Article

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

Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Kyoto 606-8501, Japan.
Molecular biology of the cell (Impact Factor: 5.98). 12/2011; 22(23):4647-56. DOI: 10.1091/mbc.E11-01-0072
Source: PubMed

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.

1 Follower
 · 
177 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Diverse environmental conditions stimulate protein " shedding " from the cell surface through proteolytic cleavage. The protease TACE [tumor necrosis factor–a (TNFa)–converting enzyme, encoded by ADAM17] mediates protein shedding, thereby regulating the maturation and release of various extracellular sub-strates, such as growth factors and cytokines, that induce diverse cellular responses. We developed a FRET (fluorescence resonance energy transfer)–based biosensor called TSen that quantitatively reports the kinetics of TACE activity in live cells. In combination with chemical biology approaches, we used TSen to probe the dependence of TACE activation on the induction of the kinases p38 and ERK (extracellular signal–regulated kinase) in various epithelial cell lines. Using TSen, we found that disruption of the actin cytoskeleton in keratinocytes induced rapid and robust TSen cleavage and the accumulation of TACE at the plasma membrane. Cytoskeletal disruption also increased the cleavage of endogenous TACE sub-strates, including transforming growth factor–a. Thus, TSen is a useful tool for unraveling the mechanisms underlying the spatiotemporal activation of TACE in live cells.
    Science Signaling 02/2015; · 7.65 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Kidney collecting duct cells are continuously exposed to the changes of extracellular pH (pHe). We aimed to study the effects of altered pHe on dDAVP-induced phosphorylation (S256, S261, S264, and S269) and apical targeting of AQP2 in rat kidney inner medullary collecting duct (IMCD) cells. When freshly prepared IMCD tubule suspensions exposed to HEPES buffer with pH 5.4, 6.4, 7.4, or 8.4 for 1 h were stimulated with dDAVP (10(-10) M, 3 min), AQP2 phosphorylation at S256, S264, and S269 was significantly attenuated in acidic conditions. Next, IMCD cells primary cultured in the transwell chambers were exposed to transepithelial pH gradient for 1 h (apical pH 6.4, 7.4 or 8.4 vs. basolateral pH 7.4 and vice versa). Immunocytochemistry and cell surface biotinylation assay revealed that exposure to either apical pH 6.4 or basolateral pH 6.4 for 1 h was associated with decreased dDAVP (10(-9) M, 15 min, basolateral)-induced apical targeting of AQP2 and surface expression of AQP2. Fluorescence resonance energy transfer analysis revealed that dDAVP (10(-9) M)-induced increase of PKA activity was significantly attenuated when LLC-PK1 cells were exposed to pHe 6.4, compared to pHe 7.4 and 8.4. In contrast, forskolin (10(-7) M)-induced PKA activation and dDAVP (10(-9) M)-induced increase of intracellular Ca(2+) were not affected. Taken together, dDAVP-induced phosphorylation and apical targeting of AQP2 are attenuated in IMCD cells under acidic pHe, likely via an inhibition of V2R-G-protein-cAMP-PKA action. Copyright © 2014, American Journal of Physiology - Renal Physiology.
    American journal of physiology. Renal physiology 01/2015; DOI:10.1152/ajprenal.00376.2014 · 3.30 Impact Factor
  • Source
    Biosensors Journal 01/2015; 4(1). DOI:10.4172/2090-4967.1000113

Full-text (2 Sources)

Download
25 Downloads
Available from
May 16, 2014