Journal: Medicines

Article Title: Discovery of Novel eEF2K Inhibitors Using HTS Fingerprint Generated from Predicted Profiling of Compound-Protein Interactions

doi: 10.3390/medicines8050023

Figure Lengend Snippet: Representative eEF2K inhibitors with the corresponding IC 50 values obtained from relevant references.

Article Snippet: Off-Chip Mobility Shift Assay (MSA) for the inhibitory activities of the compounds against eEF2K was conducted by Carna Biosciences, Inc. (Kobe, Japan) using QuickScout Screening Assist™ Mobility Shift Assay Kit.

Techniques:

Journal: Medicines

Article Title: Discovery of Novel eEF2K Inhibitors Using HTS Fingerprint Generated from Predicted Profiling of Compound-Protein Interactions

doi: 10.3390/medicines8050023

Figure Lengend Snippet: Preliminary measurements of eEF2K inhibitory activities of selected 27 compounds. Black dots indicate values for duplicate measurements and crossbars indicate mean values for each compound.

Article Snippet: Off-Chip Mobility Shift Assay (MSA) for the inhibitory activities of the compounds against eEF2K was conducted by Carna Biosciences, Inc. (Kobe, Japan) using QuickScout Screening Assist™ Mobility Shift Assay Kit.

Techniques:

Journal: Medicines

Article Title: Discovery of Novel eEF2K Inhibitors Using HTS Fingerprint Generated from Predicted Profiling of Compound-Protein Interactions

doi: 10.3390/medicines8050023

Figure Lengend Snippet: Predicted binding modes of ATP ( a ), CHEMBL1089330 ( b ), CHEMBL1090045 ( c ), compound 2 ( d ), compound 4 ( e ) and compound 13 ( f ) on eEF2K homology model. Carbon, nitrogen, oxygen and hydrogen are shown in green (ATP, compound 2, compound 4 and compound 13)/gray (eEF2K), blue, red and white, respectively. Dashed lines indicate hydrogen bond interactions. eEF2K is shown as ribbon representation.

Article Snippet: Off-Chip Mobility Shift Assay (MSA) for the inhibitory activities of the compounds against eEF2K was conducted by Carna Biosciences, Inc. (Kobe, Japan) using QuickScout Screening Assist™ Mobility Shift Assay Kit.

Techniques: Binding Assay

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

Journal: Oncogene

Article Title: eEF-2 kinase is a critical regulator of Warburg effect through controlling PP2A-A synthesis.

doi: 10.1038/onc.2016.166

Figure Lengend Snippet: Figure 2. Silencing of eEF-2K expression blunts the hypoxia-stimulated glycolysis and reduces survival of hypoxic tumor cells. (a) MDA-MB-231 or MCF-7 cells were treated by hypoxia for 24 h. Expression of hypoxia-inducible factor-1α (HIF-1α) protein was examined by western blot. Tubulin was used as a loading control. (b–e) MDA-MB-231 or MCF-7 cells were transfected with a nontargeting RNA (control) or an eEF-2K siRNA, followed by incubation in a hypoxia chamber. (b) At 24 h following hypoxia treatment, concentration of L-lactate released to the cell culture medium was measured. (c) At 0, 12 and 24 h following hypoxia treatment, cellular ATP level was assayed. (d) At 0, 24 and 48 h following hypoxia treatment, cellular viability was determined. For (b–d), each bar represents the mean ± s.d. of eight determinations from one of three independent experiments. *Po0.05, **Po0.01. (e) At 0, 24 and 48 h following hypoxia treatment, apoptosis was determined. Each bar represents the mean ± s.e. of three identical experiments.

Article Snippet: Antibodies to p-eEF-2 (2331), eEF-2 (2332), eEF-2K (3692), PKM2 (4053), c-Myc (5605), α-tubulin (2148) and PP2A-A (2041) were purchased from Cell Signaling Technologies (Danvers, MA, USA).

Techniques: Expressing, Western Blot, Control, Transfection, Incubation, Concentration Assay, Cell Culture

Journal: Oncogene

Article Title: eEF-2 kinase is a critical regulator of Warburg effect through controlling PP2A-A synthesis.

doi: 10.1038/onc.2016.166

Figure Lengend Snippet: Figure 3. Effect of eEF-2K on PKM2. (a–d) MDA-MB-231 or MCF-7 cells were transfected with a nontargeting RNA (control) or an eEF-2K siRNA. At the end of transfection, expressions of eEF-2K and PKM2 protein (a), PKM2 activity (b), pyruvate level (c) and PKM2 mRNA (d) were measured. (e, f) MDA-MB-231 or MCF-7 cells were transfected with a control plasmid or an eEF-2K expression plasmid. Expressions of eEF-2K and PKM2 protein (e) and PKM2 mRNA (f) were measured. (g, h) MDA-MB-231 or MCF-7 cells were transfected with a nontargeting RNA (control) or an eEF-2K siRNA, followed by transfection with a PKM2 expression plasmid. The concentration of glucose in the conditioned medium (g) and the amount of lactate released to the cell culture medium (h) were measured. Results shown were normalized to the control; each bar represents the mean ± s.e. of three identical experiments. *Po0.05, **Po0.01. (i–k) MDA-MB-231 or MCF-7 cells were transfected with a nontargeting RNA (control) or an eEF-2K siRNA, followed by hypoxia treatment. At the end of treatment, PKM2 mRNA (i), PKM2 activity (j) and pyruvate level (k) were measured. Each bar represents the mean ± s.e. of three identical experiments.

Article Snippet: Antibodies to p-eEF-2 (2331), eEF-2 (2332), eEF-2K (3692), PKM2 (4053), c-Myc (5605), α-tubulin (2148) and PP2A-A (2041) were purchased from Cell Signaling Technologies (Danvers, MA, USA).

Techniques: Transfection, Control, Activity Assay, Plasmid Preparation, Expressing, Concentration Assay, Cell Culture

Journal: Oncogene

Article Title: eEF-2 kinase is a critical regulator of Warburg effect through controlling PP2A-A synthesis.

doi: 10.1038/onc.2016.166

Figure Lengend Snippet: Figure 4. Overexpression of c-Myc reverses the eEF-2K-mediated downregulation of PKM2 mRNA expression. (a) MDA-MB-231 or MCF-7 cells were transfected with a nontargeting RNA (control) or an eEF-2K siRNA. At the end of transfection, expressions of eEF-2K and c-Myc protein were examined by western blot. Tubulin was used as a loading control. (b) MDA-MB-231 or MCF-7 cells were transfected with a nontargeting RNA (control) or a c-Myc siRNA. Expression of c-Myc protein was examined by western blot. Tubulin was used as a loading control. Expression of PKM2 mRNA was measured by qRT–PCR. (c) MDA-MB-231 or MCF-7 cells were transfected with a control plasmid or a His-c-Myc expression plasmid. Expressions of c-Myc protein and His were examined by western blot. Tubulin was used as a loading control. Expression of PKM2 mRNA was measured by qRT–PCR. (d) MDA-MB-231 or MCF-7 cells were transfected with a nontargeting RNA (control) or an eEF-2K siRNA, followed by transfection with a c-Myc expression plasmid. Expression of PKM2 mRNA was measured by qRT–PCR. Each bar represents the mean ± s.e. of three identical experiments. **Po0.01.

Article Snippet: Antibodies to p-eEF-2 (2331), eEF-2 (2332), eEF-2K (3692), PKM2 (4053), c-Myc (5605), α-tubulin (2148) and PP2A-A (2041) were purchased from Cell Signaling Technologies (Danvers, MA, USA).

Techniques: Over Expression, Expressing, Transfection, Control, Western Blot, Quantitative RT-PCR, Plasmid Preparation

Journal: Oncogene

Article Title: eEF-2 kinase is a critical regulator of Warburg effect through controlling PP2A-A synthesis.

doi: 10.1038/onc.2016.166

Figure Lengend Snippet: Figure 5. Silencing of eEF-2K expression promotes degradation of c-Myc protein. (a) MDA-MB-231 or MCF-7 cells were transfected with a nontargeting RNA (control) or an eEF-2K siRNA. At the end of transfection, expression of c-Myc mRNA was measured by qRT–PCR. (b) MDA- MB-231 or MCF-7 cells were transfected with a nontargeting RNA or an eEF-2K siRNA, and then subjected to cycloheximide (50 μg/ml) chase at the indicated time. Amount of c-Myc protein was examined by western blot using tubulin as a loading control. (c) MDA-MB-231 or MCF-7 cells with silencing of eEF-2K expression were treated or untreated with 10 μM of MG132 for 4 h, followed by western blot analysis of eEF-2K and c-Myc protein. The cells without silencing of eEF-2K expression were shown as a control.

Article Snippet: Antibodies to p-eEF-2 (2331), eEF-2 (2332), eEF-2K (3692), PKM2 (4053), c-Myc (5605), α-tubulin (2148) and PP2A-A (2041) were purchased from Cell Signaling Technologies (Danvers, MA, USA).

Techniques: Expressing, Transfection, Control, Quantitative RT-PCR, Western Blot

Journal: Oncogene

Article Title: eEF-2 kinase is a critical regulator of Warburg effect through controlling PP2A-A synthesis.

doi: 10.1038/onc.2016.166

Figure Lengend Snippet: Figure 6. eEF-2K controls PP2A-A protein synthesis through phosphorylating eEF-2. (a) MDA-MB-231 or MCF-7 cells were transfected with a nontargeting RNA or a PP2A-A siRNA. PP2A-A, c-Myc and phospho-c-Myc proteins were examined by western blot, using tubulin as a loading control. (b) MDA-MB-231 or MCF-7 cells were transfected with a nontargeting RNA or an eEF-2K siRNA. eEF-2K and PP2A-A proteins were examined by western blot, using tubulin as a loading control. (c) PP2A-A protein synthesis was assessed by 35S-metabolic labeling and subsequent PP2A-A immunoprecipitation, followed by gel electrophoresis and autoradiography. (d) MDA-MB-231 or MCF-7 cells were transfected with a nontargeting RNA (control) or an siRNA targeting eEF-2. eEF-2 and PP2A-A proteins were examined by western blot using tubulin as a loading control. (e) In vitro protein translation assay was performed as described in the Materials and methods. The products of reaction were examined by western blot. (f) PP2A-A mRNA or IL-6 mRNA in the eEF-2 immunoprecipitates of MDA-MB-231 cells was detected by qRT–PCR, and the results are shown as relative to that in the control IgG immunoprecipitates. Each bar represents the mean ± s.e. of three identical experiments. **Po 0.01.

Article Snippet: Antibodies to p-eEF-2 (2331), eEF-2 (2332), eEF-2K (3692), PKM2 (4053), c-Myc (5605), α-tubulin (2148) and PP2A-A (2041) were purchased from Cell Signaling Technologies (Danvers, MA, USA).

Techniques: Transfection, Western Blot, Control, Labeling, Immunoprecipitation, Nucleic Acid Electrophoresis, Autoradiography, In Vitro, Quantitative RT-PCR

Journal: Oncogene

Article Title: eEF-2 kinase is a critical regulator of Warburg effect through controlling PP2A-A synthesis.

doi: 10.1038/onc.2016.166

Figure Lengend Snippet: Figure 7. eEF-2K promotes proliferation of the Ras-transformed MEFs and human breast cancer cells. (a) Amounts of phospho-eEF-2 and Ras in the eEF-2K+/+ and eEF-2K−/−MEFs transformed with an H-RasV12 expression plasmid or a control empty plasmid were determined by western blot. Tubulin was used as a loading control. (b) The growth curve of H-Ras-MEFseEF−2K −/−and H-Ras-MEFseEF −2K+/+. (c) Anchorage-independent growth of the H-Ras-MEFseEF −2K−/−and H-Ras-MEFseEF −2K+/+, scale bar=1 mm. (d) Nude mice were inoculated subcutaneously (s.c.) with the H-Ras- MEFseEF −2K −/−or H-Ras-MEFseEF −2K+/+ (2×106 cells per inoculation). Tumor sizes were measured on the days as indicated. Data represent the mean±s.d. of tumor sizes of each group (n =6). **Po0.01, t-test. The photographs were taken 14 days after inoculation, scale bar =1 cm. (e) Colony formation of MDA-MB-231 cells stably expressing an eEF-2K-targeted shRNA or a control shRNA, scale bar=0.4 cm. (f) Growth curves of MDA-MB-231 cells stably expressing an eEF-2K-targeted shRNA or a control shRNA; nude mice were inoculated s.c. with MDA-MB-231 cells stably expressing an eEF-2K-targeted shRNA or a control shRNA (2×106 cells per inoculation). (g) Immunohistochemistry staining for Ki67 in the tumor specimens from the mice, scale bar=10 μm. (h) Tumor size was measured on the days as indicated. Data represent the mean ±s.d. of tumor sizes of each group (n =6). *Po 0.05; **Po0.01, t-test. The photographs were taken 14 days after inoculation, scale bar=1 cm. (i, j) Immunohistochemistry staining for PKM2 expression in the xenograft specimens from mice. (k, l) Immunohistochemistry staining for PP2A-A expression in the xenograft specimens from mice. Each bar represents the mean ±s.e. of three samples. *Po0.05; **Po0.01, t-test, scale bar=10 μm.

Article Snippet: Antibodies to p-eEF-2 (2331), eEF-2 (2332), eEF-2K (3692), PKM2 (4053), c-Myc (5605), α-tubulin (2148) and PP2A-A (2041) were purchased from Cell Signaling Technologies (Danvers, MA, USA).

Techniques: Transformation Assay, Expressing, Plasmid Preparation, Control, Western Blot, Stable Transfection, shRNA, Immunohistochemistry, Staining

Journal: Oncogene

Article Title: eEF-2 kinase is a critical regulator of Warburg effect through controlling PP2A-A synthesis.

doi: 10.1038/onc.2016.166

Figure Lengend Snippet: Figure 8. Knockdown of eEF-2K expression enhances sensitivity of breast cancer cells to paclitaxel and doxorubicin. (a, b) MDA-MB-231 or MCF-7 cells were transfected with a nontargeting RNA or an eEF-2K siRNA, followed by treatment with doxorubicin (a) or paclitaxel (b) for 72 h. (c) Multidrug-resistant MCF-7/ADR cells were transfected with a nontargeting RNA or an eEF-2K siRNA, followed by treatment with paclitaxel for 72 h. Cell viability was measured using MTT assay. Results shown were normalized to the control; each bar represents the mean±s.d. from three experiments. *Po0.05, **Po0.01. (d) Nude mice were inoculated subcutaneously (s.c.) with MDA-MB-231 cells stably expressing an eEF-2K-targeted shRNA or a control shRNA. The tumor-bearing mice were then treated with vehicle or 15 mg/kg paclitaxel once a week for 4 weeks. The tumor sizes were measured on the days as indicated. (e) Schematic illustration of the regulation of glycolysis by eEF-2K.

Article Snippet: Antibodies to p-eEF-2 (2331), eEF-2 (2332), eEF-2K (3692), PKM2 (4053), c-Myc (5605), α-tubulin (2148) and PP2A-A (2041) were purchased from Cell Signaling Technologies (Danvers, MA, USA).

Techniques: Knockdown, Expressing, Transfection, MTT Assay, Control, Stable Transfection, shRNA

Journal: International journal of molecular sciences

Article Title: Mallotucin D, a Clerodane Diterpenoid from Croton crassifolius , Suppresses HepG2 Cell Growth via Inducing Autophagic Cell Death and Pyroptosis.

doi: 10.3390/ijms232214217

Figure Lengend Snippet: Figure 5. MLD induced HepG2 cell mitophagy by inhibiting PI3K/AKT/mTOR pathway. (A) Con- focal detection of HepG2 cells transfected with mRFP-GFP-LC3B plasmid. Scale bar: 25 µm and 5 µm in merge and enlarged, respectively. Arrow: the colocalization of GFP-LC3B and RFP-LC3B. (B) Colocalization area statistics in A. (C) Mito staining and Lyso staining were used to analyze the fusion of mitochondria and lysosomes in HepG2 cells treated with MLD for 24 h. Scale bar: 25 µm and 5 µm in merge and enlarged, respectively. Arrow: the colocalization of mitochondria and lysosome. (D) Colocalization area statistics in C. (E) Levels of the PI3K, p-PI3K, AKT, p-AKT, mTOR, p-mTOR, P62, and LC3B proteins in the different groups were analyzed by Western blotting. (F) The quantitative analysis of relative protein levels of E. (G) Levels of the NLRP3, GSDMD, p62, and LC3B after treatment with MLD (20 µM) in the presence or absence of NAC (5 mM) for 24 h were measured by Western blotting. (H) The quantitative analysis of relative protein levels of G. The levels of IL-1β (I) and LDH (J) after treatment with MLD (20 µM) in the presence or absence of NAC (5 mM) for 24 h were measured by ELISA. The results are representative of three independent experiments and are expressed as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 compared with the control group. # p < 0.05, ## p < 0.01, and ### p < 0.001 compared with MLD-treated group.

Article Snippet: Total proteins were transferred to a polyvinylidene difluoride membrane and blocked with 5% skimmed milk for 1 h. Specific proteins were detected with primary antibodies: NLRP3 (A12694), GSDMD (A20197), IL-1β (A1112), caspase-3 (A19654), caspase-9 (A11451), BNIP3 (A5683), Cyt-c (A1561), Bax (A19684), Bcl-2 (A19693), β-actin (AC026), p-mTOR (S2448) (AP0115), mTOR (A2445), p-PI3K (AP0845), PI3K (A4992), p-Akt (AP0637), Akt (A17909), P62 (A19700), and LC3B (A19665), which were all bought from ABclonal Technology Co. Ltd., Wuhan, China.

Techniques: Transfection, Plasmid Preparation, Staining, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: International journal of molecular sciences

Article Title: Mallotucin D, a Clerodane Diterpenoid from Croton crassifolius , Suppresses HepG2 Cell Growth via Inducing Autophagic Cell Death and Pyroptosis.

doi: 10.3390/ijms232214217

Figure Lengend Snippet: Figure 8. Model of the antitumor mechanism of MLD in HepG2 cells. MLD firstly caused mito- chondrial damage and decreased the mitochondrial membrane potential, resulting in the production of ROS. The overproduction of ROS then increased BNIP3, inducing PI3K/AKT/MTOR-related mitophagy and GSDMD-related pyroptosis, leading to the induction of HepG2 cell death in vitro and in vivo.

Article Snippet: Total proteins were transferred to a polyvinylidene difluoride membrane and blocked with 5% skimmed milk for 1 h. Specific proteins were detected with primary antibodies: NLRP3 (A12694), GSDMD (A20197), IL-1β (A1112), caspase-3 (A19654), caspase-9 (A11451), BNIP3 (A5683), Cyt-c (A1561), Bax (A19684), Bcl-2 (A19693), β-actin (AC026), p-mTOR (S2448) (AP0115), mTOR (A2445), p-PI3K (AP0845), PI3K (A4992), p-Akt (AP0637), Akt (A17909), P62 (A19700), and LC3B (A19665), which were all bought from ABclonal Technology Co. Ltd., Wuhan, China.

Techniques: Membrane, In Vitro, In Vivo

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