title: The molecular mechanism of eukaryotic elongation factor 2 kinase activation.
abstract: Calmodulin (CaM)-dependent eukaryotic elongation factor 2 kinase (eEF-2K) impedes protein synthesis through phosphorylation of eukaryotic elongation factor 2 (eEF-2). It is subject to complex regulation by multiple upstream signaling pathways, through poorly described mechanisms. Precise integration of these signals is critical for eEF-2K to appropriately regulate protein translation rates. Here, an allosteric mechanism comprising two sequential conformations is described for eEF-2K activation. First, Ca(2+)/CaM binds eEF-2K with high affinity (Kd(CaM)(app) = 24 ± 5 nm) to enhance its ability to autophosphorylate Thr-348 in the regulatory loop (R-loop) by > 10(4)-fold (k(auto) = 2.6 ± 0.3 s(-1)). Subsequent binding of phospho-Thr-348 to a conserved basic pocket in the kinase domain potentially drives a conformational transition of the R-loop, which is essential for efficient substrate phosphorylation. Ca(2+)/CaM binding activates autophosphorylated eEF-2K by allosterically enhancing k(cat)(app) for peptide substrate phosphorylation by 10(3)-fold. Thr-348 autophosphorylation results in a 25-fold increase in the specificity constant (k(cat)(app)/K(m)(Pep-S) (app)), with equal contributions from k(cat)(app) and K(m)(Pep-S)(app), suggesting that peptide substrate binding is partly impeded in the unphosphorylated enzyme. In cells, Thr-348 autophosphorylation appears to control the catalytic output of active eEF-2K, contributing more than 5-fold to its ability to promote eEF-2 phosphorylation. Fundamentally, eEF-2K activation appears to be analogous to an amplifier, where output volume may be controlled by either toggling the power switch (switching on the kinase) or altering the volume control (modulating stability of the active R-loop conformation). Because upstream signaling events have the potential to modulate either allosteric step, this mechanism allows for exquisite control of eEF-2K output.
authors.affiliation: From the Graduate Program in Cell and Molecular Biology, the Division of Medicinal Chemistry, College of Pharmacy, clinttavares@utexas.edu.
authors.forename: Clint D J
authors.initials: CD
authors.lastname: Tavares
authors.affiliation: the Division of Medicinal Chemistry, College of Pharmacy.
authors.forename: Scarlett B
authors.initials: SB
authors.lastname: Ferguson
authors.affiliation: the Division of Medicinal Chemistry, College of Pharmacy.
authors.forename: David H
authors.initials: DH
authors.lastname: Giles
authors.affiliation: the Division of Medicinal Chemistry, College of Pharmacy, the Department of Biomedical Engineering, Cockrell School of Engineering, and.
authors.forename: Qiantao
authors.initials: Q
authors.lastname: Wang
authors.affiliation: the Division of Medicinal Chemistry, College of Pharmacy.
authors.forename: Rebecca M
authors.initials: RM
authors.lastname: Wellmann
authors.affiliation: the Department of Chemistry and Biochemistry, College of Natural Sciences, University of Texas, Austin, Texas 78712.
authors.forename: John P
authors.initials: JP
authors.lastname: O'Brien
authors.affiliation: the Division of Medicinal Chemistry, College of Pharmacy.
authors.forename: Mangalika
authors.initials: M
authors.lastname: Warthaka
authors.affiliation: the Department of Chemistry and Biochemistry, College of Natural Sciences, University of Texas, Austin, Texas 78712.
authors.forename: Jennifer S
authors.initials: JS
authors.lastname: Brodbelt
authors.affiliation: the Department of Biomedical Engineering, Cockrell School of Engineering, and.
authors.forename: Pengyu
authors.initials: P
authors.lastname: Ren
authors.affiliation: From the Graduate Program in Cell and Molecular Biology, the Division of Medicinal Chemistry, College of Pharmacy, dalby@austin.utexas.edu.
authors.forename: Kevin N
authors.initials: KN
authors.lastname: Dalby
mesh_terms: D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D045744:Cell Line, Tumor; D054736:Elongation Factor 2 Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D014176:Protein Biosynthesis; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013912:Threonine / Q000378:metabolism
chemical_list: D002147:Calmodulin; D013912:Threonine; C517689:EEF2K protein, human; D054736:Elongation Factor 2 Kinase; D002118:Calcium
keywords: Allosteric Regulation; CaMK-III; Calcium; Calmodulin (CaM); Phosphate-binding Pocket; Phosphorylation; Thr-348; Translation; eEF-2; eEF-2K