Mitogen-activated protein kinase (MAPK) cascades are ubiquitous signaling modules that regulate a vast array of cell-fate decisions including proliferation, differentiation, motility, stress responses, and apoptosis. The ability of this module to control diverse processes stems, in part, from its capacity for flexible activation responses. Experimental work has shown that MAPK activation can be graded or switch-like (ultrasensitive), transient or sustained, and monostable or bistable. The mechanisms underlying this plasticity are difficult to elucidate in endogenous signaling networks with multifaceted regulation. To obviate this difficulty we constructed an insulated mammalian MAPK cascade in yeast to explore how extrinsic perturbations (scaffolding and negative regulation) and intrinsic perturbations (concentration variation) affect the flexibility of this synthetic signaling module. In contrast to the prozone effect (i.e., a biphasic dependence on scaffold concentration), we observe a purely monotonic decrease in signal strength as scaffold concentration is augmented. Further, we find that increasing the concentration of sequential kinases can enhance ultrasensitivity and lower the threshold of activation. Computational analyses identify cascading as a mechanism for generating ultrasensitivity de novo and reveal that natural cascades with different kinase concentrations are innately biased toward their distinct activation profiles. In addition, we show that integrating negative regulation and concentration variation can decouple the ultrasensitivity and threshold from the strength of the response and we highlight strategies for generating a set of distinct behaviors. This work demonstrates that tunable signal processing is inherent to minimal MAPK modules and elucidates principles for rational design of more complex synthetic signaling systems.
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Tunable Signal Processing In Synthetic MAP Kinase Modules
Mitogen-activated protein kinase (MAPK) cascades are ubiquitous signaling modules that regulate a vast array of cell-fate decisions including proliferation, differentiation, motility, stress responses, and apoptosis. The ability of this module to control diverse processes stems, in part, from its capacity for flexible activation responses. Experimental work has shown that MAPK activation can be graded or switch-like (ultrasensitive), transient or sustained, and monostable or bistable. The mechanisms underlying this plasticity are difficult to elucidate in endogenous signaling networks with multifaceted regulation. To obviate this difficulty we constructed an insulated mammalian MAPK cascade in yeast to explore how extrinsic perturbations (scaffolding and negative regulation) and intrinsic perturbations (concentration variation) affect the flexibility of this synthetic signaling module. In contrast to the prozone effect (i.e., a biphasic dependence on scaffold concentration), we observe a purely monotonic decrease in signal strength as scaffold concentration is augmented. Further, we find that increasing the concentration of sequential kinases can enhance ultrasensitivity and lower the threshold of activation. Computational analyses identify cascading as a mechanism for generating ultrasensitivity de novo and reveal that natural cascades with different kinase concentrations are innately biased toward their distinct activation profiles. In addition, we show that integrating negative regulation and concentration variation can decouple the ultrasensitivity and threshold from the strength of the response and we highlight strategies for generating a set of distinct behaviors. This work demonstrates that tunable signal processing is inherent to minimal MAPK modules and elucidates principles for rational design of more complex synthetic signaling systems.