Design of an ultrabright biosensor for dynamic imaging of kinase activity in cells

Protein kinases regulate almost every major signaling pathway. Visualizing spatiotemporal dynamics of kinase activity is thus essential to understand cell signaling. Here we report a de nov o -designed a ctivity reporter of k inase, dubbed NOVARK, which contains a single polypeptide chain with multiple modular motifs that act as specific kinase substrates and reporters. NOVARK undergoes phosphorylation-induced higher order-assembly, which are detectable as ultrabright GFP droplets with a greater dynamic range than existing Förster resonance energy transfer-based kinase reporters. We designed versions of NOVARK that rapidly and reversibly report intracellular activity of protein kinase A, C, and ERK following stimulation/inhibition by upstream GPCR agonists. Our work provides a generalizable platform that enables the design of ultrabright biosensors for illuminating dynamic architecture of kinase signaling.