title: Calcium/calmodulin stimulates the autophosphorylation of elongation factor 2 kinase on Thr-348 and Ser-500 to regulate its activity and calcium dependence.
abstract: Eukaryotic elongation factor 2 kinase (eEF-2K) is an atypical protein kinase regulated by Ca(2+) 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 Ca(2+)/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 Ca(2+)/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 Ca(2+)-independent activity of eEF-2K. Mutation of Ser-500 to Asp, but not Ala, renders eEF-2K Ca(2+)-independent. Surprisingly, this Ca(2+)-independent activity requires the presence of CaM.
authors.affiliation: Graduate Program in Cell and Molecular Biology, University of Texas, Austin, Texas 78712, United States.
authors.forename: Clint D J
authors.initials: CD
authors.lastname: Tavares
authors.forename: John P
authors.initials: JP
authors.lastname: O'Brien
authors.forename: Olga
authors.initials: O
authors.lastname: Abramczyk
authors.forename: Ashwini K
authors.initials: AK
authors.lastname: Devkota
authors.forename: Kevin S
authors.initials: KS
authors.lastname: Shores
authors.forename: Scarlett B
authors.initials: SB
authors.lastname: Ferguson
authors.forename: Tamer S
authors.initials: TS
authors.lastname: Kaoud
authors.forename: Mangalika
authors.initials: M
authors.lastname: Warthaka
authors.forename: Kyle D
authors.initials: KD
authors.lastname: Marshall
authors.forename: Karin M
authors.initials: KM
authors.lastname: Keller
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
authors.forename: Jennifer S
authors.initials: JS
authors.lastname: Brodbelt
authors.forename: Bulent
authors.initials: B
authors.lastname: Ozpolat
authors.forename: Kevin N
authors.initials: KN
authors.lastname: Dalby
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D054736:Elongation Factor 2 Kinase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010766:Phosphorylation; D012694:Serine / Q000235:genetics*; D013912:Threonine / Q000235:genetics* / Q000378:metabolism
chemical_list: D002147:Calmodulin; D013912:Threonine; D012694:Serine; C517689:EEF2K protein, human; D054736:Elongation Factor 2 Kinase; D002118:Calcium