Development of an optimized backbone of FRET biosensors for kinases and GTPases

Abstract

Biosensors based on the principle of Förster (or fluorescence) resonance energy transfer (FRET) have shed new light on the spatiotemporal dynamics of signaling molecules. Among them, intramolecular FRET biosensors have been increasingly used due to their high sensitivity and user-friendliness. Time-consuming optimizations by trial and error, however, obstructed the development of intramolecular FRET biosensors. Here we report an optimized backbone for rapid development of highly sensitive intramolecular FRET biosensors. The key concept is to exclude the "orientation-dependent" FRET and to render the biosensors completely "distance-dependent" with a long, flexible linker. We optimized a pair of fluorescent proteins for distance-dependent biosensors, and then developed a long, flexible linker ranging from 116 to 244 amino acids in length, which reduced the basal FRET signal and thereby increased the gain of the FRET biosensors. Computational simulations provided insight into the mechanisms by which this optimized system was the rational strategy for intramolecular FRET biosensors. With this backbone system, we improved previously reported FRET biosensors of PKA, ERK, JNK, EGFR/Abl, Ras, and Rac1. Furthermore, this backbone enabled us to develop novel FRET biosensors for several kinases of RSK, S6K, Akt, and PKC and to perform quantitative evaluation of kinase inhibitors in living cells.

FIGURE 1:

Optimized backbone of an intramolecular FRET biosensor. (A) Mode of action of the intramolecular FRET biosensor. (B) Structure of the DNA encoding an optimized intramolecular FRET biosensor. Shown are the unique restriction enzyme sites used to exchange each domain for the development of the biosensor. (C) Schematic representation of the titration curve of FRET/CFP ratio in intramolecular FRET biosensors.

FIGURE 2:

Optimization of pairs of FPs for distance-dependent FRET biosensors. (A) Scheme of the PKA or ERK activity sensor consisting of YFPs (donor), a FHA1 or WW phosphopeptide binding domain (ligand domain), a 72-Gly linker, a PKA or ERK substrate (sensor domain), CFPs (acceptor), and nuclear export signal (NES). (B) HeLa cells expressing AKAR3 with various pairs of FPs as indicated were stimulated with 1 mM dbcAMP for 10 min. The gain in FRET/CFP is represented with the SD (n > 5). (C) HeLa cells expressing EKAR with various pairs of FPs as indicated were stimulated with 10 ng/ml EGF for 10 min. The gain in FRET/CFP is represented with the SD (n > 5).

FIGURE 3:

Effect of long linkers on the FRET gain. (A) Scheme of the PKA activity sensor consisting of YPet and ECFP. (B) HeLa cells expressing AKAR3 with a linker of various lengths were imaged by FRET microscopy to obtain the basal FRET/CFP. Each dot corresponds to the value from a single cell (n > 5). Horizontal bars are the mean values. (C) HeLa cells expressing a PKA sensor as indicated were stimulated with 1 mM dbcAMP for 10 min. The gain in FRET/CFP is represented with the SD (n > 5). (D) HeLa cells expressing AKAR3 with 52, 84, or 116 a.a. length of linker were stimulated with 1 mM dbcAMP or 1 mM dbcAMP and 50 nM Calyculin A for 10 min. Top, cell lysates were subjected to Phos-tag immunoblotting analysis with an anti-GFP antibody and a fluorescence-tagged secondary antibody. Tositions of phosphorylated (p) and nonphosphorylated biosensors (np) are indicated on the right of the representative gel image. Bottom, average values of the fraction of phosphorylated biosensors are shown with SD for three independent experiments. P value was calculated by a one-tailed paired t test.

FIGURE 4:

Improvement of FRET biosensors by the Eevee backbone. At the top of each panel, the structure of novel biosensors based on the Eevee backbone is shown. In the substrate peptide sequences, red letters indicate the phosphorylation site. Blue letters indicate amino-acid substitutions to increase the affinity to either the FHA1 or WW domain. Green letters indicate the docking site of the kinases. (A) HeLa cells expressing AKAR3EV, AKAR3, or AKAR4 were stimulated with 1 mM dbcAMP and time lapse–imaged by FRET microscopy (Supplemental Video S1). The FRET/CFP ratio of each cell was normalized by dividing by the averaged FRET/CFP value before stimulation. The mean and SD from at least 10 cells are plotted against time. (B) HeLa cells expressing EKAREV or EKAR were stimulated with 10 ng/ml EGF (Supplemental Video S2). The average of normalized FRET/CFP ratio is shown with SD (n > 10). (C) HeLa cells expressing JNKAR1EV or JNKAR1 were stimulated with 1 μg/ml Anisomycin (Supplemental Video S3). The average of normalized FRET/CFP ratio is shown with SD (n > 10). (D) HeLa cells expressing PicchuEV, an EGFR/Abl kinase sensor, or Picchu were stimulated with 25 ng/ml EGF (Supplemental Video S4). The average of normalized FRET/CFP ratio is shown with SD (n > 10).

FIGURE 5:

FRET biosensors of small GTPases based on the Eevee backbone. Structures of FRET biosensors based on the Eevee backbone, RaichuEV, are shown at the top of panels (A) and (D). RafRBD and PAK CRIB denote the Ras-binding domain of Raf1 and the Cdc42/Rac-interactive binding domain, respectively. Cos7 cells expressing RaichuEV or the prototype Raichu were stimulated with 50 ng/ml EGF and time lapse–imaged (Supplemental Videos S5 and S6). Representative FRET/CFP ratio images are shown in the intensity-modulated display mode. Scale bars are 10 μm. (B and E) The FRET/CFP ratio of each cell was normalized by dividing by the averaged FRET/CFP value before stimulation. The mean and SD from at least 10 cells are plotted against time. (C and F) Basal FRET/CFP ratios of RaichuEV and Raichu are plotted. Each dot corresponds to the value from a single cell, and at least six cells were analyzed. The horizontal bar indicates the mean.

FIGURE 6:

Novel Ser/Thr kinase FRET biosensors based on the Eevee backbone. (A, D, G, and J) Structures of Eevee-RSK, Eevee-S6K, Eevee-Akt, and Eevee-PKC, which are FRET biosensors of RSK, S6K, Akt, and classical PKC activities, respectively. Red and blue letters in the substrate peptide sequences denote the phosphorylation sites and amino-acid substitutions to increase the affinity to FHA1. (B, E, H, and K) HeLa cells expressing Eevee-RSK (B), Eevee-S6K (E), Eevee-PKC (K), or Cos7 cells expressing Eevee-Akt (H) were time lapse–imaged and stimulated with 10 ng/ml EGF (B and E), 50 ng/ml EGF (H), or 1 μM TPA (K). Cells expressing Eevee-RSK and Eevee-S6K were further treated with 10 nM BI-D1870 (B) and 100 nM rapamycin (E), respectively, at 30 min after EGF stimulation. The FRET/CFP ratio of each cell was normalized by dividing by the averaged FRET/CFP value before stimulation (Supplemental Videos S7–S10). The mean and SD from at least 10 cells are plotted against time. (C, F, I, and L) Representative FRET/CFP ratio images with Eevee-RSK (C), Eevee-S6K (F), Eevee-Akt (I), and Eevee-PKC (L) are shown in the intensity-modulated display mode. Scale bars are 10 μm.

FIGURE 7:

Quantitative evaluation of kinase inhibitors with Eevee-expressing cell lines. (A) Schematic view of the experimental design. Cells expressing EKAREV-nuc were seeded, starved, and treated with stimulant in the presence of decreasing concentrations of the indicated kinase inhibitors. After 30 min, FRET images were acquired and processed for quantification. (B) Shown here are the representative FRET/CFP ratio images of HeLa cells stably expressing EKAREV-nuc and treated with 25 ng/ml EGF and the indicated concentrations of an EGFR inhibitor, PD153035. Scale bar is 50 μm. (C) Averaged FRET/CFP ratios are plotted against the concentrations of kinase inhibitors and fitted with curves by the four-parameter logistic model. (D and E) FRET/CFP ratios in each cell at the indicated concentration of PD153035 (D) or a MEK inhibitor, PD184352 (E), were quantified and represented in histograms (n > 80 cells).