Light-gated integrator for highlighting kinase activity in living cells

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 activity distribution 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 GasR201C-induced PKA and ERK activation and dissect the mechanisms of oncogenic G protein signaling. Overall design: We generated two dual-stable cell lines: one A-KINACT effector cell line stably expressing A-KINACT and integrating TetO-driven EGFP-fused PKA inhibitory peptide (TetO-PKI-EGFP), and one A-KINACT control cell line stably expressing A-KINACT and integrating TetO-EGFP. A-KINACT-expressing HEK293T cells showing mTagBFP2-GasR201C overexpression and A-KINACT-driven PKI-EGFP expression were identified and collected via FACS to assess differences in transcription versus control cells expressing A-KINACT-driven EGFP.