Abstract Protein kinases are key signaling nodes that regulate fundamental biological and disease processes. Illuminating kinase signaling from multiple angles can provide deeper insights into disease mechanisms and improve therapeutic targeting. While fluorescent biosensors are powerful tools for visualizing live-cell kinase activity dynamics in real time, new molecular tools are needed that enable recording of transient signaling activities for post hoc analysis and targeted manipulation. Here, we develop a light-gated kinase activity coupled transcriptional integrator (KINACT) that converts dynamic kinase signals into “permanent” fluorescent marks. KINACT enables robust monitoring of kinase activity across scales, accurately recording subcellular PKA activity, highlighting PKA signaling heterogeneity in 3D cultures, and identifying PKA activators and inhibitors in high-throughput screens. We further leverage the ability of KINACT to drive signaling effector expression to allow feedback manipulation of the balance of Gα s R201C -induced PKA and ERK activation and dissect the mechanisms of oncogenic G protein signaling.

Light-gated Integrator for Highlighting Kinase Activity in Living Cells