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Sensitive Fluorescent Biosensor Reveals Differential Subcellular Regulation of PKC.

Qi Su¹, Jing Zhang^1,2, Wei Lin¹

¹Department of Pharmacology, School of Medicine, University of California San Diego, La Jolla, CA, USA.

Biorxiv : the Preprint Server for Biology

|April 8, 2024

Summary

Researchers developed a new tool, ExRai-CKAR2, to track protein kinase C (PKC) activity within cells. This reporter reveals how different PKC types are regulated at specific locations like the ER and lysosomes, offering new insights into cell signaling.

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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.