the fold-upregulation of ERK phosphatase Duvelisib (R enantiomer) activity in MG132-treated cells using the time course of MEK phosphorylation as an input. Given that MEK phosphorylation is also perturbed by MG132 treatment, our strategy was to independently fit each time course of MEK phosphorylation to a phenomenological function; then assuming those phosphorylated MEK kinetics, ERK phosphorylation kinetics were globally fit to a modified Michaelis-Menten model. All calculations were performed using MATLAB. The parameter estimation approach used is as described in detail previously. Briefly it uses a Markov chain Monte Carlo/simulated annealing-based algorithm to generate a large ensemble of good parameter sets rather than one best fit. After compiling the ensemble, the model output is recalculated for each parameter set at each time point, an ensemble mean and standard deviation are calculated. Given the potentially broad-based effects of proteasome inhibition on intracellular signaling, we hypothesized that the observed reduction of ERK phosphorylation in MG132-treated cells is not caused solely by upregulation of DUSPs. Indeed, we found that many key readouts of PDGF-stimulated signaling are systemically reduced in NIH 3T3 fibroblasts pretreated with 25 mM MG132 for 6 hours. Furthest upstream is the tyrosine phosphorylation of PDGF receptors MG132 treatment significantly reduced phosphorylated Tyr751 of PDGF b-receptor, a major phosphorylation site that contributes to the recruitment of phosphoinositide 3-kinase, in a PDGF dose-dependent manner. By contrast, PDGF-stimulated phosphorylation of Akt on the activating site Ser473, a readout of pro-survival downstream of PI3K, is significantly reduced in MG132-treated cells at both low and high PDGF concentrations total Akt levels were not perturbed by MG132 treatment. This suggests that, whereas the ability to recruit PI3K in cells stimulated with a subsaturating PDGF concentration is not affected by MG132 treatment, the ability to maintain Akt phosphorylation is reduced. A reduction in the catalytic activity of PI3K or enhancement of Akt dephosphorylation can explain this result. The Akt phosphorylation kinetics for the high PDGF dose are 934660-93-2 consistent with this interpretation; stimulated phospho-Akt levels in control cells are at all times maintained at higher levels than in MG132-treated cells, despite the eventual decay of PDGF receptor phosphorylation in control cells below the levels achieved at earlier times in MG132-treated cells.
