Correspondence to: David Ron, New York University Medical Center, SI 3-10, 540 First Ave., New York, NY 10016. Tel:(212) 263-7786 Fax:(212) 263-8951 E-mail:ron{at}saturn.med.nyu.edu.
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Abstract |
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Phosphorylation of the subunit of eukaryotic translation initiation factor 2 (eIF2
) on serine 51 integrates general translation repression with activation of stress-inducible genes such as ATF4, CHOP, and BiP in the unfolded protein response. We sought to identify new genes active in this phospho-eIF2
dependent signaling pathway by screening a library of recombinant retroviruses for clones that inhibit the expression of a CHOP::GFP reporter. A retrovirus encoding the COOH terminus of growth arrest and DNA damage gene (GADD)34, also known as MYD116 (Fornace, A.J., D.W. Neibert, M.C. Hollander, J.D. Luethy, M. Papathanasiou, J. Fragoli, and N.J. Holbrook. 1989. Mol. Cell. Biol. 9:41964203; Lord K.A., B. Hoffman-Lieberman, and D.A. Lieberman. 1990. Nucleic Acid Res. 18:2823), was isolated and found to attenuate CHOP (also known as GADD153) activation by both protein malfolding in the endoplasmic reticulum, and amino acid deprivation. Despite normal activity of the cognate stress-inducible eIF2
kinases PERK (also known as PEK) and GCN2, phospho-eIF2
levels were markedly diminished in GADD34-overexpressing cells. GADD34 formed a complex with the catalytic subunit of protein phosphatase 1 (PP1c) that specifically promoted the dephosphorylation of eIF2
in vitro. Mutations that interfered with the interaction with PP1c prevented the dephosphorylation of eIF2
and blocked attenuation of CHOP by GADD34. Expression of GADD34 is stress dependent, and was absent in PERK-/- and GCN2-/- cells. These findings implicate GADD34-mediated dephosphorylation of eIF2
in a negative feedback loop that inhibits stress-induced gene expression, and that might promote recovery from translational inhibition in the unfolded protein response.
Key Words: endoplasmic reticulum, translation, gene expression regulation, signal transduction, molecular cloning
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Introduction |
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Translation initiation is inhibited in cells exposed to different stressful conditions. The phosphorylation of the subunit of eukaryotic translation initiation factor 2 (eIF2
)1 plays an important role in this stereotyped response, and is mediated by distinct kinases that are activated by specific stress signals. The interferon-inducible, double-stranded RNAactivated kinase (PKR) phosphorylates eIF2
in response to viral infection (
in cells experiencing stress from protein malfolding in the ER (
phosphorylation, but the responsible kinase(s) has not been identified (
When phosphorylated on serine 51, eIF2 binds to and inhibits the guanine nucleotide exchange factor, eIF2B. The latter is required for the formation of the eukaryotic translational preinitiation complexes, and its sequestration in an inactive complex with phosphorylated eIF2
inhibits the initiation step of protein synthesis. In the case of viral infection, the physiological significance of PKR-induced eIF2
phosphorylation is revealed by the observation that nearly all successful viruses have adapted a mechanism for circumventing the activity of this upstream kinase or the downstream response (
We have recently discovered that in addition to its role in regulating protein synthesis, eIF2 phosphorylation is also required for stress-induced gene expression. Cells lacking the upstream kinases PERK or GCN2 are impaired in the induction of the C/EBP homologous protein (CHOP) and immunoglobulin binding protein of B cells (BiP) genes by cognate stress signals (
phosphorylation and the reduced formation of translational preinitiation complexes activate the translation of a transcription factor, ATF4 (
kinase PERK (
on serine 51 integrates transcriptional and translational responses in mammalian cells. We tentatively refer to this pathway as the integrated stress response.
We sought to identify new components of the integrated stress response by screening for genes or gene fragments that, when expressed ectopically in either their sense or antisense orientation, would block the response. We report here on the isolation of one such genetic suppressor element (GSE) of the integrated stress response that encodes the COOH terminus of the stress-inducible growth arrest and DNA damage gene (GADD)34 protein. Our studies suggest that GADD34 participates in a negative feedback loop that attenuates signaling in the integrated stress response.
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Materials and Methods |
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Identifying GSEs That Impair CHOP::GFP Activation
CHO-K1 cells were stably transformed with a CHOP::GFP reporter plasmid. The plasmid was constructed by fusing an 8.5-kb 5' murine CHOP gene fragment, whose 3' end is at the PmlI site in exon 3, nine nucleotides 5' of the CHOP coding region, to enhanced green fluorescent protein (GFP) (CLONTECH Laboratories, Inc.) and termination sequences from the SV-40 virus (
A random primed cDNA library from CHO-K1 cells was constructed in the retroviral plasmid pBabe Puro® (
Retroviruses were packed into vesicular stomatitis virus glycoprotein (VSV-G) envelope pseudotyped viruses (
Cloning, Validation, and Functional Characterization of the A1 GSE, GADD34, and Mutant Derivatives
Genomic DNA was prepared from clones defective in CHOP::GFP expression, and the cDNA insert was recovered by PCR of the integrated retrovirus. The insert was ligated into the parental preretroviral plasmid, packaged into infectious particles, and transduced into parental CHOP::GFP cells. Reconstitution of the defect in CHOP::GFP expression was taken as evidence for the presence of a GSE in the retroviral clone.
The cDNA insert was also introduced into a pCDNA3 plasmid (Invitrogen) in which the Neo® marker was replaced by the human CD2 cell surface marker, creating pCDNA3-CD2. This plasmid was used in transient transfection (Lipofectamine Plus; Life Technologies) to transduce the GSE into parental CHOP::GFP cells. A full-length GADD34 EST (GenBank/EMBL/DDBJ accession no. AW320574) was obtained (Incyte), and a combination of PCR-based manipulations and restriction digests were used to introduce a FLAG epitope tag at the NH2 terminus, and to create the deletion mutants and the point mutations indicated in the text.
The eIF2 cDNA was a gift of R. Schneider (New York University, New York, NY). The S51D mutation was introduced by PCR mutagenesis, and the wild-type and mutant cDNA were introduced into the pCDNA3-CD2 plasmid.
Transfected cells were treated with tunicamycin (Calbiochem) or cultured in media lacking amino acids to activate CHOP::GFP. Treated cells were recovered by trypsinization, stained with a Tricolor antihuman CD2 antibody (Caltag Laboratories) to report on transfected cells, and analyzed by dual channel FACS® analysis for the GFP and antiCD2-Tricolor signals.
Immunoprecipitation, Immunoblotting, and Detection of CHOP, eIF2, PERK, GCN2, ATF4, and PP1c
Cell treatment, lysate preparation, immunoprecipitation, and immunoblotting followed procedures described previously ( and eIF2
phosphorylated on serine 51 have been described previously (
In Vitro Dephosphorylation of eIF2
Bacterially expressed GST-PERK was used to phosphorylate eIF2 in vitro in rabbit reticulocyte lysate as described previously (
-32P]ATP, 1-µl aliquots of the radiolabeled proteins in the reticulocyte lysate were placed in a 10-µl dephosphorylation reaction (dephosphorylation buffer: 20 mM Tris-HCl, pH 7.4, 50 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.8 mM ATP;
Northern Blot Analysis
The PERK-/- and GCN2-/- cells have been described previously (
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Results |
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Isolating a GSE That Interferes with CHOP Expression in Stressed Cells
We have described previously a stable CHO cell line carrying a CHOP::GFP transgene that faithfully reports on the activity of the endogenous CHOP gene; the reporter is tightly repressed in unstressed cells, and is strongly activated by the UPR or amino acid deprivation (Fig 1 A, and
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Pools of cells transduced with the retroviral library were exposed to tunicamycin, an agent that causes ER stress by inhibiting protein glycosylation, and cells that failed to activate the resident CHOP::GFP transgene were selected by FACS®. The GFP-dull population was enriched by successive cycles of sorting. Clones of GFP-dull cells were isolated by limit dilution, expanded, and the cDNA insert was recovered by PCR of the integrated viral genome. The insert containing the putative GSE was used to construct a new recombinant retrovirus to recapitulate the inhibition of CHOP::GFP expression. Fig 1 A shows the effect on CHOP::GFP expression of an inhibitory retrovirus containing one such GSE isolated by this screen that we refer to as A1. Amino acid deprivation and ER stress, manipulations that activate the eIF2 kinases GCN2 and PERK, respectively (
The A1 GSE Interferes with eIF2 Phosphorylation and Blocks the Integrated Stress Response
ATF4 is a transcription factor that activates CHOP in the integrated stress response (
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Phosphorylation of eIF2 on serine 51 activates ATF4 translation, and is a key step in signaling cell stress. Therefore, we compared the phosphorylation of eIF2
in parental CHO cells with that of the A1-transduced cells. Immunoblotting of lysates from stressed cells with an antiserum that specifically detects eIF2
phosphorylated on serine 51 revealed a profound defect in eIF2
phosphorylation in response to both ER stress and amino acid starvation in A1-transduced cells (Fig 3A and Fig B). The protein kinases PERK and GCN2 are responsible for eIF2
phosphorylation by ER stress and amino acid deprivation, respectively (
phosphorylation, acting downstream of the eIF2
kinases PERK and GCN2.
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ER stress and the attendant PERK-induced eIF2 phosphorylation inhibits protein synthesis (
phosphorylation induced by the A1 GSE, we compared new protein synthesis rates in ER-stressed parental CHO cells with A1-transduced cells. Cells were treated with DTT, an agent that strongly activates PERK and inhibits protein synthesis in a strictly PERK-dependent manner (
phosphorylation induced by the A1 GSE leads to loss of translational regulation.
Some of the effects of phosphorylation of eIF2 can be mimicked by an activating substitution (serine 51 to aspartic acid, S51D;
could rescue the defect in CHOP::GFP expression in A1-transduced cells, we transfected cells with a plasmid expressing the mutant eIF2
. In addition to the effector protein, the plasmid expressed the cell surface marker CD2 that can be detected with a specific antibody identifying the transfected cells. The effect of eIF2
S51D on the activity of the integrated CHOP::GFP reporter was measured by dual channel FACS® analysis of transfected cells. Plasmids expressing CD2 alone or CD2 and wild-type eIF2
had no effect on CHOP::GFP activity, whereas the S51D mutant eIF2
reproducibly activated CHOP::GFP in a small subset of transfected cells. The relatively small number of transfected cells expressing CHOP::GFP may be due to the toxicity of the activated eIF2
protein. This was equally apparent in parental and A1 GSEtransduced cells (Fig 3 D). Rescue by the activated eIF2
S51D suggests that the A1 GSE exerts its effect on CHOP expression at the level of eIF2
phosphorylation.
The A1 GSE Encodes the COOH Terminus of GADD34 and Its Function Is Dependent on Interaction with the Catalytic Subunit of PP1
The insert of retroviral clone A1 was sequenced and found to encode the COOH-terminal 299 amino acids (aa 292590) of the hamster GADD34 protein (also known as MYD116) (N) profoundly inhibited CHOP::GFP expression in this transient transfection assay (Fig 4 A). The normal induction of CHOP::GFP in the CD2-negative, untransfected portion of the cells provided a built-in positive control for the induction of ER stress in each experiment.
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Full-length murine GADD34 reproducibly inhibited CHOP::GFP, but the level of inhibition was significantly less than that induced by the A1 GSE and the comparable murine COOH-terminal fragment (Fig 4 A). Deletion of the 121 COOH-terminal residues (GADD34C, aa 1536) abolished the inhibitory effect, attesting to the importance of COOH-terminal region of GADD34 in the inhibition of the integrated stress response (Fig 4 A).
To determine if GADD34 and its derivatives were also able to inhibit endogenous CHOP activation by stress, we treated transfected mouse embryonic fibroblast cells with tunicamycin and stained them for endogenous CHOP and the recombinant GADD34 proteins (the latter were detected by an NH2-terminal FLAG epitope tag). Endogenous CHOP expression was significantly attenuated by the A1 GSE, the homologous murine fragment (aa 241657), and by the full-length GADD34, but not by the COOH-terminal deletion mutant that also failed to attenuate the CHOP::GFP reporter (aa 1536; Fig 4 B). The fraction of CHOP-positive cells among the cells expressing the recombinant proteins was reduced from 69.7% positive cells among those transfected with the inactive GADD34C fragment (aa 1536) to 3.5% in GADD34 transfectants, 4.7% in the A1 GSE transfectants, and 4% in GADD34 fragment 241657 transfectants. In this assay we could not detect differences between the activity of the A1 GSElike COOH-terminal fragments of GADD34 and the full-length protein. This may be due to the nonquantitative nature of the immunocytochemical assay compared with the highly quantitative FACS® analysis.
In the course of these experiments we noticed that the staining pattern of the A1-like COOH-terminal fragment of mouse GADD34 was very different from that of the full-length GADD34: the former exhibited a diffuse cytosolic pattern, whereas the latter was distributed in a reticular pattern (Fig 4 C). The possible implications of this finding are discussed below.
The COOH terminus of GADD34 is related in sequence to the COOH terminus of the herpes simplex virus (HSV)-encoded protein 134.5 (
134.5 plays an important role in evading the consequences of PKR-mediated shutdown of host protein synthesis in virally infected cells, and the COOH-terminal fragment of GADD34 can substitute for this activity of
134.5 (
134.5 is dependent on its ability to associate with the catalytic subunit of PP1 (PP1c), and correlates with an increase in a cellular phosphatase activity that dephosphorylates eIF2
(
phosphorylation levels by promoting dephosphorylation, we compared the phosphatase activity directed against eIF2
in lysates of cells expressing the A1 GSE or GADD34 with that of parental cells. The eIF2
in reticulocyte lysate was phosphorylated in vitro on serine 51 using bacterially expressed GST-PERK (
was efficiently dephosphorylated in lysates from cells expressing the GADD34 derivatives, whereas the dephosphorylation activity in lysates from parental cells was considerably lower (Fig 5 A). Expression of the GADD34 fragments specifically promoted the dephosphorylation of eIF2
, as the dephosphorylation of phosphoGST-PERK and an unidentified phosphoprotein of 110 kD that are both present in the radiolabeled reticulocyte lysate progressed to similar degree in lysates of parental CHO cells and cells expressing GADD34 (Fig 5 A).
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To determine if the eIF2 phosphatase activity induced by GADD34 is physically associated with the GADD34 proteins, we performed the same in vitro eIF2
dephosphorylation assay using purified immune complexes recovered from transfected cells. Immunopurified complexes containing FLAG epitopetagged A1 GSE or full-length mouse GADD34 were both able to efficiently dephosphorylate eIF2
. The phosphatase activity of the immune complex was reduced by okadaic acid, an inhibitor of PP1 and PP2A (Fig 5 B). Okadaic acid also inhibited eIF2
dephosphorylation by crude lysates of cells expressing GADD34 (data not shown). Proteins immunoprecipitated from mock-transfected cells had only background eIF2
phosphatase activity. Phosphatase activity directed towards the 110-kD phosphoprotein and phosphoGST-PERK was similar in all three immunecomplexes, providing a built-in control for the activity of the eIF2
phosphatase. These results implicate GADD34 in dephosphorylation of eIF2
through its physical association with a PP1- (or PP2A-) like activity.
To determine if the inhibitory effect of GADD34 on the integrated stress response correlated with its binding to PP1c, we constructed mutant derivatives of mouse GADD34 that would be predicted not to bind PP1c (Fig 6 A); the predictions were based on the crystal structure of PP1c ( phosphatase activity in vitro (Fig 6 D), supporting a role for eIF2
dephosphorylation in the inhibition of the integrated stress response.
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Immediately NH2 terminal to the PP1c-binding site, GADD34 has a series of conserved repetitive elements (hamster, 3.5 repeats; humans, 4 repeats; mouse, 4.5 repeats; Fig 6 A). A 172-residue fragment of GADD34 (aa 485657) that lacks the repeats still bound to PP1c and also inhibited CHOP::GFP activity, albeit less strongly than the A1 fragment that contains some of the repeats (Fig 6 B). This result indicates that the repeats may play a role in inactivating the integrated stress response, but are not absolutely required.
Expression of GADD34 Is Mediated by the Integrated Stress Response
GADD34 is a stress-inducible gene whose expression profile parallels that of CHOP ( kinase PERK, whereas GADD34 induction by amino acid starvation was dependent on GCN2 (Fig 7). These results indicate that the integrated stress response plays an important role in GADD34 activation. There are also PERK- and GCN2-independent pathways to activate GADD34, as the induction of the gene by the alkylating agent methyl methanesulfonate (MMS) was preserved in PERK-/- and partially preserved in GCN2-/- cells. We do not know if this stress-causing agent induced GADD34 by promoting eIF2
phosphorylation through an alternative kinase or by other activating signaling pathways that are independent of eIF2
phosphorylation.
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Discussion |
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Signaling in the general control response (the yeast counterpart of the mammalian integrated stress response) is dependent on phosphorylation of eIF2, as substitution of the residue corresponding to serine 51 in yeast eIF2
prevents activation of genes by amino acid starvation (
kinases PERK and GCN2, and is mediated by translational regulation of ATF4 (
is able to block the integrated stress response. Activation of the stress-induced eIF2
kinases is unaffected by this GSE, indicating that the latter does not interfere with the development of the proximal stress signal. Our findings further support the notion that the activation of gene expression by the integrated stress response is mediated by eIF2
phosphorylation in mammalian cells.
The GSE we identified is a product of the GADD34 gene. Its ability to interfere with signaling in the integrated stress response correlates with its ability to bind the catalytic subunit of PP1 and to dephosphorylate eIF2. Binding to PP1c is shared by the structurally related HSV protein
134.5, and lysates of cells infected with
134.5-expressing virus also have markedly increased activity of an eIF2
phosphatase (
. It seems likely that HSV has coopted this cellular mechanism to prevent host protein synthesis shutdown when the eIF2
kinase PKR is activated by viral infection, as suggested originally by Roizman and colleagues (
134.5's essential role in viral infectivity is well established, it is not known if inhibition of host gene activation is part of this essential role. In this regard, it is interesting to note that a dominant negative mutation in the yeast PP1c homologue, GLC7, is able to derepress a transcriptional program that is dependent on signaling by the eIF2
kinase, Gcn2p (
Suppression of the integrated stress response by the COOH-terminal fragment of GADD34 is significantly greater than that by the full-length protein. This difference does not appear to be due to the higher level of expression of the smaller fragment, or to significantly reduced ability to associate with the catalytic subunit of the phosphatase (Fig 6 C). Furthermore, the full-length GADD34 was able to impart high levels of phosphatase activity to lysates of transfected cells or to immune complexes obtained from transfected cells (Fig 5 B and Fig 6 D). This suggests that its ability to promote the dephosphorylation of eIF2 was unmasked by cell lysis and detergent extraction. We noted that the COOH-terminal active fragment of GADD34 is distributed throughout the cytoplasm, whereas the full-length protein is localized to a reticular structure costained with antiserum to ER markers (Fig 4 C and data not shown). It is tempting to speculate that the reduced activity of the full-length protein may be due to its sequestration at an inactive site. If correct, this suggests a means to regulate GADD34 activity posttranslationally as well as at the level of the gene's expression.
What might be the physiological significance of GADD34's potential activity as an inhibitor of the integrated stress response? It had previously been noted that eIF2 phosphorylation and protein synthesis inhibition are transient in stressed cells (
kinases PERK and GCN2 (Fig 7B and Fig C). Therefore, it seems likely that transcriptional induction of GADD34 is part of a negative feedback loop that attenuates signaling in the integrated stress response. Other stress-responsive signaling pathways have elaborated components that serve similar negative feedback functions. For example, the mitogen-activated protein kinase pathway is inhibited by dual-specificity phosphatases that are transcriptionally induced by mitogen-activated protein kinase signaling (
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There are several theoretical reasons why the phosphorylation of eIF2 may have evolved as a transient signal in stressed cells. Sustained eIF2
phosphorylation is lethal to cells in culture (
phosphorylation correlates with death in vivo (
phosphorylation inhibit translation of most proteins, including some like BiP that are induced later in the course of the response (
provide a means for cells to translate mRNAs like BiP, whose induction occurred earlier, during the active phase of the integrated stress response.
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Footnotes |
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1 Abbreviations used in this paper: ATF, activating transcription factor; BiP, immunoglobulin binding protein of B cells; CHOP, C/EBP homologous protein; eIF2, eukaryotic translation initiation factor 2
; GADD, growth arrest and DNA damage gene; GAPDH, glutaraldehyde 3-phosphate dehydrogenase; GCN, general control nonrepressed; GFP, green fluorescent protein; GSE, genetic suppressor element; GST, glutathione S-transferase; HSV, herpes simplex virus; MMS, methyl methanesulfonate; PERK, PKR-like ER kinase; PKR, double-stranded RNAactivated-kinase; PP1, protein phosphatase 1; UPR, unfolded protein response.
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Acknowledgements |
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We thank J. Hirst (New York University) for expert assistance in fluorescent activated cell sorting, D. Littman (New York University) for plasmids used in retroviral production and for use of the FACScanTM, and I. Mohr, D. Levy, and E.Y. Skolnik for useful discussions.
This study was supported by grants ES08681 and DK47119 from the National Institutes of Health. I. Novoa was supported by a Basque Government Fellowship. D. Ron is a Scholar of the Leukemia and Lymphoma Society of America.
Submitted: 20 February 2001
Revised: 6 April 2001
Accepted: 6 April 2001
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References |
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