Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1998 Aug 15;334(Pt 1):261–267. doi: 10.1042/bj3340261

Amino acid availability regulates p70 S6 kinase and multiple translation factors.

X Wang 1, L E Campbell 1, C M Miller 1, C G Proud 1
PMCID: PMC1219687  PMID: 9693128

Abstract

Incubation of Chinese hamster ovary cells without amino acids for up to 60 min caused a rapid marked decrease in p70 S6 kinase activity and increased binding of initiation factor eIF4E to its inhibitory regulator protein 4E-BP1. This was associated with dephosphorylation of 4E-BP1 and eIF4E and dissociation of eIF4E from eIF4G. All these effects were rapidly reversed by resupplying a mixture of amino acids and this was blocked by rapamycin and by inhibitors of phosphatidylinositol 3-kinase, implying a role for phosphatidylinositol 3-kinase in the signalling pathway linking amino acids with the control of p70 S6 kinase activity and the phosphorylation of these translation factors. Amino acid withdrawal also led to changes in the phosphorylation of other translation factors; phosphorylation of eIF4E decreased whereas elongation factor eEF2 became more heavily phosphorylated, each of these changes being associated with decreased activity of the factor in question. Earlier studies have suggested that protein kinase B (PKB) may act upstream of p70 S6 kinase. However, amino acids did not affect the activity of PKB, indicating that amino acids activate p70 S6 kinase through a pathway independent of this enzyme. Studies with individual amino acids suggested that the effects on p70 S6 kinase activity and translation-factor phosphorylation were independent of cell swelling. The data show that amino acid supply regulates multiple translation factors in mammalian cells.

Full Text

The Full Text of this article is available as a PDF (322.4 KB).

Selected References

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

  1. Alessi D. R., Andjelkovic M., Caudwell B., Cron P., Morrice N., Cohen P., Hemmings B. A. Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J. 1996 Dec 2;15(23):6541–6551. [PMC free article] [PubMed] [Google Scholar]
  2. Alessi D. R., Deak M., Casamayor A., Caudwell F. B., Morrice N., Norman D. G., Gaffney P., Reese C. B., MacDougall C. N., Harbison D. 3-Phosphoinositide-dependent protein kinase-1 (PDK1): structural and functional homology with the Drosophila DSTPK61 kinase. Curr Biol. 1997 Oct 1;7(10):776–789. doi: 10.1016/s0960-9822(06)00336-8. [DOI] [PubMed] [Google Scholar]
  3. Alessi D. R., Kozlowski M. T., Weng Q. P., Morrice N., Avruch J. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates and activates the p70 S6 kinase in vivo and in vitro. Curr Biol. 1998 Jan 15;8(2):69–81. doi: 10.1016/s0960-9822(98)70037-5. [DOI] [PubMed] [Google Scholar]
  4. Blommaart E. F., Luiken J. J., Blommaart P. J., van Woerkom G. M., Meijer A. J. Phosphorylation of ribosomal protein S6 is inhibitory for autophagy in isolated rat hepatocytes. J Biol Chem. 1995 Feb 3;270(5):2320–2326. doi: 10.1074/jbc.270.5.2320. [DOI] [PubMed] [Google Scholar]
  5. Burgering B. M., Coffer P. J. Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature. 1995 Aug 17;376(6541):599–602. doi: 10.1038/376599a0. [DOI] [PubMed] [Google Scholar]
  6. Burnett P. E., Barrow R. K., Cohen N. A., Snyder S. H., Sabatini D. M. RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1. Proc Natl Acad Sci U S A. 1998 Feb 17;95(4):1432–1437. doi: 10.1073/pnas.95.4.1432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Carlberg U., Nilsson A., Nygård O. Functional properties of phosphorylated elongation factor 2. Eur J Biochem. 1990 Aug 17;191(3):639–645. doi: 10.1111/j.1432-1033.1990.tb19169.x. [DOI] [PubMed] [Google Scholar]
  8. Cheatham B., Vlahos C. J., Cheatham L., Wang L., Blenis J., Kahn C. R. Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation. Mol Cell Biol. 1994 Jul;14(7):4902–4911. doi: 10.1128/mcb.14.7.4902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chung J., Grammer T. C., Lemon K. P., Kazlauskas A., Blenis J. PDGF- and insulin-dependent pp70S6k activation mediated by phosphatidylinositol-3-OH kinase. Nature. 1994 Jul 7;370(6484):71–75. doi: 10.1038/370071a0. [DOI] [PubMed] [Google Scholar]
  10. Clemens M. J., Galpine A., Austin S. A., Panniers R., Henshaw E. C., Duncan R., Hershey J. W., Pollard J. W. Regulation of polypeptide chain initiation in Chinese hamster ovary cells with a temperature-sensitive leucyl-tRNA synthetase. Changes in phosphorylation of initiation factor eIF-2 and in the activity of the guanine nucleotide exchange factor GEF. J Biol Chem. 1987 Jan 15;262(2):767–771. [PubMed] [Google Scholar]
  11. Cross D. A., Alessi D. R., Cohen P., Andjelkovich M., Hemmings B. A. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature. 1995 Dec 21;378(6559):785–789. doi: 10.1038/378785a0. [DOI] [PubMed] [Google Scholar]
  12. Cross D. A., Watt P. W., Shaw M., van der Kaay J., Downes C. P., Holder J. C., Cohen P. Insulin activates protein kinase B, inhibits glycogen synthase kinase-3 and activates glycogen synthase by rapamycin-insensitive pathways in skeletal muscle and adipose tissue. FEBS Lett. 1997 Apr 7;406(1-2):211–215. doi: 10.1016/s0014-5793(97)00240-8. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Dahl J., Freund R., Blenis J., Benjamin T. L. Studies of partially transforming polyomavirus mutants establish a role for phosphatidylinositol 3-kinase in activation of pp70 S6 kinase. Mol Cell Biol. 1996 Jun;16(6):2728–2735. doi: 10.1128/mcb.16.6.2728. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Dickens M., Chin J. E., Roth R. A., Ellis L., Denton R. M., Tavaré J. M. Characterization of insulin-stimulated protein serine/threonine kinases in CHO cells expressing human insulin receptors with point and deletion mutations. Biochem J. 1992 Oct 1;287(Pt 1):201–209. doi: 10.1042/bj2870201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Diggle T. A., Moule S. K., Avison M. B., Flynn A., Foulstone E. J., Proud C. G., Denton R. M. Both rapamycin-sensitive and -insensitive pathways are involved in the phosphorylation of the initiation factor-4E-binding protein (4E-BP1) in response to insulin in rat epididymal fat-cells. Biochem J. 1996 Jun 1;316(Pt 2):447–453. doi: 10.1042/bj3160447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Flynn A., Proud C. G. Insulin and phorbol ester stimulate initiation factor eIF-4E phosphorylation by distinct pathways in Chinese hamster ovary cells overexpressing the insulin receptor. Eur J Biochem. 1996 Feb 15;236(1):40–47. doi: 10.1111/j.1432-1033.1996.00040.x. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Flynn A., Proud C. G. The role of eIF4 in cell proliferation. Cancer Surv. 1996;27:293–310. [PubMed] [Google Scholar]
  21. Flynn A., Proud G. Insulin-stimulated phosphorylation of initiation factor 4E is mediated by the MAP kinase pathway. FEBS Lett. 1996 Jul 1;389(2):162–166. doi: 10.1016/0014-5793(96)00564-9. [DOI] [PubMed] [Google Scholar]
  22. Fox H. L., Kimball S. R., Jefferson L. S., Lynch C. J. Amino acids stimulate phosphorylation of p70S6k and organization of rat adipocytes into multicellular clusters. Am J Physiol. 1998 Jan;274(1 Pt 1):C206–C213. doi: 10.1152/ajpcell.1998.274.1.C206. [DOI] [PubMed] [Google Scholar]
  23. Franke T. F., Kaplan D. R., Cantley L. C., Toker A. Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-3,4-bisphosphate. Science. 1997 Jan 31;275(5300):665–668. doi: 10.1126/science.275.5300.665. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Hinnebusch A. G. Translational control of GCN4: an in vivo barometer of initiation-factor activity. Trends Biochem Sci. 1994 Oct;19(10):409–414. doi: 10.1016/0968-0004(94)90089-2. [DOI] [PubMed] [Google Scholar]
  26. Hinnebusch A. G. Translational regulation of yeast GCN4. A window on factors that control initiator-trna binding to the ribosome. J Biol Chem. 1997 Aug 29;272(35):21661–21664. doi: 10.1074/jbc.272.35.21661. [DOI] [PubMed] [Google Scholar]
  27. Jefferies H. B., Fumagalli S., Dennis P. B., Reinhard C., Pearson R. B., Thomas G. Rapamycin suppresses 5'TOP mRNA translation through inhibition of p70s6k. EMBO J. 1997 Jun 16;16(12):3693–3704. doi: 10.1093/emboj/16.12.3693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Jefferies H. B., Reinhard C., Kozma S. C., Thomas G. Rapamycin selectively represses translation of the "polypyrimidine tract" mRNA family. Proc Natl Acad Sci U S A. 1994 May 10;91(10):4441–4445. doi: 10.1073/pnas.91.10.4441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Klippel A., Kavanaugh W. M., Pot D., Williams L. T. A specific product of phosphatidylinositol 3-kinase directly activates the protein kinase Akt through its pleckstrin homology domain. Mol Cell Biol. 1997 Jan;17(1):338–344. doi: 10.1128/mcb.17.1.338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Krause U., Rider M. H., Hue L. Protein kinase signaling pathway triggered by cell swelling and involved in the activation of glycogen synthase and acetyl-CoA carboxylase in isolated rat hepatocytes. J Biol Chem. 1996 Jul 12;271(28):16668–16673. doi: 10.1074/jbc.271.28.16668. [DOI] [PubMed] [Google Scholar]
  32. Lane H. A., Fernandez A., Lamb N. J., Thomas G. p70s6k function is essential for G1 progression. Nature. 1993 May 13;363(6425):170–172. doi: 10.1038/363170a0. [DOI] [PubMed] [Google Scholar]
  33. Lawrence J. C., Jr, Abraham R. T. PHAS/4E-BPs as regulators of mRNA translation and cell proliferation. Trends Biochem Sci. 1997 Sep;22(9):345–349. doi: 10.1016/s0968-0004(97)01101-8. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. 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]
  36. Minden A., Lin A., Claret F. X., Abo A., Karin M. Selective activation of the JNK signaling cascade and c-Jun transcriptional activity by the small GTPases Rac and Cdc42Hs. Cell. 1995 Jun 30;81(7):1147–1157. doi: 10.1016/s0092-8674(05)80019-4. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. 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]
  39. Moule S. K., Edgell N. J., Welsh G. I., Diggle T. A., Foulstone E. J., Heesom K. J., Proud C. G., Denton R. M. Multiple signalling pathways involved in the stimulation of fatty acid and glycogen synthesis by insulin in rat epididymal fat cells. Biochem J. 1995 Oct 15;311(Pt 2):595–601. doi: 10.1042/bj3110595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Peterson R. T., Schreiber S. L. Translation control: connecting mitogens and the ribosome. Curr Biol. 1998 Mar 26;8(7):R248–R250. doi: 10.1016/s0960-9822(98)70152-6. [DOI] [PubMed] [Google Scholar]
  42. Petritsch C., Woscholski R., Edelmann H. M., Parker P. J., Ballou L. M. Selective inhibition of p70 S6 kinase activation by phosphatidylinositol 3-kinase inhibitors. Eur J Biochem. 1995 Jun 1;230(2):431–438. doi: 10.1111/j.1432-1033.1995.0431h.x. [DOI] [PubMed] [Google Scholar]
  43. Pollard J. W., Galpine A. R., Clemens M. J. A novel role for aminoacyl-tRNA synthetases in the regulation of polypeptide chain initiation. Eur J Biochem. 1989 Jun 1;182(1):1–9. doi: 10.1111/j.1432-1033.1989.tb14793.x. [DOI] [PubMed] [Google Scholar]
  44. Price N., Proud C. The guanine nucleotide-exchange factor, eIF-2B. Biochimie. 1994;76(8):748–760. doi: 10.1016/0300-9084(94)90079-5. [DOI] [PubMed] [Google Scholar]
  45. Proud C. G. Protein phosphorylation in translational control. Curr Top Cell Regul. 1992;32:243–369. doi: 10.1016/b978-0-12-152832-4.50008-2. [DOI] [PubMed] [Google Scholar]
  46. Proud C. G. p70 S6 kinase: an enigma with variations. Trends Biochem Sci. 1996 May;21(5):181–185. [PubMed] [Google Scholar]
  47. Pullen N., Dennis P. B., Andjelkovic M., Dufner A., Kozma S. C., Hemmings B. A., Thomas G. Phosphorylation and activation of p70s6k by PDK1. Science. 1998 Jan 30;279(5351):707–710. doi: 10.1126/science.279.5351.707. [DOI] [PubMed] [Google Scholar]
  48. Redpath N. T., Foulstone E. J., Proud C. G. Regulation of translation elongation factor-2 by insulin via a rapamycin-sensitive signalling pathway. EMBO J. 1996 May 1;15(9):2291–2297. [PMC free article] [PubMed] [Google Scholar]
  49. Redpath N. T. High-resolution one-dimensional polyacrylamide gel isoelectric focusing of various forms of elongation factor-2. Anal Biochem. 1992 May 1;202(2):340–343. doi: 10.1016/0003-2697(92)90115-n. [DOI] [PubMed] [Google Scholar]
  50. Redpath N. T., Price N. T., Severinov K. V., Proud C. G. Regulation of elongation factor-2 by multisite phosphorylation. Eur J Biochem. 1993 Apr 15;213(2):689–699. doi: 10.1111/j.1432-1033.1993.tb17809.x. [DOI] [PubMed] [Google Scholar]
  51. Redpath N. T., Proud C. G. Molecular mechanisms in the control of translation by hormones and growth factors. Biochim Biophys Acta. 1994 Jan 13;1220(2):147–162. doi: 10.1016/0167-4889(94)90130-9. [DOI] [PubMed] [Google Scholar]
  52. Reinhard C., Fernandez A., Lamb N. J., Thomas G. Nuclear localization of p85s6k: functional requirement for entry into S phase. EMBO J. 1994 Apr 1;13(7):1557–1565. doi: 10.1002/j.1460-2075.1994.tb06418.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Rhoads R. E. Regulation of eukaryotic protein synthesis by initiation factors. J Biol Chem. 1993 Feb 15;268(5):3017–3020. [PubMed] [Google Scholar]
  54. 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]
  55. Terada N., Patel H. R., Takase K., Kohno K., Nairn A. C., Gelfand E. W. Rapamycin selectively inhibits translation of mRNAs encoding elongation factors and ribosomal proteins. Proc Natl Acad Sci U S A. 1994 Nov 22;91(24):11477–11481. doi: 10.1073/pnas.91.24.11477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Vries R. G., Flynn A., Patel J. C., Wang X., Denton R. M., Proud C. G. Heat shock increases the association of binding protein-1 with initiation factor 4E. J Biol Chem. 1997 Dec 26;272(52):32779–32784. doi: 10.1074/jbc.272.52.32779. [DOI] [PubMed] [Google Scholar]
  57. Walker K. S., Deak M., Paterson A., Hudson K., Cohen P., Alessi D. R. Activation of protein kinase B beta and gamma isoforms by insulin in vivo and by 3-phosphoinositide-dependent protein kinase-1 in vitro: comparison with protein kinase B alpha. Biochem J. 1998 Apr 1;331(Pt 1):299–308. doi: 10.1042/bj3310299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. 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]
  59. Weng Q. P., Andrabi K., Klippel A., Kozlowski M. T., Williams L. T., Avruch J. Phosphatidylinositol 3-kinase signals activation of p70 S6 kinase in situ through site-specific p70 phosphorylation. Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5744–5748. doi: 10.1073/pnas.92.12.5744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Weng Q. P., Andrabi K., Kozlowski M. T., Grove J. R., Avruch J. Multiple independent inputs are required for activation of the p70 S6 kinase. Mol Cell Biol. 1995 May;15(5):2333–2340. doi: 10.1128/mcb.15.5.2333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Wilson M., Burt A. R., Milligan G., Anderson N. G. Wortmannin-sensitive activation of p70s6k by endogenous and heterologously expressed Gi-coupled receptors. J Biol Chem. 1996 Apr 12;271(15):8537–8540. doi: 10.1074/jbc.271.15.8537. [DOI] [PubMed] [Google Scholar]
  62. Xu G., Marshall C. A., Lin T. A., Kwon G., Munivenkatappa R. B., Hill J. R., Lawrence J. C., Jr, McDaniel M. L. Insulin mediates glucose-stimulated phosphorylation of PHAS-I by pancreatic beta cells. An insulin-receptor mechanism for autoregulation of protein synthesis by translation. J Biol Chem. 1998 Feb 20;273(8):4485–4491. doi: 10.1074/jbc.273.8.4485. [DOI] [PubMed] [Google Scholar]
  63. von Manteuffel S. R., Dennis P. B., Pullen N., Gingras A. C., Sonenberg N., Thomas G. The insulin-induced signalling pathway leading to S6 and initiation factor 4E binding protein 1 phosphorylation bifurcates at a rapamycin-sensitive point immediately upstream of p70s6k. Mol Cell Biol. 1997 Sep;17(9):5426–5436. doi: 10.1128/mcb.17.9.5426. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES