SIRT1 Suppresses Activating Transcription Factor 4 (ATF4) Expression in Response to Proteasome Inhibition
SIRT1 Suppresses Activating Transcription Factor 4 (ATF4) Expression in Response to Proteasome Inhibition
Journal of Microbiology and Biotechnology. 2013. Dec, 23(12): 1785-1790
Copyright © 2013, The Korean Society For Microbiology And Biotechnology
  • Received : September 11, 2013
  • Accepted : October 01, 2013
  • Published : December 30, 2013
Export by style
Cited by
About the Authors
Seon Rang Woo
Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul 139-706, Republic of Korea
Jeong-Eun Park
Dongguk University Research Institute of Biotechnology, Dongguk University, Seoul 100-715, Republic of Korea
Yang Hyun Kim
Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul 139-706, Republic of Korea
Yeun-Jin Ju
Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul 139-706, Republic of Korea
Hyun-Jin Shin
Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul 139-706, Republic of Korea
Hyun-Yoo Joo
Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul 139-706, Republic of Korea
Eun-Ran Park
Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul 139-706, Republic of Korea
Sung Hee Hong
Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul 139-706, Republic of Korea
Gil Hong Park
Department of Biochemistry, College of Medicine, Korea University, Seoul 136-705, Republic of Korea
Kee-Ho Lee
Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul 139-706, Republic of Korea

The synthetic machinery of ATF4 (activating transcription factor 4) is activated in response to various stress conditions involved in nutrient restriction, endoplasmic reticulum homeostasis, and oxidation. Stress-induced inhibition of proteasome activity triggers the unfolded protein response and endoplasmic reticulum stress, where ATF4 is crucial for consequent biological events. In the current study, we showed that the NAD + -dependent deacetylase, SIRT1, suppresses ATF4 synthesis during proteasome inhibition. SIRT1 depletion via transfection of specific siRNA into HeLa cells resulted in a significant increase in ATF4 protein, which was observed specifically in the presence of the proteasome inhibitor MG132. Consistent with SIRT1 depletion data, transient transfection of cells with SIRT1-overexpressing plasmid induced a decrease in the ATF4 protein level in the presence of MG132. Interestingly, however, ATF4 mRNA was not affected by SIRT1, even in the presence of MG132, indicating that SIRT1-induced suppression of ATF4 synthesis occurs under post-transcriptional control. Accordingly, we propose that SIRT1 serves as a negative regulator of ATF4 protein synthesis at the post-transcriptional level, which is observed during stress conditions, such as proteasome inhibition.
Unfolded and misfolded proteins that are inappropriately processed in the endoplasmic reticulum (ER) are eliminated via proteasomal degradation. Attenuation or inhibition of proteasomal degradation activity leads to the accumulation of abnormal proteins in the ER, thereby eliciting ER stress [6 , 17 , 19] . Activating transcription factor 4 (ATF4), a member of the ATF/CREB (activating transcription factor/cyclic AMP response element binding protein) family, mediates responses to unfolded protein and ER stress [6] .
The majority of normal cells exhibit not only inefficient translation of ATF4 mRNA but also active post-translational degradation of ATF4 protein [1] . Therefore, the basal level of ATF4 protein in normal cells remains too low for detection, despite the ubiquitous expression of ATF4 mRNA [1] . With the development of stress conditions, such as deprivation of amino acids, glucose, or oxygen, cells respond by accumulating ATF4 protein, mainly due to changes in translation and post-translational degradation, and partly due to transcription [2 , 5 , 11] . Under these stress conditions, translational inhibition of ATF4 mRNA by its upstream open reading frame is bypassed [1 , 10] and degradation of protein by E3 ligase β-TRCP reduced [1] , leading to accumulation of ATF4 protein. ATF4 translation is basically regulated via phosphorylation of eIF2α [10] . Phosphorylation of eIF2α, induced during stress, preferentially increases ATF4 translation while suppressing that of most other mRNA m olecules [11 , 16 , 24 , 28] . Based o n the t ype of stress, different kinases are activated to trigger eIF2α phosphorylation, including PERK for endoplasmic reticulum stress, GCN2 for amino acid deprivation [26] , PKR for viral infection [8] , and HRI for heme deficiency [18] . Posttranslational modification of ATF4 contributes to protein degradation via modulation of β-TRCP interactions with the “DSGXXXS” motif on ATF4 [15] . As discussed above, the regulatory factors and mechanisms contributing to cellular ATF4 levels are diverse, depending on the type of stress and regulation steps. The regulatory factors underlying the complex physiologies associated with ATF4 remain to be established.
In the last decade, SIRT1, an NAD + -dependent deacetylase, has been identified as a sensor for detecting internal and external stress and delivering information to target proteins [20] . Accumulating evidence has shown that transcription factors are the main downstream targets for SIRT1. In addition, SIRT1 controls the activity of transcription factors by regulating the nuclear translocation of metabolic enzymes such as GAPDH [13 , 25]
The regulation of transcription factors by SIRT1 is achieved through the modulation of chromatin structure via direct interactions with histone leading to deacetylation [21] . Moreover, p300 acetyltransferase participates in these cascade pathways counteracting SIRT1. As indicated above, ATF4 is regulated at the transcriptional, translational, and post-translational levels. Identification of the controller molecules involved in each step is a key challenge. In addition, it is important to understand the precise mechanisms by which proteasome inhibition regulates ER stress [23] . Based on the finding that both SIRT1 and ATF4 control the same physiological processes (such as nutrient restriction and proteasome degradation) and interact with p300, we hypothesized that SIRT1 functions as a controller of ATF4 expression. Data from the present study showed that SIRT1 acts as a negative regulator of ATF4 expression under conditions in which the proteasome pathway is inhibited.
Materials and Methods
- Cell Culture and Reagents
The media used for culture of HeLa, HT1080, H460, and HCT116 cell lines were Minimum Essential Medium (Cat. No. LM 007-7; Welgene Inc, Korea), Dulbecco’s Modified Eagle’s Medium (Cat. LM001-05; Welgene), RPMI 1640 Medium (Cat. No. LM 011- 01; Welgene), and McCoy’s 5A Medium (Cat. No. LM 005-01; Welgene), respectively. The cell lines were cultured in the media supplemented with 10% fetal bovine serum (Cat. No. 43640; JRS, USA) and 1% penicillin/streptomycin (Cat. No. 15140; GIBCO, USA) at 37℃ in a humidified atmosphere containing 5% CO 2 (v/v). The reagents used were resveratrol (Cat. No. R5010; Sigma, USA) and MG132 (Cat. No. C2211; Sigma).
- Gene Silencing with RNA Interference
For transient silencing experiments, cells were transfected with either negative control siRNA (Cat. No. 12935-300; Invitrogen, USA) or SIRT1 siRNA using Lipofectamine RNAiMAX (Cat. No. 13778-150; Invitrogen), according to the manufacturer’s instructions. The SIRT1 siRNA oligonucleotide was 5’-ACUUUGCUGUAACCCUGUA-3’. To obtain cell lines expressing SIRT1, cells were transfected with MFG-puro-SIRT1 plasmid, and puromycin-resistant cells were collected.
- Western Blot Analysis
HeLa cells were lysed in TNN buffer (120 mM NaCl, 40 mM Tris-HCl, pH 8.0, 0.5% NP-40, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 100 mM sodium fluoride, and 2.5 μg/ml leupeptin, aprotinin, and pepstatin). Lysate samples for western blot analysis were separated using SDS-polyacrylamide gel electrophoresis. Proteins on the gel were electrophoretically transferred to nitrocellulose membranes, followed by immunoblotting with antibodies against ATF4 (Cat. No. sc-200; Santa Cruz Biotechnology), SIRT1 (Cat. No. sc-55404; Santa Cruz Biotechnology), or β-actin (Cat. No. sc-47778; Santa Cruz Biotechnology). Each protein band was detected using a luminal reagent (Cat. No. sc- 2048; Santa Cruz Biotechnology).
- Total RNA Isolation and Reverse Transcriptase-Polymerase Chain Reaction
Total RNA was extracted from cells using the RNeasy Mini kit (Cat. No. 74106; Qiagen, Germany) according to the manufacturer’s protocol. For cDNA synthesis, total RNA was reverse-transcribed using the iScript cDNA synthesis kit (Cat. No. 170-8890; Bio-Rad, USA). Reverse transcriptase polymerase chain reaction (RT-PCR) was performed using the Maxime PCR PreMix kit (i-StarTaq) (Cat. No. #25167; Intron Biotechnology, Korea). The primer sequences used were as follows: ATF4 forward primer (5’-TCA AAC CTC ATG GGT TCT CC-3’) and reverse primer (5’-GGG CTC ATA CAG ATG CCA CT-3’), SIRT1 forward primer (5’-CAA ACT TTG CTG TAA CCC TGT-3’) and reverse primer (5’-CAG CCA CTG AAG TTC TTT CAT-3’), and β-actin forward primer (5’-AAG GAT TCC TAT GTC GGC) and reverse primer (5’-CAT CTC TTG CTC GAA GTC-3’).
- SIRT1 Overexpression Leads to Suppression of the ATF4 Protein Level During Proteasome Inhibition
To ascertain whether SIRT1 regulates ATF4 in association with the proteasome pathway, we examined the effect of SIRT1 on cellular ATF4 protein levels under conditionswhere the proteasome pathway is inhibited. To this end, we employed MG132, an inhibitor of 26S proteasomal degradation, which induces accumulation of unfolded protein and ER stress [22 , 23] . An initial experiment was performed with established HeLa cells stably transfected with a plasmid overexpressing exogenous SIRT1 [12] . Consistent with the earlier finding that ATF4 protein is short-lived and normally degraded by β-TRCP E3 ligase [15] , we observed an extremely low basal ATF4 level ( Fig. 1 A). However, the minimal detectable ATF4 protein level was increased in the presence of MG132. Comparison of the ATF4 protein levels in the presence of MG132 between HeLa cells with and without SIRT1 transfection revealed that SIRT1 induces a decrease in the amount of ATF4 protein. The ATF4 protein level was significantly lower in cells transfected with SRT1 than those with empty vector. Under conditions where the MG132 concentration was increased from 5 to 20 μM, resulting in higher accumulation of ATF4, SIRT1-mediated downregulation of ATF4 protein was consistently observed. To further evaluate this finding, we examined ATF4 protein expression in HeLa cells transiently transfected with Myc-tagged SIRT1. In the presence of MG132, increasing the amount of transfected Myc-tagged SIRT1 led to a decrease in the ATF4 protein level ( Fig. 1 B). Based on these results, we conclude that SIRT1 suppresses the synthesis of ATF4 protein, which is detectable during proteasome inhibition.
PPT Slide
Lager Image
ATF protein synthesis is decreased upon SIRT1 overexpression. (A) The amounts of ATF4 protein were determined in the presence or absence of MG132 in HeLa cells with and without SIRT1 overexpression, established via transfection of SIRT1 (S) and empty vector (V), respectively. (B) The amount of ATF4 protein in the presence of MG132 was determined in HeLa cells transiently transfected with increasing concentrations of Myc-tagged SIRT1 plasmid (+, 1 μg; ++, 2 μg; +++, 3 μg).
PPT Slide
Lager Image
ATF4 protein synthesis is increased upon SIRT1 depletion. SIRT1 siRNA was transiently transfected into established HeLa cells previously transfected with SIRT1 (HeLa S) or empty vector (HeLa V), and the amount of ATF4 protein was determined in the presence or absence of MG132.
- SIRT1 Depletion Induces ATF4 Protein Expression During Proteasome Inhibition
To further assess the SIRT1 suppression of ATF4 protein synthesis observed during proteasome inhibition, we examined the effect of SIRT1 depletion on the ATF4 protein level. Consistent with the data obtained with HeLa cells overexpressing SIRT1, SIRT1 depletion with siRNA in HeLa cells led to a significant increase in ATF4 protein in the presence of MG132 (lane 3 vs 4, Fig. 2 ). The effect of SIRT1 depletion on the ATF4 protein level was additionally observed in HeLa cells overexpressing SIRT1. Notably, ATF4 protein expression suppressed by SIRT1 overexpression (lane 3 vs 7, Fig. 2 ) was conversely increased with SIRT1 depletion (lane 7 vs 8, Fig. 2 ). However, in the absence of MG132, SIRT1 depletion did not affect the ATF4 protein level in HeLa cells transfected with either empty vector or SIRT1 (lane 1 vs 2 or lane 5 vs 6, respectively, Fig. 2 ). Similarly, SIRT1 overexpression had no impact on the ATF4 protein level in the absence of MG132 (lanes 1 vs 2, Fig. 1 A). These findings further support SIRT1 suppression of ATF4 protein synthesis during proteasome inhibition.
- SIRT1 Regulation of ATF4 Synthesis is Achieved at the Post-Transcriptional Level
Next, we focused on determining whether SIRT1 regulation of the ATF4 protein is mediated by transcriptional control of the gene. In contrast to the increased ATF4 protein expression observed in the presence of MG132, the ATF4 mRNA level was not altered upon SIRT1 depletion ( Figs. 3 A and 3 B). The MG132 concentration did not affect ATF4 transcription (2 and 5 μM in Figs. 3 A and 3 B, respectively). Moreover, SIRT1 did not affect the ATF4 mRNA level in the absence of MG132 ( Figs. 3 A and 3 B). The changes detected at the protein, but not mRNA level, in the presence of MG132 indicate that SIRT1 regulates ATF4 protein synthesis via a post-transcriptional mechanism.
PPT Slide
Lager Image
ATF4 protein synthesis, but not mRNA, is increased upon SIRT1 depletion. Levels of ATF4 protein and mRNA were determined with western blot assay and semiquantitative RT-PCR, respectively, in HeLa cell lines in the presence of 2 μM (A) and 5 μM (B) MG132.
- SIRT1 Suppression of ATF4 Synthesis is Found in Various Human Cells
As shown above, the SIRT1 suppression of ATF4 synthesis was observed in HeLa cervical carcinoma cells. To further determine whether our present finding generally occurs in human cells, we examined it using three more cell lines, including HT1080 human fibrosarcoma cells, H460 human lung carcinoma cells, and HCT116 human colon carcinoma cells. Similar to the result shown in HeLa cells, all these cell lines examined, HT1080 ( Fig. 4 A), H460 ( Fig. 4 B), and HCT116 cells ( Fig. 4 C), showed that SIRT1 depletion increased ATF4 synthesis upon treatment of MG132. Consistently, this was not observed in these cell lines under condition without MG132 ( Figs. 4 A- 4 C). Thus, the SIRT1 suppression of ATF4 synthesis during proteasome inhibition is generally found in human cells, not specific to a certain cell type.
PPT Slide
Lager Image
SIRT1 suppression of ATF4 synthesis is generally found in human cell lines. SIRT1 siRNA was transiently transfected into HT1080 (A), H460 (B), and HCT116 cell lines (C) in the presence (+) or absence (-) of MG132. ATF4 protein level was determined by western blot assay.
In the present study, we identified SIRT1 as a novel negative regulator of ATF4 protein synthesis, which is detected during proteasome inhibition. Moreover, SIRT1 suppresses ATF4 synthesis under post-transcriptional control. This conclusion was derived from the following findings: (1) ATF4 protein accumulation in the presence of MG132, a proteasome inhibitor, was reduced upon SIRT1 overexpression, but enhanced by SIRT1 depletion; (2) in the absence of MG132, SIRT1 did not affect the ATF4 protein level; and (3) the ATF4 mRNA level was not affected by SIRT1, irrespective of the presence or absence of MG132.
MG132 inhibits the proteasomal degradation of unfolded proteins, in turn promoting the unfolded protein response and ER stress [22 , 23] . Therefore, SIRT1 may be involved in ATF4 synthesis when proteasomal degradation of unfolded and misfolded proteins is inhibited and ER stress increasingly develops owing to the accumulation of inappropriate proteins. The finding that SIRT1 does not affect the ATF4 mRNA level indicates that SIRT1-mediated regulation is not associated with transcriptional activation of the gene. SIRT1 may thus regulate ATF4 protein synthesis at the level of translation or post-translational degradation. Recent studies have shown that SIRT1 interacts with and suppresses the phosphorylation of eIF2α [9] , raising the possibility that SIRT1-mediated regulation of ATF4 protein synthesis occurs via eIF2α phosphorylation. Phosphorylation of eIF2α attenuates the inhibitory action of the open reading frame on the 5’-untranslated region located upstream of ATF4 mRNA, leading to protein translation [4 , 16 , 28] . Several kinases, including PERK, GCN2, PKR, and HRI, are responsible for eIF2α phosphorylation. Under stress conditions where these kinases are activated, SIRT1 may inhibit their activities, leading to decreased eIF2α phosphorylation and subsequent ATF4 translation. Recently, a study closely related to ours reported that ATF4 upregulates SIRT1 expression through direct binding to the SIRT1 promoter [29] . These data, taken together with our presented data, provide evidence that SIRT1 regulates ATF expression in a post-transcriptional negative feedback loop.
The ATF4 protein is stabilized via dissociation from β-TRCP E3 ligase, and affinity between the two proteins is also dependent on the phosphorylation of ATF4. RSK2, CK1, and CK2 are the suggested kinases responsible for phosphorylation of ATF4 protein [3 , 7 , 30 , 31] . P300 inhibits the interactions between ATF4 and β-TRCP E3 ligase, leading to dissociation of the two proteins [14] . Since SIRT1 counteracts p300 and regulates β-TRCP (unpublished data), we cannot rule out the possibility that SIRT1 participates in ATF4 protein stability. The facts that, under stress condition, SIRT1 controls nuclear translocation of transcription modulators including GAPDH [13] and ATF4 is translocated into the nucleus [27] further suggest the association of SIRT1 with ATF4. Thus, further efforts are required to solve the mechanisms underlying the modulation of cellular levels of ATF4 protein.
This study was supported by grants from the National Research Foundation of Korea (NRF-2012R1A1A2008457 and NRF-2012M3A9B6055346), and the nuclear R&D program of Korea Ministry of Sciences and Technology.
Ameri K , Harris AL 2008 Activating transcription factor 4. Int. J. Biochem. Cell Biol. 40 14 - 21    DOI : 10.1016/j.biocel.2007.01.020
Ameri K , Lewis CE , Raida M , Sowter H , Hai T , Harris AL 2004 Anoxic induction of ATF4 through Hif1-independent pathways of protein stabilization in human cancer cells. Blood 103 1876 - 1882    DOI : 10.1182/blood-2003-06-1859
Ampofo E , Sokolowsky T , Gotz C , Montenarh M 2013 Functional interaction of protein kinase CK2 and activating transcription factor 4 (ATF4), a key player in the cellular stress response. Biochim. Biophys. Acta 1833 439 - 451    DOI : 10.1016/j.bbamcr.2012.10.025
Baird TD , Wek RC 2012 Eukaryotic initiation factor 2 phosphorylation and translational control in metabolism. Adv. Nutr. 3 307 - 321    DOI : 10.3945/an.112.002113
Blais JD , Filipenko V , Bi M , Harding HP , Ron D , Koumenis C 2004 Activating transcription factor 4 is translationally regulated by hypoxic stress. Mol. Cell Biol. 24 7469 - 7482    DOI : 10.1128/MCB.24.17.7469-7482.2004
Clarke R , Cook KL , Hu R 2012 Endoplasmic reticulum stress, the unfolded protein response, autophagy, and the integrated regulation of breast cancer cell fate. Cancer Res. 72 1321 - 1331    DOI : 10.1158/1538-7445.AM2012-1321
Frank CL , Ge X , Xie Z , Zhou Y , Tsai LH 2010 Control of activating transcription factor 4 (ATF4) persistence by multisite phosphorylation impacts cell cycle progression and neurogenesis. J. Biol. Chem. 285 33324 - 33337    DOI : 10.1074/jbc.M110.140699
Garcia MA , Meurs EF , Esteban M 2007 The dsRNA protein kinase PKR: virus and cell control. Biochimie 89 799 - 811    DOI : 10.1016/j.biochi.2007.03.001
Ghosh HS , Reizis B , Robbins PD 2011 SIRT1 associates with eIF2-alpha and regulates the cellular stress response. Sci. Rep. 1 150 -    DOI : 10.1038/srep00150
Harding HP , Novoa I , Zhang Y , Zeng H , Wek R , Schapira M 2000 Regulated translation initiation controls stressinduced gene expression in mammalian cells. Mol. Cell 6 1099 - 1108    DOI : 10.1016/S1097-2765(00)00108-8
Harding HP , Zhang Y , Zeng H , Novoa I , Lu PD , Calfon M 2003 An integrated stress response regulates amino acid metabolism and resistance to oxidative stress. Mol. Cell 11 619 - 633    DOI : 10.1016/S1097-2765(03)00105-9
Jeong J , Juhn K , Lee H , Kim SH , Min BH , Lee KM 2007 SIRT1 promotes DNA repair activity and deacetylation of Ku70. Exp. Mol. Med. 39 8 - 13    DOI : 10.1038/emm.2007.2
Joo HY , Woo SR , Shen YN , Yun MY , Shin HJ , Park ER 2012 SIRT1 interacts with and protects glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from nuclear translocation: implications for cell survival after irradiation. Biochem. Biophys. Res. Commun. 424 681 - 686    DOI : 10.1016/j.bbrc.2012.07.006
Lassot I , Estrabaud E , Emiliani S , Benkirane M , Benarous R , Marqottin-Goquet F 2005 P300 modulates ATF4 stability and transcriptional activity independently of its acetyltransferase domain. J. Biol. Chem. 280 41537 - 41545    DOI : 10.1074/jbc.M505294200
Lassot I , Segeral E , Berlioz-Torrent C , Durand H , Groussin L , Hai T 2001 ATF4 degradation relies on a phosphorylation-dependent interaction with the SCF(beta TrCP) ubiquitin ligase. Mol. Cell Biol. 21 2192 - 2202    DOI : 10.1128/MCB.21.6.2192-2202.2001
Lu PD , Harding HP , Ron D 2004 Translation reinitiation at alternative open reading frames regulates gene expression in an integrated stress response. J. Cell Biol. 167 27 - 33    DOI : 10.1083/jcb.200408003
Malhotra JD , Kaufman RJ 2007 The endoplasmic reticulum and the unfolded protein response. Semin. Cell Dev. Biol. 18 716 - 731    DOI : 10.1016/j.semcdb.2007.09.003
McEwen E , Kedersha N , Song B , Scheuner D , Gilks N , Han A 2005 Heme-regulated inhibitor kinase-mediated phosphorylation of eukaryotic translation initiation factor 2 inhibits formation, and mediates survival upon arsenite exposure. J. Biol. Chem. 280 16925 - 16933    DOI : 10.1074/jbc.M412882200
Meusser B , Hirsch C , Jarosch E , Sommer T 2005 ERAD: the long road to destruction. Nat. Cell Biol. 7 766 - 772    DOI : 10.1038/ncb0805-766
Michan S , Sinclair D 2007 Sirtuins in mammalian: insights into their biological function. Biochem. J. 404 1 - 13
Moazed D 2001 Enzymatic activities of Sir2 and chromatin silencing. Curr. Opin. Cell Biol. 13 232 - 238    DOI : 10.1016/S0955-0674(00)00202-7
Nakajima S , Kato H , Takahashi S , Johno H , Kitamura M 2011 Inhibition of NF-kB by MG132 through ER stressmediated induction of LAP and LIP. FEBS Lett. 585 2249 - 2254    DOI : 10.1016/j.febslet.2011.05.047
Park HS , Jun do Y , Han CR , Woo HJ , Kim YH 2011 Proteasome inhibitor MG132-induced apoptosis via ER stress-mediated apoptotic pathway and its potentiation by protein tyrosine kinase p56lck in human jurkat T cells. Biochem. Pharmacol. 82 1110 - 1125    DOI : 10.1016/j.bcp.2011.07.085
Rutkowski DT , Kaufman RJ 2003 All roads lead to ATF4. Dev. Cell 4 442 - 444    DOI : 10.1016/S1534-5807(03)00100-X
Sen N , Hara MR , Kornberg MD , Cascio MB , Bae BI , Shahani N 2008 Nitric oxide-induced nuclear GAPDH activates p300/CBP and mediates apoptosis. Nat. Cell Biol. 10 866 - 873    DOI : 10.1038/ncb1747
Sood R , Porter AC , Olsen DA , Cavener DR , Wek RC 2000 A mammalian homologue of GCN2 protein kinase important for translational control by phosphorylation of eukaryotic initiation factor-2 alpha. Genetics 154 787 - 801
Takahata Y , Hinoi E , Takarada T , Nakamura Y , Ogawa S , Yoneda Y 2012 Positive regulation by γ-aminobutyric acid B receptor subunit-1 of chondrogenesis through acceleration of nuclear translocation of activating transcription factor-4. J. Biol. Chem. 287 33293 - 33303    DOI : 10.1074/jbc.M112.344051
Vattem KM , Wek RC 2004 Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells. Proc. Natl. Acad. Sci. USA 101 11269 - 11274    DOI : 10.1073/pnas.0400541101
Zhu H , Xia L , Zhang Y , Wang H , Xu W , Hu H 2012 Activating transcription factor 4 confers a multidrug resistance phenotype to gastric cancer cells through transactivation of SIRT1 expression. PLoS One 7 e31431 -    DOI : 10.1371/journal.pone.0031431
Yang X , Matsuda K , Bialek P , Jacquot S , Masuoka HC , Schinke T 2004 ATF4 is a substrate of RSK2 and an essential regulator of osteoblast biology; implication for Coffin-Lowry Syndrome. Cell 117 387 - 398    DOI : 10.1016/S0092-8674(04)00344-7
Zong ZH , Du Zx , Li N , Li C , Zhang Q , Iu BQ 2012 Implication of Nrf2 and ATF4 in differential induction of CHOP by proteasome inhibition in thyroid cancer cells. Biochim. Biophys. Acta 1823 1395 - 1404    DOI : 10.1016/j.bbamcr.2012.06.001