UC San Diego

Protein kinases control nearly every facet of cellular function, integrating diverse inputs to regulate complex physiological processes, and aberrant kinase signaling is linked to numerous diseases. While fluorescent protein-based biosensors have revolutionized the study of kinase signaling, powerful new molecular tools capable of robustly tracking kinase activity dynamics across diverse experimental contexts are needed to fully dissect the role of kinase signaling in physiology and pathology To study PKA, we developed an ultrasensitive, second-generation excitation-ratiometric protein kinase A (PKA) activity reporter (ExRai-AKAR2), obtained via high-throughput linker library screening, which enables sensitive and rapid monitoring of live-cell PKA activity across multiple fluorescence detection modalities, including plate reading, cell sorting, one- or two-photon imaging and visual cortex imaging in awake mice. UsingExRai-AKAR2, we performed a plate reader based screen of the Johns Hopkins Drug Library, which consists of over 4000 FDA approved drugs or other clinically relevant compounds, to uncover unknown mechanisms of PKA regulation and promising drugs that could be repurposed to perturb PKA signaling pathways. We successfully discovered several novel PKA activators and inhibitors, and discovered that the inhibition of respiratory chain in mitochondria leads to the activation of soluble adenylyl cyclase and PKA. In addition, we also engineered new FRET-based AKARs with enhanced dynamic range via linker engineering. At the same time, we identified the nonspecific phosphorylation of AKARs by a nucleus localized kinase, MSK1 and engineered a new variant, AKAR6-s, to abolish the nonspecific phosphorylation by re-engineering the sensor substrate. We introduced these improved FRET-based AKARs with better sensitivity and specificity as a tool kit for studying PKA activities under specific requirements and in different imaging modalities.