Journal: Nature Communications

Article Title: Receptor-interacting protein kinase 2 (RIPK2) stabilizes c-Myc and is a therapeutic target in prostate cancer metastasis

doi: 10.1038/s41467-022-28340-6

Figure Lengend Snippet: a Representative images (left) and quantification (right) of PLA detecting the association of endogenous RIPK2 and MKK7 in HEK293T cells ( n = 4 biologically independent samples per group). Scale bars, 10 μm. α-: anti-. b Bar plot of PLA detecting the association of endogenous RIPK2 and MKK7 in parental PC cells ( n = 4 biologically independent samples per group). c Representative images of fluorescence colocalization of RIPK2 and MKK7 ectopically expressed in RIPK2 / MKK7 -DKO HEK293T cells. Scale bars, 10 μm. eGFP: enhanced green fluorescent protein. d Representative images of Co-IP analysis detecting the association of RIPK2 and MKK7 ectopically expressed in RIPK2 / MKK7 -DKO HEK293T cells. e Three-dimensional structural model of direct RIPK2 and MKK7 association in ribbon representation. f Representative images of radioactive in vitro RIPK2 kinase assay detecting RIPK2 phosphorylation of MKK7, using kinase-dead (K149A) MKK7 as substrate. The upper and lower panels show representative autoradiography and Coomassie blue-stained gel images, respectively. g – k Representative immunoblots of the indicated proteins in total lysates of g RIPK2 -KO HEK293T cells transiently transfected with the indicated amounts of RIPK2m4 plasmid, with 0.5 µg MKK7 being co-transfected to enable reliable quantification of p-MKK7, h control or RIPK2 -KO PC3 cells, i RIPK2 -KO HEK293T cells under the indicated conditions (g#1 and g#2: two independent gRNAs for gene knockout), j RIPK2 -KO 22Rv1 cells under the indicated conditions, and k RIPK2 / MKK7 -DKO 22Rv1 cells under the indicated conditions. Nominal p- values were determined by unpaired two-tailed Student’s t -test ( a , b ). Data are shown as mean ± SEM ( a , b ). Experiments were repeated three times independently with similar results ( c , d , f – k ).

Article Snippet: Membranes were probed with antibodies against RIPK2 (1:1000, Cell Signaling Technology (CST) #4142 or Santa Cruz Biotechnology #sc-166765), phospho-NF-κB p65 (Ser536) (1;1000, CST #3033), NF-κB p65 (1:1000, CST #8242), IκBα (1:1000, CST #4814), c-Myc (1:5000, Abcam #ab32072), phospho-c-Myc (S62) (1:1000, CST #13748S), ubiquitin (K48-linkage specific) (1:1000, CST #12805), FLAG (1:5000, Sigma #F1804), MKK7 (1:1000, CST #4172S or 1:2000, Santa Cruz #sc-25288), MKK7 (phospho-Ser271) (1:1000, Aviva Systems Biology #OAAF05547), JNK (1:1000, CST #9252S or 1:2000 Santa Cruz sc-7345), JNK (phospho-T183/Y185) (1:1000, CST #9251S), mCherry (1:1000, Abcam #ab213511), GFP (1:1000, Abcam #ab290), β-actin (1:5000, Sigma Aldrich #5441), or GAPDH (1:1000, CST #3683).

Techniques: Fluorescence, Co-Immunoprecipitation Assay, In Vitro, Kinase Assay, Phospho-proteomics, Autoradiography, Staining, Western Blot, Transfection, Plasmid Preparation, Control, Gene Knockout, Two Tailed Test

Journal: Nature Communications

Article Title: Receptor-interacting protein kinase 2 (RIPK2) stabilizes c-Myc and is a therapeutic target in prostate cancer metastasis

doi: 10.1038/s41467-022-28340-6

Figure Lengend Snippet: a Representative immunoblots of the indicated proteins in total lysates of RIPK2 / MKK7 -DKO HEK293T cells, which were transiently co-transfected with 0.5 µg MYC , 0.5 µg RIPK2m4 , and 0.1 µg MAP2K7 or vector plasmids and then treated with vehicle control, MKK7-COV-3 (25 µM), or JNK-IN-8 (1 µM) in 10% FBS-containing medium for 2 h. b Representative images (upper) and quantification (lower) of PLA detecting the association of endogenous MKK7 and c-Myc in PC3 cells ( n = 7 biologically independent samples per group). Scale bars, 10 μm. c Representative images of immunofluorescence colocalization of MKK7 and c-Myc in RIPK2 / MKK7 -DKO 22Rv1 cells, which were transiently co-transfected with RIPK2m4 , MKK7-His , and MYC plasmids. Scale bars, 10 μm. d Representative immunoblots of the indicated proteins after in vitro kinase reactions. e Representative immunoblots of the indicated proteins in total lysates of RIPK2 / MKK7 -DKO HEK293T cells, which were transiently transfected with the indicated forms of MKK7 plasmids or vector control. f Representative immunoblots of the indicated proteins in total lysates, cytoplasmic fraction, and nuclear fraction of RIPK2 / MKK7 -DKO HEK293T cells, which were transiently co-transfected with RIPK2m4 , MYC , and MKK7 or vector control. WCL: whole-cell lysate; Cyto: cytoplasmic; Nuc: nuclear. g Representative images (upper) and quantification (lower) of PLA detecting the MKK7-c-Myc complex in RIPK2 -KO HEK293T cells, which were transiently transfected with MYC and RIPK2m4 or vector control ( n = 5 biologically independent samples per group). Scale bars, 20 μm. Nominal p- values were determined by unpaired two-tailed Student’s t -test ( b , g ). Data are Mean ± SEM ( b , g ). Experiments were repeated three times ( a , d ) or twice ( e , f ) independently with similar results.

Article Snippet: Membranes were probed with antibodies against RIPK2 (1:1000, Cell Signaling Technology (CST) #4142 or Santa Cruz Biotechnology #sc-166765), phospho-NF-κB p65 (Ser536) (1;1000, CST #3033), NF-κB p65 (1:1000, CST #8242), IκBα (1:1000, CST #4814), c-Myc (1:5000, Abcam #ab32072), phospho-c-Myc (S62) (1:1000, CST #13748S), ubiquitin (K48-linkage specific) (1:1000, CST #12805), FLAG (1:5000, Sigma #F1804), MKK7 (1:1000, CST #4172S or 1:2000, Santa Cruz #sc-25288), MKK7 (phospho-Ser271) (1:1000, Aviva Systems Biology #OAAF05547), JNK (1:1000, CST #9252S or 1:2000 Santa Cruz sc-7345), JNK (phospho-T183/Y185) (1:1000, CST #9251S), mCherry (1:1000, Abcam #ab213511), GFP (1:1000, Abcam #ab290), β-actin (1:5000, Sigma Aldrich #5441), or GAPDH (1:1000, CST #3683).

Techniques: Western Blot, Transfection, Plasmid Preparation, Control, Immunofluorescence, In Vitro, Two Tailed Test

Journal: Nature Communications

Article Title: Receptor-interacting protein kinase 2 (RIPK2) stabilizes c-Myc and is a therapeutic target in prostate cancer metastasis

doi: 10.1038/s41467-022-28340-6

Figure Lengend Snippet: a Representative immunoblots (left) and quantification (right) of p-MKK7-S271 in total lysates of RIPK2 -KO HEK293T cells under the indicated conditions (2 h treatment in the presence of 10% FBS) ( n = 3 biologically independent samples per group). b , c Representative immunoblots of the indicated proteins in total lysates of b RIPK2 -KO HEK293T cells or c 22Rv1 cells under the indicated conditions (2 h treatment). Experiments were repeated three times independently with similar results ( b , c ). d Quantification of 3D spheroid invasion assays of PC3 cells treated with vehicle control, 10 µM GSK583, or 5 µM ponatinib ( n = 6 biologically independent samples per group). e Bar plot of anchorage-dependent colony formation of PC3, DU145, and 22Rv1 cells treated with vehicle control or 10 µM GSK583 ( n = 6 biologically independent samples per group). f Drug response curve of anchorage-dependent colony formation of PC3 cells treated with vehicle control or the indicated concentrations of ponatinib ( n = 6 biologically independent samples per group). g Schematic diagram of drug treatment for the inhibition of PC metastasis in vivo. Created with BioRender.com. h Effect of GSK583 treatment (10 mg/kg/day) on total BLI intensities in biologically independent mice (vehicle, n = 5; GSK583, n = 7), starting on day 9 following the i.c. injection. i Representative BLI images of the ventral (upper) and dorsal (lower) sides of mice treated with vehicle control or GSK583 for 4 weeks. j Effect of low-dose (6 mg/kg/day) or high dose (30 mg/kg/day for 10 days followed by 15 mg/kg/day for 18 days) ponatinib treatment on total BLI intensities in biologically independent mice (vehicle, n = 6; low-dose ponatinib, n = 8; high-dose ponatinib, n = 7), starting on day 9 following the i.c. injection. Pon: ponatinib. k Representative BLI images of the ventral (upper) and dorsal (lower) sides of mice treated with vehicle control, low-dose ponatinib, or high-dose ponatinib for 4 weeks. Nominal p- values were determined by unpaired two-tailed Student’s t -test ( a , d – f ) or two-way ANOVA ( h , j ). Data are Mean ± SEM ( a , d – f , h , j ).

Article Snippet: Membranes were probed with antibodies against RIPK2 (1:1000, Cell Signaling Technology (CST) #4142 or Santa Cruz Biotechnology #sc-166765), phospho-NF-κB p65 (Ser536) (1;1000, CST #3033), NF-κB p65 (1:1000, CST #8242), IκBα (1:1000, CST #4814), c-Myc (1:5000, Abcam #ab32072), phospho-c-Myc (S62) (1:1000, CST #13748S), ubiquitin (K48-linkage specific) (1:1000, CST #12805), FLAG (1:5000, Sigma #F1804), MKK7 (1:1000, CST #4172S or 1:2000, Santa Cruz #sc-25288), MKK7 (phospho-Ser271) (1:1000, Aviva Systems Biology #OAAF05547), JNK (1:1000, CST #9252S or 1:2000 Santa Cruz sc-7345), JNK (phospho-T183/Y185) (1:1000, CST #9251S), mCherry (1:1000, Abcam #ab213511), GFP (1:1000, Abcam #ab290), β-actin (1:5000, Sigma Aldrich #5441), or GAPDH (1:1000, CST #3683).

Techniques: Western Blot, Control, Inhibition, In Vivo, Injection, Two Tailed Test

Journal: Nature Communications

Article Title: Receptor-interacting protein kinase 2 (RIPK2) stabilizes c-Myc and is a therapeutic target in prostate cancer metastasis

doi: 10.1038/s41467-022-28340-6

Figure Lengend Snippet: RIPK2 binds to MKK7 and activates the MKK7(/JNK)/c-Myc phosphorylation cascades, thereby stabilizing the c-Myc oncoprotein and enhancing c-Myc activity, which is required for PC metastasis. The non-canonical RIPK2/MKK7/c-Myc signaling pathways can be blocked by genetic or pharmacological inhibition of RIPK2, resulting in the proteasomal degradation of c-Myc and the inhibition of c-Myc activity and PC metastasis. Of clinical relevance, RIPK2 and MYC are frequently co-amplified/gained in PC and several other cancer types, and their activity scores are strongly correlated across many cancers. Created with BioRender.com.

Article Snippet: Membranes were probed with antibodies against RIPK2 (1:1000, Cell Signaling Technology (CST) #4142 or Santa Cruz Biotechnology #sc-166765), phospho-NF-κB p65 (Ser536) (1;1000, CST #3033), NF-κB p65 (1:1000, CST #8242), IκBα (1:1000, CST #4814), c-Myc (1:5000, Abcam #ab32072), phospho-c-Myc (S62) (1:1000, CST #13748S), ubiquitin (K48-linkage specific) (1:1000, CST #12805), FLAG (1:5000, Sigma #F1804), MKK7 (1:1000, CST #4172S or 1:2000, Santa Cruz #sc-25288), MKK7 (phospho-Ser271) (1:1000, Aviva Systems Biology #OAAF05547), JNK (1:1000, CST #9252S or 1:2000 Santa Cruz sc-7345), JNK (phospho-T183/Y185) (1:1000, CST #9251S), mCherry (1:1000, Abcam #ab213511), GFP (1:1000, Abcam #ab290), β-actin (1:5000, Sigma Aldrich #5441), or GAPDH (1:1000, CST #3683).

Techniques: Phospho-proteomics, Activity Assay, Protein-Protein interactions, Inhibition, Amplification

Journal: Oncotarget

Article Title: Inhibition of the MAP2K7-JNK pathway with 5Z-7-oxozeaenol induces apoptosis in T-cell acute lymphoblastic leukemia.

doi: 10.18632/oncotarget.28040

Figure Lengend Snippet: Figure 3: 5Z7O inhibits MAP2K7 activity and downstream activation of JNK. (A) Immunoblot of the MAP2K7 pathway signaling in a panel of T-ALL and non-leukemic LCL cell lines following incubation with 3 µM 5Z7O for 21 hours. (B) Biochemical kinase assay using purified MAP2K7 protein, dead JNK2 substrate, and two methods of kinase activity based on ATP consumption or ADP production. (C) ADP-GLO in vitro kinase activity of purified TAK1-MBP protein following 30-minute pre-treatment with 5Z7O. (D) Immunoblot of KOPTK1 cells treated with 3 µM 5Z7O to evaluate TAK1 inhibition. Diagram indicating effects of 5Z7O in the MKK signaling pathway in T-ALL cells. (E) KOPTK1 cells were treated with sorbitol to evaluate capacity of 5Z7O to inhibit activated MAP2K7. (F) Jurkat and P12-Ichikawa cells transduced with retrovirus expressing the fusion MAP2K7-JNK2 (MKK7-JNK2) protein were treated with 5Z7O to inhibit activated MAP2K7. For phosphorylated ATF2, immunoblot in Jurkat cells required longer exposure.

Article Snippet: In vitro kinase assay Purified human MAP2K7 (Origene) or TAK1TAB protein (Promega V4088) was plated with the corresponding substrate (dead JNK2 fragment for MAP2K7 and Myelin Basic Protein for TAK1) along with vehicle (DMSO) or 5Z7O.

Techniques: Activity Assay, Activation Assay, Western Blot, Incubation, Kinase Assay, Purification, In Vitro, Inhibition, Transduction, Expressing

Journal: bioRxiv

Article Title: RIPK2 Stabilizes c-Myc and is an Actionable Target for Inhibiting Prostate Cancer Metastasis

doi: 10.1101/2020.05.14.096867

Figure Lengend Snippet: a, Western blot analysis of c-Myc, FLAG-tag, and RIPK2 in RIPK2-KO HEK293T cells under the indicated conditions. β-actin was used as a loading control. Vec, vector control; WT, wild-type; NLS, nuclear localization signal; NES, nuclear export signal; Δkinase, deletion of the kinase domain; ΔCARD, deletion of the CARD domain. b, Heatmap of the 219 protein candidates specifically associated with the kinase domain of cytoplasmic RIPK2. G5 and G3 were experimental groups, whereas G1, G2, and G4 were control groups (see Extended Data Fig. 26 for more details). The heatmap cells in gray indicate zero label-free quantification (LFQ) intensities. c, Bar graph of the average activity change of inferred kinases downstream of RIPK2. A dot represents kinase activity in control PC3 cells relative to that in a RIPK2-KO PC3 single-cell clone (#4 or #12). d, Activation network connecting RIPK2-interacting proteins with direct c-Myc-Ser62 kinases downstream of RIPK2. The numbers indicate kinase activities in control PC3 cells relative to those in RIPK2-KO PC3 clone#4 (left) and clone#12 (right). e, Western blot analysis of p-MKK7-S271, total MKK7, p-JNK-T183/Y185, total JNK, and RIPK2 in RIPK2-KO HEK293T cells under the indicated conditions. β-actin was used as a loading control. In the upper panel, cells were co-transfected with MKK7 to enable the detection of p-MKK7-S271. f, Western blot analysis of target proteins in RIPK2-KO HEK293T cells under the indicated conditions. β-actin was used as a loading control. Vec and RIPK2m4, 0.1 μg; GSK583, 10 μM; Ponatinib, 5 μM. In the left panel, cells were co-transfected with MKK7 to enable the detection of p-MKK7-S271. g, Representative images (left) and a bar graph (right) of proximity ligation assay detecting the association of endogenous RIPK2 and MKK7 in HEK293T cells. h, Representative images of fluorescence colocalization assay of RIPK2 and MKK7 in HEK293T cells. i , Representative Western blots of RIPK2 and MKK7 in co-immunoprecipitation products and input samples. GAPDH was used as a loading control. j , Structural model showing that RIPK2 directly binds to MKK7 in a head-to-tail fashion. Three-dimensional structures of kinase domains are shown as ribbon representation. k-m , Representative Western blots of target proteins in HEK293T cells under the indicated conditions. Cells were co-transfected with 0.5 μg c-Myc plasmid to facilitate the detection of p-c-Myc-Ser62. β-actin was used as a loading control. R KO , RIPK2-knockout; R KO M KO , knockout of both RIPK2 and MKK7 genes; R KO P KO , knockout of both RIPK2 and PRKDC genes.

Article Snippet: The canonical RIPK2 coding sequence (NM_003821) and MKK7 (i.e., MAP2K7) coding sequence (NM_145185.4) were amplified by PCR from a RIPK2 vector (Origene, #RG202530) and a MKK7 vector (GenScript, Ohu28940), respectively, and cloned into the pLenti-puro vector.

Techniques: Western Blot, FLAG-tag, Control, Plasmid Preparation, Quantitative Proteomics, Activity Assay, Activation Assay, Transfection, Proximity Ligation Assay, Fluorescence, Immunoprecipitation, Knock-Out

Journal: Blood advances

Article Title: Antileukemic properties of the kinase inhibitor OTSSP167 in T-cell acute lymphoblastic leukemia.

doi: 10.1182/bloodadvances.2022008548

Figure Lengend Snippet: Figure 4. Inhibition of the MAP2K7-JNK pathway in T-ALL. (A) The inhibition of MELK and the MAP2K7-JNK pathway in T-ALL cell lines by OTSSP167 (50 nM, 48 hours)

Article Snippet: Purified human MAP2K7 (Origene, 320 nM) was preincubated with a dead-JNK2 fragment (350 nM) in the presence of different concentrations of OTSSP167 or vehicle for 30 minutes.

Techniques: Inhibition