Live-cell Imaging with Genetically Encoded Protein Kinase Activity Reporters

Abstract

Protein kinases play pivotal roles in intracellular signal transduction, and dysregulation of kinases leads to pathological results such as malignant tumors. Kinase activity has hitherto been measured by biochemical methods such as in vitro phosphorylation assay and western blotting. However, these methods are less useful to explore spatial and temporal changes in kinase activity and its cell-to-cell variation. Recent advances in fluorescent proteins and live-cell imaging techniques enable us to visualize kinase activity in living cells with high spatial and temporal resolutions. Several genetically encoded kinase activity reporters, which are based on the modes of action of kinase activation and phosphorylation, are currently available. These reporters are classified into single-fluorophore kinase activity reporters and Förster (or fluorescence) resonance energy transfer (FRET)-based kinase activity reporters. Here, we introduce the principles of genetically encoded kinase activity reporters, and discuss the advantages and disadvantages of these reporters.

Key words: kinase, FRET, phosphorylation, KTR

Introduction

Protein phosphorylation is a post-translational modification in which a specific enzyme, namely a protein kinase (hereinafter referred to simply as kinase), catalyzes the transfer of γ-phosphate of ATP to specific phosphoacceptor amino acids of target substrate proteins (Ubersax and Ferrell, 2007). More than 500 genes encoding protein kinases exist in the human genome, including genes for Ser/Thr kinases and Tyr kinases, and approximately 30% of proteins are estimated to be phosphorylated on at least one residue (Cohen, 2000; Ptacek et al., 2005). Protein phosphorylation is involved in a wide range of cellular processes, such as proliferation, differentiation, survival, and cytoskeletal reorganization (Ubersax and Ferrell, 2007), and aberrant activation of kinases leads to pathological consequences, notably in malignant tumors (Frémin and Meloche, 2010). Indeed, small-molecule inhibitors targeting oncogenic kinases have become an important therapeutic paradigm for the treatment of cancer patients (Gross et al., 2015; Zhang et al., 2009).

Kinase and phosphorylation signaling have mostly been studied by means of population-level experiments such as western blotting. However, it is unclear whether individual cells behave in conformity with the averaged data. Live-cell imaging with fluorescent proteins (FPs) is a key technique to address this issue. Green fluorescent proteins (GFP) were discovered in the jellyfish Aequorea victoria in 1962 by Dr. Shimomura (Shimomura et al., 1962). Over the past 50 years, the evolution of live-cell imaging with FPs has opened a door to the probing of a variety of biological processes. Currently, a broad range of fluorescent proteins beyond GFP are available from other marine organisms (Chudakov et al., 2010). One advantage of FPs is that they are encoded by responsible genes, and thus researchers in the field of life science can easily introduce FPs and biosensors fused with FPs into cells and animals of interest by conventional gene delivery methods. Further, with the advent of newly developed fluorescence microscopes, it has become possible to apply FPs not only to label proteins, but also to more precisely visualize the subcellular localization of proteins beyond the optical diffraction limit (Huang et al., 2009), to observe deeper tissues in living animals (Kamioka et al., 2012), and to measure kinetic parameters (Aoki et al., 2011; Sadaie et al., 2014). Moreover, through the efforts of many groups, various genetically encoded fluorescent reporters based on the principle of Förster (or fluorescence) resonance energy transfer (FRET) have been developed, such as intracellular ions (Chen et al., 2013; Miyawaki et al., 1997), small GTPases (Aoki and Matsuda, 2009; Kiyokawa et al., 2011; Mochizuki et al., 2001), phospholipids (Nishioka et al., 2008; Sato et al., 2003), enzymatic reactions (Komatsu et al., 2011; Ni et al., 2006) and so on. Among them, kinase activation is one of the most intensively studied targets for FRET reporters.

Here, we focus on the applications of FPs to the monitoring of kinase activity in living cells; this review is primarily intended as a guide to the development and use of genetically encoded kinase activity reporters. Many excellent reviews provide details on the aspects of FP and FRET-based biosensors, and we encourage the interested reader to refer to them (Miyawaki, 2003; Muscheler et al., 2007; Oldach and Zhang, 2014; Zhang et al., 2002).

The mode of action of kinase activation and substrate phosphorylation

In order to develop a genetically encoded fluorescent reporter for kinase activity, we first must understand where and how the kinase is regulated and activated within a cell, and how the substrate behaves before and after phosphorylation. In many cases, the enzymatic activity of kinases is turned off when the kinases are at a steady state level, and turned on when they are externally and/or internally stimulated by growth factors, cytokines, or cell cycle progression (Fig. 1A). The kinase activation is often accompanied by a combination of several kinds of reactions. The simplest of these is the gene expression of the kinase itself, which serves as an activation mechanism per se (Fig. 1B). Translocation to different subcellular compartments such as the plasma membrane also triggers kinase activation (Fig. 1C). Under basal condition, kinases are often self-inactivated by steric constraints. For instance, a closed-inactive form of a kinase is incapable of binding to substrates, and thus the release of the auto-inhibition can be an essential process of kinase activation (Fig. 1D). Post-translational modification including phosphorylation is also a common mechanism as an activation switch for kinases (Fig. 1E). Finally, not only proteins but also secondary messengers such as calcium and cAMP act as regulatory factors that activate or inactivate kinases (Fig. 1F). Importantly, these events are not mutually exclusive, but rather complement each other to accomplish full activation of a kinase. Thereafter, the activated kinases then catalyze their substrates, generating phosphorylated proteins as a product. Some of the phosphorylated proteins are also known to be translocated to different subcellular spaces or degraded (Fig. 1G).

Fig. 1

Mechanisms of kinase activation. (A) Inactive kinase (left) is activated (right) by stimulation, leading to the phosphorylation of substrates. (B) Gene expression increases the amount of constitutively active kinase protein. (C) Subcellular translocation from the cytoplasm to plasma membrane induces kinase activation. (D) Closed-inactive kinase is activated upon stimulation by releasing intramolecular auto-inhibition and exposing the substrate-binding site. (E) Post-translational modification such as phosphorylation increases kinase activity. (F) Dissociation or association of the regulatory protein through ligand binding regulates kinase activation. (G) The phosphorylated substrate undergoes subcellular translocation such as nuclear import or degradation.

Most of the kinase reporters exploit, to a greater or lesser degree, the aforementioned changes in the kinase itself or its substrates in the process of kinase activation and inactivation. The kinase activity reporters can be classified into two categories: the single-fluorophore kinase activity reporters and the FRET-based kinase activity reporters (Miyawaki and Niino, 2015). We will introduce examples and discuss the pros and cons of these two types of reporters.

Single-fluorophore kinase activity reporters

A single-fluorophore kinase activity reporter is defined as a biosensor that harbors only a single FP. An advantage of the single-fluorophore kinase activity reporters is that they are often easier to develop than the FRET-based kinase activity reporters. In addition, single-fluorophore kinase activity reporters enable multiplex imaging with differently colored FPs, while FRET-based reporters are often associated with the difficulty in multiplexed imaging. There are also some drawbacks to the use of single-fluorophore kinase activity reporters. First, these reporters are more susceptible to the focus drift effect and object motion than ratiometric FRET-based imaging. Second, it is difficult to monitor subcellular kinase activation by most of the single-fluorophore kinase activity reporters.

Table I lists the most commonly used single-fluorophore kinase activity reporters.

Table I Single fluorophore-based kinase activity reporters

Target kinase	Name of probe	Reference
Localization-based reporter (substrate, if the substrate is fused with FP)
Akt	GFP-AktPH	(Haugh et al., 2000)
Akt (FoxO1, 3)	FoxO1-Clover	(Gross and Rotwein, 2016)
BTK	BTK-PH-GFP	(Varnai, 2005)
CDK2	DHB	(Spencer et al., 2013)
DGK	GFP-DGK	(Shirai et al., 2000)
ERK	GFP-ERK2, GFP-ERK5	(Carter et al., 2009; Rubinfeld et al., 1999)
p38	EGFP-p38	(Gong et al., 2010)
p38 (MAPKAPK2/MK2)	EGFP-MK2	(Gong et al., 2010)
JAK (STAT)	STAT-GFP	(Samsonov et al., 2013)
JNK	GFP-JNK3	(McDonald et al., 2000)
JNK (PDK-1)	PDX-1-GFP	(Kawamori et al., 2003)
Lats (YAP)	GFP-YAP	(Bao et al., 2011)
MEKK2	MEKK2-GFP	(Schaefer et al., 1999)
PKA	PKAcat-GFP	(Almholt et al., 2004b)
PKC	γ-PKC-GFP	(Sakai et al., 1997)
PKC (KRas, MARCKS)	GFP-KRas, MARCKS-GFP	(Bivona et al., 2006; Ohmori et al., 2000)
PKD (HDAK5)	GFP-HDAK5	(Vega et al., 2004)
Raf	Raf-GFP	(Knight et al., 2006; Rocks et al., 2006)
TGFbR (Smad)	EGFP-Smad	(Schmierer and Hill, 2005)
Degradation-based reporter (substrate)
ERK (FosL)	FIRE	(Albeck et al., 2013)
IKK (IκB)	IκBα-EGFP	(Nelson et al., 2002)
Susceptible cpFP-based reporter (substrate)
Src (IRS-1)	cyan-, green-, yellow-sinphos	(Kawai et al., 2004)
Kinase translocation reporter (KTR) system
JNK	JNK-KTR	(Regot et al., 2014)
ERK	ERK-KTR	(Regot et al., 2014)
p38	p38-KTR	(Regot et al., 2014)
PKA	PKA-KTR	(Regot et al., 2014)
Akt	Akt-FoxO3A-KTR	(Maryu et al., 2016)

Localization- or translocation-based kinase activity reporters

In extreme cases, the translocation of proteins upon activation has been employed to probe kinase activity with a single-fluorophore. Upon activation, some kinases move to different subcellular compartments, because the redistribution of kinases is required for their activation and/or substrate phosphorylation. In either case, we can trace kinase activation by tagging the kinases with an FP and observing their subcellular distribution. For instance, Akt, also known as protein kinase B, and CRaf are localized at the cytosol in the basal state, and translocated to the plasma membrane by binding to phosphatidylinositol 3,4,5-trisphosphate (PIP₃) or to GTP-loaded active Ras, respectively (Haugh et al., 2000; Rocks et al., 2006) (Fig. 2A, left). Therefore, membrane recruitment of Akt and CRaf fused with FP functions as a marker of their activation, respectively. Of note, Akt and CRaf carrying FP can also be interpreted as a reporter of PIP₃ and Ras activity, respectively. In another example, the nuclear import of ERK MAP kinase has been widely used as a marker of ERK activation (Lidke et al., 2010; Raisner et al., 2005). MEK binds to and anchors ERK at the cytoplasm in the resting state. Upon stimulation, ERK is phosphorylated by MEK and is subsequently dissociated from MEK, followed by the nuclear translocation (Fig. 2A, right). In both cases, the expression level of reporters is of critical importance in order to accurately quantify kinase activation; the expression level must be comparable with the number of the binding partners, namely Ras, PIP₃, and MEK. Therefore, co-expression of binding proteins might help to increase the dynamic range of reporter translocation in response to stimulation (Fujioka et al., 2006; Terai and Matsuda, 2005). To prevent unnecessary signal transduction by the reporters themselves, kinase-dead mutants or truncated mutants containing the responsible domain for the translocation would work better than full-length kinases fused with FPs.

Fig. 2

Single-fluorophore kinase activity reporters. (A) Translocation-based kinase activity reporter. Kinase activation often takes place in a specific subcellular compartment such as the plasma membrane (upper left) and nucleus (upper right), and this allows us to illuminate kinase activation by the translocation of kinase fused with FP. CRaf proteins fused with FP are recruited to the plasma membrane upon EGF stimulation (lower left). Nuclear accumulation of ERK2 proteins fused with FP is evident after ERK activation (lower right). Scale bar, 15 μm. (B) Degradation-based kinase activity reporter. An activated kinase phosphorylates a substrate (yellow), leading to increased protein stability (upper). The abundance of FIRE, a degradation-based ERK activation reporter, is increased by ERK activation induced by EGF (lower). Scale bar, 15 μm. (C) Susceptible cpFP-based reporter. cpFP is fused with the phosphorylated peptide and its binding domain (orange). Once the peptide (green) is phosphorylated by activated kinase, the intramolecular binding between the peptide and binding domain undergoes a change in the chromophore of cpFP, increasing or decreasing the fluorescence. (D) KTR system. Shown here is the basic design of the KTR (left). The unphosphorylated form of KTR is retained in the nucleus by the function of bipartite NLS (bNLS). Upon phosphorylation, the bNLS and NES are inactivated and activated, respectively, and therefore the phosphorylated KTR is exported to the cytosol. EGF-induced Akt and ERK activation are manifested as nuclear export of Akt-FoxO3a-KTR (upper right) and ERK-KTR (lower right), respectively. Scale bar, 15 μm.

Many substrates phosphorylated by protein kinases are also subjected to changes in subcellular distribution. The transcription factors known as Forkhead box Os (FOXOs) are phosphorylated by Akt, and then translocated from nucleus to cytoplasm (Burgering, 2003). Intriguingly, Akt phosphorylates serine/threonine amino acid residues located at or near the nuclear localization signal (NLS), resulting in their nuclear export and cytoplasmic retention. Based on this idea, GFP-fused FOXOs are used as Akt activity reporters (Katsura et al., 2015; Maryu et al., 2016). In a similar way, p38 activity has been investigated by quantifying the subcellular distribution of MK2/MAPKAPK2, which is a substrate of p38 MAP kinase (Almholt et al., 2004a); the phosphorylation of MK2 by p38 induces the translocation from nucleus to cytoplasm.

Degradation-based kinase activity reporters

Protein stability and degradation can be altered by phosphorylation, providing us with other principles for kinase activity reporters (Fig. 2B). Brugge and her colleagues reported FIRE (Fra-1-based integrative reporter of ERK), which consists of YFP fused to the PEST domain of Fra-1 (Albeck et al., 2013). Because phosphorylation of Fra-1 by ERK (or downstream kinase RSK) suppresses degradation of the Fra-1 PEST domain (Casalino et al., 2003; Vial and Marshall, 2003), the fluorescence intensity of FIRE increases upon ERK activation (Fig. 2B). Fusion of the phosphorylation-regulated degradation domains to a fluorescent protein, such as in the FIRE reporter, paves the way to the generation of single-fluorophore kinase activity reporters. It is noteworthy that the dynamics of FIRE intensity exhibit a much slower time scale (~ 12 hours) than the fluctuations of ERK activity (~ 30 min) (Albeck et al., 2013; Aoki et al., 2013). This could be attributable to the mechanism of this reporter, which is based on protein degradation and synthesis.

Inflammatory signaling is also investigated by the degradation of inhibitors of κB (IκBs) and nuclear localization of transcription factor NF-κB (Nelson et al., 2002). Under an unstimulated condition, NF-κB is sequestered in the cytoplasm by binding to IκBs, which masks the NLS of NF-κB. Upon stimulation, IκB kinase (IKK) phosphorylates IκBs, leading to their ubiquitination and degradation. Consequently, the NF-κB is released from the IκB to enter the nucleus, and initiates its transcription. Therefore, the degradation of IκBs indeed reports IKK activity.

Susceptible circular permutated-GFP (cpGFP)-based kinase activity reporter

The fluorescence of FPs is usually robust and unaffected by the external change, because the β-barrel fold of FPs reduces the accessibility of outside solvent. Interestingly, however, the chromophore of circular permutated GFP at residues 144-145 (cp145-GFP) is more sensitive to neighboring events than normal GFPs (Baird et al., 1999; Miyawaki and Niino, 2015). Based on this principle, single fluorophore-based genetically encoded calcium indicators (GECIs) have been developed by many laboratories (Lin and Schnitzer, 2016; Nagai et al., 2001; Nakai et al., 2001; Pérez Koldenkova and Nagai, 2013). This approach is potentially applicable to the development of kinase activity reporters. Indeed, the pioneering work of Umezawa and co-workers has demonstrated proof-of-concept for the design of kinase activity reporters with cp145-FPs (Kawai et al., 2004). In their tyrosine kinase reporters, which are known as “sinphoses”, the cp145-FPs are sandwiched between the substrate domain and phosphorylation recognition domain together with a localization signal domain (Fig. 2C). It is anticipated that, upon tyrosine kinase activation, the phosphorylation of the substrate domain brings it into close proximity to the phosphorylation recognition domain, leading to the change in fluorescence of cp145-FPs. Interestingly, cp145-EGFP shows an increase in green fluorescence, whereas cp145-ECFP exhibits a decrease in cyan fluorescence (Kawai et al., 2004). Although the development and improvement of susceptible cpGFP-based reporters requires time-consuming and labor-intensive steps, this approach has great potential for overcoming the limitations of the dynamic range and signal-to-noise ratio of kinase activity reporters.

Kinase translocation reporter (KTR) system

Recently, Covert and his colleagues reported a rationally designed kinase reporter system, KTR (kinase translocation reporter) (Regot et al., 2014). The KTR system is comprised of a kinase docking site, bipartite NLS (bNLS), and NES, and phosphorylation sites are located within the bNLS and NES (Fig. 2D). The bNLS sequences are defined as KRX_10-12K(K/R)(K/R), and acidic residues (D and E) should be enriched in the central linker region. The authors optimized the bNLS sequence to be negatively regulated by phosphorylation. Introducing negatively charged amino acids or phosphorylation within the NES sequence increases its export activity. Therefore, the phosphorylation of KTR by kinases induces the translocation from nucleus to cytoplasm. The kinase specificity is achieved by adding a kinase docking site and optimizing the phosphorylation site. For MAP kinases, the addition of a specific docking site (e.g., FQFP for ERK) is sufficient to change the kinase specificity of KTR, and in this way, the authors could develop JNK-, ERK-, and p38-KTRs. For AGC kinases such as PKA, the specificity of KTR should be achieved by optimizing the phosphorylation sequence, though there is limited flexibility for the sequence space. Finally, the authors showed the great potential of the KTR system for multiplexed imaging in living cells. Based on this idea, we have developed an Akt activation reporter, Akt-FoxO3a-KTR, and demonstrated multiplexed imaging of Akt and ERK activity in proliferating cells (Maryu et al., 2016). The KTR system opens the possibility of developing a single-fluorophore activity reporter for kinases that do not show any characteristic changes in subcellular distribution described above.

FRET-based kinase activity reporters

FRET is a non-radiative process in which the excitation energy of a donor fluorophore is transferred to an acceptor fluorophore (Förster, 1946). In principle, FRET efficiency denoted as E is described as follows:

  

$$E=\frac{R_0{\text{}}^6}{R_0{\text{}}^6+r^6},$$(1)

  

$$R_0{\text{}}^6=c_0{\kappa }^{2}J{n}^{-4}{Q}_0,$$(2)

  

$$J=\int \overline{f_D}\left(\lambda \right){\epsilon }_A\left(\lambda \right){\lambda }^{4}d\lambda ,$$(3)

where r is the distance between donor and acceptor (over the range of 1–10 nm), R₀ is the Förster distance, κ² is the parameter of the relative-orientation of the donor absorption and acceptor transition moments (range 0–4), n is the refractive index, Q₀ is the quantum yield of the donor in the absence of the acceptor, and J is the spectral overlap integrated over the wavelength λ with the normalized donor emission spectrum f_D and the acceptor molar extinction coefficient ε_A; c₀ = 8.8 * 10^–28 for R₀ in nm (Jares-Erijman and Jovin, 2003; Padilla-Parra and Tramier, 2012; Ueda et al., 2013). The orientation factor, κ², can be approximated as 2/3, when the donor and acceptor fluorophores are randomly oriented. However, as mentioned later, this approximation is available only when highly mobile, small molecules are used for donor and acceptor fluorophores, and is not applied to the case where FPs are used. Based on these equations, FRET efficiency is inversely proportional to the sixth power of distance between donor and acceptor fluorophores, and therefore FRET is very sensitive to the distance. This is the reason why FRET is known as a spectroscopic ruler (Stryer, 1978).

In practical terms, for the development of a FRET biosensor, the distance and relative orientation between donor and acceptor FPs are the main factors that influence the FRET efficiency once a pair of donor and acceptor fluorophores is fixed. Cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) are preferably used for the sensitized FRET, because YFP allows the easy acceptor photo-bleaching, which provides a simple and practical measure of FRET (Kiyokawa et al., 2011; Miyawaki, 2003). Meanwhile, the pair of GFP and red fluorescent protein (RFP) is applied to fluorescence lifetime microscopy (FLIM)-FRET, because of the high photostability and single component of the fluorescence lifetime in GFP.

The sensitized-FRET and FLIM-FRET methods are two major approaches for visualizing FRET signals in living cells (Miyawaki, 2011; Padilla-Parra and Tramier, 2012; Yasuda, 2006). Under the FRET condition, the donor fluorescence and lifetime are decreased, while the acceptor emits fluorescence through energy transfer. The sensitized-FRET method utilizes these reciprocal changes in the donor and acceptor fluorescence for evaluating to what extent FRET takes place. Therefore, the spectral overlaps, such as bleed-through of the donor in the acceptor detection channel and cross-excitation of the acceptor excited by donor excitation light, should be carefully corrected (Aoki and Matsuda, 2009; Fujioka et al., 2006). Meanwhile, the FLIM-FRET method measures the fluorescence decay of a donor fluorophore on the order of nano-seconds, and calculates its lifetime. To accomplish this, the FLIM-FRET method requires special equipment, e.g., a pulsed-laser and a high-sensitive detector (Day and Davidson, 2012; Sun et al., 2013). The sensitized-FRET method is technically easier but less quantitative than the FLIM-FRET method (van Munster and Gadella, 2005; Yasuda, 2006). The FRET-based kinase activity reporters are listed in Table II.

Table II FRET-based kinase activity reporters

Target	Name of probe	Reference
Intermolecular FRET
Akt, PDK1	Akt-Venus, CFP-PDK1;	(Yoshizaki et al., 2006)
AMPK	bimABKAR, bimABKAR-Kras	(Depry et al., 2015)
CRaf MEK	CFP-c-Raf-pm MEK-YFP	(Terai and Matsuda, 2005)
ERK	bimEKAR	(Depry et al., 2015)
ERK MEK	mVenus-MEK1 mCFP-ERK2;CFP-ERK MEK-YFP	(Aoki et al., 2011; Fujioka et al., 2006)
JNK	bimJNKAR	(Depry et al., 2015)
Intramolecular FRET: (1) kinase type
Akt	Akind, ReAktion	(Ananthanarayanan et al., 2007; Yoshizaki et al., 2006)
Aurora A kinase	AURKA biosensor	(Bertolin et al., 2016)
B-Raf	Prin-BRaf	(Terai and Matsuda, 2006)
C-Raf	Prin-CRaf	(Terai and Matsuda, 2005)
CAMKII	Camuiα, Camk2a(314)-F40	(Piljić et al., 2011; Takao et al., 2005)
DAPK	DAPK1(334)-F40	(Piljić et al., 2011)
EGFR	FLAME	(Offterdinger et al., 2004)
ERK	Miu2	(Fujioka et al., 2006)
FAK	FERM sensor, SPASM sensor	(Papusheva et al., 2009; Ritt et al., 2013)
MELK	YFP-xMELK K/R-CFP	(Le Page et al., 2011)
MK2	GMB	(Neininger et al., 2001)
MLCK	MLCK-FIP	(Chew et al., 2002)
PAK1	Pakabi	(Parrini et al., 2009)
PDK1	Lyn-PARE	(Gao et al., 2011)
PKC	CY-PKCδ	(Braun et al., 2005)
Intramolecular FRET: (2) substrate type
Abl	Abl indicator, Pickles	(Mizutani et al., 2010; Ting et al., 2001)
Akt	Aktus, BKAR, AktAR, Eevee-Akt, Eevee-iAkt	(Gao and Zhang, 2008; Komatsu et al., 2011; Kunkel et al., 2005; Miura et al., 2014; Sasaki et al., 2003)
AMPK	AMPKAR, ABKAR, T2AMPKAR, AMPKAR-EV	(Chennell et al., 2016; Konagaya et al., 2017; Sample et al., 2015; Tsou et al., 2011)
ATM kinase	Atomic	(Johnson et al., 2007)
Aurora B kinase	Aurora B sensor, Aurora B kinase sensor	(Chu et al., 2011; Fuller et al., 2008)
Cdk1	Cyclin B1-Cdk1 biosensor	(Gavet and Pines, 2010)
ERK	Erkus, EKAR, EKAREV, ERKy, EKAR2G1, EKAR-TVV	(Fritz et al., 2013; Harvey et al., 2008; Komatsu et al., 2011; Sato et al., 2007; Tomida et al., 2012; Vandame et al., 2014)
EGFR	Picchu, EGFR indicator, Picchu-Z, PicchuEV, FLAME	(Itoh et al., 2005; Komatsu et al., 2011; Kurokawa et al., 2001; Ting et al., 2001)
FAK	FAK biosensor	(Seong et al., 2011)
Insulin receptor	Phocus	(Sato et al., 2002)
JNK	JNKAR1, JNKAR1-EV	(Fosbrink et al., 2010; Komatsu et al., 2011)
MARK	MARK-AR1	(Timm et al., 2011)
MLCK, Rho kinase	CRCit	(Yamada et al., 2005)
mTORC1	TORCAR	(Zhou et al., 2015)
p38	PerKy-p38	(Tomida et al., 2015)
PKA	AKAR1, AKAR2, AKAR3, AKAR3EV	(Allen and Zhang, 2006; Komatsu et al., 2011; Zhang et al., 2001, 2005)
PKC	CKAR, KCP-1, Eevee-PKC	(Komatsu et al., 2011; Schleifenbaum et al., 2004; Violin et al., 2003)
PKD	DKAR	(Kunkel et al., 2007)
PLK1	PLK1 FRET-probe	(Macůrek et al., 2008)
ROCK/Rho kinase	Eevee-ROCK	(Li et al., 2017)
RSK	Eevee-RSK	(Komatsu et al., 2011)
S6K	Eevee-S6K	(Komatsu et al., 2011)
SAP3K	SAP3K reporter	(Tomida et al., 2009)
Src	Src indicator, Src reporter, Srcus, Src biosensor, BG-Src biosensor	(Hitosugi et al., 2007; Ouyang et al., 2008; Su et al., 2013; Ting et al., 2001; Wang et al., 2005)
SYK	SYK biosensor	(Xiang et al., 2011)
TAK1	Eevee-TAK1	(Takaoka et al., 2016)
ZAP70	ROZA, ROZA-XL	(Cadra et al., 2015; Randriamampita et al., 2008)

Intermolecular, or bimolecular, FRET biosensors

FP-based FRET biosensors are roughly classified into two categories: intermolecular (or bimolecular) FRET biosensors and intramolecular (or unimolecular) FRET biosensors (Aoki et al., 2008; Oldach and Zhang, 2014). In the former case, a polypeptide fused with a donor FP is expressed along with a separate polypeptide attached to an acceptor FP. The increase in FRET signals is observed upon binding between the donor and acceptor FP-fused proteins (Fig. 3A). Under the dissociation condition of the intermolecular FRET biosensor, the distance between donor and acceptor fluorophores can be quite a bit greater than the Förster distance, which is a few nm. For this reason, the relative distance between donor and acceptor FPs is mainly attributed to the increase in FRET in the intermolecular FRET. The advantage of this intermolecular FRET system is the simplicity of development of the biosensor in comparison to the intramolecular FRET biosensor. The possible configurations of FPs and binding domains (for example, A and B) are limited to 4 types: FP_donor-A and FP_acceptor-B, FP_donor-A and B-FP_acceptor, A-FP_donor and FP_acceptor-B, and A-FP_donor and B-FP_acceptor (Fig. 3A). The combination of FPs showing the highest response should be selected as an intermolecular FRET biosensor.

Fig. 3

FRET-based kinase activity reporters. (A) Schematic representation of possible combinations of intermolecular FRET. Donor (cyan FP) and acceptor (yellow FP) are fused with A and B domains, respectively, at the N- or C-terminus of each FP. (B) Bimolecular kinase activity reporter (BimKAR). An activated kinase phosphorylates a substrate peptide fused with FP. Thereafter, a phospho-peptide binding domain (orange) with FP interacts with the phosphorylated peptide (green), bringing the donor FP in close proximity to the acceptor FP. (C) Intramolecular FRET biosensors can be roughly divided into two types. The first one is the conformation type, which monitors the conformation change in a kinase upon activation (left). An example of a conformation-type FRET biosensor, Miu2 (ERK2 FRET biosensor), is depicted before and after EGF treatment, showing the induction of open conformation and nuclear accumulation (middle). The second one is the substrate type, which serves as a substrate, indicating FRET signals in a phosphorylation-dependent manner (right). Scale bar, 15 μm. (D) Optimized backbone for the intramolecular FRET biosensor, called the Eevee system, which includes a long flexible linker and sticky FP pair (left). Stochastic ERK activation is observed in proliferating cells with an Eevee biosensor, the EKAREV-nuclear export sequence (NES) (ERK FRET biosensor), which is a substrate-type intramolecular FRET biosensor. Scale bar, 20 μm.

The bimolecular kinase activity reporter (bimKAR) is a good example of an intermolecular FRET biosensor for monitoring kinase activity (Depry et al., 2015; Herbst et al., 2011). The bimKAR is comprised of two polypeptides, i.e., the FP_donor-FHA1 domain or FP_donor-WW domain and the substrate peptide-FP_acceptor (Fig. 3B). The FHA1 domain and WW domain are phosphorylated peptide-binding domains that recognize the pT-X-X-D motif and pS/pT-P motif, respectively. The CMGC kinases (cyclin-dependent kinases (CDKs), mitogen-activated protein kinases (MAP kinases), glycogen synthase kinases (GSK) and CDK-like kinases) phosphorylate the pS/pT-P motif, and therefore the WW domain is suitable for the phosphorylated peptide-binding domain. In other kinases, the phosphorylated peptide motif should be optimized to pT-X-X-D for recognition of the FHA1 domain. The target kinase, which is activated upon stimulation, phosphorylates the substrate peptide-FP_acceptor, followed by the binding of the phosphorylated substrate-FP_acceptor to FP_donor-WW or FP_donor-FHA1. Thus, the FRET signal is increased upon kinase activation. The expression levels of donor and acceptor fluorophores usually differ from each other within individual cells, and therefore careful spectral corrections such as bleed-through and cross-excitation are necessary to obtain accurate FRET signals. This image correction process usually reduces the signal-to-noise ratio by the propagation of error. This issue may be partly overcome by using a self-cleaving 2A peptide to express almost similar amounts of donor and acceptor fluorophores. Because the donor-to-acceptor molar ratio is fixed, the bleed-through and cross-excitation are cancelled out by simple ratiometry (Szymczak et al., 2004). In addition, the binding domain for the intermolecular FRET biosensor often acts as a competitor binding to phosphorylated endogenous proteins, resulting in a possible perturbation of intracellular signaling. For these reasons, less effort has been devoted to developing intermolecular FRET biosensors than intramolecular FRET biosensors.

Intramolecular, or unimolecular, FRET biosensors

Intramolecular (or unimolecular) FRET biosensors bear both donor and acceptor FPs in a single polypeptide, and therefore FRET occurs primarily within the biosensor (Fig. 3C). Unlike in intermolecular FRET biosensors, the relative orientation between donor and acceptor FPs plays an important role in the FRET efficiency. Intramolecular FRET biosensors for monitoring kinase activity typically have one of two designs, namely, the conformation-type or substrate-type design (Fig. 3C). The former type detects conformational changes in a kinase or its substrate upon activation by FRET (Fig. 3C, left). For example, Miu2, a FRET biosensor for conformational change in ERK2, demonstrates an open and active conformation after EGF stimulation, concomitant with the nuclear translocation (Fujioka et al., 2006) (Fig. 3C, middle). Meanwhile, in the latter type, the FRET biosensor includes not only a substrate peptide that is phosphorylated by the target kinase, but also a phosphorylation recognition domain such as a WW domain or FHA1 domain (Fig. 3C, right). Intramolecular FRET biosensors have clear advantages—that is, they permit easier introduction of the expression vector, they do not require spectral corrections because the donor-to-acceptor molar ratio is always 1:1 (Sabouri-Ghomi et al., 2008), and they exhibit empirically higher sensitivity and gain as compared with the intermolecular FRET biosensors. Therefore, the intramolecular FRET biosensors have been widely utilized in cell biology.

The serious caveat is the difficulty associated with the development of intramolecular FRET biosensors (Aoki et al., 2012). For instance, it must be decided which FPs, sensor and ligand domains should be included, how the order of these domains should be configured, and how long the optimal linker between the domains should be. Most of these difficulties are derived from the orientation-dependent features of the intramolecular FRET biosensor (Jares-Erijman and Jovin, 2003). The precise structures of the sensor and ligand domains are rarely available. In practical terms, the addition or removal of only one amino acid of linker length has a major effect on the FRET efficiencies (Horikawa et al., 2010). It is almost impossible to predict the effect of the orientation-dependent factor of intramolecular FRET biosensors, and therefore the development of an intramolecular FRET biosensor requires labor-intensive, trial-and-error characterizations.

To circumvent the difficulties inherent in the development of intramolecular FRET biosensors, a number of FP variants that are optimized for FRET applications have been generated by several research groups (Bajar et al., 2016; Karasawa et al., 2004; Lam et al., 2012; Nguyen and Daugherty, 2005; Rizzo et al., 2004). In addition, we have reported a rationally designed optimal backbone for intramolecular FRET biosensors (Komatsu et al., 2011). This backbone is named the extension for enhanced visualization by evading extra FRET (Eevee) system. This Eevee system has two characteristic features: a long flexible linker, called an EV linker, and a “sticky” FP pair (Fig. 3D, left). The long flexible linker renders the biosensor completely “distance-dependent”, thereby precluding the “orientation-dependent” effect on FRET. In addition, the long flexible linker lowers basal FRET levels. The “sticky” FP pair—for example YPet and ECFP—enhances the gain of the FRET signal induced by stimulation (Kotera et al., 2010). Applying the Eevee system to the previously reported kinase activity reporters has improved them several-fold. By using the highly sensitive ERK FRET biosensor, EKAREV, stochastic ERK activations were visualized in proliferating cultured cells (Albeck et al., 2013; Aoki et al., 2013) (Figure 3D, right). Further, the use of the Eevee system enabled us to develop new biosensors for S6K and RSK without many optimization steps. Thus, the Eevee system reduces the laborious steps required for prototyping biosensors. Despite advances in the rational backbone for FRET biosensors, much work is still needed for the development and characterization of a FRET biosensor for protein kinases, because many peptide sequences that are phosphorylated by a given kinase are not expected to be phosphorylated when they are included in a FRET biosensor.

Future perspectives

The development of genetically encoded kinase activity reporters is still challenging. However, by employing a broad range of techniques, the development of these reporters has become feasible. For instance, Pertz and his colleagues reported versatile tool kits for the development of intramolecular FRET biosensors (Fritz et al., 2013). Their techniques allowed efficient and comprehensive characterization of intramolecular FRET biosensors. Further, the FLIM-FRET with a non-fluorescent protein enables multiplexed imaging of molecular activity (Laviv et al., 2016). While one of the remaining difficulties is to find the peptide sequences to be phosphorylated by a target kinase, a recent advance in phosphoproteomics, kinase-interacting substrate screening (KISS) (Amano et al., 2015), facilitated the development of a novel ROCK FRET biosensor (Li et al., 2017).

Finally, we will consider the future perspectives related to the imaging of kinase activity. First, the manipulation of kinase activity in cells or animals is becoming an indispensable technique for the evaluation of kinase functions in vitro and in vivo. Chemically induced dimerization has been reported to modulate kinase activity by small chemical compounds (Aoki et al., 2011; Derose et al., 2013). More recently, an optogenetic approach has become an indispensable tool for controlling kinase activity in space and time by light (Schlipalius et al., 2012; Wilson et al., 2017), clearly dissecting the causal relationship between kinase activation and its physiological function (Aoki et al., 2013, 2017; Wilson et al., 2017). Second, the in vivo imaging of kinase activity sheds new light on the role of kinase activation in a more physiological and pathological context (Hiratsuka et al., 2015; Kamioka et al., 2012; Mizuno et al., 2014; Tomida et al., 2012). Further progress will require a deeper understanding of the mechanisms of kinase activation, a development of improved FPs for live-cell imaging, and continued efforts to combine these different technologies.

Acknowledgments

We thank the members of the Matsuda and Aoki Laboratory for their helpful discussions. MM and KA were supported by CREST and JST (JPMJCR1654). KA was supported by JSPS KAKENHI Grants (nos. JP16H01447, 26290053, 16H01425 “Resonance Bio”, 16KT0069), the Hori Sciences and Arts Foundation, the Takeda Science Foundation and The Nakajima Foundation.

References

•  Albeck,  J.G.,  Mills,  G.B., and  Brugge,  J.S. 2013. Frequency-Modulated Pulses of ERK Activity Transmit Quantitative Proliferation Signals. Mol. Cell, 49: 249–261.
•  Allen,  M.D. and  Zhang,  J. 2006. Subcellular dynamics of protein kinase A activity visualized by FRET-based reporters. Biochem. Biophys. Res. Commun., 348: 716–721.
•  Almholt,  D.L.C.,  Loechel,  F.,  Nielsen,  S.J.,  Krog-Jensen,  C.,  Terry,  R.,  Bjørn,  S.P.,  Pedersen,  H.C.,  Praestegaard,  M.,  Møller,  S.,  Heide,  M., et al. 2004a. Nuclear Export Inhibitors and Kinase Inhibitors Identified Using a MAPK-Activated Protein Kinase 2 Redistribution^® Screen. Assay Drug Dev. Technol., 2: 7–20.
•  Almholt,  K.,  Tullin,  S.,  Skyggebjerg,  O.,  Scudder,  K.,  Thastrup,  O., and  Terry,  R. 2004b. Changes in intracellular cAMP reported by a Redistribution^® assay using a cAMP-dependent protein kinase-green fluorescent protein chimera. Cell. Signal., 16: 907–920.
•  Amano,  M.,  Hamaguchi,  T.,  Shohag,  M.H.,  Kozawa,  K.,  Kato,  K.,  Zhang,  X.,  Yura,  Y.,  Matsuura,  Y.,  Kataoka,  C.,  Nishioka,  T., et al. 2015. Kinase-interacting substrate screening is a novel method to identify kinase substrates. J. Cell Biol., 209: 895–912.
•  Ananthanarayanan,  B.,  Fosbrink,  M.,  Rahdar,  M., and  Zhang,  J. 2007. Live-cell molecular analysis of Akt activation reveals roles for activation loop phosphorylation. J. Biol. Chem., 282: 36634–36641.
•  Aoki,  K.,  Kiyokawa,  E.,  Nakamura,  T., and  Matsuda,  M. 2008. Visualization of growth signal transduction cascades in living cells with genetically encoded probes based on Forster resonance energy transfer. Philos. Trans. R. Soc. B Biol. Sci., 363: 2143–2151.
•  Aoki,  K. and  Matsuda,  M. 2009. Visualization of small GTPase activity with fluorescence resonance energy transfer-based biosensors. Nat. Protoc., 4: 1623–1631.
•  Aoki,  K.,  Yamada,  M.,  Kunida,  K.,  Yasuda,  S., and  Matsuda,  M. 2011. Processive phosphorylation of ERK MAP kinase in mammalian cells. Proc. Natl. Acad. Sci., 108: 12675–12680.
•  Aoki,  K.,  Komatsu,  N.,  Hirata,  E.,  Kamioka,  Y., and  Matsuda,  M. 2012. Stable expression of FRET biosensors: A new light in cancer research. Cancer Sci., 103: 614–619.
•  Aoki,  K.,  Kumagai,  Y.,  Sakurai,  A.,  Komatsu,  N.,  Fujita,  Y.,  Shionyu,  C., and  Matsuda,  M. 2013. Stochastic ERK activation induced by noise and cell-to-cell propagation regulates cell density-dependent proliferation. Mol. Cell, 52: 529–540.
•  Aoki,  K.,  Kondo,  Y.,  Naoki,  H.,  Hiratsuka,  T.,  Itoh,  R.E., and  Matsuda,  M. 2017. Propagating Wave of ERK Activation Orients Collective Cell Migration. Dev. Cell, 43: 305–317.e5.
•  Baird,  G.S.,  Zacharias,  D.A., and  Tsien,  R.Y. 1999. Circular permutation and receptor insertion within green fluorescent proteins. Proc. Natl. Acad. Sci., 96: 11241–11246.
•  Bajar,  B.T.,  Wang,  E.S.,  Zhang,  S.,  Lin,  M.Z., and  Chu,  J. 2016. A guide to fluorescent protein FRET pairs. Sensors (Switzerland), 16.
•  Bao,  Y.,  Nakagawa,  K.,  Yang,  Z.,  Ikeda,  M.,  Withanage,  K.,  Ishigami-Yuasa,  M.,  Okuno,  Y.,  Hata,  S.,  Nishina,  H., and  Hata,  Y. 2011. A cell-based assay to screen stimulators of the Hippo pathway reveals the inhibitory effect of dobutamine on the YAP-dependent gene transcription. J. Biochem., 150: 199–208.
•  Bertolin,  G.,  Sizaire,  F.,  Herbomel,  G.,  Reboutier,  D.,  Prigent,  C., and  Tramier,  M. 2016. A FRET biosensor reveals spatiotemporal activation and functions of aurora kinase A in living cells. Nat. Commun., 7: 12674.
•  Bivona,  T.G.,  Quatela,  S.E.,  Bodemann,  B.O.,  Ahearn,  I.M.,  Soskis,  M.J.,  Mor,  A.,  Miura,  J.,  Wiener,  H.H.,  Wright,  L.,  Saba,  S.G., et al. 2006. PKC regulates a farnesyl-electrostatic switch on K-Ras that promotes its association with Bcl-XL on mitochondria and induces apoptosis. Mol. Cell, 21: 481–493.
•  Braun,  D.C.,  Garfield,  S.H., and  Blumberg,  P.M. 2005. Analysis by fluorescence resonance energy transfer of the interaction between ligands and protein kinase Cδ in the intact cell. J. Biol. Chem., 280: 8164–8171.
•  Burgering,  B.M.T. 2003. Decisions on life and death: FOXO Forkhead transcription factors are in command when PKB / Akt is off duty. J. Leukoc. Biol., 73: 689–701.
•  Cadra,  S.,  Gucciardi,  A.,  Valignat,  M.-P.,  Theodoly,  O.,  Vacaflores,  A.,  Houtman,  J.C.D., and  Lellouch,  A.C. 2015. ROZA-XL, an improved FRET based biosensor with an increased dynamic range for visualizing zeta associated protein 70 kD (ZAP-70) tyrosine kinase activity in live T cells. Biochem. Biophys. Res. Commun., 459: 405–410.
•  Carter,  E.J.,  Cosgrove,  R.A.,  Gonzalez,  I.,  Eisemann,  J.H.,  Lovett,  F.A.,  Cobb,  L.J., and  Pell,  J.M. 2009. MEK5 and ERK5 are mediators of the pro-myogenic actions of IGF-2. J. Cell Sci., 122: 3104–3112.
•  Casalino,  L.,  De Cesare,  D., and  Verde,  P. 2003. Accumulation of Fra-1 in ras-Transformed Cells Depends on Both Transcriptional Autoregulation and MEK-Dependent Posttranslational Stabilization. Mol. Cell. Biol., 23: 4401–4415.
•  Chen,  T.W.,  Wardill,  T.J.,  Sun,  Y.,  Pulver,  S.R.,  Renninger,  S.L.,  Baohan,  A.,  Schreiter,  E.R.,  Kerr,  R.A.,  Orger,  M.B.,  Jayaraman,  V., et al. 2013. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature, 499: 295–300.
•  Chennell,  G.,  Willows,  R.J.W.,  Warren,  S.C.,  Carling,  D.,  French,  P.M.W.,  Dunsby,  C., and  Sardini,  A. 2016. Imaging of metabolic status in 3D cultures with an improved AMPK FRET biosensor for FLIM. Sensors (Switzerland), 16.
•  Chew,  T.L.,  Wolf,  W.A.,  Gallagher,  P.J.,  Matsumura,  F., and  Chisholm,  R.L. 2002. A fluorescent resonant energy transfer-based biosensor reveals transient and regional myosin light chain kinase activation in lamella and cleavage furrows. J. Cell Biol., 156: 543–553.
•  Chu,  Y.,  Yao,  P.Y.,  Wang,  W.,  Wang,  D.,  Wang,  Z.,  Zhang,  L.,  Huang,  Y.,  Ke,  Y.,  Ding,  X., and  Yao,  X. 2011. Aurora B kinase activation requires survivin priming phosphorylation by PLK1. J. Mol. Cell Biol., 3: 260–267.
•  Chudakov,  D.M.,  Matz,  M.V.,  Lukyanov,  S., and  Lukyanov,  K.A. 2010. Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues. Physiol. Rev., 90: 1103–1163.
•  Cohen,  P. 2000. The regulation of protein function by multisite phosphorylation - A 25 year update. Trends Biochem. Sci., 25: 596–601.
•  Day,  R.N. and  Davidson,  M.W. 2012. Fluorescent proteins for FRET microscopy: Monitoring protein interactions in living cells. BioEssays, 34: 341–350.
•  Depry,  C.,  Mehta,  S.,  Li,  R., and  Zhang,  J. 2015. Visualization of Compartmentalized Kinase Activity Dynamics Using Adaptable BimKARs. Chem. Biol., 22: 1470–1480.
•  Derose,  R.,  Miyamoto,  T., and  Inoue,  T. 2013. Manipulating signaling at will: Chemically-inducible dimerization (CID) techniques resolve problems in cell biology. Pflugers Arch. Eur. J. Physiol., 465: 409–417.
•  Förster,  T. 1946. Energiewanderung und Fluoreszenz. Naturwissenschaften, 33: 166–175.
•  Fosbrink,  M.,  Aye-Han,  N.-N.,  Cheong,  R.,  Levchenko,  A., and  Zhang,  J. 2010. Visualization of JNK activity dynamics with a genetically encoded fluorescent biosensor. Proc. Natl. Acad. Sci., 107: 5459–5464.
•  Frémin,  C. and  Meloche,  S. 2010. From basic research to clinical development of MEK1/2 inhibitors for cancer therapy. J. Hematol. Oncol., 3: 8.
•  Fritz,  R.D.,  Letzelter,  M.,  Reimann,  A.,  Martin,  K.,  Fusco,  L.,  Ritsma,  L.,  Ponsioen,  B.,  Fluri,  E.,  Schulte-Merker,  S.,  Van Rheenen,  J., et al. 2013. A versatile toolkit to produce sensitive FRET biosensors to visualize signaling in time and space. Sci. Signal., 6: rs12-rs12.
•  Fujioka,  A.,  Terai,  K.,  Itoh,  R.E.,  Aoki,  K.,  Nakamura,  T.,  Kuroda,  S.,  Nishida,  E., and  Matsuda,  M. 2006. Dynamics of the Ras/ERK MAPK cascade as monitored by fluorescent probes. J. Biol. Chem., 281: 8917–8926.
•  Fuller,  B.G.,  Lampson,  M.A.,  Foley,  E.A.,  Rosasco-Nitcher,  S., Le, K.V,  Tobelmann,  P.,  Brautigan,  D.L.,  Stukenberg,  P.T., and  Kapoor,  T.M. 2008. Midzone activation of aurora B in anaphase produces an intracellular phosphorylation gradient. Nature, 453: 1132–1136.
•  Gao,  X. and  Zhang,  J. 2008. Spatiotemporal Analysis of Differential Akt Regulation in Plasma Membrane Microdomains. Mol. Biol. Cell, 19: 4366–4373.
•  Gao,  X.,  Lowry,  P.R.,  Zhou,  X.,  Depry,  C.,  Wei,  Z.,  Wong,  G.W., and  Zhang,  J. 2011. PI3K/Akt signaling requires spatial compartmentalization in plasma membrane microdomains. Proc. Natl. Acad. Sci., 108: 14509–14514.
•  Gavet,  O. and  Pines,  J. 2010. Progressive activation of CyclinB1-Cdk1 coordinates entry to mitosis. Dev. Cell, 18: 533–543.
•  Gong,  X.,  Ming,  X.,  Deng,  P., and  Jiang,  Y. 2010. Mechanisms regulating the nuclear translocation of p38 MAP kinase. J. Cell. Biochem., 110: 1420–1429.
•  Gross,  S.M. and  Rotwein,  P. 2016. Mapping growth-factor-modulated Akt signaling dynamics. J. Cell Sci., 129: 2052–2063.
•  Gross,  S.,  Rahal,  R.,  Stransky,  N.,  Lengauer,  C., and  Hoeflich,  K.P. 2015. Targeting cancer with kinase inhibitors. J. Clin. Invest., 125: 1780–1789.
•  Harvey,  C.D.,  Ehrhardt,  A.G.,  Cellurale,  C.,  Zhong,  H.,  Yasuda,  R.,  Davis,  R.J., and  Svoboda,  K. 2008. A genetically encoded fluorescent sensor of ERK activity. Proc. Natl. Acad. Sci. USA, 105: 19264–19269.
•  Haugh,  J.M.,  Codazzi,  F.,  Teruel,  M., and  Meyer,  T. 2000. Spatial sensing in fibroblasts mediated by 3' phosphoinositides. J. Cell Biol., 151: 1269–1279.
•  Herbst,  K.J.,  Allen,  M.D., and  Zhang,  J. 2011. Luminescent kinase activity biosensors based on a versatile bimolecular switch. J. Am. Chem. Soc., 133: 5676–5679.
•  Hiratsuka,  T.,  Fujita,  Y.,  Naoki,  H.,  Aoki,  K.,  Kamioka,  Y., and  Matsuda,  M. 2015. Intercellular propagation of extracellular signal-regulated kinase activation revealed by in vivo imaging of mouse skin. Elife 2015.
•  Hitosugi,  T.,  Sato,  M.,  Sasaki,  K., and  Umezawa,  Y. 2007. Lipid raft specific knockdown of SRC family kinase activity inhibits cell adhesion and cell cycle progression of breast cancer cells. Cancer Res., 67: 8139–8148.
•  Horikawa,  K.,  Yamada,  Y.,  Matsuda,  T.,  Kobayashi,  K.,  Hashimoto,  M.,  Matsu-Ura,  T.,  Miyawaki,  A.,  Michikawa,  T.,  Mikoshiba,  K., and  Nagai,  T. 2010. Spontaneous network activity visualized by ultrasensitive Ca 2+ indicators, yellow Cameleon-Nano. Nat. Methods, 7: 729–732.
•  Huang,  B.,  Bates,  M., and  Zhuang,  X. 2009. Super-Resolution Fluorescence Microscopy. Annu. Rev. Biochem., 78: 993–1016.
•  Itoh,  R.E.,  Kurokawa,  K.,  Fujioka,  A.,  Sharma,  A.,  Mayer,  B.J., and  Matsuda,  M. 2005. A FRET-based probe for epidermal growth factor receptor bound non-covalently to a pair of synthetic amphipathic helixes. Exp. Cell Res., 307: 142–152.
•  Jares-Erijman,  E.A. and  Jovin,  T.M. 2003. FRET imaging. Nat. Biotechnol., 21: 1387–1395.
•  Johnson,  S.A.,  You,  Z., and  Hunter,  T. 2007. Monitoring ATM kinase activity in living cells. DNA Repair (Amst), 6: 1277–1284.
•  Kamioka,  Y.,  Sumiyama,  K.,  Mizuno,  R.,  Sakai,  Y.,  Hirata,  E.,  Kiyokawa,  E., and  Matsuda,  M. 2012. Live Imaging of Protein Kinase Activities in Transgenic Mice Expressing FRET Biosensors. Cell Struct. Funct., 37: 65–73.
•  Karasawa,  S.,  Araki,  T.,  Nagai,  T.,  Mizuno,  H., and  Miyawaki,  A. 2004. Cyan-emitting and orange-emitting fluorescent proteins as a donor/acceptor. Biochem. J., 312: 307–312.
•  Katsura,  Y.,  Kubota,  H.,  Kunida,  K.,  Kanno,  A.,  Kuroda,  S., and  Ozawa,  T. 2015. An optogenetic system for interrogating the temporal dynamics of Akt. Sci. Rep., 5: 14589.
•  Kawai,  Y.,  Sato,  M., and  Umezawa,  Y. 2004. Single color fluorescent indicators of protein phosphorylation for multicolor imaging of intracellular signal flow dynamics. Anal. Chem., 76: 6144–6149.
•  Kawamori,  D.,  Kajimoto,  Y.,  Kaneto,  H.,  Umayahara,  Y.,  Fujitani,  Y.,  Miyatsuka,  T.,  Watada,  H.,  Leibiger,  I.B.,  Yamasaki,  Y., and  Hori,  M. 2003. Oxidative Stress Induces Nucleo-Cytoplasmic Translocation of Pancreatic Transcription Factor PDX-1 Through Activation of c-Jun NH2-terminal Kinase. Diabetes, 52: 2896–2904.
•  Kiyokawa,  E.,  Aoki,  K.,  Nakamura,  T., and  Matsuda,  M. 2011. Spatiotemporal Regulation of Small GTPases as Revealed by Probes Based on the Principle of Förster Resonance Energy Transfer (FRET): Implications for Signaling and Pharmacology. Annu. Rev. Pharmacol. Toxicol., 51: 337–358.
•  Knight,  Z.A.,  Gonzalez,  B.,  Feldman,  M.E.,  Zunder,  E.R.,  Goldenberg,  D.D.,  Williams,  O.,  Loewith,  R.,  Stokoe,  D.,  Balla,  A.,  Toth,  B., et al. 2006. A Pharmacological Map of the PI3-K Family Defines a Role for p110α in Insulin Signaling. Cell, 125: 733–747.
•  Komatsu,  N.,  Aoki,  K.,  Yamada,  M.,  Yukinaga,  H.,  Fujita,  Y.,  Kamioka,  Y., and  Matsuda,  M. 2011. Development of an optimized backbone of FRET biosensors for kinases and GTPases. Mol. Biol. Cell, 22: 4647–4656.
•  Konagaya,  Y.,  Terai,  K.,  Hirao,  Y.,  Takakura,  K.,  Imajo,  M.,  Kamioka,  Y.,  Sasaoka,  N.,  Kakizuka,  A.,  Sumiyama,  K.,  Asano,  T., et al. 2017. A Highly Sensitive FRET Biosensor for AMPK Exhibits Heterogeneous AMPK Responses among Cells and Organs. Cell Rep., 21: 2628–2638.
•  Kotera,  I.,  Iwasaki,  T.,  Imamura,  H.,  Noji,  H., and  Nagai,  T. 2010. Reversible dimerization of aequorea victoria fluorescent proteins increases the dynamic range of FRET-based indicators. ACS Chem. Biol., 5: 215–222.
•  Kunkel,  M.T.,  Ni,  Q.,  Tsien,  R.Y.,  Zhang,  J., and  Newton,  A.C. 2005. Spatio-temporal dynamics of protein kinase B/Akt signaling revealed by a genetically encoded fluorescent reporter. J. Biol. Chem., 280: 5581–5587.
•  Kunkel,  M.T.,  Toker,  A.,  Tsien,  R.Y., and  Newton,  A.C. 2007. Calcium-dependent regulation of protein kinase D revealed by a genetically encoded kinase activity reporter. J. Biol. Chem., 282: 6733–6742.
•  Kurokawa,  K.,  Mochizuki,  N.,  Ohba,  Y.,  Mizuno,  H.,  Miyawaki,  A., and  Matsuda,  M. 2001. A Pair of Fluorescent Resonance Energy Transfer-based Probes for Tyrosine Phosphorylation of the CrkII Adaptor Protein in Vivo. J. Biol. Chem., 276: 31305–31310.
•  Lam,  A.J.,  St-Pierre,  F.,  Gong,  Y.,  Marshall,  J.D.,  Cranfill,  P.J.,  Baird,  M.A.,  McKeown,  M.R.,  Wiedenmann,  J.,  Davidson,  M.W.,  Schnitzer,  M.J., et al. 2012. Improving FRET dynamic range with bright green and red fluorescent proteins. Nat. Methods, 9: 1005–1012.
•  Laviv,  T.,  Kim,  B.B.,  Chu,  J.,  Lam,  A.J.,  Lin,  M.Z., and  Yasuda,  R. 2016. Simultaneous dual-color fluorescence lifetime imaging with novel red-shifted fluorescent proteins. Nat. Methods, 13: 989–992.
•  Li,  C.,  Imanishi,  A.,  Komatsu,  N.,  Terai,  K.,  Amano,  M.,  Kaibuchi,  K., and  Matsuda,  M. 2017. A FRET Biosensor for ROCK Based on a Consensus Substrate Sequence Identified by KISS Technology. Cell Struct. Funct., 42: 1–13.
•  Lidke,  D.S.,  Huang,  F.,  Post,  J.N.,  Rieger,  B.,  Wilsbacher,  J.,  Thomas,  J.L.,  Pouysségur,  J.,  Jovin,  T.M., and  Lenormand,  P. 2010. ERK nuclear translocation is dimerization-independent but controlled by the rate of phosphorylation. J. Biol. Chem., 285: 3092–3102.
•  Lin,  M.Z. and  Schnitzer,  M.J. 2016. Genetically encoded indicators of neuronal activity. Nat. Neurosci., 19: 1142–1153.
•  Macůrek,  L.,  Lindqvist,  A.,  Lim,  D.,  Lampson,  M.A.,  Klompmaker,  R.,  Freire,  R.,  Clouin,  C.,  Taylor,  S.S.,  Yaffe,  M.B., and  Medema,  R.H. 2008. Polo-like kinase-1 is activated by aurora A to promote checkpoint recovery. Nature, 455: 119–123.
•  Maryu,  G.,  Matsuda,  M., and  Aoki,  K. 2016. Multiplexed Fluorescence Imaging of ERK and Akt Activities and Cell-cycle Progression. Cell Struct. Funct., 41: 81–92.
•  McDonald,  P.H.,  Chow,  C.W.,  Miller,  W.E.,  Laporte,  S.A.,  Field,  M.E.,  Lin,  F.T.,  Davis,  R.J., and  Lefkowitz,  R.J. 2000. Beta-arrestin 2: a receptor-regulated MAPK scaffold for the activation of JNK3. Science (80-. ), 290: 1574–1577.
•  Miura,  H.,  Matsuda,  M., and  Aoki,  K. 2014. Development of a FRET Biosensor with High Specificity for Akt. Cell Struct. Funct., 39: 9–20.
•  Miyawaki,  A.,  Llopis,  J.,  Heim,  R.,  Michael McCaffery,  J.,  Adams,  J.A.,  Ikura,  M., and  Tsien,  R.Y. 1997. Fluorescent indicators for Ca2+based on green fluorescent proteins and calmodulin. Nature, 388: 882–887.
•  Miyawaki,  A. 2003. Visualization of the spatial and temporal dynamics of intracellular signaling. Dev. Cell, 4: 295–305.
•  Miyawaki,  A. 2011. Development of Probes for Cellular Functions Using Fluorescent Proteins and Fluorescence Resonance Energy Transfer. Annu. Rev. Biochem., 80: 357–373.
•  Miyawaki,  A. and  Niino,  Y. 2015. Molecular Spies for Bioimaging-Fluorescent Protein-Based Probes. Mol. Cell, 58: 632–643.
•  Mizuno,  R.,  Kamioka,  Y.,  Kabashima,  K.,  Imajo,  M.,  Sumiyama,  K.,  Nakasho,  E.,  Ito,  T.,  Hamazaki,  Y.,  Okuchi,  Y.,  Sakai,  Y., et al. 2014. In vivo imaging reveals PKA regulation of ERK activity during neutrophil recruitment to inflamed intestines. J. Exp. Med., 211: 1123–1136.
•  Mizutani,  T.,  Kondo,  T.,  Darmanin,  S.,  Tsuda,  M.,  Tanaka,  S.,  Tobiume,  M.,  Asaka,  M., and  Ohba,  Y. 2010. A novel FRET-based biosensor for the measurement of BCR-ABL activity and its response to drugs in living cells. Clin. Cancer Res., 16: 3964–3975.
•  Mochizuki,  N.,  Yamashita,  S.,  Kurokawa,  K.,  Ohba,  Y.,  Nagai,  T.,  Miyawaki,  A., and  Matsuda,  M. 2001. Spatio-temporal images of growth-factor-induced activation of Ras and Rap1. Nature, 411: 1065–1068.
•  van Munster,  E.B. and  Gadella,  T.W.J. 2005. Fluorescence Lifetime Imaging Microscopy (FLIM). Adv. Biochem. Eng. Biotechnol., 95: 143–175.
•  Muscheler,  R.,  Joos,  F.,  Beer,  J.,  Müller,  S.A.,  Vonmoos,  M., and  Snowball,  I. 2007. Solar activity during the last 1000 yr inferred from radionuclide records. Quat. Sci. Rev., 26: 82–97.
•  Nagai,  T.,  Sawano,  A.,  Park,  E.S., and  Miyawaki,  A. 2001. Circularly permuted green fluorescent proteins engineered to sense Ca2+. Proc. Natl. Acad. Sci., 98: 3197–3202.
•  Nakai,  J.,  Ohkura,  M., and  Imoto,  K. 2001. A high signal-to-noise ca2+ probe composed of a single green fluorescent protein. Nat. Biotechnol., 19: 137–141.
•  Neininger,  A.,  Thielemann,  H., and  Gaestel,  M. 2001. FRET-based detection of different conformations of MK2. EMBO Rep., 2: 703–708.
•  Nelson,  G.,  Paraoan,  L.,  Spiller,  D.G.,  Wilde,  G.J.C., Browne, M. a,  Djali,  P.K.,  Unitt,  J.F.,  Sullivan,  E.,  Floettmann,  E., and  White,  M.R.H. 2002. Multi-parameter analysis of the kinetics of NF-kappaB signalling and transcription in single living cells. J. Cell Sci., 115: 1137–1148.
•  Nguyen,  A.W. and  Daugherty,  P.S. 2005. Evolutionary optimization of fluorescent proteins for intracellular FRET. Nat. Biotechnol., 23: 355–360.
•  Ni,  Q.,  Titov,  D.V., and  Zhang,  J. 2006. Analyzing protein kinase dynamics in living cells with FRET reporters. Methods, 40: 279–286.
•  Nishioka,  T.,  Aoki,  K.,  Hikake,  K.,  Yoshizaki,  H.,  Kiyokawa,  E., and  Matsuda,  M. 2008. Rapid Turnover Rate of Phosphoinositides at the Front of Migrating MDCK Cells. Mol. Biol. Cell, 19: 4213–4223.
•  Offterdinger,  M.,  Georget,  V.,  Girod,  A., and  Bastiaens,  P.I.H. 2004. Imaging Phosphorylation Dynamics of the Epidermal Growth Factor Receptor. J. Biol. Chem., 279: 36972–36981.
•  Ohmori,  S.,  Sakai,  N.,  Shirai,  Y.,  Yamamoto,  H.,  Miyamoto,  E.,  Shimizu,  N., and  Saito,  N. 2000. Importance of Protein Kinase C Targeting for the Phosphorylation of Its Substrate, Myristoylated Alanine-rich C-kinase Substrate. J. Biol. Chem., 275: 26449–26457.
•  Oldach,  L. and  Zhang,  J. 2014. Genetically encoded fluorescent biosensors for live-cell visualization of protein phosphorylation. Chem. Biol., 21: 186–197.
•  Ouyang,  M.,  Sun,  J.,  Chien,  S., and  Wang,  Y. 2008. Determination of hierarchical relationship of Src and Rac at subcellular locations with FRET biosensors. Proc. Natl. Acad. Sci. USA, 105: 14353–14358.
•  Padilla-Parra,  S. and  Tramier,  M. 2012. FRET microscopy in the living cell: Different approaches, strengths and weaknesses. BioEssays, 34: 369–376.
•  Le Page,  Y.,  Chartrain,  I.,  Badouel,  C., and  Tassan,  J.-P. 2011. A functional analysis of MELK in cell division reveals a transition in the mode of cytokinesis during Xenopus development. J. Cell Sci., 124: 958–968.
•  Papusheva,  E.,  de Queiroz,  F.M.,  Dalous,  J.,  Han,  Y.,  Esposito,  A.,  Jares-Erijmanxa,  E.A.,  Jovin,  T.M., and  Bunt,  G. 2009. Dynamic conformational changes in the FERM domain of FAK are involved in focal-adhesion behavior during cell spreading and motility. J. Cell Sci., 122: 656–666.
•  Parrini,  M.C.,  Camonis,  J.,  Matsuda,  M., and  de Gunzburg,  J. 2009. Dissecting activation of the PAK1 kinase at protrusions in living cells. J. Biol. Chem., 284: 24133–24143.
•  Pérez Koldenkova,  V. and  Nagai,  T. 2013. Genetically encoded Ca2+ indicators: Properties and evaluation. Biochim. Biophys. Acta - Mol. Cell Res., 1833: 1787–1797.
•  Piljić,  A.,  de Diego,  I.,  Wilmanns,  M., and  Schultz,  C. 2011. Rapid development of genetically encoded FRET reporters. ACS Chem. Biol., 6: 685–691.
•  Ptacek,  J.,  Devgan,  G.,  Michaud,  G.,  Zhu,  H.,  Zhu,  X.,  Fasolo,  J.,  Guo,  H.,  Jona,  G.,  Breitkreutz,  A.,  Sopko,  R., et al. 2005. Global analysis of protein phosphorylation in yeast. Nature, 438: 679–684.
•  Raisner,  R.M.,  Hartley,  P.D.,  Meneghini,  M.D.,  Bao,  M.Z.,  Liu,  C.L.,  Schreiber,  S.L.,  Rando,  O.J., and  Madhani,  H.D. 2005. Histone variant H2A.Z Marks the 5' ends of both active and inactive genes in euchromatin. Cell, 123: 233–248.
•  Randriamampita,  C.,  Mouchacca,  P.,  Malissen,  B.,  Marguet,  D.,  Trautmann,  A., and  Lellouch,  A.C. 2008. A novel ZAP-70 dependent FRET based biosensor reveals kinase activity at both the immunological synapse and the antisynapse. PLoS One, 3: e1521.
•  Regot,  S.,  Hughey,  J.J.,  Bajar,  B.T.,  Carrasco,  S., and  Covert,  M.W. 2014. High-sensitivity measurements of multiple kinase activities in live single cells. Cell, 157: 1724–1734.
•  Ritt,  M.,  Guan,  J.L., and  Sivaramakrishnan,  S. 2013. Visualizing and manipulating Focal Adhesion Kinase regulation in live cells. J. Biol. Chem., 288: 8875–8886.
•  Rizzo,  M.A.,  Springer,  G.H.,  Granada,  B., and  Piston,  D.W. 2004. An improved cyan fluorescent protein variant useful for FRET. Nat. Biotechnol., 22: 445–449.
•  Rocks,  O.,  Peyker,  A., and  Bastiaens,  P.I.H. 2006. Spatio-temporal segregation of Ras signals: one ship, three anchors, many harbors. Curr. Opin. Cell Biol., 18: 351–357.
•  Rubinfeld,  H.,  Hanoch,  T., and  Seger,  R. 1999. Identification of a cytoplasmic-retention sequence in ERK2. J. Biol. Chem., 274: 30349–30352.
•  Sabouri-Ghomi,  M.,  Wu,  Y.,  Hahn,  K., and  Danuser,  G. 2008. Visualizing and quantifying adhesive signals. Curr. Opin. Cell Biol., 20: 541–550.
•  Sadaie,  W.,  Harada,  Y.,  Matsuda,  M., and  Aoki,  K. 2014. Quantitative In Vivo Fluorescence Cross-Correlation Analyses Highlight the Importance of Competitive Effects in the Regulation of Protein-Protein Interactions. Mol. Cell. Biol., 34: 3272–3290.
•  Sakai,  N.,  Sasaki,  K.,  Ikegaki,  N.,  Shirai,  Y.,  Ono,  Y., and  Saito,  N. 1997. Direct visualization of the translocation of the gamma-subspecies of protein kinase C in living cells using fusion proteins with green fluorescent protein. J. Cell Biol., 139: 1465–1476.
•  Sample,  V.,  Ramamurthy,  S.,  Gorshkov,  K., Ronnett, G.V, and  Zhang,  J. 2015. Polarized activities of AMPK and BRSK in primary hippocampal neurons. Mol. Biol. Cell, 26: 1935–1946.
•  Samsonov,  A.,  Zenser,  N.,  Zhang,  F.,  Zhang,  H.,  Fetter,  J., and  Malkov,  D. 2013. Tagging of Genomic STAT3 and STAT1 with Fluorescent Proteins and Insertion of a Luciferase Reporter in the Cyclin D1 Gene Provides a Modified A549 Cell Line to Screen for Selective STAT3 Inhibitors. PLoS One, 8: e68391.
•  Sasaki,  K.,  Sato,  M., and  Umezawa,  Y. 2003. Fluorescent indicators for Akt/protein kinase B and dynamics of Akt activity visualized in living cells. J. Biol. Chem., 278: 30945–30951.
•  Sato,  M.,  Ozawa,  T.,  Inukai,  K.,  Asano,  T., and  Umezawa,  Y. 2002. Fluorescent indicators for imaging protein phosphorylation in single living cells. Nat. Biotechnol., 20: 287–294.
•  Sato,  M.,  Ueda,  Y.,  Takagi,  T., and  Umezawa,  Y. 2003. Production of PtdInsP3 at endomembranes is triggered by receptor endocytosis. Nat. Cell Biol., 5: 1016–1022.
•  Sato,  M.,  Kawai,  Y., and  Umezawa,  Y. 2007. Genetically encoded fluorescent indicators to visualize protein phosphorylation by extracellular signal-regulated kinase in single living cells. Anal. Chem., 79: 2570–2575.
•  Schaefer,  B.C.,  Ware,  M.F.,  Marrack,  P.,  Fanger,  G.R.,  Kappler,  J.W.,  Johnson,  G.L., and  Monks,  C.R. 1999. Live cell fluorescence imaging of T cell MEKK2: redistribution and activation in response to antigen stimulation of the T cell receptor. Immunity, 11: 411–421.
•  Schleifenbaum,  A.,  Stier,  G.,  Gasch,  A.,  Sattler,  M., and  Schultz,  C. 2004. Genetically encoded FRET probe for PKC activity based on pleckstrin. J. Am. Chem. Soc., 126: 11786–11787.
•  Schlipalius,  D.I.,  Valmas,  N.,  Tuck,  A.G.,  Jagadeesan,  R.,  Ma,  L.,  Kaur,  R.,  Goldinger,  A.,  Anderson,  C.,  Kuang,  J.,  Zuryn,  S., et al. 2012. A Core Metabolic Enzyme Mediates Resistance to Phosphine Gas. Science (80-.), 338: 807–810.
•  Schmierer,  B. and  Hill,  C.S. 2005. Kinetic Analysis of Smad Nucleocytoplasmic Shuttling Reveals a Mechanism for Transforming Growth Factor -Dependent Nuclear Accumulation of Smads. Mol. Cell. Biol., 25: 9845–9858.
•  Seong,  J.,  Ouyang,  M.,  Kim,  T.,  Sun,  J.,  Wen,  P.C.,  Lu,  S.,  Zhuo,  Y.,  Llewellyn,  N.M.,  Schlaepfer,  D.D.,  Guan,  J.L., et al. 2011. Detection of focal adhesion kinase activation at membrane microdomains by fluorescence resonance energy transfer. Nat. Commun., 2: 406.
•  Shimomura,  O.,  Johnson,  F.H., and  Saiga,  Y. 1962. Extraction, Purification and Properties of Aequorin, a Bioluminescent Protein from the Luminous Hydromedusan,Aequorea. J. Cell. Comp. Physiol., 59: 223–239.
•  Shirai,  Y.,  Segawa,  S.,  Kuriyama,  M.,  Goto,  K.,  Sakai,  N., and  Saito,  N. 2000. Subtype-specific translocation of diacylglycerol kinase alpha and gamma and its correlation with protein kinase C. J. Biol. Chem., 275: 24760–24766.
•  Spencer,  S.L.,  Cappell,  S.D.,  Tsai,  F.-C.,  Overton,  K.W.,  Wang,  C.L., and  Meyer,  T. 2013. The proliferation-quiescence decision is controlled by a bifurcation in CDK2 activity at mitotic exit. Cell, 155: 369–383.
•  Stryer,  L. 1978. Fluorescence Energy Transfer as a Spectroscopic Ruler. Annu. Rev. Biochem., 47: 819–846.
•  Su,  T.,  Pan,  S.,  Luo,  Q., and  Zhang,  Z. 2013. Monitoring of dual bio-molecular events using FRET biosensors based on mTagBFP/sfGFP and mVenus/mKOκ fluorescent protein pairs. Biosens. Bioelectron., 46: 97–101.
•  Sun,  Y.,  Rombola,  C.,  Jyothikumar,  V., and  Periasamy,  A. 2013. Förster Resonance Energy Transfer Microscopy and Spectroscopy for Localizing Protein – Protein Interactions in Living Cells. Cytom. Part A, 83: 780–793.
•  Szymczak,  A.L.,  Workman,  C.J.,  Wang,  Y.,  Vignali,  K.M.,  Dilioglou,  S.,  Vanin,  E.F., and  Vignali,  D.A.A. 2004. Correction of multi-gene deficiency in vivo using a single “self-cleaving” 2A peptide-based retroviral vector. Nat. Biotechnol., 22: 589–594.
•  Takao,  K.,  Okamoto,  K.-I.,  Nakagawa,  T.,  Neve,  R.L.,  Nagai,  T.,  Miyawaki,  A.,  Hashikawa,  T.,  Kobayashi,  S., and  Hayashi,  Y. 2005. Visualization of synaptic Ca2+ /calmodulin-dependent protein kinase II activity in living neurons. J. Neurosci., 25: 3107–3112.
•  Takaoka,  S.,  Kamioka,  Y.,  Takakura,  K.,  Baba,  A.,  Shime,  H.,  Seya,  T., and  Matsuda,  M. 2016. Live imaging of transforming growth factor-β activated kinase 1 activation in Lewis lung carcinoma 3LL cells implanted into syngeneic mice and treated with polyinosinic:polycytidylic acid. Cancer Sci., 107: 644–652.
•  Terai,  K. and  Matsuda,  M. 2005. Ras binding opens c-Raf to expose the docking site for mitogen-activated protein kinase kinase. EMBO Rep., 6: 251–255.
•  Terai,  K. and  Matsuda,  M. 2006. The amino-terminal B-Raf-specific region mediates calcium-dependent homo- and hetero-dimerization of Raf. EMBO J., 25: 3556–3564.
•  Timm,  T.,  von Kries,  J.P.,  Li,  X.,  Zempel,  H.,  Mandelkow,  E., and  Mandelkow,  E.-M. 2011. Microtubule affinity regulating kinase activity in living neurons was examined by a genetically encoded fluorescence resonance energy transfer/fluorescence lifetime imaging-based biosensor: inhibitors with therapeutic potential. J. Biol. Chem., 286: 41711–41722.
•  Ting,  A.Y.,  Kain,  K.H.,  Klemke,  R.L., and  Tsien,  R.Y. 2001. Genetically encoded fluorescent reporters of protein tyrosine kinase activities in living cells. Proc. Natl. Acad. Sci., 98: 15003–15008.
•  Tomida,  T.,  Takekawa,  M.,  O’Grady,  P., and  Saito,  H. 2009. Stimulus-specific distinctions in spatial and temporal dynamics of stress-activated protein kinase kinase kinases revealed by a fluorescence resonance energy transfer biosensor. Mol. Cell. Biol., 29: 6117–6127.
•  Tomida,  T.,  Oda,  S.,  Takekawa,  M.,  Iino,  Y., and  Saito,  H. 2012. The Temporal Pattern of Stimulation Determines the Extent and Duration of MAPK Activation in a Caenorhabditis elegans Sensory Neuron. Sci. Signal., 5: ra76-ra76.
•  Tomida,  T.,  Takekawa,  M., and  Saito,  H. 2015. Oscillation of p38 activity controls efficient pro-inflammatory gene expression. Nat. Commun., 6: 8350.
•  Tsou,  P.,  Zheng,  B.,  Hsu,  C.-H.,  Sasaki,  A.T., and  Cantley,  L.C. 2011. A fluorescent reporter of AMPK activity and cellular energy stress. Cell Metab., 13: 476–486.
•  Ubersax,  J.A. and  Ferrell,  J.E. 2007. Mechanisms of specificity in protein phosphorylation. Nat. Rev. Mol. Cell Biol., 8: 530–541.
•  Ueda,  Y.,  Kwok,  S., and  Hayashi,  Y. 2013. Application of FRET probes in the analysis of neuronal plasticity. Front. Neural Circuits, 7: 163.
•  Vandame,  P.,  Spriet,  C.,  Riquet,  F.,  Trinel,  D.,  Cailliau-Maggio,  K., and  Bodart,  J.-F. 2014. Optimization of ERK activity biosensors for both ratiometric and lifetime FRET measurements. Sensors (Basel), 14: 1140–1154.
•  Varnai,  P. 2005. Selective cellular effects of overexpressed pleckstrin-homology domains that recognize PtdIns(3,4,5)P3 suggest their interaction with protein binding partners. J. Cell Sci., 118: 4879–4888.
•  Vega,  R.B.,  Harrison,  B.C.,  Meadows,  E.,  Roberts,  C.R.,  Papst,  P.J.,  Olson,  E.N., and  McKinsey,  T.A. 2004. Protein kinases C and D mediate agonist-dependent cardiac hypertrophy through nuclear export of histone deacetylase 5. Mol. Cell. Biol., 24: 8374–8385.
•  Vial,  E. and  Marshall,  C.J. 2003. Elevated ERK-MAP kinase activity protects the FOS family member FRA-1 against proteasomal degradation in colon carcinoma cells. J. Cell Sci., 116: 4957–4963.
•  Violin,  J.D.,  Zhang,  J.,  Tsien,  R.Y., and  Newton,  A.C. 2003. A genetically encoded fluorescent reporter reveals oscillatory phosphorylation by protein kinase C. J. Cell Biol., 161: 899–909.
•  Wang,  Y.,  Botvinick,  E.L.,  Zhao,  Y.,  Berns,  M.W.,  Usami,  S.,  Tsien,  R.Y., and  Chien,  S. 2005. Visualizing the mechanical activation of Src. Nature, 434: 1040–1045.
•  Wilson,  M.Z.,  Ravindran,  P.T.,  Lim,  W.A., and  Toettcher,  J.E. 2017. Tracing Information Flow from Erk to Target Gene Induction Reveals Mechanisms of Dynamic and Combinatorial Control. Mol. Cell, 67: 757–769.e5.
•  Xiang,  X.,  Sun,  J.,  Wu,  J.,  He,  H.T.,  Wang,  Y., and  Zhu,  C. 2011. A fret-based biosensor for imaging syk activities in living cells. Cell. Mol. Bioeng., 4: 670–677.
•  Yamada,  A.,  Hirose,  K.,  Hashimoto,  A., and  Iino,  M. 2005. Real-time imaging of myosin II regulatory light-chain phosphorylation using a new protein biosensor. Biochem. J., 385: 589–594.
•  Yasuda,  R. 2006. Imaging spatiotemporal dynamics of neuronal signaling using fluorescence resonance energy transfer and fluorescence lifetime imaging microscopy. Curr. Opin. Neurobiol., 16: 551–561.
•  Yoshizaki,  H.,  Mochizuki,  N.,  Gotoh,  Y., and  Matsuda,  M. 2006. Akt-PDK1 Complex Mediates Epidermal Growth Factor-induced Membrane Protrusion through Ral Activation. Mol. Biol. Cell, 18: 119–128.
•  Zhang,  J.,  Ma,  Y.,  Taylor,  S.S., and  Tsien,  R.Y. 2001. Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering. Proc. Natl. Acad. Sci. USA, 98: 14997–15002.
•  Zhang,  J.,  Campbell,  R.E.,  Ting,  A.Y., and  Tsien,  R.Y. 2002. Creating new fluorescent probes for cell biology. Nat. Rev. Mol. Cell Biol., 3: 906–918.
•  Zhang,  J.,  Hupfeld,  C.J.,  Taylor,  S.S.,  Olefsky,  J.M., and  Tsien,  R.Y. 2005. Insulin disrupts beta-adrenergic signalling to protein kinase A in adipocytes. Nature, 437: 569–573.
•  Zhang,  J.,  Yang,  P.L., and  Gray,  N.S. 2009. Targeting cancer with small molecule kinase inhibitors. Nat. Rev. Cancer, 9: 28–39.
•  Zhou,  X.,  Clister,  T.L.,  Lowry,  P.R.,  Seldin,  M.M.,  Wong,  G.W., and  Zhang,  J. 2015. Dynamic Visualization of mTORC1 Activity in Living Cells. Cell Rep., 10: 1767–1777.