Synthetic Condensed-Phase Signaling Expands Kinase Specificity and Responds to Macromolecular Crowding

Liquid–liquid phase separation (LLPS) can concentrate biomolecules and accelerate reactions within membraneless organelles. For example, the nucleolus and PML-nuclear bodies are thought to create network hubs by bringing signaling molecules such as kinases and substrates together. However, the mechanisms and principles connecting mesoscale organization to signaling dynamics are difficult to dissect due to the pleiotropic effects associated with disrupting endogenous condensates. Here, we recruited multiple distinct kinases and substrates into synthetic LLPS systems to create new phosphorylation reactions within condensates, and generally found increased activity and broadened specificity. Dynamic phosphorylation within condensates could drive cell-cycle-dependent localization changes. Detailed comparison of phosphorylation of clients with varying recruitment valency and affinity into condensates comprised of either flexible or rigid scaffolds revealed unexpected principles. First, high client concentration within condensates is important, but is not the main factor for efficient multi-site phosphorylation. Rather, the availability of a large number of excess client binding sites, together with a flexible scaffold is crucial. Finally, phosphorylation within a suboptimal, flexible condensate was modulated by changes in macromolecular crowding. Thus, condensates readily generate new signaling connections and can create sensors that respond to perturbations to the biophysical properties of the cytoplasm.