|Mutation Type||splice site (6 bp from exon)|
|Coordinate||118,400,653 bp (GRCm38)|
|Base Change||T ⇒ C (forward strand)|
|Gene Name||eukaryotic translation initiation factor 2 alpha kinase 4|
|Chromosomal Location||118,388,618-118,475,234 bp (+)|
FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] This gene encodes a member of a family of kinases that phosphorylate the alpha subunit of eukaryotic translation initiation factor-2 (EIF2), resulting in the downregulaton of protein synthesis. The encoded protein responds to amino acid deprivation by binding uncharged transfer RNAs. It may also be activated by glucose deprivation and viral infection. Mutations in this gene have been found in individuals suffering from autosomal recessive pulmonary venoocclusive-disease-2. [provided by RefSeq, Mar 2014]
PHENOTYPE: Homozygotes for a null allele have altered feeding behavior, synaptic plasticity and dendritic cell function. Homozygotes for another null allele show enhanced muscle loss and morbidity after amino acid deprivation. Homozygotes for an ENU-induced allele show higher susceptibility to viral infection. [provided by MGI curators]
NCBI RefSeq: NM_013719 (isoform 1), NM_001177806 (isoform 2); MGI: 1353427
|Amino Acid Change|
|Institutional Source||Beutler Lab|
|Gene Model||not available|
|AlphaFold||no structure available at present|
|Predicted Effect||probably benign|
|Predicted Effect||probably benign|
|Predicted Effect||probably benign|
|Predicted Effect||probably benign|
|Predicted Effect||probably benign|
|Meta Mutation Damage Score||Not available|
|Is this an essential gene?||Non Essential (E-score: 0.000)|
|Phenotypic Category||Autosomal Recessive|
|Candidate Explorer Status||loading ...|
Linkage Analysis Data
|Alleles Listed at MGI|
All alleles(7) : Targeted, knock-out(2) Targeted, other(1) Gene trapped(4)
|Mode of Inheritance||Autosomal Recessive|
|Local Stock||Live Mice, Sperm|
|Last Updated||2016-05-13 3:09 PM by Stephen Lyon|
|Other Mutations in This Stock||
Stock #: A9681 Run Code:
Validation Efficiency: 88/103
Stock #: ZE80 Run Code:
Validation Efficiency: 82/103
The atchoum phenotype was identified among ENU-mutagenized G3 mice in an ex vivo macrophage screen for control of viral infection. Macrophages from the index atchoum mouse were highly susceptible to infection by the adenoviral vector Ad5-F16-GFP (1), as indicated by an increased percentage of GFP+ cells relative to wild type 72 hours after infection (2). The percentage of infected cells was similar to that seen for the STAT1 mutant domino (2;3). However, type I IFN production after infection was completely normal, unlike that of domino macrophages (2). Atchoum macrophages may be slightly susceptible to MCMV (4) infection, while both influenza and Rift Valley Fever Virus (RVFV) are completely controlled (Figure 1) (2). The phenotype is even stronger when macrophages are pre-treated overnight with IFNγ.
The function of the protein encoded by atchoum was assayed by exposure of Eif2ak4atc/atc mouse embryonic fibroblasts with ultraviolet (UV) radiation, a known activator of GCN2 (5;6). In the atchoum fibroblasts an increase in eIF2α phosphorylation was not observed upon UVB exposure; a drastic increase in phosphorylation of eIF2α is observed at 40 hours post-viral infection in normal fibroblasts (2). This indicates that the atchoum mutation results in a complete loss of GCN2 function (2). Injection of atchoum mice with MCMV led to the deaths of several on or before nine days post-infection, indicating that viral susceptibility stems from a defect in the innate immune response (2). The atchoum mice were tested for defects in the adaptive immune response; they mounted a normal IgG response to stimulation with β-galactosidase and a normal IgM response to NP-Ficoll (2).
Macrophages from the index atchoum mouse were also found to have a defective TNF-α response to the TLR7 ligand R-848 in the TLR Signaling Screen. However, testing of offspring from a cross of the index male and his sister demonstrated that the defective response to R-848 and susceptibility to adenovirus are unrelated, and caused by independently segregating mutations. The TLR signaling phenotype is now called rsq3.
|Nature of Mutation|
The atchoum mutation was mapped by bulk segregation analysis (BSA) of F2 backcross offspring using C57BL/10J as the mapping strain (n=40 with mutant phenotype, 9 with normal phenotype). The mutation showed strongest linkage with marker B10SNPS0032 at position 103735349 bp on Chromosome 2 (synthetic LOD=6.0) (2). Whole genome SOLiD sequencing of an atchoum homozygote was carried out: no Chromosome 2 mutations were validated among successful nucleotide calls covered 3 or more times. However, among 327 “N pattern” nucleotides (those covered 1 or 2 times) on Chromosome 2 (validation efficiency 281/327), a single mutation was identified at position 118226389 bp, 14.5 Mb from the marker with peak linkage (2). The mutation corresponds to a T to C transition at position 12038 of the genomic DNA sequence of Eif2ak4 (NC_000068), encoding GCN2. The mutation affects the sixth nucleotide of Eif2ak4 intron 2:
<--exon 2 intron 2--> 12016 CCTACATACCCAGATGT GTGAGTACCTGTAT 80 -P--T--Y--P--D--V…
The mutated nucleotide is indicated in red; the splice donor sequence is shown in blue. cDNA sequencing of four major Gcn2 transcripts in atchoum mice revealed that the mutation invariably results in skipping of exon 2, in some cases along with skipping of other exons (2); splicing from exon 1 to exon 3, exon 4, exon 5, or exon 6 was observed in the four sequenced transcripts. The most abundant transcript lacked exons 2, 3, and 4; this would result in an in-frame deletion of 123 amino acids.
|Illustration of Mutations in
Gene & Protein
Four kinases, GCN2, PKR, PERK, and HRI, regulate protein translation in response to distinct environmental stresses (7). They do so by phosphorylating serine 51 of the translation initiation factor eIF2α, leading to inhibition of translation initiation and thereby, protein synthesis (see Background). Initial cloning of mouse GCN2 revealed the presence of three isoforms (α, β, and γ) that differed in their N-terminal sequences and were derived by alternative splicing/transcription start of the GCN2 transcript (8;9). GCN2β is the longest isoform (1648 aa) and is the form discussed here unless otherwise specified. Amino acids 1-278 of GCN2β are missing from GCN2α, resulting in a protein 1370 aa in length. The first 86 amino acids of GCN2β are replaced with eight unique residues in GCN2γ, resulting in a protein 1570 aa in length. Six protein coding Eif2ak4 transcripts are annotated in the Vega database, encoding proteins from 347 to 1648 amino acids in length. Mouse and human GCN2 are 91% identical in sequence.
The kinase activity of GCN2 is regulated by several domains that serve to couple eIF2α phosphorylation to amino acid or serum starvation (Figure 2). Near the C terminus of GCN2 is a histidyl-tRNA synthetase (HisRS)-like domain to which uncharged tRNA binds, activating GCN2 (10-13). The HisRS domain binds to various uncharged tRNAs with similar affinities, discriminating only against their aminoacylated forms (14). Two segments within the N- and C-terminal halves of the HisRS domain (HisRS-N and HisRS-C) have been shown to mediate dimerization of GCN2 (15). Both HisRS-N and HisRS-C are required for GCN2 function in vivo, but neither is essential for dimerization of the full length protein, likely because of the presence of other dimerization domains in GCN2 (within the kinase domain and RB/DD; see below). It appears that dimerization is critical to the function of GCN2, and some evidence suggests that dimerization of the HisRS domain is required for tRNA binding (15). In addition, HisRS-N physically interacts with the kinase domain, and select mutations in HisRS-N abolished kinase function without impairing tRNA binding, suggesting that the HisRS and kinase domains participate in stable intramolecular interactions that, upon binding of tRNA, stimulate kinase activity through a conformational change propagated from HisRS to kinase domain (15).
The C-terminal 156 amino acids of GCN2 form the RB/DD domain, which contains ribosome binding (16) and homodimerization activities (17). The domain is characterized by three hydrophobic segments and a predicted amphipathic helix (18). RB/DD is required for GCN2 function in vivo (19), being necessary to induce eIF2α phosphorylation in response to amino acid limitation (18). In addition to binding the translation machinery, the isolated RB/DD can bind to double-stranded RNA in vitro, suggesting that interaction with rRNA mediates ribosome targeting (16). Homodimerization via the RB/DD and kinase domain is thought to permit trans-autophosphorylation of Thr882 and Thr887 in the activation loop, activating GCN2 kinase activity. The RB/DD also participates in direct physical contacts with the kinase domain that are thought to inhibit kinase activity (15;17). Binding of uncharged tRNA to the HisRS domain may stabilize a stimulatory conformation in which the C-terminal portion of GCN2 dissociates from the kinase domain and also becomes more accessible to tRNA. Another proposed role for the extreme carboxyl terminus of GCN2 is to facilitate binding of the HisRS domain to uncharged tRNA (10).
The serine/threonine kinase domain of GCN2 is highly similar to those of PKR, PERK, and HRI. Analysis of the X-ray crystal structure of the isolated GCN2 kinase domain (insert deleted) demonstrated a typical, bi-lobed kinase fold comprised of N- and C-lobes linked by a flexible hinge and the ATP binding cleft positioned between the two lobes (Figure 4; PDB 1ZYC) (see iconoclast, a mutation in the Src family kinase Lck) (29). The GCN2 kinase domain crystallized as a homodimer. The smaller N-lobe is preceded by a short α-helix and contains a five-stranded β sheet with two α-helices (αB and αC) linking β3 and β4. In the apo form of the structure, the position of the important αC helix is shifted outward, as typically seen in the inactive conformation of kinases. In this position, the αC helix is unable to coordinate ATP within the catalytic cleft. The kinase domains of each of the eIF2α kinase domains is divided by a large insert; in GCN2 the insert is 139 amino acids in length and occurs in the N-lobe, between sheets β4 and β5. The larger C-lobe of the kinase contains amino acids implicated in catalysis, activation, and substrate recognition; it is predominantly α-helical.
Comparison of the structures of apo and ATP-bound GCN2 revealed that partial closure of the active site cleft in the apo form restricts ATP binding by restraining the conformation of the hinge region and strongly coupling the two kinase lobes (29). In contrast, a tRNA-independent constitutively active GCN2 point mutant (R794G) (30) had a relatively open conformation resembling ATP-bound GCN2, and lacked multiple interactions that rigidify the hinge of GCN2. In GCN2R794G, Asn793, which normally blocks ATP entry into the active site cleft, was displaced away from the active site permitting ATP binding. Thus, it was proposed that increased flexibility of the hinge region induced by uncharged tRNA binding to the HisRS domain would permit productive binding of ATP to the active site and autophosphorylation of Thr887 and Thr882 in the activation loop, thereby activating GCN2. 3D modeling of a dimeric kinase-HisRS segment supports the idea that the hinge interacts directly with HisRS, such that structural changes in HisRS upon tRNA binding could propagate to the hinge region of the kinase domain. In addition to modulating the flexibility of the kinase domain hinge, tRNA binding to the HisRS domain relieves a network of hydrophobic interactions that constrains rotation of helix αC into a stimulatory conformation for kinase activity (31).
The atchoum mutation occurs in intron 2. The GCN2β isoform would be affected by the mutation; neither GCN2α nor GCN2γ would be affected.
GCN2 mRNA was detected in all tissues examined, including heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testis, with the highest expression in liver and brain (8;9). GCN2 isoforms were differentially expressed among tissues.
The atchoum mutation results in no detectable protein expression even though splicing from exon1 to exon 4, 5 or 6 would maintain the correct translational reading fram, suggesting that the mutation leads to an unstable protein that is subsequently degraded (2).
One way that gene expression is regulated is through control of translation of mRNAs into proteins. Translational control is typically more rapid than transcriptional control because it exerts its effects on existing mRNAs, and can therefore meet the cell’s immediate needs to maintain homeostasis or adapt to stress in a changing environment. The process of translation can be divided into four stages: initiation, elongation, termination, and ribosome recycling. Most regulation occurs at the stage of initiation, in which the AUG start codon is identified and decoded within the P site of the ribosome by the methionyl tRNA specialized for initiation (Met-tRNAi) [reviewed in (32)].
In eukaryotes, the start codon is generally identified by a scanning mechanism, where the small (40S) ribosomal subunit loaded with Met-tRNAi in a preinitiation complex (PIC) binds to the mRNA near the 5’ end and scans the 5’ untranslated region (UTR) for an AUG codon (Figure 5) (32). The mRNA itself is activated for PIC binding by the eukaryotic initiation factor 4F complex (eIF4F, composed of eIF4E, eIF4G, and eIF4A) binding to the 5’ cap, and poly A binding protein (PABP) binding to the poly(A) tail, circularizing the mRNA. The PIC is formed when eIF1, eIF1A, and eIF3 promote dissociation of the 80S ribosome into 60S and 40S subunits, and together with eIF5, ternary complex (composed of eIF2•GTP bound to Met-tRNAi), and the 40S subunit, assemble into the 43S PIC (33). This 43S PIC binds near the 5’ cap, facilitated by eIF3/eIF5 interactions with eIF4G/eIF4B, and scans the leader for complementarity to the anticodon of Met-tRNAi in an ATP-dependent reaction. Once the start codon has been identified, hydrolysis of GTP in the ternary complex leads to release of eIF2•GDP, Pi, and other eIFs from the 40S platform (34). Another factor, GTP-bound eIF5B, catalyzes the joining of the 60S ribosomal subunit to the 40S subunit, and subsequent release of eIF5B•GDP and eIF1A yields the final 80S initiation complex, ready to accept the appropriate aminoacyl-tRNA into the A site and synthesize the first peptide bond (35-38).
Cells use several mechanisms to regulate the efficiency of translation initiation. For example, RNA structures that impede the ability of ribosomes to interact with the 5′UTR in single-stranded form, or to scan the 5′UTR, reduce the efficiency of initiation. Decoy AUG codons in the 5′UTR can also slow a ribosome in finding the correct start codon. mRNA activation can be inhibited by inactivating components of the eIF4F complex. Some viral RNAs as well as eukaryotic mRNAs bypass the scanning process and the requirement for one or more eIFs by using specialized sequence elements called internal ribosome entry sites (IRESs) to recruit the 40S subunit directly to the start codon (39). Another prominent mechanism for blocking translation initiation is to reduce the activities of eIFs, such as eIF2, that stimulate Met-tRNAi recruitment to the 40S subunit.
Translational control by eIF2 phosphorylation
eIF2 is a heterotrimeric G protein consisting of α, β, and γ subunits that binds charged Met-tRNAi in a GTP-dependent manner and delivers it, in the context of the ternary complex, to the PIC (Figure 5). The eIF2 GTPase cycle begins when eIF2 is first activated by binding GTP, and the resulting eIF2•GTP interacts with Met-tRNAi to form a ternary complex, which is transferred to a 40S ribosomal subunit along with other eIFs to form the PIC. The assembled 43S PIC scans the mRNA, and upon AUG codon recognition, the factor eIF5 interacts with the ternary complex and functions as a GTPase activating protein (GAP) for eIF2•GTP, stimulating hydrolysis of GTP by eIF2 (35;40;41). eIF2•GDP, Pi, and other initiation factors are released, and the 60S ribosomal subunit joins to form the elongation competent 80S initiation complex. The inactive eIF2•GDP is then recycled by exchange of GTP for GDP, catalyzed by the guanine nucleotide exchange factor (GEF) eIF2B. A complex of eIF5/eIF2 antagonizes guanine nucleotide exchange by eIF2B (42;43).
Whereas the β and γ subunits of eIF2 mediate interactions with guanine nucleotide, Met-tRNAi, eIF2B, and eIF5 that permit ternary complex formation, GTP hydrolysis, and GTP/GDP exchange (44-51), the α subunit regulates these activities. Phosphorylation of the α subunit at a conserved serine residue (S51) converts eIF2•GDP into a competitive inhibitor of eIF2B (52;53), resulting in functional sequestration of the limiting amounts of eIF2B present in the cell, and thereby decreasing levels of eIF2•GTP, ternary complex formation, and global protein synthesis. Regulation of eIF2 in this manner is conserved across species, and is controlled by four eIF2 kinases that respond to distinct environmental stresses: PERK is activated by misfolded proteins in the ER (ER stress); PKR by double-stranded RNA in virus-infected cells; HRI by heme deprivation, and oxidative and heat stresses in erythroid tissues; and GCN2 by amino acid deprivation, UV irradiation, and proteasome inhibition.
Amino acid limitation results in an increase in uncharged tRNAs, which in turn activates GCN2. Paradoxically, phosphorylation of eIF2α reduces translation initiation generally while at the same time inducing translation of particular mRNAs through specialized mechanisms that enable the cell to turn on stress-specific response pathways. GCN2 was in fact first identified as an inducer of GCN4, a transcriptional activator of amino acid biosynthesis in yeast (54-56). Four short open reading frames (upstream ORFs, uORFs) in the GCN4 mRNA leader that support ribosomal reinitiation act as inhibitors of GCN4 translation under conditions of amino acid sufficiency (i.e. when eIF2•GTP is plentiful) (57;58). After translating the 5’ uORF (uORF1), post-termination 40S subunits rapidly re-acquire the activated, GTP-bound ternary complex, form a new PIC, and resume scanning. They re-initiate translation downstream at uORF2, uORF3, or uORF4; scanning does not resume after termination at these uORFs. Under conditions of amino acid starvation leading to GCN2 activation, eIF2α phosphorylation, and downregulation of eIF2•GTP, the rate of reassembly of the PIC after translation of uORF1 is slowed, thus delaying re-initiation of translation. The consequence is that start codons of uORFs 2-4 are bypassed by the ribosome, which instead re-initiates at the GCN4 start codon. Thus, eIF2α phosphorylation by GCN2 in amino acid-starved yeast cells translationally induces GCN4, which in turn transcriptionally activates amino acid biosynthesis.
In mammals as in yeast, GCN2 is required for adaptation to amino acid limitation, with the exception that non-essential amino acids (i.e. those that are synthesized by mammals) are also regulated in an eIF2 phosphorylation-independent manner in mammals. Although mice deficient in GCN2 and freely fed a normal diet developed and reproduced as well as wild type mice, prenatal and neonatal lethality were significantly increased in Gcn2–/– mice whose mothers were reared on leucine-, tryptophan-, or glycine-deficient diets during gestation (59). Cells from Gcn2–/– mice failed to induce phosphorylation of eIF2α and repression of eIF2B activity during histidine or leucine deprivation. Gcn2–/– mice maintained on a leucine-free diet for six days also lost more body mass than wild type mice, and 40% died or required euthanasia after 3-5 days (60). In the liver, in response to six days of leucine deprivation, wild type mice decreased protein synthesis concomitant with increased phoshphorylation of eIF2, whereas Gcn2–/– mice showed reduced eIF2 phosphorylation and levels of protein synthesis similar to those of mice fed complete diets. As a result, liver weights were preserved while skeletal muscle mass was sacrificially reduced in Gcn2–/– mice fed a leucine-free diet. GCN2 is also required for lipid homeostasis in the liver in response to amino acid deprivation: Gcn2–/– mice fed leucine-free diets for seven days developed steatosis and failed to repress lipogenic genes and the activity of fatty acid synthase (FAS) in the liver, as well as failed to mobilize lipid stores in adipose tissue (61). Finally, GCN2 participates in the sensing of nutritional imbalance that guides food selection for survival. Cells in the anterior piriform cortex of the brain are sensitive to essential amino acid deficiency and signal aversion to otherwise nutritious foods lacking deficient amino acids. GCN2 deficiency impaired this aversive response and phosphorylation of eIF2α in the anterior piriform cortex in response to an imbalanced diet (62-64).
GCN2 is though to act in the brain to inhibit long term memory formation through activation of ATF4. Although local protein synthesis at individual synapses is required for sustained increases in synaptic transmission, excessive translation is deleterious to memory formation. Thus, Gcn2–/– mice exhibited enhanced memory under a weak training protocol relative to wild type mice (65), as did eIF2αSer51Ala/+ heterozygous knock-in mice (66). The mechanism of enhanced memory formation is reduced translation of ATF4 as a result of decreased eIF2α phosphorylation. ATF4 inhibits CREB-mediated gene expression, which is critical for long term enhancement of synaptic strength (67). How GCN2 activity is regulated in response to synaptic stimulation is unknown.
In mammalian cells, upregulated translation of the bZIP transcription factor ATF4 is induced by GCN2 phosphorylation of eIF2α following a similar mechanism as used in control of yeast GCN4 translation upon amino acid starvation (68-71). Two uORFs in the 5’ leader of the ATF4 mRNA act to inhibit its translation during amino acid sufficiency, but promote translation during amino acid deficiency. Elevated levels of ATF4 lead to induction of transcription factors including ATF3 and CHOP, proteins that activate additional stress response genes, and GADD34, a regulatory subunit of an eIF2α phosphatase that provides feedback control of GCN2 activity [(70;72;73); reviewed in (74)].
GCN2 phosphorylation of eIF2 is reportedly required for activation of NF-κB in response to either amino acid starvation or UV irradiation of MEFs cultured in vitro (6;75). However, the activation of NF-κB in response to eIF2α phosphorylation was not dependent on IKK phosphorylation and proteasome-mediated degradation of IκB. Instead, GCN2 activation leads to decreased translation of IκB as part of the general inhibition of translation, contributing to the release of NF-κB from IκB and subsequent NF-κB translocation to the nucleus. GCN2 may downregulate caspase activity through NF-κB in order to protect against UV irradiation-induced apoptosis. GCN2 also mediates translation inhibition in response to UV irradiation (5). GCN2 is also activated and induces eIF2 phosphorylation in MEFs in response to treatment with proteasome inhibitors (76). NF-κB is not induced by proteasome inhibition. However, in this case abrogation of eIF2 phosphorylation blocked caspase activation and apoptosis.
GCN2 during viral infection
Viruses depend on the host translational machinery to synthesize their proteins. One mechanism mammalian cells use to counter viruses is to downregulate protein translation in infected cells through phosphorylation of eIF2α. The eIF2 kinase PKR is specialized to sense viral infection, being induced by interferon and activated mainly by dsRNA, which is synthesized in virus-infected cells as a byproduct of viral replication or transcription. Phosphorylation of eIF2α by PKR leads to inhibition of host and viral mRNA translation; PKR can also trigger apoptosis of virally infected cells [reviewed in (77)]. Consequently, viruses have developed a variety of strategies to gain access to the host translational machinery and avoid the deleterious effects of PKR activation. These include proteins that interfere with PKR activation, sequester dsRNA, inhibit PKR dimerization, act as PKR pseudosubstrates, activate antagonist phosphatases, and degrade PKR. Even in the face of a general inhibition of protein synthesis through downregulation of translation initiation, viruses can selectively promote translation of their own mRNAs through the use of IRESs and RNA structures that correctly position ribosomes without the function of eIF2.
Although PKR plays a major role in defense against viruses, other eIF2α kinases may have redundant functions in antagonizing viral infection. Knock-in mice or cells expressing a kinase-deficient PKR protein show normal antiviral responses to influenza, Sindbis, vaccinia, and encephalomyocarditis viruses, and normal levels of phosphorylated eIF2α under non-infected conditions (78-80). PERK and GCN2 have been implicated in antiviral responses to Sindbis virus, vesicular stomatitis virus (81;82) and herpes simplex virus 1 (HSV1) (83) through their phosphorylation of eIF2α. Upon HSV1 infection, PERK may be activated by accumulation of viral proteins in the ER (83), whereas GCN2 is directly activated in vitro by binding of the Sindbis virus genomic RNA (two noncontiguous regions) to the histidyl-tRNA synthetase-like domain (81). GCN2-deficient mice display increased susceptibility to Sindbis virus replication relative to wild type mice. However, one report suggests that phosphorylation of substrates other than eIF2α may be important for GCN2- and PERK-mediated antiviral responses (84). Vaccinia virus K3L protein acts as a pseudosubstrate of GCN2 to inhibit GCN2 phosphorylation of eIF2α, supporting the idea that GCN2 contributes to the eIF2-dependent antiviral response (85).
Adenovirus contains a double-stranded DNA genome and infects cells of epithelial and lymphocytic origin. During the late phase of infection following DNA replication (after 18 hours post-infection (?)), adenovirus uses several mechanisms to promote viral protein synthesis while inhibiting cellular protein synthesis. One of these, targeted towards prevention of eIF2α phosphorylation, is the expression of the virus-associated RNA molecule I (VAI RNA), a noncoding, 160-nucleotide RNA transcribed by RNA polymerase III and highly expressed during the late phase of infection (86;87). VAI RNA directly binds PKR and prevents its activation (88). Through an unknown mechanism involving the ubiquitin ligase activity of the associated complex, the adenovirus E1B 55-kilodalton (E1B-55K) and E4 open reading frame 6 (E4orf6) proteins also help to prevent activation of PKR during the late phase of infection (89).
Activation of GCN2 in response to adenovirus infection has not been reported; neither have mechanisms utilized by adenovirus to counteract GCN2 been reported. Direct binding of adenoviral dsRNA species, such as VAI RNA, may directly activate GCN2 in a manner analogous to Sindbis virus activation of GCN2. The mechanisms by which GCN2 contributes to the antiviral response to adenovirus, and possibly MCMV, are unknown.
It is proposed that viral infection may restrict amino acid availability and that the pool fo free amino acids within the cell is depleted during MCMV and adenovirus infection. GCN2 would subsequently downregulate further translation which would affect both cellular and viral proteins (2)
|Primers||Primers cannot be located by automatic search.|
Atchoum genotyping is performed by amplifying the region containing the mutation using PCR, followed by sequencing of the amplified region to detect the single nucleotide change.
atchoum (F): 5’-AATTGGCTGGGACGGTGTCAAG -3’
atchoum (R): 5’- GGAAGCACTTTAAATGCTCGCCAC -3’
1) 95°C 2:00
2) 95°C 0:30
3) 56°C 0:30
4) 72°C 1:00
5) repeat steps (2-4) 29X
6) 72°C 7:00
7) 4°C ∞
Primers for sequencing
atchoum_seq(F): 5’- GCTGTAACTTAGTGTACAGGGAC -3’
atchoum_seq(R): 5’- CATCGGTAGTGAGCACTCTACAG -3’
The following sequence of 581 nucleotides (from Genbank genomic region NC_000068.6 for linear genomic sequence of Eif2ak4, sense strand) is amplified:
aattggctg ggacggtgtc aagcagctgt aacttagtgt acagggacag gtggggagaa
11881 ggaaaaaccc tacgagatgt gttctttttc tcttttaggt gagagagcct cctgaaatca
11941 acttagttct ttaccctcag ggcctagctg gtgaagaagt atacgtgcaa gtggaactga
12001 gggttaaatg cccacctaca tacccagatg tgtgagtacc tgtataagaa gcttccgtgt
12061 catttcgctt ttctgttttg acagcatagg aatattacag ttgggagcat ttcctctttc
12121 cttcctttgt ctgccaatgt aaagtttcat ttcccatcac ctatatctca gtaacactat
12181 atccgcccta gactttttgc aaagcagcct ttaagtctgc atatagctgt agagtgctca
12241 ctaccgatgc taatattcac tcattgaccg gatgctagta tttactcatt gaccaccttt
12301 ttgcttttgc aactttcatg cttatttaat tctttgtaat ctagagagaa ggaaggtcta
12361 tacatatagg ataaagatgt ggcgagcatt taaagtgctt cc
Primer binding sites are underlined; sequencing primer binding sites are highlighted in gray; the mutated T is indicated in red.
1. Hsu, C., Boysen, M., Gritton, L. D., Frosst, P. D., Nemerow, G. R., and Von Seggern, D. J. (2005) In Vitro Dendritic Cell Infection by Pseudotyped Adenoviral Vectors does Not Correlate with their in Vivo Immunogenicity. Virology. 332, 1-7.
2. Won, S., Eidenschenk, C., Arnold, C. N., Siggs, O. M., Sun, L., Brandl, K., Mullen, T. M., Nemerow, G. R., Moresco, E. M., and Beutler, B. (2011) Increased Susceptibility to DNA Virus Infection in Mice with a GCN2 Mutation. J. Virol.. .
3. Crozat, K., Georgel, P., Rutschmann, S., Mann, N., Du, X., Hoebe, K., and Beutler, B. (2006) Analysis of the MCMV Resistome by ENU Mutagenesis. Mamm. Genome. 17, 398-406.
4. Mathys, S., Schroeder, T., Ellwart, J., Koszinowski, U. H., Messerle, M., and Just, U. (2003) Dendritic Cells Under Influence of Mouse Cytomegalovirus have a Physiologic Dual Role: To Initiate and to Restrict T Cell Activation. J. Infect. Dis.. 187, 988-999.
5. Deng, J., Harding, H. P., Raught, B., Gingras, A. C., Berlanga, J. J., Scheuner, D., Kaufman, R. J., Ron, D., and Sonenberg, N. (2002) Activation of GCN2 in UV-Irradiated Cells Inhibits Translation. Curr. Biol.. 12, 1279-1286.
6. Jiang, H. Y., and Wek, R. C. (2005) GCN2 Phosphorylation of eIF2alpha Activates NF-kappaB in Response to UV Irradiation. Biochem. J.. 385, 371-380.
7. Wek, R. C., Jiang, H. Y., and Anthony, T. G. (2006) Coping with Stress: EIF2 Kinases and Translational Control. Biochem. Soc. Trans.. 34, 7-11.
8. Sood, R., Porter, A. C., Olsen, D. A., Cavener, D. R., and Wek, R. C. (2000) A Mammalian Homologue of GCN2 Protein Kinase Important for Translational Control by Phosphorylation of Eukaryotic Initiation Factor-2alpha. Genetics. 154, 787-801.
9. Berlanga, J. J., Santoyo, J., and De Haro, C. (1999) Characterization of a Mammalian Homolog of the GCN2 Eukaryotic Initiation Factor 2alpha Kinase. Eur. J. Biochem.. 265, 754-762.
10. Dong, J., Qiu, H., Garcia-Barrio, M., Anderson, J., and Hinnebusch, A. G. (2000) Uncharged tRNA Activates GCN2 by Displacing the Protein Kinase Moiety from a Bipartite tRNA-Binding Domain. Mol. Cell. 6, 269-279.
11. Zhu, S., Sobolev, A. Y., and Wek, R. C. (1996) Histidyl-tRNA Synthetase-Related Sequences in GCN2 Protein Kinase Regulate in Vitro Phosphorylation of eIF-2. J. Biol. Chem.. 271, 24989-24994.
12. Wek, S. A., Zhu, S., and Wek, R. C. (1995) The Histidyl-tRNA Synthetase-Related Sequence in the eIF-2 Alpha Protein Kinase GCN2 Interacts with tRNA and is Required for Activation in Response to Starvation for Different Amino Acids. Mol. Cell. Biol.. 15, 4497-4506.
13. Wek, R. C., Jackson, B. M., and Hinnebusch, A. G. (1989) Juxtaposition of Domains Homologous to Protein Kinases and Histidyl-tRNA Synthetases in GCN2 Protein Suggests a Mechanism for Coupling GCN4 Expression to Amino Acid Availability. Proc. Natl. Acad. Sci. U. S. A.. 86, 4579-4583.
14. Zaborske, J. M., Narasimhan, J., Jiang, L., Wek, S. A., Dittmar, K. A., Freimoser, F., Pan, T., and Wek, R. C. (2009) Genome-Wide Analysis of tRNA Charging and Activation of the eIF2 Kinase Gcn2p. J. Biol. Chem.. 284, 25254-25267.
15. Qiu, H., Dong, J., Hu, C., Francklyn, C. S., and Hinnebusch, A. G. (2001) The tRNA-Binding Moiety in GCN2 Contains a Dimerization Domain that Interacts with the Kinase Domain and is Required for tRNA Binding and Kinase Activation. EMBO J.. 20, 1425-1438.
16. Zhu, S., and Wek, R. C. (1998) Ribosome-Binding Domain of Eukaryotic Initiation Factor-2 Kinase GCN2 Facilitates Translation Control. J. Biol. Chem.. 273, 1808-1814.
17. Qiu, H., Garcia-Barrio, M. T., and Hinnebusch, A. G. (1998) Dimerization by Translation Initiation Factor 2 Kinase GCN2 is Mediated by Interactions in the C-Terminal Ribosome-Binding Region and the Protein Kinase Domain. Mol. Cell. Biol.. 18, 2697-2711.
18. Narasimhan, J., Staschke, K. A., and Wek, R. C. (2004) Dimerization is Required for Activation of eIF2 Kinase Gcn2 in Response to Diverse Environmental Stress Conditions. J. Biol. Chem.. 279, 22820-22832.
19. Wek, R. C., Ramirez, M., Jackson, B. M., and Hinnebusch, A. G. (1990) Identification of Positive-Acting Domains in GCN2 Protein Kinase Required for Translational Activation of GCN4 Expression. Mol. Cell. Biol.. 10, 2820-2831.
20. Garcia-Barrio, M., Dong, J., Ufano, S., and Hinnebusch, A. G. (2000) Association of GCN1-GCN20 Regulatory Complex with the N-Terminus of eIF2alpha Kinase GCN2 is Required for GCN2 Activation. EMBO J.. 19, 1887-1899.
21. Vazquez de Aldana, C. R., Marton, M. J., and Hinnebusch, A. G. (1995) GCN20, a Novel ATP Binding Cassette Protein, and GCN1 Reside in a Complex that Mediates Activation of the eIF-2 Alpha Kinase GCN2 in Amino Acid-Starved Cells. EMBO J.. 14, 3184-3199.
22. Kubota, H., Ota, K., Sakaki, Y., and Ito, T. (2001) Budding Yeast GCN1 Binds the GI Domain to Activate the eIF2alpha Kinase GCN2. J. Biol. Chem.. 276, 17591-17596.
23. Kubota, H., Sakaki, Y., and Ito, T. (2000) GI Domain-Mediated Association of the Eukaryotic Initiation Factor 2alpha Kinase GCN2 with its Activator GCN1 is Required for General Amino Acid Control in Budding Yeast. J. Biol. Chem.. 275, 20243-20246.
24. Doerks, T., Copley, R. R., Schultz, J., Ponting, C. P., and Bork, P. (2002) Systematic Identification of Novel Protein Domain Families Associated with Nuclear Functions. Genome Res.. 12, 47-56.
25. Nameki, N., Yoneyama, M., Koshiba, S., Tochio, N., Inoue, M., Seki, E., Matsuda, T., Tomo, Y., Harada, T., Saito, K., Kobayashi, N., Yabuki, T., Aoki, M., Nunokawa, E., Matsuda, N., Sakagami, N., Terada, T., Shirouzu, M., Yoshida, M., Hirota, H., Osanai, T., Tanaka, A., Arakawa, T., Carninci, P., Kawai, J., Hayashizaki, Y., Kinoshita, K., Guntert, P., Kigawa, T., and Yokoyama, S. (2004) Solution Structure of the RWD Domain of the Mouse GCN2 Protein. Protein Sci.. 13, 2089-2100.
26. Alpi, A. F., Pace, P. E., Babu, M. M., and Patel, K. J. (2008) Mechanistic Insight into Site-Restricted Monoubiquitination of FANCD2 by Ube2t, FANCL, and FANCI. Mol. Cell. 32, 767-777.
27. Cole, A. R., Lewis, L. P., and Walden, H. (2010) The Structure of the Catalytic Subunit FANCL of the Fanconi Anemia Core Complex. Nat. Struct. Mol. Biol.. 17, 294-298.
28. Carbia-Nagashima, A., Gerez, J., Perez-Castro, C., Paez-Pereda, M., Silberstein, S., Stalla, G. K., Holsboer, F., and Arzt, E. (2007) RSUME, a Small RWD-Containing Protein, Enhances SUMO Conjugation and Stabilizes HIF-1alpha during Hypoxia. Cell. 131, 309-323.
29. Padyana, A. K., Qiu, H., Roll-Mecak, A., Hinnebusch, A. G., and Burley, S. K. (2005) Structural Basis for Autoinhibition and Mutational Activation of Eukaryotic Initiation Factor 2alpha Protein Kinase GCN2. J. Biol. Chem.. 280, 29289-29299.
30. Qiu, H., Hu, C., Dong, J., and Hinnebusch, A. G. (2002) Mutations that Bypass tRNA Binding Activate the Intrinsically Defective Kinase Domain in GCN2. Genes Dev.. 16, 1271-1280.
31. Garriz, A., Qiu, H., Dey, M., Seo, E. J., Dever, T. E., and Hinnebusch, A. G. (2009) A Network of Hydrophobic Residues Impeding Helix alphaC Rotation Maintains Latency of Kinase Gcn2, which Phosphorylates the Alpha Subunit of Translation Initiation Factor 2. Mol. Cell. Biol.. 29, 1592-1607.
32. Sonenberg, N., and Hinnebusch, A. G. (2009) Regulation of Translation Initiation in Eukaryotes: Mechanisms and Biological Targets. Cell. 136, 731-745.
33. Asano, K., Clayton, J., Shalev, A., and Hinnebusch, A. G. (2000) A Multifactor Complex of Eukaryotic Initiation Factors, eIF1, eIF2, eIF3, eIF5, and Initiator tRNA(Met) is an Important Translation Initiation Intermediate in Vivo. Genes Dev.. 14, 2534-2546.
34. Unbehaun, A., Borukhov, S. I., Hellen, C. U., and Pestova, T. V. (2004) Release of Initiation Factors from 48S Complexes during Ribosomal Subunit Joining and the Link between Establishment of Codon-Anticodon Base-Pairing and Hydrolysis of eIF2-Bound GTP. Genes Dev.. 18, 3078-3093.
35. Pestova, T. V., Lomakin, I. B., Lee, J. H., Choi, S. K., Dever, T. E., and Hellen, C. U. (2000) The Joining of Ribosomal Subunits in Eukaryotes Requires eIF5B. Nature. 403, 332-335.
36. Acker, M. G., Shin, B. S., Dever, T. E., and Lorsch, J. R. (2006) Interaction between Eukaryotic Initiation Factors 1A and 5B is Required for Efficient Ribosomal Subunit Joining. J. Biol. Chem.. 281, 8469-8475.
37. Fringer, J. M., Acker, M. G., Fekete, C. A., Lorsch, J. R., and Dever, T. E. (2007) Coupled Release of Eukaryotic Translation Initiation Factors 5B and 1A from 80S Ribosomes Following Subunit Joining. Mol. Cell. Biol.. 27, 2384-2397.
38. Shin, B. S., Maag, D., Roll-Mecak, A., Arefin, M. S., Burley, S. K., Lorsch, J. R., and Dever, T. E. (2002) Uncoupling of Initiation Factor eIF5B/IF2 GTPase and Translational Activities by Mutations that Lower Ribosome Affinity. Cell. 111, 1015-1025.
40. Das, S., Ghosh, R., and Maitra, U. (2001) Eukaryotic Translation Initiation Factor 5 Functions as a GTPase-Activating Protein. J. Biol. Chem.. 276, 6720-6726.
41. Paulin, F. E., Campbell, L. E., O'Brien, K., Loughlin, J., and Proud, C. G. (2001) Eukaryotic Translation Initiation Factor 5 (eIF5) Acts as a Classical GTPase-Activator Protein. Curr. Biol.. 11, 55-59.
42. Jennings, M. D., and Pavitt, G. D. (2010) EIF5 has GDI Activity Necessary for Translational Control by eIF2 Phosphorylation. Nature. 465, 378-381.
43. Singh, C. R., Lee, B., Udagawa, T., Mohammad-Qureshi, S. S., Yamamoto, Y., Pavitt, G. D., and Asano, K. (2006) An eIF5/eIF2 Complex Antagonizes Guanine Nucleotide Exchange by eIF2B during Translation Initiation. EMBO J.. 25, 4537-4546.
44. Gaspar, N. J., Kinzy, T. G., Scherer, B. J., Humbelin, M., Hershey, J. W., and Merrick, W. C. (1994) Translation Initiation Factor eIF-2. Cloning and Expression of the Human cDNA Encoding the Gamma-Subunit. J. Biol. Chem.. 269, 3415-3422.
45. Kurzchalia, T. V., Bommer, U. A., Babkina, G. T., and Karpova, G. G. (1984) GTP Interacts with the Gamma-Subunit of Eukaryotic Initiation Factor eIF-2. FEBS Lett.. 175, 313-316.
46. Alone, P. V., and Dever, T. E. (2006) Direct Binding of Translation Initiation Factor eIF2gamma-G Domain to its GTPase-Activating and GDP-GTP Exchange Factors eIF5 and eIF2B Epsilon. J. Biol. Chem.. 281, 12636-12644.
47. Asano, K., Krishnamoorthy, T., Phan, L., Pavitt, G. D., and Hinnebusch, A. G. (1999) Conserved Bipartite Motifs in Yeast eIF5 and eIF2Bepsilon, GTPase-Activating and GDP-GTP Exchange Factors in Translation Initiation, Mediate Binding to their Common Substrate eIF2. EMBO J.. 18, 1673-1688.
48. Kimball, S. R., Heinzinger, N. K., Horetsky, R. L., and Jefferson, L. S. (1998) Identification of Interprotein Interactions between the Subunits of Eukaryotic Initiation Factors eIF2 and eIF2B. J. Biol. Chem.. 273, 3039-3044.
49. Chaudhuri, J., Das, K., and Maitra, U. (1994) Purification and Characterization of Bacterially Expressed Mammalian Translation Initiation Factor 5 (eIF-5): Demonstration that eIF-5 Forms a Specific Complex with eIF-2. Biochemistry. 33, 4794-4799.
50. Flynn, A., Oldfield, S., and Proud, C. G. (1993) The Role of the Beta-Subunit of Initiation Factor eIF-2 in Initiation Complex Formation. Biochim. Biophys. Acta. 1174, 117-121.
51. Bommer, U. A., and Kurzchalia, T. V. (1989) GTP Interacts through its Ribose and Phosphate Moieties with Different Subunits of the Eukaryotic Initiation Factor eIF-2. FEBS Lett.. 244, 323-327.
52. Krishnamoorthy, T., Pavitt, G. D., Zhang, F., Dever, T. E., and Hinnebusch, A. G. (2001) Tight Binding of the Phosphorylated Alpha Subunit of Initiation Factor 2 (eIF2alpha) to the Regulatory Subunits of Guanine Nucleotide Exchange Factor eIF2B is Required for Inhibition of Translation Initiation. Mol. Cell. Biol.. 21, 5018-5030.
53. Rowlands, A. G., Panniers, R., and Henshaw, E. C. (1988) The Catalytic Mechanism of Guanine Nucleotide Exchange Factor Action and Competitive Inhibition by Phosphorylated Eukaryotic Initiation Factor 2. J. Biol. Chem.. 263, 5526-5533.
54. Dever, T. E., Feng, L., Wek, R. C., Cigan, A. M., Donahue, T. F., and Hinnebusch, A. G. (1992) Phosphorylation of Initiation Factor 2 Alpha by Protein Kinase GCN2 Mediates Gene-Specific Translational Control of GCN4 in Yeast. Cell. 68, 585-596.
55. Hinnebusch, A. G. (1985) A Hierarchy of Trans-Acting Factors Modulates Translation of an Activator of Amino Acid Biosynthetic Genes in Saccharomyces Cerevisiae. Mol. Cell. Biol.. 5, 2349-2360.
56. Hinnebusch, A. G. (1984) Evidence for Translational Regulation of the Activator of General Amino Acid Control in Yeast. Proc. Natl. Acad. Sci. U. S. A.. 81, 6442-6446.
57. Abastado, J. P., Miller, P. F., Jackson, B. M., and Hinnebusch, A. G. (1991) Suppression of Ribosomal Reinitiation at Upstream Open Reading Frames in Amino Acid-Starved Cells Forms the Basis for GCN4 Translational Control. Mol. Cell. Biol.. 11, 486-496.
58. Mueller, P. P., and Hinnebusch, A. G. (1986) Multiple Upstream AUG Codons Mediate Translational Control of GCN4. Cell. 45, 201-207.
59. Zhang, P., McGrath, B. C., Reinert, J., Olsen, D. S., Lei, L., Gill, S., Wek, S. A., Vattem, K. M., Wek, R. C., Kimball, S. R., Jefferson, L. S., and Cavener, D. R. (2002) The GCN2 eIF2alpha Kinase is Required for Adaptation to Amino Acid Deprivation in Mice. Mol. Cell. Biol.. 22, 6681-6688.
60. Anthony, T. G., McDaniel, B. J., Byerley, R. L., McGrath, B. C., Cavener, D. R., McNurlan, M. A., and Wek, R. C. (2004) Preservation of Liver Protein Synthesis during Dietary Leucine Deprivation Occurs at the Expense of Skeletal Muscle Mass in Mice Deleted for eIF2 Kinase GCN2. J. Biol. Chem.. 279, 36553-36561.
61. Guo, F., and Cavener, D. R. (2007) The GCN2 eIF2alpha Kinase Regulates Fatty-Acid Homeostasis in the Liver during Deprivation of an Essential Amino Acid. Cell. Metab.. 5, 103-114.
62. Maurin, A. C., Jousse, C., Averous, J., Parry, L., Bruhat, A., Cherasse, Y., Zeng, H., Zhang, Y., Harding, H. P., Ron, D., and Fafournoux, P. (2005) The GCN2 Kinase Biases Feeding Behavior to Maintain Amino Acid Homeostasis in Omnivores. Cell. Metab.. 1, 273-277.
63. Hao, S., Sharp, J. W., Ross-Inta, C. M., McDaniel, B. J., Anthony, T. G., Wek, R. C., Cavener, D. R., McGrath, B. C., Rudell, J. B., Koehnle, T. J., and Gietzen, D. W. (2005) Uncharged tRNA and Sensing of Amino Acid Deficiency in Mammalian Piriform Cortex. Science. 307, 1776-1778.
64. Gietzen, D. W., Ross, C. M., Hao, S., and Sharp, J. W. (2004) Phosphorylation of eIF2alpha is Involved in the Signaling of Indispensable Amino Acid Deficiency in the Anterior Piriform Cortex of the Brain in Rats. J. Nutr.. 134, 717-723.
65. Costa-Mattioli, M., Gobert, D., Harding, H., Herdy, B., Azzi, M., Bruno, M., Bidinosti, M., Ben Mamou, C., Marcinkiewicz, E., Yoshida, M., Imataka, H., Cuello, A. C., Seidah, N., Sossin, W., Lacaille, J. C., Ron, D., Nader, K., and Sonenberg, N. (2005) Translational Control of Hippocampal Synaptic Plasticity and Memory by the eIF2alpha Kinase GCN2. Nature. 436, 1166-1173.
66. Costa-Mattioli, M., Gobert, D., Stern, E., Gamache, K., Colina, R., Cuello, C., Sossin, W., Kaufman, R., Pelletier, J., Rosenblum, K., Krnjevic, K., Lacaille, J. C., Nader, K., and Sonenberg, N. (2007) EIF2alpha Phosphorylation Bidirectionally Regulates the Switch from Short- to Long-Term Synaptic Plasticity and Memory. Cell. 129, 195-206.
67. Barco, A., Alarcon, J. M., and Kandel, E. R. (2002) Expression of Constitutively Active CREB Protein Facilitates the Late Phase of Long-Term Potentiation by Enhancing Synaptic Capture. Cell. 108, 689-703.
68. Lu, P. D., Harding, H. P., and Ron, D. (2004) Translation Reinitiation at Alternative Open Reading Frames Regulates Gene Expression in an Integrated Stress Response. J. Cell Biol.. 167, 27-33.
69. Vattem, K. M., and Wek, R. C. (2004) Reinitiation Involving Upstream ORFs Regulates ATF4 mRNA Translation in Mammalian Cells. Proc. Natl. Acad. Sci. U. S. A.. 101, 11269-11274.
70. Jiang, H. Y., Wek, S. A., McGrath, B. C., Lu, D., Hai, T., Harding, H. P., Wang, X., Ron, D., Cavener, D. R., and Wek, R. C. (2004) Activating Transcription Factor 3 is Integral to the Eukaryotic Initiation Factor 2 Kinase Stress Response. Mol. Cell. Biol.. 24, 1365-1377.
71. Harding, H. P., Novoa, I., Zhang, Y., Zeng, H., Wek, R., Schapira, M., and Ron, D. (2000) Regulated Translation Initiation Controls Stress-Induced Gene Expression in Mammalian Cells. Mol. Cell. 6, 1099-1108.
72. Marciniak, S. J., Yun, C. Y., Oyadomari, S., Novoa, I., Zhang, Y., Jungreis, R., Nagata, K., Harding, H. P., and Ron, D. (2004) CHOP Induces Death by Promoting Protein Synthesis and Oxidation in the Stressed Endoplasmic Reticulum. Genes Dev.. 18, 3066-3077.
73. Novoa, I., Zeng, H., Harding, H. P., and Ron, D. (2001) Feedback Inhibition of the Unfolded Protein Response by GADD34-Mediated Dephosphorylation of eIF2alpha. J. Cell Biol.. 153, 1011-1022.
74. Kilberg, M. S., Shan, J., and Su, N. (2009) ATF4-Dependent Transcription Mediates Signaling of Amino Acid Limitation. Trends Endocrinol. Metab.. 20, 436-443.
75. Jiang, H. Y., Wek, S. A., McGrath, B. C., Scheuner, D., Kaufman, R. J., Cavener, D. R., and Wek, R. C. (2003) Phosphorylation of the Alpha Subunit of Eukaryotic Initiation Factor 2 is Required for Activation of NF-kappaB in Response to Diverse Cellular Stresses. Mol. Cell. Biol.. 23, 5651-5663.
76. Jiang, H. Y., and Wek, R. C. (2005) Phosphorylation of the Alpha-Subunit of the Eukaryotic Initiation Factor-2 (eIF2alpha) Reduces Protein Synthesis and Enhances Apoptosis in Response to Proteasome Inhibition. J. Biol. Chem.. 280, 14189-14202.
77. Garcia, M. A., Meurs, E. F., and Esteban, M. (2007) The dsRNA Protein Kinase PKR: Virus and Cell Control. Biochimie. 89, 799-811.
78. Gorchakov, R., Frolova, E., Williams, B. R., Rice, C. M., and Frolov, I. (2004) PKR-Dependent and -Independent Mechanisms are Involved in Translational Shutoff during Sindbis Virus Infection. J. Virol.. 78, 8455-8467.
79. Abraham, N., Stojdl, D. F., Duncan, P. I., Methot, N., Ishii, T., Dube, M., Vanderhyden, B. C., Atkins, H. L., Gray, D. A., McBurney, M. W., Koromilas, A. E., Brown, E. G., Sonenberg, N., and Bell, J. C. (1999) Characterization of Transgenic Mice with Targeted Disruption of the Catalytic Domain of the Double-Stranded RNA-Dependent Protein Kinase, PKR. J. Biol. Chem.. 274, 5953-5962.
80. Yang, Y. L., Reis, L. F., Pavlovic, J., Aguzzi, A., Schafer, R., Kumar, A., Williams, B. R., Aguet, M., and Weissmann, C. (1995) Deficient Signaling in Mice Devoid of Double-Stranded RNA-Dependent Protein Kinase. EMBO J.. 14, 6095-6106.
81. Berlanga, J. J., Ventoso, I., Harding, H. P., Deng, J., Ron, D., Sonenberg, N., Carrasco, L., and de Haro, C. (2006) Antiviral Effect of the Mammalian Translation Initiation Factor 2alpha Kinase GCN2 Against RNA Viruses. EMBO J.. 25, 1730-1740.
82. Baltzis, D., Qu, L. K., Papadopoulou, S., Blais, J. D., Bell, J. C., Sonenberg, N., and Koromilas, A. E. (2004) Resistance to Vesicular Stomatitis Virus Infection Requires a Functional Cross Talk between the Eukaryotic Translation Initiation Factor 2alpha Kinases PERK and PKR. J. Virol.. 78, 12747-12761.
83. Cheng, G., Feng, Z., and He, B. (2005) Herpes Simplex Virus 1 Infection Activates the Endoplasmic Reticulum Resident Kinase PERK and Mediates eIF-2alpha Dephosphorylation by the Gamma(1)34.5 Protein. J. Virol.. 79, 1379-1388.
84. Krishnamoorthy, J., Mounir, Z., Raven, J. F., and Koromilas, A. E. (2008) The eIF2alpha Kinases Inhibit Vesicular Stomatitis Virus Replication Independently of eIF2alpha Phosphorylation. Cell. Cycle. 7, 2346-2351.
85. Qian, W., Zhu, S., Sobolev, A. Y., and Wek, R. C. (1996) Expression of Vaccinia Virus K3L Protein in Yeast Inhibits Eukaryotic Initiation Factor-2 Kinase GCN2 and the General Amino Acid Control Pathway. J. Biol. Chem.. 271, 13202-13207.
86. Soderlund, H., Pettersson, U., Vennstrom, B., Philipson, L., and Mathews, M. B. (1976) A New Species of Virus-Coded Low Molecular Weight RNA from Cells Infected with Adenovirus Type 2. Cell. 7, 585-593.
87. Weinmann, R., Raskas, H. J., and Roeder, R. G. (1974) Role of DNA-Dependent RNA Polymerases II and III in Transcription of the Adenovirus Genome Late in Productive Infection. Proc. Natl. Acad. Sci. U. S. A.. 71, 3426-3439.
88. Mathews, M. B., and Shenk, T. (1991) Adenovirus Virus-Associated RNA and Translation Control. J. Virol.. 65, 5657-5662.
|Science Writers||Eva Marie Y. Moresco|
|Illustrators||Diantha La Vine|
|Authors||Celine Eidenschenk, Sungyong Won, Bruce Beutler|