title: Luminescent kinase activity biosensors based on a versatile bimolecular switch.
abstract: Real-time tracking of kinase activity in living systems has revealed new modes of encoding signaling information into spatiotemporal activity patterns and opened new avenues for screening kinase modulators. However, the sensitivity of kinase activity detection, which is commonly coupled to a fluorescence resonance energy transfer (FRET)-based readout, has often been a limiting factor. Here we show that a kinase-inducible bimolecular switch consisting of a substrate for the kinase of interest and a phosphoamino acid binding domain can be designed to sense different kinase activities and coupled to various readouts, thereby allowing for examination of dynamic kinase activity with increased sensitivity and versatility. Specifically, we demonstrate that bimolecular switches designed to sense protein kinase A (PKA) or protein kinase C (PKC) activities can turn on FRET as well as bioluminescence signals. Notably, the FRET-based sensors gain larger dynamic ranges in comparison with their unimolecular counterparts; the novel bioluminescence-based reporters for PKA and PKC show high sensitivity and a unique capability to detect basal kinase activities and should enable new applications in in vivo imaging of kinase activity and high-throughput compound screening. Thus, this generalizable design advances the molecular toolkit of kinase activity detection and provides a means for versatile and sensitive detection of kinase activity in various biological systems.
authors: Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United States.
authors: Katie J
authors: KJ
authors: Herbst
authors: Michael D
authors: MD
authors: Allen
authors: Jin
authors: J
authors: Zhang
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D049449:Luminescence; D019606:Phosphoamino Acids / Q000378:metabolism; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary
chemical_list: D019606:Phosphoamino Acids; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C