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

Abstract

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.

Rotenone inhibits axonogenesis via an Lfc/RhoA/ROCK pathway in cultured hippocampal neurons

“…Therefore, to examine this with more detail we evaluated whether or not rotenone‐induced RhoA activation involves an increase in ROCK activity using the Eevee‐ROCK biosensor (Li et al . ).…”

“…) and the Eevee‐ROCK was a kind gift of Dr. Matsuda (Li et al . ). Both constructs are unimolecular forster resonance energy transfer (FRET)‐based biosensor, which consists of genetically encoded fusions of a sensing module flanked with a donor fluorophore [a cyan fluorescent protein (CFP)] and an acceptor fluorophore (a yellow fluorescent protein) that allow ratiometric measurements (Fritz et al .…”

“…) or the Eevee ROCK biosensor (Li et al . ). The neurons were electroporated with 4 μg of RhoA or Eevee ROCK biosensors.…”

A Novel Morphological Marker for the Analysis of Molecular Activities at the Single-cell Level

“…Abe et al previously compared the distribution of the membrane markers lyn-EGFP, EGFP-KRas, GAP43-EGFP, Display-Venus, and Display-mCherry, and concluded that lyn-EGFP performed best . We previously compared the ROCK biosensor tagged with the myristoylation signal of lyn, the KRas CAAX domain, and the HRas CAAX domain and found that the KRas CAAX domain worked best (Li et al, 2017). We need to further explore the best membrane marker for the in vivo imaging.…”

Live-cell Imaging with Genetically Encoded Protein Kinase Activity Reporters

“…Further, the FLIM-FRET with a non-fluorescent protein enables multi-plexed imaging of molecular activity (Laviv et al, 2016). While one of the remaining difficulties is to find the peptide sequences to be phosphorylated by a target kinase, a recent advance in phosphoproteomics, kinase-interacting substrate screening (KISS) (Amano et al, 2015), facilitated the development of a novel ROCK FRET biosensor (Li et al, 2017).…”