Uncovering Aberrant Mutant PKA Function with Flow Cytometric FRET
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 cont...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2016-03-01
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| Series: | Cell Reports |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2211124716302121 |
| _version_ | 1819084094184620032 |
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| author | Shin-Rong Lee Lingjie Sang David T. Yue |
| author_facet | Shin-Rong Lee Lingjie Sang David T. Yue |
| author_sort | Shin-Rong Lee |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-21T20:42:59Z |
| format | Article |
| id | doaj.art-0d9403b9f8d54c9c9e42da8e3a31e835 |
| institution | Directory Open Access Journal |
| issn | 2211-1247 |
| language | English |
| last_indexed | 2024-12-21T20:42:59Z |
| publishDate | 2016-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Cell Reports |
| spelling | doaj.art-0d9403b9f8d54c9c9e42da8e3a31e8352022-12-21T18:50:55ZengElsevierCell Reports2211-12472016-03-0114123019302910.1016/j.celrep.2016.02.077Uncovering Aberrant Mutant PKA Function with Flow Cytometric FRETShin-Rong Lee0Lingjie Sang1David T. Yue2Calcium Signals Laboratory, Department of Biomedical Engineering, The Johns Hopkins University School of Medicine, Ross Building, Room 713, 720 Rutland Avenue, Baltimore, MD 21205, USACalcium Signals Laboratory, Department of Biomedical Engineering, The Johns Hopkins University School of Medicine, Ross Building, Room 713, 720 Rutland Avenue, Baltimore, MD 21205, USACalcium Signals Laboratory, Department of Biomedical Engineering, The Johns Hopkins University School of Medicine, Ross Building, Room 713, 720 Rutland Avenue, Baltimore, MD 21205, USABiology 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.http://www.sciencedirect.com/science/article/pii/S2211124716302121 |
| spellingShingle | Shin-Rong Lee Lingjie Sang David T. Yue Uncovering Aberrant Mutant PKA Function with Flow Cytometric FRET Cell Reports |
| title | Uncovering Aberrant Mutant PKA Function with Flow Cytometric FRET |
| title_full | Uncovering Aberrant Mutant PKA Function with Flow Cytometric FRET |
| title_fullStr | Uncovering Aberrant Mutant PKA Function with Flow Cytometric FRET |
| title_full_unstemmed | Uncovering Aberrant Mutant PKA Function with Flow Cytometric FRET |
| title_short | Uncovering Aberrant Mutant PKA Function with Flow Cytometric FRET |
| title_sort | uncovering aberrant mutant pka function with flow cytometric fret |
| url | http://www.sciencedirect.com/science/article/pii/S2211124716302121 |
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