Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1998 Dec 15;336(Pt 3):525–529. doi: 10.1042/bj3360525

Regulation of protein-synthesis elongation-factor-2 kinase by cAMP in adipocytes.

T A Diggle 1, N T Redpath 1, K J Heesom 1, R M Denton 1
PMCID: PMC1219899  PMID: 9841860

Abstract

Treatment of primary rat epididymal adipocytes or 3T3-L1 adipocytes with various agents which increase cAMP led to the phosphorylation of eukaryotic translation elongation factor-2 (eEF-2). The increase in eEF-2 phosphorylation was a consequence of the activation of eEF-2 kinase (eEF-2K), which is a Ca2+/calmodulin-dependent kinase. eEF-2K was shown to be essentially inactive at less than 0.1 microM free Ca2+ when measured in cell-free extracts. Treatment of adipocytes with isoproterenol induced Ca2+-independent eEF-2K activity, and an 8-10-fold activation of eEF-2K was observed at Ca2+ concentrations of less than 0.1 microM. Increased cAMP in 3T3-L1 adipocytes led to the inhibition of total protein synthesis and decreased the rate of polypeptide-chain elongation. We also show that the phosphorylation of eEF-2 and the activity of eEF-2K are insulin-regulated in adipocytes. These results demonstrate a novel mechanism for the control of protein synthesis by hormones which act by increasing cytoplasmic cAMP.

Full Text

The Full Text of this article is available as a PDF (123.1 KB).

Selected References

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

  1. Diggle T. A., Bloomberg G. B., Denton R. M. Further characterization of the acid-soluble phosphoprotein (SDS/PAGE apparent molecular mass of 22 kDa) in rat fat-cells by peptide sequencing and immuno-analysis: effects of insulin and isoprenaline. Biochem J. 1995 Feb 15;306(Pt 1):135–139. doi: 10.1042/bj3060135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Diggle T. A., Denton R. M. Comparison of the effects of insulin and adrenergic agonists on the phosphorylation of an acid-soluble 22 kDa protein in rat epididymal fat-pads and isolated fat-cells. Biochem J. 1992 Mar 15;282(Pt 3):729–736. doi: 10.1042/bj2820729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Frost V., Morley S. J., Mercep L., Meyer T., Fabbro D., Ferrari S. The phosphodiesterase inhibitor SQ 20006 selectively blocks mitogen activation of p70S6k and transition to S phase of the cell division cycle without affecting the steady state phosphorylation of eIF-4E. J Biol Chem. 1995 Nov 3;270(44):26698–26706. doi: 10.1074/jbc.270.44.26698. [DOI] [PubMed] [Google Scholar]
  4. Graves L. M., Bornfeldt K. E., Argast G. M., Krebs E. G., Kong X., Lin T. A., Lawrence J. C., Jr cAMP- and rapamycin-sensitive regulation of the association of eukaryotic initiation factor 4E and the translational regulator PHAS-I in aortic smooth muscle cells. Proc Natl Acad Sci U S A. 1995 Aug 1;92(16):7222–7226. doi: 10.1073/pnas.92.16.7222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Kelly K. L., Deeney J. T., Corkey B. E. Cytosolic free calcium in adipocytes. Distinct mechanisms of regulation and effects on insulin action. J Biol Chem. 1989 Aug 5;264(22):12754–12757. [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. McKenzie F. R., Pouysségur J. cAMP-mediated growth inhibition in fibroblasts is not mediated via mitogen-activated protein (MAP) kinase (ERK) inhibition. cAMP-dependent protein kinase induces a temporal shift in growth factor-stimulated MAP kinases. J Biol Chem. 1996 Jun 7;271(23):13476–13483. doi: 10.1074/jbc.271.23.13476. [DOI] [PubMed] [Google Scholar]
  9. Monfar M., Lemon K. P., Grammer T. C., Cheatham L., Chung J., Vlahos C. J., Blenis J. Activation of pp70/85 S6 kinases in interleukin-2-responsive lymphoid cells is mediated by phosphatidylinositol 3-kinase and inhibited by cyclic AMP. Mol Cell Biol. 1995 Jan;15(1):326–337. doi: 10.1128/mcb.15.1.326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Moule S. K., Welsh G. I., Edgell N. J., Foulstone E. J., Proud C. G., Denton R. M. Regulation of protein kinase B and glycogen synthase kinase-3 by insulin and beta-adrenergic agonists in rat epididymal fat cells. Activation of protein kinase B by wortmannin-sensitive and -insensitive mechanisms. J Biol Chem. 1997 Mar 21;272(12):7713–7719. doi: 10.1074/jbc.272.12.7713. [DOI] [PubMed] [Google Scholar]
  11. Nairn A. C., Palfrey H. C. Identification of the major Mr 100,000 substrate for calmodulin-dependent protein kinase III in mammalian cells as elongation factor-2. J Biol Chem. 1987 Dec 25;262(36):17299–17303. [PubMed] [Google Scholar]
  12. Ovchinnikov L. P., Motuz L. P., Natapov P. G., Averbuch L. J., Wettenhall R. E., Szyszka R., Kramer G., Hardesty B. Three phosphorylation sites in elongation factor 2. FEBS Lett. 1990 Nov 26;275(1-2):209–212. doi: 10.1016/0014-5793(90)81473-2. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Petritsch C., Woscholski R., Edelmann H. M., Ballou L. M. Activation of p70 S6 kinase and erk-encoded mitogen-activated protein kinases is resistant to high cyclic nucleotide levels in Swiss 3T3 fibroblasts. J Biol Chem. 1995 Nov 3;270(44):26619–26625. doi: 10.1074/jbc.270.44.26619. [DOI] [PubMed] [Google Scholar]
  15. Price N. T., Redpath N. T., Severinov K. V., Campbell D. G., Russell J. M., Proud C. G. Identification of the phosphorylation sites in elongation factor-2 from rabbit reticulocytes. FEBS Lett. 1991 May 6;282(2):253–258. doi: 10.1016/0014-5793(91)80489-p. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Proud C. G. Peptide-chain elongation in eukaryotes. Mol Biol Rep. 1994 May;19(3):161–170. doi: 10.1007/BF00986958. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. 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]
  21. Redpath N. T., Price N. T., Proud C. G. Cloning and expression of cDNA encoding protein synthesis elongation factor-2 kinase. J Biol Chem. 1996 Jul 19;271(29):17547–17554. [PubMed] [Google Scholar]
  22. 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]
  23. Redpath N. T., Proud C. G. Activity of protein phosphatases against initiation factor-2 and elongation factor-2. Biochem J. 1990 Nov 15;272(1):175–180. doi: 10.1042/bj2720175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Redpath N. T., Proud C. G. Cyclic AMP-dependent protein kinase phosphorylates rabbit reticulocyte elongation factor-2 kinase and induces calcium-independent activity. Biochem J. 1993 Jul 1;293(Pt 1):31–34. doi: 10.1042/bj2930031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Redpath N. T., Proud C. G. The tumour promoter okadaic acid inhibits reticulocyte-lysate protein synthesis by increasing the net phosphorylation of elongation factor 2. Biochem J. 1989 Aug 15;262(1):69–75. doi: 10.1042/bj2620069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Requero A. M., Díaz J. P., Ayuso-Parrilla M. S., Parrilla R. On the mechanism of the glucagon-induced inhibition of hepatic protein synthesis. Arch Biochem Biophys. 1979 Jun;195(1):223–234. doi: 10.1016/0003-9861(79)90344-8. [DOI] [PubMed] [Google Scholar]
  28. Ryazanov A. G., Shestakova E. A., Natapov P. G. Phosphorylation of elongation factor 2 by EF-2 kinase affects rate of translation. Nature. 1988 Jul 14;334(6178):170–173. doi: 10.1038/334170a0. [DOI] [PubMed] [Google Scholar]
  29. Ryazanov A. G., Ward M. D., Mendola C. E., Pavur K. S., Dorovkov M. V., Wiedmann M., Erdjument-Bromage H., Tempst P., Parmer T. G., Prostko C. R. Identification of a new class of protein kinases represented by eukaryotic elongation factor-2 kinase. Proc Natl Acad Sci U S A. 1997 May 13;94(10):4884–4889. doi: 10.1073/pnas.94.10.4884. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES