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

Area of Science:

• Molecular Biology
• Cell Signaling
• Biotechnology

Background:

• Förster/fluorescence resonance energy transfer (FRET) biosensors are crucial for studying signaling molecule dynamics.
• Developing kinase-specific FRET biosensors is challenging due to the difficulty in identifying suitable substrate peptides.

Purpose of the Study:

• To develop a sensitive and specific FRET biosensor for Rho-associated kinase (ROCK) activity.
• To utilize kinase-interacting substrate screening (KISS) technology for identifying ROCK-specific substrate sequences.

Main Methods:

• Kinase-interacting substrate screening (KISS) technology was employed to deduce a consensus substrate sequence for ROCK.
• A FRET biosensor, Eevee-ROCK, was constructed using the identified substrate sequence.
• HeLa cells were treated with ROCK inhibitors/siRNAs and stimulated with various agents to validate the biosensor's specificity and sensitivity.
• Time-lapse imaging was performed on cells stably expressing Eevee-ROCK to monitor ROCK activity during cellular processes.

Main Results:

• The KISS-derived substrate sequence successfully generated a highly sensitive and specific FRET biosensor for ROCK (Eevee-ROCK).
• Basal Eevee-ROCK FRET signals were significantly reduced by ROCK1/2 inhibition, confirming specificity.
• Eevee-ROCK detected ROCK activation in response to epidermal growth factor, lysophosphatidic acid, and serum.
• ROCK activity increased post-cytokinesis and during apoptosis, as revealed by live-cell imaging with Eevee-ROCK.

Conclusions:

• Eevee-ROCK, developed using KISS technology, is a valuable tool for real-time monitoring of ROCK activity.
• This biosensor facilitates a deeper understanding of ROCK's physiological roles in cellular processes like cytokinesis and apoptosis.
• The KISS approach offers a promising strategy for developing FRET biosensors for other protein kinases.