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. 2002 Oct 15;367(Pt 2):525–532. doi: 10.1042/BJ20020916

Stress-induced regulation of eukaryotic elongation factor 2 kinase by SB 203580-sensitive and -insensitive pathways.

Axel Knebel 1, Claire E Haydon 1, Nick Morrice 1, Philip Cohen 1
PMCID: PMC1222910  PMID: 12171600

Abstract

Eukaryotic elongation factor 2 (eEF2) kinase, the enzyme that inactivates eEF2, is controlled by phosphorylation. Previous work showed that stress-activated protein kinase 4 (SAPK4, also called p38delta) inhibits eEF2 kinase in vitro by phosphorylating Ser-359, while ribosomal protein S6 kinases inhibit eEF2 kinase by phosphorylating Ser-366 [Knebel, Morrice and Cohen (2001) EMBO J. 20, 4360-4369; Wang, Li, Williams, Terada, Alessi and Proud (2001) EMBO J. 20, 4370-4379]. In the present study we have examined the effects of the protein synthesis inhibitor anisomycin and tumour necrosis factor-alpha (TNF-alpha) on the phosphorylation of eEF2 kinase. We demonstrate that Ser-359, Ser-366 and two novel sites (Ser-377 and Ser-396) are all phosphorylated in human epithelial KB cells, but only the phosphorylation of Ser-359 and Ser-377 increases in response to these agonists and correlates with the dephosphorylation (activation) of eEF2. Ser-377 is probably a substrate of MAPKAP-K2/K3 (mitogen-activated protein kinase-activated protein kinase 2/kinase 3) in cells, because eEF2 kinase is phosphorylated efficiently by these protein kinases in vitro and phosphorylation of this site, induced by TNF-alpha and low (but not high) concentrations of anisomycin, is prevented by SB 203580, which inhibits SAPK2a/p38, their "upstream" activator. The phosphorylation of Ser-359 induced by high concentrations of anisomycin is probably catalysed by SAPK4/p38delta in cells, because no other stress-activated, proline-directed protein kinase tested phosphorylates this site in vitro and phosphorylation is insensitive to SB 203580. Interestingly, the phosphorylation of Ser-359 induced by TNF-alpha or low concentrations of anisomycin is suppressed by SB 203580, indicating that phosphorylation is also mediated by a novel pathway. Since the phosphorylation of Ser-377 does not inhibit eEF2 kinase in vitro, our results suggest that anisomycin or TNF-alpha inhibit eEF2 kinase via the phosphorylation of Ser-359.

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Selected References

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  1. 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]
  2. Clauser K. R., Baker P., Burlingame A. L. Role of accurate mass measurement (+/- 10 ppm) in protein identification strategies employing MS or MS/MS and database searching. Anal Chem. 1999 Jul 15;71(14):2871–2882. doi: 10.1021/ac9810516. [DOI] [PubMed] [Google Scholar]
  3. Clifton A. D., Young P. R., Cohen P. A comparison of the substrate specificity of MAPKAP kinase-2 and MAPKAP kinase-3 and their activation by cytokines and cellular stress. FEBS Lett. 1996 Sep 2;392(3):209–214. doi: 10.1016/0014-5793(96)00816-2. [DOI] [PubMed] [Google Scholar]
  4. Diggle T. A., Subkhankulova T., Lilley K. S., Shikotra N., Willis A. E., Redpath N. T. Phosphorylation of elongation factor-2 kinase on serine 499 by cAMP-dependent protein kinase induces Ca2+/calmodulin-independent activity. Biochem J. 2001 Feb 1;353(Pt 3):621–626. doi: 10.1042/0264-6021:3530621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Eyers P. A., Craxton M., Morrice N., Cohen P., Goedert M. Conversion of SB 203580-insensitive MAP kinase family members to drug-sensitive forms by a single amino-acid substitution. Chem Biol. 1998 Jun;5(6):321–328. doi: 10.1016/s1074-5521(98)90170-3. [DOI] [PubMed] [Google Scholar]
  6. Eyers P. A., van den IJssel P., Quinlan R. A., Goedert M., Cohen P. Use of a drug-resistant mutant of stress-activated protein kinase 2a/p38 to validate the in vivo specificity of SB 203580. FEBS Lett. 1999 May 21;451(2):191–196. doi: 10.1016/s0014-5793(99)00552-9. [DOI] [PubMed] [Google Scholar]
  7. Gum R. J., McLaughlin M. M., Kumar S., Wang Z., Bower M. J., Lee J. C., Adams J. L., Livi G. P., Goldsmith E. J., Young P. R. Acquisition of sensitivity of stress-activated protein kinases to the p38 inhibitor, SB 203580, by alteration of one or more amino acids within the ATP binding pocket. J Biol Chem. 1998 Jun 19;273(25):15605–15610. doi: 10.1074/jbc.273.25.15605. [DOI] [PubMed] [Google Scholar]
  8. Hovland R., Eikhom T. S., Proud C. G., Cressey L. I., Lanotte M., Døskeland S. O., Houge G. cAMP inhibits translation by inducing Ca2+/calmodulin-independent elongation factor 2 kinase activity in IPC-81 cells. FEBS Lett. 1999 Feb 5;444(1):97–101. doi: 10.1016/s0014-5793(99)00039-3. [DOI] [PubMed] [Google Scholar]
  9. Kigoshi T., Uchida K., Morimoto S. Elongation factor 2 as the major substrate for Ca2+/calmodulin-dependent protein kinase in rat adrenal glomerulosa cells. J Steroid Biochem. 1989 Mar;32(3):381–385. doi: 10.1016/0022-4731(89)90210-0. [DOI] [PubMed] [Google Scholar]
  10. Knebel A., Morrice N., Cohen P. A novel method to identify protein kinase substrates: eEF2 kinase is phosphorylated and inhibited by SAPK4/p38delta. EMBO J. 2001 Aug 15;20(16):4360–4369. doi: 10.1093/emboj/20.16.4360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. McLaughlin M. M., Kumar S., McDonnell P. C., Van Horn S., Lee J. C., Livi G. P., Young P. R. Identification of mitogen-activated protein (MAP) kinase-activated protein kinase-3, a novel substrate of CSBP p38 MAP kinase. J Biol Chem. 1996 Apr 5;271(14):8488–8492. doi: 10.1074/jbc.271.14.8488. [DOI] [PubMed] [Google Scholar]
  12. Nairn A. C., Bhagat B., Palfrey H. C. Identification of calmodulin-dependent protein kinase III and its major Mr 100,000 substrate in mammalian tissues. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7939–7943. doi: 10.1073/pnas.82.23.7939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. New L., Jiang Y., Zhao M., Liu K., Zhu W., Flood L. J., Kato Y., Parry G. C., Han J. PRAK, a novel protein kinase regulated by the p38 MAP kinase. EMBO J. 1998 Jun 15;17(12):3372–3384. doi: 10.1093/emboj/17.12.3372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ni H., Wang X. S., Diener K., Yao Z. MAPKAPK5, a novel mitogen-activated protein kinase (MAPK)-activated protein kinase, is a substrate of the extracellular-regulated kinase (ERK) and p38 kinase. Biochem Biophys Res Commun. 1998 Feb 13;243(2):492–496. doi: 10.1006/bbrc.1998.8135. [DOI] [PubMed] [Google Scholar]
  16. Palfrey H. C. Presence in many mammalian tissues of an identical major cytosolic substrate (Mr 100 000) for calmodulin-dependent protein kinase. FEBS Lett. 1983 Jun 27;157(1):183–190. doi: 10.1016/0014-5793(83)81142-9. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Rouse J., Cohen P., Trigon S., Morange M., Alonso-Llamazares A., Zamanillo D., Hunt T., Nebreda A. R. A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins. Cell. 1994 Sep 23;78(6):1027–1037. doi: 10.1016/0092-8674(94)90277-1. [DOI] [PubMed] [Google Scholar]
  20. Ryazanov A. G., Natapov P. G., Shestakova E. A., Severin F. F., Spirin A. S. Phosphorylation of the elongation factor 2: the fifth Ca2+/calmodulin-dependent system of protein phosphorylation. Biochimie. 1988 May;70(5):619–626. doi: 10.1016/0300-9084(88)90245-3. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Stokoe D., Caudwell B., Cohen P. T., Cohen P. The substrate specificity and structure of mitogen-activated protein (MAP) kinase-activated protein kinase-2. Biochem J. 1993 Dec 15;296(Pt 3):843–849. doi: 10.1042/bj2960843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Wang X., Li W., Williams M., Terada N., Alessi D. R., Proud C. G. Regulation of elongation factor 2 kinase by p90(RSK1) and p70 S6 kinase. EMBO J. 2001 Aug 15;20(16):4370–4379. doi: 10.1093/emboj/20.16.4370. [DOI] [PMC free article] [PubMed] [Google Scholar]

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