A novel method to identify protein kinase substrates: eEF2 kinase is phosphorylated and inhibited by SAPK4/p38delta.
We have developed a method of general application for identifying putative substrates of protein kinases in cell extracts. Using this procedure, we identified the physiological substrates of several mitogen-activated protein kinase kinases and an authentic substrate of stress-activated protein kinase (SAPK) 2a/p38. A 120 kDa protein was detected in skeletal muscle extracts that was phosphorylated rapidly by SAPK4/p38delta, but poorly by SAPK2/p38, SAPK3/p38gamma, SAPK1/JNK or extracellular signal-regulated kinase 2 (ERK2). It was purified and identified as eukaryotic elongation factor 2 kinase (eEF2K). SAPK4/p38delta phosphorylated eEF2K at Ser359 in vitro, causing its inactivation. eEF2K became phosphorylated at Ser359 and its substrate eEF2 became dephosphorylated (activated) when KB cells were exposed to anisomycin, an agonist that activates all SAPKs, including SAPK4/p38delta. The anisomycin-induced phosphorylation of Ser359 was unaffected by SB 203580, U0126 or rapamycin, and was prevented by overexpression of a catalytically inactive SAPK4/p38delta mutant, suggesting that SAPK4/p38delta may mediate the inhibition of eEF2K by this stress. The phosphorylation of eEF2K at Ser359 was also induced by insulin-like growth factor-1. However, this was blocked by rapamycin, indicating that Ser359 is targeted by at least two signalling pathways.
Knebel
A
A
MRC Protein Phosphorylation Unit, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
Morrice
N
N
Cohen
P
P
D000595:Amino Acid Sequence; D000841:Anisomycin / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054736:Elongation Factor 2 Kinase; D004847:Epithelial Cells; D006367:HeLa Cells; D006801:Humans; D048369:MAP Kinase Kinase 1; D048670:MAP Kinase Kinase 4; D048669:MAP Kinase Kinase 6; D048291:Mitogen-Activated Protein Kinase 12; D048292:Mitogen-Activated Protein Kinase 13; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D013379:Substrate Specificity; D048051:p38 Mitogen-Activated Protein Kinases
D000841:Anisomycin; D011494:Protein Kinases; D048291:Mitogen-Activated Protein Kinase 12; D048292:Mitogen-Activated Protein Kinase 13; C517689:EEF2K protein, human; D017346:Protein Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D054736:Elongation Factor 2 Kinase; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; D048670:MAP Kinase Kinase 4; D048669:MAP Kinase Kinase 6; C482125:MAP2K1 protein, human; C482559:MAP2K4 protein, human; C482566:MAP2K6 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases