Sensitive Fluorescent Biosensor Reveals Differential Subcellular Regulation of PKC

Area of Science:

• Cellular Biology
• Molecular Signaling
• Biochemistry

Background:

• Protein Kinase C (PKC) enzymes are crucial for diverse cellular functions, with three distinct subfamilies.
• Regulation of PKC isoforms at specific subcellular locations, including the ER, lysosomes, and Par complexes, remains poorly understood.
• Understanding subcellular PKC dynamics is essential for deciphering complex cellular signaling pathways.

Approach:

• Developed a sensitive Excitation Ratiometric (ExRai) C Kinase Activity Reporter (ExRai-CKAR2) for detecting minute changes in subcellular PKC activity.
• Utilized ExRai-CKAR2 alongside an enhanced diacylglycerol (DAG) biosensor to monitor intracellular DAG dynamics.
• Applied the reporter system to investigate differential PKC isoform regulation at distinct subcellular compartments and in 3D organoids.

Key Points:

• G-protein coupled receptor (GPCR) stimulation induces sustained PKC activity at the ER (conventional PKC) and lysosomes (novel PKC).
• Novel PKC (nPKC) exhibits high basal activity on lysosome membranes due to elevated basal DAG levels.
• ExRai-CKAR2 enables detection of previously inaccessible atypical PKC (aPKC) activity in the cytosol and Par-complexes within 3D organoids.

Conclusions:

• ExRai-CKAR2 is a highly sensitive tool for precisely measuring subcellular PKC activity in response to physiological stimuli.
• The study reveals differential subcellular regulation of PKC isoforms, highlighting the distinct roles of cPKC, nPKC, and aPKC.
• This reporter system provides a powerful method for interrogating kinase dynamics in complex cellular environments and disease models.