Signal Integration at Elongation Factor 2 Kinase: THE ROLES OF CALCIUM, CALMODULIN, AND SER-500 PHOSPHORYLATION.

Autor: Tavares CDJ; From the Graduate Program in Cell and Molecular Biology, University of Texas, Austin, Texas 78712; Division of Chemical Biology and Medicinal Chemistry, University of Texas, Austin, Texas 78712. Electronic address: clinttavares@utexas.edu., Giles DH; Division of Chemical Biology and Medicinal Chemistry, University of Texas, Austin, Texas 78712., Stancu G; Division of Chemical Biology and Medicinal Chemistry, University of Texas, Austin, Texas 78712., Chitjian CA; From the Graduate Program in Cell and Molecular Biology, University of Texas, Austin, Texas 78712; Division of Chemical Biology and Medicinal Chemistry, University of Texas, Austin, Texas 78712., Ferguson SB; Division of Chemical Biology and Medicinal Chemistry, University of Texas, Austin, Texas 78712., Wellmann RM; Division of Chemical Biology and Medicinal Chemistry, University of Texas, Austin, Texas 78712., Kaoud TS; Division of Chemical Biology and Medicinal Chemistry, University of Texas, Austin, Texas 78712., Ghose R; the Department of Chemistry, City College of New York, New York, New York 10031; the Graduate Center, City University of New York, New York, New York 10016., Dalby KN; From the Graduate Program in Cell and Molecular Biology, University of Texas, Austin, Texas 78712; Division of Chemical Biology and Medicinal Chemistry, University of Texas, Austin, Texas 78712. Electronic address: dalby@austin.utexas.edu.
Jazyk: angličtina
Zdroj: The Journal of biological chemistry [J Biol Chem] 2017 Feb 03; Vol. 292 (5), pp. 2032-2045. Date of Electronic Publication: 2016 Dec 12.
DOI: 10.1074/jbc.M116.753277
Abstrakt: Eukaryotic elongation factor 2 kinase (eEF-2K), the only calmodulin (CaM)-dependent member of the unique α-kinase family, impedes protein synthesis by phosphorylating eEF-2. We recently identified Thr-348 and Ser-500 as two key autophosphorylation sites within eEF-2K that regulate its activity. eEF-2K is regulated by Ca 2+ ions and multiple upstream signaling pathways, but how it integrates these signals into a coherent output, i.e. phosphorylation of eEF-2, is unclear. This study focuses on understanding how the post-translational phosphorylation of Ser-500 integrates with Ca 2+ and CaM to regulate eEF-2K. CaM is shown to be absolutely necessary for efficient activity of eEF-2K, and Ca 2+ is shown to enhance the affinity of CaM toward eEF-2K. Ser-500 is found to undergo autophosphorylation in cells treated with ionomycin and is likely also targeted by PKA. In vitro, autophosphorylation of Ser-500 is found to require Ca 2+ and CaM and is inhibited by mutations that compromise binding of phosphorylated Thr-348 to an allosteric binding pocket on the kinase domain. A phosphomimetic Ser-500 to aspartic acid mutation (eEF-2K S500D) enhances the rate of activation (Thr-348 autophosphorylation) by 6-fold and lowers the EC 50 for Ca 2+ /CaM binding to activated eEF-2K (Thr-348 phosphorylated) by 20-fold. This is predicted to result in an elevation of the cellular fraction of active eEF-2K. In support of this mechanism, eEF-2K knock-out MCF10A cells reconstituted with eEF-2K S500D display relatively high levels of phospho-eEF-2 under basal conditions. This study reports how phosphorylation of a regulatory site (Ser-500) integrates with Ca 2+ and CaM to influence eEF-2K activity.
(© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)
Databáze: MEDLINE
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