Fig. 1. JNK-ATF2 binding depends on an intact Zn-finger + D-motif module.

a Mapping of JNK1 binding to ATF2 by GST pull-down. Interaction of recombinant expressed and purified bait and prey was detected on Coomassie stained SDS-PAGE. GST control was used as negative control to assess unspecific binding of the prey (GST; M—molecular weight marker, JNK—load). The experiment was repeated independently at least three times with similar results. b GST pull-down experiment with JNK1 (prey) and GST-ATF2(19–58) (bait). The binding mix contained 2 mM EDTA or the bait had a lysine to glutamate mutation (K48E). c Result of a competitive fluorescent polarization-based binding assay. Error bars on the binding isotherms show SD based on three independent measurements (n = 3). Data are represented as mean values ± SD. d Binding of JNK1 to MBP-ATF2(19–100) and MBP-c-Jun(1–78) fusion proteins in the presence of EDTA, or when lysine residues (K48E or K35E in ATF2 and c-Jun, respectively) were mutated. Error bars on the binding isotherms show SD based on three independent measurements (n = 3). Date are represented as mean values ± SD. (MBP: maltose-binding protein fusion tag.) e ITC measurements of JNK1 and ATF2(19–58) binding in the absence or presence of 2 mM EDTA. Representative ITC data (upper panels) are shown with raw injection heats and the corresponding specific binding isotherms. Lower panels show the enthalpic (ΔH) and entropic (−TΔS) contributions to binding free energy (ΔG). f Phosphorylation of ATF2 reporter constructs. GST constructs containing different docking motif cassettes were dotted on a membrane and incubated with activated JNK1 (pp-JNK1). Equal load was confirmed by Anti-His6 antibody, while phosphorylation of the probe was determined by Anti-p-T71-ATF2 immunoblots. (empty: no docking motif; NFAT4: the D-motif from NFAT4 was used as reference to former work¹⁶). Error bars show SD from three technical replicates. Data points are represented as mean values ± SD. The experiment was repeated twice with similar results. Source data are provided as a Source data file.