A novel method to identify protein kinase substrates: eEF2 kinase is phosphorylated and inhibited by SAPK4/p38 delta

We have developed a method of general application for identifying putative substrates of protein kinases in cell extracts. Using this procedure, we id entified the physiological substrates of several mitogen-activated protein kinase kinases and an authentic substrate of stress-activated protein kinas e (SAPK) 2a/p38. A 120 kDa protein was detected in skeletal muscle extracts that was phosphorylated rapidly by SAPK4/p386, but poorly by SA-PK2/p38, S A-PK3/p38 gamma, SAPK1/JNK or extracellular signal-regulated kinase 2 (ERK2 ). It was purified and identified as eukaryotic elongation factor 2 kinase (eEF2K). SAPK4/p38 delta phosphorylated eEF2K at Ser359 in vitro, causing i ts inactivation. eEF2K became phosphorylated at Ser359 and its substrate eE F2 became dephosphorylated (activated) when KB cells were exposed to anisom ycin, an agonist that activates all SAPKs, including SAPK4/p38 delta. The a nisomycin-induced phosphorylation of Ser359 was unaffected by SB 203580, U0 126 or rapamycin, and was prevented by overexpression of a catalytically in active SA-PK4/p38 delta mutant, suggesting that SAPK4/p38 delta may mediate the inhibition of eEF2K by this stress. The phosphorylation of eEF2K at Se r359 was also induced by insulin-like growth factor-1. However, this was bl ocked by rapamycin, indicating that Ser359 is targeted by at least two sign alling pathways.