Uncovering Aberrant Mutant PKA Function with Flow Cytometric FRET

Shin Rong Lee, Lingjie Sang, David T. Yue

Research output: Contribution to journalArticlepeer-review

Abstract

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
Volume14
Issue number12
DOIs
StatePublished - Mar 29 2016

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Fingerprint Dive into the research topics of 'Uncovering Aberrant Mutant PKA Function with Flow Cytometric FRET'. Together they form a unique fingerprint.

Cite this