Enhanced kinase translocation reporters for simultaneous real-time measurement of PKA, ERK, and calcium.

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 PKA and extracellular signal-regulated kinase (ERK) by (i) increasing KTR size, which reduces the confounding effect of KTR diffusion through the nuclear pore and (ii) modulating the strength of the bipartite nuclear localization signal in their kinase sensor domains, to ensure that the relative distribution of the KTR between the nucleus and cytoplasmic is determined by active nuclear import, active nuclear export, and relative activity of their cognate kinase. The resultant sets of ePKA-KTRs and eERK-KTRs display high sensitivity, broad dynamic range, and cell type-specific tuning. Moreover, co-expression of optically separable ePKA-KTRs and eERK-KTRs allowed us to simultaneously monitor the activation and inhibition of PKA and ERK, together with calcium levels, in live cells. These eKTRs responded as expected to direct agonists and inhibitors, and also confirmed that crosstalk between the PKA and ERK pathways is highly unbalanced, with the activation of PKA suppressing ERK activity, while activation of ERK induces PKA activity. Taken together, our findings highlight the importance of KTR size and bipartite nuclear localization signal strength to KTR sensitivity and dynamic range, show that different cell types require different eKTRs, and identify ePKA-KTR1.4 and eERK-KTR1.2 as particularly well-suited for monitoring PKA and ERK in primary sensory neurons.