Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1999 Sep 15;342(Pt 3):519–526.

Cellular stress in xenopus kidney cells enhances the phosphorylation of eukaryotic translation initiation factor (eIF)4E and the association of eIF4F with poly(A)-binding protein.

C S Fraser 1, V M Pain 1, S J Morley 1
PMCID: PMC1220492  PMID: 10477262

Abstract

Eukaryotic initiation factor (eIF) 4E binds to the 5'-cap structure of eukaryotic mRNA and has a central role in the control of cell proliferation. We have shown previously that the stimulation of cultured Xenopus kidney cells with serum resulted in the activation of protein synthesis, enhanced phosphorylation of eIF4E and increased binding of the adapter protein, eIF4G, and poly(A)-binding protein (PABP) to eIF4E to form the functional initiation factor complex, eIF4F/PABP. We now show that cellular stresses such as arsenite, anisomycin and heat shock also result in increased phosphorylation of eIF4E, eIF4F complex formation and the association of PABP with eIF4G, in conditions under which the rate of protein synthesis is severely inhibited. In contrast with reported effects on mammalian cells, the stress-induced increase in eIF4F complex formation occurs in the absence of changes in the association of eIF4E with its binding proteins 4E-BP1 or 4E-BP2. The stress-induced changes in eIF4E phosphorylation were totally abrogated by the p38 mitogen-activated protein (MAP) kinase inhibitor SB203580, and were partly inhibited by the phosphoinositide 3-kinase inhibitor LY294002 and the mammalian target of rapamycin (mTOR) inhibitor rapamycin. However, eIF4E phosphorylation was unaffected by extracellular signal-regulated protein kinase (MAP kinase) inhibitor PD98059. These results indicate that cellular stresses activate multiple signalling pathways that converge at the level of eIF4F complex formation to influence the interactions between eIF4E, eIF4G and PABP.

Full Text

The Full Text of this article is available as a PDF (328.7 KB).

Selected References

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

  1. Alessi D. R., Cohen P. Mechanism of activation and function of protein kinase B. Curr Opin Genet Dev. 1998 Feb;8(1):55–62. doi: 10.1016/s0959-437x(98)80062-2. [DOI] [PubMed] [Google Scholar]
  2. Carr M. D., Wollborn U., McIntosh P. B., Frenkiel T. A., McCormick J. E., Bauer C. J., Klempnauer K. H., Feeney J. Structure of the B-Myb DNA-binding domain in solution and evidence for multiple conformations in the region of repeat-2 involved in DNA binding: implications for sequence-specific DNA binding by Myb proteins. Eur J Biochem. 1996 Feb 1;235(3):721–735. doi: 10.1111/j.1432-1033.1996.00721.x. [DOI] [PubMed] [Google Scholar]
  3. Cuenda A., Rouse J., Doza Y. N., Meier R., Cohen P., Gallagher T. F., Young P. R., Lee J. C. SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1. FEBS Lett. 1995 May 8;364(2):229–233. doi: 10.1016/0014-5793(95)00357-f. [DOI] [PubMed] [Google Scholar]
  4. Duncan R. F., Hershey J. W. Protein synthesis and protein phosphorylation during heat stress, recovery, and adaptation. J Cell Biol. 1989 Oct;109(4 Pt 1):1467–1481. doi: 10.1083/jcb.109.4.1467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Flynn A., Proud C. G. Serine 209, not serine 53, is the major site of phosphorylation in initiation factor eIF-4E in serum-treated Chinese hamster ovary cells. J Biol Chem. 1995 Sep 15;270(37):21684–21688. doi: 10.1074/jbc.270.37.21684. [DOI] [PubMed] [Google Scholar]
  6. Fraser C. S., Pain V. M., Morley S. J. The association of initiation factor 4F with poly(A)-binding protein is enhanced in serum-stimulated Xenopus kidney cells. J Biol Chem. 1999 Jan 1;274(1):196–204. doi: 10.1074/jbc.274.1.196. [DOI] [PubMed] [Google Scholar]
  7. Gingras A. C., Kennedy S. G., O'Leary M. A., Sonenberg N., Hay N. 4E-BP1, a repressor of mRNA translation, is phosphorylated and inactivated by the Akt(PKB) signaling pathway. Genes Dev. 1998 Feb 15;12(4):502–513. doi: 10.1101/gad.12.4.502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Haghighat A., Mader S., Pause A., Sonenberg N. Repression of cap-dependent translation by 4E-binding protein 1: competition with p220 for binding to eukaryotic initiation factor-4E. EMBO J. 1995 Nov 15;14(22):5701–5709. doi: 10.1002/j.1460-2075.1995.tb00257.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Haghighat A., Sonenberg N. eIF4G dramatically enhances the binding of eIF4E to the mRNA 5'-cap structure. J Biol Chem. 1997 Aug 29;272(35):21677–21680. doi: 10.1074/jbc.272.35.21677. [DOI] [PubMed] [Google Scholar]
  10. Joshi B., Cai A. L., Keiper B. D., Minich W. B., Mendez R., Beach C. M., Stepinski J., Stolarski R., Darzynkiewicz E., Rhoads R. E. Phosphorylation of eukaryotic protein synthesis initiation factor 4E at Ser-209. J Biol Chem. 1995 Jun 16;270(24):14597–14603. doi: 10.1074/jbc.270.24.14597. [DOI] [PubMed] [Google Scholar]
  11. Kleijn M., Scheper G. C., Voorma H. O., Thomas A. A. Regulation of translation initiation factors by signal transduction. Eur J Biochem. 1998 May 1;253(3):531–544. doi: 10.1046/j.1432-1327.1998.2530531.x. [DOI] [PubMed] [Google Scholar]
  12. Kumar S. The apoptotic cysteine protease CPP32. Int J Biochem Cell Biol. 1997 Mar;29(3):393–396. doi: 10.1016/s1357-2725(96)00146-x. [DOI] [PubMed] [Google Scholar]
  13. Lamphear B. J., Kirchweger R., Skern T., Rhoads R. E. Mapping of functional domains in eukaryotic protein synthesis initiation factor 4G (eIF4G) with picornaviral proteases. Implications for cap-dependent and cap-independent translational initiation. J Biol Chem. 1995 Sep 15;270(37):21975–21983. doi: 10.1074/jbc.270.37.21975. [DOI] [PubMed] [Google Scholar]
  14. Lamphear B. J., Panniers R. Heat shock impairs the interaction of cap-binding protein complex with 5' mRNA cap. J Biol Chem. 1991 Feb 15;266(5):2789–2794. [PubMed] [Google Scholar]
  15. Lewis T., Groom L. A., Sneddon A. A., Smythe C., Keyse S. M. XCL100, an inducible nuclear MAP kinase phosphatase from Xenopus laevis: its role in MAP kinase inactivation in differentiated cells and its expression during early development. J Cell Sci. 1995 Aug;108(Pt 8):2885–2896. doi: 10.1242/jcs.108.8.2885. [DOI] [PubMed] [Google Scholar]
  16. Lin T. A., Kong X., Haystead T. A., Pause A., Belsham G., Sonenberg N., Lawrence J. C., Jr PHAS-I as a link between mitogen-activated protein kinase and translation initiation. Science. 1994 Oct 28;266(5185):653–656. doi: 10.1126/science.7939721. [DOI] [PubMed] [Google Scholar]
  17. Lin T. A., Lawrence J. C., Jr Control of the translational regulators PHAS-I and PHAS-II by insulin and cAMP in 3T3-L1 adipocytes. J Biol Chem. 1996 Nov 22;271(47):30199–30204. doi: 10.1074/jbc.271.47.30199. [DOI] [PubMed] [Google Scholar]
  18. Mader S., Lee H., Pause A., Sonenberg N. The translation initiation factor eIF-4E binds to a common motif shared by the translation factor eIF-4 gamma and the translational repressors 4E-binding proteins. Mol Cell Biol. 1995 Sep;15(9):4990–4997. doi: 10.1128/mcb.15.9.4990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Marcotrigiano J., Gingras A. C., Sonenberg N., Burley S. K. Cocrystal structure of the messenger RNA 5' cap-binding protein (eIF4E) bound to 7-methyl-GDP. Cell. 1997 Jun 13;89(6):951–961. doi: 10.1016/s0092-8674(00)80280-9. [DOI] [PubMed] [Google Scholar]
  20. Matsuo H., Li H., McGuire A. M., Fletcher C. M., Gingras A. C., Sonenberg N., Wagner G. Structure of translation factor eIF4E bound to m7GDP and interaction with 4E-binding protein. Nat Struct Biol. 1997 Sep;4(9):717–724. doi: 10.1038/nsb0997-717. [DOI] [PubMed] [Google Scholar]
  21. Minich W. B., Balasta M. L., Goss D. J., Rhoads R. E. Chromatographic resolution of in vivo phosphorylated and nonphosphorylated eukaryotic translation initiation factor eIF-4E: increased cap affinity of the phosphorylated form. Proc Natl Acad Sci U S A. 1994 Aug 2;91(16):7668–7672. doi: 10.1073/pnas.91.16.7668. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Morley S. J., Curtis P. S., Pain V. M. eIF4G: translation's mystery factor begins to yield its secrets. RNA. 1997 Oct;3(10):1085–1104. [PMC free article] [PubMed] [Google Scholar]
  23. Morley S. J., Dever T. E., Etchison D., Traugh J. A. Phosphorylation of eIF-4F by protein kinase C or multipotential S6 kinase stimulates protein synthesis at initiation. J Biol Chem. 1991 Mar 15;266(8):4669–4672. [PubMed] [Google Scholar]
  24. Morley S. J. Intracellular signalling pathways regulating initiation factor eIF4E phosphorylation during the activation of cell growth. Biochem Soc Trans. 1997 May;25(2):503–509. doi: 10.1042/bst0250503. [DOI] [PubMed] [Google Scholar]
  25. Morley S. J., McKendrick L. Involvement of stress-activated protein kinase and p38/RK mitogen-activated protein kinase signaling pathways in the enhanced phosphorylation of initiation factor 4E in NIH 3T3 cells. J Biol Chem. 1997 Jul 11;272(28):17887–17893. doi: 10.1074/jbc.272.28.17887. [DOI] [PubMed] [Google Scholar]
  26. Morley S. J., Pain V. M. Hormone-induced meiotic maturation in Xenopus oocytes occurs independently of p70s6k activation and is associated with enhanced initiation factor (eIF)-4F phosphorylation and complex formation. J Cell Sci. 1995 Apr;108(Pt 4):1751–1760. doi: 10.1242/jcs.108.4.1751. [DOI] [PubMed] [Google Scholar]
  27. Morley S. J., Pain V. M. Translational regulation during activation of porcine peripheral blood lymphocytes: association and phosphorylation of the alpha and gamma subunits of the initiation factor complex eIF-4F. Biochem J. 1995 Dec 1;312(Pt 2):627–635. doi: 10.1042/bj3120627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Morley S. J. Signal transduction mechanisms in the regulation of protein synthesis. Mol Biol Rep. 1994 May;19(3):221–231. doi: 10.1007/BF00986964. [DOI] [PubMed] [Google Scholar]
  29. Morley S. J. Signalling through either the p38 or ERK mitogen-activated protein (MAP) kinase pathway is obligatory for phorbol ester and T cell receptor complex (TCR-CD3)-stimulated phosphorylation of initiation factor (eIF) 4E in Jurkat T cells. FEBS Lett. 1997 Dec 1;418(3):327–332. doi: 10.1016/s0014-5793(97)01405-1. [DOI] [PubMed] [Google Scholar]
  30. Morley S. J., Traugh J. A. Stimulation of translation in 3T3-L1 cells in response to insulin and phorbol ester is directly correlated with increased phosphate labelling of initiation factor (eIF-) 4F and ribosomal protein S6. Biochimie. 1993;75(11):985–989. doi: 10.1016/0300-9084(93)90149-m. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Panniers R. Translational control during heat shock. Biochimie. 1994;76(8):737–747. doi: 10.1016/0300-9084(94)90078-7. [DOI] [PubMed] [Google Scholar]
  33. Pause A., Belsham G. J., Gingras A. C., Donzé O., Lin T. A., Lawrence J. C., Jr, Sonenberg N. Insulin-dependent stimulation of protein synthesis by phosphorylation of a regulator of 5'-cap function. Nature. 1994 Oct 27;371(6500):762–767. doi: 10.1038/371762a0. [DOI] [PubMed] [Google Scholar]
  34. Pause A., Sonenberg N. Mutational analysis of a DEAD box RNA helicase: the mammalian translation initiation factor eIF-4A. EMBO J. 1992 Jul;11(7):2643–2654. doi: 10.1002/j.1460-2075.1992.tb05330.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Poulin F., Gingras A. C., Olsen H., Chevalier S., Sonenberg N. 4E-BP3, a new member of the eukaryotic initiation factor 4E-binding protein family. J Biol Chem. 1998 May 29;273(22):14002–14007. doi: 10.1074/jbc.273.22.14002. [DOI] [PubMed] [Google Scholar]
  36. Proud C. G., Denton R. M. Molecular mechanisms for the control of translation by insulin. Biochem J. 1997 Dec 1;328(Pt 2):329–341. doi: 10.1042/bj3280329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Pyronnet S., Imataka H., Gingras A. C., Fukunaga R., Hunter T., Sonenberg N. Human eukaryotic translation initiation factor 4G (eIF4G) recruits mnk1 to phosphorylate eIF4E. EMBO J. 1999 Jan 4;18(1):270–279. doi: 10.1093/emboj/18.1.270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Rowlands A. G., Montine K. S., Henshaw E. C., Panniers R. Physiological stresses inhibit guanine-nucleotide-exchange factor in Ehrlich cells. Eur J Biochem. 1988 Jul 15;175(1):93–99. doi: 10.1111/j.1432-1033.1988.tb14170.x. [DOI] [PubMed] [Google Scholar]
  39. Scheper G. C., Mulder J., Kleijn M., Voorma H. O., Thomas A. A., van Wijk R. Inactivation of eIF2B and phosphorylation of PHAS-I in heat-shocked rat hepatoma cells. J Biol Chem. 1997 Oct 24;272(43):26850–26856. doi: 10.1074/jbc.272.43.26850. [DOI] [PubMed] [Google Scholar]
  40. Scheper G. C., Thomas A. A., van Wijk R. Inactivation of eukaryotic initiation factor 2B in vitro by heat shock. Biochem J. 1998 Sep 1;334(Pt 2):463–467. doi: 10.1042/bj3340463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Scott P. H., Brunn G. J., Kohn A. D., Roth R. A., Lawrence J. C., Jr Evidence of insulin-stimulated phosphorylation and activation of the mammalian target of rapamycin mediated by a protein kinase B signaling pathway. Proc Natl Acad Sci U S A. 1998 Jun 23;95(13):7772–7777. doi: 10.1073/pnas.95.13.7772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Stokoe D., Campbell D. G., Nakielny S., Hidaka H., Leevers S. J., Marshall C., Cohen P. MAPKAP kinase-2; a novel protein kinase activated by mitogen-activated protein kinase. EMBO J. 1992 Nov;11(11):3985–3994. doi: 10.1002/j.1460-2075.1992.tb05492.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Stokoe D., Engel K., Campbell D. G., Cohen P., Gaestel M. Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins. FEBS Lett. 1992 Nov 30;313(3):307–313. doi: 10.1016/0014-5793(92)81216-9. [DOI] [PubMed] [Google Scholar]
  44. Wakiyama M., Saigoh M., Shiokawa K., Miura K. mRNA encoding the translation initiation factor eIF-4E is expressed early in Xenopus embryogenesis. FEBS Lett. 1995 Feb 27;360(2):191–193. doi: 10.1016/0014-5793(95)00081-j. [DOI] [PubMed] [Google Scholar]
  45. Wang X., Flynn A., Waskiewicz A. J., Webb B. L., Vries R. G., Baines I. A., Cooper J. A., Proud C. G. The phosphorylation of eukaryotic initiation factor eIF4E in response to phorbol esters, cell stresses, and cytokines is mediated by distinct MAP kinase pathways. J Biol Chem. 1998 Apr 17;273(16):9373–9377. doi: 10.1074/jbc.273.16.9373. [DOI] [PubMed] [Google Scholar]
  46. Wang X., Proud C. G. p70 S6 kinase is activated by sodium arsenite in adult rat cardiomyocytes: roles for phosphatidylinositol 3-kinase and p38 MAP kinase. Biochem Biophys Res Commun. 1997 Sep 8;238(1):207–212. doi: 10.1006/bbrc.1997.7273. [DOI] [PubMed] [Google Scholar]
  47. Waskiewicz A. J., Cooper J. A. Mitogen and stress response pathways: MAP kinase cascades and phosphatase regulation in mammals and yeast. Curr Opin Cell Biol. 1995 Dec;7(6):798–805. doi: 10.1016/0955-0674(95)80063-8. [DOI] [PubMed] [Google Scholar]
  48. Waskiewicz A. J., Flynn A., Proud C. G., Cooper J. A. Mitogen-activated protein kinases activate the serine/threonine kinases Mnk1 and Mnk2. EMBO J. 1997 Apr 15;16(8):1909–1920. doi: 10.1093/emboj/16.8.1909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Waskiewicz A. J., Johnson J. C., Penn B., Mahalingam M., Kimball S. R., Cooper J. A. Phosphorylation of the cap-binding protein eukaryotic translation initiation factor 4E by protein kinase Mnk1 in vivo. Mol Cell Biol. 1999 Mar;19(3):1871–1880. doi: 10.1128/mcb.19.3.1871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wells S. E., Hillner P. E., Vale R. D., Sachs A. B. Circularization of mRNA by eukaryotic translation initiation factors. Mol Cell. 1998 Jul;2(1):135–140. doi: 10.1016/s1097-2765(00)80122-7. [DOI] [PubMed] [Google Scholar]
  51. Wickens M., Anderson P., Jackson R. J. Life and death in the cytoplasm: messages from the 3' end. Curr Opin Genet Dev. 1997 Apr;7(2):220–232. doi: 10.1016/s0959-437x(97)80132-3. [DOI] [PubMed] [Google Scholar]
  52. von Manteuffel S. R., Gingras A. C., Ming X. F., Sonenberg N., Thomas G. 4E-BP1 phosphorylation is mediated by the FRAP-p70s6k pathway and is independent of mitogen-activated protein kinase. Proc Natl Acad Sci U S A. 1996 Apr 30;93(9):4076–4080. doi: 10.1073/pnas.93.9.4076. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES