Phosphorylation of eIF2 Directs ATF5 Translational Control in Response to Diverse Stress Conditions
2008; Elsevier BV; Volume: 283; Issue: 11 Linguagem: Inglês
10.1074/jbc.m708530200
ISSN1083-351X
AutoresDonghui Zhou, Lakshmi Reddy Palam, Li Jiang, Jana Narasimhan, Kirk A. Staschke, Ronald C. Wek,
Tópico(s)CRISPR and Genetic Engineering
ResumoPhosphorylation of eukaryotic initiation factor 2 (eIF2) is an important mechanism regulating global and gene-specific translation in response to different environmental stresses. Central to the eIF2 kinase response is the preferential translation of ATF4 mRNA, encoding a transcriptional activator of genes involved in stress remediation. In this report, we addressed whether there are additional transcription factors whose translational expression is regulated by eIF2 kinases. We show that the expression of the basic zipper transcriptional regulator ATF5 is induced in response to many different stresses, including endoplasmic reticulum stress, arsenite exposure, and proteasome inhibition, by a mechanism requiring eIF2 phosphorylation. ATF5 is subject to translational control as illustrated by the preferential association of ATF5 mRNA with large polyribosomes in response to stress. ATF5 translational control involves two upstream open reading frames (uORFs) located in the 5′-leader of the ATF5 mRNA, a feature shared with ATF4. Mutational analyses of the 5′-leader of ATF5 mRNA fused to a luciferase reporter suggest that the 5′-proximal uORF1 is positive-acting, allowing scanning ribosomes to reinitiate translation of a downstream ORF. During non-stressed conditions, when eIF2 phosphorylation is low, ribosomes reinitiate translation at the next ORF, the inhibitory uORF2. Phosphorylation of eIF2 during stress delays translation reinitiation, allowing scanning ribosomes to bypass uORF2, and instead translate the ATF5 coding region. In addition to translational control, ATF5 mRNA levels are significantly reduced in ATF4-/- mouse embryo fibroblasts, suggesting that ATF4 contributes to basal ATF5 transcription. These results demonstrate that eIF2 kinases direct the translational expression of multiple transcription regulators by a mechanism involving delayed translation reinitiation. Phosphorylation of eukaryotic initiation factor 2 (eIF2) is an important mechanism regulating global and gene-specific translation in response to different environmental stresses. Central to the eIF2 kinase response is the preferential translation of ATF4 mRNA, encoding a transcriptional activator of genes involved in stress remediation. In this report, we addressed whether there are additional transcription factors whose translational expression is regulated by eIF2 kinases. We show that the expression of the basic zipper transcriptional regulator ATF5 is induced in response to many different stresses, including endoplasmic reticulum stress, arsenite exposure, and proteasome inhibition, by a mechanism requiring eIF2 phosphorylation. ATF5 is subject to translational control as illustrated by the preferential association of ATF5 mRNA with large polyribosomes in response to stress. ATF5 translational control involves two upstream open reading frames (uORFs) located in the 5′-leader of the ATF5 mRNA, a feature shared with ATF4. Mutational analyses of the 5′-leader of ATF5 mRNA fused to a luciferase reporter suggest that the 5′-proximal uORF1 is positive-acting, allowing scanning ribosomes to reinitiate translation of a downstream ORF. During non-stressed conditions, when eIF2 phosphorylation is low, ribosomes reinitiate translation at the next ORF, the inhibitory uORF2. Phosphorylation of eIF2 during stress delays translation reinitiation, allowing scanning ribosomes to bypass uORF2, and instead translate the ATF5 coding region. In addition to translational control, ATF5 mRNA levels are significantly reduced in ATF4-/- mouse embryo fibroblasts, suggesting that ATF4 contributes to basal ATF5 transcription. These results demonstrate that eIF2 kinases direct the translational expression of multiple transcription regulators by a mechanism involving delayed translation reinitiation. Phosphorylation of the α subunit of eukaryotic initiation factor-2 (eIF2) 2The abbreviations used are:eIF2eukaryotic initiation factor 2ERendoplasmic reticulumbZIPbasic zipperORFopen reading frameuORFupstream open reading frameMEFmouse embryonic fibroblastRTreverse transcriptionRACErapid amplification of cDNA ends.2The abbreviations used are:eIF2eukaryotic initiation factor 2ERendoplasmic reticulumbZIPbasic zipperORFopen reading frameuORFupstream open reading frameMEFmouse embryonic fibroblastRTreverse transcriptionRACErapid amplification of cDNA ends. is an important mechanism regulating protein synthesis in response to a diverse range of environmental stresses (1Dever T.E. Cell. 2002; 108: 545-556Abstract Full Text Full Text PDF PubMed Scopus (611) Google Scholar, 2Holcik M. Sonenberg N. Nat. Rev. Mol. Cell. Biol. 2005; 6: 318-327Crossref PubMed Scopus (1029) Google Scholar, 3Wek R.C. Jiang H.Y. Anthony T.G. Biochem. Soc. Trans. 2006; 34: 7-11Crossref PubMed Scopus (1013) Google Scholar). Four eIF2α kinases have been described in mammals, each responding to different stress arrangements through their unique regulatory regions. For example, phosphorylation of eIF2α by PEK (also known as Perk or EIF2AK3) is induced by accumulation of malfolded proteins in the endoplasmic reticulum (ER) (4Ron D. Walter P. Nat. Rev. Mol. Cell. Biol. 2007; 8: 519-529Crossref PubMed Scopus (4851) Google Scholar, 5Marciniak S.J. Ron D. Physiol. Rev. 2006; 86: 1133-1149Crossref PubMed Scopus (777) Google Scholar, 6Schroder M. Kaufman R.J. Annu. Rev. Biochem. 2005; 74: 739-789Crossref PubMed Scopus (2432) Google Scholar). Phosphorylation of eIF2α during this so-called ER stress inhibits global translation by lowering the levels of eIF2-GTP that are central for binding of initiator Met-tRNAiMet to the translational machinery (1Dever T.E. Cell. 2002; 108: 545-556Abstract Full Text Full Text PDF PubMed Scopus (611) Google Scholar, 2Holcik M. Sonenberg N. Nat. Rev. Mol. Cell. Biol. 2005; 6: 318-327Crossref PubMed Scopus (1029) Google Scholar, 3Wek R.C. Jiang H.Y. Anthony T.G. Biochem. Soc. Trans. 2006; 34: 7-11Crossref PubMed Scopus (1013) Google Scholar). Together with reduced protein synthesis, eIF2α phosphorylation increases the preferential translation of ATF4 mRNA, encoding a basic zipper (bZIP) transcription activator that is important for directing the expression of genes involved in metabolism, the redox status of cells, and apoptosis (7Harding H.P. Zhang Y. Zeng H. Novoa I. Lu P.D. Calfon M. Sadri N. Yun C. Popko B. Paules R. Stojdl D.F. Bell J.C. Hettmann T. Leiden J.M. Ron D. Mol. Cell. 2003; 11: 619-633Abstract Full Text Full Text PDF PubMed Scopus (2364) Google Scholar, 8Vattem K.M. Wek R.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11269-11274Crossref PubMed Scopus (1122) Google Scholar, 9Lu P.D. Harding H.P. Ron D. J. Cell Biol. 2004; 167: 27-33Crossref PubMed Scopus (654) Google Scholar). Decreased protein synthesis conserves energy and provides sufficient time for ATF4, and other stress-responsive transcription factors, to reconfigure gene expression that would block or ameliorate damage elicited by the underlying stress. Other members of the eIF2α kinase family include GCN2 (EIF2AK4), whose activity is enhanced by amino acid depletion, UV irradiation or proteasome inhibition; HRI (EIF2AK1), which is regulated by heme deficiency and oxidative stress; and PKR (EIF2AK2), which functions in an antiviral pathway (2Holcik M. Sonenberg N. Nat. Rev. Mol. Cell. Biol. 2005; 6: 318-327Crossref PubMed Scopus (1029) Google Scholar, 3Wek R.C. Jiang H.Y. Anthony T.G. Biochem. Soc. Trans. 2006; 34: 7-11Crossref PubMed Scopus (1013) Google Scholar, 6Schroder M. Kaufman R.J. Annu. Rev. Biochem. 2005; 74: 739-789Crossref PubMed Scopus (2432) Google Scholar, 10Wek R.C. Cavener D.R. Antioxid. Redox Signal. 2007; 9: 2357-2371Crossref PubMed Scopus (234) Google Scholar). Aberrations in these eIF2α kinase pathways are associated with a number of diseases, including diabetes, stroke, eating disorders, viral infection, anemia, and neurological disorders. eukaryotic initiation factor 2 endoplasmic reticulum basic zipper open reading frame upstream open reading frame mouse embryonic fibroblast reverse transcription rapid amplification of cDNA ends. eukaryotic initiation factor 2 endoplasmic reticulum basic zipper open reading frame upstream open reading frame mouse embryonic fibroblast reverse transcription rapid amplification of cDNA ends. Enhanced ATF4 expression during stress-induced eIF2α phosphorylation occurs primarily by translational control, as illustrated by increased association of ATF4 mRNA with polysomes (11Harding H.P. Novoa I. Zhang Y. Zeng H. Wek R. Schapira M. Ron D. Mol. Cell. 2000; 6: 1099-1108Abstract Full Text Full Text PDF PubMed Scopus (2404) Google Scholar). Central to ATF4 translational control is the 5′-leader of the ATF4 mRNA, which encodes two uORFs that have opposing functions (8Vattem K.M. Wek R.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11269-11274Crossref PubMed Scopus (1122) Google Scholar, 9Lu P.D. Harding H.P. Ron D. J. Cell Biol. 2004; 167: 27-33Crossref PubMed Scopus (654) Google Scholar). ATF4 translation begins with the 40 S ribosomal subunit bound to eIF2/GTP/Met-tRNAiMet scanning from the 5′-end of the ATF4 mRNA and initiating translation at the positive-acting uORF1. Following uORF1 translation, ribosomes are thought to retain association with ATF4 mRNA and reinitiate translation at a downstream coding region (2Holcik M. Sonenberg N. Nat. Rev. Mol. Cell. Biol. 2005; 6: 318-327Crossref PubMed Scopus (1029) Google Scholar, 8Vattem K.M. Wek R.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11269-11274Crossref PubMed Scopus (1122) Google Scholar, 10Wek R.C. Cavener D.R. Antioxid. Redox Signal. 2007; 9: 2357-2371Crossref PubMed Scopus (234) Google Scholar). In non-stressed cells when eIF2α phosphorylation is low and there is abundant eIF2-GTP, ribosomes scanning downstream from uORF1 readily reinitiate translation at the next available ORF, the inhibitory uORF2. Following translation of uORF2, ribosomes are suggested to dissociate from the ATF4 transcript, leading to lowered translation of the ATF4 coding region. During stress conditions, elevated phosphorylation of eIF2α reduces eIF2-GTP levels, thus increasing the time required for scanning ribosomes to become competent to reinitiate translation. Following translation of uORF1, delayed reinitiation would allow for a portion of the ribosomes to bypass the uORF2 initiation codon, and instead translate the ATF4 coding region. The central feature in ATF4 translational control-delayed translation reinitiation in response to eIF2α phosphorylation, is shared with the mechanism that induces translation of a related bZIP transcriptional regulator GCN4 in yeast Saccharomyces cerevisiae (12Hinnebusch A.G. Annu. Rev. Microbiol. 2005; 59: 407-450Crossref PubMed Scopus (897) Google Scholar). ATF4 can form homodimers or heterodimers with other bZIP transcription factors, and elevated ATF4 synthesis directly contributes to increased binding of this transcription activator to the promoters of targeted genes (4Ron D. Walter P. Nat. Rev. Mol. Cell. Biol. 2007; 8: 519-529Crossref PubMed Scopus (4851) Google Scholar, 6Schroder M. Kaufman R.J. Annu. Rev. Biochem. 2005; 74: 739-789Crossref PubMed Scopus (2432) Google Scholar, 13Vinson C. Myakishev M. Acharya A. Mir A.A. Moll J.R. Bonovich M. Mol. Cell. Biol. 2002; 22: 6321-6335Crossref PubMed Scopus (343) Google Scholar, 14Kilberg M.S. Pan Y.X. Chen H. Leung-Pineda V. Annu. Rev. Nutr. 2005; 25: 59-85Crossref PubMed Scopus (220) Google Scholar). Among these genes is CHOP/GADD153, a bZIP transcriptional regulator that facilitates apoptosis during stress conditions (5Marciniak S.J. Ron D. Physiol. Rev. 2006; 86: 1133-1149Crossref PubMed Scopus (777) Google Scholar, 6Schroder M. Kaufman R.J. Annu. Rev. Biochem. 2005; 74: 739-789Crossref PubMed Scopus (2432) Google Scholar, 15Zinszner H. Kuroda M. Wang X.Z. Batchvarova N. Lightfoot R.T. Remotti H. Stevens J.L. Ron D. Genes Dev. 1998; 12: 982-995Crossref PubMed Scopus (1675) Google Scholar, 16Marciniak S.J. Yun C.Y. Oyadomari S. Novoa I. Zhang Y. Jungreis R. Nagata K. Harding H.P. Ron D. Genes Dev. 2004; 18: 3066-3077Crossref PubMed Scopus (1489) Google Scholar). ATF4 and CHOP contribute to the transcriptional expression of GADD34, encoding a targeting subunit for a type 1 Ser/Thr protein phosphatase that dephosphorylates eIF2α (5Marciniak S.J. Ron D. Physiol. Rev. 2006; 86: 1133-1149Crossref PubMed Scopus (777) Google Scholar, 16Marciniak S.J. Yun C.Y. Oyadomari S. Novoa I. Zhang Y. Jungreis R. Nagata K. Harding H.P. Ron D. Genes Dev. 2004; 18: 3066-3077Crossref PubMed Scopus (1489) Google Scholar). Therefore, the ATF4/GADD34 path is important for directing feedback control of the eIF2α kinase pathway, allowing for translation of stress-related mRNAs induced via ATF4. Microarray studies utilizing PEK/PERK-/- and ATF4-/- mouse embryo fibroblast (MEF) cells reported that of the genes requiring eIF2α phosphorylation for their induction in response to ER stress, about half required ATF4 function (7Harding H.P. Zhang Y. Zeng H. Novoa I. Lu P.D. Calfon M. Sadri N. Yun C. Popko B. Paules R. Stojdl D.F. Bell J.C. Hettmann T. Leiden J.M. Ron D. Mol. Cell. 2003; 11: 619-633Abstract Full Text Full Text PDF PubMed Scopus (2364) Google Scholar). These results suggest that there may be additional transcription factors that are important for directing the eIF2α kinase pathway and are subject to translational control. We reasoned that, given the central role for bZIP transcription activators in the eIF2α kinase response, there may be additional bZIP proteins that are subject to translational control in response to eIF2α phosphorylation. One candidate is ATF5, a bZIP transcriptional regulator that is encoded by an mRNA that contains two uORFs with analogous proximity to that described in the ATF4 transcript (13Vinson C. Myakishev M. Acharya A. Mir A.A. Moll J.R. Bonovich M. Mol. Cell. Biol. 2002; 22: 6321-6335Crossref PubMed Scopus (343) Google Scholar, 17Hansen M.B. Mitchelmore C. Kjaerulff K.M. Rasmussen T.E. Pedersen K.M. Jensen N.A. Genomics. 2002; 80: 344-350Crossref PubMed Scopus (47) Google Scholar). ATF5 mRNA is expressed in many different tissues, with the highest levels present in liver (17Hansen M.B. Mitchelmore C. Kjaerulff K.M. Rasmussen T.E. Pedersen K.M. Jensen N.A. Genomics. 2002; 80: 344-350Crossref PubMed Scopus (47) Google Scholar). ATF5 is suggested to play a role in cell survival, and reduced ATF5 function by siRNA approaches was shown to lead to selective destruction of certain cultured cancer cells, such as glioblastomas (18Angelastro J.M. Canoll P.D. Kuo J. Weicker M. Costa A. Bruce J.N. Greene L.A. Oncogene. 2005; 25: 907-916Crossref Scopus (72) Google Scholar). Because ATF5 is suggested to be highly expressed in neuroprogenitor cells, and is diminished in postmitotic neurons, appropriate expression of ATF5 is proposed to be critical for neural differentiation (18Angelastro J.M. Canoll P.D. Kuo J. Weicker M. Costa A. Bruce J.N. Greene L.A. Oncogene. 2005; 25: 907-916Crossref Scopus (72) Google Scholar, 19Angelastro J.M. Mason J.L. Ignatova T.N. Kukekov V.G. Stengren G.B. Goldman J.E. Greene L.A. J. Neurosci. 2005; 25: 3889-3899Crossref PubMed Scopus (67) Google Scholar, 20Angelastro J.M. Ignatova T.N. Kukekov V.G. Steindler D.A. Stengren G.B. Mendelsohn C. Greene L.A. J. Neurosci. 2003; 23: 4590-4600Crossref PubMed Google Scholar, 21Mason J.L. Angelastro J.M. Ignatova T.N. Kukekov V.G. Lin G. Greene L.A. Goldman J.E. Mol. Cell Neurosci. 2005; 29: 372-380Crossref PubMed Scopus (57) Google Scholar). In this study we find that expression of ATF5 protein is induced in response to eIF2α phosphorylation during a range of different stress conditions. Increased ATF5 expression occurs by a translational mechanism involving ribosomal reinitiation and uORFs located in the 5′-leader of the ATF5 mRNA. Superimposed with this translational control, we find that ATF5 mRNA levels are significantly reduced in ATF4-/- MEF cells, suggesting a role for ATF4-directed transcription of ATF5. Together, these results suggest that eIF2α phosphorylation directs ATF5 expression, and this transcription factor is integral to the eIF2α kinase response. Expression of Recombinant ATF5 and Antibody Production—Human ATF5 cDNA was inserted between the BamHI and XhoI restriction sites of plasmid pET28, yielding p834 that encodes an N-terminal polyhistidine-tagged version of full-length ATF5 expressed from an inducible T7 promoter. This plasmid was introduced and expressed in Escherichia coli strain BL21(DE3) (F- ompTrB- containing lysogen DE3), and bacterial cells were grown at 37 °C with shaking in Luria-Bertani medium supplemented with 100 μg/ml ampicillin until an A600 of between 0.4 and 0.6. 1 mm isopropyl β-d-thiogalactoside was added to the cultures, and after further incubation at 37 °C for 6 h, cells were collected by centrifugation. The cell pellet was suspended in Buffer A solution (20 mm Tris (pH 7.9), 500 mm NaCl, and 10% glycerol) containing 10 mm imidazole and lysed using a French press. Proteins in the soluble and insoluble portions of the lysates were separated by SDS-PAGE and visualized by staining with Coomassie R-250. The majority of recombinant ATF5 protein was found to be in the insoluble fraction. The soluble lysate portion was applied to nickel-nitrilotriacetic acid-agarose (Qiagen) equilibrated with Buffer A and incubated at 4 °C. The agarose was washed with Buffer A solution containing 50 mm imidazole, and the ATF5 protein was eluted with buffer A solution containing 200 mm imidazole. The purified recombinant ATF5 protein was Mr ∼ 35,000 and was specifically recognized by antibody recognizing the polyhistidine tag. To prepare ATF5-specific antibody, insoluble lysate containing recombinant ATF5 was separated by SDS-PAGE, and the ATF5 protein was sliced from the polyacrylamide gel and injected into rabbits. ATF5 polyclonal antibody was affinity-purified using recombinant ATF5 protein. As described further below in the immunoblot analyses, the ATF5-specific antibody recognized purified recombinant ATF5 protein and ATF5 in mouse and human cell lines that was induced by different stress conditions. Cell Culture and Stress Conditions—MEF cells that were derived from S/S (wild-type eIF2α) and A/A (mutant eIF2α-Ser51A) mice were previously described (22Scheuner D. Song B. McEwen E. Liu C. Laybutt R. Gillespie P. Saunders T. Bonner-Weir S. Kaufman R.J. Mol. Cell. 2001; 7: 1165-1176Abstract Full Text Full Text PDF PubMed Scopus (1085) Google Scholar, 23Jiang H.Y. Wek S.A. McGrath B.C. Scheuner D. Kaufmann R.J. Cavener D.R. Wek R.C. Mol. Cell. Biol. 2003; 23: 5651-5663Crossref PubMed Scopus (355) Google Scholar). PEK-/- and GCN2-/- MEF cells, and their wild-type counterparts, were reported previously (24Jiang H.Y. Wek S.A. McGrath B.C. Lu D. Hai T. Harding H.P. Wang X. Ron D. Cavener D.R. Wek R.C. Mol. Cell. Biol. 2004; 24: 1365-1377Crossref PubMed Scopus (387) Google Scholar). MEF cells were cultured in Dulbecco's modified Eagle's medium supplemented with 1 mm non-essential amino acids, 100 units/ml penicillin, 10% fetal bovine serum, and 100 μg/ml streptomycin. ER stress was elicited in MEF cells by the addition of either 0.1 μm or 1 μm thapsigargin to the medium, followed by incubation for up to 6 h, as indicated. Alternatively, 20 μm arsenite or 1 μm of the proteasome inhibitor, MG132, was added to the culture medium, and the MEF cells were cultured for up to 6 h, as indicated. To block transcription, 10 μm actinomycin D was added to MEF cells, as indicated. Human HepG2 hepatoma cells were cultured as described (25Chen H. Pan Y.X. Dudenhausen E.E. Kilberg M.S. J. Biol. Chem. 2004; 279: 50829-50839Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar) and treated with either 20 μm arsenite or 1 μm thapsigargin. Preparation of Protein Lysates and Immunoblot Analyses—MEF and human HepG2 cells cultured in stressed or non-stressed conditions were washed two times with chilled phosphate-buffered solution, and lysed in a solution containing 50 mm Tris-HCl (pH 7.9), 150 mm NaCl, 1% Nonidet P-40, 0.1% SDS, 100 mm NaF, 17.5 mm β-glycerolphosphate, 10% glycerol supplemented with protease inhibitors (100 μm of phenylmethylsulfonyl fluoride, 0.15 μm aprotinin, 1 μm leupeptin, and 1 μm of pepstatin) and sonication for 30 s. Cell lysates were clarified by centrifugation, and protein content was determined by the Bio-Rad protein quantitation kit for detergent lysis following the manufacturer's instructions. Equal amounts of each protein sample were separated by SDS-PAGE, and proteins were then transferred to nitrocellulose filters. Polypeptide markers of known molecular weights (Bio-Rad) were included to determine the size of proteins identified in the immunoblot analysis. Transferred filters were then incubated in TBS-T solution containing 20 mm Tris-HCl (pH 7.9), 150 mm NaCl, and 0.2% Tween 20 supplemented with 4% nonfat milk, followed by incubation with TBS-T solution with antibody that specifically recognized the indicated proteins. ATF5 antibody was prepared against recombinant human ATF5 protein, as described above. ATF4 antibody was prepared against an affinity-purified rabbit polyclonal antibody prepared against purified polyhistidine-tagged human ATF4. CHOP (sc-7351) antibody was obtained from Santa Cruz Biotechnology, and β-actin monoclonal antibody (A5441) was purchased from Sigma. Polyclonal antibody that specifically recognized phosphorylated eIF2α at Ser-51 was purchased from BioSource (44-728G). Monoclonal antibodies that recognizes either phosphorylated or nonphosphorylated forms of eIF2α was provided by Dr. Scot Kimball (Pennsylvania State University, College of Medicine, Hershey, PA). Filters were then washed three times in TBS-T, and the protein-antibody complexes were visualized using horseradish peroxidase-labeled secondary antibody and chemiluminescent substrate. Autoradiograms shown in the figures are representative of three independent experiments. RNA Isolation and Analyses—Northern analyses were carried out as previously described (26Kevil C.G. Walsh L. Laroux F.S. Kalogeris T. Grisham M.B. Alexander J.S. Biochem. Biophys. Res. Commun. 1997; 238: 277-279Crossref PubMed Scopus (96) Google Scholar). Total cellular RNA was isolated from S/S and A/A MEF cells treated with 1 μm thapsigargin, 20 μm sodium arsenite, or no stress, for the indicated number of hours using the TRIzol reagent (Invitrogen) following the manufacturer's instructions. 10 μg of total RNA from each sample preparation was separated by electrophoresis using a 1.2% agarose gel and visualized by using ethidium bromide staining and UV light. RNA was transferred onto nylon filters and hybridized to 32P-labeled-DNA probes specific for the indicated genes. Filters were washed using high stringency conditions and visualized by autoradiography. Levels of ATF5-luciferase mRNA expressed in S/S and A/A MEF cells transfected with the ATF5-Luc fusion constructs were treated with 0.1 μm thapsigargin for 6 h, or no stress. A 32P-labeled probe complementary to the luciferase reporter gene was used in a Northern blot analysis to measure ATF5-Luc transcripts. Plasmid Constructions and Luciferase Assays—PCR was used to generate a HindIII-PagI fragment DNA encoding the full-length ATF5 mRNA leader and ATF5 initiation codon, which was inserted between HindIII and NcoI restriction sites in a derivative of plasmid pGL3. The resulting plasmid contains the 5′-portion of the ATF5 coding sequence fused to the luciferase reporter gene downstream of a minimal TK promoter. The ATG initiation codons in each of the uORFs in the ATF5 mRNA were mutated individually or in combination to AGG using the site-directed mutagenesis kit (Stratagene), following the manufacturer's instructions. All mutations were sequenced to ensure that there were only the desired changes. Plasmid transfections were performed using the S/S and A/A MEF cells grown to 40% confluency and the FuGENE 6 transfection reagent (Roche Applied Science). Co-transfections were carried out in triplicates using wild type or mutant versions of the ATF5-Luc fusion plasmids and a Renilla luciferase plasmid serving as an internal control (Promega, Madison, WI). 24 h after transfection, MEF cells were treated with 0.1 μm thapsigargin for 6 h, or with no ER stress. Dual luciferase assays were carried out as described by the Promega instruction manual. Values are a measure of a ratio of firefly versus Renilla luciferase units (relative light units) and represent the mean values of three independent transfections. Results are presented as means ± S.E. that were derived from three independent experiments. The Student's t test was used to determine the statistical significance. Transcriptional Start Site of ATF5 Transcripts—The cDNAs corresponding to the 5′-ends of the ATF5-Luc transcripts expressed in S/S MEF cells treated with 0.1 μm thapsigargin, or no stress, were amplified using a RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE kit, Ambion) following the manufacturer's instructions. Alternatively, there was amplification of cDNAs corresponding to the 5′-ends of ATF5 transcripts prepared from human HepG2 hepatoma cells treated with this ER stress condition, or no stress. Briefly, 10 μg of total RNA was treated with calf intestinal phosphatase, resulting in the removal of free 5′-phosphates from RNAs other than mRNAs containing intact 5′-cap structures. The RNA preparations were then treated with tobacco acid pyrophosphatase to remove the cap structure, leaving a 5′-monophosphate that was ligated using T4 RNA ligase to a 45-base RNA adapter oligonucleotide that was supplied in the kit. A random-primed reverse transcription (RT) reaction and nested PCR were then carried out to amplify the 5′-end of endogenous ATF5 transcripts, as well as transfected thymidine kinase-minimal promoter driven ATF5-Luc mRNAs. The primers corresponding to the 5′-RACE adapter sequence were provided by the manufacturer. The sequences of the two nested antisense primers specific to endogenous ATF5 mRNA were the outer primer 5′-TTCCCATAGTCTACGAGCCATCCC-3′ and inner primer 5′-ACATGGCTGTAGCACAGGTGCT-3′. The outer primer used for amplifying the 5′-ends of ATF5-Luc transcript was 5′-CCATCTTCCAGCGGATAGAA-3′, which was combined with the same inner primer described earlier. A portion of the amplified DNA products were analyzed by agarose gel electrophoresis, and the DNA was visualized by ethidium bromide staining and UV irradiation. The major DNA band was excised from the gel and sequenced (Fig. 1). The transcriptional start site was determined as the first nucleotide residue that was 3′- to the adapter sequence that was ligated to 5′- of the cDNA. Polysome Analysis of ATF5 Translational Control—S/S cells were cultured in Dulbecco's modified Eagle's medium, as highlighted above, in the presence of 1.0 μm thapsigargin, or to no stress, for 6 h. 10 μg/ml cycloheximide was added to the medium prior to collection and analysis. Cells were washed in cold phosphate-buffered-saline solution supplemented with 10 μg/ml cycloheximide, and then lysed with ice-cold lysis buffer containing 20 mm Tris-HCl (pH 7.5), 5 mm MgCl2, 100 mm NaCl, 0.4% Nonidet P-40, and 10 μg/ml cycloheximide. The extracts were passed through a 23-gauge needle for proper lysis of cells, incubated for 10 min on ice, and insoluble material was collected by microcentrifugation at 10,000 rpm for 10 min at 4 °C. The resulting supernatant was then applied onto a 15-45% sucrose gradient containing 20 mm Tris-HCl (pH 7.5), 5 mm MgCl2, 100 mm NaCl, 10 μg/ml cycloheximide, and centrifuged for 2 h at 40,000 rpm in a Beckman SW-41Ti rotor. Following centrifugation, the gradients were fractionated, and the absorbance of cytosolic RNA at 254 nm was recorded by an in-line UV monitor. Total RNA was isolated from a portion of each fraction using TRIzol reagent as described by the manufacturer's instructions (Invitrogen). The mRNAs from fractions collected were amplified using RT-PCR kit (Invitrogen) and the following primers: ATF4 forward (5′-TCACGAAATCCAGCAGCAGTG-3′), ATF4 reverse (5′-CAAGCCATCATCCATAGCCG-3′), ATF5 forward (5′-CTACCCCTCCATTCCACTTTCC-3′, ATF5 reverse (5′-TTCTTGACTGGCTTCTCACTTGTG-3′), β-actin forward (5′-TTCTTTGCAGCTCCTTCGTTGCCG-3′), and β-actin reverse (5′-TGGATGGCTACGTACATGGCTGGG-3′). For RT-PCR analysis of cytosolic mRNA levels, equal volumes were reverse transcribed using oligo(dT), then 50 ng of cDNA from each fraction was amplified with Bullseye R-Taq (MIDSCI). PCR was carried out for 25, 27, 30, 33, and 35 cycles to determine the linear range of amplification. In this study, 25 cycles were used for PCR for ATF4, and 27 cycles for ATF5 and β-actin. Densitometry was performed using the software provided with Quantity One imaging system (Bio-Rad). Phosphorylation of eIF2α Is Required for Increased ATF5 Protein Levels in Response to Diverse Stress Conditions—The ATF5 mRNA has two uORFs that are conserved among many different vertebrates, including human, mouse, rat, cow, and frogs (Fig. 1). The 5′-proximal uORF1 encodes a polypeptide that is only three amino acid residues in length, Met-Ala-Leu, that is conserved among the different ATF5 orthologs. The downstream uORF2 encodes a polypeptide ranging from 59 residues in length in human and cow, to 53 residues in the frog ATF5 mRNA. In each example, the uORF2 overlaps, out of frame, with the ATF5 coding region (Fig. 1). Given the importance of uORFs in ATF4 translational control in response to eIF2α phosphorylation, we addressed whether the levels of ATF5 protein were increased in response to environmental stresses by a mechanism requiring eIF2α phosphorylation. Wild-type MEF cells, designated S/S, and a mutant version containing alanine substituted for the serine-51 phosphorylation site in eIF2α, termed A/A, were exposed to three different stress conditions known
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