In eukaryotic cells, networks of signaling proteins are responsible for converting environmental inputs into the appropriate regulatory outputs. The proteins that comprise signaling networks are highly modular, and are typically made up of multiple, functionally distinct domains. These domains are of two varieties: 1) catalytic domains (eg. kinases, GTPases), which leave a chemical imprint on a downstream target, and 2) interaction domains, which facilitate recognition between network members. As such, information flow in signaling networks is potentiated by catalytic domain function, while protein-protein interaction domains direct this catalytic function to specific targets, thereby defining network connectivity.
In signaling networks, groups of proteins that mediate a particular response (pathways) are often found co-localized with one another into complexes. These complexes are coordinated by scaffolds proteins, which typically are composed of multiple, modular interaction domains. By defining which catalytic components are co-localized within a pathway complex, the interaction domains in a scaffold encode the connectivity for that pathway. It has been hypothesized that scaffolding may have facilitated the evolutionary elaboration of signaling by acting as a modular hub for the addition of new linkages in a pathway: since scaffolds create specificity by co-localization, addition of new components to a pathway may be easily accomplished by addition of a new interaction domain to the scaffold.
We reasoned that the same modularity that may have made scaffolded signaling pathways highly evolvable might also make them engineerable. In the present work we provide evidence that supports this premise: scaffolding can be exploited to generate novel, synthetic pathway linkages, and thus can be used to reshape the I/O of a signaling pathway. We reprogrammed the I/O behavior of the mating response pathway in S. cerevisiea, a classically studied MAP kinase signaling pathway that utilizes a scaffold protein, Ste5, to coordinate members of the pathway. We demonstrate that Ste5 can be used as an assembly platform for the synthetic recruitment of factors that positively and negatively regulate mating pathway activity. By incorporating these recruited modulators into simple transcriptional feedback loops, we demonstrate that the scaffold can be used as a junction to incorporate artificial feedback control into the pathway's response, dramatically altering its I/O. Using a limited set of molecular components, we constructed a variety of feedback circuit architectures and were able to generate diverse classes of behavior.
The results of the work described herein demonstrate the essential plasticity of a signaling network that utilizes scaffolding to define its connectivity. By exploiting these features, we were able to easily introduce new network linkages into a signaling pathway and dramatically reshape its signaling behavior. The variability of circuit behaviors achieved from a small collection of constituent parts supports the idea that the facile rewiring of regulatory linkages using scaffolding may have played an important role in facilitating the evolution of protein signaling networks. Furthermore, the approaches we used for rewiring the mating pathway may be generalizable to a variety of other signaling pathways both in yeast and other organisms.