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

Biochem J. 2002 Oct 15;367(Pt 2):525-32. doi: 10.1042/BJ20020916.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Anisomycin / pharmacology
  • Calcium-Calmodulin-Dependent Protein Kinases / drug effects
  • Calcium-Calmodulin-Dependent Protein Kinases / metabolism*
  • Dose-Response Relationship, Drug
  • Elongation Factor 2 Kinase
  • Enzyme Inhibitors / pharmacology*
  • Humans
  • Imidazoles / pharmacology*
  • Intracellular Signaling Peptides and Proteins
  • Mitogen-Activated Protein Kinase 11
  • Mitogen-Activated Protein Kinase 13
  • Mitogen-Activated Protein Kinases / drug effects
  • Mitogen-Activated Protein Kinases / metabolism
  • Molecular Sequence Data
  • Peptide Elongation Factor 2 / metabolism
  • Phosphorylation
  • Protein-Serine-Threonine Kinases / metabolism
  • Pyridines / pharmacology*
  • Serine / metabolism
  • Signal Transduction
  • Stress, Physiological
  • Tumor Cells, Cultured
  • Tumor Necrosis Factor-alpha / pharmacology
  • p38 Mitogen-Activated Protein Kinases

Substances

  • Enzyme Inhibitors
  • Imidazoles
  • Intracellular Signaling Peptides and Proteins
  • Peptide Elongation Factor 2
  • Pyridines
  • Tumor Necrosis Factor-alpha
  • Serine
  • Anisomycin
  • MAP-kinase-activated kinase 5
  • MAP-kinase-activated kinase 2
  • MAP-kinase-activated kinase 3
  • Mitogen-Activated Protein Kinase 13
  • EEF2K protein, human
  • Protein-Serine-Threonine Kinases
  • Calcium-Calmodulin-Dependent Protein Kinases
  • Elongation Factor 2 Kinase
  • Mitogen-Activated Protein Kinase 11
  • Mitogen-Activated Protein Kinases
  • p38 Mitogen-Activated Protein Kinases
  • SB 203580