Journal article
Ultrasensitive bioluminescent reporters of Protein Kinase A based on Luciferase Activity Modulated by Phosphorylation (PKA LAMP)
The Journal of biological chemistry, Vol.301(8), 110445
08/2025
DOI: 10.1016/j.jbc.2025.110445
PMCID: PMC12329089
PMID: 40609788
Abstract
We developed the novel biosensor platform Luciferase Activity Modulated by Phosphorylation (LAMP) to monitor, with unprecedented sensitivity and dynamic range, reversible protein phosphorylation in cells. Based on NanoLuc luciferase complementation (NanoBiT), LAMP sensors are small (22 kD) and provide stable and bright light output that decreases upon phosphorylation and increases upon dephosphorylation. In this report, we designed LAMP biosensors to report spatial and temporal dynamics of cAMP-dependent protein kinase A (PKA) signaling. By incorporating both PKA phosphorylation and protein phosphatase 2A (PP2A) dephosphorylation motifs into the small component of split NanoLuc (generating PKABiT/pBiT), we achieved a 7-fold dynamic range. LAMP sensors are modular and flexible, allowing the two components, LgBiT and pBiT, to be expressed as part of the same polypeptide (cis) or as part of separate, but interacting polypeptides (trans). With trans LAMP, we show that RIα and RIβ form heterodimers with activities indistinguishable from homodimers of the two PKA regulatory subunit isoforms. Cis PKA LAMP sensors revealed different activation and inactivation kinetics of endogenous, membrane anchored PKA/RI and PKA/RII holoenzymes. They also allowed us to measure kinetics of cAMP diffusion and PKA catalytic subunit translocation to the nucleus. Lastly, we used a regeneratively phosphorylated PKA LAMP sensor to identify an autoinhibitory sequence in the PP2A regulatory subunit B56δ. By tailoring the sequence of pBiT, the LAMP platform can be extended to track the activity of other protein kinases and phosphatases and second messengers they respond to, thus providing new tools for cell signaling research and drug discovery.
Details
- Title: Subtitle
- Ultrasensitive bioluminescent reporters of Protein Kinase A based on Luciferase Activity Modulated by Phosphorylation (PKA LAMP)
- Creators
- Yufang Kong - University of IowaStefan Strack - University of Iowa
- Resource Type
- Journal article
- Publication Details
- The Journal of biological chemistry, Vol.301(8), 110445
- DOI
- 10.1016/j.jbc.2025.110445
- PMID
- 40609788
- PMCID
- PMC12329089
- NLM abbreviation
- J Biol Chem
- ISSN
- 0021-9258
- eISSN
- 1083-351X
- Publisher
- ELSEVIER
- Grant note
- NIH: R21 AG080472-01 Ataxia Charlevoix-Saguenay FoundationRegents of the University of California, A22: 2853-S004 DoD/CDMRP: AR230307 Eagles Autism FoundationSimons Foundation Autism Research Initiative (SFARI)Department of Defense/Congressionally Directed Medical Research Programs (DoD/CDMRP)
This work was supported by NIH R21 AG080472-01, the Ataxia Charlevoix-Saguenay Foundation, Regents of the University of California, A22 to 2853-S004 (Jordan's Guardian Angels), DoD/CDMRP (AR230307), the Eagles Autism Foundation, and the Simons Foundation Autism Research Initiative (SFARI) to S. S. The Department of Defense/Congressionally Directed Medical Research Programs (DoD/CDMRP) is an additional funding source. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
- Language
- English
- Electronic publication date
- 07/01/2025
- Date published
- 08/2025
- Academic Unit
- Pathology; Iowa Neuroscience Institute; Fraternal Order of Eagles Diabetes Research Center; Neuroscience and Pharmacology
- Record Identifier
- 9984843596302771
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