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Sensitive fluorescent biosensor reveals differential subcellular regulation of PKC

Nature Chemical Biology volume 21, pages 501–511 (2025)Cite this article

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Abstract

The protein kinase C (PKC) family of serine and threonine kinases, consisting of three distinctly regulated subfamilies, has been established as critical for various cellular functions. However, how PKC enzymes are regulated at different subcellular locations, particularly at emerging signaling hubs, is unclear. Here we present a sensitive excitation ratiometric C kinase activity reporter (ExRai-CKAR2) that enables the detection of minute changes (equivalent to 0.2% of maximum stimulation) in subcellular PKC activity. Using ExRai-CKAR2 with an enhanced diacylglycerol (DAG) biosensor, we uncover that G-protein-coupled receptor stimulation triggers sustained PKC activity at the endoplasmic reticulum and lysosomes, differentially mediated by Ca2+-sensitive conventional PKC and DAG-sensitive novel PKC, respectively. The high sensitivity of ExRai-CKAR2, targeted to either the cytosol or partitioning defective complexes, further enabled us to detect previously inaccessible endogenous atypical PKC activity in three-dimensional organoids. Taken together, ExRai-CKAR2 is a powerful tool for interrogating PKC regulation in response to physiological stimuli.

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Fig. 1: Development and characterization of ExRai-CKAR2.
Fig. 2: Subcellular targeting of ExRai-CKAR2.
Fig. 3: PKC regulation at the ER and lysosome.
Fig. 4: Lysosomal DAG is critical for lysosomal PKC activity.
Fig. 5: ExRai-CKAR2 reports endogenous aPKC activity in organoids.
Fig. 6: Targeted biosensor reports aPKC activity in lumenogenesis.

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Data availability

The data that support the findings of this study are available within the main text and Supplementary Information. Data are also available from the corresponding author upon request. Source data are provided with this paper.

Code availability

Custom ImageJ macros and MATLAB code used to analyze imaging data are available on GitHub (https://github.com/jinzhanglab-ucsd/MatScopeSuite)32.

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Acknowledgements

We thank all members of the Zhang lab, especially Y. Zhong, for technical help and discussion of the paper and J. Hardy for help with artwork. We thank E. Griffis and P. Guo of the UCSD Nikon Imaging Center for assistance with confocal imaging, T. Hoang for his technical help with cloning and G. Lorden Losada from the A.C.N. lab for providing purified PKCα and technical support with in vitro characterization of ExRai-CKAR2. This work is supported by National Institutes of Health grants R35 CA197622, R01 CA262815 and RF1 MH126707 (to J.Z.), R35 GM122523 (to A.C.N.) and R01 CA236386, R01 CA174869 and R01 CA262794 (to J.Y.), as well as by a TRDRP Postdoctoral Fellowship (T32FT5342) to Q.S.

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  1. Jin-Fan Zhang

    Present address: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA

  2. These authors contributed equally: Jing Zhang, Wei Lin.

Authors and Affiliations

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

    Qi Su, Jing Zhang, Wei Lin, Alexandra C. Newton, Sohum Mehta, Jing Yang & Jin Zhang

  2. Moores Cancer Center, University of California San Diego, La Jolla, CA, USA

    Jing Zhang, Alexandra C. Newton, Jing Yang & Jin Zhang

  3. Shu Chien-Gene Lay Department of Bioengineering, University of California San Diego, La Jolla, CA, USA

    Jin-Fan Zhang & Jin Zhang

  4. Department of Pediatrics, University of California San Diego, La Jolla, CA, USA

    Jing Yang

  5. Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA, USA

    Jin Zhang

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Contributions

Q.S., S.M., J.Y. and Jin Zhang conceptualized the project. J.-F.Z. and S.M. performed the linker screening and generated ExRai-CKAR2. Q.S. performed the in vitro characterization of ExRai-CKAR2, generated the other constructs and performed all live-cell and organoid imaging. Jing Zhang and Q.S. generated ExRai-CKAR2 and Cyto-ExRai-CKAR2 stable MDCK cell lines. Jing Zhang generated MDCK organoids. Q.S. generated HEK293T organoids. A.C.N., J.Y. and Jin Zhang supervised the project and coordinated experiments. Q.S. and Jing Zhang analyzed the data. Q.S., Jing Zhang, W.L., S.M. and Jin Zhang wrote the paper. All authors read and agreed on the final paper.

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Correspondence to Jin Zhang.

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Su, Q., Zhang, J., Lin, W. et al. Sensitive fluorescent biosensor reveals differential subcellular regulation of PKC. Nat Chem Biol 21, 501–511 (2025). https://doi.org/10.1038/s41589-024-01758-3

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