title: Visualization of Compartmentalized Kinase Activity Dynamics Using Adaptable BimKARs.
abstract: 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.
authors.affiliation: Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: Charlene
authors.initials: C
authors.lastname: Depry
authors.affiliation: Department of Pharmacology, University of California, San Diego, 9500 Gilman Drive, BRF2 1120, La Jolla, CA 92093, USA.
authors.forename: Sohum
authors.initials: S
authors.lastname: Mehta
authors.affiliation: Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: Ruojing
authors.initials: R
authors.lastname: Li
authors.affiliation: Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; The Solomon H. Snyder Department of Neuroscience and Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Pharmacology, University of California, San Diego, 9500 Gilman Drive, BRF2 1120, La Jolla, CA 92093, USA. Electronic address: jzhang32@ucsd.edu.
authors.forename: Jin
authors.initials: J
authors.lastname: Zhang
mesh_terms: D000091162:AMP-Activated Protein Kinase Kinases; D055372:AMP-Activated Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D017346:Protein Serine-Threonine Kinases; C109790:STK11 protein, human; D017868:Cyclic AMP-Dependent Protein Kinases; D000091162:AMP-Activated Protein Kinase Kinases; D055372:AMP-Activated Protein Kinases
keywords: AMPK; FRET; PKA; biosensor; compartmentalized signaling; live-cell imaging