Year: 2026
Type: preprint
Abstract: The cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway regulates diverse cellular processes through precise spatiotemporal control across subcellular compartments. Förster resonance energy transfer (FRET)-based A-kinase activity reporters (AKARs) have enabled live-cell visualization of PKA activity, but their limited dynamic range constrains detection of subtle or compartment-specific signaling events. Here, we present a suite of sixth-generation cyan/yellow FRET-based PKA sensors (the AKAR6 series) with substantially enhanced sensitivity and improved selectivity. Systematic optimization of the FRET donor-acceptor pair and sensor backbone yields superior performance versus previous best-in-class FRET-based AKARs, enabling robust detection of subtle PKA activity changes across diverse experimental modalities, including flow cytometry, fluorescence lifetime-based FRET, and two-photon imaging in brain slices. We further leverage kinome atlas data to engineer a variant with improved selectivity for more accurate visualization of nuclear PKA activity. Using the AKAR6 toolkit, we showed that in contrast to strong GPCR-induced PKA activities across all tested compartments in PC12 cells, growth factors stimulated a significant PKA activity at the trans-Golgi network but no detectable activity at cis-Golgi, signifying highly compartmentalized PKA signaling at the sub-organelle level. Furthermore, NGF and EGF induced sustained and transient PKA activity, respectively, across various intracellular compartments, including the nucleus, suggesting that growth factor-specific temporal controls are maintained across subcellular compartments. Together, the AKAR6 toolkit provides a sensitive, selective, and versatile platform for dissecting compartmentalized PKA signaling across cells and tissues.
Language: English
Subfield: Biophysics
Domain: Life Sciences
Open Access status: green
Funders: National Science Foundation, American Heart Association, Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung, National Institutes of Health, University of California, San Diego, National Institute of Mental Health, National Institute of Neurological Disorders and Stroke
Awards: Circuit architecture and dynamics of the insular cortex underlying motivational behaviors, G3033040839, G4869884297, Spying on kinase activity architecture by super-resolution microscopy, Ultrasensitive kinase biosensors for multiplex imaging of coordinated spatiotemporal signaling in cancer-immune interactions, University of California San Diego Neuroscience Microscopy Imaging Core, G639426234, Regulation of ß-AR stimulated cAMP signaling in cardiomyocytes via RIa phase separation, Sensing and manipulating neuromodulatory signaling in vivo, G7624441509, G8448096725, G8971701765, G9000013793, G9000022920, G9000050626, G9000130922, G9000153585, G9000230046, G9000255603, G9000284303
OpenAlex ID: W7128536652