Fig. 7: Modeling the impact of JNK and/or p38 MAPK pathway flux on ATF2 TAD phosphorylation.
From: Co-regulation of the transcription controlling ATF2 phosphoswitch by JNK and p38
a Mechanistic scheme of MAPK mediated ATF2 TAD phosphorylation. The panel shows the architecture of the rule-based BioNetGen model. Dashed lines represent binding rules (with kon and koff) and lines with an arrow indicate enzymatic reactions (with reaction rates of kinases, k, and phosphatases, dp). Signaling is initiated by increasing k6 and k7 activity (keq6 → kstim6, keq7 → kstim7). D: D-motif; DRS: D-motif recruitment site; F: SPFENEF or F-motif; FRS: F-motif recruitment site; S90-ON: S90 is phosphorylated by JNK blocking p38(FRS):F-motif binding. P: phosphorylation on T69, T71 or S90. b Determination of model parameters. HEK293T cells were stimulated by anisomycin and pp-JNK, pp-p38 and pp-ATF2-T69/T71 were monitored in western-blots (JNK-IN-8: in the presence of JNK inhibitor; CTR: control, without the inhibitor; “calc” denotes simulated plots). The parallel p38-ATF2 NanoBit assay signal was also used to determine the parameters that could not be directly measured (i.e., kstim6, kstim7, dp1, dp2, dp3 and dp4). c Simulated ATF2 TAD phosphorylation levels 40 min after stimulation. The model was stimulated by different kstim6 and kstim7 (evenly distributed from 10−6 to 10−2 in the logarithmic scale). 3D plots show ATF2 TAD phosphorylation as the function of different amounts of pp-p38 and pp-JNK (from 0.001 to 1 µM; from blue to red, respectively), where pp-JNK and pp-p38 levels denote the activated state of the MAPKs. (Total in-cell MAPK and ATF2 concentrations were set to 1 µM for each protein.) ATF2-S90N S90-OFF: ATF2 TAD with a non-phosphorylatable serine to asparagine replacement at 90 (invertebrates); ATF2-WT S90-ON: results of the simulation with intact vertebrate ATF2 TAD. 2D panels show projections of the corresponding 3D plots. Black line indicates where pp-ATF2 level is equally sensitive to the activation of both MAPKs. The region above this line corresponds to an area where pp-ATF2 is more sensitive to JNK, while the region below the line is more sensitive to changes in p38 pathway flux. d The impact of MAPK binding affinity and specificity on pp-ATF2 levels. JNK-ATF2 or p38-ATF2 binding affinities were arbitrarily changed in the model and the impact of these on pp-ATF2 level was simulated. Binding affinity was increased by 100-fold by increasing the kon (left and middle panels) or changed by 10-fold (lowered for JNK but increased for p38; right panel). Note that koff1/kon1, koff2/kon2, and koff3/kon3 describe the affinity of JNK(DRS)-, p38(FRS)-, and p38(DRS)-ATF2 TAD binding, respectively (FRS: F-motif recruitment site; DRS: D-motif recruitment site).
