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Calcium/Calmodulin Stimulates the Autophosphorylation of Elongation Factor 2 Kinase on Thr-348 and Ser-500 To Regulate Its Activity and Calcium Dependence

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posted on 2016-02-21, 17:06 authored by Clint D. J. Tavares, John P. O’Brien, Olga Abramczyk, Ashwini K. Devkota, Kevin S. Shores, Scarlett B. Ferguson, Tamer S. Kaoud, Mangalika Warthaka, Kyle D. Marshall, Karin M. Keller, Yan Zhang, Jennifer S. Brodbelt, Bulent Ozpolat, Kevin N. Dalby
Eukaryotic elongation factor 2 kinase (eEF-2K) is an atypical protein kinase regulated by Ca2+ and calmodulin (CaM). Its only known substrate is eukaryotic elongation factor 2 (eEF-2), whose phosphorylation by eEF-2K impedes global protein synthesis. To date, the mechanism of eEF-2K autophosphorylation has not been fully elucidated. To investigate the mechanism of autophosphorylation, human eEF-2K was coexpressed with λ-phosphatase and purified from bacteria in a three-step protocol using a CaM affinity column. Purified eEF-2K was induced to autophosphorylate by incubation with Ca2+/CaM in the presence of MgATP. Analyzing tryptic or chymotryptic peptides by mass spectrometry monitored the autophosphorylation over 0–180 min. The following five major autophosphorylation sites were identified: Thr-348, Thr-353, Ser-445, Ser-474, and Ser-500. In the presence of Ca2+/CaM, robust phosphorylation of Thr-348 occurs within seconds of addition of MgATP. Mutagenesis studies suggest that phosphorylation of Thr-348 is required for substrate (eEF-2 or a peptide substrate) phosphorylation, but not self-phosphorylation. Phosphorylation of Ser-500 lags behind the phosphorylation of Thr-348 and is associated with the Ca2+-independent activity of eEF-2K. Mutation of Ser-500 to Asp, but not Ala, renders eEF-2K Ca2+-independent. Surprisingly, this Ca2+-independent activity requires the presence of CaM.

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