The GCN2 eIF2{alpha} Kinase Is Required for Adaptation to Amino Acid Deprivation in Mice

The GCN2 eIF2 ${alpha}$ kinase is essential for activation of the generalamino acid control pathway in yeast when one or more amino acidsbecome limiting for growth. GCN2's function in mammals is unknown,but must differ, since mammals, unlike yeast, can synthesizeonly half of the standard 20 amino acids. To investigate thefunction of mammalian GCN2, we have generated a Gcn2^-/- knockoutstrain of mice. Gcn2^-/- mice are viable, fertile, and exhibitno phenotypic abnormalities under standard growth conditions.However, prenatal and neonatal mortalities are significantlyincreased in Gcn2^-/- mice whose mothers were reared on leucine-,tryptophan-, or glycine-deficient diets during gestation. Leucinedeprivation produced the most pronounced effect, with a 63%reduction in the expected number of viable neonatal mice. Culturedembryonic stem cells derived from Gcn2^-/- mice failed to showthe normal induction of eIF2 ${alpha}$ phosphorylation in cells deprivedof leucine. To assess the biochemical effects of the loss ofGCN2 in the whole animal, liver perfusion experiments were conducted.Histidine limitation in the presence of histidinol induced atwofold increase in the phosphorylation of eIF2 ${alpha}$ and a concomitantreduction in eIF2B activity in perfused livers from wild-typemice, but no changes in livers from Gcn2^-/- mice.

INTRODUCTION

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Introduction

All organisms utilize externally available amino acids in lieuof synthesizing amino acids de novo. Consequently, amino acidbiosynthetic pathways are generally repressed until externalsources become limiting. The bacterium Escherichia coli is capableof assessing specific amino acid limitations and respondingselectively to express enzymes involved in the synthesis ofthe limiting amino acid (36, 42). In contrast, the eukaryoticyeast Saccharomyces cerevisiae relies upon a general amino acidcontrol system, whereby starvation for any single amino acidelicits derepression of all of the amino acid biosynthetic pathways(10, 41). Activation of the yeast general amino acid controlsystem is mediated by GCN2 protein kinase induction of GCN4translational expression. GCN4 is a transcriptional activatorof genes involved in amino acid synthesis and related metabolicpathways (12, 24, 25, 39-41). Amino acid limitation resultsin an increase in uncharged tRNAs, which in turn activates theGCN2 eIF2 ${alpha}$ kinase. Phosphorylation of Ser51 of eIF2 ${alpha}$ by GCN2 inhibitsthe activity of the eIF2B guanylate exchange factor, resultingin reduced formation of the eIF2 ${alpha}$ -GTP-Met-tRNAi ternary complexnecessary for translation initiation (22). Although decreasedavailability of the eIF2 ${alpha}$ -GTP-Met-tRNAi ternary complex can resultin the repression of global protein synthesis, the yeast GCN4mRNA contains a unique cluster of upstream open reading frames(uORFs) that mediate translational derepression of the GCN4coding sequence only when the ternary complex is limiting. Thus,it is clear that phosphorylation of eIF2 ${alpha}$ by GCN2 under conditionsof amino acid starvation allows yeast to adapt to this stressby down-regulating global protein synthesis while promotingthe translation of a transcription factor, which in turn activatesthe transcription of genes encoding amino acid biosyntheticenzymes.

Clues to the mechanism by which amino acid starvation is coupledto GCN2 activation were found in the predicted domain structureof the GCN2 protein. GCN2 contains a domain homologous to histidyl-tRNAsynthetases (HisRS), an enzyme normally responsible for charginghistidyl-tRNA with histidine. The HisRS-related domain of GCN2lacks this normal synthetase activity, and residues criticalfor histidine-specific binding are missing in the GCN2 HisRSdomain (40). Wek and coworkers (40) proposed that unchargedtRNAs, which increase in concentration concomitant with aminoacid deprivation, may activate the eIF2 ${alpha}$ kinase activity of GCN2through binding the modified GCN2 HisRS-related domain. Thishypothesis is supported by the demonstration that a varietyof uncharged tRNAs can bind the modified HisRS domain of GCN2,resulting in activation of the catalytic domain (6, 28, 38,41, 44).

Regulation of amino acid biosynthetic pathways in metazoans,including mammals, entails a further complication in that theydo not have the biosynthetic capacity to synthesize 10 of theamino acids (i.e., the so-called "essential amino acids"). Regulationof the biosynthesis of the nonessential amino acids appearsto be dependent upon a general amino acid control system similarto that of yeast (14), but also appears to be independent ofeIF2B activity and eIF2 ${alpha}$ phosphorylation. In contrast, deprivationof essential amino acids induces phosphorylation of eIF2 ${alpha}$ , reductionin eIF2B activity, and repression of global protein synthesis(13, 21, 37). Recently, homologues of yeast GCN2 have been discoveredand characterized in Neurospora (31), Drosophila (26, 30), andmice (2, 34). The major domains of GCN2, including catalyticand HisRS-related domains, are conserved among these species.However, analysis of the complete genomic sequence of Drosophila,Caenorhabditis elegans, mice, and humans has failed to detectan apparent homolog of GCN4, the regulatory target of yeastGCN2 (D.R.C., unpublished observations). Nonetheless, upon introductionin yeast cells, Drosophila and mouse GCN2 are capable of rescuingGcn2 mutants by phosphorylating yeast eIF2 ${alpha}$ and thereby derepressingthe translation initiation of GCN4 (26, 34).

Yeast GCN2 has been implicated as an important regulator ofgrowth in response to the stress of limiting amino acids. GCN2mutants not only are incapable of mounting the general controlresponse when starved for amino acids, but also are unable togrow when amino acids are limiting or when inhibitors of aminoacid biosynthesis are present in the growth medium (5, 12, 41).The role of GCN2 in growth and development in higher eukaryotesunder conditions of nutritional stress is currently unknown.

To determine the potential role of GCN2 in the regulation ofamino acid biosynthesis in higher eukaryotes, we have isolatedthe mouse Gcn2 gene and have generated a Gcn2^-/- knockout (loss-of-function)mutant mouse line. Gcn2^-/- mice are viable and fertile whenreared under standard conditions, but have a decreased probabilityof completing development under conditions in which amino acidsare deprived. Limiting specific amino acids is correlated withan increase in phosphorylation of eIF2 ${alpha}$ and a concomitant reductionof eIF2B activity in wild-type liver and cultured cells; however,these responses are ablated in the absence of GCN2.

MATERIALS AND METHODS

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Materials and Methods

Cloning and targeted disruption of the Gcn2 gene. Genomic clones of the Gcn2 gene were isolated from a BAC genomiclibrary (Genome Systems, St. Louis, Mo.) and from a lambda genomiclibrary constructed from genomic DNA isolated from the mouseTL1 129 SvEvTac strain (a gift from Christopher Wright, VanderbiltUniversity School of Medicine). Extensive DNA sequencing wasperformed throughout the 39 exons and flanking intronic regionsof GCN2. Exon-intron boundaries were determined by comparisonof a GCN2 cDNA clone (34) with the genomic DNA sequence determinedherein. To generate a targeted substitution or deletion of essentialdomains of the Gcn2 gene, the neomycin resistance (Neo^r) genewas substituted for most of exon 12 (corresponding to residues606 to 748 of the mGCN2ß isoform [GenBank accessionno. AF193343]). Specifically, the targeting vector was constructedin the pPNT-1 plasmid and contained a 3.0-kb HindIII genomicGCN2 fragment comprised of intron 11 and the first 15 nucleotidesof exon 12, a 1.1-kb HindIII-XhoI fragment containing the Neo^rgene, and a 9-kb XhoI-HindIII genomic GCN2 fragment comprisedof introns 12 to 15 and exons 13 to 15. The genomic DNA fragmentsof GCN2 were all derived from the lambda genomic library ofthe TL1 129 SvEvTac mouse strain. The GCN2-neo targeting vectorwas electroporated into TL1 129 SvEvTac mouse embryonic stem(ES) cells (Vanderbilt University Medical Center, TransgenicMouse Core Facility). Homologous recombinants were identifiedby SacI digestion followed by Southern blot analysis and furtherverified by PCR. Primer 1 (5'-GGTCTGAAGTAGAAGGATGTCAAGTTC-3')and primer 2 (5'-TCCATCAGTTTGCTTCTCTC-3') will amplify an 890-bpfragment from the wild-type Gcn2 allele, while primer 1 andthe neo-specific primer 3 (5'-TACTGTGGTTTCCAAATGTGTCAGTTT-3')will amplify a 550-bp fragment when exon 12 has been replacedby the Neo^r marker. Homologous recombinant cells were transplantedinto C57BL/6 blastocysts. Two chimeric founder mice were shownto have the Gcn2-neo gene in their germ lines (data not shown).The heterozygous F₁ hybrid mice were inbred to produce F₂ progeny.

Homozygous Gcn2^-/- ES cells were generated by incubating Gcn2^+/-heterozygous ES cells with a high (1 µg/ml) concentrationof G418 (neomycin) and selecting for resistant colonies. Onehighly resistant colony was subjected to further molecular analysisand shown to be homozygous for the target Neo^r substitutionof exon 12 of mGCN2.

RT-PCR analysis of Gcn2 mRNA. Total RNA was isolated with TRI reagent (Sigma). The followingprimers were used for reverse transcription-PCR (RT-PCR) analysisof the mGCN2 isoforms: RCW 150 (3' primer for all three isoforms),5'-ATGGAGGATGTCACACGAGCCAGGAGAG; RCW 285, 5'-AAGTTGAGTCTGGTTGTTACACTGT;RCW 244, 5'-ATACCCAGATGTAGTTCCCGAAA; and RCW 201, 5'-GACCAGGTGGTACAGGGTT.The 5' primer for mGCN2 ${alpha}$ was RCW 285, corresponding to sequencesin the extended 5'-exon specific for the ${alpha}$ isoform. The 5' primerfor mGCN2ß was RCW 244, corresponding to sequencescontiguous between exons 1 and 2 of the ß isoform.In addition, RCW 201, the 5' primer for mGCN2 ${gamma}$ , was within aportion of the 314-bp exon unique of the ${gamma}$ isoform, located betweenexons 1 and 2 of mGCN2ß (34).

For analysis of Gcn2 knockout mutants, 1 µg of total RNAwas subjected to RT-PCR with the Access RT-PCR kit (Promega)with primers flanking the targeted exon. Forward primer fromthe upstream exon 11 (positions 1768 to 1787, 5'-ACCGTCATTCCCAGCAACCA-3')and reverse primer from the downstream exon 15 (positions 2509to 2488, 5'-GCAGCGTGCTCTTCTCGCAGTA-3') will amplify a 742-bpproduct from the wild-type Gcn2 allele, whereas both Gcn2^-/-and Gcn2^+/- display an 812-bp fragment. RT-PCR products werecloned for sequencing. Sequence analysis revealed that the 812-bpmutant Gcn2 mRNA corresponded to the RNA generated from a crypticsplice located on the antisense strand of the Neo^r gene, whereasthe other splicing events remain intact. RT-PCR products werefurther analyzed by Southern hybridization with part of exon12 as the probe to demonstrate that the Gcn2^-/- mice completelylacked the RT-PCR product (742 bp) containing exon 12 codingsequences.

Amino acid limitation diet experiments. For 10 days prior to the beginning of dietary experiments, maleand female mice were provided with a low-fat synthetic dietfor 3 h. Food was weighed both prior to and after the 3-h feedingperiod. Water was provided ad libitum throughout the experiment.Mice were weighed daily, and their food intake and weight wereplotted versus time. After the 10-day meal-training regimen,animals were switched to the synthetic control diet containinga complete complement of amino acids, and matings were establishedbetween several replicate cages each containing one Gcn2^+/-and two Gcn2^-/- females. After 4 days, the males were removed.On day 12 of gestation, each cage was randomly assigned oneof the four diets: glycine-deficient, leucine-deficient, tryptophan-deficient,or complete-synthetic diet. Pregnant females remained on thediet until day 17 of gestation or until weight loss exceeded1 g for multiple days and then switched back to the completeamino acid control diet. Preliminary experiments indicated thatthis was the longest period of time amino-acid-limited dietscould be imposed during gestation without causing an unacceptablerate of spontaneous abortions. Cages were checked daily fornew litters, which were removed immediately. The date of birth,genotype of parents, general health, and number of pups wererecorded for each litter. Live pups were euthanized, and tailDNA was isolated from all pups for the purpose of genotyping.The genotype and numbers of alive and dead pups were recordedfor each diet.

Liver perfusion. Livers were perfused in situ as described previously (3, 16),with the following modifications. Livers were perfused for 15min with a nonrecirculating medium delivered at a flow rateof 4 ml/min. The perfusate contained amino acids present at10 times the concentrations in rat arterial plasma (controlmedium) (35) or 10 times the concentration found in rat arterialplasma except for histidine and additionally containing 4 mMhistidinol (histidinol medium). Livers were then removed, rinsedin ice-cold saline, and homogenized as described below.

Determination of eIF2 ${alpha}$ phosphorylation state. Livers were homogenized in 7 volumes of buffer consisting of20 mM HEPES (pH 7.4), 100 mM KCl, 0.2 mM EDTA, 2 mM EGTA, 1mM dithiothreitol, 50 mM NaF, 50 mM ß-glycerolphosphate,0.1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, and0.5 mM sodium vanadate and then centrifuged at 10,000 x g for10 min at 4°C. The relative amount of eIF2 ${alpha}$ in the phosphorylatedform was quantitated by protein immunoblot analysis with anaffinity-purified antibody that specifically recognizes eIF2 ${alpha}$ phosphorylated at Ser⁵¹ (eIF2 ${alpha}$ [P]; Biosource International).The total amount of eIF2 ${alpha}$ in the samples was determined by reprobingthe blot with a monoclonal antibody that recognizes equallythe phosphorylated and unphosphorylated forms of eIF2 ${alpha}$ followedby an antimouse secondary antibody. Values obtained with theanti-eIF2 ${alpha}$ [P] antibody were normalized for the total amount ofeIF2 ${alpha}$ present in the sample as described previously (20).

Measurement of eIF2B activity. Livers were homogenized in 4 volumes of buffer consisting of45 mM HEPES (pH 7.4), 95 mM potassium acetate, 375 µMmagnesium acetate, 75 µM EDTA, 10% glycerol, 2.5 mg ofdigitonin per ml, and 1 µM microcystin and then centrifugedat 10,000 x g for 10 min at 4°C. The guanine nucleotideexchange activity of eIF2B was measured in the 10,000 x g supernatantexactly as described previously (17). Briefly, eIF2 purifiedfrom rat liver (18) was incubated with [³H]GDP to form the substratefor the reaction. Liver supernatant was mixed with assay buffercontaining nonradiolabeled GDP at a final concentration of 1µM and incubated at 30°C for 1 min. The eIF2-[³H]GDPcomplex was added, and at various times, aliquots of the reactionmixture were removed and collected on nitrocellulose filterdisks. Radioactivity bound to the disks was quantitated by liquidscintillation spectrometry. eIF2B activity was calculated asthe rate of loss of [³H]GDP from the eIF2-[³H]GDP complex.

Measurements of rates of protein synthesis. Rates of protein synthesis were determined by the incorporationof [³H]leucine into protein during the final 10 min of liverperfusion as described previously (7).

GCN2 immunoblot analysis. Liver or kidney tissue derived from Gcn2^+/+ or Gcn2^-/- micewas frozen in liquid nitrogen, pulverized, and homogenized in10 volumes (wt/vol) of 50 mM Tris-HCl (pH 7.5), 1% Nonidet P-40,1 mM dithiothreitol, 10% glycerol, 1 mM EDTA, and protease inhibitors(100 µM phenylmethylsulfonyl fluoride [PMSF], 0.15 µMaprotinin, 1 µM pepstatin, 1 µM leupeptin). Thehomogenate was subjected to a 5-s burst with a Polytron homogenizerand clarified by centrifugation at 14,000 x g. Seventy microgramsof protein for each sample were fractionated on 7.5% SDS-polyacrylamidegels and transferred to a nitrocellulose filter, which was thenincubated with an antibody prepared against the carboxy terminusof mouse GCN2 (1/1,000 dilution). After incubation with a horseradishperoxidase-coupled antirabbit secondary antibody, the antibody-proteincomplexes were visualized with a chemiluminescent substrate.

Stress analysis of ES cells. ES cells were cultured in Dulbecco's modified Eagle's medium(BioWhittaker), supplemented with 2 mM glutamine, 0.1 mM eachnonessential amino acid, 50 µg of gentamicin per ml, 5.5µM ß-mercaptoethanol, 10% fetal bovine serum(HyClone), and 10 ng of mouse leukemia inhibitory factor (LIF)per ml in humidified air with 5% CO₂ at 37°C. ES cells weremaintained on irradiated mouse embryonic fibroblast feeder cellsand passed twice without feeders on gelatinized plates beforeeach experiment. Cells were cultured to about 50% confluenceand subjected to the presence or absence of stress for the indicatedtimes. Stress conditions were brought about by growth in completemedium supplemented with 1 µM thapsigargin or in mediadepleted of leucine. In the leucine depletion study, the mediumwas supplemented with dialyzed fetal bovine serum. Lysates wereprepared in a solution of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl,1% NP-400, and 0.5% sodium deoxycholate supplemented with proteaseinhibitors (100 µM PMSF, 0.15 µM aprotinin, 1 µMpepstatin, 1 µM leupeptin), 50 mM NaF, and 40 mM ß-glycerolphosphatefor Western blot analysis to detect the phosphorylated formof eIF2 ${alpha}$ at Ser⁵¹ (antisera from Research Genetics) or totaleIF2 ${alpha}$ with a monoclonal antibody that recognizes either phosphorylatedor nonphosphorylated forms.

RESULTS

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Results

Isolation and characterization of the mouse Gcn2 gene. The Gcn2 gene was isolated from mouse genomic libraries, andall of the exons were identified by sequencing and comparisonto cDNA clones of GCN2 (Fig. 1A). Three major mRNAs ( ${alpha}$ , ß,and ${gamma}$ ) that arise from three alternative promoters (34) wereidentified. Mouse Gcn2 is comprised of 39 exons that span 75kb of the mouse genome. Drosophila Gcn2 is composed of 10 exons(26) and does not share any exon-intron boundaries with themouse Gcn2 gene, suggesting that the extant introns were insertedinto the Gcn2 genes of each of these species after they divergedfrom a common ancestor. The ß GCN2 mRNA is the mostabundant GCN2 isoform and is expressed in a variety of tissues(Fig. 1B). The ${alpha}$ GCN2 mRNA isoform is most abundantly expressedin the brain, whereas the ${gamma}$ isoform is expressed in nearly equivalentlevels in six of the eight tissues examined, but is completelyabsent in skeletal muscle and kidney. All three GCN2 mRNA isoformsencode the critical catalytic and HisRS regulatory domains,but are predicted to have distinct amino-terminal domains. Theamino terminus of the GCN2 ß isoform contains an evolutionarilyconserved domain that interacts with GCN1 in yeast (8, 23).Recent studies show that GCN1 is required to mediate the activationof GCN2 by uncharged tRNAs (8). The ${alpha}$ and ${gamma}$ isoforms of GCN2 aremissing all or part of the GCN1 binding domain, suggesting thatthey may be activated by a different mechanism.

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FIG. 1. Structure and expression of the mouse Gcn2 gene. (A) Three isoforms of mouse GCN2 RNA transcripts are encoded within a 75-kb region of the mouse. The first exon of the ${alpha}$ isoform (denoted 6a) is inclusive of exon 6b present in the ß and ${gamma}$ isoforms and shares a common 3' end with exon 6b. The first exon of the ß isoform (denoted 1a) is inclusive of exon 1b present in the ${gamma}$ isoform and shares a common 3' end with exon 1b. Each of the three isoforms contains a unique translation initiation site (34), but terminates translation at a common UGA stop codon in exon 39. All of the exon-intron junctions conform to the eukaryotic consensus splice sites. Restriction sites mapped in genomic DNA: X, XbaI; and R, EcoRI. (B) Expression of mGCN2 mRNA isoforms. Primers specific for each of the three GCN2 mRNA isoforms were used in RT-PCRs with RNA isolated from various mouse tissues to detect their relative tissue-specific expression. Further quantitative RT-PCR experiments indicated that the ß isoform is considerably more abundant than the ${alpha}$ and ${gamma}$ isoforms. Diagrams depicting the domains included in each isoform are shown to the right.

Generation of Gcn2 knockout mutant mice. To generate a loss-of-function mutation of GCN2, we createda targeted disruption of exon 12, which encodes the criticalkinase subdomains II to IV and part of the insert region betweensubdomains IV and V (Fig. 2). Targeted disruption of exon 12was verified by Southern hybridization (Fig. 3A) and PCR (Fig.3B). Several Gcn2^-/- mutant strains were isolated and shownto produce a single-mutant GCN2 mRNA (Fig. 3C), and they donot express GCN2 protein (Fig. 3D). Sequence analysis of RT-PCRproduct from mutant GCN2 mRNA showed that cryptic splicing sitesexist in the Neo^r gene, and the splicing product contains aframeshift mutation. Matings between heterozygous Gcn2^+/- miceresult in the expected frequency of viable Gcn2^-/- mutants.Similar results were obtained from another Gcn2 mutant mousestrain, in which exon 12 was entirely deleted by using the Cre/loxPsystem (unpublished data).

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FIG. 2. Gcn2 targeting knockout vector. The approximate locations of the mouse Gcn2 exons 10 to 15 are shown below a restriction endonuclease map of genomic DNA. Xb, XbaI; Sac, SacI; Bam, BamHI; R, EcoRI; H, HindIII; and Xh, XhoI. The left arm of the target vector included a 3.0-kb HindIII fragment including a small part of exon 12, whereas the right arm included a 5.0-kb XhoI-HindIII fragment. The Neo^r gene was inserted between the right arm and left arm of the targeting vector, resulting in a deletion and substitution of most of exon 12.

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FIG. 3. Targeted disruption of the mouse Gcn2 gene. (A) Southern hybridization was performed on SacI-restricted genomic DNA to verify the targeted disruption. The Southern blot was probed with a combination of mGCN2 cDNA derived from exons 9 to 12 and a BamHI 1.3-kb genomic fragment corresponding to the 3' end of the left arm of the targeting vector. The sizes of the fragments are indicated. +/+, wild type; +/-, heterozygote; -/-, homozygous mutant. (B) Transgenic animals were verified by PCR amplification of genomic DNA. The wild-type mGCN2 allele was detected as a 550-bp fragment from exon 12, whereas the mutant allele was detected as an 890-bp product from Neo^r gene substitution. (C) RT-PCR products (exons 11 to 15) derived from liver RNA were analyzed by Southern hybridization with Neo^r gene or a fragment of exon 12 as the probe. Gcn2^-/- and Gcn2^+/- mice display an 812-bp fragment detected by the Neo^r probe. Sequence analysis of this fragment revealed that it corresponded to an RNA generated from a cryptic splice located on the antisense strand of the Neo^r gene. As expected, the Gcn2^-/- mice completely lacked the RT-PCR product (742 bp) containing exon 12 coding sequences. (D) Western blot analysis of mouse GCN2 with antisera prepared against the carboxyl terminus of mouse GCN2 indicated the absence of GCN2 in the homozygous mutant liver and kidney.

Mutation of Gcn2 has significant developmental affects when essential amino acids are absent in the maternal diet. In yeast, GCN2 is required for growth in the absence of aminoacids or in the presence of inhibitors of amino acid biosynthesis(5, 12, 41). Prenatal and postnatal growth in mammals is influencedby maternal health and diet, as well as the genetic compositionof the progeny. To determine if GCN2 influences mouse growthand development under conditions of limiting amino acids, Gcn2^-/-homozygous mutant females were mated with Gcn2^+/- heterozygousmales to produce litters with an expected Mendelian 1:1 ratioof Gcn2^-/- and Gcn2^+/- genotypes. During days 12 to 17 of gestation,pregnant females were fed one of three synthetic diets lackinga single amino acid. Two of the diets lacked the essential aminoacids leucine or tryptophan, while the other lacked the nonessentialamino acid glycine. Control groups were fed a synthetic dietreplete with all amino acids.

Mice fed a complete synthetic diet during gestation showed theexpected 1:1 ratio of Gcn2^-/- to Gcn2^+/- progeny. In contrast,mice fed a leucine-deficient diet showed a nearly threefoldreduction in the number of Gcn2^-/- progeny (Table 1). The numberof Gcn2^-/- progeny was also reduced in the glycine- and tryptophan-deficientdiet experiments, although the reduction did not quite reachstatistical significance in either case. Furthermore, the aminoacid-deficient diets adversely affect prenatal viability, asshown by the relatively large number of stillborn pups. Thenegative impact of the amino acid-deficient diets was most pronouncedin the leucine-deficient diet, with which 24% of the pups werestillborn.

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TABLE 1. Affects of amino acid deprivation on progeny number and genotype

Gcn2^-/- mice exhibit normal liver glycogen content. Scheuner and coworkers (32) have mutated the regulatory phosphorylationsite of eIF2 ${alpha}$ in mice by substituting the Ser⁵¹ residue withalanine. Ser51Ala knock-in mice die within the first day afterbirth due to hypoglycemia associated with the lack of glycogenstorage in the liver, leading Scheuner et al. (32) to speculatethat the Ser51Ala lethal phenotype was caused by blocking theregulatory activity of GCN2. However, as noted, our Gcn2^-/-mice are normal at birth and have survived for more than 1 yearunder standard laboratory conditions. We have examined the liverglycogen content of adult mice (4 to 10 months old) and foundthat no difference occurs between Gcn2^-/- knockout mice andwild-type mice (Table 2).

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TABLE 2. Liver glycogen levels in Gcn2^+/+ versus Gcn2^-/- mice

Gcn2^-/- mice fail to show induction of eIF2 ${alpha}$ [P] and repression of eIF2B activity during histidine deprivation. In a previous study, it was shown that perfusion of rat liverswith medium lacking histidine and containing the amino acidanalog histidinol resulted in an increase in the levels of phosphorylatedeIF2 ${alpha}$ and a reduction in eIF2B activity in liver extracts (16).In the present study, the effect of histidinol on eIF2 ${alpha}$ phosphorylationin livers from wild-type and Gcn2^-/- mice was examined. In theabsence of stress, the relative phosphorylation of eIF2 ${alpha}$ in Gcn2^+/+mice was significantly higher than that in Gcn2^-/- littermates.Upon perfusion with histidinol, eIF2 ${alpha}$ phosphorylation in theGcn2^+/+ livers was increased about twofold, with no detectablephosphorylation of this initiation factor in the Gcn2^-/- tissue(Fig. 4A and B).

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FIG. 4. Effect of histidinol treatment on relative eIF2 ${alpha}$ phosphorylation, eIF2B activity, and protein synthesis. (A) Mouse livers were perfused with histidinol in situ as described in Materials and Methods. Total eIF2 ${alpha}$ and eIF2 ${alpha}$ [P] were then detected by Western blot analysis. (B) Quantitative analysis of eIF2 ${alpha}$ [P] relative to total eIF2 ${alpha}$ is shown as the mean of seven livers per condition. (C) The guanine nucleotide exchange activity of eIF2B in mouse liver homogenates was measured as described in Materials and Methods. The results represent the mean ± standard error of three to seven livers per condition. (D) The eIF2 ${alpha}$ phosphorylation and eIF2B activity data presented in panels B and C were subjected to linear regression analysis and are presented in graphic form. (E) Rates of protein synthesis were determined by the incorporation of [³H]leucine into protein during the final 10 min of the perfusion and are expressed as milligrams of protein synthesized per gram of tissue protein per hour. The values represent the mean ± standard error for three to six livers per condition: a complete mixture of amino acids (open bars) or medium containing all amino acids except histidine and additionally containing 4 mM histidinol (solid bars). *, P < 0.01 versus complete amino acid condition. The perfusate contained amino acids present at 10 times the concentrations typically found in arterial plasma (10X control medium) or 10 times the concentration found in arterial plasma except for histidine and additionally containing 4 mM histidinol (Hisol medium).

The guanine nucleotide exchange activity of eIF2B was measuredin extracts of livers from wild-type and Gcn2^-/- mice perfusedin the presence or absence of histidinol. The eIF2B activitywas reduced to approximately 30% of the control value in liversfrom wild-type mice perfused with histidinol medium (Fig. 4C).In contrast, histidinol had no affect on eIF2B activity in liversfrom Gcn2^-/- mice. Interestingly, eIF2B activity was significantlygreater in livers from Gcn2^-/- mice perfused with control mediumthan in livers from wild-type animals (P = 0.05). The latterfinding correlates with the reduction in eIF2 ${alpha}$ phosphorylationobserved in livers from Gcn2^-/- mice compared to wild-type mice.In fact, linear regression analysis revealed that eIF2 ${alpha}$ phosphorylationand eIF2B activity were inversely proportional (r² = 0.99; Fig.4D) for the four conditions examined here. Despite the factthat eIF2B activity remained high in Gcn2^-/- livers perfusedwith histidinol, global protein synthesis in Gcn2^-/- liverswas repressed by histidinol treatment to the same extent asthat seen in Gcn2^+/+ mice (Fig. 4E).

GCN2 phosphorylates eIF2 ${alpha}$ in response to leucine depletion. Leucine deprivation results in the repression of global proteinsynthesis via the reduction in eIF2B activity (19). The reductionin eIF2B activity is caused in part by the phosphorylation ofSer⁵¹ of eIF2 ${alpha}$ by one or more of the eIF2 ${alpha}$ kinases. To assessthe importance of GCN2 in the induction of eIF2 ${alpha}$ phosphorylationduring leucine deprivation, Gcn2^-/- ES cells were incubatedin leucine-deficient media for 1 to 12 h. A 3.5-fold enhancementof eIF2 ${alpha}$ phosphorylation in Gcn2^+/+ ES cells was observed afterthe first hour of leucine deprivation and was sustained throughoutthe 12-h incubation period (Fig. 5). In contrast, Gcn2^-/- EScells failed to show any induction in eIF2 ${alpha}$ phosphorylation duringthe first 3 h of leucine deprivation, and only after a 6-h stresswas there a two- to fourfold induction of eIF2 ${alpha}$ phosphorylation.In contrast, thapsigargin, an endoplasmic reticulum stress agentthat inhibits Ca²⁺-ATPase and activates the PERK eIF2 ${alpha}$ kinase,showed a strong and rapid induction of eIF2 ${alpha}$ phosphorylation.This increase in eIF2 ${alpha}$ phosphorylation was observed independentof the Gcn2 genotype.

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FIG. 5. Phosphorylation of eIF2 ${alpha}$ in leucine-deprived ES cell is dependent upon GCN2. Gcn2^+/+ (A) and Gcn2^-/- (B) ES cells were grown in leucine-replete (lane C) or leucine-deficient (-Leu) media as described in Materials and Methods. Cells were harvested at 1, 3, 6, 9, and 12 h and analyzed by immunoblotting with antisera directed against either eIF2 ${alpha}$ or eIF2 ${alpha}$ [P]. The ratio of eIF2 ${alpha}$ [P] to eIF2 ${alpha}$ is indicated at the bottom of each panel. For comparison, Gcn2^+/+ and Gcn2^-/- ES cells were also treated for 1 or 3 h with 1 µM thapsigargin (Tg) to induce eIF2 ${alpha}$ phosphorylation via the PERK-dependent unfolded protein response.

DISCUSSION

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Discussion

The control of translation in response to external signalingis an important mechanism by which cells and organisms respondto changes in their environment. Yeast cells have evolved amechanism exquisitely adapted to sense amino acid deprivationthat results in the selective translational derepression ofGCN4, a transcriptional activator of the amino acid biosyntheticgenes (12). Yeast has a single eIF2 ${alpha}$ kinase, GCN2, which actsas the sensor in this regulatory pathway. Regulation of aminoacid biosynthesis in multicellular higher eukaryotes is considerablymore complex owing to the diverse needs of different tissues,the transport of amino acids through the circulatory system,and the inability of higher eukaryotes to synthesize 10 of theamino acids. Nonetheless, GCN2 is also present in higher eukaryotesand is functionally equivalent to yeast GCN2 (26, 34).

Similar to loss-of-function mutations in yeast Gcn2, mouse Gcn2knockout mutants have no observable mutant phenotype in a nonstressedstate. The loss of GCN2 function, however, negatively impactsfetal development when specific amino acids, particularly leucine,are missing in the maternal diet. This developmental defectis analogous to the slow-growth phenotype of yeast Gcn2 mutantsgrown on amino acid-deprived media or in the presence of inhibitorsof amino acid biosynthesis. The developmental defects that weobserve in the Gcn2^-/- knockout mice reared on leucine-deficientdiets are likely to be due to the loss of signaling via thephosphorylation of eIF2 ${alpha}$ . Gcn2^-/- ES cells do not exhibit thenormal rapid induction of phosphorylation of eIF2 ${alpha}$ when shiftedto media lacking leucine. In contrast, eIF2 ${alpha}$ phosphorylationis induced in Gcn2^-/- cells by thapsigargin, a known inducerof the unfolded protein response and an indirect activator ofthe PERK (PEK) eIF2 ${alpha}$ kinase (10, 33). Leucine deprivation doeseventually result in the induction of eIF2 ${alpha}$ phosphorylation inGcn2^-/- ES cells, suggesting that one or more of the other eIF2 ${alpha}$ kinases are activated by a secondary effect of the leucine deprivation.We have observed a similar effect in Perk^-/- cells treated withthapsigargin (D.R.C., unpublished data), which suggests thatalthough PERK is the major eIF2 ${alpha}$ kinase responding to Ca²⁺ depletionin the endoplasmic reticulum, one or more of the other eIF2 ${alpha}$ kinases can also respond. We propose that although each of theeIF2 ${alpha}$ kinases may play a dominant role in responding to a specificphysiological change or stress, each may also contribute toeIF2 ${alpha}$ phosphorylation signaling in response to a diverse arrayof perturbations.

Deprivation of essential amino acids in cultured mammalian cellsand in liver perfusion experiments has shown that phosphorylationof eIF2 ${alpha}$ is induced concomitant with a decrease in eIF2B activityand global protein synthesis (15, 37). We show that phosphorylationof eIF2 ${alpha}$ and reduction of eIF2B activity is strikingly dependentupon GCN2 when the liver is deprived of histidine. Surprisingly,however, global protein synthesis is still repressed in Gcn2^-/-mutant liver under histidine deprivation, suggesting that therepression of global protein synthesis is not mediated by thephosphorylation of eIF2 ${alpha}$ and the reduction in eIF2B activity.The repression of global protein synthesis under these conditionsmay instead be mediated by changes in eIF4EBP, as has been suggestedpreviously (15). In yeast, deprivation of a single amino aciddoes not result in the repression of global protein synthesis,although GCN2 is activated, phosphorylates eIF2 ${alpha}$ , and leads tothe reduction in eIF2B activity and the translational derepressionof the amino acid biosynthetic transcriptional activator GCN4.Although mammalian cells apparently lack an orthologue of GCN4,we propose that mammalian GCN2 is likely to be important intranslational control of specific mRNAs. Over half of the mRNAsencoding regulatory proteins of diverse nature in mammals containmultiple uORFs in their 5' untranslated leader sequence (29),and these transcripts are candidates for similar translationalcontrol as described for yeast GCN4. One candidate for suchregulation in mammals is the transcription factor ATF4 (alsocalled CREB-2), which contains conserved uORFs. Harding andcoworkers (10) have shown that derepression of ATF4 in mouseES cells deprived of leucine is dependent upon GCN2 and the5' leader of ATF4 mRNA.

GCN2 exhibits a distinct tissue-specific pattern of expression,suggesting that it may have specific developmental or physiologicalfunctions (reference 34 and data shown herein). The highestlevel of GCN2 mRNA is seen in the brain of both mouse (reference34 and data shown herein) and Drosophila (26, 30). ATF4, a knowntarget of GCN2 regulation in cell culture, is intimately involvedin regulating long-term memory in mammals and invertebrates,including Drosophila (1). In addition to their primary functionin protein synthesis, specific amino acids, including glutamateand aspartate, function as excitatory signals in the centralnervous system. In part because of these dual functions, aminoacid pools in the brain are rigorously regulated (4, 9, 27).

In addition to amino acid deprivation, yeast GCN2 is known tobe activated by purine and glucose deprivation. In mammaliancells, glucose-induced secretion of insulin is dependent uponamino acids, and individual amino acids such as arginine arepotent stimulators of insulin secretion and thereby play importantroles in glucose homeostasis. Regulation of translation initiationvia phosphorylation of eIF2 ${alpha}$ appears to play multiple roles inglucose homeostasis, as revealed by mutating the regulatoryphosphorylation site (Ser⁵¹) of eIF2 ${alpha}$ and by knockout mutationsof the PERK eIF2 ${alpha}$ kinase. Ser51Ala knock-in mutant mice exhibitneonatal loss of insulin-secreting pancreatic ß cellsand deficiency in liver glycogen storage. These mice typicallydie within the first day after birth because of severe hypoglycemiaassociated with defects in gluconeogenesis. The defect in insulin-secretingpancreatic ß cells is also seen in the Perk knockoutmutant mice, which succumb to severe hyperglycemia after thethird postnatal week (11, 43). Zhang and coworkers have alsoshown that the glucagon-secreting pancreatic ${alpha}$ cells are lostin the Perk^-/- mutants after the loss of the insulin-secretingß cells (43). Scheuner and coworkers (32) speculatedthat the severe hypoglycemia observed in their Ser51Ala knock-inmice was due to the lack of regulation of eIF2 ${alpha}$ phosphorylationin the liver, as mediated by GCN2. However, we show herein thatGcn2^-/- mice exhibit normal glycogen levels in the liver. Inasmuchas none of the single-knockout mice for the four eIF2 ${alpha}$ kinases(GCN2, PERK, HRI, and PKR) display the severe neonatal hypoglycemiaseen in the Ser51Ala knock-in mutant, we suggest that two ormore of the eIF2 ${alpha}$ kinases participate jointly in liver glycogenmetabolism. We are currently generating double- and triple-mutantcombinations of Gcn2^-/-, Perk^-/-, and Pkr^-/- mice to test thishypothesis.

ACKNOWLEDGMENTS

We thank Mark Magnuson and Cathy Pettepher for help in establishingthe mouse Gcn2 knockout strain and Christopher Wright for providingthe TL1 129 SvEvTac lambda genomic library. We thank Scott Myers,Keri Merritt, Zhao Lin, Rui Peng, and Adam Harris for technicalassistance in isolation and characterization of the Gcn2 geneand Gcn2 knockout mice.

This work was supported by the Culpeper Foundation; the IngramCancer Center and Clinical Nutrition Research Unit of the VanderbiltUniversity School of Medicine; the Pennsylvania State University;and National Institutes of Health grants GM56957 to D.R.C.,DK13499 to L.S.J., and GM49164 and GM643540 to R.C.W.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biology, 208 Mueller Laboratory, The Pennsylvania State University, University Park, PA 16802. Phone: (814) 865-9790. Fax: (814) 865-6193. E-mail: drc9{at}psu.edu.

REFERENCES

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