Uncovering Aberrant Mutant PKA Function with Flow Cytometric FRET

Shin Rong Lee, Lingjie Sang, David T. Yue

Research output: Contribution to journalArticlepeer-review

15 Scopus citations


Biology has been revolutionized by tools that allow the detection and characterization of protein-protein interactions (PPIs). Förster resonance energy transfer (FRET)-based methods have become particularly attractive as they allow quantitative studies of PPIs within the convenient and relevant context of living cells. We describe here an approach that allows the rapid construction of live-cell FRET-based binding curves using a commercially available flow cytometer. We illustrate a simple method for absolutely calibrating the cytometer, validating our binding assay against the gold standard isothermal calorimetry (ITC), and using flow cytometric FRET to uncover the structural and functional effects of the Cushing-syndrome-causing mutation (L206R) on PKA's catalytic subunit. We discover that this mutation not only differentially affects PKAcat's binding to its multiple partners but also impacts its rate of catalysis. These findings improve our mechanistic understanding of this disease-causing mutation, while illustrating the simplicity, general applicability, and power of flow cytometric FRET.

Original languageEnglish (US)
Pages (from-to)3019-3029
Number of pages11
JournalCell Reports
Issue number12
StatePublished - Mar 29 2016

ASJC Scopus subject areas

  • General Biochemistry, Genetics and Molecular Biology


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