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A FRET Biosensor for ROCK Based on a Consensus Substrate Sequence Identified by KISS Technology.

Authors
  • Li, Chunjie1
  • Imanishi, Ayako
  • Komatsu, Naoki
  • Terai, Kenta
  • Amano, Mutsuki
  • Kaibuchi, Kozo
  • Matsuda, Michiyuki
  • 1 Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University.
Type
Published Article
Journal
Cell Structure and Function
Publisher
Japan Society for Cell Biology
Publication Date
Jan 11, 2017
Volume
42
Issue
1
Pages
1–13
Identifiers
DOI: 10.1247/csf.16016
PMID: 27885213
Source
Medline
License
Unknown

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.

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