Journal: Scientific Reports

Article Title: Prevention of calpain-dependent degradation of STK38 by MEKK2-mediated phosphorylation

doi: 10.1038/s41598-019-52435-8

Figure Lengend Snippet: MEKK2 inhibits heat- and calpain-mediated degradation of STK38. ( A ) COS-7 cells were transfected with human STK38-V5 alone, or with FLAG-MEKK1 or FLAG-MEKK2. Forty-eight hours after transfection, the cells were heated to 44 °C for 20 min or left untreated as controls and harvested. ( B ) COS-7 cells were transfected with human STK38-V5 alone, or with FLAG-MEKK2 (WT) or FLAG-MEKK2 (KM). Forty-eight hours after transfection, the cells were harvested. ( C ) HeLa cells were transfected with the scramble oligonucleotides control (scr) or MEKK2 -specific siRNA. Forty-eight hours after transfection, the cells were heated to 44 °C for the indicated times or left untreated as controls and harvested. ( D ) HeLa cells were transfected with FLAG-MEKK2. Forty-eight hours after transfection, the cells were heated to 44 °C for the indicated times or left untreated as controls and harvested. The MEKK2 activity was measured by immune complex kinase assay with an anti-FLAG antibody using GST-MKK4 as the substrate. ( E ) GST-STK38 was incubated with calpain I (0.07 units of calpain I in each lane) in the absence or presence of GST-active MEKK2 for 15 min at 30 °C. Cell lysates or in vitro reaction products were analysed by western blotting with the antibodies against the indicated proteins. A representative image of western blot is shown (see Supplementary Fig for corresponding full-length image). Relative levels of STK38 were determined from the western blot using Image J software. Data are presented as the mean ± standard deviation of three independent experiments. Statistical significance was determined by the Student’s t -test (** P < 0.05).

Article Snippet: For the MEKK2 kinase assay, V5-STK38 immunoprecipitates were incubated with 10 ng of active-MEKK2 (Signal Chem, Richmond, BC) in MEKK2 kinase buffer containing 0.37 MBq ml −1 [γ- 32 P] ATP at 30 °C for 15 min. For in vitro kinase assays, active MEKK2 was incubated with 1.0 μg of wild-type inactive STK38 (Signal Chem) or inactive MKK4 (Signal Chem) in MEKK2 kinase buffer containing 0.37 MBq ml −1 [γ- 32 P] ATP for 30 min at 30 °C.

Techniques: Transfection, Control, Activity Assay, Immune Complex Kinase Assay, Incubation, In Vitro, Western Blot, Software, Standard Deviation

Journal: Scientific Reports

Article Title: Prevention of calpain-dependent degradation of STK38 by MEKK2-mediated phosphorylation

doi: 10.1038/s41598-019-52435-8

Figure Lengend Snippet: Maintenance of STK38 stability requires its phosphorylation. ( A ) MEKK2 phosphorylates STK38 in vitro . GST-tagged STK38 (unactive) or MKK4 (unactive) was incubated with or without active MEKK2 in the presence of [γ- 32 P] ATP for 30 min at 30 °C. ( B , upper) Putative phosphorylation sites identified within STK38. Inactive GST-STK38 was incubated with or without active MEKK2, and the kinase reaction products were subjected to SDS-PAGE. GST-STK38 was excised and processed by tryptic cleavage for MS analysis. *T74 is endogenously phosphorylated. ( B , bottom) In vitro kinase reaction was performed by incubating active MEKK2 alone or with the V5-immunopurified wild-type STK38, STK38 (S91A), STK38 (T243A), STK38 (T270A) from the transfected 293 T cells for 15 min at 30 °C. The kinase reaction products were subjected to SDS-PAGE and then visualised by autoradiography ( 32 P, top panel) or Coomassie Brilliant Blue staining (CBB, bottom panel). ( C ) COS-7 cells were transfected with human STK38-V5 (WT) or STK38-V5 (S91A). Forty-eight hours after transfection, the cells were harvested. In vitro cleavage reaction was performed by incubating calpain I (0.07 units) with lysates (30 μg) of the transfected cells from STK38-V5 (WT) or STK38-V5 (S91A) for 30–60 min at 30 °C. Reaction products were subjected to western blotting analysis with antibodies against the indicated proteins. ( D ) COS-7 cells were transfected with human STK38-V5 (WT) or STK38-V5 (S91A). Forty-eight hours after transfection, the cells were treated as described in Fig. . Cell lysates were analysed by western blotting with antibodies against the indicated proteins. A representative image of western blot, CBB-stained gel, or autoradiography is shown (see Supplementary Fig for corresponding full-length image). Relative levels of STK38 were determined from the western blot by using Image J software. Data are presented as the mean ± standard deviation of three independent experiments. Statistical significance was determined by the Student’s t -test (* P < 0.05; ** P < 0.01).

Article Snippet: For the MEKK2 kinase assay, V5-STK38 immunoprecipitates were incubated with 10 ng of active-MEKK2 (Signal Chem, Richmond, BC) in MEKK2 kinase buffer containing 0.37 MBq ml −1 [γ- 32 P] ATP at 30 °C for 15 min. For in vitro kinase assays, active MEKK2 was incubated with 1.0 μg of wild-type inactive STK38 (Signal Chem) or inactive MKK4 (Signal Chem) in MEKK2 kinase buffer containing 0.37 MBq ml −1 [γ- 32 P] ATP for 30 min at 30 °C.

Techniques: Phospho-proteomics, In Vitro, Incubation, SDS Page, Transfection, Autoradiography, Staining, Western Blot, Software, Standard Deviation

Journal: Molecular cancer

Article Title: A novel polypeptide CAPG-171aa encoded by circCAPG plays a critical role in triple-negative breast cancer.

doi: 10.1186/s12943-023-01806-x

Figure Lengend Snippet: Fig. 6 CAPG-171aa interacts with STK38 activating the downstream MEK1/2-ERK1/2 pathway via MEKK2. (A) MDA-MB-231 and MDA-MB-468 cell lysates were IP with anti-MEKK2 antibody followed by detection with anti-MEKK2, STK38, and SMURF1 antibody. (B) MDA-MB-231 and MDA-MB-468 were trans fected with CAPG-171aa-FLAG. Whole-cell lysates were IP with anti-SMURF1 and IgG antibodies followed by detection with anti-FLAG, STK38, SMURF1, and GAPDH antibodies. (C-D) Before being treated with MG132, MDA-MB-231, and MDA-MB-468 were transfected with CAPG-171aa-FLAG and circCAPG KD plasmids. Ubiquitination and protein expression levels of MEKK2 were assayed in CAPG-171aa OE (C) and circCAPG KD (D) MDA-MB-231 and MDA- MB-468. (E) Ubiquitination and protein expression levels of MEKK2 were assayed in circCAPG KD MDA-MB-231 and MDA-MB-468 through pulse-chase experiments with cycloheximide. (F) IB of MEKK2, p-MEK1/2, MEK1/2, p-ERK1/2 and ERK1/2 in circCAPG KD MDA-MB-231 and MDA-MB-468. (G) IB of p-MEK1/2, MEK1/2, p-ERK1/2 and ERK1/2 in OE STK38 MDA-MB-231 and MDA-MB-468. (H) IB of MEKK2, p-MEK1/2, MEK1/2, p-ERK1/2 and ERK1/2 in CAPG-171aa, OE-STK38 and OE-STK38/CAPG-171aa transfected MDA-MB-231 and MDA-MB-468. All data were representative of at least three biological replicates and shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: MDA-MB-231 and MDA-MB-468 cells were treated with 10 μg/mL MG132, respectively, (Solarbio, IM0310) for 12 h. Cell lysates were obtained using PierceTM IP lysis buffer (Thermo Fisher Scientific, USA) supplemented with a cocktail (Thermo Fisher Scientific, USA) and then incubated with anti-MEKK2 antibody (Proteintech, USA) and Protein A/G beads overnight at 4°C.

Techniques: Transfection, Ubiquitin Proteomics, Expressing, Pulse Chase

Journal: Frontiers in Genetics

Article Title: MiR-372-3p Functions as a Tumor Suppressor in Colon Cancer by Targeting MAP3K2

doi: 10.3389/fgene.2022.836256

Figure Lengend Snippet: Sequence primers designed for real-time qPCR.

Article Snippet: Anti-MAP3K2 (BA3634-2) polyclonal antibody was purchased from BOSTER Biological Technology Co. Ltd. (Wuhan, China).

Techniques: Sequencing

Journal: Frontiers in Genetics

Article Title: MiR-372-3p Functions as a Tumor Suppressor in Colon Cancer by Targeting MAP3K2

doi: 10.3389/fgene.2022.836256

Figure Lengend Snippet: MAP3K2 may be the potential target of miR-372-3p in colon cancer tissues. (A–C) mRNA expression levels of the potential targets of miR-372-3p including p21, HDAC4, and Wee1, are displayed as 2E-deltaCT (normalized to GAPDH) in colon cancer or matched normal tissues. ** p < 0.01 vs. normal tissues (Mann Whitney test). (D–F) Correlation between miR-372-3p expression and its potential targets p21, HDAC4, and Wee1 in colon cancer. The Y-axis exhibits the log2 value of the ratio of miR-372-3p (orange triangle) and p21, HDAC4, or Wee1 (blue circle) expression levels between colon cancer and matched normal tissues, and the X-axis exhibits the number of samples. The correlation between miR-372-3p and p21, HDAC4, or Wee1 was statistically analyzed using the GraphPad Prism software. (G) MAP3K2 mRNA expression (normalized to GAPDH) in colon cancer, matched adjacent and normal tissues. *** p < 0.0001 vs. normal tissues (one-way analysis of variance). (H) Expression patterns of MAP3K2 in colon cancer and matched adjacent tissues. Each bar presents the log2 value of the ratio of MAP3K2 expression levels between colon cancer and matched normal tissues or adjacent and normal tissues from the same patients. (I) Correlation between miR-372-3p and MAP3K2 expression levels in colon cancer tissues. The Y-axis exhibits the log2 value of the ratio of miR-372-3p (orange triangle) and MAP3K2 (blue circle) expression levels, and the X-axis exhibits the number of samples. All qPCRs were performed in three independent experiments with three replicates per group.

Article Snippet: Anti-MAP3K2 (BA3634-2) polyclonal antibody was purchased from BOSTER Biological Technology Co. Ltd. (Wuhan, China).

Techniques: Expressing, MANN-WHITNEY, Software

Journal: Frontiers in Genetics

Article Title: MiR-372-3p Functions as a Tumor Suppressor in Colon Cancer by Targeting MAP3K2

doi: 10.3389/fgene.2022.836256

Figure Lengend Snippet: miR-372-3p suppressed cell proliferation in SW480 colon cancer cells, and also negatively regulated MAP3K2 expression. (A) SW480 cell proliferation was inhibited by miR-372-3p mimics. SW480 cells were transiently transfected with miR-372-3p mimics in the presence or absence of miR-372-3p inhibitors. The cell viability was then detected using a CCK-8 assay kit at 24 and 48 h. Data are presented using mean ± standard deviation values. * p < 0.05, ** p < 0.001, vs. the vector group, # p < 0.05, ## p < 0.01, vs. the miR-372-3p 3 µg group (Mann Whitney test). (B) Colony-formation assay. The colony-formation ability of SW480 cells was analyzed with a colony formation assay (upper), and the quantified numbers of colonies for each group are displayed as a bar graph (lower). Data are presented using mean ± standard deviation values. ** p < 0.01, *** p < 0.001, vs. The vector group, ## p < 0.01, ### p < 0.001, vs. the miR-372-3p 3 µg group (Mann Whitney test). (C,D) Effects of miR-372-3p on the MAP3K2 expression in SW480 cells. Cells were transfected with miR-372-3p mimics (0, 1, 3 µg). MAP3K2 mRNA was detected using RT-qPCR (at 48 h) (C) , and the protein levels were analyzed with western bloting (at 48 and 72 h) approach (D) . GAPDH was used as an internal control. (E) The MAP3K2 protein level reduced by miR-372-3p was restored by co-transfection with miR-372-3p inhibitors in SW480 cells. (F) Binding sites of miR-372-3p on the MAP3K2 3′-UTR. The 3′-UTR fragments of human MAP3K2 (+21 to +472 bp, +6394 to +6656 bp) were cloned downstream of the luciferase between the MulI and HindIII sites.

Article Snippet: Anti-MAP3K2 (BA3634-2) polyclonal antibody was purchased from BOSTER Biological Technology Co. Ltd. (Wuhan, China).

Techniques: Expressing, Transfection, CCK-8 Assay, Standard Deviation, Plasmid Preparation, MANN-WHITNEY, Colony Assay, Quantitative RT-PCR, Western Blot, Control, Cotransfection, Binding Assay, Clone Assay, Luciferase

Journal: Frontiers in Genetics

Article Title: MiR-372-3p Functions as a Tumor Suppressor in Colon Cancer by Targeting MAP3K2

doi: 10.3389/fgene.2022.836256

Figure Lengend Snippet: miR-372-3p modulated the expression of MAP3K2 by targeting its 3′-UTR in SW480 colon cancer cells. (A,B) Relative luciferase activities of pMIR-MAP3K2 3′-UTR wild type (wt) and mutants (mt) were detected in pMIR-Vector and miR-372-3p mimics (0, 1, and 2 µg) groups. * p < 0.05, ** p < 0.01, *** p < 0.001, vs. vector group (Mann Whitney test). Three biological replicates were conducted.

Article Snippet: Anti-MAP3K2 (BA3634-2) polyclonal antibody was purchased from BOSTER Biological Technology Co. Ltd. (Wuhan, China).

Techniques: Expressing, Luciferase, Plasmid Preparation, MANN-WHITNEY