Fig. 6: Modeling the effects of ORF45 on ERK and RSK phosphorylation.

a Schematic of the MKK-ERK-RSK Network Model involving phosphatases and ORF45. The inset shows how the Biophysical Model was extended towards Network Model in steps. The full list of reaction parameters, directly measured or introduced into the model based on in vitro or in-cell fits, is shown in Supplementary Table 3. Dashed gray lines indicate that ORF45 binding to RSK or ERK competes with the binding of their respective phosphatases (PP2 or MKP). b Results of the parameter fit to in vitro experiments in the absence (in blue) and presence of ORF45 (in red). Panels show the impact of ORF45 on the MKK1-ERK2-RSK2 reaction (+MKK, see Fig. 5a) or on the dephosphorylation of ppERK2 by the MKP3 phosphatase (+MKP, see Fig. 5c; blue: ppERK2 + ERK2, gray: ppERK2 + RSK2 + MKP3; red: ppERK2 + RSK2 + ORF45). c Results of the global parameter fit to the cell-based experiments. The initial phase (up to 20 min; unaffected by transcription level effects) of the EGF mediated ERK pathway response in HEK293T or in HEK293T-ΔRSK1/2 cells was used to fit the Network Model (see Fig. 1; blue: ORF45 is not expressed; red: ORF45 expression is turned on). In-cell ERK and RSK levels were set to 0.7 and 2 µM, respectively, and plots show the ratio of phosphorylated kinase versus total amount. The level of ppERK at 15 minutes after EGF treatment in WT cells was estimated to be ~15% (see Supplementary Fig. 6). d Simulation results with intact (red) and modified Network Models (magenta or green). D/F-SUBe: ERK substrates dependent on D- or F-motif binding, shown with a yellow or magenta ellipsoid on Panel a, respectively; SUBr: RSK substrates independent of docking; VF-SUBr: RSK substrates dependent on VF-motif binding and thus on access to the RSK VF-groove, shown with a green ellipsoid on Panel a. The in-cell concentration of ORF45 was systematically changed between 0.01 and 100 µM and the steady-state level of activated kinases were calculated upon pathway activation (pERK: phosphorylated active ERK normalized to total in-cell ERK concentration). Furthermore, the fraction of kinase competent for substrate phosphorylation (normalized to the total kinase concentration) is also plotted. The dashed vertical line on the graphs indicates the in-cell concentration of ORF45 used in the original Network Model (1 µM). Arrows indicate how the artificial parameter change affects the phosphorylation levels compared to the intact model.