A novel method to identify protein kinase substrates: eEF2 kinase is phosphorylated and inhibited by SAPK4/p38delta
Open Access
- 15 August 2001
- journal article
- research article
- Published by Springer Nature in The EMBO Journal
- Vol. 20 (16) , 4360-4369
- https://doi.org/10.1093/emboj/20.16.4360
Abstract
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/p38δ, but poorly by SAPK2/p38, SAPK3/p38γ, SAPK1/JNK or extracellular signal‐regulated kinase 2 (ERK2). It was purified and identified as eukaryotic elongation factor 2 kinase (eEF2K). SAPK4/p38δ 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/p38δ. The anisomycin‐induced phosphorylation of Ser359 was unaffected by SB 203580, U0126 or rapamycin, and was prevented by overexpression of a catalytically inactive SAPK4/p38δ mutant, suggesting that SAPK4/p38δ 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.Keywords
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