Visualization of Compartmentalized Kinase Activity Dynamics Using Adaptable BimKARs

• Published in: Chemistry & biology Vol. 22; no. 11; pp. 1470 - 1479
• Main Authors: Depry, Charlene, Mehta, Sohum, Li, Ruojing, Zhang, Jin
• Format: Journal Article
• Language: English
• Published: United States Elsevier Ltd 19.11.2015
• Subjects: AMP-Activated Protein Kinases - chemistry; AMP-Activated Protein Kinases - genetics; AMP-Activated Protein Kinases - metabolism; AMPK; Animals; biosensor; Cell Membrane - metabolism; Cercopithecus aethiops; compartmentalized signaling; COS Cells; Cyclic AMP-Dependent Protein Kinases - genetics; Cyclic AMP-Dependent Protein Kinases - metabolism; Fluorescence Resonance Energy Transfer; FRET; Genes, Reporter; HeLa Cells; Humans; live-cell imaging; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Microscopy, Fluorescence; Phosphorylation; PKA; Protein-Serine-Threonine Kinases - metabolism; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - genetics; compartmentalized signaling; AMPK; FRET; biosensor; PKA; live-cell imaging
• ISSN: 1074-5521; 1879-1301
• DOI: 10.1016/j.chembiol.2015.10.004

The ability to monitor kinase activity dynamics in live cells greatly aids the study of how signaling events are spatiotemporally regulated. Here, we report on the adaptability of bimolecular kinase activity reporters (bimKARs) as molecular tools to enhance the real-time visualization of kinase activity. We demonstrate that the bimKAR design is truly versatile and can be used to monitor a variety of kinases, including JNK, ERK, and AMPK. Furthermore, bimKARs can have significantly enhanced dynamic ranges over their unimolecular counterparts, allowing the elucidation of previously undetectable kinase activity dynamics. Using these newly designed bimKARs, we investigate the regulation of AMPK by protein kinase A (PKA) in the plasma membrane, and demonstrate that PKA can both negatively and positively regulate AMPK activity in the same cell. [Display omitted] •The versatile bimKAR design is easily adapted to monitor various kinase activities•A membrane-targeted bimKAR improves the detection of local AMPK activity•Membrane-localized AMPK activity undergoes opposing bidirectional regulation by PKA Depry et al. demonstrate the versatility of FRET-based bimolecular kinase activity reporters (bimKARs) as tools for improving the visualization of signaling dynamics in living cells, and reveal that PKA acts simultaneously as both a positive and negative regulator of AMPK signaling at the plasma membrane.