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

Shang-Jui Tsai; Yijing Gong; Austin Dabbs; Fiddia Zahra; Junhao Xu; Aleksander Geske; Michael J Caterina; Stephen J Gould

doi:10.1016/j.jbc.2025.108183

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

J Biol Chem. 2025 Jan 13:108183. doi: 10.1016/j.jbc.2025.108183. Online ahead of print.

Authors

Shang-Jui Tsai¹, Yijing Gong², Austin Dabbs², Fiddia Zahra³, Junhao Xu², Aleksander Geske⁴, Michael J Caterina², Stephen J Gould⁵

Affiliations

¹ Department of Neurosurgery, Johns Hopkins University School of Medicine, Baltimore, MD 21205 USA; Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205; Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205 USA. Electronic address: [email protected].
² Department of Neurosurgery, Johns Hopkins University School of Medicine, Baltimore, MD 21205 USA; Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205; Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205 USA.
³ Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205 USA.
⁴ Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
⁵ Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205 USA. Electronic address: [email protected].

PMID: 39814226
DOI: 10.1016/j.jbc.2025.108183

Abstract

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) 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 (bNLS) in their kinase sensor domains, to ensures 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 in live cells, which verified that these eKTRs respond as expected to direct agonists and inhibitors, and that crosstalk between these pathways is highly unbalanced, with activation of PKA suppressing ERK activity, while activation of ERK induces PKA activity. Taken together, our findings highlight the importance of KTR size and bNLS 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.