Calcium/calmodulin stimulates the autophosphorylation of elongation factor 2 kinase on Thr-348 and Ser-500 to regulate its activity and calcium dependence.

Biochemistry
Clint D J TavaresKevin N Dalby

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 act...Continue Reading

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Citations

May 24, 2014·Advances in Biological Regulation·Justin W KenneyChristopher G Proud
Oct 16, 2012·Biological Psychiatry·Lisa M MonteggiaEge T Kavalali
Dec 25, 2015·Biochemical and Biophysical Research Communications·Satoshi KameshimaHideyuki Yamawaki
Sep 17, 2014·Chembiochem : a European Journal of Chemical Biology·Ashwini K DevkotaKevin N Dalby
Jul 23, 2014·Bioorganic & Medicinal Chemistry·Ramakrishna EdupugantiKevin N Dalby
Oct 19, 2019·Protein Science : a Publication of the Protein Society·Kwangwoon LeeRanajeet Ghose
May 8, 2020·Journal of Molecular Medicine : Official Organ of the Gesellschaft Deutscher Naturforscher Und Ärzte·Didem Karakas, Bulent Ozpolat
Mar 15, 2015·American Journal of Physiology. Heart and Circulatory Physiology·Satoshi KameshimaHideyuki Yamawaki
Jul 12, 2014·The Journal of Biological Chemistry·Clint D J TavaresKevin N Dalby
Aug 30, 2020·Nature Communications·Piero GiansantiFrank J M van Kuppeveld
May 10, 2017·Nihon yakurigaku zasshi. Folia pharmacologica Japonica·Satoshi KameshimaHideyuki Yamawaki
Dec 14, 2016·The Journal of Biological Chemistry·Clint D J TavaresKevin N Dalby
Apr 17, 2021·ACS Pharmacology & Translational Science·Ferah Comert OnderBulent Ozpolat

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