Skip to main content
PMC home page
Genetics logoLink to Genetics
. 2000 Feb;154(2):787–801. doi: 10.1093/genetics/154.2.787

A mammalian homologue of GCN2 protein kinase important for translational control by phosphorylation of eukaryotic initiation factor-2alpha.

R Sood 1, A C Porter 1, D A Olsen 1, D R Cavener 1, R C Wek 1
PMCID: PMC1460965  PMID: 10655230

Abstract

A family of protein kinases regulates translation in response to different cellular stresses by phosphorylation of the alpha subunit of eukaryotic initiation factor-2 (eIF-2alpha). In yeast, an eIF-2alpha kinase, GCN2, functions in translational control in response to amino acid starvation. It is thought that uncharged tRNA that accumulates during amino acid limitation binds to sequences in GCN2 homologous to histidyl-tRNA synthetase (HisRS) enzymes, leading to enhanced kinase catalytic activity. Given that starvation for amino acids also stimulates phosphorylation of eIF-2alpha in mammalian cells, we searched for and identified a GCN2 homologue in mice. We cloned three different cDNAs encoding mouse GCN2 isoforms, derived from a single gene, that vary in their amino-terminal sequences. Like their yeast counterpart, the mouse GCN2 isoforms contain HisRS-related sequences juxtaposed to the kinase catalytic domain. While GCN2 mRNA was found in all mouse tissues examined, the isoforms appear to be differentially expressed. Mouse GCN2 expressed in yeast was found to inhibit growth by hyperphosphorylation of eIF-2alpha, requiring both the kinase catalytic domain and the HisRS-related sequences. Additionally, lysates prepared from yeast expressing mGCN2 were found to phosphorylate recombinant eIF-2alpha substrate. Mouse GCN2 activity in both the in vivo and in vitro assays required the presence of serine-51, the known regulatory phosphorylation site in eIF-2alpha. Together, our studies identify a new mammalian eIF-2alpha kinase, GCN2, that can mediate translational control.

Full Text

The Full Text of this article is available as a PDF (542.5 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abastado J. P., Miller P. F., Jackson B. M., Hinnebusch A. G. Suppression of ribosomal reinitiation at upstream open reading frames in amino acid-starved cells forms the basis for GCN4 translational control. Mol Cell Biol. 1991 Jan;11(1):486–496. doi: 10.1128/mcb.11.1.486. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Andrulis I. L., Hatfield G. W., Arfin S. M. Asparaginyl-tRNA aminoacylation levels and asparagine synthetase expression in cultured Chinese hamster ovary cells. J Biol Chem. 1979 Nov 10;254(21):10629–10633. [PMC free article] [PubMed] [Google Scholar]
  3. Arnez J. G., Harris D. C., Mitschler A., Rees B., Francklyn C. S., Moras D. Crystal structure of histidyl-tRNA synthetase from Escherichia coli complexed with histidyl-adenylate. EMBO J. 1995 Sep 1;14(17):4143–4155. doi: 10.1002/j.1460-2075.1995.tb00088.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barber G. N., Jagus R., Meurs E. F., Hovanessian A. G., Katze M. G. Molecular mechanisms responsible for malignant transformation by regulatory and catalytic domain variants of the interferon-induced enzyme RNA-dependent protein kinase. J Biol Chem. 1995 Jul 21;270(29):17423–17428. doi: 10.1074/jbc.270.29.17423. [DOI] [PubMed] [Google Scholar]
  5. Brandl C. J., Struhl K. Yeast GCN4 transcriptional activator protein interacts with RNA polymerase II in vitro. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2652–2656. doi: 10.1073/pnas.86.8.2652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bruhat A., Jousse C., Wang X. Z., 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. J Biol Chem. 1997 Jul 11;272(28):17588–17593. doi: 10.1074/jbc.272.28.17588. [DOI] [PubMed] [Google Scholar]
  7. Chen J. J., London I. M. Regulation of protein synthesis by heme-regulated eIF-2 alpha kinase. Trends Biochem Sci. 1995 Mar;20(3):105–108. doi: 10.1016/s0968-0004(00)88975-6. [DOI] [PubMed] [Google Scholar]
  8. Cigan A. M., Pabich E. K., Feng L., Donahue T. F. Yeast translation initiation suppressor sui2 encodes the alpha subunit of eukaryotic initiation factor 2 and shares sequence identity with the human alpha subunit. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2784–2788. doi: 10.1073/pnas.86.8.2784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. DeGracia D. J., Sullivan J. M., Neumar R. W., Alousi S. S., Hikade K. R., Pittman J. E., White B. C., Rafols J. A., Krause G. S. Effect of brain ischemia and reperfusion on the localization of phosphorylated eukaryotic initiation factor 2 alpha. J Cereb Blood Flow Metab. 1997 Dec;17(12):1291–1302. doi: 10.1097/00004647-199712000-00004. [DOI] [PubMed] [Google Scholar]
  10. Dever T. E., Chen J. J., Barber G. N., Cigan A. M., Feng L., Donahue T. F., London I. M., Katze M. G., Hinnebusch A. G. Mammalian eukaryotic initiation factor 2 alpha kinases functionally substitute for GCN2 protein kinase in the GCN4 translational control mechanism of yeast. Proc Natl Acad Sci U S A. 1993 May 15;90(10):4616–4620. doi: 10.1073/pnas.90.10.4616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dever T. E., Feng L., Wek R. C., Cigan A. M., Donahue T. F., Hinnebusch A. G. Phosphorylation of initiation factor 2 alpha by protein kinase GCN2 mediates gene-specific translational control of GCN4 in yeast. Cell. 1992 Feb 7;68(3):585–596. doi: 10.1016/0092-8674(92)90193-g. [DOI] [PubMed] [Google Scholar]
  12. Drysdale C. M., Jackson B. M., McVeigh R., Klebanow E. R., Bai Y., Kokubo T., Swanson M., Nakatani Y., Weil P. A., Hinnebusch A. G. The Gcn4p activation domain interacts specifically in vitro with RNA polymerase II holoenzyme, TFIID, and the Adap-Gcn5p coactivator complex. Mol Cell Biol. 1998 Mar;18(3):1711–1724. doi: 10.1128/mcb.18.3.1711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Feng G. S., Chong K., Kumar A., Williams B. R. Identification of double-stranded RNA-binding domains in the interferon-induced double-stranded RNA-activated p68 kinase. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5447–5451. doi: 10.1073/pnas.89.12.5447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Guerrini L., Gong S. S., Mangasarian K., Basilico C. Cis- and trans-acting elements involved in amino acid regulation of asparagine synthetase gene expression. Mol Cell Biol. 1993 Jun;13(6):3202–3212. doi: 10.1128/mcb.13.6.3202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hanks S. K., Hunter T. Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. FASEB J. 1995 May;9(8):576–596. [PubMed] [Google Scholar]
  16. Harding H. P., Zhang Y., Ron D. Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase. Nature. 1999 Jan 21;397(6716):271–274. doi: 10.1038/16729. [DOI] [PubMed] [Google Scholar]
  17. Heal R., McGivan J. Induction of calreticulin expression in response to amino acid deprivation in Chinese hamster ovary cells. Biochem J. 1998 Jan 15;329(Pt 2):389–394. doi: 10.1042/bj3290389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hutson R. G., Kilberg M. S. Cloning of rat asparagine synthetase and specificity of the amino acid-dependent control of its mRNA content. Biochem J. 1994 Dec 15;304(Pt 3):745–750. doi: 10.1042/bj3040745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kilberg M. S., Hutson R. G., Laine R. O. Amino acid-regulated gene expression in eukaryotic cells. FASEB J. 1994 Jan;8(1):13–19. doi: 10.1096/fasebj.8.1.8299885. [DOI] [PubMed] [Google Scholar]
  20. Kimball S. R., Antonetti D. A., Brawley R. M., Jefferson L. S. Mechanism of inhibition of peptide chain initiation by amino acid deprivation in perfused rat liver. Regulation involving inhibition of eukaryotic initiation factor 2 alpha phosphatase activity. J Biol Chem. 1991 Jan 25;266(3):1969–1976. [PubMed] [Google Scholar]
  21. Koromilas A. E., Roy S., Barber G. N., Katze M. G., Sonenberg N. Malignant transformation by a mutant of the IFN-inducible dsRNA-dependent protein kinase. Science. 1992 Sep 18;257(5077):1685–1689. doi: 10.1126/science.1382315. [DOI] [PubMed] [Google Scholar]
  22. Marton M. J., Crouch D., Hinnebusch A. G. GCN1, a translational activator of GCN4 in Saccharomyces cerevisiae, is required for phosphorylation of eukaryotic translation initiation factor 2 by protein kinase GCN2. Mol Cell Biol. 1993 Jun;13(6):3541–3556. doi: 10.1128/mcb.13.6.3541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Marton M. J., Vazquez de Aldana C. R., Qiu H., Chakraburtty K., Hinnebusch A. G. Evidence that GCN1 and GCN20, translational regulators of GCN4, function on elongating ribosomes in activation of eIF2alpha kinase GCN2. Mol Cell Biol. 1997 Aug;17(8):4474–4489. doi: 10.1128/mcb.17.8.4474. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. McGivan J. D., Pastor-Anglada M. Regulatory and molecular aspects of mammalian amino acid transport. Biochem J. 1994 Apr 15;299(Pt 2):321–334. doi: 10.1042/bj2990321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Meurs E. F., Galabru J., Barber G. N., Katze M. G., Hovanessian A. G. Tumor suppressor function of the interferon-induced double-stranded RNA-activated protein kinase. Proc Natl Acad Sci U S A. 1993 Jan 1;90(1):232–236. doi: 10.1073/pnas.90.1.232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Moras D. Structural and functional relationships between aminoacyl-tRNA synthetases. Trends Biochem Sci. 1992 Apr;17(4):159–164. doi: 10.1016/0968-0004(92)90326-5. [DOI] [PubMed] [Google Scholar]
  27. Natarajan K., Jackson B. M., Rhee E., Hinnebusch A. G. yTAFII61 has a general role in RNA polymerase II transcription and is required by Gcn4p to recruit the SAGA coactivator complex. Mol Cell. 1998 Nov;2(5):683–692. doi: 10.1016/s1097-2765(00)80166-5. [DOI] [PubMed] [Google Scholar]
  28. Olsen D. S., Jordan B., Chen D., Wek R. C., Cavener D. R. Isolation of the gene encoding the Drosophila melanogaster homolog of the Saccharomyces cerevisiae GCN2 eIF-2alpha kinase. Genetics. 1998 Jul;149(3):1495–1509. doi: 10.1093/genetics/149.3.1495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pavitt G. D., Yang W., Hinnebusch A. G. Homologous segments in three subunits of the guanine nucleotide exchange factor eIF2B mediate translational regulation by phosphorylation of eIF2. Mol Cell Biol. 1997 Mar;17(3):1298–1313. doi: 10.1128/mcb.17.3.1298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Pohjanpelto P., Hölttä E. Deprivation of a single amino acid induces protein synthesis-dependent increases in c-jun, c-myc, and ornithine decarboxylase mRNAs in Chinese hamster ovary cells. Mol Cell Biol. 1990 Nov;10(11):5814–5821. doi: 10.1128/mcb.10.11.5814. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Qiu H., Garcia-Barrio M. T., Hinnebusch A. G. 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. 1998 May;18(5):2697–2711. doi: 10.1128/mcb.18.5.2697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ramirez M., Wek R. C., Hinnebusch A. G. Ribosome association of GCN2 protein kinase, a translational activator of the GCN4 gene of Saccharomyces cerevisiae. Mol Cell Biol. 1991 Jun;11(6):3027–3036. doi: 10.1128/mcb.11.6.3027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ramirez M., Wek R. C., Vazquez de Aldana C. R., Jackson B. M., Freeman B., Hinnebusch A. G. Mutations activating the yeast eIF-2 alpha kinase GCN2: isolation of alleles altering the domain related to histidyl-tRNA synthetases. Mol Cell Biol. 1992 Dec;12(12):5801–5815. doi: 10.1128/mcb.12.12.5801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rolfes R. J., Hinnebusch A. G. Translation of the yeast transcriptional activator GCN4 is stimulated by purine limitation: implications for activation of the protein kinase GCN2. Mol Cell Biol. 1993 Aug;13(8):5099–5111. doi: 10.1128/mcb.13.8.5099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Romano P. R., Garcia-Barrio M. T., Zhang X., Wang Q., Taylor D. R., Zhang F., Herring C., Mathews M. B., Qin J., Hinnebusch A. G. Autophosphorylation in the activation loop is required for full kinase activity in vivo of human and yeast eukaryotic initiation factor 2alpha kinases PKR and GCN2. Mol Cell Biol. 1998 Apr;18(4):2282–2297. doi: 10.1128/mcb.18.4.2282. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Romano P. R., Green S. R., Barber G. N., Mathews M. B., Hinnebusch A. G. Structural requirements for double-stranded RNA binding, dimerization, and activation of the human eIF-2 alpha kinase DAI in Saccharomyces cerevisiae. Mol Cell Biol. 1995 Jan;15(1):365–378. doi: 10.1128/mcb.15.1.365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Romano P. R., Green S. R., Barber G. N., Mathews M. B., Hinnebusch A. G. Structural requirements for double-stranded RNA binding, dimerization, and activation of the human eIF-2 alpha kinase DAI in Saccharomyces cerevisiae. Mol Cell Biol. 1995 Jan;15(1):365–378. doi: 10.1128/mcb.15.1.365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Romano P. R., Zhang F., Tan S. L., Garcia-Barrio M. T., Katze M. G., Dever T. E., Hinnebusch A. G. Inhibition of double-stranded RNA-dependent protein kinase PKR by vaccinia virus E3: role of complex formation and the E3 N-terminal domain. Mol Cell Biol. 1998 Dec;18(12):7304–7316. doi: 10.1128/mcb.18.12.7304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Santoyo J., Alcalde J., Méndez R., Pulido D., de Haro C. Cloning and characterization of a cDNA encoding a protein synthesis initiation factor-2alpha (eIF-2alpha) kinase from Drosophila melanogaster. Homology To yeast GCN2 protein kinase. J Biol Chem. 1997 May 9;272(19):12544–12550. doi: 10.1074/jbc.272.19.12544. [DOI] [PubMed] [Google Scholar]
  41. Sattlegger E., Hinnebusch A. G., Barthelmess I. B. cpc-3, the Neurospora crassa homologue of yeast GCN2, encodes a polypeptide with juxtaposed eIF2alpha kinase and histidyl-tRNA synthetase-related domains required for general amino acid control. J Biol Chem. 1998 Aug 7;273(32):20404–20416. doi: 10.1074/jbc.273.32.20404. [DOI] [PubMed] [Google Scholar]
  42. Scorsone K. A., Panniers R., Rowlands A. G., Henshaw E. C. Phosphorylation of eukaryotic initiation factor 2 during physiological stresses which affect protein synthesis. J Biol Chem. 1987 Oct 25;262(30):14538–14543. [PubMed] [Google Scholar]
  43. Shi Y., Vattem K. M., Sood R., An J., Liang J., Stramm L., Wek R. C. Identification and characterization of pancreatic eukaryotic initiation factor 2 alpha-subunit kinase, PEK, involved in translational control. Mol Cell Biol. 1998 Dec;18(12):7499–7509. doi: 10.1128/mcb.18.12.7499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Srivastava S. P., Kumar K. U., Kaufman R. J. Phosphorylation of eukaryotic translation initiation factor 2 mediates apoptosis in response to activation of the double-stranded RNA-dependent protein kinase. J Biol Chem. 1998 Jan 23;273(4):2416–2423. doi: 10.1074/jbc.273.4.2416. [DOI] [PubMed] [Google Scholar]
  45. Vazquez de Aldana C. R., Marton M. J., Hinnebusch A. G. 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. 1995 Jul 3;14(13):3184–3199. doi: 10.1002/j.1460-2075.1995.tb07321.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wek R. C., Ramirez M., Jackson B. M., Hinnebusch A. G. Identification of positive-acting domains in GCN2 protein kinase required for translational activation of GCN4 expression. Mol Cell Biol. 1990 Jun;10(6):2820–2831. doi: 10.1128/mcb.10.6.2820. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wek S. A., Zhu S., Wek R. C. 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. 1995 Aug;15(8):4497–4506. doi: 10.1128/mcb.15.8.4497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zhu S., Romano P. R., Wek R. C. Ribosome targeting of PKR is mediated by two double-stranded RNA-binding domains and facilitates in vivo phosphorylation of eukaryotic initiation factor-2. J Biol Chem. 1997 May 30;272(22):14434–14441. doi: 10.1074/jbc.272.22.14434. [DOI] [PubMed] [Google Scholar]
  49. Zhu S., Sobolev A. Y., Wek R. C. Histidyl-tRNA synthetase-related sequences in GCN2 protein kinase regulate in vitro phosphorylation of eIF-2. J Biol Chem. 1996 Oct 4;271(40):24989–24994. doi: 10.1074/jbc.271.40.24989. [DOI] [PubMed] [Google Scholar]
  50. Zhu S., Wek R. C. Ribosome-binding domain of eukaryotic initiation factor-2 kinase GCN2 facilitates translation control. J Biol Chem. 1998 Jan 16;273(3):1808–1814. doi: 10.1074/jbc.273.3.1808. [DOI] [PubMed] [Google Scholar]
  51. de Haro C., Méndez R., Santoyo J. The eIF-2alpha kinases and the control of protein synthesis. FASEB J. 1996 Oct;10(12):1378–1387. doi: 10.1096/fasebj.10.12.8903508. [DOI] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES