Enhanced kinase translocation reporters for simultaneous real-time measurement of PKA, ERK, and Ca ²⁺

Kinase translocation reporters (KTRs) are powerful tools for single-cell measurement of time-integrated kinase activity but suffer from restricted dynamic range and limited sensitivity, particularly in neurons. To address these limitations, we developed enhanced KTRs (eKTRs) for protein kinase A (PKA) and extracellular signal-regulated kinase (ERK) that display high sensitivity, rapid response kinetics, broad dynamic range, cell type-specific tuning, and an ability to detect PKA and ERK activity in primary sensory neurons. Moreover, co-expression of optically separable eKTRs for PKA and ERK revealed the kinetics of expected and unexpected crosstalk between PKA, ERK, protein kinase C, and calcium signaling pathways, demonstrating the utility of eKTRs for live cell monitoring of diverse and interacting signaling pathways. These results open the door to improved live-cell and in vivo measurements of key signaling pathways in neurons, while at the same time demonstrating the importance of KTR size and NLS strength to KTR dynamics.