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

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
52 Reads
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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.
    Journal of Cell Science 06/2015; 128(14). DOI:10.1242/jcs.168773 · 5.43 Impact Factor
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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.
    Frontiers in Genetics 08/2014; 5:263. DOI:10.3389/fgene.2014.00263
  • Source
    • "Coexpression of Bcr, but not a GAP-dead Bcr mutant (Bcr-GD), inhibited both Tiam1-induced Rac1 activation (Figure S1D) and autophosphorylation of Pak (Figures 1F and 1G), a Rac1 effector that regulates spine morphogenesis (Nikoli c, 2008). To measure the effect of Bcr on Tiam1-mediated Rac1 signaling in neurons, we utilized the Fö rster resonance energy transfer (FRET) Rac1 activation biosensor RaichuEV-Rac1 (Komatsu et al., 2011). Bcr overexpression decreased Rac1 activation in dendrites and spines both basally and when coexpressed with Tiam1 Figure 1. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The small GTPase Rac1 orchestrates actin-dependent remodeling essential for numerous cellular processes including synapse development. While precise spatiotemporal regulation of Rac1 is necessary for its function, little is known about the mechanisms that enable Rac1 activators (GEFs) and inhibitors (GAPs) to act in concert to regulate Rac1 signaling. Here, we identify a regulatory complex composed of a Rac-GEF (Tiam1) and a Rac-GAP (Bcr) that cooperate to control excitatory synapse development. Disruption of Bcr function within this complex increases Rac1 activity and dendritic spine remodeling, resulting in excessive synaptic growth that is rescued by Tiam1 inhibition. Notably, EphB receptors utilize the Tiam1-Bcr complex to control synaptogenesis. Following EphB activation, Tiam1 induces Rac1-dependent spine formation, whereas Bcr prevents Rac1-mediated receptor internalization, promoting spine growth over retraction. The finding that a Rac-specific GEF/GAP complex is required to maintain optimal levels of Rac1 signaling provides an important insight into the regulation of small GTPases.
    Developmental Cell 06/2014; 29(6):701-715. DOI:10.1016/j.devcel.2014.05.011 · 9.71 Impact Factor
Show more