Fig 1: PD-L1 directly interacts with PPM1B and inhibits its phosphatase activity, thereby activating p38 MAPK pathway and TGFβ production.a HEK293T cells were transfected with Flag-tagged PD-L1 and Myc-tagged PPM1B for 48 h and then subjected to immunoprecipitation followed by immunoblotting. b, c A549 and H460 cells were transfected with PD-L1 overexpressing vector or PD-L1 siRNA. At 24 h of transfection, cells were submitted to co-immunoprecipitation assay with PD-L1 to evaluate the protein complex formation with PPM1B and p38 MAPK. d Full length (FL), extracellular domain (ECD), and intracellular domain (ICD) of GST-tagged PD-L1 were purified from E. coli. GST pull-down assays were performed using 1 μg of each indicated protein. e In vitro phosphatase assay was performed using recombinant p-p38 as a substrate and recombinant PPM1B as a phosphatase in the presence and absence of recombinant PD-L1 and phosphatase inhibitor (PPase inhibitor). Phosphatase activity was evaluated by measurement of free phosphate (upper) and immunoblotting for p-p38 (lower). Relative intensity of p-p38 was estimated on immunoblot. f H460 cells were transfected with PD-L1 siRNA and/or PPM1B siRNA. At 24 h after transfection, cell were submitted to Western blotting for indicated molecules. g H460 cells were transfected with PD-L1 siRNA and/or PPM1B siRNA for 24 h and then transfected with luciferase-expressing vector with wild-type (wt) promoter sequence of TGFβ containing ATF2- and c-Jun-binding motif. At 12 h after transfection, luciferase activity was measured. As a negative control, the plasmid with truncated mutation (mut) of the TGFβ promoter sequence were used. h A549 and H460 cells were incubated with rhPD-1 (1 μg/ml) for 24 h, and TGFβ production in the culture supernatant was measured using an ELISA. i A549 and H460 cells were treated with rhPD-1 (1 μg/ml) in the presence or absence of anti-TGFβ neutralizing antibody (1 μg/mL). At 24 h after treatment, total protein was extracted and submitted to Western blotting for EMT markers. j, k After 24 h treatment of rhPD-1, protein complex of endogenous PD-L1, PPM1B, and p38-MAPK was analyzed by co-immunoprecipitation assay (j), and phosphorylation of p38, c-Jun, and ATF2 was evaluated using Western blotting (k) in A549 and H460 cells. l, m A549 and H460 cells were treated with rhPD-1 in the presence or absence of p38 inhibitor (SB203580, 10 μM). Cells were then transfected with TGFβ-luc vector and at the 12 h of transfection luciferase activity of promoter sequence of TGFβ containing ATF2- and c-Jun-binding motif were measured. Data are presented as mean ± S.E.M. of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001.
Fig 2: CRC organoids are resistant to type I but sensitive to type 1.5 p38α inhibitors.a, Generation of KAP organoids expressing Mapk14 shRNAs. b, Knockdown test of Mapk14 shRNAs. Representative western blot analysis of p38α in KAP2D cells upon 6 days of treatment with doxycycline (Dox; cropped blot images, n = 3 biologically independent experiments). c, Cell viability analysis in KAP-shMapk14 and KAP-shNC organoids upon 12 days of treatment with doxycycline (n = 4 biologically independent experiments; data are presented as the mean ± s.d.). Statistical significance was calculated using an ANOVA and Dunnett’s multiple-comparisons test (P < 0.0001). d, Cell viability analysis in KAP organoids upon 4 days of treatment with SKL, PH-797804, LY2228820 or DMSO (n = 3 biologically independent experiments; data are presented as the mean ± s.d.). e, Representative western blot analysis of KAP2D cells upon 1 day of treatment with 5 µM SKL, PH-797804 or DMSO (cropped blot images; n = 3 biologically independent experiments). f, Schematic picture showing the binding of 1639 to HRI, HRII and R-spine of the p38α kinase. g, Cell viability analysis of KAP organoids upon 4 days of treatment with SKL, 1639 or DMSO (n = 3 biologically independent experiments; data are presented as the mean ± s.d.). Statistical significance was calculated using a two-tailed Student’s t-test (P < 0.0001). Conc., concentration. h, Generation of a CRC mouse model based on subcutaneous injection of KAP organoids into wild-type (WT) mice. i, Representative pictures of hematoxylin and eosin (H&E) and immunohistochemical staining for pan-CK and CDX2 in KAP subcutaneous tumors, 19 days after tumor initiation (n = 4 tumors per group). Scale bars, 100 µm. j, Treatment of subcutaneous KAP CRCs with SKL, 1639 or carrier (n = 10 tumors per group; data are presented as the mean ± s.e.m.). Statistical significance was calculated using an ANOVA and Dunnett’s multiple-comparisons test (P = 0.0192). NS, not significant. Treatment was started 1 week after organoid transplantation. The experiments in b, d, e and g were independently performed three times, the experiment in c was independently performed four times and the stainings in i were independently performed twice, all with similar results.Source data
Fig 3: Validation of nilotinib as a p38/MK2 PPI Inhibitor. (A) Thermal shift assay (TSA) showing dose-dependent stabilization of recombinant His-tagged p38 by nilotinib (ΔT max = 8.22 °C), consistent with direct binding. (B) TSA profile for SR318, a type II ATP-competitive p38 inhibitor, used as a positive control (ΔT max = 13.47 °C). (C) Nilotinib competes with His-MK2 346–400 fragment for VF-p38 in a cell lysate-based TR-FRET assay. (D) Quantitative qRT-PCR analysis showing that nilotinib significantly (p-value <0.05) suppresses LPS-induced TNF-α, IL-6, and IL-1β expression in HMC3 microglial cells. P38 inhibitors SR318 and VX-745 were used as positive controls. (E) Nilotinib disrupts the endogenous p38/MK2 complex in HMC3 cells, as shown by coimmunoprecipitation, correlating with cytokine suppression. (F) qRT-PCR analysis showing that nilotinib suppresses LPS/IFNγ-induced TNF-α expression in the human iPSC-derived microglia (iMGL). (G) TR-FRET assay with recombinant p38 and MK2 proteins purified from E. coli demonstrated direct inhibition of the complex by nilotinib (IC50 = 2.2 μM). In contrast, ATP-site inhibitors VX-745 and SR318 failed to disrupt the interaction, supporting a non-ATP-competitive mechanism for nilotinib activity. (H) Nilotinib demonstrates a weak inhibition of p38/ATF2 PPI (IC50 > 30 μM, maximal inhibition ∼ 37%) in a TR-FRET assay with recombinant purified His-p38 and GST-ATF2. The inhibition of His-p38/GST-MK2 PPI by nilotinib was monitored in parallel.
Fig 4: Structure-guided mapping and peptide validation of the p38/MK2 interaction interface. (A) The cocrystal structure of the p38/MK2 complex (PDB ID: 6TCA). The molecular surface of p38 is shown in gray. The p38 docking groove is highlighted in yellow. MK2 is shown as green ribbons. (B) The MK2 D345-H400 docking motif bound to the p38 docking groove is colored based on its fragments tested in this study: D345-H400 is colored in green, I370–L393 in blue, and I370-L382 in red. (C) The binding curve from a fluorescence polarization assay showing high-affinity binding of FITC-labeled MK2 370–393 peptide to His-tagged p38 (EC50 = 26.9 nM). (D) Dose–response curves from TR-FRET inhibition assays demonstrating that both MK2 370–393 and 369–382 peptides disrupt the p38/MK2 complex (IC50 = 0.42 μM and 4.26 μM, respectively).
Fig 5: Development of a lysate-based TR-FRET platform for high-throughput screening of p38/MK2 PPI inhibitors. (A) The preferential binding of MK2 to p38α and p38β isoforms was determined by Flag-immunoprecipitation in HEK293T cells. (B) Isoform selectivity of MK2 binding was validated by TR-FRET using lysates coexpressing GST-tagged MK2 and Venus-Flag (VF)-tagged p38 isoforms. Robust signal was observed for p38α and p38β, with negligible interaction detected for p38γ and p38δ. (C) TR-FRET assay shows stable signal over 48 h postantibody addition, indicating excellent temporal stability. (D) The platform tolerates up to 10% DMSO without signal degradation, supporting its suitability for screening applications. (E) Pilot screen of 2036 compounds from the Emory Enriched Library (EEL) in 1536-well format identified 48 compounds that inhibited the p38/MK2 interaction by ≥ 50% relative to vehicle control. Gray dots indicate fluorescence assay-interfering compounds.
Supplier Page from Abcam for Recombinant human p38 alpha/MAPK14 protein (Active) (His tag N-Terminus)