Subcellular dynamics of protein kinase A activity visualized by FRET-based reporters.
cAMP-dependent protein kinase (PKA) mediates key cellular processes via compartmentalized activity, and the ability to track its activity in living cells should help increase our understanding of this precise regulation. Here, through systematic testing of new fluorescent proteins, we developed a new FRET-based A-kinase activity reporter (AKAR), AKAR3, with a dynamic range of 31-41%, twice that of predecessors. Visualization of PKA activity at plasma membrane, cytoplasm, nucleus, and mitochondria was achieved. Targeting AKAR3 to outer mitochondrial membrane revealed that basal PKA activity at mitochondria differs from that in the cytoplasm, indicating differential regulation of PKA activity at different subcellular locations.
Allen
Michael D
MD
Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Zhang
Jin
J
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000201:enzymology; D002467:Cell Nucleus / Q000201:enzymology; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003593:Cytoplasm / Q000201:enzymology; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry; D008928:Mitochondria / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry
D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D008164:Luminescent Proteins; D011994:Recombinant Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D017868:Cyclic AMP-Dependent Protein Kinases