Genetically encoded FRET probe for PKC activity based on pleckstrin

Area of Science:

• Biochemistry
• Cell Biology
• Molecular Imaging

Background:

• Protein kinase C (PKC) plays a crucial role in various cellular processes.
• Monitoring PKC activity in living cells is essential for understanding signal transduction.
• Existing methods for PKC activity assessment often lack real-time or in vivo capabilities.

Purpose of the Study:

• To develop and validate a novel Förster Resonance Energy Transfer (FRET)-based probe for real-time monitoring of protein kinase C (PKC) activity.
• To demonstrate the probe's efficacy in vitro and in vivo for tracking PKC modulation.
• To assess the probe's responsiveness to physiological stimuli and inhibitors.

Main Methods:

• Design and synthesis of a FRET-based probe utilizing a pleckstrin fragment flanked by FRET-capable fluorophores.
• In vitro characterization of probe response to PKC activation and inhibition.
• In vivo validation in living cells, monitoring FRET changes upon stimulation with phorbol ester, histamine, or bradykinin, and subsequent addition of PKC inhibitors.

Main Results:

• The developed probe reliably detected changes in PKC activity through FRET variations.
• The probe exhibited rapid responses to PKC activation induced by phorbol ester.
• Reversible FRET changes were observed upon administration of PKC inhibitors, confirming probe specificity.
• Physiologically relevant PKC activation was successfully triggered and monitored using histamine and bradykinin stimulation.

Conclusions:

• A novel, sensitive, and responsive FRET-based probe for real-time monitoring of PKC activity in living cells has been successfully developed.
• The probe allows for dynamic tracking of PKC signaling in response to pharmacological activators and physiological stimuli.
• This tool provides a valuable method for investigating the role of PKC in cellular signaling pathways and disease states.