follow analogous patterns to those of PDGF receptor and Akt, respectively; phosphorylation of ERK1/2, but not of MEK1/2, is significantly reduced in GSK583 MG132-treated versus control cells stimulated with the low PDGF dose, whereas phosphorylation of both MEK1/2 and ERK1/2 are dramatically reduced in MG132-treated cells stimulated with the high PDGF dose. Although it would appear that MEK1/2 phosphorylation stimulated at low PDGF concentration is minimally perturbed by MG132 treatment, it should be noted that total MEK1/2 levels are modestly increased in MG132-treated cells furthermore, the accompanying ablation of ERK1/2 activation is expected to relieve a potent negative feedback affecting MEK1/2 phosphorylation in these cells. A qualitatively similar pattern of MEK and ERK phosphorylation was found in FGF-stimulated cells, except that the effect of MG132 treatment on ERK phosphorylation elicited by a low dose of FGF-2 is not statistically significant by two-way ANOVA. The interpretation is that while the MEK and ERK phosphorylation kinetics are certainly consistent with upregulation of ERK dephosphorylation activity in MG132-treated cells, suppression of ERK signaling is also affected by reduced activation of the upstream kinase. Based on the suggestion that MEK activation is reduced in combination with increased ERK dephosphorylation activity in MG132-treated cells, we sought to parse these two effects quantitatively. To accomplish this, we devised a kinetic modeling scheme. Given the potentially complex effects of MG132 treatment on growth factor receptor-mediated signaling upstream of ERK1/2, our strategy was to fit each MEK1/2 phosphorylation time course to an empirical function, which serves then as the input to a modified Michaelis-Menten model of ERK phosphorylation and dephosphorylation on its two activating sites. In the case of the time courses with MG132, this model tests the consistency of the simplest hypothesis that the phosphatase activity of the enzyme catalyzing dephosphorylation of the two sites on ERK1/2 is 852808-04-9 enhanced by a constant factor, while the rest of the parameters affecting ERK phosphorylation kinetics were constrained to have the same values in MG132-treated and control cells. This model was iteratively fit to the ERK data set by Monte Carlo sampling of the model parameters to obtain a large ensemble of parameter sets that produce nearly equivalent qualities of fit, allowing us to evaluate the degree to which each parameter was properly constrained. As a central estimate of the model output, the mean of the ensemble is quantitatively consistent with the corresponding ERK phosphorylation data.