eef 2k  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Ser500 phosphorylation acts as a conformational switch to prime eEF-2K for activation

doi: 10.1101/2025.06.30.662482

Figure Lengend Snippet:

Article Snippet: 10 nM GST-tagged eEF-2K (SignalChem, #E01-10G-320) and 2 nM His 6 -tagged CaM (EMD Millipore, #208670) with 1 mM excess CaCl 2 were incubated with 0 – 50 nM of eEF-2K λ , S500D-eEF-2K λ , eEF-2K p or S500D-eEF-2K p , or 0 – 5 μM W85A-eEF-2K λ for 30 min at room temperature.

Techniques: Recombinant, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: The critical role of the C-terminal lobe of calmodulin in activating eukaryotic elongation factor 2 kinase

doi: 10.1016/j.jbc.2025.110650

Figure Lengend Snippet: CaM C stimulates eEF-2K autophosphorylation. A , eEF-2K (300 nM) activity toward the primary autophosphorylation site T348 was measured with 1 μM CaM, 1 μM CaM C , 1 or 10 μM CaM N or no CaM, in 50 μM free Ca 2+ . Reactions were initiated with 1 mM Mg 2+ •ATP and quenched at various times by addition of hot SDS-loading buffer. Western blot detected p T348 and total eEF-2K. Representative data from two independent experiments are shown. B - C , rapid quench-flow analysis measured T348 autophosphorylation rates. eEF-2K (200 nM) was preincubated with 2 μM CaM ( B ) or 2 μM CaM C ( C ) at 50 μM free Ca 2+ , rapidly mixed with 1 mM Mg 2+ •ATP and quenched at different times. Western blots quantified phosphorylation as fraction of maximal control values (2 or 60 s for CaM or CaM C , respectively). Data (mean ± SD, n = 2) were fit to Equation yielding apparent autophosphorylation rate constants ( k auto app ) of 3.8 ± 0.38 s −1 for CaM and 1.4 ± 0.09 s −1 for CaM C . CaM, calmodulin; CaM C , C-terminal lobe of CaM; CaM N , N-terminal lobe of CaM; eEF-2K, eukaryotic elongation factor 2 kinase.

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

Techniques: Activity Assay, Western Blot, Phospho-proteomics, Control

Journal: The Journal of Biological Chemistry

Article Title: The critical role of the C-terminal lobe of calmodulin in activating eukaryotic elongation factor 2 kinase

doi: 10.1016/j.jbc.2025.110650

Figure Lengend Snippet: CaM C binds and activates eEF-2K in a Ca 2+ -sensitive manner. A , dose-dependent activation of eEF-2K (1 nM) by CaM or CaM C was measured using 150 μM peptide substrate in 50 μM free Ca 2+ and 1 mM [γ- 32 P]-ATP. Observed rate constants ( k obs , mean ± SD, n = 2) were plotted versus CaM concentration and fit to Equation to derive k obs max and K CaM app . Parameters: k obs max = 20 ± 0.6 s −1 and K CaM app = 67 ± 7 nM for CaM; k obs max = 20 ± 0.7 s −1 and K CaM app = 85 ± 10 nM for CaM C . B , eEF-2K (0.5 nM) activity toward 5 μM yeast eEF-2 was measured with varied CaM or CaM C concentrations in 50 μM free Ca 2+ and 1 mM [γ- 32 P]-ATP. Reactions were quenched by hot SDS-loading buffer and analyzed by SDS-PAGE. Phosphorimaging visualized 32 P incorporation (n = 4). Gel bands were quantified by scintillation counting. k obs values were plotted and fit to Equation , yielding k obs max = 6.1 ± 1.5 s −1 and K CaM app = 121 ± 41 nM for CaM; k obs max = 5.6 ± 1.4 s −1 and K CaM app = 156 ± 64 nM for CaM C . C , activity of 2 nM eEF-2K toward 150 μM PepS was measured in the presence of 1 μM CaM or CaM C with varying free Ca 2+ (0–1000 nM). k obs values (mean ± SD, n = 3) are shown as bars. Individual data from ( A ) at 50 μM free Ca 2+ and 1 μM CaM or CaM C are replotted for comparison. D , activity of 2 nM eEF-2K with varying CaM or CaM C concentrations in the absence of Ca 2+ was measured using 150 μM PepS. Corresponding k obs values are shown (mean ± SD, n = 2). CaM, calmodulin; CaM C , C-terminal lobe of CaM; eEF-2K, eukaryotic elongation factor 2 kinase; PepS, peptide substrate.

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

Techniques: Activation Assay, Concentration Assay, Activity Assay, SDS Page, Comparison

Journal: The Journal of Biological Chemistry

Article Title: The critical role of the C-terminal lobe of calmodulin in activating eukaryotic elongation factor 2 kinase

doi: 10.1016/j.jbc.2025.110650

Figure Lengend Snippet: In vitro characterization of C-LiNK. A , schematic of C-LiNK construct: CaM C (residues 76–148) linked via two glycines to N terminally truncated eEF-2K (residues 71–725). Structural elements include CaM-targeting motif (CTM), α-kinase domain (KD), and regulatory loop (R-loop) with activating (T348, S500) and inhibitory (S359) phosphorylation sites. B , multiangle light scattering (MALS) of purified C-LiNK shows a monomeric species with molar mass ∼82.4 kDa, consistent with predicted ∼83 kDa. C , activity of 1 nM eEF-2K (with 1 μM CaM) or C-LiNK (without added CaM) was measured at varying PepS concentrations with 1 mM [γ- 32 P]-ATP and 50 μM free Ca 2+ . k obs values (mean ± SD, n = 2) were fit to Equation to yield k cat app and K m app : eEF-2K (19 ± 1 s −1 , 59 ± 12 μM) and C-LiNK (26 ± 1 s −1 , 61 ± 9 μM). D , activity of 1 nM C-LiNK was measured against 150 μM PepS with 1 mM [γ- 32 P]-ATP, 10 mM Mg 2+ , 1 mM EGTA, and varying free Ca 2+ . Independent data points (n = 3) are shown as open circles ; mean ± SD as bars with error lines . E , autophosphorylation of C-LiNK (300 nM) at S500 was measured at 30 °C with 0 or 1 μM free Ca 2+ . Reactions were initiated with 1 mM ATP and samples collected over 0 to 120 min. Western blots quantified p S500 normalized to total protein; fraction phosphorylated was normalized to 1 μM Ca 2+ at 120 min. Data were fit to Equation . Apparent autophosphorylation rate constant ( k auto app ) was 0.00057 ± 0.00006 s −1 (t 1/2 ∼20 min) at 1 μM Ca 2+ ; k auto app was not determined at 0 μM Ca 2+ . C-LiNK, CaM C is linked to N-truncated eEF-2K; eEF-2K, eukaryotic elongation factor 2 kinase; PepS, peptide substrate.

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

Techniques: In Vitro, Construct, Phospho-proteomics, Multi-Angle Light Scattering, Purification, Activity Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The critical role of the C-terminal lobe of calmodulin in activating eukaryotic elongation factor 2 kinase

doi: 10.1016/j.jbc.2025.110650

Figure Lengend Snippet: Structure of C-LiNK TR . A , the organization of the C-LiNK TR construct is shown schematically on the left panel . This construct is derived from C-LiNK ( A ), with an R-loop segment comprising residues 359 to 489 replaced by six 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 , the conformations of key catalytic site elements in the C-LiNK TR ( left ) and the CaM• p eEF-2K TR complex (PDB: 8FNY , right ) structures show no substantial differences. D284 in the C-LiNK TR structure is phosphorylated and a Mg 2+ ion is bound at the catalytic site. 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 TR . The geometry of the spine in C-LiNK TR ( 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 (three-letter codes are used for CaM residues), 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 TR ( left ) and the 8FNY structure ( right ) is identical. CaM C , C-terminal lobe of CaM; C-LiNK, CaM C is linked to N-truncated eEF-2K; eEF-2K, eukaryotic elongation factor 2 kinase; KD, kinase domain; R-loop, regulatory loop.

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

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

Journal: The Journal of Biological Chemistry

Article Title: The critical role of the C-terminal lobe of calmodulin in activating eukaryotic elongation factor 2 kinase

doi: 10.1016/j.jbc.2025.110650

Figure Lengend Snippet: C-LiNK activity in MCF10A eef2k −/− cells is resistant to inhibitory signaling. A , MCF10A eef2k −/− cells were transfected with indicated amounts of pcDNA3 encoding eEF-2K or C-LiNK. After 16 h, lysates were analyzed by Western blot for eEF-2K, eEF2, phospho-eEF2, and actin (loading control). B , cells transfected with empty vector, eEF-2K, or C-LiNK were treated for 2 h with complete media (NT) or starved with DPBS (STRV). A representative Western blot shown on the left . The graph ( right ) displays data from ( B ) with the phospho-eEF2 signal normalized to total eEF2 and expressed as a fraction of eEF-2K STRV signal. The mean ± SD (n = 3) is indicated. C , cells expressing eEF-2K or C-LiNK were starved or given fresh media; inhibitory phosphorylation at S359 was assessed by Western blot. C-LiNK, CaM C is linked to N-truncated eEF-2K; DPBS, Dulbecco’s PBS; eEF-2, eukaryotic elongation factor 2; eEF-2K, eukaryotic elongation factor 2 kinase.

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

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

Journal: bioRxiv

Article Title: Ser500 phosphorylation acts as a conformational switch to prime eEF-2K for activation

doi: 10.1101/2025.06.30.662482

Figure Lengend Snippet: (A) The ability of recombinant wild-type eEF-2K (WT) and the phosphomimetic S500D to bind CaM was assessed via their association with a CaM labeled with the fluorophore IAEDANS (CaM IAE ) on a nondenaturing gel. The purified proteins were either co-expressed with l-phosphatase (indicated by “ λ ”; unphosphorylated at T348) or not (“ p ” indicates phosphorylated on T348). Each construct was incubated with the indicated concentrations of CaM IAE . The +Ca 2+ samples contained 150 μM free Ca 2+ . The samples were run on a native gel to separate the unbound CaM AE from the eEF-2K/CaM AE complexes. The fluorescence of the bound CaM IAE was visualized with a UV imager, and the total protein was determined by Coomassie staining. (B) An AlphaScreen assay was used to monitor competition between GST-eEF-2K (10 nM) and untagged eEF-2K mutants (0–50 nM) for His-tagged CaM (2 nM) in the presence of 1 mM Ca 2+ . (C) IC 50 values from the plot in (B) were determined for WT p (0.78 ± 0.044 nM), S500D p (0.47 ± 0.026 nM), WT λ (2.1 ± 0.14 nM), and S500D λ (0.86 ± 0.054 nM). Two replicates were used in each case with the error bars representing the standard deviation.

Article Snippet: The native proteins were transferred to a PVDF membrane for 16 h at 30V in 25 mM Tris, 192 mM glycine, 2 mM CaCl 2 , and 20% methanol and then probed with CaM (4830, Cells Signaling Technology), eEF-2K (C-12, sc-390710, Santa Cruz Biotechnology), or p T348 eEF-2K (EP4411, ECM Biosciences) specific antibodies.

Techniques: Recombinant, Labeling, Purification, Construct, Incubation, Fluorescence, Staining, Amplified Luminescent Proximity Homogenous Assay, Standard Deviation

Journal: bioRxiv

Article Title: Ser500 phosphorylation acts as a conformational switch to prime eEF-2K for activation

doi: 10.1101/2025.06.30.662482

Figure Lengend Snippet: (A) The entire gel from , showing bound and unbound CaM IAE fluorescence (512 nm excitation). The bound CaM IAE comigrates with recombinant eEF-2K (∼82 KDa) on the native gel, while the excess free CaM (∼17 KDa) migrates more rapidly through the gel. Addition of Ca 2+ causes a slight upward shift in free CaM. (B) The ability of recombinant eEF-2K proteins (“ p ” denotes presence of p T348, while “ λ ” denotes coexpression with λ-phosphatase, i.e., T348 is not phosphorylated) to bind CaM IAE on a native gel was assessed similarly to , except the incubation tubes contained 1 mM ADP and the enzyme concentration was increased from 0.67 to 2.5 µM. The CaM IAE fluorescence was measured using two different exposure times, 8 and 20 sec, to visualize low fluorescence signals. (C) Assessment of binding of recombinant eEF-2K proteins (0.2 µM) to CaM (0.2 µM) by native gel followed by western blotting with antibodies for CaM and eEF-2K. The concentrations of free Ca 2+ are indicated. (D) S500D-eEF-2K p was pre-incubated with or without purified λ-phosphatase (λ-pp) before combining with either 25 µM calcium-free CaM or 2 µM CaM with 150 µM free Ca 2+ . Samples were loaded onto a native gel and analyzed by Western blot using CaM, eEF-2K, and pT348-eEF-2K-specific antibodies.

Article Snippet: The native proteins were transferred to a PVDF membrane for 16 h at 30V in 25 mM Tris, 192 mM glycine, 2 mM CaCl 2 , and 20% methanol and then probed with CaM (4830, Cells Signaling Technology), eEF-2K (C-12, sc-390710, Santa Cruz Biotechnology), or p T348 eEF-2K (EP4411, ECM Biosciences) specific antibodies.

Techniques: Fluorescence, Recombinant, Incubation, Concentration Assay, Binding Assay, Western Blot, Purification

Journal: bioRxiv

Article Title: Ser500 phosphorylation acts as a conformational switch to prime eEF-2K for activation

doi: 10.1101/2025.06.30.662482

Figure Lengend Snippet: Schematic representation of the various eEF-2K constructs, including wild-type full-length eEF-2K, the eEF-2K TR (a 6-glycine linker has replaced missing 70 N-terminal residues, and the 359-490 segment; the linker is indicated in purple), eEF-2K ΔN (missing 70 N-terminal residues but containing an intact regulatory loop), eEF-2K Δ490-520 (a 6-glycine linker has replaced 490-520 segment), and eEF-2K Δ497-502 (a 6-glycine linker has replaced 490-502 segment). The N-terminal calmodulin-targeting motif (CTM), the α−kinase domain (KD), the regulatory loop, and the C-terminal domain (CTD) are indicated. The activating T348 and S500 sites are located at the two ends of the regulatory loop.

Article Snippet: The native proteins were transferred to a PVDF membrane for 16 h at 30V in 25 mM Tris, 192 mM glycine, 2 mM CaCl 2 , and 20% methanol and then probed with CaM (4830, Cells Signaling Technology), eEF-2K (C-12, sc-390710, Santa Cruz Biotechnology), or p T348 eEF-2K (EP4411, ECM Biosciences) specific antibodies.

Techniques: Construct

Journal: bioRxiv

Article Title: Ser500 phosphorylation acts as a conformational switch to prime eEF-2K for activation

doi: 10.1101/2025.06.30.662482

Figure Lengend Snippet: (A) Hydrogen-deuterium exchange mass spectrometry (HXMS) experiments were performed on eEF-2K ΔN alone or in a complex with CaM. T348 was dephosphorylated in all cases. The only region within eEF-2K ΔN that showed statistically significant differences between its CaM-free and CaM-bound states involved the V502-D513 segment proximal to S500. Two overlapping peptides corresponding to this region are shown. These peptides illustrate reduced 2 H incorporation in the CaM-free compared to the CaM-bound state. ( B) MCF10A eEF-2K -/- were transfected with the indicated eEF-2K mutants. eEF-2K expression and eEF-2 phosphorylation were assessed via western blotting. (C) The intrinsic activities of eEF-2K p , S500D-eEF-2K p , and eEF-2K Δ497-502 p were measured (in duplicate) using 450 μM PepS. k obs values of eEF-2K p , S500D-eEF-2K p , and eEF-2K Δ497-502 p are displayed in bar graph form. The mean values in the three cases were 0.04 s -1 , 0.9 s -1 , and 0.8 s -1 , respectively. (D) The association of recombinant eEF-2K p and corresponding mutants with the fluorescent CaM IAE via native gel.

Article Snippet: The native proteins were transferred to a PVDF membrane for 16 h at 30V in 25 mM Tris, 192 mM glycine, 2 mM CaCl 2 , and 20% methanol and then probed with CaM (4830, Cells Signaling Technology), eEF-2K (C-12, sc-390710, Santa Cruz Biotechnology), or p T348 eEF-2K (EP4411, ECM Biosciences) specific antibodies.

Techniques: Mass Spectrometry, Transfection, Expressing, Phospho-proteomics, Western Blot, Recombinant

Journal: bioRxiv

Article Title: Ser500 phosphorylation acts as a conformational switch to prime eEF-2K for activation

doi: 10.1101/2025.06.30.662482

Figure Lengend Snippet: Left panel: Wild-type eEF-2K (E, black) has low activity until bound to CaM (E•CaM). The formation of this complex (step 1) promotes autophosphorylation at T348 (step 2) to yield the active complex (E p T348 •CaM). The E p T348 form has a higher affinity for CaM than E and partially stabilizes the active complex (step 3). Right panel: Phosphorylation of S500 (mimicked by the S500D mutant, E, purple) increases the amount of active complex (E p T348 •CaM) by increasing the affinity of E for CaM (step 1), enhancing the rate of T348 autophosphorylation (step 2) and increasing the affinity of the corresponding E p T348 form for CaM (step 3). Cellular eEF-2K is phosphorylated at S500 either through CaM-induced autophosphorylation or by PKA. In the former case, phosphorylation on S500 occurs once T348 is phosphorylated and serves to trap eEF-2K in the E p T348 •CaM complex by significantly enhancing CaM affinity, i.e., affecting only step 3. PKA-mediated phosphorylation of S500, however, can influence all three steps to drive the formation of a stable E p T348 •CaM complex.

Article Snippet: The native proteins were transferred to a PVDF membrane for 16 h at 30V in 25 mM Tris, 192 mM glycine, 2 mM CaCl 2 , and 20% methanol and then probed with CaM (4830, Cells Signaling Technology), eEF-2K (C-12, sc-390710, Santa Cruz Biotechnology), or p T348 eEF-2K (EP4411, ECM Biosciences) specific antibodies.

Techniques: Activity Assay, Phospho-proteomics, Mutagenesis