Development of an optimized backbone of FRET biosensors for kinases and GTPases. Mol Biol Cell

Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Kyoto 606-8501, Japan.
Molecular biology of the cell (Impact Factor: 4.47). 12/2011; 22(23):4647-56. DOI: 10.1091/mbc.E11-01-0072
Source: PubMed


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.

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Available from: Kazuhiro Aoki, Mar 14, 2014
    • "peptide, (GGSGG)3, between YPet-GL and the Rab7-binding domain of Rabring7, the EV linker of 116 a.a. (Komatsu et al., 2011) between the RBD domain of Rabring7 and SECFP, and the 15 a.a. peptide, (GGSGG)3, between SECFP and Rab7. "
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    ABSTRACT: Rab GTPases act as molecular switches regulating various aspects of membrane trafficking. Among them, Rab5 and Rab7 play central roles in the endolysosomal network. Although many effectors downstream of Rab7 have been elucidated, our present understanding of the mechanism regulating Rab7 activity is extremely limited. It has only recently been accepted that Mon1-Ccz1 is a Rab7 guanine nucleotide exchange factor, but the question where Mon1-Ccz1 works with Rab7 is hardly answered. To address what kind of change/switch exists in the regulatory mechanism upstream of Rab7 during the transition from late endosome to lysosome, we examined Rab7 activity in steady-state cells and EGF-induced macropinocytosis using a newly developed FRET sensor. A combination of a Rab7 sensor and confocal FRET imaging techniques revealed that the activation of Rab7 on late endosomes depends on Mon1-Ccz1 and is implicated in late endosome-lysosome fusion. In contrast, Rab7 activity on lysosomes was independent of Mon1-Ccz1 and active Rab7 played a role in perinuclear clustering of lysosomes.
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    • "These substrate proteins include mediators of immediate changes in cell shape, movement and intermediary metabolism, and components of longer-term effects on gene expression, cell viability, division or differentiation (Hay, 2011; Manning and Cantley, 2007; Toker, 2012). A variety of FRET-based reporters have been developed to track Akt by live-cell imaging (Gao and Zhang, 2008; Komatsu et al., 2011; Kunkel et al., 2005; Miura et al., 2014; Yoshizaki et al., 2007). Collectively, they have yielded data demonstrating rapid induction of enzymatic function in response to signaling by different growth factors, but have provided little information about how Akt activity is encoded into signaling outputs or about the dynamics of responses within a cell population. "
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    ABSTRACT: The protein kinase Akt is a key intracellular mediator of many biological processes, yet knowledge of Akt signaling dynamics is limited. Here we have constructed a fluorescent reporter molecule in a lentiviral delivery system to assess Akt kinase activity at the single cell level. The reporter, a fusion between a modified FoxO1 transcription factor and clover, a green fluorescent protein, rapidly translocates from the nucleus to the cytoplasm in response to Akt stimulation. Because of its long half-life and the intensity of clover fluorescence, the sensor provides a robust readout that can be tracked for days under a range of biological conditions. Using this reporter, we find that stimulation of Akt activity by IGF-I is encoded into stable and reproducible analog responses at the population level, but that single cell signaling outcomes are variable. This reporter, which provides a simple and dynamic measure of Akt activity, should be compatible with many cell types and experimental platforms, and thus opens the door to new insights into how Akt regulates its biological responses. © 2015. Published by The Company of Biologists Ltd.
    Full-text · Article · Jun 2015 · Journal of Cell Science
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    • "The basic modular design described above has been used to create activity reporters for a number of protein kinases, including PKA, protein kinase C (PKC), ataxia telangiectasia mutated (ATM), Akt, Abl, Src, aurora kinase B, ERK, c-Jun N-terminal kinase (JNK), cyclin-dependent kinase 1 (CDK1), AMP-activated protein kinase (AMPK), and the epidermal growth factor receptor (EGFR) (Zhang and Allen, 2007; Newman et al., 2011; Tsou et al., 2011; Belal et al., 2014). Using these reporters, researchers have uncovered important details about both the kinetics and the spatial distribution of endogenous kinase action in a variety of cellular contexts (Zhang et al., 2005a; Zhang and Allen, 2007; Erickson et al., 2011; Gao et al., 2011; Komatsu et al., 2011; Mehta and Zhang, 2011; Newman et al., 2011; Tsou et al., 2011; Arencibia et al., 2013; Ritt et al., 2013; Belal et al., 2014). However, despite their unique ability to track kinase activity in real time and at single cell resolution, to date, kinase activity reporters are available for <3% of the 518 human kinases in the human kinome. "
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    ABSTRACT: To better understand how cells sense and respond to their environment, it is important to understand the organization and regulation of the phosphorylation networks that underlie most cellular signal transduction pathways. These networks, which are composed of protein kinases, protein phosphatases and their respective cellular targets, are highly dynamic. Importantly, to achieve signaling specificity, phosphorylation networks must be regulated at several levels, including at the level of protein expression, substrate recognition, and spatiotemporal modulation of enzymatic activity. Here, we briefly summarize some of the traditional methods used to study the phosphorylation status of cellular proteins before focusing our attention on several recent technological advances, such as protein microarrays, quantitative mass spectrometry, and genetically-targetable fluorescent biosensors, that are offering new insights into the organization and regulation of cellular phosphorylation networks. Together, these approaches promise to lead to a systems-level view of dynamic phosphorylation networks.
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