Both amino acid starvation and thapsigargin treatment caused a rapid transient increase in phosphorylated eIF2α levels (Fig. 3,A and B). The amount was increased within 30–60 min of treatment, was maximal at 1 h, and returned to base-line levels by 2–6 h. The amount of total eIF2α protein did not significantly change during these treatments, indicating that there was a transient increase in the extent of eIF2α phosphorylation. poly(IC) treatment also caused induction of eIF2α phosphorylation (Fig. 3 C). However, in this case, there was a sustained induction, which began after 2 h of treatment and was still evident after 24 h. Significantly, for both ER stress and dsRNA, the increases in eIF2α phosphorylation and translation mediated by the cat-1 IRES follow different time courses (Fig. 1). For ER stress, the increase in IRES-mediated translation did not occur until eIF2α phosphorylation had increased and then returned to base-line levels. These kinetics are similar to those observed previously for amino acid starvation (12Fernandez J. Yaman I. Mishra R. Merrick W.C. Snider M.D. Lamers W.H. Hatzoglou M. J. Biol. Chem. 2001; 276: 12285-12291Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). For dsRNA, IRES-mediated translation also increased several hours after the increase in phosphorylation of eIF2α. It has been suggested that IRES-mediated translation may increase when the translation initiation factor eIF4F, which is important in cap-dependent translation initiation, is inactivated (30Gingras A.C. Raught B. Sonenberg N. Annu. Rev. Biochem. 1999; 68: 913-963Crossref PubMed Scopus (1766) Google Scholar). The activity of the cap-binding protein eIF4E, an important constituent of eIF4F, is regulated by phosphorylation (30Gingras A.C. Raught B. Sonenberg N. Annu. Rev. Biochem. 1999; 68: 913-963Crossref P" @default.
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