title: A FRET Biosensor for ROCK Based on a Consensus Substrate Sequence Identified by KISS Technology.
abstract: Genetically-encoded biosensors based on Förster/fluorescence resonance energy transfer (FRET) are versatile tools for studying the spatio-temporal regulation of signaling molecules within not only the cells but also tissues. Perhaps the hardest task in the development of a FRET biosensor for protein kinases is to identify the kinase-specific substrate peptide to be used in the FRET biosensor. To solve this problem, we took advantage of kinase-interacting substrate screening (KISS) technology, which deduces a consensus substrate sequence for the protein kinase of interest. Here, we show that a consensus substrate sequence for ROCK identified by KISS yielded a FRET biosensor for ROCK, named Eevee-ROCK, with high sensitivity and specificity. By treating HeLa cells with inhibitors or siRNAs against ROCK, we show that a substantial part of the basal FRET signal of Eevee-ROCK was derived from the activities of ROCK1 and ROCK2. Eevee-ROCK readily detected ROCK activation by epidermal growth factor, lysophosphatidic acid, and serum. When cells stably-expressing Eevee-ROCK were time-lapse imaged for three days, ROCK activity was found to increase after the completion of cytokinesis, concomitant with the spreading of cells. Eevee-ROCK also revealed a gradual increase in ROCK activity during apoptosis. Thus, Eevee-ROCK, which was developed from a substrate sequence predicted by the KISS technology, will pave the way to a better understanding of the function of ROCK in a physiological context.
authors.affiliation: Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University.
authors.forename: Chunjie
authors.initials: C
authors.lastname: Li
authors.forename: Ayako
authors.initials: A
authors.lastname: Imanishi
authors.forename: Naoki
authors.initials: N
authors.lastname: Komatsu
authors.forename: Kenta
authors.initials: K
authors.lastname: Terai
authors.forename: Mutsuki
authors.initials: M
authors.lastname: Amano
authors.forename: Kozo
authors.initials: K
authors.lastname: Kaibuchi
authors.forename: Michiyuki
authors.initials: M
authors.lastname: Matsuda
mesh_terms: D000595:Amino Acid Sequence; D015374:Biosensing Techniques; D015153:Blotting, Western; D031541:Fluorescence Resonance Energy Transfer; D015870:Gene Expression / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D013379:Substrate Specificity; D059008:Time-Lapse Imaging; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; D054460:rho-Associated Kinases