Stress-induced regulation of eukaryotic elongation factor 2 kinase by SB 203580-sensitive and -insensitive pathways.
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.
Knebel
Axel
A
MRC Protein Phosphorylation Unit, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
Haydon
Claire E
CE
Morrice
Nick
N
Cohen
Philip
P
D000595:Amino Acid Sequence; D000841:Anisomycin / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000187:drug effects / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D054736:Elongation Factor 2 Kinase; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D047908:Intracellular Signaling Peptides and Proteins; D048290:Mitogen-Activated Protein Kinase 11; D048292:Mitogen-Activated Protein Kinase 13; D020928:Mitogen-Activated Protein Kinases / Q000187:drug effects / Q000378:metabolism; D008969:Molecular Sequence Data; D020652:Peptide Elongation Factor 2 / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology*; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction; D013312:Stress, Physiological; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases; D000099190:MAP-Kinase-Activated Kinase 2
D000841:Anisomycin; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D054736:Elongation Factor 2 Kinase; D004791:Enzyme Inhibitors; D007093:Imidazoles; D047908:Intracellular Signaling Peptides and Proteins; D048290:Mitogen-Activated Protein Kinase 11; D048292:Mitogen-Activated Protein Kinase 13; D020928:Mitogen-Activated Protein Kinases; D020652:Peptide Elongation Factor 2; D017346:Protein Serine-Threonine Kinases; D011725:Pyridines; D012694:Serine; D014409:Tumor Necrosis Factor-alpha; D048051:p38 Mitogen-Activated Protein Kinases; D000099190:MAP-Kinase-Activated Kinase 2; C111049:MAP-kinase-activated kinase 5; C098416:MAP-kinase-activated kinase 3; C517689:EEF2K protein, human; C093642:SB 203580