EIF2AK4 (GCN2) senses amino acid deficiency by binding uncharged tRNAs near the ribosome and responds by phosphorylating EIF2S1, the alpha subunit of the translation initiation factor EIF2 (inferred from yeast homologs and mouse homologs, reviewed in Chaveroux et al. 2010, Castilho et al. 2014, Gallinetti et al. 2013, Bröer and Bröer 2017, Wek 2018). Phosphorylated EIF2S1 reduces translation of most mRNAs but increases translation of downstream ORFs in mRNAs such as ATF4 that contain upstream ORFs (inferred from mouse homologs in Vattem and Wek 2004, reviewed in Hinnebusch et al. 2016, Sonenberg and Hinnebusch 2009). ATF4, in turn, activates expression of genes involved in responding to amino acid deficiency such as DDIT3 (CHOP), ASNS (asparagine synthetase), CEBPB, and ATF3 (reviewed in Kilberg et al. 2012, Wortel et al. 2017). In mice, EIF2AK4 in the brain may responsible for avoidance of diets lacking essential amino acids (Hao et al. 2005, Maurin et al. 2005, see also Leib and Knight 2015, Gietzen et al. 2016, reviewed in Dever and Hinnebusch 2005). EIF2AK4 is bound to both the ribosome and GCN1, which is required for activation of EIF2AK4 and may act by shuttling uncharged tRNAs from the A site of the ribosome to EIF2AK4. Upon binding tRNA, EIF2AK4 trans-autophosphorylates. Phosphorylated EIF2AK4 then phosphorylates EIF2S1 on serine-52, the same serine residue phosphorylated by other kinases of the integrated stress response: EIF2AK1 (HRI, activated by heme deficiency and other stresses), EIF2AK2 (PKR, activated by double-stranded RNA), and EIF2AK3 (PERK, activated by unfolded proteins) (reviewed in Hinnebusch 1994, Wek et al. 2006, Donnelly et al. 2013, Pakos-Zebrucka et al. 2016, Wek 2018),
View original pathway at Reactome.
Guo L, Guo ZX, Gong HP, Shang YY, Zhong M, Zhang Y, Zhang W.; ''Tribbles homolog 3 is induced by high glucose and associated with apoptosis in human endothelial cells.''; PubMedEurope PMCScholia
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Blais JD, Filipenko V, Bi M, Harding HP, Ron D, Koumenis C, Wouters BG, Bell JC.; ''Activating transcription factor 4 is translationally regulated by hypoxic stress.''; PubMedEurope PMCScholia
Gallinetti J, Harputlugil E, Mitchell JR.; ''Amino acid sensing in dietary-restriction-mediated longevity: roles of signal-transducing kinases GCN2 and TOR.''; PubMedEurope PMCScholia
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Hayner JN, Shan J, Kilberg MS.; ''Regulation of the ATF3 gene by a single promoter in response to amino acid availability and endoplasmic reticulum stress in human primary hepatocytes and hepatoma cells.''; PubMedEurope PMCScholia
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Chen H, Pan YX, Dudenhausen EE, Kilberg MS.; ''Amino acid deprivation induces the transcription rate of the human asparagine synthetase gene through a timed program of expression and promoter binding of nutrient-responsive basic region/leucine zipper transcription factors as well as localized histone acetylation.''; PubMedEurope PMCScholia
Bartlett JD, Luethy JD, Carlson SG, Sollott SJ, Holbrook NJ.; ''Calcium ionophore A23187 induces expression of the growth arrest and DNA damage inducible CCAAT/enhancer-binding protein (C/EBP)-related gene, gadd153. Ca2+ increases transcriptional activity and mRNA stability.''; PubMedEurope PMCScholia
Huggins CJ, Mayekar MK, Martin N, Saylor KL, Gonit M, Jailwala P, Kasoji M, Haines DC, Quiñones OA, Johnson PF.; ''C/EBPγ Is a Critical Regulator of Cellular Stress Response Networks through Heterodimerization with ATF4.''; PubMedEurope PMCScholia
Armstrong JL, Flockhart R, Veal GJ, Lovat PE, Redfern CP.; ''Regulation of endoplasmic reticulum stress-induced cell death by ATF4 in neuroectodermal tumor cells.''; PubMedEurope PMCScholia
Pakos-Zebrucka K, Koryga I, Mnich K, Ljujic M, Samali A, Gorman AM.; ''The integrated stress response.''; PubMedEurope PMCScholia
Carlson SG, Fawcett TW, Bartlett JD, Bernier M, Holbrook NJ.; ''Regulation of the C/EBP-related gene gadd153 by glucose deprivation.''; PubMedEurope PMCScholia
Leib DE, Knight ZA.; ''Re-examination of Dietary Amino Acid Sensing Reveals a GCN2-Independent Mechanism.''; PubMedEurope PMCScholia
Hua F, Mu R, Liu J, Xue J, Wang Z, Lin H, Yang H, Chen X, Hu Z.; ''TRB3 interacts with SMAD3 promoting tumor cell migration and invasion.''; PubMedEurope PMCScholia
Hinnebusch AG.; ''The eIF-2 alpha kinases: regulators of protein synthesis in starvation and stress.''; PubMedEurope PMCScholia
Xu J, Lv S, Qin Y, Shu F, Xu Y, Chen J, Xu BE, Sun X, Wu J.; ''TRB3 interacts with CtIP and is overexpressed in certain cancers.''; PubMedEurope PMCScholia
Hao S, Sharp JW, Ross-Inta CM, McDaniel BJ, Anthony TG, Wek RC, Cavener DR, McGrath BC, Rudell JB, Koehnle TJ, Gietzen DW.; ''Uncharged tRNA and sensing of amino acid deficiency in mammalian piriform cortex.''; PubMedEurope PMCScholia
Bruhat A, Jousse C, Wang XZ, Ron D, Ferrara M, Fafournoux P.; ''Amino acid limitation induces expression of CHOP, a CCAAT/enhancer binding protein-related gene, at both transcriptional and post-transcriptional levels.''; PubMedEurope PMCScholia
Kilberg MS, Balasubramanian M, Fu L, Shan J.; ''The transcription factor network associated with the amino acid response in mammalian cells.''; PubMedEurope PMCScholia
Sonenberg N, Hinnebusch AG.; ''Regulation of translation initiation in eukaryotes: mechanisms and biological targets.''; PubMedEurope PMCScholia
Bruhat A, Chérasse Y, Maurin AC, Breitwieser W, Parry L, Deval C, Jones N, Jousse C, Fafournoux P.; ''ATF2 is required for amino acid-regulated transcription by orchestrating specific histone acetylation.''; PubMedEurope PMCScholia
Örd D, Örd T, Biene T, Örd T.; ''TRIB3 increases cell resistance to arsenite toxicity by limiting the expression of the glutathione-degrading enzyme CHAC1.''; PubMedEurope PMCScholia
Fu L, Kilberg MS.; ''Elevated cJUN expression and an ATF/CRE site within the ATF3 promoter contribute to activation of ATF3 transcription by the amino acid response.''; PubMedEurope PMCScholia
Siu F, Chen C, Zhong C, Kilberg MS.; ''CCAAT/enhancer-binding protein-beta is a mediator of the nutrient-sensing response pathway that activates the human asparagine synthetase gene.''; PubMedEurope PMCScholia
Vattem KM, Wek RC.; ''Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells.''; PubMedEurope PMCScholia
Jousse C, Bruhat A, Carraro V, Urano F, Ferrara M, Ron D, Fafournoux P.; ''Inhibition of CHOP translation by a peptide encoded by an open reading frame localized in the chop 5'UTR.''; PubMedEurope PMCScholia
Hinnebusch AG, Ivanov IP, Ivanov IP, Sonenberg N.; ''Translational control by 5'-untranslated regions of eukaryotic mRNAs.''; PubMedEurope PMCScholia
Dever TE, Hinnebusch AG.; ''GCN2 whets the appetite for amino acids.''; PubMedEurope PMCScholia
Averous J, Bruhat A, Jousse C, Carraro V, Thiel G, Fafournoux P.; ''Induction of CHOP expression by amino acid limitation requires both ATF4 expression and ATF2 phosphorylation.''; PubMedEurope PMCScholia
Bröer S, Bröer A.; ''Amino acid homeostasis and signalling in mammalian cells and organisms.''; PubMedEurope PMCScholia
Bruhat A, Jousse C, Carraro V, Reimold AM, Ferrara M, Fafournoux P.; ''Amino acids control mammalian gene transcription: activating transcription factor 2 is essential for the amino acid responsiveness of the CHOP promoter.''; PubMedEurope PMCScholia
Chaveroux C, Lambert-Langlais S, Cherasse Y, Averous J, Parry L, Carraro V, Jousse C, Maurin AC, Bruhat A, Fafournoux P.; ''Molecular mechanisms involved in the adaptation to amino acid limitation in mammals.''; PubMedEurope PMCScholia
Balasubramanian MN, Shan J, Kilberg MS.; ''Dynamic changes in genomic histone association and modification during activation of the ASNS and ATF3 genes by amino acid limitation.''; PubMedEurope PMCScholia
Lee JI, Dominy JE, Sikalidis AK, Hirschberger LL, Wang W, Stipanuk MH.; ''HepG2/C3A cells respond to cysteine deprivation by induction of the amino acid deprivation/integrated stress response pathway.''; PubMedEurope PMCScholia
Gietzen DW, Anthony TG, Fafournoux P, Maurin AC, Koehnle TJ, Hao S.; ''Measuring the Ability of Mice to Sense Dietary Essential Amino Acid Deficiency: The Importance of Amino Acid Status and Timing.''; PubMedEurope PMCScholia
Wortel IMN, van der Meer LT, Kilberg MS, van Leeuwen FN.; ''Surviving Stress: Modulation of ATF4-Mediated Stress Responses in Normal and Malignant Cells.''; PubMedEurope PMCScholia
Lee SH, Min KW, Zhang X, Baek SJ.; ''3,3'-diindolylmethane induces activating transcription factor 3 (ATF3) via ATF4 in human colorectal cancer cells.''; PubMedEurope PMCScholia
Ohoka N, Yoshii S, Hattori T, Onozaki K, Hayashi H.; ''TRB3, a novel ER stress-inducible gene, is induced via ATF4-CHOP pathway and is involved in cell death.''; PubMedEurope PMCScholia
Maurin AC, Jousse C, Averous J, Parry L, Bruhat A, Cherasse Y, Zeng H, Zhang Y, Harding HP, Ron D, Fafournoux P.; ''The GCN2 kinase biases feeding behavior to maintain amino acid homeostasis in omnivores.''; PubMedEurope PMCScholia
Chérasse Y, Maurin AC, Chaveroux C, Jousse C, Carraro V, Parry L, Deval C, Chambon C, Fafournoux P, Bruhat A.; ''The p300/CBP-associated factor (PCAF) is a cofactor of ATF4 for amino acid-regulated transcription of CHOP.''; PubMedEurope PMCScholia
Pan YX, Chen H, Thiaville MM, Kilberg MS.; ''Activation of the ATF3 gene through a co-ordinated amino acid-sensing response programme that controls transcriptional regulation of responsive genes following amino acid limitation.''; PubMedEurope PMCScholia
The Asparagine Synthetase (ASNS) gene is transcribed to yield mRNA and the mRNA is translated to yield protein (Chen et al. 2004, Lee et al. 2008, Gjymishka et al. 2009, Sikalidis et al. 2011, Balasubramanian et al. 2013, inferred from the mouse homolog). Transcription of ASNS is activated by the unfolded protein response (Gjymishka et al. 2009), amino acid deficiency (Chen et al. 2004, Lee et al. 2008, Sikalidis et al. 2011, Balasubramanian et al. 2013, inferred from the mouse homolog), and heme deficiency (inferred from the mouse homolog).
The ATF3 gene is transcribed to yield mRNA and the mRNA is translated to yield protein (Chen et al. 2004, Pan et al. 2007, Lee et al. 2008, Armstrong et al. 2010, Sikalidis et al. 2011, Fu and Kilberg 2013, Lee et al. 2013, Hayner et al. 2018). Transcription of ATF3 is enhanced in response to amino acid deficiency (Chen et al. 2004, Pan et al. 2007, Lee et al. 2008, Sikaldis et al. 2011, Fu and Kilberg 2013, Hayner et al. 2018). ATF4 binds a CEBP-ATF response element (CARE) and an additional upstream element in the promoter of the ATF3 gene, resulting in enhanced transcription (Pan et al. 2007, Armstrong et al. 2010, Fu and Kilberg 2013, Lee et al. 2013, Hayner et al. 2018, and inferred from mouse homologs). CEBPB and ATF3 bind later and correlate with reduced expression of ATF4 (Pan et al. 2007)
ATF4 mRNA is translated to yield ATF4 protein, which then transits to the nucleus (Blais et al. 2004, Ross et al. 2018). The mRNA of ATF4 contains 2 upstream ORFs (uORFs) (Ross et al. 2018 and inferred from the mouse homolog). The second uORF overlaps the ORF encoding ATF4 and thus prevents translation of ATF4. When EIF2S1 (eIF2-alpha) is phosphorylated, translation initiation is decreased overall, translation of the uORFs is suppressed, and translation of the ORF encoding ATF4 is increased (Blais et al. 2004, Ross et al. 2018, and inferred from mouse homologs).
The histidyl-tRNA synthetase-like domain of EIF2AK4 (GCN2) binds uncharged tRNA, resulting in activation of the protein kinase domain of EIF2AK4 (Inglis et al. 2019 and inferred from yeast homologs and mouse homologs). In the absence of tRNA, EIF2AK4 appears to exist in an equilibrium between antiparallel and parallel dimers. Upon binding tRNA, the parallel dimer is stabilized and the C-terminal domain shifts away from the protein kinase domain, resulting in activation of the kinase activity of EIF2AK4 (inferred from GCN2, the yeast homolog). EIF2AK4 interacts with GCN1 and the P-stalk of ribosomes (Inglis et al. 2019), though the interaction between mammalian EIF2AK4 and ribosomes is not as stable as the interaction between yeast GCN2 and ribosomes (inferred from yeast homologs and mouse homologs). By such transient interactions, a population of EIF2AK4 may sample a larger population of ribosomes for uncharged tRNAs. The interaction between EIF2AK4 and GCN1 is required for efficient phosphorylation of EIF2S1 by EIF2AK4 and GCN1 may act to transfer uncharged tRNAs from the A site of the ribosome to EIF2AK4 (inferred from yeast homologs and mouse homologs).
After binding uncharged tRNA and autophosphorylating, EIF2AK4 (GCN2) phosphorylates EIF2S1 (eIF2 alpha subunit) on serine-52 (serine-51 in the rabbit homolog, inferred from mouse homologs and yeast homologs), which inhibits the guanine nucleotide exchange factor eIF2B, impairs exchange of GDP for GTP, and reduces recycling of EIF2 for initiation of translation. This causes downregulation of translation of most mRNAs, however translation of certain mRNAs possessing upstream ORFs, such as ATF4, is upregulated (inferred from mouse homologs and yeast homologs).
After binding uncharged tRNA, the EIF2AK4 (GCN2) dimer trans-autophosphorylates on threonine-899, resulting in activation of the kinase domain of EIF2AK4 (Harding et al. 2000, Deng et al. 2002, Cambiaghi et al. 2014, and inferred from mouse homologs and yeast homologs).
IMPACT, a mammalian homolog of yeast YIH1, competes with EIF2AK4 (GCN2) for binding to GCN1, which is required for activation of EIF2AK4 and may act by transferring unacylated tRNAs from the ribosome to EIF2AK4 (inferred from mouse homologs). IMPACT thereby inhibits phosphorylation of EIF2A by EIF2AK4 in response to amino acid deficiency (inferred from mouse homologs). IMPACT is preferentially expressed in neurons, associates with translating ribosomes, enhances translation initiation, and promotes neuritogenesis (inferred from mouse homologs).
ATF4 binds an amino acid response element (AARE) in the promoter of the ATF3 gene (Chen et al. 2004, Pan et al. 2007, Fu and Kilberg 2013, Hayner et al. 2018). ATF4 initially binds the ATF3 promoter with phosphorylated ATF2, then with JUN (c-Jun), then with CEBPB (Fu and Kilberg 2013, Hayner et al. 2018). ATF3 and CEBPB bind later and correlate with reduced expression of ATF3 (Pan et al. 2007, Fu and Kilberg 2013, Hayner et al. 2018).
The TRIB3 (TRB3, NIPK) gene is transcribed to yield mRNA and the mRNA is translated to yield TRIB3 protein (Ohoka et al. 2005, Ord and Ord 2005, Lee et al. 2008, Sikalidis et al. 2011, Ord et al. 2016, and inferred from the mouse homolog). Transcription of TRIB3 is enhanced in response to amino acid deficiency (Lee et al. 2008, Sikalidis et al. 2011, and inferred from mouse homologs), endoplasmic reticulum stress (Ohoka et al. 2005, Ord and Ord 2005), oxidative stress (Ord and Ord 2005, Ord et al. 2016) and heme deficiency (inferred from mouse homologs). ATF4 bound with a CEBP family protein to the promoter of TRIB3 (NIPK, TRB3) enhances transcription of TRIB3 (Ohoka et al. 2005, Ord and Ord 2005, Lee et al. 2008, Sikalidis et al. 2011, Ord et al. 2016, and inferred from mouse homologs).
ATF4 and CEBPB or CEBPG bind a CEBP-ATF regulatory element (CARE) in the promoter of the ASNS gene (Siu et al 2001, Chen et al. 2004, inferred from mouse homologs). ATF4 binds rapidly during the first 2 hours after amino acid deprivation (Chen et al. 2004). ATF3 and CEBPB accumulate on the ASNS promoter more slowly and appear to correlate with decreasing transcription of ASNS (Chen et al. 2004). EIF2AK1 acts via ATF4 to activate transcription of ASNS in response to heme deficiency (inferred from mouse homologs).
The CEBPB gene is transcribed to yield mRNA and the mRNA is translated to yield protein (Chen et al. 2005, Lee et al. 2008, Sikalidis et al. 2011). Transcription of CEBPB is activated in response to amino acid deficiency (Chen et al. 2005, Lee et al. 2008, Sikalidis et al. 2011). ATF4 bound to an enhancer downstream of the CEBPB coding region (Chen et al. 2005) increases transcription of CEBPB approximately 4-fold (Chen et al. 2005, Lee et al. 2008, Sikalidis et al. 2011).
ATF4 binds composite CEBP-ATF elements located in three 33-bp tandem repeats in the promoter of the TRIB3 (TRB3, NIPK) gene (Ohoka et al. 2005, Ord and Ord 2005). ATF4 cooperates with DDIT3 to activate TRIB3 promoter activity (Ohoka et al. 2005). ATF4 also appears to bind as a heterodimer with CEBPB or CEBPG, which is required for full response to amino acid deficiency (inferred from mouse homologs).
ATF4 binds an enhancer downstream of the protein coding region of the CEBPB gene (Chen et al. 2005). The binding site resembles a composite CEBP-ATF element. Therefore ATF4 may form a heterodimer with a CEBP protein at the element (Chen et al. 2005).
The DDIT3 (CHOP) gene is transcribed to yield mRNA and the mRNA is translated to yield protein (Bartlett et al. 1992, Carlson et al. 1993, Bruhat et al. 1997, Yoshida et al. 2000, Lee et al. 2008, Sikalidis et al. 2011). In response to amino acid starvation, transcription of DDIT3 s enhanced by ATF4 and phosphorylated ATF2 (Bruhat et al. 2000, Averous et al. 2004, Bruhat et al. 2007)
The DDIT3 mRNA is translated to yield DDIT3 (CHOP) protein (Jousse et al. 2001, and inferred from the mouse homolog), which is then imported into the nucleus. The mRNA of DDIT3 contains an upstream ORF (uORF) which has a start codon in an unfavorable context (Jousse et al. 2001, and inferred from the mouse homolog), resulting in low expression of the downstream DDIT3 coding region. When EIF2S1 (eIF2-alpha) is phosphorylated in response to stress, translation of the uORF is suppressed and translation of DDIT3 is increased (inferred from the mouse homolog).
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EIF2AK4 interacts with GCN1 and the P-stalk of ribosomes (Inglis et al. 2019), though the interaction between mammalian EIF2AK4 and ribosomes is not as stable as the interaction between yeast GCN2 and ribosomes (inferred from yeast homologs and mouse homologs). By such transient interactions, a population of EIF2AK4 may sample a larger population of ribosomes for uncharged tRNAs. The interaction between EIF2AK4 and GCN1 is required for efficient phosphorylation of EIF2S1 by EIF2AK4 and GCN1 may act to transfer uncharged tRNAs from the A site of the ribosome to EIF2AK4 (inferred from yeast homologs and mouse homologs).