Skip to main content
SpringerLink
Log in

GCN2- and eIF2α-phosphorylation-independent, but ATF4-dependent, induction of CARE-containing genes in methionine-deficient cells

  • Original Article
  • Published:
Amino Acids Aims and scope Submit manuscript

Abstract

Amino-acid deprivation is sensed by the eIF2α kinase GCN2. Under conditions of essential amino-acid limitation, GCN2 phosphorylates eIF2α, inhibiting the formation of a new ternary complex and hence mRNA translation initiation. While decreasing global mRNA translation, eIF2α phosphorylation also increases the translation of the integrated stress response (ISR) transcription factor ATF4, which increases the expression of many stress response genes that contain a C/EBP-ATF response element (CARE), including Atf4, 4Ebp1, Asns, and Chop. Using wild-type as well as Gcn2 knockout and unphosphorylatable eIF2α mutant MEFs, we characterized a novel GCN2/eIF2α phosphorylation-independent, but ATF4-dependent, pathway that upregulates the expression of CARE-containing genes in MEFs lacking GCN2 or phosphorylatable eIF2α when these cells are exposed to methionine-deficient, and to a lesser extent arginine- or histidine-deficient, medium. Thus, we demonstrate a GCN2/eIF2α phosphorylation-independent pathway that converges with the GCN2/eIF2α kinase-dependent pathway at the level of ATF4 and similarly results in the upregulation of CARE-containing genes. We hypothesize that the essential role of methionine-charged initiator tRNA in forming ternary complex is responsible for the robust ability of methionine deficiency to induce ATF4 and the ISR even in the absence of GCN2 or eIF2α kinase activity.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Anthony TG, Reiter AK, Anthony JC et al (2001) Deficiency of dietary EAA preferentially inhibits mRNA translation of ribosomal proteins in liver of meal-fed rats. Am J Physiol Endocrinol Metab 281:E430–E439

    CAS  PubMed  Google Scholar 

  • B’Chir W, Maurin AC, Carraro V et al (2013) The eIF2α/ATF4 pathway is essential for stress-induced autophagy gene expression. Nucleic Acids Res 41:7683–7699

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • B’chir W, Chaveroux C, Carraro V et al (2014) Dual role for CHOP in the crosstalk between autophagy and apoptosis to determine cell fate in response to amino acid deprivation. Cell Signal 26:1385–1391

    Article  CAS  PubMed  Google Scholar 

  • Baird T, Wek R (2012) Eukaryotic initiation factor 2 phosphorylation and translational control in metabolism. Adv Nutr 3:307–321

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dang Do AN, Kimball SR, Cavener DR, Jefferson LS (2009) eIF2alpha kinases GCN2 and PERK modulate transcription and translation of distinct sets of mRNAs in mouse liver. Physiol Genomics 38:328–341

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Deval C, Chaveroux C, Maurin AC et al (2009) Amino acid limitation regulates the expression of genes involved in several specific biological processes through GCN2-dependent and GCN2-independent pathways. FEBS J 276:707–718

    Article  CAS  PubMed  Google Scholar 

  • Dever TE, Hinnebusch AG (2005) GCN2 whets the appetite for amino acids. Mol Cell 18:141–142

    Article  CAS  PubMed  Google Scholar 

  • Dever TE, Yang W, Aström S et al (1995) Modulation of tRNA(iMet), eIF-2, and eIF-2B expression shows that GCN4 translation is inversely coupled to the level of eIF-2.GTP.Met-tRNA(iMet) ternary complexes. Mol Cell Biol 15:6351–6363

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dey S, Baird TD, Zhou D et al (2010) Both transcriptional regulation and translational control of ATF4 are central to the integrated stress response. J Biol Chem 285:33165–33174

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dong J, Qiu H, Garcia-Barrio M et al (2000) Uncharged tRNA activates GCN2 by displacing the protein kinase moiety from a bipartite tRNA-binding domain. Mol Cell 6:269–279

    Article  CAS  PubMed  Google Scholar 

  • Donnelly N, Gorman AM, Gupta S, Samali A (2013) The eIF2alpha kinases: their structures and functions. Cell Mol Life Sci 70:3493–3511

    Article  CAS  PubMed  Google Scholar 

  • Drabkin HJ, Estrella M, Rajbhandary UL (1998) Initiator-elongator discrimination in vertebrate tRNAs for protein synthesis. Mol Cell Biol 18:1459–1466

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Francis M, Rajbhandary UL (1990) Expression and function of a human initiator tRNA gene in the yeast Saccharomyces cerevisiae. Mol Cell Biol 10:4486–4494

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Grolleau A, Sonenberg N, Wietzerbin J, Beretta L (1999) Differential regulation of 4E-BP1 and 4E-BP2, two repressors of translation initiation, during human myeloid cell differentiation. J Immunol 162:3491–3497

    CAS  PubMed  Google Scholar 

  • Harding HP, Novoa I, Zhang Y et al (2000) Regulated translation initiation controls stress-induced gene expression in mammalian cells. Mol Cell 6:1099–1108

    Article  CAS  PubMed  Google Scholar 

  • Hinnebusch AG (2014) The scanning mechanism of eukaryotic translation initiation. Annu Rev Biochem 83:779–812

    Article  CAS  PubMed  Google Scholar 

  • Jousse C, Averous J, Bruhat A et al (2004) Amino acids as regulators of gene expression: molecular mechanisms. Biochem Biophys Res Commun 313:447–452

    Article  CAS  PubMed  Google Scholar 

  • Kapp LD, Lorsch JR (2004) GTP-dependent recognition of the methionine moiety on initiator tRNA by translation factor eIF2. J Mol Biol 335:923–936

    Article  CAS  PubMed  Google Scholar 

  • Kilberg MS, Shan J, Su N (2009) ATF4-dependent transcription mediates signaling of amino acid limitation. Trends Endocrinol Metab 20:436–443

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kolitz SE, Lorsch JR (2010) Eukaryotic initiator tRNA: finely tuned and ready for action. FEBS Lett 584:396–404

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Krishnamoorthy T, Pavitt G (2001) Tight binding of the phosphorylated α subunit of initiation factor 2 (eIF2α) to the regulatory subunits of guanine nucleotide exchange factor eIF2B is required for inhibition of translation. Mol Cell Biol 21:5018–5030

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Narasimhan J, Staschke KA, Wek RC (2004) Dimerization is required for activation of eIF2 kinase Gcn2 in response to diverse environmental stress conditions. J Biol Chem 279:22820–22832

    Article  CAS  PubMed  Google Scholar 

  • Palii SS, Kays CE, Deval C et al (2009) Specificity of amino acid regulated gene expression: analysis of genes subjected to either complete or single amino acid deprivation. Amino Acids 37:79–88

    Article  CAS  PubMed  Google Scholar 

  • Pavitt GD, Ramaiah KVA, Kimball SR, Hinnebusch AG (1998) eIF2 independently binds two distinct eIF2b subcomplexes that catalyze and regulate guanine-nucleotide exchange. Genes Dev 12:514–526

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Qiu H, Dong J, Hu C et al (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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Scheuner D, Song B, McEwen E et al (2001) Translational control is required for the unfolded protein response and in vivo glucose homeostasis. Mol Cell 7:1165–1176

    Article  CAS  PubMed  Google Scholar 

  • Shan J, Ord D, Ord T, Kilberg MS (2009) Elevated ATF4 expression, in the absence of other signals, is sufficient for transcriptional induction via CCAAT enhancer-binding protein-activating transcription factor response elements. J Biol Chem 284:21241–21248

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Shan J, Lopez M-C, Baker HV, Kilberg MS (2010) Expression profiling after activation of amino acid deprivation response in HepG2 human hepatoma cells. Physiol Genomics 41:315–327

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sikalidis AK, Lee JI, Stipanuk MH (2011) Gene expression and integrated stress response in HepG2/C3A cells cultured in amino acid deficient medium. Amino Acids 41:159–171

    Article  CAS  PubMed  Google Scholar 

  • Sikalidis AK, Mazor KM, Kang M et al (2013) Total 4EBP1 is elevated in liver of rats in response to low sulfur amino acid intake. J Amino Acids 2013:864757

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Siu F, Bain PJ, Leblanc-Chaffin R et al (2002) ATF4 is a mediator of the nutrient-sensing response pathway that activates the human asparagine synthetase gene. J Biol Chem 277:24120–24127

    Article  CAS  PubMed  Google Scholar 

  • Sood R, Porter AC, Olsen D et al (2000) A mammalian homologue of GCN2 protein kinase important for translational control by phosphorylation of eukaryotic initiation factor-2α. Genetics 154:787–801

    CAS  PubMed  PubMed Central  Google Scholar 

  • Stolboushkina EA, Garber MB (2011) Eukaryotic type translation initiation factor 2: structure-functional aspects. Biochemistry (Mosc) 76:283–294

    Article  CAS  Google Scholar 

  • Tsukiyama-Kohara K, Vidal SM, Gingras C et al (1996) Tissue distribution, genomic structure, and chromosome mapping of mouse and human eukaryotic initiation factor 4E-binding proteins 1 and 2. Genomics 38:353–363

    Article  CAS  PubMed  Google Scholar 

  • Tsukiyama-Kohara K, Poulin F, Kohara M et al (2001) Adipose tissue reduction in mice lacking the translational inhibitor 4E-BP1. Nat Med 7:1128–1132

    Article  CAS  PubMed  Google Scholar 

  • Unbehaun A, Borukhov SI, Hellen CUT, Pestova T V (2004) The 40S subunit in 48S complexes formed at the initiation codon of mRNA is bound to eukaryotic initiation factor (eIF) 3, eIF1, eIF1A, and an eIF2/GTP/Met-tRNA. Genes Dev 3078–3093

  • Van Der Kelen K, Beyaert R, Inzé D, De Veylder L (2009) Translational control of eukaryotic gene expression. Crit Rev Biochem Mol Biol 44:143–168

    Article  CAS  Google Scholar 

  • Vattem KM, Wek RC (2004) Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells. Proc Natl Acad Sci USA 101:11269–11274

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Walter P, Ron D (2011) The unfolded protein response: from stress pathway to homeostatic regulation. Science 334:1081–1086

    Article  CAS  PubMed  Google Scholar 

  • Wanders D, Stone KP, Forney LA, Cortez CC, Dille KN, Simon J, Xu M, Hotard EC, Nikonorova IA, Pettit AP, Anthony TG, Gettys TW (2016) Role of GCN2-independent signaling through a noncanonical PERK/NRF2 pathway in the physiological responses to dietary methionine restriction. Diabetes 65:1499–1510

    Article  CAS  PubMed  Google Scholar 

  • Webb BLJ, Proud CG (1997) Eukaryotic initiation factor 2B (eIF2B). Int J Biochem Cell Biol 29:1127–1131

    Article  CAS  PubMed  Google Scholar 

  • Wek RC, Jackson BM, Hinnebusch AG (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 USA 86:4579–4583

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wek SA, Zhu S, Wek RC (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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wek RC, Jiang H-Y, Anthony TG (2006) Coping with stress: eIF2 kinases and translational control. Biochem Soc Trans 34:7–11

    Article  CAS  PubMed  Google Scholar 

  • Yamaguchi S, Ishihara H, Yamada T et al (2008) ATF4-mediated induction of 4E-BP1 contributes to pancreatic beta cell survival under endoplasmic reticulum stress. Cell Metab 7:269–276

    Article  CAS  PubMed  Google Scholar 

  • Zaborske JM, Narasimhan J, Jiang L et al (2009) Genome-wide analysis of tRNA charging and activation of the eIF2 kinase Gcn2p. J Biol Chem 284:25254–25267

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang P, McGrath BC, Reinert J et al (2002) The GCN2 eIF2α kinase is required for adaptation to amino acid deprivation in mice. Mol Cell Biol 22:6681–6688

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhu S, Sobolev AY, Wek RC (1996) Histidyl-tRNA synthetase-related sequences in GCN2 protein kinase regulate in vitro phosphorylation of eIF-2. J Biol Chem 271:24989–24994

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Division of Nutritional Sciences, Cornell University, Savage Hall, Rm. 227, Ithaca, NY, 14853, USA

    Kevin M. Mazor & Martha H. Stipanuk

Authors
  1. Kevin M. Mazor
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Martha H. Stipanuk
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Martha H. Stipanuk.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Funding

This work was supported by Grants DK-083473 and DK-056649 from the National Institute of Diabetes and Digestive and Kidney Diseases.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Additional information

Handling Editor: H. Jakubowski.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Mazor, K.M., Stipanuk, M.H. GCN2- and eIF2α-phosphorylation-independent, but ATF4-dependent, induction of CARE-containing genes in methionine-deficient cells. Amino Acids 48, 2831–2842 (2016). https://doi.org/10.1007/s00726-016-2318-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00726-016-2318-9

Keywords

Search

Navigation