Fig. 4: JNK mediated phosphorylation affects p38-ATF2 TAD binding.
From: Co-regulation of the transcription controlling ATF2 phosphoswitch by JNK and p38
a p38-ATF2 TAD binary interaction after anisomycin treatment. Binding was monitored in time after the addition of anisomycin using a luciferase complementation based protein–protein binding assay (NanoBit) in live cells (left panel). Anisomycin treatment turns on p38 and JNK pathway activity in HEK293T cells (pp-p38 or pp-JNK—or p-c-Jun—western-blot signal, respectively; right panel; min—minutes). Wild-type (WT), S90N, and FENEF region mutated (MUT4) TAD constructs interacted with the p38 luciferase probe differently after turning on p38 phosphorylation by anisomycin (10µg/mL). Luminescence signal was normalized to cells not treated by anisomycin (treated/untreated). The panel on the right shows results of western-blots with different antibodies; exo: luciferase fragment tagged exogenous p38 probe; endo: endogenous p38; tubulin: anti-tubulin antibody used as the load control. DMSO CTR: cells treated only by anisomycin. JNK-IN-8: cells treated with anisomycin and a JNK-specific inhibitor. MAPK mediated ATF2 TAD phosphorylation at the phosphoswitch was monitored with the pp-ATF2(T69/71) antibody. Error bars show SD from three technical replicates. Data are represented as mean values ± SD. The experiment was repeated twice with similar results. b Inhibition of pp-p38 mediated TAD phosphorylation by SPFENEF motif-containing peptides in vitro. WT, S90N and S90 phosphorylated (pS90) peptides (83–102) were added in increasing amounts to an in vitro pp-p38 → TAD(19–100) phosphorylation reaction. The Ki values for the three peptides are highlighted. c Schematic of MAPK binding to different ATF2 TAD regions and phosphorylation of critical S/TP sites. Source data are provided as a Source data file.
