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

Journal: Molecular Cancer

Article Title: PP2A inhibition overcomes acquired resistance to HER2 targeted therapy

doi: 10.1186/1476-4598-13-157

Figure Lengend Snippet: Phospho-proteomic analysis reveals decreased levels of p-eEF2 in SKBR3-L cells. (A) Example of a 3D view of p-eEF2 protein abundance with graphs of protein abundance analyzed by DeCyder software in SKBR3-par and SKBR3-L cells. The solid line represents the average of three replicate measurements (dotted lines) of protein abundance. (B) Schematic depiction of mTOR-mediated activation of eEF2; active mTOR phosphorylates and activates p70S6k, which in turn phosphorylates and deactivates eEF2k thus preventing the phosphorylation of eEF2 resulting in active eEF2. (C) Immunoblot analysis of total and phosphorylated eEF2 (Thr56) in SKBR3-par and SKBR3-L cells following 24 hr lapatinib treatment.

Article Snippet: Membranes were incubated at 4°C overnight with primary antibody at a 1:1000 dilution in blocking solution (unless otherwise stated) against p-HER2 tyr1221/1222 , p-EGFR tyr1173 , AKT, p-AKT ser473 , ERK, pERK thr202/tyr204 , eEF2, p-eEF2 thr56 , mTOR, p-mTOR ser2448 , eEF2k, p-eEF2k ser366 (Cell Signaling Technology), p-eEF2k ser359 (1:200) (SantaCruzBiotechnology), HER2 (Calbiochem), EGFR (1:250) (Neomarkers), and α-tubulin, anti-mouse and anti-rabbit secondary antibodies (Sigma-Aldrich).

Techniques: Quantitative Proteomics, Software, Activation Assay, Phospho-proteomics, Western Blot

Journal: Molecular Cancer

Article Title: PP2A inhibition overcomes acquired resistance to HER2 targeted therapy

doi: 10.1186/1476-4598-13-157

Figure Lengend Snippet: mTOR and eEF2k mediated regulation of eEF2 phosphorylation. (A) Immunoblot analysis of total and phosphorylated mTOR (Ser2448) in SKBR3-par and SKBR3-L cells following 24 hr. lapatinib treatment. (B) Effect of rapamycin on growth of SKBR3-par and SKBR3-L cells. Error bars represent the mean ± SD (n = 3). (C) Immunoblot analysis of total and phosphorylated eEF2 (Thr56) following 24 hr. treatment with lapatinib and/or rapamycin. (D) Immunoblot analysis of total and phosphorylated eEF2k (Ser366, 359) in SKBR3-par and SKBR3-L cells following 24 hr. lapatinib treatment. (E) Immunoblot examining the effect of NH125 alone and in combination with lapatinib on the phosphorylation of eEF2 (Thr56) in SKBR3-par cells. *denotes p ≤ 0.05.

Article Snippet: Membranes were incubated at 4°C overnight with primary antibody at a 1:1000 dilution in blocking solution (unless otherwise stated) against p-HER2 tyr1221/1222 , p-EGFR tyr1173 , AKT, p-AKT ser473 , ERK, pERK thr202/tyr204 , eEF2, p-eEF2 thr56 , mTOR, p-mTOR ser2448 , eEF2k, p-eEF2k ser366 (Cell Signaling Technology), p-eEF2k ser359 (1:200) (SantaCruzBiotechnology), HER2 (Calbiochem), EGFR (1:250) (Neomarkers), and α-tubulin, anti-mouse and anti-rabbit secondary antibodies (Sigma-Aldrich).

Techniques: Phospho-proteomics, Western Blot

Journal: Acta Neuropathologica Communications

Article Title: Activity of translation regulator eukaryotic elongation factor-2 kinase is increased in Parkinson disease brain and its inhibition reduces alpha synuclein toxicity

doi: 10.1186/s40478-018-0554-9

Figure Lengend Snippet: eEF2K expression and activity in PD brain. a Quantitation of p-eEF2 (T56) IHC (3,3′-Diaminobenzidine-DAB staining) in postmortem hippocampus- Hip (CA1 and CA2 fields) and midbrain- MB (SN-substantia nigra, and PAG-peri-aqueductal gray matter) sections from 3 control and 6 PD cases (Additional file : Table S1; counts from at least 6 high power fields from each control or PD section; Mann–Whitney test, * p < 0.05, *** p < 0.005; error bars indicate Mean ± S.D.). b - d eEF2K mRNA expression in control and PD striatum ( b ), medial substantia nigra ( b ) and dorsal nucleus of vagus nerve ( d ). The following publicly available transcriptomic profile datasets were analyzed on the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) platform: Striatum ( b )- dataset GEO accession # GSE28894, Illumina human Ref-8 v2.0 expression beadchip platform, probe ID ILMN_1789171, controls n = 15 and PD n = 15; Medial substantia nigra ( c )- dataset GEO accession # GSE8397, Affymetrix Human Genome U133B Array, probe ID 225546_at, controls n = 8 and PD n = 15; Dorsal nucleus of vagus ( d )- dataset GEO accession # GSE43490, Agilent-014850 Whole Human Genome Microarray, probe ID A_24_P716162, controls n = 6 and PD n = 7. (Mann–Whitney test, * p < 0.05; error bars in 3b-d indicate Mean ± S.D.). e Relative eEF2K mRNA expression in human iPSCs derived cultured midbrain control (WT, n = 3) or A53T ( n = 2) mutation carrying organoids (T-test, * p < 0.05; error bars indicate Mean ± S.D.)

Article Snippet: Additional reagents and biochemical assays employed during these studies include: pool of small interference RNAs (SiRNAs) targeting mouse eEF2K (Santa Cruz, #sc-39,012), Cell Titer Glo ATP measurement kit (Promega, #G7570), Lactate dehydrogenase (LDH) fluorometric assay (Novus Biologicals, #NBP2–54851), Seahorse Mito stress test kit (Agilent, #103015–100), 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescent ROS reagent (ThermoFisher, #D399), and MitoTracker Green fluorescent reagent for mitochondrial mass (ThermoFisher, #M7514).

Techniques: Expressing, Activity Assay, Quantitation Assay, Staining, Control, MANN-WHITNEY, Gene Expression, Microarray, Derivative Assay, Cell Culture, Mutagenesis

Journal: Acta Neuropathologica Communications

Article Title: Activity of translation regulator eukaryotic elongation factor-2 kinase is increased in Parkinson disease brain and its inhibition reduces alpha synuclein toxicity

doi: 10.1186/s40478-018-0554-9

Figure Lengend Snippet: Brain eEF2K expression and activity in transgenic M83 +/+ PD mice. a eEF2K mRNA levels in whole brain homogenates from transgenic M83 +/+ PD mice intramuscularly (IM) injected bilaterally with phosphate buffered saline (PBS, n = 10) or pre-formed fibrillar (PFF, n = 13) mouse wild type AS. (Mann–Whitney test, *** p < 0.005; error bars indicate Mean ± S.D.). b - c Western blot analysis of p-eEF2 (T56) and p-ASyn (S129) in whole brain homogenates from transgenic M83 +/+ PD mice intramuscularly (IM) injected bilaterally with phosphate buffered saline (PBS) or pre-formed fibrillar (PFF) mouse wild type AS ( b ), and corresponding densitometry analysis ( c ) ( n = 7/group; Mann–Whitney test, * p < 0.05, *** p < 0.005; error bars indicate Mean ± S.D.)

Article Snippet: Additional reagents and biochemical assays employed during these studies include: pool of small interference RNAs (SiRNAs) targeting mouse eEF2K (Santa Cruz, #sc-39,012), Cell Titer Glo ATP measurement kit (Promega, #G7570), Lactate dehydrogenase (LDH) fluorometric assay (Novus Biologicals, #NBP2–54851), Seahorse Mito stress test kit (Agilent, #103015–100), 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescent ROS reagent (ThermoFisher, #D399), and MitoTracker Green fluorescent reagent for mitochondrial mass (ThermoFisher, #M7514).

Techniques: Expressing, Activity Assay, Transgenic Assay, Injection, Saline, MANN-WHITNEY, Western Blot

Journal: Acta Neuropathologica Communications

Article Title: Activity of translation regulator eukaryotic elongation factor-2 kinase is increased in Parkinson disease brain and its inhibition reduces alpha synuclein toxicity

doi: 10.1186/s40478-018-0554-9

Figure Lengend Snippet: Effects of eEF2K inhibition on human AS cytotoxicity in differentiated N2A cells. a - b Western blot analysis of p-eEF2 (T56) levels in N2A cells subsequent to transient overexpression of human wild type or mutant A53T AS, with or without siRNA mediated eEF2K knockdown ( a ), and corresponding densitometry analysis ( b ) ( n = 6–9/group from three independent experiments; One-way ANOVA post-hoc Bonferroni test, * p < 0.05, *** p < 0.005; error bars indicate Mean ± S.E.M). c Measurements of cytotoxicity by lactate dehydrogenase-LDH release in the culture medium ( c ) and FACS analysis of propidium iodide-PI staining ( d ) in N2A cells subsequent to transient overexpression of human wild type or mutant A53T AS, with or without siRNA mediated eEF2K knockdown ( n = 9–12/group from three independent experiments; One-way ANOVA post-hoc Bonferroni test, * p < 0.05, ** p < 0.01, *** p < 0.005, NS = not significant; error bars indicate Mean ± S.D.)

Article Snippet: Additional reagents and biochemical assays employed during these studies include: pool of small interference RNAs (SiRNAs) targeting mouse eEF2K (Santa Cruz, #sc-39,012), Cell Titer Glo ATP measurement kit (Promega, #G7570), Lactate dehydrogenase (LDH) fluorometric assay (Novus Biologicals, #NBP2–54851), Seahorse Mito stress test kit (Agilent, #103015–100), 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescent ROS reagent (ThermoFisher, #D399), and MitoTracker Green fluorescent reagent for mitochondrial mass (ThermoFisher, #M7514).

Techniques: Inhibition, Western Blot, Over Expression, Mutagenesis, Knockdown, Staining

Journal: Acta Neuropathologica Communications

Article Title: Activity of translation regulator eukaryotic elongation factor-2 kinase is increased in Parkinson disease brain and its inhibition reduces alpha synuclein toxicity

doi: 10.1186/s40478-018-0554-9

Figure Lengend Snippet: Effects of eEF2K inhibition on mitochondrial dysfunction and oxidative stress induced by human AS in differentiated N2A cells. a - b Measurements of basal oxygen consumption rate-OCR ( b ) and ATP levels ( c ) in N2A cells subsequent to transient overexpression of human wild type or mutant A53T AS, with or without siRNA mediated eEF2K knockdown ( n = 9–12/group from three independent experiments; Unpaired T-test, * p < 0.05, ** p < 0.01, *** p < 0.005; error bars indicate Mean ± S.D.). c Flow cytometry analysis of reactive oxygen species (ROS), measured by DCFDA staining, in N2A cells subsequent to transient overexpression of human wild type or mutant A53T AS, with or without siRNA mediated eEF2K knockdown ( n = 9/group from three independent experiments; Unpaired T-test, * p < 0.05, ** p < 0.01, *** p < 0.005; error bars indicate Mean ± S.D.)

Article Snippet: Additional reagents and biochemical assays employed during these studies include: pool of small interference RNAs (SiRNAs) targeting mouse eEF2K (Santa Cruz, #sc-39,012), Cell Titer Glo ATP measurement kit (Promega, #G7570), Lactate dehydrogenase (LDH) fluorometric assay (Novus Biologicals, #NBP2–54851), Seahorse Mito stress test kit (Agilent, #103015–100), 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescent ROS reagent (ThermoFisher, #D399), and MitoTracker Green fluorescent reagent for mitochondrial mass (ThermoFisher, #M7514).

Techniques: Inhibition, Over Expression, Mutagenesis, Knockdown, Flow Cytometry, Staining