TY - JOUR
T1 - A FRET biosensor for ROCK based on a consensus substrate sequence identified by KISS technology
AU - Li, Chunjie
AU - Imanishi, Ayako
AU - Komatsu, Naoki
AU - Terai, Kenta
AU - Amano, Mutsuki
AU - Kaibuchi, Kozo
AU - Matsuda, Michiyuki
N1 - Funding Information:
Acknowledgments. This study was funded by the Platform for Dynamic Approaches to Living Systems from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, (M.M.), by JSPS KAKENHI Grant Nos. 15H0594 "Resonance Bio" and 15H02397, and by the Nakatani Foundation. We are grateful to the members of the Matsuda Laboratory for their helpful input, to K. Hirano, K. Takakura and A. Kawagishi for their technical assistance, and to the Kyoto University Live Imaging Center for fluorescence imaging.
PY - 2017
Y1 - 2017
N2 - 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.
AB - 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.
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U2 - 10.1247/csf.16016
DO - 10.1247/csf.16016
M3 - Article
C2 - 27885213
AN - SCOPUS:85011579520
VL - 42
SP - 1
EP - 13
JO - Cell Structure and Function
JF - Cell Structure and Function
SN - 0386-7196
IS - 1
ER -