Tunable signal processing in synthetic mitogen-activated protein kinase cascades

Mitogen-activated protein kinase (MAPK) cascades regulate a vast array of cell-fate decisions. The ability of this module to control diverse processes stems, in part, from its capacity for flexible activation responses. The mechanisms underlying the plasticity of this response have not been fully elucidated and are difficult to parse out given the multifaceted regulation found in signaling networks. We have constructed a synthetic mammalian MAPK cascade in yeast to explore the effects of intrinsic perturbations (concentration variation) and extrinsic perturbations (scaffolding and negative regulation) on the flexibility of this isolated signaling module. Experimentally, increasing the concentration of sequential kinases augmented ultrasensitivity and lowered the threshold of activation. Computational analyses of these intrinsic perturbations indicate that two, well characterized, natural cascades in X. laevis and S. cerevisiae with distinct activation profiles are innately biased toward their respective behaviors by kinase concentration. We also showed that signal propagation through this catalytically efficient cascade is unaltered by moderate scaffold co-expression yet it is greatly attenuated at high scaffold levels. Further, over-expression of the scaffold resulted in a biphasic Hill response. We hypothesized that this complex activation profile arises from sequestration and we tested this computationally with a compartmental model. We showed that negative regulation by both enzymatic phosphatase expression and small-molecule inhibitor binding attenuated the ultrasensitivity and increased the threshold of activation over the range of experimental parameters. Interestingly, a computational model integrating negative regulation and concentration variation revealed parameter regimes in which these different modes of regulation enable distinct activation responses. This integrated computational model also predicted parameters for which specific signal characteristics can be decoupled and we demonstrated this effect experimentally by tuning the ultrasensitivity and threshold independently from the strength of the response. This work demonstrates that tunable signal processing is an inherent feature of minimal MAPK modules and elucidates principles for rational design of more complex synthetic signaling systems.