Abstract

The ability of cells to rapidly sense and respond to changes in their external environment via networks of phosphorylation-based signaling proteins is essential for their survival and function. Despite the importance of these networks, and the clear potential for harnessing them to create cell-based technology, the ability to engineer artificial phospho-signaling networks remains underexplored. To address this deficit, we recently developed a framework for the bottom-up engineering of artificial phospho-signaling networks in human cells. Our approach is based on the construction of reversible enzymatic amplifiers—or “push-pull” motifs—from protein domain building blocks. By interlinking and tuning push-pulls, we demonstrate the ability to construct phosphorylation-based signaling networks in human cells. By linking circuits to synthetic receptors, push-pull networks can be engineered to sense and respond on a fast timescale to the presence of extracellular ligands, while downstream connections can enable regulation of gene expression, secretion, and molecular condensate formation. This work defines a broadly applicable framework for engineering post-translational signaling networks that can be used to create cell-based theranostic devices capable of sensing disease markers and responding with a therapeutic output.