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Cell Signaling Technology Inc eef2k antibody
Eef2k Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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eef2k antibody - by Bioz Stars, 2026-03

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(A) Left: Phosphorylation of eIF2α on Ser52 converts eIF2 into an inhibitor of its guanine nucleotide exchange factor (eIF2B), blocking ternary complex regeneration and suppressing translation initiation. Right: Phosphorylation of eEF2 on Thr57 by eEF2 kinase (eEF2K) prevents ribosomal translocation, thereby slowing peptide elongation. (B) Phosphorylation of eIF2α (Ser52) by stress-activated kinases (PKR, GCN2, PERK, HRI) converts active eIF2 into an inhibitor of its guanine nucleotide exchange factor eIF2B, thereby preventing ternary complex formation and suppressing global initiation. (C) The eEF2 cycle: eEF2K phosphorylates eEF2 at Thr57, which prevents ribosomal translocation; dephosphorylation by PP2A restores elongation. (D) Illustration of the experimental workflow for lysate generation and in vitro translation (IVT). Suspension Expi293F cells were harvested, lysed under native conditions, and the resulting extracts programmed with a Nanoluciferase (NanoLuc) reporter mRNA to quantify translational efficiency. (E) Schematic representation of human Expi293F cells engineered by prime editing (PE) to introduce phospho-null substitutions in EIF2S1 (eIF2α S52A) and EEF2 (eEF2 T57A). All amino acid residue numbers correspond to the human reference sequences according to the UniProt database, entries P05198 and P13639 for EIF2S1 and EEF2, respectively. (F) Comparison of translational output in IVT reactions programmed with NanoLuc mRNA using extracts prepared from wild-type Expi293F cells or genome-edited Expi293F eIF2α-S52A and eEF2-T57A lines. All experiments were performed in biological triplicates.

Journal: bioRxiv

Article Title: Overcoming the eIF2α Brake in Human Cell-Derived Translation Systems

doi: 10.1101/2025.11.16.688697

Figure Lengend Snippet: (A) Left: Phosphorylation of eIF2α on Ser52 converts eIF2 into an inhibitor of its guanine nucleotide exchange factor (eIF2B), blocking ternary complex regeneration and suppressing translation initiation. Right: Phosphorylation of eEF2 on Thr57 by eEF2 kinase (eEF2K) prevents ribosomal translocation, thereby slowing peptide elongation. (B) Phosphorylation of eIF2α (Ser52) by stress-activated kinases (PKR, GCN2, PERK, HRI) converts active eIF2 into an inhibitor of its guanine nucleotide exchange factor eIF2B, thereby preventing ternary complex formation and suppressing global initiation. (C) The eEF2 cycle: eEF2K phosphorylates eEF2 at Thr57, which prevents ribosomal translocation; dephosphorylation by PP2A restores elongation. (D) Illustration of the experimental workflow for lysate generation and in vitro translation (IVT). Suspension Expi293F cells were harvested, lysed under native conditions, and the resulting extracts programmed with a Nanoluciferase (NanoLuc) reporter mRNA to quantify translational efficiency. (E) Schematic representation of human Expi293F cells engineered by prime editing (PE) to introduce phospho-null substitutions in EIF2S1 (eIF2α S52A) and EEF2 (eEF2 T57A). All amino acid residue numbers correspond to the human reference sequences according to the UniProt database, entries P05198 and P13639 for EIF2S1 and EEF2, respectively. (F) Comparison of translational output in IVT reactions programmed with NanoLuc mRNA using extracts prepared from wild-type Expi293F cells or genome-edited Expi293F eIF2α-S52A and eEF2-T57A lines. All experiments were performed in biological triplicates.

Article Snippet: Antibodies for RPS19 (A304-002A) and eEF2K (A301-686A-T) were purchased from Bethyl Laboratories Inc. Anti-mouse IgG-HRP (sc-525409) and GADD34 (sc-373815) were from Santa Cruz Biotechnology.

Techniques: Phospho-proteomics, Blocking Assay, Translocation Assay, De-Phosphorylation Assay, In Vitro, Suspension, Introduce, Residue, Comparison

(A) Schematic of the EIF2K (encoding eEF2 kinase) genomic locus and CRISPR–Cas9 targeting strategy. Exons and intron structure are shown with the chromosomal position (16p12.2). A single guide RNA (sgRNA) was designed to target exon 3, introducing a frameshift mutation predicted to disrupt kinase catalytic function. Sequence is according to Homo sapience reference genome assembly GRCh38.p14 (GenBank assembly accession: GCA_000001405.29). (B) Validation of eEF2K knockout clones by immunoblotting. Whole-cell lysates from three independent eEF2K-KO clones and wild-type (WT) Expi293F cells were analyzed by Western blot using an anti-eEF2K antibody. All KO clones showed complete loss of eEF2K protein, while eS19 served as a loading control. Gels are representative of two independent experiments. (C) Growth characteristics of eEF2K-KO clones. All three knockout clones proliferated at rates comparable to WT Expi293F cells, indicating that eEF2K loss does not affect suspension-culture viability or growth kinetics.

Journal: bioRxiv

Article Title: Overcoming the eIF2α Brake in Human Cell-Derived Translation Systems

doi: 10.1101/2025.11.16.688697

Figure Lengend Snippet: (A) Schematic of the EIF2K (encoding eEF2 kinase) genomic locus and CRISPR–Cas9 targeting strategy. Exons and intron structure are shown with the chromosomal position (16p12.2). A single guide RNA (sgRNA) was designed to target exon 3, introducing a frameshift mutation predicted to disrupt kinase catalytic function. Sequence is according to Homo sapience reference genome assembly GRCh38.p14 (GenBank assembly accession: GCA_000001405.29). (B) Validation of eEF2K knockout clones by immunoblotting. Whole-cell lysates from three independent eEF2K-KO clones and wild-type (WT) Expi293F cells were analyzed by Western blot using an anti-eEF2K antibody. All KO clones showed complete loss of eEF2K protein, while eS19 served as a loading control. Gels are representative of two independent experiments. (C) Growth characteristics of eEF2K-KO clones. All three knockout clones proliferated at rates comparable to WT Expi293F cells, indicating that eEF2K loss does not affect suspension-culture viability or growth kinetics.

Techniques: CRISPR, Mutagenesis, Sequencing, Biomarker Discovery, Knock-Out, Clone Assay, Western Blot, Control, Suspension

Western blot analysis of extracts prepared from WT, eEF2 T57A mutant, and eEF2K knockout Expi293F cells. Immunoblotting with phospho-specific antibodies shows that eEF2 phosphorylation is completely abolished in both mutant and knockout strains, confirming loss of eEF2K-dependent modification at Thr57. Total eEF2 levels remain unchanged across all samples, as detected by pan-eEF2 antibody. Ribosomal protein eS19 serves as a loading control. Gels are representative of two independent experiments.

Journal: bioRxiv

Article Title: Overcoming the eIF2α Brake in Human Cell-Derived Translation Systems

doi: 10.1101/2025.11.16.688697

Figure Lengend Snippet: Western blot analysis of extracts prepared from WT, eEF2 T57A mutant, and eEF2K knockout Expi293F cells. Immunoblotting with phospho-specific antibodies shows that eEF2 phosphorylation is completely abolished in both mutant and knockout strains, confirming loss of eEF2K-dependent modification at Thr57. Total eEF2 levels remain unchanged across all samples, as detected by pan-eEF2 antibody. Ribosomal protein eS19 serves as a loading control. Gels are representative of two independent experiments.

Techniques: Western Blot, Mutagenesis, Knock-Out, Phospho-proteomics, Modification, Control

CaMactivates eEF-2K through a two-step process. In the first step , CaM binds the eEF-2K that is in the inactive state (E I ), leading to a state (E A’ ) that has high activity toward T348. In the second step , rapid autophosphorylation in T348 and its subsequent engagement in a phosphate binding leads to a fully activated state (E A ) that can phosphorylate the substrate eEF-2 (S) on Thr-56 with high efficiency.

Journal: bioRxiv

Article Title: The Critical Role of the C-terminal Lobe of Calmodulin in Activating Eukaryotic Elongation Factor 2 Kinase

doi: 10.1101/2025.05.13.653565

Figure Lengend Snippet: CaMactivates eEF-2K through a two-step process. In the first step , CaM binds the eEF-2K that is in the inactive state (E I ), leading to a state (E A’ ) that has high activity toward T348. In the second step , rapid autophosphorylation in T348 and its subsequent engagement in a phosphate binding leads to a fully activated state (E A ) that can phosphorylate the substrate eEF-2 (S) on Thr-56 with high efficiency.

Article Snippet: Samples were analyzed for autophosphorylation by western blotting using specific antibodies for eEF-2K (Santa Cruz Biotechnology) and its p T348, p S445, or p S500 forms (ECM Biosciences).

Techniques: Activity Assay, Binding Assay

Full-length eEF-2K consists of 725 residues organized in an N-terminal calmodulin targeting motif (CTM), an α−kinase domain (KD), a regulatory loop (R-loop) containing multiple phosphorylation sites that terminates in an α−helical C-terminal domain (CTD). The stimulatory autophosphorylation sites, T348 and S500, and the inhibitory phosphorylation site, S359, discussed in the text, are indicated in green and red, respectively. The truncated eEF-2K construct (eEF-2K TR ) is missing 70 N-terminal residues, and 6 glycines have replaced the segment of the R-loop between 359 and 489.

Journal: bioRxiv

Article Title: The Critical Role of the C-terminal Lobe of Calmodulin in Activating Eukaryotic Elongation Factor 2 Kinase

doi: 10.1101/2025.05.13.653565

Figure Lengend Snippet: Full-length eEF-2K consists of 725 residues organized in an N-terminal calmodulin targeting motif (CTM), an α−kinase domain (KD), a regulatory loop (R-loop) containing multiple phosphorylation sites that terminates in an α−helical C-terminal domain (CTD). The stimulatory autophosphorylation sites, T348 and S500, and the inhibitory phosphorylation site, S359, discussed in the text, are indicated in green and red, respectively. The truncated eEF-2K construct (eEF-2K TR ) is missing 70 N-terminal residues, and 6 glycines have replaced the segment of the R-loop between 359 and 489.

Techniques: Phospho-proteomics, Construct

$(A) eEF-2K (300 nM) activity was measured against its primary autophosphorylation site, T348. The enzyme was incubated with CaM WT (1 µM), CaM C (1 µM), CaM N (1 or 10 µM), or no CaM, and 50 µM free Ca 2+ before initiating the reaction with 1 mM Mg 2+ •ATP. The reaction was quenched at various time points by adding 2.3 volumes of hot SDS-loading buffer. 150 ng of eEF-2K was loaded onto a gel, and p T348 and total eEF-2K were detected by western blotting. ( B-C ) Rapid quench flow was utilized to measure the rate of T348 autophosphorylation in the presence of CaM WT or CaM C . eEF-2K (200 nM) was pre-incubated with ( B ) 2 µM CaM WT or ( C ) 2 µM CaM C in the presence of 50 µM free Ca 2+ , then rapidly mixed (2 ms) with 1 mM Mg 2+ •ATP. The reaction was quenched at various times with 200 mM KCl, 50 mM EDTA, and 10 mM EGTA. Western blotting was used to detect phosphorylation on T348, and phosphate incorporation was recorded as the fraction of their maximal control values (2 or 60 sec for CaM WT or CaM C , respectively). The experimental data, shown as circles representing the mean with standard deviation (n = 2), were fit to to obtain best-fit values of ; CaM WT = 3.8 ± 0.38 s -1 and CaM C = 1.4 ± 0.09 s -1 .$

Journal: bioRxiv

Article Title: The Critical Role of the C-terminal Lobe of Calmodulin in Activating Eukaryotic Elongation Factor 2 Kinase

doi: 10.1101/2025.05.13.653565

Figure Lengend Snippet: (A) eEF-2K (300 nM) activity was measured against its primary autophosphorylation site, T348. The enzyme was incubated with CaM WT (1 µM), CaM C (1 µM), CaM N (1 or 10 µM), or no CaM, and 50 µM free Ca 2+ before initiating the reaction with 1 mM Mg 2+ •ATP. The reaction was quenched at various time points by adding 2.3 volumes of hot SDS-loading buffer. 150 ng of eEF-2K was loaded onto a gel, and p T348 and total eEF-2K were detected by western blotting. ( B-C ) Rapid quench flow was utilized to measure the rate of T348 autophosphorylation in the presence of CaM WT or CaM C . eEF-2K (200 nM) was pre-incubated with ( B ) 2 µM CaM WT or ( C ) 2 µM CaM C in the presence of 50 µM free Ca 2+ , then rapidly mixed (2 ms) with 1 mM Mg 2+ •ATP. The reaction was quenched at various times with 200 mM KCl, 50 mM EDTA, and 10 mM EGTA. Western blotting was used to detect phosphorylation on T348, and phosphate incorporation was recorded as the fraction of their maximal control values (2 or 60 sec for CaM WT or CaM C , respectively). The experimental data, shown as circles representing the mean with standard deviation (n = 2), were fit to to obtain best-fit values of ; CaM WT = 3.8 ± 0.38 s -1 and CaM C = 1.4 ± 0.09 s -1 .

Techniques: Activity Assay, Incubation, Western Blot, Phospho-proteomics, Control, Standard Deviation

Journal: bioRxiv

Article Title: The Critical Role of the C-terminal Lobe of Calmodulin in Activating Eukaryotic Elongation Factor 2 Kinase

doi: 10.1101/2025.05.13.653565

Figure Lengend Snippet:

Techniques:

(A) The dose-dependence of p eEF-2K (1 nM) activity on CaM WT or CaM C was measured against 150 µM peptide substrate (PepS) with Ca 2+ (50 µM free) and 1 mM [γ- 32 P]-ATP. The k obs values (mean with standard deviation, n = 2) were plotted against the concentration of the CaM construct, and data were fit to to obtain best-fit values for k obs,max (CaM WT = 20 ± 0.6 s -1 ; CaM C = 20 ± 0.7 s -1 ) and (CaM WT = 67 ± 7 nM; CaM C = 85 ± 10 nM). (B) The activity of 0.5 nM eEF-2K was measured against 5 µM of yeast eEF-2 in the presence of Ca 2+ (50 µM free) with 1 mM [γ- 32 P]-ATP and varied CaM WT or CaM C . Samples were quenched in addition to a hot SDS-loading buffer and then analyzed using SDS-PAGE gels. A phosphorimager visualized the incorporation of 32 P into eEF-2. Samples were extracted from the gel and measured by scintillation counting. The k obs at each CaM concentration were plotted against the CaM concentration, and data were fit to to obtain best-fit values for k obs , max (CaM WT = 6.1 ± 1.5 s -1 ; CaM C = 5.6 ± 1.4 s -1 ) and (CaM WT = 121 ± 41 s -1 ; CaM C = 156 ± 64 s -1 ). Also see . ( C ) The activity of 2 nM eEF-2K was measured against 150 µM PepS with 1 mM [γ- 32 P]-ATP in the presence of 1000 nM CaM WT or CaM C with varying concentrations of free Ca 2+ (0-1000 nM). The k obs values obtained at various Ca 2+ concentrations are indicated. On the right, Data from (A), 50 µM free Ca 2+ and 1000 nM CaM WT or CaM C are re-plotted for comparison. (D) The activity of 2 nM eEF-2K with varying concentrations of CaM WT or CaM C in the absence of Ca 2+ was measured against 150 µM PepS with 1 mM [γ- 32 P]-ATP; the corresponding k obs values are shown.

Journal: bioRxiv

Article Title: The Critical Role of the C-terminal Lobe of Calmodulin in Activating Eukaryotic Elongation Factor 2 Kinase

doi: 10.1101/2025.05.13.653565

Figure Lengend Snippet: (A) The dose-dependence of p eEF-2K (1 nM) activity on CaM WT or CaM C was measured against 150 µM peptide substrate (PepS) with Ca 2+ (50 µM free) and 1 mM [γ- 32 P]-ATP. The k obs values (mean with standard deviation, n = 2) were plotted against the concentration of the CaM construct, and data were fit to to obtain best-fit values for k obs,max (CaM WT = 20 ± 0.6 s -1 ; CaM C = 20 ± 0.7 s -1 ) and (CaM WT = 67 ± 7 nM; CaM C = 85 ± 10 nM). (B) The activity of 0.5 nM eEF-2K was measured against 5 µM of yeast eEF-2 in the presence of Ca 2+ (50 µM free) with 1 mM [γ- 32 P]-ATP and varied CaM WT or CaM C . Samples were quenched in addition to a hot SDS-loading buffer and then analyzed using SDS-PAGE gels. A phosphorimager visualized the incorporation of 32 P into eEF-2. Samples were extracted from the gel and measured by scintillation counting. The k obs at each CaM concentration were plotted against the CaM concentration, and data were fit to to obtain best-fit values for k obs , max (CaM WT = 6.1 ± 1.5 s -1 ; CaM C = 5.6 ± 1.4 s -1 ) and (CaM WT = 121 ± 41 s -1 ; CaM C = 156 ± 64 s -1 ). Also see . ( C ) The activity of 2 nM eEF-2K was measured against 150 µM PepS with 1 mM [γ- 32 P]-ATP in the presence of 1000 nM CaM WT or CaM C with varying concentrations of free Ca 2+ (0-1000 nM). The k obs values obtained at various Ca 2+ concentrations are indicated. On the right, Data from (A), 50 µM free Ca 2+ and 1000 nM CaM WT or CaM C are re-plotted for comparison. (D) The activity of 2 nM eEF-2K with varying concentrations of CaM WT or CaM C in the absence of Ca 2+ was measured against 150 µM PepS with 1 mM [γ- 32 P]-ATP; the corresponding k obs values are shown.

Techniques: Activity Assay, Standard Deviation, Concentration Assay, Construct, SDS Page, Comparison

The dose-dependence of p eEF-2K (1 nM) activity on CaM WT or CaM C was measured against 150 µM PepS with 1 mM [γ- 32 P]-ATP in the presence of 1 mM free Ca 2+ and 10 mM free Mg 2+ . The k obs values were plotted against the concentration of CaM, and data were fit to to obtain best-fit values for k obs,max (CaM WT = 8.8 ± 0.3 s -1 ; CaM C = 8.5 ± 0.3 s -1 ) and (CaM WT = 41 ± 5 nM; CaM C = 73 ± 9 nM). Compare values for’ CaM WT (67 ± 7 nM) and CaM C (85 ± 10 nM) at a Ca 2+ : Mg 2+ ratio of 1:200.

Journal: bioRxiv

Article Title: The Critical Role of the C-terminal Lobe of Calmodulin in Activating Eukaryotic Elongation Factor 2 Kinase

doi: 10.1101/2025.05.13.653565

Figure Lengend Snippet: The dose-dependence of p eEF-2K (1 nM) activity on CaM WT or CaM C was measured against 150 µM PepS with 1 mM [γ- 32 P]-ATP in the presence of 1 mM free Ca 2+ and 10 mM free Mg 2+ . The k obs values were plotted against the concentration of CaM, and data were fit to to obtain best-fit values for k obs,max (CaM WT = 8.8 ± 0.3 s -1 ; CaM C = 8.5 ± 0.3 s -1 ) and (CaM WT = 41 ± 5 nM; CaM C = 73 ± 9 nM). Compare values for’ CaM WT (67 ± 7 nM) and CaM C (85 ± 10 nM) at a Ca 2+ : Mg 2+ ratio of 1:200.

Techniques: Activity Assay, Concentration Assay

$(A) A schematic representation of the C-LiNK construct in which CaM C (CaM residues 76-148) is connected via two glycine residues to an N-terminally truncated eEF-2K (71–725). The various key elements of eEF-2K, including the CaM-targeting motif (CTM), the α−kinase domain (KD), the regulatory loop (R-loop) with activating (T348, S500) and inhibiting (S359) phosphorylation sites are indicated schematically. (B) Multi-angle light scattering (MALS) analysis of purified C-LiNK is shown. The curve represents the sample’s refractive index, while the horizontal data points represent the estimated molar mass across the peak. C-LiNK is found to be monomeric, with a molar mass of ∼82.4 kDa. (C) The activity dependence of 1 nM wild-type eEF-2K (with 1 µM CaM) or C-LiNK (with 0 µM added CaM) on PepS concentration was measured using 1 mM [γ- 32 P]-ATP in the presence of 50 µM free Ca 2+ . The k obs was plotted against PepS concentration and fit to to obtain best-fit values for (eEF-2K = 19 ± 1 sec -1 ; C-LiNK = 26 ± 1 sec -1 ) and K m (eEF-2K = 59 ± 12; C-LiNK= 61 ± 9 µM). (D) The activity of 1 nM C-LiNK was measured against 150 µM PepS using 1 mM [γ- 32 P]-ATP, 10 mM free Mg 2+ , 1 mM EGTA, and various amounts of CaCl 2 . The open circles indicate experimental data points (n=3). (E) Measurement of C-LiNK (300 nM) activity towards the secondary autophosphorylation site, S500. Reactions were incubated with 0 or 1 µM free Ca 2+ at 30 °C before initiating with 1 mM ATP. Samples were subject to western blotting to detect total enzyme and p S500 (ECM Biosciences) levels; 0.125 µg of protein was loaded in each case. Signals for p S500 and total C-LiNK were quantified, and p S500 levels were corrected for their corresponding total C-LiNK signals, then converted to fraction phosphorylated by normalizing data to the 1 µM Ca 2+ 120 min sample. The experimental data, shown as circles representing the mean (n = 2) and standard deviation, were fit to (lower panel). The solid line indicates the best fit through the data. The best fit for 0 µM free Ca 2+ could not be ascertained (experimental data shown as black filled circles); for 1 µM free Ca 2+ (experimental data shown as red filled circles) was 0.00057 ± 0.6 ξ 10 -4 sec -1 ( t 1/2 ∼20 min).$

Journal: bioRxiv

Article Title: The Critical Role of the C-terminal Lobe of Calmodulin in Activating Eukaryotic Elongation Factor 2 Kinase

doi: 10.1101/2025.05.13.653565

Figure Lengend Snippet: (A) A schematic representation of the C-LiNK construct in which CaM C (CaM residues 76-148) is connected via two glycine residues to an N-terminally truncated eEF-2K (71–725). The various key elements of eEF-2K, including the CaM-targeting motif (CTM), the α−kinase domain (KD), the regulatory loop (R-loop) with activating (T348, S500) and inhibiting (S359) phosphorylation sites are indicated schematically. (B) Multi-angle light scattering (MALS) analysis of purified C-LiNK is shown. The curve represents the sample’s refractive index, while the horizontal data points represent the estimated molar mass across the peak. C-LiNK is found to be monomeric, with a molar mass of ∼82.4 kDa. (C) The activity dependence of 1 nM wild-type eEF-2K (with 1 µM CaM) or C-LiNK (with 0 µM added CaM) on PepS concentration was measured using 1 mM [γ- 32 P]-ATP in the presence of 50 µM free Ca 2+ . The k obs was plotted against PepS concentration and fit to to obtain best-fit values for (eEF-2K = 19 ± 1 sec -1 ; C-LiNK = 26 ± 1 sec -1 ) and K m (eEF-2K = 59 ± 12; C-LiNK= 61 ± 9 µM). (D) The activity of 1 nM C-LiNK was measured against 150 µM PepS using 1 mM [γ- 32 P]-ATP, 10 mM free Mg 2+ , 1 mM EGTA, and various amounts of CaCl 2 . The open circles indicate experimental data points (n=3). (E) Measurement of C-LiNK (300 nM) activity towards the secondary autophosphorylation site, S500. Reactions were incubated with 0 or 1 µM free Ca 2+ at 30 °C before initiating with 1 mM ATP. Samples were subject to western blotting to detect total enzyme and p S500 (ECM Biosciences) levels; 0.125 µg of protein was loaded in each case. Signals for p S500 and total C-LiNK were quantified, and p S500 levels were corrected for their corresponding total C-LiNK signals, then converted to fraction phosphorylated by normalizing data to the 1 µM Ca 2+ 120 min sample. The experimental data, shown as circles representing the mean (n = 2) and standard deviation, were fit to (lower panel). The solid line indicates the best fit through the data. The best fit for 0 µM free Ca 2+ could not be ascertained (experimental data shown as black filled circles); for 1 µM free Ca 2+ (experimental data shown as red filled circles) was 0.00057 ± 0.6 ξ 10 -4 sec -1 ( t 1/2 ∼20 min).

Techniques: Construct, Phospho-proteomics, Multi-Angle Light Scattering, Purification, Refractive Index, Activity Assay, Concentration Assay, Incubation, Western Blot, Standard Deviation

( A ) 2 µg of purified C-LiNK protein was run on a 10% polyacrylamide denaturing gel and stained for total protein content with Coomassie blue. ( B ) 0.5 µg of wild-type eEF-2K or C-LiNK protein were probed using eEF-2K and CaM-specific antibodies that recognize the C-termini of eEF-2K (Santa Cruz, residues 436-725) and CaM (Cell Signaling) ( C ) Purified wild-type eEF-2K (+/-co-expression with λ-phosphatase) or C-LiNK (co-expressed with λ-phosphatase) were incubated with 1 mM ATP for 0, 30, or 60 mins in the presence of 50 µM Ca 2+ before quenching with hot SDS-loading buffer. Samples were subject to western blotting to detect total enzyme and phosphate incorporation through autophosphorylation at T348 and S500 (0.125 µg of protein was loaded). ( D-E) C-LiNK was expressed in the presence of λ-phosphatase and either treated or not during the purification process with λ-phosphatase (+/-λ-PP treatment). The autophosphorylation reaction was performed in assay buffer H [25 mM HEPES (pH 7.5), 50 mM KCl, 10 mM MgCl 2 , 100 µM EGTA, 2 mM DTT, 20 µg/mL BSA, 0.005% Brij-35] at 30 °C with 200 nM enzyme in the presence of 50 µM free CaCl 2 . The reaction was initiated with 1 mM ATP, then quenched at various time points by adding hot SDS loading buffer. 150 ng of enzyme was run on SDS-PAGE, and phosphate incorporation was detected by western blotting for ( D ) p T348 or ( E ) p S445 and ( D-E ) total eEF-2K.

Journal: bioRxiv

Article Title: The Critical Role of the C-terminal Lobe of Calmodulin in Activating Eukaryotic Elongation Factor 2 Kinase

doi: 10.1101/2025.05.13.653565

Figure Lengend Snippet: ( A ) 2 µg of purified C-LiNK protein was run on a 10% polyacrylamide denaturing gel and stained for total protein content with Coomassie blue. ( B ) 0.5 µg of wild-type eEF-2K or C-LiNK protein were probed using eEF-2K and CaM-specific antibodies that recognize the C-termini of eEF-2K (Santa Cruz, residues 436-725) and CaM (Cell Signaling) ( C ) Purified wild-type eEF-2K (+/-co-expression with λ-phosphatase) or C-LiNK (co-expressed with λ-phosphatase) were incubated with 1 mM ATP for 0, 30, or 60 mins in the presence of 50 µM Ca 2+ before quenching with hot SDS-loading buffer. Samples were subject to western blotting to detect total enzyme and phosphate incorporation through autophosphorylation at T348 and S500 (0.125 µg of protein was loaded). ( D-E) C-LiNK was expressed in the presence of λ-phosphatase and either treated or not during the purification process with λ-phosphatase (+/-λ-PP treatment). The autophosphorylation reaction was performed in assay buffer H [25 mM HEPES (pH 7.5), 50 mM KCl, 10 mM MgCl 2 , 100 µM EGTA, 2 mM DTT, 20 µg/mL BSA, 0.005% Brij-35] at 30 °C with 200 nM enzyme in the presence of 50 µM free CaCl 2 . The reaction was initiated with 1 mM ATP, then quenched at various time points by adding hot SDS loading buffer. 150 ng of enzyme was run on SDS-PAGE, and phosphate incorporation was detected by western blotting for ( D ) p T348 or ( E ) p S445 and ( D-E ) total eEF-2K.

Techniques: Purification, Staining, Expressing, Incubation, Western Blot, SDS Page

(A) The organization of the C-LiNK TR construct is shown schematically on the left panel. This construct is derived from C-LiNK with an R-loop segment comprising residues 359-489 replaced by 6 glycines in analogy to eEF-2K TR . The structure of C-LiNK TR is shown on the right panel, with the key structural modules indicated and colored as on the left panel. Two ADP molecules, one bound at the catalytic site and a second bound to the interface between the CaM C module and the N-lobe of the KD, are shown as spheres. The phosphorylated T348 ( p T348) is also indicated. (B) Conformation of key catalytic site elements in C-LiNK (left) and the CaM• p eEF-2K TR complex (PDB: 8FNY, right) show no significant variation in conformation. D284 in the C-LiNK structure is phosphorylated and contains a bound Mg 2+ ion. Hydrogen bonds are indicated by the green dashed lines in all cases; the gold dashed lines denote heteroatoms with 3.2 Å of the metal center. (C) The activation spine that links CaM C to the kinase catalytic site through the bound nucleotide is fully formed in C-LiNK. The geometry of the spine in C-LiNK (top panel; eEF-2K modules in light blue, CaM C in yellow) is identical to that seen in the structures of the CaM•eEF-2K TR complex (bottom panel; a representative heterodimeric complex, PDB: 8FNY; eEF-2K in pink, CaM C in orange). Key spine residues are labeled (3-letter code for the CaM residue), the nucleotide bound to the catalytic site is shown in both cases, and the active site is circled. (D) The coordination of p T348 at the phosphate-binding pocket in C-LiNK (left) and the 8FNY structure (right) is identical.

Journal: bioRxiv

Article Title: The Critical Role of the C-terminal Lobe of Calmodulin in Activating Eukaryotic Elongation Factor 2 Kinase

doi: 10.1101/2025.05.13.653565

Figure Lengend Snippet: (A) The organization of the C-LiNK TR construct is shown schematically on the left panel. This construct is derived from C-LiNK with an R-loop segment comprising residues 359-489 replaced by 6 glycines in analogy to eEF-2K TR . The structure of C-LiNK TR is shown on the right panel, with the key structural modules indicated and colored as on the left panel. Two ADP molecules, one bound at the catalytic site and a second bound to the interface between the CaM C module and the N-lobe of the KD, are shown as spheres. The phosphorylated T348 ( p T348) is also indicated. (B) Conformation of key catalytic site elements in C-LiNK (left) and the CaM• p eEF-2K TR complex (PDB: 8FNY, right) show no significant variation in conformation. D284 in the C-LiNK structure is phosphorylated and contains a bound Mg 2+ ion. Hydrogen bonds are indicated by the green dashed lines in all cases; the gold dashed lines denote heteroatoms with 3.2 Å of the metal center. (C) The activation spine that links CaM C to the kinase catalytic site through the bound nucleotide is fully formed in C-LiNK. The geometry of the spine in C-LiNK (top panel; eEF-2K modules in light blue, CaM C in yellow) is identical to that seen in the structures of the CaM•eEF-2K TR complex (bottom panel; a representative heterodimeric complex, PDB: 8FNY; eEF-2K in pink, CaM C in orange). Key spine residues are labeled (3-letter code for the CaM residue), the nucleotide bound to the catalytic site is shown in both cases, and the active site is circled. (D) The coordination of p T348 at the phosphate-binding pocket in C-LiNK (left) and the 8FNY structure (right) is identical.

Techniques: Construct, Derivative Assay, Activation Assay, Labeling, Residue, Binding Assay

An overlay of all available CaM• p eEF-2K TR complex structures with C-LiNK TR suggests that the overall conformations of its eEF-2K TR and the CaM C modules remain unchanged. The 7SHQ structure shows a slightly different CTD conformation than all other structures, including C-LiNK TR (see inset for an expanded view; the CTDs of C-LiNK TR , 8FNY, and 7SHQ are shown). The 7SHQ structure was solved using different crystallization conditions, and its slightly altered CTD conformation is likely a reflection of interdomain flexibility.

Journal: bioRxiv

Article Title: The Critical Role of the C-terminal Lobe of Calmodulin in Activating Eukaryotic Elongation Factor 2 Kinase

doi: 10.1101/2025.05.13.653565

Figure Lengend Snippet: An overlay of all available CaM• p eEF-2K TR complex structures with C-LiNK TR suggests that the overall conformations of its eEF-2K TR and the CaM C modules remain unchanged. The 7SHQ structure shows a slightly different CTD conformation than all other structures, including C-LiNK TR (see inset for an expanded view; the CTDs of C-LiNK TR , 8FNY, and 7SHQ are shown). The 7SHQ structure was solved using different crystallization conditions, and its slightly altered CTD conformation is likely a reflection of interdomain flexibility.

Techniques: Crystallization Assay

$(A) MCF10A eEF-2K-/- cells were transfected with the indicated amount of vector encoding either wild-type eEF-2K or C-LiNK. After 16 hours, cells were lysed and analyzed by western blotting using specific antibodies for eEF-2K, eEF2, p eEF-2, and actin (loading control). (B) MCF10A eEF-2K-/- cells transfected with a vector encoding no gene, WT eEF-2K, or C-LiNK. After 16 hours, cells were treated for 2 hours with complete media (NT) or starved with DPBS (STRV). The experiment was performed in triplicate, and a representative western blot is shown. The graph shows the p eEF-2 signal corrected by the total eEF-2 signal, then normalized as the fraction of the signal for wild-type eEF-2K STRV. (C) Wild-type eEF-2K or C-LiNK expressing cells were starved or given fresh media, and the abundance of the inhibitory phosphorylation on S359 was assessed via western blotting.$

Journal: bioRxiv

Article Title: The Critical Role of the C-terminal Lobe of Calmodulin in Activating Eukaryotic Elongation Factor 2 Kinase

doi: 10.1101/2025.05.13.653565

Figure Lengend Snippet: (A) MCF10A eEF-2K-/- cells were transfected with the indicated amount of vector encoding either wild-type eEF-2K or C-LiNK. After 16 hours, cells were lysed and analyzed by western blotting using specific antibodies for eEF-2K, eEF2, p eEF-2, and actin (loading control). (B) MCF10A eEF-2K-/- cells transfected with a vector encoding no gene, WT eEF-2K, or C-LiNK. After 16 hours, cells were treated for 2 hours with complete media (NT) or starved with DPBS (STRV). The experiment was performed in triplicate, and a representative western blot is shown. The graph shows the p eEF-2 signal corrected by the total eEF-2 signal, then normalized as the fraction of the signal for wild-type eEF-2K STRV. (C) Wild-type eEF-2K or C-LiNK expressing cells were starved or given fresh media, and the abundance of the inhibitory phosphorylation on S359 was assessed via western blotting.

Techniques: Transfection, Plasmid Preparation, Western Blot, Control, Expressing, Phospho-proteomics

MCF10A eEF- 2K-/- cells were transfected with 0.1 µg of either wild-type (WT) or D284A HAF-eEF-2K, unmodified (UNM, D284) or D284A C-LiNK. After 16 hours, cells were lysed and 40 µg of cell lysate was analyzed by western blotting using specific antibodies for eEF-2K, p eEF-2, and actin (loading control).

Journal: bioRxiv

Article Title: The Critical Role of the C-terminal Lobe of Calmodulin in Activating Eukaryotic Elongation Factor 2 Kinase

doi: 10.1101/2025.05.13.653565

Figure Lengend Snippet: MCF10A eEF- 2K-/- cells were transfected with 0.1 µg of either wild-type (WT) or D284A HAF-eEF-2K, unmodified (UNM, D284) or D284A C-LiNK. After 16 hours, cells were lysed and 40 µg of cell lysate was analyzed by western blotting using specific antibodies for eEF-2K, p eEF-2, and actin (loading control).

Techniques: Transfection, Western Blot, Control

400 nM eEF-2K was incubated with the indicated concentrations of CaM labeled with the fluorescent dye IAEDANS (I-CaM; the dye was covalently attached to position 75 in a K75C mutant of CaM) and 100 µM free CaCl 2 for 20 min at room temperature before running samples on a gradient (4-15%) native gel. Fluorescence was detected by exposing the gel to ultraviolet light for 0.5 (bottom) or 34.5 (top) seconds before capturing the image. The gel was Coomassie stained to detect total protein.

Journal: bioRxiv

Article Title: The Critical Role of the C-terminal Lobe of Calmodulin in Activating Eukaryotic Elongation Factor 2 Kinase

doi: 10.1101/2025.05.13.653565

Figure Lengend Snippet: 400 nM eEF-2K was incubated with the indicated concentrations of CaM labeled with the fluorescent dye IAEDANS (I-CaM; the dye was covalently attached to position 75 in a K75C mutant of CaM) and 100 µM free CaCl 2 for 20 min at room temperature before running samples on a gradient (4-15%) native gel. Fluorescence was detected by exposing the gel to ultraviolet light for 0.5 (bottom) or 34.5 (top) seconds before capturing the image. The gel was Coomassie stained to detect total protein.

Techniques: Incubation, Labeling, Mutagenesis, Fluorescence, Staining

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