Engineering synthetic phosphorylation signaling networks in human cells

Protein phosphorylation signaling networks play a central role in how cells sense and respond to their environment. Here, we describe the engineering of artificial phosphorylation networks in which “push-pull” motifs—reversible enzymatic phosphorylation cycles consisting of opposing kinase and phosphatase activities—are assembled from modular protein domain parts and then wired together to create synthetic phosphorylation circuits in human cells. We demonstrate that the composability of our design scheme enables model-guided tuning of circuit function and the ability to make diverse network connections; synthetic phosphorylation circuits can be coupled to upstream cell surface receptors to enable fast-timescale sensing of extracellular ligands, while downstream connections can regulate gene expression. We leverage these capabilities to engineer cell-based cytokine controllers that dynamically sense and suppress activated T cells. Our work introduces a generalizable approach for designing and building phosphorylation signaling circuits that enable user-defined sense-and-respond function for diverse biosensing and therapeutic applications.