Skip to main content
Biochemical Journal logoLink to Biochemical Journal
. 1999 Aug 15;342(Pt 1):65–70.

Caspase-mediated cleavage of eukaryotic translation initiation factor subunit 2alpha.

S Satoh 1, M Hijikata 1, H Handa 1, K Shimotohno 1
PMCID: PMC1220437  PMID: 10432301

Abstract

Eukaryotic translation initiation factor 2alpha (eIF-2alpha), a target molecule of the interferon-inducible double-stranded-RNA-dependent protein kinase (PKR), was cleaved in apoptotic Saos-2 cells on treatment with poly(I).poly(C) or tumour necrosis factor alpha. This cleavage occurred with a time course similar to that of poly(ADP-ribose) polymerase, a well-known caspase substrate. In addition, eIF-2alpha was cleaved by recombinant active caspase-3 in vitro. By site-directed mutagenesis, the cleavage site was mapped to an Ala-Glu-Val-Asp(300) downward arrowGly(301) sequence located in the C-terminal portion of eIF-2alpha. PKR phosphorylates eIF-2alpha on Ser(51), resulting in the suppression of protein synthesis. PKR-mediated translational suppression was repressed when the C-terminally cleaved product of eIF-2alpha was overexpressed in Saos-2 cells, even though PKR can phosphorylate this cleaved product. These results suggest that caspase-3 or related protease(s) can modulate the efficiency of protein synthesis by cleaving the alpha subunit of eIF-2, a key component in the initiation of translation.

Full Text

The Full Text of this article is available as a PDF (183.5 KB).

Selected References

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

  1. Akagi T., Ono H., Nyunoya H., Shimotohno K. Characterization of peripheral blood T-lymphocytes transduced with HTLV-I Tax mutants with different trans-activating phenotypes. Oncogene. 1997 May 1;14(17):2071–2078. doi: 10.1038/sj.onc.1201045. [DOI] [PubMed] [Google Scholar]
  2. Alnemri E. S., Livingston D. J., Nicholson D. W., Salvesen G., Thornberry N. A., Wong W. W., Yuan J. Human ICE/CED-3 protease nomenclature. Cell. 1996 Oct 18;87(2):171–171. doi: 10.1016/s0092-8674(00)81334-3. [DOI] [PubMed] [Google Scholar]
  3. Casciola-Rosen L. A., Miller D. K., Anhalt G. J., Rosen A. Specific cleavage of the 70-kDa protein component of the U1 small nuclear ribonucleoprotein is a characteristic biochemical feature of apoptotic cell death. J Biol Chem. 1994 Dec 9;269(49):30757–30760. [PubMed] [Google Scholar]
  4. Chen J. J., Throop M. S., Gehrke L., Kuo I., Pal J. K., Brodsky M., London I. M. Cloning of the cDNA of the heme-regulated eukaryotic initiation factor 2 alpha (eIF-2 alpha) kinase of rabbit reticulocytes: homology to yeast GCN2 protein kinase and human double-stranded-RNA-dependent eIF-2 alpha kinase. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7729–7733. doi: 10.1073/pnas.88.17.7729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Choi S. Y., Scherer B. J., Schnier J., Davies M. V., Kaufman R. J., Hershey J. W. Stimulation of protein synthesis in COS cells transfected with variants of the alpha-subunit of initiation factor eIF-2. J Biol Chem. 1992 Jan 5;267(1):286–293. [PubMed] [Google Scholar]
  6. Chong K. L., Feng L., Schappert K., Meurs E., Donahue T. F., Friesen J. D., Hovanessian A. G., Williams B. R. Human p68 kinase exhibits growth suppression in yeast and homology to the translational regulator GCN2. EMBO J. 1992 Apr;11(4):1553–1562. doi: 10.1002/j.1460-2075.1992.tb05200.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Faleiro L., Kobayashi R., Fearnhead H., Lazebnik Y. Multiple species of CPP32 and Mch2 are the major active caspases present in apoptotic cells. EMBO J. 1997 May 1;16(9):2271–2281. doi: 10.1093/emboj/16.9.2271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hale A. J., Smith C. A., Sutherland L. C., Stoneman V. E., Longthorne V. L., Culhane A. C., Williams G. T. Apoptosis: molecular regulation of cell death. Eur J Biochem. 1996 Feb 15;236(1):1–26. doi: 10.1111/j.1432-1033.1996.00001.x. [DOI] [PubMed] [Google Scholar]
  9. Hijikata M., Mizushima H., Akagi T., Mori S., Kakiuchi N., Kato N., Tanaka T., Kimura K., Shimotohno K. Two distinct proteinase activities required for the processing of a putative nonstructural precursor protein of hepatitis C virus. J Virol. 1993 Aug;67(8):4665–4675. doi: 10.1128/jvi.67.8.4665-4675.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hijikata M., Mizushima H., Tanji Y., Komoda Y., Hirowatari Y., Akagi T., Kato N., Kimura K., Shimotohno K. Proteolytic processing and membrane association of putative nonstructural proteins of hepatitis C virus. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10773–10777. doi: 10.1073/pnas.90.22.10773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hirota M., Satoh S., Asabe S., Kohara M., Tsukiyama-Kohara K., Kato N., Hijikata M., Shimotohno K. Phosphorylation of nonstructural 5A protein of hepatitis C virus: HCV group-specific hyperphosphorylation. Virology. 1999 Apr 25;257(1):130–137. doi: 10.1006/viro.1999.9658. [DOI] [PubMed] [Google Scholar]
  12. Kato N., Ikeda M., Mizutani T., Sugiyama K., Noguchi M., Hirohashi S., Shimotohno K. Replication of hepatitis C virus in cultured non-neoplastic human hepatocytes. Jpn J Cancer Res. 1996 Aug;87(8):787–792. doi: 10.1111/j.1349-7006.1996.tb02101.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kaufman R. J., Davies M. V., Pathak V. K., Hershey J. W. The phosphorylation state of eucaryotic initiation factor 2 alters translational efficiency of specific mRNAs. Mol Cell Biol. 1989 Mar;9(3):946–958. doi: 10.1128/mcb.9.3.946. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kimball S. R., Heinzinger N. K., Horetsky R. L., Jefferson L. S. Identification of interprotein interactions between the subunits of eukaryotic initiation factors eIF2 and eIF2B. J Biol Chem. 1998 Jan 30;273(5):3039–3044. doi: 10.1074/jbc.273.5.3039. [DOI] [PubMed] [Google Scholar]
  15. King P., Goodbourn S. STAT1 is inactivated by a caspase. J Biol Chem. 1998 Apr 10;273(15):8699–8704. doi: 10.1074/jbc.273.15.8699. [DOI] [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Lazebnik Y. A., Kaufmann S. H., Desnoyers S., Poirier G. G., Earnshaw W. C. Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE. Nature. 1994 Sep 22;371(6495):346–347. doi: 10.1038/371346a0. [DOI] [PubMed] [Google Scholar]
  18. Lazebnik Y. A., Takahashi A., Moir R. D., Goldman R. D., Poirier G. G., Kaufmann S. H., Earnshaw W. C. Studies of the lamin proteinase reveal multiple parallel biochemical pathways during apoptotic execution. Proc Natl Acad Sci U S A. 1995 Sep 26;92(20):9042–9046. doi: 10.1073/pnas.92.20.9042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lee S. B., Esteban M. The interferon-induced double-stranded RNA-activated protein kinase induces apoptosis. Virology. 1994 Mar;199(2):491–496. doi: 10.1006/viro.1994.1151. [DOI] [PubMed] [Google Scholar]
  20. Lee S. B., Melkova Z., Yan W., Williams B. R., Hovanessian A. G., Esteban M. The interferon-induced double-stranded RNA-activated human p68 protein kinase potently inhibits protein synthesis in cultured cells. Virology. 1993 Jan;192(1):380–385. doi: 10.1006/viro.1993.1048. [DOI] [PubMed] [Google Scholar]
  21. MacFarlane M., Cain K., Sun X. M., Alnemri E. S., Cohen G. M. Processing/activation of at least four interleukin-1beta converting enzyme-like proteases occurs during the execution phase of apoptosis in human monocytic tumor cells. J Cell Biol. 1997 Apr 21;137(2):469–479. doi: 10.1083/jcb.137.2.469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Marissen W. E., Lloyd R. E. Eukaryotic translation initiation factor 4G is targeted for proteolytic cleavage by caspase 3 during inhibition of translation in apoptotic cells. Mol Cell Biol. 1998 Dec;18(12):7565–7574. doi: 10.1128/mcb.18.12.7565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Martins L. M., Kottke T., Mesner P. W., Basi G. S., Sinha S., Frigon N., Jr, Tatar E., Tung J. S., Bryant K., Takahashi A. Activation of multiple interleukin-1beta converting enzyme homologues in cytosol and nuclei of HL-60 cells during etoposide-induced apoptosis. J Biol Chem. 1997 Mar 14;272(11):7421–7430. doi: 10.1074/jbc.272.11.7421. [DOI] [PubMed] [Google Scholar]
  24. Meurs E. F., Watanabe Y., Kadereit S., Barber G. N., Katze M. G., Chong K., Williams B. R., Hovanessian A. G. Constitutive expression of human double-stranded RNA-activated p68 kinase in murine cells mediates phosphorylation of eukaryotic initiation factor 2 and partial resistance to encephalomyocarditis virus growth. J Virol. 1992 Oct;66(10):5805–5814. doi: 10.1128/jvi.66.10.5805-5814.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pain V. M. Initiation of protein synthesis in eukaryotic cells. Eur J Biochem. 1996 Mar 15;236(3):747–771. doi: 10.1111/j.1432-1033.1996.00747.x. [DOI] [PubMed] [Google Scholar]
  26. Pain V. M. Initiation of protein synthesis in mammalian cells. Biochem J. 1986 May 1;235(3):625–637. doi: 10.1042/bj2350625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pathak V. K., Schindler D., Hershey J. W. Generation of a mutant form of protein synthesis initiation factor eIF-2 lacking the site of phosphorylation by eIF-2 kinases. Mol Cell Biol. 1988 Feb;8(2):993–995. doi: 10.1128/mcb.8.2.993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Petryshyn R., Chen J. J., London I. M. Detection of activated double-stranded RNA-dependent protein kinase in 3T3-F442A cells. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1427–1431. doi: 10.1073/pnas.85.5.1427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Petryshyn R., Chen J. J., London I. M. Growth-related expression of a double-stranded RNA-dependent protein kinase in 3T3 cells. J Biol Chem. 1984 Dec 10;259(23):14736–14742. [PubMed] [Google Scholar]
  30. Piedrafita F. J., Pfahl M. Retinoid-induced apoptosis and Sp1 cleavage occur independently of transcription and require caspase activation. Mol Cell Biol. 1997 Nov;17(11):6348–6358. doi: 10.1128/mcb.17.11.6348. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pochampally R., Fodera B., Chen L., Shao W., Levine E. A., Chen J. A 60 kd MDM2 isoform is produced by caspase cleavage in non-apoptotic tumor cells. Oncogene. 1998 Nov 19;17(20):2629–2636. doi: 10.1038/sj.onc.1202206. [DOI] [PubMed] [Google Scholar]
  32. Ray R. B., Meyer K., Steele R., Shrivastava A., Aggarwal B. B., Ray R. Inhibition of tumor necrosis factor (TNF-alpha)-mediated apoptosis by hepatitis C virus core protein. J Biol Chem. 1998 Jan 23;273(4):2256–2259. doi: 10.1074/jbc.273.4.2256. [DOI] [PubMed] [Google Scholar]
  33. Samuel C. E. The eIF-2 alpha protein kinases, regulators of translation in eukaryotes from yeasts to humans. J Biol Chem. 1993 Apr 15;268(11):7603–7606. [PubMed] [Google Scholar]
  34. Song Q., Lees-Miller S. P., Kumar S., Zhang Z., Chan D. W., Smith G. C., Jackson S. P., Alnemri E. S., Litwack G., Khanna K. K. DNA-dependent protein kinase catalytic subunit: a target for an ICE-like protease in apoptosis. EMBO J. 1996 Jul 1;15(13):3238–3246. [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Takahashi A., Hirata H., Yonehara S., Imai Y., Lee K. K., Moyer R. W., Turner P. C., Mesner P. W., Okazaki T., Sawai H. Affinity labeling displays the stepwise activation of ICE-related proteases by Fas, staurosporine, and CrmA-sensitive caspase-8. Oncogene. 1997 Jun 12;14(23):2741–2752. doi: 10.1038/sj.onc.1201131. [DOI] [PubMed] [Google Scholar]
  37. Thompson C. B. Apoptosis in the pathogenesis and treatment of disease. Science. 1995 Mar 10;267(5203):1456–1462. doi: 10.1126/science.7878464. [DOI] [PubMed] [Google Scholar]
  38. Vazquez de Aldana C. R., Dever T. E., Hinnebusch A. G. Mutations in the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha) that overcome the inhibitory effect of eIF-2 alpha phosphorylation on translation initiation. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):7215–7219. doi: 10.1073/pnas.90.15.7215. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES