title: Signal Integration at Elongation Factor 2 Kinase: THE ROLES OF CALCIUM, CALMODULIN, AND SER-500 PHOSPHORYLATION.
abstract: 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 Ca2+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 Ca2+and CaM to regulate eEF-2K. CaM is shown to be absolutely necessary for efficient activity of eEF-2K, and Ca2+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 Ca2+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 EC50for Ca2+/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 Ca2+and CaM to influence eEF-2K activity.
authors.affiliation: 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.
authors.forename: Clint D J
authors.initials: CDJ
authors.lastname: Tavares
authors.affiliation: Division of Chemical Biology and Medicinal Chemistry, University of Texas, Austin, Texas 78712.
authors.forename: David H
authors.initials: DH
authors.lastname: Giles
authors.affiliation: Division of Chemical Biology and Medicinal Chemistry, University of Texas, Austin, Texas 78712.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Stancu
authors.affiliation: 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.
authors.forename: Catrina A
authors.initials: CA
authors.lastname: Chitjian
authors.affiliation: Division of Chemical Biology and Medicinal Chemistry, University of Texas, Austin, Texas 78712.
authors.forename: Scarlett B
authors.initials: SB
authors.lastname: Ferguson
authors.affiliation: Division of Chemical Biology and Medicinal Chemistry, University of Texas, Austin, Texas 78712.
authors.forename: Rebecca M
authors.initials: RM
authors.lastname: Wellmann
authors.affiliation: Division of Chemical Biology and Medicinal Chemistry, University of Texas, Austin, Texas 78712.
authors.forename: Tamer S
authors.initials: TS
authors.lastname: Kaoud
authors.affiliation: 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.
authors.forename: Ranajeet
authors.initials: R
authors.lastname: Ghose
authors.affiliation: 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.
authors.forename: Kevin N
authors.initials: KN
authors.lastname: Dalby
mesh_terms: D019943:Amino Acid Substitution; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D054736:Elongation Factor 2 Kinase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D020125:Mutation, Missense; D010766:Phosphorylation / Q000235:genetics; D012694:Serine / Q000235:genetics / Q000378:metabolism
chemical_list: D002147:Calmodulin; D012694:Serine; C517689:EEF2K protein, human; D054736:Elongation Factor 2 Kinase; D002118:Calcium
keywords: CaMK-III; S500D; Ser-500; Thr-348; calcium; calmodulin (CaM); eEF-2K; phosphorylation; translation; translation elongation factor