rabbit polyclonal anti eef2k  (Proteintech)

Journal: Biochemistry

Article Title: Calcium/Calmodulin Stimulates the Autophosphorylation of Elongation Factor 2 Kinase on Thr-348 and Ser-500 to Regulate its Activity and Calcium Dependence

doi: 10.1021/bi201788e

Figure Lengend Snippet: (A) Characterization of anti-phospho-eEF-2K (Ser-500) antibody. The antibody was characterized against 50 ng recombinant eEF-2K by immunoblotting as described under ‘Experimental Procedures’. Lanes: 1 – untreated eEF-2K WT; 2 – autophosphorylated eEF-2K WT; 3 – untreated eEF-2K S500A; 4 – autophosphorylated eEF-2K S500A; 5 – untreated eEF-2K S500D; 6 – autophosphorylated eEF-2K S500D. (B) Time course of incorporation of phosphate at Ser-500. eEF-2K (500 nM) was allowed to autophosphorylate in the presence of 5 µM CaM and 50 µM free Ca2+. At the indicated times, 50 ng of eEF-2K were removed and the reaction quenched with hot SDS-PAGE sample loading buffer. The samples were then analyzed by Western blotting using the anti-phospho-eEF-2K (Ser-500) antibody as described under ‘Experimental Procedures’. (C) Graphical representation of (B). Western blots were quantified using ImageJ, and data then plotted as the percent phosphorylation of Ser-500 against autophosphorylation time. Inset: Expansion of the data for 0–70 min. Experiments were performed in duplicate, and error bars represent the standard deviation. (D) Buffers used are described under ‘Experimental Procedures’. Assays were performed with eEF-2K enzyme, ± 50 µM free Ca2+ and ± 2 µM calmodulin. EGTA (1 mM) was added to all assays conducted in the absence of Ca2+. For eEF-2K WT, S500A and S500D assayed in the presence of both Ca2+ and CaM, and eEF-2K S500D assayed in the presence of only CaM, activities were much higher than the basal level of kinase activity, and hence only 5 nM of kinase was used. For all the other assays, 50 nM eEF-2K was used in order to detect an increase in kinase activity over the basal level. Kinase activity was determined by measuring the rate of phosphorylation of the peptide (µM.s−1). Activities of the mutants are reported as the percentage of the wild type activity.

Article Snippet: Monitoring incorporation of phosphate at Ser-500 by immunoblotting To analyze the time course of phosphate incorporation at Ser-500, ECM Biosciences generated affinity-purified rabbit polyclonal anti-eEF-2K (Ser-500) phospho-specific antibodies, which were used in Western blotting.

Techniques: Recombinant, Western Blot, SDS Page, Standard Deviation, Activity Assay

Journal: Biochemistry

Article Title: Calcium/Calmodulin Stimulates the Autophosphorylation of Elongation Factor 2 Kinase on Thr-348 and Ser-500 to Regulate its Activity and Calcium Dependence

doi: 10.1021/bi201788e

Figure Lengend Snippet: Summary of the various phosphorylated residues on eEF-2K. Components are color coded as follows: ( - red) – suggested to be involved in the negative regulation of eEF-2K activity through an inhibitory phosphorylation (these sites include Ser-78, Ser-359, Ser-366 and Ser-396). Regulation through the mTOR pathway involves the phosphorylation of Ser-366 by p70 S6 kinase, and the phosphorylation of Ser-359 and Ser-78 by at least two additional unknown kinases (22–24). It has been postulated that the Ser-78 phosphorylation acts to hinder the binding of CaM to eEF-2K (24). The cdc2-cyclin B complex has been shown to modulate eEF-2K activity via Ser-359 in a manner that is dependent on the cell cycle as well as amino acid availability, and is perhaps controlled by mTOR (25). Regulation through the MAPK cascade occurs via the phosphorylation of Ser-366 by p90RSK1 in an ERK-dependent fashion (22). In addition, the stress-activated protein kinases p38α and p38δ inhibit eEF2K via phosphorylation on Ser-396 (23). p38δ is also known to phosphorylate eEF-2K on Ser-359 (21); ( - green) – suggested to be involved in the positive regulation of eEF-2K activity through an activating phosphorylation (these sites include Ser-398 and Ser-500). Phosphorylation of Ser-398 by the energy-supply regulator AMPK is known to activate eEF-2K (29). The cAMP-dependent PKA has also been shown to activate eEF-2K via a phosphorylation on Ser-500, and in the process imparts Ca2+-independent activity to the kinase (26–28); ( - blue) – involved in autophosphorylation of eEF-2K (these sites include Thr-348, Thr-353, Ser-445, Ser-474 and Ser-500). Of the 5 autophosphorylation sites, only Thr-348 appears to be essential for activity against its substrate. Ser-500 is an autophosphorylation site and is also known to be phosphorylated by PKA, and could be the key residue responsible for autophosphorylation-induced Ca2+-independent (CaM-dependent – this work) activity (16, 17). The role of the phosphorylation at Ser-377 by MAPKAP-K2 has not yet been determined (23).

Article Snippet: Monitoring incorporation of phosphate at Ser-500 by immunoblotting To analyze the time course of phosphate incorporation at Ser-500, ECM Biosciences generated affinity-purified rabbit polyclonal anti-eEF-2K (Ser-500) phospho-specific antibodies, which were used in Western blotting.

Techniques: Activity Assay, Binding Assay