Delineation of a Negative Feedback Regulatory Loop That Controls Protein Translation during Endoplasmic Reticulum Stress
2003; Elsevier BV; Volume: 278; Issue: 37 Linguagem: Inglês
10.1074/jbc.m301107200
ISSN1083-351X
AutoresYanjun Ma, Linda M. Hendershot,
Tópico(s)Autophagy in Disease and Therapy
ResumoTransient protein synthesis inhibition is an important protective mechanism used by cells during various stress conditions including endoplasmic reticulum (ER) stress. This response centers on the phosphorylation state of eukaryotic initiation factor (eIF)-2α, which is induced by kinases like protein kinase R-like ER kinase (PERK) and GCN2 to suppress translation and is later reversed so translation resumes. GADD34 was recently identified as the factor that activates the type 1 protein serine/threonine phosphatase (PP1), which dephosphorylates eIF-2α during cellular stresses. Our study delineates a negative feedback regulatory loop in which the eIF-2α-controlled inhibition of protein translation leads to GADD34 induction, which promotes translational recovery. We show that activating transcription factor-4 (ATF4), which is paradoxically translated during the eIF-2α-mediated translational block, is required for the transactivation of the GADD34 promoter in response to ER stress and amino acid deprivation. ATF4 directly binds to and trans-activates a conserved ATF site in the GADD34 promoter during ER stress. Examination of ATF4–/– MEFs revealed an absence of GADD34 induction, prolonged eIF-2α phosphorylation, delayed protein synthesis recovery, and diminished translational up-regulation of BiP during ER stress. These studies demonstrate the essential role of GADD34 in the resumption of protein synthesis, define the pathway for its induction, and reveal that cytoprotective unfolded protein response targets like BiP are sensitive to the eIF-2α-mediated block in translation. Transient protein synthesis inhibition is an important protective mechanism used by cells during various stress conditions including endoplasmic reticulum (ER) stress. This response centers on the phosphorylation state of eukaryotic initiation factor (eIF)-2α, which is induced by kinases like protein kinase R-like ER kinase (PERK) and GCN2 to suppress translation and is later reversed so translation resumes. GADD34 was recently identified as the factor that activates the type 1 protein serine/threonine phosphatase (PP1), which dephosphorylates eIF-2α during cellular stresses. Our study delineates a negative feedback regulatory loop in which the eIF-2α-controlled inhibition of protein translation leads to GADD34 induction, which promotes translational recovery. We show that activating transcription factor-4 (ATF4), which is paradoxically translated during the eIF-2α-mediated translational block, is required for the transactivation of the GADD34 promoter in response to ER stress and amino acid deprivation. ATF4 directly binds to and trans-activates a conserved ATF site in the GADD34 promoter during ER stress. Examination of ATF4–/– MEFs revealed an absence of GADD34 induction, prolonged eIF-2α phosphorylation, delayed protein synthesis recovery, and diminished translational up-regulation of BiP during ER stress. These studies demonstrate the essential role of GADD34 in the resumption of protein synthesis, define the pathway for its induction, and reveal that cytoprotective unfolded protein response targets like BiP are sensitive to the eIF-2α-mediated block in translation. When mammalian cells encounter physiological or chemical stresses that impinge upon the normal folding of proteins in the endoplasmic reticulum (ER), 1The abbreviations used are: ER, endoplasmic reticulum; eIF, eukaryotic initiation factor; ORF, open reading frame; UTR, untranslated region; UPR, unfolded protein response; WT, wild type; MEFs, mouse embryonic fibroblasts; Tg, thapsigargin; EMSA, electrophoretic mobility shift assays; PERK, protein kinase R-like ER kinase; ATF4, activating transcription factor-4; CHIP, chromatin immunoprecipitation; CAT, chloramphenicol acetyltransferase. rapid and complex signal transduction cascades, known collectively as the unfolded protein response (UPR), are put into play that are responsible for a number of cytoprotective measures (1Ma Y. Hendershot L.M. Cell. 2001; 107: 827-830Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar, 2Patil C. Walter P. Curr. Opin. Cell Biol. 2001; 13: 349-355Crossref PubMed Scopus (677) Google Scholar, 3Kaufman R.J. Genes Dev. 1999; 13: 1211-1233Crossref PubMed Scopus (1934) Google Scholar, 4Lee A.S. Trends Biochem. Sci. 2001; 26: 504-510Abstract Full Text Full Text PDF PubMed Scopus (922) Google Scholar). If homeostasis of the ER cannot be re-established, apoptotic pathways are activated to destroy the damaged cell in order to ultimately protect the organism (5Nakagawa T. Zhu H. Morishima N. Li E. Xu J. Yankner B.A. Yuan J. Nature. 2000; 403: 98-103Crossref PubMed Scopus (2955) Google Scholar). The protective components of the UPR include the transcriptional up-regulation of ER chaperones and folding enzymes. These proteins bind to unfolded proteins that begin accumulating in the ER and prevent their aggregation. In addition, the increased expression of the chaperones allows for the rapid refolding of nascent secretory pathway proteins once homeostasis has been re-established. A second protective response is the transient block in protein synthesis (6Brostrom C.O. Brostrom M.A. Prog. Nucleic Acids Res. Mol. Biol. 1998; 58: 79-125Crossref PubMed Scopus (250) Google Scholar). This limits the load of proteins being introduced into a hostile environment where they would be unable to fold. The inhibition of protein synthesis is not limited to ER proteins and is responsible for a third aspect of the response, which is the arrest of cells in the G1 phase of cell cycle. The inhibition of the synthesis of the short lived cyclin D1 protein results in a rapid depletion of this protein and a concomitant cell cycle arrest (7Brewer J.W. Hendershot L.M. Sherr C.J. Diehl J.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8505-8510Crossref PubMed Scopus (233) Google Scholar). Finally, the up-regulation of proteins associated with proteasomal degradation helps to dispose of the accumulated unfolded proteins, further reducing the load on the ER (8Friedlander R. Jarosch E. Urban J. Volkwein C. Sommer T. Nat. Cell Biol. 2000; 2: 379-384Crossref PubMed Scopus (391) Google Scholar, 9Travers K.J. Patil C.K. Wodicka L. Lockhart D.J. Weissman J.S. Walter P. Cell. 2000; 101: 249-258Abstract Full Text Full Text PDF PubMed Scopus (1594) Google Scholar). In the past 5 years, many components of the signaling pathways controlling these cytoprotective responses have been described. The ER-localized transmembrane protein, ATF6, encodes a cytosolic transcription factor tethered to an ER luminal “stress sensing” domain (10Haze K. Yoshida H. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. 1999; 10: 3787-3799Crossref PubMed Scopus (1552) Google Scholar). ER stress allows ATF6 to travel to the Golgi where it is cleaved by the S1P and S2P proteases, thus liberating the cytosolic transcription factor domain (11Ye J. Rawson R.B. Komuro R. Chen X. Dave U.P. Prywes R. Brown M.S. Goldstein J.L. Mol. Cell. 2000; 6: 1355-1364Abstract Full Text Full Text PDF PubMed Scopus (1367) Google Scholar, 12Shen J. Chen X. Hendershot L. Prywes R. Dev. Cell. 2002; 3: 99-111Abstract Full Text Full Text PDF PubMed Scopus (1074) Google Scholar). Cleaved ATF6 binds to ER stress response elements in the chaperone promoters and up-regulates their transcription. In addition, three transmembrane ER kinases have been identified that also possess luminal “sensing” domains in addition to their cytosolic kinase domains. Two of the kinases, Ire1α and β (13Tirasophon W. Welihinda A.A. Kaufman R.J. Genes Dev. 1998; 12: 1812-1824Crossref PubMed Scopus (747) Google Scholar, 14Wang X.Z. Harding H.P. Zhang Y. Jolicoeur E.M. Kuroda M. Ron D. EMBO J. 1998; 17: 5708-5717Crossref PubMed Scopus (658) Google Scholar), are orthologues of the sole ER stress transducer in yeast, Ire1p. Like yeast Ire1p, these proteins possess a unique C-terminal endonuclease domain, which upon activation of the kinase domains targets a specific RNA sequence. Most recently, the XBP-1 transcription factor was shown to be the target of endonuclease activity of the mammalian Ire1 (15Yoshida H. Matsui T. Yamamoto A. Okada T. Mori K. Cell. 2001; 107: 881-891Abstract Full Text Full Text PDF PubMed Scopus (2981) Google Scholar, 16Shen X. Ellis R.E. Lee K. Liu C.Y. Yang K. Solomon A. Yoshida H. Morimoto R. Kurnit D.M. Mori K. Kaufman R.J. Cell. 2001; 107: 893-903Abstract Full Text Full Text PDF PubMed Scopus (561) Google Scholar, 17Calfon M. Zeng H. Urano F. Till J.H. Hubbard S.R. Harding H.P. Clark S.G. Ron D. Nature. 2002; 415: 92-96Crossref PubMed Scopus (2143) Google Scholar). The Ire1-catalyzed removal of 26 nucleotides from XBP-1 mRNA followed by a religation reaction alters the XBP-1 reading frame. The remodeled XBP-1 protein encodes a novel transcription factor with the original DNA binding domain tethered to a new transactivation domain. The targets of spliced XBP-1 are not clear but may be involved in ER-associated degradation (18Yoshida H. Matsui T. Hosokawa N. Kaufman R.J. Nagata K. Mori K. Dev. Cell. 2003; 4: 265-271Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar). The ATF6 transcription factor up-regulates XBP-1 mRNA providing a convergence of the Ire1-ATF6 pathways. The third ER stress-transducing kinase is pancreatic eIF-2α kinase (PEK)/PERK (19Harding H.P. Zhang Y. Ron D. Nature. 1999; 397: 271-274Crossref PubMed Scopus (2533) Google Scholar, 20Shi Y. Vattem K.M. Sood R. An J. Liang J. Stramm L. Wek R.C. Mol. Cell. Biol. 1998; 18: 7499-7509Crossref PubMed Google Scholar), which shares homology with the luminal domains of Ire1α and -β but does not encode an endonuclease domain. The target of the kinase activity of PERK is the eucaryotic translation initiation factor, eIF-2α. Phosphorylation of eIF-2α during ER stress interferes with the assembly of the translation initiation complex, thereby dramatically diminishing translation (21Scheuner 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 (1090) Google Scholar, 22Harding H.P. Zeng H. Zhang Y. Jungries R. Chung P. Plesken H. Sabatini D.D. Ron D. Mol. Cell. 2001; 7: 1153-1163Abstract Full Text Full Text PDF PubMed Scopus (1008) Google Scholar). This aspect of the UPR is shared by a number of other cellular stresses, which activate different eIF-2α kinases. Interestingly, although most cellular protein synthesis is shut down, translation of the ATF4 transcription factor is specifically induced. ATF4 is constitutively transcribed in non-stressed cells, even though its transcripts cannot be efficiently translated due to the presence of short open reading frames (5′-ORF) in its 5′-untranslated region (5′-UTR) that interfere with initiation at the proper start codon. During ER stress, these short ORFs are no longer utilized which leads to a dramatic increase in ATF4 protein synthesis (23Harding 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 (2415) Google Scholar). One target of the ATF4 transcription factor is the CHOP promoter (24Ma Y. Brewer J.W. Diehl J.A. Hendershot L.M. J. Mol. Biol. 2002; 318: 1351-1365Crossref PubMed Scopus (553) Google Scholar, 25Fawcett T.W. Martindale J.L. Guyton K.Z. Hai T. Holbrook N.J. Biochem. J. 1999; 339: 135-141Crossref PubMed Scopus (368) Google Scholar), which appears to play a role in the induction of apoptosis during ER stress. Unlike the up-regulation of ER chaperones, the inhibition of protein synthesis during the UPR and other cellular stresses is transient. This is accomplished by inhibition of the PERK kinase itself and dephosphorylation of eIF-2α. Very recently, it was demonstrated that a constitutively expressed double-stranded RNA-activated protein kinase (PKR) inhibitor, p58IPK, is induced during ER stress and plays an important role in down-regulating PERK phosphorylation later during ER stress (26Yan W. Frank C.L. Korth M.J. Sopher B.L. Novoa I. Ron D. Katze M.G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15920-15925Crossref PubMed Scopus (303) Google Scholar, 27Van Huizen R. Martindale J.L. Gorospe M. Holbrook N.J. J. Biol. Chem. 2003; 278: 15558-15564Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). GADD34 was originally identified in a screen for genes induced during growth arrest and DNA damage (28Fornace Jr., A.J. Alamo Jr., I. Hollander M.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8800-8804Crossref PubMed Scopus (564) Google Scholar). Later study reveals that the GADD34 protein directly interacts with the catalytic subunit of type 1 protein serine/threonine phosphatase (PP1) and activates the ability of PP1 to dephosphorylate eIF-2α, allowing most protein synthesis to resume (29Novoa I. Zeng H. Harding H.P. Ron D. J. Cell Biol. 2001; 153: 1011-1022Crossref PubMed Scopus (1040) Google Scholar, 30Connor J.H. Weiser D.C. Li S. Hallenbeck J.M. Shenolikar S. Mol. Cell. Biol. 2001; 21: 6841-6850Crossref PubMed Scopus (224) Google Scholar, 31Novoa I. Zhang Y. Zeng H. Jungreis R. Harding H.P. Ron D. EMBO J. 2003; 22: 1180-1187Crossref PubMed Scopus (349) Google Scholar). The transcription induction of GADD34 during ER and other cellular stresses is dependent on the presence and activity of the respective eIF-2α kinases (29Novoa I. Zeng H. Harding H.P. Ron D. J. Cell Biol. 2001; 153: 1011-1022Crossref PubMed Scopus (1040) Google Scholar). The mechanism of GADD34 induction during cellular stress is not presently known. In this study, we demonstrate that the translational induction of ATF4 in response to eIF-2α phosphorylation is immediately upstream of GADD34 induction. ATF4 null MEFs are unable to induce GADD34 mRNA during ER or other cellular stresses, which leads to prolonged eIF-2α phosphorylation and diminished protein synthesis recovery. ATF4 is able to bind specifically to and trans-activate a conserved ATF site in the GADD34 promoter in a stress-dependent manner. Through the chromatin immunoprecipitation assay, we further demonstrate that ATF4 directly binds to the GADD34 promoter region encompassing the ATF site in an ER stress-dependent manner. Together, our study delineates an autoregulatory loop in which PERK (or presumably other stress-specific eIF-2α kinases) phosphorylates eIF-2α, in turn inhibits protein synthesis, and allows ATF4 translation. This then leads to GADD34 induction, eIF-2α dephosphorylation, and finally the resumption of protein synthesis. Furthermore, our data revealed that BiP up-regulation at the translational level is significantly diminished in ATF4 null cells where protein synthesis inhibition persists, demonstrating that the cytoprotective UPR targets are not immune to the eIF-2α-dependent translational inhibition and thus underscore the importance of translational recovery early in the response. Isolation, Genotyping, Culture, and Treatment of WT and ATF4 Null Mouse Embryonic Fibroblasts—Our ATF4 heterozygous mice were generated by Tanaka et al. (32Tanaka T. Tsujimura T. Takeda K. Sugihara A. Maekawa A. Terada N. Yoshida N. Akira S. Genes Cells. 1998; 3: 801-810Crossref PubMed Scopus (133) Google Scholar) and were further maintained by Dr. Adrian F. Gombart, Cedars-Sinai Medical Center, who kindly provided our breeding pairs. In keeping with previous reports, homozygous ATF4 null mice are born at significantly less than expected Mendelian ratios and are blind due to the lack of lens development (32Tanaka T. Tsujimura T. Takeda K. Sugihara A. Maekawa A. Terada N. Yoshida N. Akira S. Genes Cells. 1998; 3: 801-810Crossref PubMed Scopus (133) Google Scholar, 33Hettmann T. Barton K. Leiden J.M. Dev. Biol. 2000; 222: 110-123Crossref PubMed Scopus (111) Google Scholar). Mouse embryos were explanted at day 13.5–14.5 of gestation from ATF4 +/– animals to obtain embryonic fibroblasts (MEFs) (34Kamijo T. Zindy F. Roussel M.F. Quelle D.E. Downing J.R. Ashmun R.A. Grosveld G. Sherr C.J. Cell. 1997; 91: 649-659Abstract Full Text Full Text PDF PubMed Scopus (1385) Google Scholar). MEFs were propagated in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 2 mm glutamine, 0.1 mm nonessential amino acids, 55 μm 2-mercaptoethanol, and 10 μg/ml gentamicin (Invitrogen). Both the amniotic membrane of the embryo and the cultured MEFs were genotyped as illustrated before (32Tanaka T. Tsujimura T. Takeda K. Sugihara A. Maekawa A. Terada N. Yoshida N. Akira S. Genes Cells. 1998; 3: 801-810Crossref PubMed Scopus (133) Google Scholar) with primer pair “a” and “b” for the WT allele and primer pair “b” and “c” for the mutant allele. a is 5′-AAAGGAATGCTCTGGAGTGGAAGACAG; b is 5′-AAGCAAAGCTAAGCCTCCATCTTGTGC; and c is 5′-ATCGCCTTCTATCGCCTTCTTGACGAG. Passage 5–7 MEFs were plated for experiments and were left untreated, treated with Tg (1 μm), or cultured in media that lacked leucine for the indicated periods of time (components of the minus leucine media are based on Dulbecco's modified Eagle's medium (Invitrogen 11960) with only leucine left out). Isolation and Analysis of Cytoplasmic RNA—Cytoplasmic RNA was harvested using the Qiagen RNeasy miniprep kit following the protocol provided by the supplier. 1 μg of RNA was used to generate 50 μl of cDNA using a poly(dT) primer (1 mm). Semi-quantitative PCR was performed with the fail-safe PCR system from Epicenter using 2 μl of cDNA, 0.25 mm of each primer pair, 2.5 units of Taq DNA polymerase, and 33 nm [α-32P]dCTP (Amersham Biosciences) for 18–20 cycles. The PCR products were run on a 4% TBE-acrylamide gel, dried, and exposed to film. For quantification data, each radioactive band was quantified using an Amersham Biosciences PhosphorImager and normalized to the level of γ-actin transcript measured from the same sample. The linear range was monitored by using serial dilutions (1:5 and 1:25) of the sample that showed the highest radioactive signal in each experimental group. The primer pair for mouse γ-actin was 5′-CCGACGGGCAGGTGATCAC (upstream) and 5′-GAGCAGTTAACTTGAATACAAGG (downstream) and for mouse BiP (GRP78) was 5′-GATTCCAAGGAACACTGTGGTA (upstream) and 5′-CAGTAAACAGCCACTTGGGC (downstream). 20 μg of cytoplasmic mRNA from each experimental group was run on a Northern blot as described previously (35Brewer J.W. Cleveland J.L. Hendershot L.M. EMBO J. 1997; 16: 7207-7216Crossref PubMed Scopus (77) Google Scholar). The probe for BiP was an 800-bp piece amplified from mouse cDNA with oligonucleotides mentioned above. The probe for ATF4 was a 600-bp piece amplified from mouse cDNA with oligonucleotides 5′-CCAGGGGTTCTGTCTTCCAC and 5′-AGCAAACACAGCAACACAAGCA. The probe for GADD34 was an 825-bp piece amplified from mouse cDNA with oligonucleotides 5′-CCCAGACACATGGCCCCG and 5′-TTGTCTCAGGTCCTCCTTCC. The probe for GAPDH is a human fragment purchased from Clontech (Palo Alto, CA). Western Blot, Metabolic Labeling, and Immunoprecipitation—For direct Western blotting, cells were lysed in Nonidet P-40 lysing buffer and separated on a reducing 10% SDS-acrylamide gel, transferred to a nitrocellulose membrane, and probed with the indicated primary antibody as described previously (24Ma Y. Brewer J.W. Diehl J.A. Hendershot L.M. J. Mol. Biol. 2002; 318: 1351-1365Crossref PubMed Scopus (553) Google Scholar): polyclonal rabbit anti-BiP (36Hendershot L.M. Wei J.Y. Gaut J.R. Lawson B. Freiden P.J. Murti K.G. Mol. Biol. Cell. 1995; 6: 283-296Crossref PubMed Scopus (88) Google Scholar), rabbit anti-ATF4, goat anti-Hsc70 (Santa Cruz Biotechnology, Santa Cruz CA), rabbit anti-phosphorylated eIF-2α, and monoclonal mouse anti-total eIF-2α (BIOSOURCE, Camarillo, CA). Metabolic labeling and immunoprecipitation were performed as described previously (37Lawson B. Brewer J.W. Hendershot L.M. J. Cell. Physiol. 1998; 174: 170-178Crossref PubMed Scopus (84) Google Scholar). In brief, WT or ATF4–/– MEFs were labeled for 30 min with [35S]methionine and -cysteine (Translabel, ICN, Irvine, CA). Cells were then harvested, lysed directly in Nonidet P-40 lysing buffer, and immunoprecipitated with polyclonal rabbit anti-BiP antiserum before electrophoresing on a 10% denaturing SDS gel. 35S signals were quantified using a Amersham Biosciences PhosphorImager. Nuclear Extract Purification, Electrophoretic Mobility Shift Assay, and Supershift Assay—WT or ATF4 null MEFs treated with Tg (1 μm) for the indicated times were harvested and washed with phosphate-buffered saline twice before nuclear extracts were purified as described (24Ma Y. Brewer J.W. Diehl J.A. Hendershot L.M. J. Mol. Biol. 2002; 318: 1351-1365Crossref PubMed Scopus (553) Google Scholar). The EMSA reaction was performed as described previously (24Ma Y. Brewer J.W. Diehl J.A. Hendershot L.M. J. Mol. Biol. 2002; 318: 1351-1365Crossref PubMed Scopus (553) Google Scholar) with 10 μg of nuclear extract, 1 μg of dI-dC, 10-fold excess of cold mutant oligonucleotides, and [γ-32P]ATP-labeled probe (10,000 cpm) or 2 μg of supershift antibody when indicated (polyclonal rabbit anti-C/EBPβ and anti-ATF4 antisera were purchased from Santa Cruz Biotechnology). The double strand synthetic oligonucleotide probes were annealed and end-labeled with [γ-32P]ATP as described (24Ma Y. Brewer J.W. Diehl J.A. Hendershot L.M. J. Mol. Biol. 2002; 318: 1351-1365Crossref PubMed Scopus (553) Google Scholar). A 29-bp CHOP C/EBP-ATF WT probe was designed as described previously (24Ma Y. Brewer J.W. Diehl J.A. Hendershot L.M. J. Mol. Biol. 2002; 318: 1351-1365Crossref PubMed Scopus (553) Google Scholar). Similarly, a 29-bp sequence containing –77 to –53 bp of the mouse GADD34 promoter (5′-GATCGCTCTCCGGTGACGTCAGCACAGCA) was designated mGADD34-ATF. For the mutant oligonucleotides, the GTGACGTCAG sequence of the WT probe containing the ATF site was altered to acttatgtca. Chromatin Immunoprecipitation (CHIP) Assay—The CHIP assay was performed with the CHIP kit from Upstate Biotechnology, Cell Signaling Solutions (Lake Placid, NY), and according to the protocol provided by Upstate Biotechnology with the following modifications. Instead of using whole cell lysates, nuclear extracts were harvested from non-stressed and Tg-treated (1 μm, 6 h) NIH3T3 cells using method described before (24Ma Y. Brewer J.W. Diehl J.A. Hendershot L.M. J. Mol. Biol. 2002; 318: 1351-1365Crossref PubMed Scopus (553) Google Scholar). The cross-linked chromatin-protein complexes were sonicated to obtain chromatin fragments with an average length of ∼600–700 bp. Polyclonal rabbit anti-YY-1 antibody was used in the negative control immunoprecipitation, and a polyclonal rabbit anti-ATF4 antibody was used for ATF4 immunoprecipitation (both from Santa Cruz Biotechnology). Two targeted sequences were amplified by 25 cycles of PCR in the presence of 33 nm [α-32P]dCTP. The primer pair used to amplify region A of the mouse GADD34 promoter was 5′-GCTCGGAAATTACGTGAGATCG and 5′-GCGCCAACATCGTCCACGCG. The pair for the amplification of region B was 5′-GGGCAGCCAGGGCTATACTG and 5′-GGTGTACAAGGAGGTCGGAAG. Chloramphenicol Acetyltransferase Assay—Sequences encompassing –721 to +130 bp of the mouse GADD34 promoter was cloned into a CAT reporter vector using PCR-based method as mentioned before (24Ma Y. Brewer J.W. Diehl J.A. Hendershot L.M. J. Mol. Biol. 2002; 318: 1351-1365Crossref PubMed Scopus (553) Google Scholar). The ATF site (TGACGTCA) alone was deleted in the mutant promoter construct with the Stratagene Quickchange™ site-directed mutagenesis kit (Cedar Creek, TX). 1 μg of WT or mutant construct was transfected into COS-1 cells, and cell lysates were analyzed for CAT activity as described before (24Ma Y. Brewer J.W. Diehl J.A. Hendershot L.M. J. Mol. Biol. 2002; 318: 1351-1365Crossref PubMed Scopus (553) Google Scholar). Each experimental group was analyzed in triplicate, normalized against protein concentration, and then repeated twice. The error bars shown in Fig. 6D were calculations of standard deviations for each triplicate experiment. Isolation and Analysis of ATF4 Null Mouse Embryonic Fibroblasts—Recently, the stress-induced GADD34 protein was shown to bind to the PP1 phosphatase and activate the dephosphorylation of eIF-2α, thereby triggering the resumption of protein translation during cellular stresses (29Novoa I. Zeng H. Harding H.P. Ron D. J. Cell Biol. 2001; 153: 1011-1022Crossref PubMed Scopus (1040) Google Scholar, 30Connor J.H. Weiser D.C. Li S. Hallenbeck J.M. Shenolikar S. Mol. Cell. Biol. 2001; 21: 6841-6850Crossref PubMed Scopus (224) Google Scholar, 31Novoa I. Zhang Y. Zeng H. Jungreis R. Harding H.P. Ron D. EMBO J. 2003; 22: 1180-1187Crossref PubMed Scopus (349) Google Scholar). Induction of GADD34 during ER stress and amino acid deprivation stress did not occur in PERK–/– or GCN2–/– cells, respectively, which are defective in the initial block in protein synthesis during ER stress or amino acid deprivation (29Novoa I. Zeng H. Harding H.P. Ron D. J. Cell Biol. 2001; 153: 1011-1022Crossref PubMed Scopus (1040) Google Scholar). This suggests the presence of a feedback regulatory loop, in which the eIF-2α kinases block protein synthesis and by an unknown mechanism induce GADD34, which then allows translation to resume. Ron and co-workers (29Novoa I. Zeng H. Harding H.P. Ron D. J. Cell Biol. 2001; 153: 1011-1022Crossref PubMed Scopus (1040) Google Scholar) suggested that this might occur either via a mechanism similar to the induction of ATF4 where small 5′-ORFs that interfere with correct translation initiation are suppressed by the inhibition of protein synthesis thus allowing initiation at the correct site or, conversely, that GADD34 might be a target of the ATF4 transcription factor. To address the role of ATF4 in the activation of GADD34 directly, we isolated WT and ATF4 null mouse embryonic fibroblasts (MEFs) by mating ATF4 +/– mice. To confirm the lack of ATF4 transcription and translation in these cells, cytoplasmic mRNA and whole cell lysates were harvested from WT and ATF4–/– cells that were left untreated or treated with Tg to induce ER stress. The ATF4 transcript was detected using Northern blotting (Fig. 1B), and ATF4 protein expression was detected via Western blotting (Fig. 1A). ATF4 message was detected only in the WT MEFs but not in the ATF4 null MEFs (Fig. 1B). Consistent with published data, ATF4 was constitutively transcribed but not translated in WT cells under non-stressed conditions. ATF4 protein, as detected by Western blotting, was induced in WT MEFs as early as 2 h after Tg treatment but could not be detected even after 8 h of UPR activation in ATF4 null cells (Fig. 1A). When nuclear extracts harvested from either WT or ATF4–/– cells were added in gel shift assays, ATF4 binding was detected only with those from WT cells (Fig. 6C). These data confirm the lack of ATF4 transcripts and protein in our null MEFs both before and after ER stress. The targeted disruption of the ATF4 allele was generated by replacing the second exon of ATF4 gene containing the basic leucine zipper domain, which is essential for the dimerization of ATF4 and DNA binding ability, with the neomycin resistance gene (32Tanaka T. Tsujimura T. Takeda K. Sugihara A. Maekawa A. Terada N. Yoshida N. Akira S. Genes Cells. 1998; 3: 801-810Crossref PubMed Scopus (133) Google Scholar). Thus, even if a partial ATF4 protein was expressed that was not recognized by our antibody, it would not be functional due to the lack of the leucine zipper domain. Induction of BiP Transcription Was Intact in ATF4–/– Cells—To begin characterizing the ER stress response in the ATF4 null cells, we first analyzed the transcriptional up-regulation of the ER chaperone BiP. BiP transcription is a target of the ATF6 transcription factor (38Yoshida H. Okada T. Haze K. Yanagi H. Yura T. Negishi M. Mori K. Mol. Cell. Biol. 2001; 21: 1239-1248Crossref PubMed Scopus (260) Google Scholar) and may be partially regulated by Ire1-altered XBP-1 (15Yoshida H. Matsui T. Yamamoto A. Okada T. Mori K. Cell. 2001; 107: 881-891Abstract Full Text Full Text PDF PubMed Scopus (2981) Google Scholar, 39Lee K. Tirasophon W. Shen X. Michalak M. Prywes R. Okada T. Yoshida H. Mori K. Kaufman R.J. Genes Dev. 2002; 16: 452-466Crossref PubMed Scopus (833) Google Scholar) and thus represents a read-out of these arms of the UPR. We harvested cytoplasmic mRNA from WT as well as ATF4–/– cells that had been treated with Tg for the indicated times, and we performed both Northern blotting and semi-quantitative reverse transcriptase-PCRs to detect the BiP transcript (Fig. 1, B–D). As early as 2 h after Tg treatment, an increase in BiP transcripts could be detected in both cells, with BiP mRNA levels continuing to build throughout the time course (Fig. 1B). Results from three independent reverse transcriptase-PCRs were quantified, normalized to the actin signal, and summarized in Fig. 1D, demonstrating that ATF4 null MEFs responded to Tg treatment by inducing BiP transcripts equally well as their wild type counterparts. This verifies that both WT and ATF4 null cells experienced and responded to ER stress as expected and suggests that at least the ATF6-ER stress response element signaling pathway is intact in the null cells. Furthermore, our data directly demonstrate that ATF4 is not required for the induction of BiP transcription during ER stress. Prolonged eIF-2α Phosphorylation in ATF4–/– Cells—To determine whether the PERK activation signal was intact in these cells, we monitored the phosphorylation status of its target protein, eIF-2α, during the course of ER stress using an antiserum that is specific for the phospho-form. WT and ATF4–/– cells were treated with Tg for the indicated periods of time, and protein blots were analyzed (Fig. 2A). The level of total eIF-2α was also measured as a loading control. Phosphorylation of eIF-2α peaked from 0.5 to 1.5 h after Tg treatment in both WT and ATF4–/– cells, although that in the
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