In summary, using scaffold-mediated signaling as an example, we have demonstrated that various components in the EGF signaling pathway have distinct contributions and they respond and act in concert to execute the final signal output as a function of varying EGF concentrations and times after stimulation. While the actual physiological significance of this multi-level and cross-regulation effects remain to be verified experimentally, the current model provides an attractive platform to further integrate the input of other scaffolds and regulators such as the Sef, Mek, GEFs and GAPs as well as a higher level of control via scaffold dimerization and the interactions among different scaffold proteins. These serve as a regulatory hub to finetune ERK signaling in response to different fluxes of physiological or pathophysiological stimuli. Understanding the Reversine Aurora Kinase inhibitor intricate interplay and their differences in normal and pathophysiological conditions should help shed light to the possible mechanism of their involvement in cancer, inflammation, adipogenesis, cardiovascular disease, urinary bladder dysfunction, and in the response to anti-proliferative agents targeting these proteins and pathways. The pathway model used here is illustrated schematically in Figure 1. Two cascades were added to our earlier EGFR-ERK simulation model. These are the KSR and MP1 cascades based on the published models of the MAPK cascade with generic scaffold proteins as illustrated in Figures 2 and 3, GDC-0449 Hedgehog inhibitor respectively. These models were based on the following assumptions made by Levchenko et al and supported by experimental data: These scaffold proteins do not bind partially or fully activated kinases, based on the observation that MP1 has no effect on MEK-1 previously activated by B-Raf. Kinase activation by a scaffold protein is processive rather than distributive, based on two observations that MP1 increases B-raf activation of MEK-1 and that dual phosphorylation of MAPK at two sites take place simultaneously in the presence of MEF whereas phosphorylation at the second site is delayed by about 20 min in the absence of MEF. The catalytic activity of a scaffold protein can be precluded from the model, as supported by the finding that the scaffolding function of KSR is independent of its kinase activity. Kinases bind to scaffold proteins independent of each other, as revealed by some experimental studies. There is no inter-scaffold protein interaction, based on the fact that although p14 and MP1 were suggested to be able to weakly self-associate in vitro, there has been no reports about such homodimers being detected in experimental systems to date. The constituent molecular interactions, their kinetic constants, and molecular concentrations are detailed in Supplementary Tables S1 and S2.