Calcium/Calmodulin Stimulates the Autophosphorylation of Elongation Factor 2 Kinase on Thr-348 and Ser-500 to Regulate its Activity and Calcium Dependence
| Autor: | Jennifer S. Brodbelt, Kevin S. Shores, Yan Zhang, Ashwini K. Devkota, Scarlett B. Ferguson, Mangalika Warthaka, Kevin N. Dalby, Karin M. Keller, John P. O'Brien, Bulent Ozpolat, Olga Abramczyk, Tamer S. Kaoud, Clint D.J. Tavares, Kyle D. Marshall |
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| Jazyk: | angličtina |
| Rok vydání: | 2012 |
| Předmět: |
Elongation Factor 2 Kinase
Threonine Calmodulin Molecular Sequence Data Biology Biochemistry Article Mass Spectrometry Ca2+/calmodulin-dependent protein kinase Casein kinase 2 alpha 1 Serine Humans Amino Acid Sequence Phosphorylation education MAPK14 education.field_of_study Binding Sites Cyclin-dependent kinase 2 Autophosphorylation Elongation factor biology.protein Calcium Elongation Factor-2 Kinase |
| Popis: | Eukaryotic elongation factor 2 kinase (eEF-2K) is an atypical protein kinase regulated by Ca2+ 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 co-expressed with λ-phosphatase, and purified from bacteria in a three-step protocol using a calmodulin-affinity column. Purified eEF-2K was induced to autophosphorylate by incubation with Ca2+/CaM in the presence of MgATP. Analyzing tryptic or chymotryptic peptides by mass spectrometry monitored the autophosphorylation over 0–180 minutes. The following five major autophosphorylation sites were identified, Thr-348, Thr-353, Ser-445, Ser-474 and Ser-500. In the presence of Ca2+/CaM, robust phosphorylation of Thr-348 occurs within seconds of adding 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 calcium-independent activity of eEF-2K. Mutation of Ser-500 to Asp, but not Ala, renders eEF-2K calcium-independent. Surprisingly, this calcium-independent activity requires the presence of calmodulin. |
| Databáze: | OpenAIRE |
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