Fig. 3: p38 mediated binding and phosphorylation of ATF2 TAD.
From: Co-regulation of the transcription controlling ATF2 phosphoswitch by JNK and p38
a Luciferase complementation assay (NanoBit) on p38-ATF2 and JNK1-ATF2 binding in HEK293T cells. The impact of p38 and JNK activation by anisomycin and the K48E mutation were examined. Luminescence signal change upon MAPK activation was monitored in time after addition of anisomycin (see inset for p38:WT-ATF2 binding). Complex formation between p38α or JNK1 and ATF2(19–100) was measured after the addition of the luciferase substrate Coelenterazine h (at 30 min) and luminescence values obtained in the presence of anisomycin (red) were normalized to control (−; blue). MAPK activation was confirmed by anti-phosphoMAPK (anti-pp-p38 or anti-pp-JNK) western-blots. (endo: endogenous MAPKs; exo: luciferase fragment tagged MAPK probe). Anti-tubulin antibody was used to demonstrate equal sample load. Error bars show SD calculated based on three technical replicates. Bar charts show data points with mean ± SD and p-value, two-sided unpaired t-test. b Mapping critical amino acid residues for pp-p38-ATF2 TAD binding in HEK293T cells. The luciferase complementation based NanoBit assay was used to test the binding of p38 and different ATF2 TAD (19–100) mutants. Selective activation of p38 was achieved by doxycycline (DOX) addition in HT-MKK6-EE cells: DOX induces the expression of MKK6EE, which is the upstream activator kinase of p38. Error bars show SD calculated based on three technical replicates. Bar charts show data points with mean ± SD and p-value, two-sided unpaired t-test. c In vitro phosphorylation of ATF2 TAD constructs. JNK1 or p38α mediated phosphorylation of two ATF2 TAD (19–100) constructs (K48E and MUT4) were compared to wild-type (WT). ATF2 TAD phosphorylation was detected by using anti-phosphoT69/71 antibody in western-blots. d Interaction region mapping by NMR. 15N-labeled ATF2(19–106; 100 µM) was mixed with pp-p38 (upper panel) or nonphosphorylated p38 (lower panel) in different molar ratio. Histograms show the 1H/15N chemical shift perturbations (CSPs) vs residue number (for intensity changes upon binding see Supplementary Fig. 5b). Dashed lines indicate CSPs corresponding to 1ϭ or 2ϭ changes. Residues displaying line broadening are indicated by gray bars. ‘x’ indicates resonances of residues that were not resolved in the spectra and therefore could not be quantified or prolines. Source data are provided as a Source data file.
