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. 2016 Mar 29;14(12):3019-29.
doi: 10.1016/j.celrep.2016.02.077. Epub 2016 Mar 17.

Uncovering Aberrant Mutant PKA Function with Flow Cytometric FRET

Shin-Rong Lee  1 , Lingjie Sang  2 , David T Yue  3
Affiliations

Uncovering Aberrant Mutant PKA Function with Flow Cytometric FRET

Shin-Rong Lee et al. Cell Rep. .

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.

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Conflict of interest statement

We have no conflicts of interests to report.

Figures

Figure 1
Figure 1. Flow cytometric FRET
A. Cartoon depicting binding partners A and B tagged with fluorescent proteins Ven (monomeric Venus) and Cer (monomeric Cerulean). B. FRET binding curves are obtained by plotting 〈E〉 as a function of either Afree or Bfree. C.—E. HEK293(T) cells are cultured and transfected in 6 well plates, harvested into cytometer-compatible round bottom tubes, and loaded into the flow cytometer. F. Fluorescence signals are analyzed offline using custom software to yield the concentration of Cer, Ven, and 〈E〉, from which binding curves can be constructed.
Figure 2
Figure 2. Calibrating the flow cytometer for FRET
A. The ratios gCer/gVen and fVen/fCer can be discerned from Cer-Ven dimers of varying linker lengths using the following linear equation. B. Each cell expressing a particular Cer-Ven dimer is plotted as a single colored dot on the graph. Contour lines are overlaid on top of each Cer-Ven dimer to show the areas of highest density. C. The mean ± SD of NVen/NCer for each dimer is plotted. D. Plots of 〈E〉Ven and 〈E〉Cer for the five Cer-Ven dimers, with each point representing data from a single cell.
Figure 3
Figure 3. Flow cytometric FRET binding curves
A. Diagram of the binding reaction between Ven-SH3 (Mona/Gads) and Cer-X, where X represents one of several binding partners of Mona/Gads. B. FRET binding curves for Ven-SH3 in complex with SLP-76, aa.231-243 (black), Gab1, aa.515-527 (green), USP8, aa.403-415 (blue), SLP-76 (D236K), aa.231-243 (cyan), and SLP-76 (K240R), aa.231-243 (red). Their respective binding affinities were Kd = 0.8, 1, 4, 7 and 24 μM, with Emax = 0.39, 0.4, 0.37, 0.35 and 0.35 respectively. C. Comparison between dissociation constants measured by flow cytometry (y-axis) and those measured by ITC (x-axis). On average, affinities measured by flow cytometry are ~3-fold less.
Figure 4
Figure 4. Differential effects on binding by PKAcat L206R
A. Fluorescent-protein tagged PKAcat and PKAreg fall on a FRET binding curve, with Emax = 0.46 and Kd ≈ 1.7 μM. B. Binding curves between PKAcat (L206R) and PKAreg show complete lack of binding. C. and D. Similarly, these scatter plots show binding curves between PKI and either PKAcat or PKAcat L206R, both of which bind with Emax = 0.16, and Kd = 3.5 and 10 μM respectively.
Figure 5
Figure 5. Enzyme kinetics of PKA L206R revealed through FRET-based PKA sensor
A. Diagram illustrating the different states of the PKA activity sensor AKAR4. In its unphosphorylated state, it has low FRET (Emin). Phosphorylation through a Michaelis-Menten scheme (k-1, k1, k2) results in a state of high FRET (Emax). Dephosphorylation (k3) occurs through endogenous phosphatases. B. Scatter plot of single cell FRET efficiency as a function of AKAR4 expression, either with PKAcat overexpressed (red) or with H-89 (100 μM) in the solution (black). C. H-89 (100 μM) was added to cells expressing AKAR4 and PKAcat at time zero. While measurements of Cer and Ven concentrations were unaffected by addition of H-89 (top), 〈E〉 declined exponentially with τ = 33 sec (red curve). D. Scatter plot of single cell FRET efficiency vs concentration of mCherry-tagged proteins. PKI-expressing cells (black) have 〈E〉 near Emin. Expression of mCherry-PKAcat (red) resulted in 〈E〉 increasing to near Emax, while expression of mCherry-PKAcat(L206R) resulted in a gentler ascent of 〈E〉 to a lower plateau. E. Scheme outlining the addition of an inhibitor. KI represents the dissociation constant of the inhibitor, whereas Ke represents the “aggregate dissociation constant” for the AKAR4 system. F. Solution to the model diagrammed in E. where P (percent of AKAR4 phosphorylated) is plotted versus Ctot/Itot (PKAcat to inhibitor ratio) for different values of KI/Ke. G. and H. Single cell FRET efficiencies of cells transfected with PKAreg together with either PKAcat or PKAcat(L206R). Mean results of either PKAcat (L206R in H.) or PKAreg alone are represented by thick black lines, with their kernel density estimates overlaid in red.
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References

    1. Banning C, Votteler J, Hoffmann D, Koppensteiner H, Warmer M, Reimer R, Kirchhoff F, Schubert U, Hauber J, Schindler M. A flow cytometry-based FRET assay to identify and analyse protein-protein interactions in living cells. PloS one. 2010;5:e9344. - PMC - PubMed
    1. Berney C, Danuser G. FRET or no FRET: a quantitative comparison. Biophysical journal. 2003;84:3992–4010. - PMC - PubMed
    1. Beuschlein F, Fassnacht M, Assie G, Calebiro D, Stratakis CA, Osswald A, Ronchi CL, Wieland T, Sbiera S, Faucz FR, et al. Constitutive activation of PKA catalytic subunit in adrenal Cushing’s syndrome. The New England journal of medicine. 2014;370:1019–1028. - PMC - PubMed
    1. Bruckner A, Polge C, Lentze N, Auerbach D, Schlattner U. Yeast two-hybrid, a powerful tool for systems biology. International journal of molecular sciences. 2009;10:2763–2788. - PMC - PubMed
    1. Calebiro D, Hannawacker A, Lyga S, Bathon K, Zabel U, Ronchi C, Beuschlein F, Reincke M, Lorenz K, Allolio B, et al. PKA catalytic subunit mutations in adrenocortical Cushing’s adenoma impair association with the regulatory subunit. Nature communications. 2014;5:5680. - PubMed

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