Development of a FRET Biosensor with High Specificity for Akt

Abstract

The serine/threonine kinase Akt plays a critical role in cell proliferation, survival, and tumorigenesis. As a central kinase in the phosphatidylinositol 3-kinase pathway, its activation mechanism at the plasma membrane has been well characterized. However, the subcellular Akt activity in living cells is still largely unknown. Fluorescence resonance energy transfer (FRET)-based biosensors have emerged as indispensable tools to visualize the subcellular activities of signaling molecules. In this study, we developed a highly specific FRET biosensor for Akt based on the Eevee backbone, called Eevee-iAkt. Using inhibitors targeting kinases upstream and downstream of Akt, we showed that Eevee-iAkt specifically monitors Akt activity in living cells. To visualize Akt activity at different subcellular compartments, we targeted Eevee-iAkt to raft and non-raft regions of the plasma membrane, mitochondria, and nucleus in HeLa and Cos7 cells. Interestingly, we revealed substantial differences in Akt activation between HeLa and Cos7 cells upon epidermal growth factor (EGF) stimulation: Akt was transiently activated in HeLa cells with comparable levels at the plasma membrane, cytosol, and mitochondria. In contrast, sustained and spatially localized Akt activation was observed in EGF-stimulated Cos7 cells. We found high Akt activity at the plasma membrane, low activity in the cytosol, and no detectable activity at the mitochondria and nucleus in Cos7 cells. The Eevee-iAkt biosensor was shown to be a valuable tool to study the functional relationship between subcellular Akt activation and its anti-apoptotic role in living cells.

Introduction

The serine/threonine kinase Akt, also known as protein kinase B, is a critical mediator of signaling pathways regulating diverse cellular processes such as cell growth, glucose metabolism, motility, proliferation, and survival (Manning and Cantley, 2007). Abnormal Akt signaling causes a wide variety of disorders, including cancer (Nicholson and Anderson, 2002). All three mammalian isoforms, Akt1, Akt2, and Akt3, share a conserved domain structure consisting of an N-terminal pleckstrin homology (PH) domain, a central catalytic domain, and a C-terminal hydrophobic motif (Brazil et al., 2004; Hanada et al., 2004). The activation of Akt is a multi-step process initiated by various stimuli, including growth factors (Burgering and Coffer, 1995). Growth factors bind to their cognate receptor tyrosine kinases, which turns phosphatidylinositol 3-kinase (PI3K) on. Activated PI3K then generates the second messenger phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P₃] (Auger et al., 1989; Carpenter and Cantley, 1990). Because the PH domain of Akt recognizes PI(3,4,5)P₃ and its derivative PI(3,4)P₂, PI3K activation triggers recruitment of Akt to the plasma membrane, where Akt becomes activated via two phosphorylation events (Franke et al., 1997): Phosphorylation of Thr308 of Akt1 in the activation loop of the catalytic domain is a pre-requisite for activation and is mediated by 3-phosphoinositide-dependent protein kinase-1 (PDK1), which also binds to PI(3,4,5)P₃ via the PH domain (Alessi et al., 1997). Meanwhile, Ser473 of Akt1 in the hydrophobic motif is phosphorylated by the mTOR complex 2 (mTORC2), leading to full activation of Akt (Sarbassov et al., 2005). Furthermore, a tumor suppressor, phosphatase and tensin homologue deleted on chromosome 10 (PTEN), dephosphorylates PI(3,4,5)P₃ to produce PI(4,5)P₂, and thereby suppresses Akt activation (Maehama and Dixon, 1998). The SH2 domain-containing inositol 5′-phosphatase (SHIP), however, dephosphorylates PI(3,4,5)P₃ to generate PI(3,4)P₂, which has the potential to activate Akt (Damen et al., 1996).

Subcellular localization is critical for the activation of and signal transduction from Akt. At the plasma membrane, lipid rafts have been suggested to be the preferential Akt activation site (Gao et al., 2011; Gao and Zhang, 2008; Lasserre et al., 2008). After leaving the plasma membrane, Akt phosphorylates and regulates numerous substrates in the cytosol: Akt phosphorylates glycogen synthase kinase 3β (GSK3β) to block the kinase activity on glycogen synthase and β-catenin (Cross et al., 1995). Akt Phosphorylation of tuberous sclerosis complex 2 (TSC2) and proline-rich Akt substrate of 40 kDa (PRAS40) induces the activation of mammalian target of rapamycin complex 1 (mTORC1) signaling, which potentiates the translational activity (Inoki et al., 2002; Sancak et al., 2007). Moreover, Akt phosphorylates an E3 ubiquitin ligase, Mdm2, leading to the cytoplasmic retention and inhibition of Mdm2-mediated p53 degradation (Mayo and Donner, 2001; Zhou et al., 2001). The phosphorylated Akt is also found in the nucleus in various cell lines (Borgatti et al., 2000, 2003; Wang and Brattain, 2006). Upon growth factor stimulation the phosphorylated Akt is translocated from the cytoplasm to the nucleus via an active process (Andjelkovic et al., 1997; Borgatti et al., 2000, 2003; Xuan Nguyen et al., 2006). Alternatively, Akt may be phosphorylated and activated in the nucleus (Wang and Brattain, 2006). The Akt in the nucleus phosphorylates members of the FoxO subfamily of forkhead transcription factors, promoting nuclear exclusion and thereby inhibition of transcription of death genes (Biggs et al., 1999; Brunet et al., 1999; Kops et al., 1999). Akt has also been found to rapidly translocate to mitochondria upon PI3K activation (Bijur and Jope, 2003). Phosphorylation of a pro-apoptotic BH3-only protein, BAD, by Akt causes BAD to dissociate from the prosurvival Bcl-2/Bcl-X complex, which is localized to the mitochondrial outer membrane, and thus inhibits its cell death functions (Datta et al., 1997; Yang et al., 1995).

Genetically encoded biosensors based on fluorescent proteins and Förster/fluorescence resonance energy transfer (FRET) have been developed in order to visualize the subcellular activities of signaling molecules (Aoki et al., 2008; Miyawaki, 2003). These FRET biosensors have enabled visualization of the spatio-temporal dynamics of signaling events in living cells, which could not be adequately investigated using the techniques of conventional biochemistry. Several FRET-based Akt biosensors have previously been developed, such as Akind (Yoshizaki et al., 2007), which monitors the conformational change of Akt upon activation, and Aktus (Sasaki et al., 2003), BKAR (Kunkel et al., 2005), and Eevee-Akt (Komatsu et al., 2011), which detect substrate phosphorylation by Akt. However, these biosensors were found to have technical drawbacks: Akind expression induced excess Akt signaling from the biosensor, overexpression of Akt was required to see clear signals from Aktus (Sasaki et al., 2003), and BKAR exhibited low sensitivity (Kunkel et al., 2005).

Here, we present a novel Akt biosensor with improved sensitivity and specificity based on the optimized Eevee backbone (Komatsu et al., 2011), called Eevee-iAkt (the “i” stands for improved). Using inhibitors targeting the PI3K-Akt-mTOR pathway, we show that Eevee-iAkt specifically monitors Akt activity in living cells. To visualize Akt activity at different subcellular compartments, we further targeted Eevee-iAkt to raft and non-raft regions of the plasma membrane, the nucleus and mitochondria in various cell lines. Interestingly, we found that Akt was rapidly and transiently activated upon epidermal growth factor (EGF) stimulation in HeLa cells with similar amplitudes at the plasma membrane, the cytosol and the mitochondria. In contrast, sustained and spatially-localized Akt activation was observed in EGF-stimulated Cos7 cells, with high Akt activity at the plasma membrane, low activity in the cytosol, and no detectable activity at the mitochondria and nucleus. This is the first comparative study of Akt activity across the main locations where Akt is known to be localized in living cells.

Materials and Methods

Materials

Blasticidin S was purchased from InvivoGen (San Diego, CA). Calyculin A and EGF were purchased from Sigma-Aldrich (St. Louis, MO). LY294002, PI-103, dibutyryl cyclic AMP (dbcAMP), and 12-O-tetradecanoylphorbol-13-acetate (TPA) were purchased from Calbiochem (La Jolla, CA). Rapamycin was obtained from LC Laboratories (Woburn, MA). Torin1 was purchased from Tocris Bioscience (Bristol, UK). The indole-modified rapamycin analog, iRAP, was the kind gift of T. Inoue (Inoue et al., 2005). Anti-Akt (pan) (C67E7) rabbit monoclonal antibody and anti-phospho-Akt (Ser473) (587F11) mouse monoclonal antibody were purchased from Cell Signaling Technology (Danvers, MA). The IRDye680LT- and IRDye800CW-conjugated anti-rabbit and anti-mouse immunoglobulin G secondary antibodies were obtained from LI-COR (Lincoln, NE).

FRET biosensors

All Eevee-iAkt constructs were generated through substitution of the kinase substrate sequence and the localization sequence in the previously described Eevee backbone (Komatsu et al., 2011). From the N-terminus, Eevee-iAkt consists of YPet, a spacer (Leu-Glu), the FHA1 domain of yeast Rad53, a spacer (Gly-Thr), the EV Linker, a spacer (Ser-Gly), an optimized Akt substrate sequence derived from human glycogen synthase kinase 3β (GSK3β) (SGRPRTTTFADSCKP), a spacer (Gly-Gly-Arg), enhanced CFP (ECFP), a spacer (Ser-Arg), and nuclear export sequence of HIV-1 rev protein (LQLPPLERLTLD). The substrate sequence of GSK3β was modified in such a way that the Ser residue at the phosphorylation site was replaced by Thr and the residue at the +3 position from the said Thr was substituted with Asp for optimal FHA1 binding. In Eevee-iAkt-T/A, the phosphoacceptor of Thr was mutated to Ala. In the constructs Eevee-iAkt-nls, Eevee-iAkt-pm (KRas), and Eevee-iAkt-pm (HRas), the C-terminal localization signal was substituted by the nuclear localization signal of the SV40 large T-antigen (PKKKRKV), the C-terminal region of human KRas (KMSKDGKKKKKKSKTKCVIM), and the C-terminal region of human HRas (KLNPPDESGPGCMSCKCVLS), respectively. In the case of Eevee-iAkt-pm (Lyn) and Eevee-iAkt-mito, additional localization signals were N-terminally added; these were the first 13 amino acids of human Lyn kinase (MGCIKSKGKDSLS), and the mitochondrial localization sequence of human phospholipase D6 (MGRLSWQVAAAAAVGLALTLEALPWVLRWLRSRRRRPRR). The cDNAs of the biosensors were inserted into the eukaryotic expression vectors pCAGGS (Niwa et al., 1991) for transient expression and/or pPBbsr for stable expression (Yusa et al., 2009). The FRET biosensors Eevee-Akt-cyt, BKAR, Aktus, and Akind were reported previously (Komatsu et al., 2011; Kunkel et al., 2005; Sasaki et al., 2003; Yoshizaki et al., 2007).

Cell culture and transfection

HeLa cells were purchased from the Human Science Research Resources Bank (Sennanshi, Japan). The Cos7 cells used in this study were Cos7/E3, a subclone of Cos7 cells established by Y. Fukui (National Health Research Institutes, Zhunan, Republic of China). HeLa and Cos7 cells were maintained in DMEM (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum at 37°C, 5% CO₂. For transient expression of the FRET biosensors, HeLa and Cos7 cells were transfected using 293fectin (Invitrogen, San Diego, CA) and FugeneHD (Promega, Madison, WI), respectively, according to the manufacturers’ instructions. For stable expression, the pPBbsr-based FRET biosensor and pCMV-mPBase(neo-) encoding the piggyBac transposase (provided by A. Bradley, Welcome Trust Sanger Institute, Cambridge, UK (Yusa et al., 2009)) were cotransfected into HeLa cells using 293fectin at a ratio of 1:4, and cells were selected with 20 μg/ml blasticidin S for at least 10 days. The rapamycin-inducible PI3K activation system was constructed according to Suh et al. (2006); HeLa cells stably expressing Eevee-iAkt were cotransfected with pCAGGS-GST-FKBP-iSH2 and pERedNLS-LDR using 293fectin according to the manufacturers’ instructions.

Time-lapse FRET imaging

FRET imaging was essentially performed as previously reported (Aoki and Matsuda, 2009). Cells were plated on 35 mm glass base dishes, transfected, and after 24 h starved for 3–6 h in phenol red–free M199 (Invitrogen, Carlsbad, CA), 20 mM HEPES (Invitrogen, Carlsbad, CA), and 0.1% BSA. Cells were treated with stimuli and inhibitors, if necessary. Imaging was performed with an IX81 inverted microscope (Olympus, Tokyo, Japan) equipped with a UPlanSApo 40×/0.95 or PlanApo 60×/1.40 oil objective lens (Olympus), a CoolSNAP K4 CCD camera (Roper Scientific, Tucson, AZ), a CoolLED precisExcite LED illumination system (Molecular Devices, Sunnyvale, CA), an IX2-ZDC laser-based autofocusing system (Olympus), and an MD-XY30100T-Meta automatically programmable XY stage (SIGMA KOKI, Tokyo, Japan). The following filters used for the dual-emission imaging studies were obtained from Omega Optical (Brattleboro, VT): an XF1071 440AF21 excitation filter, an XF2034 455DRLP dichroic mirror, and an XF3075 480AF30 emission filter for CFP, and a XF3079 535AF26 emission filter for FRET. After background subtraction, FRET/CFP ratio images were created with Meta-Morph software (Universal Imaging, West Chester, PA), and represented in the intensity modulated display mode. In the intensity-modulated display mode, eight colors from red to blue are used to represent the FRET/CFP ratio, with the intensity of each color indicating the mean intensity of FRET and CFP. For the quantification, the FRET and CFP intensities were averaged over the whole cell area, and the results were exported to Excel software (Microsoft Corporation, Redmond, WA). The FRET/CFP values before stimulation were averaged and used as a reference. The ratio of the raw FRET/CFP value to the reference value was defined as the normalized FRET/CFP value.

Confocal imaging

Cells were plated on 35 mm glass base dishes and transfected, and after 24 h the localization of the biosensors was analyzed in phenol red–free M199, 20 mM HEPES, and 0.1% BSA. Imaging was performed with an FV1000-IX81 inverted confocal microscope equipped with a UPlanSApo 60x/1.35 oil lens. The excitation and fluorescence filter settings were as follows: 440 nm excitation laser, DM 405–440/515 excitation dichroic mirror, SDM 510 CFP channel PMT dichroic mirror, CFP channel PMT spectral setting 460–500 nm, FRET channel PMT dichroic mirror, FRET channel spectral setting 515–615 nm.

Immunoblotting

HeLa and Cos7 cells were starved for 3 h in phenol red–free M199, 20 mM HEPES, and 0.1% BSA and stimulated with 10 ng/ml EGF and 50 ng/ml EGF, respectively, for the indicated times. Cells were lysed in 1x SDS sample buffer (1M Tris-HCl pH 6.8, 50% glycerol, 10% SDS, 0.2% Bromo Phenol Blue, and 10% 2-mercaptoethanol). After sonication, the samples were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA). After blocking with Odyssey blocking buffer (LI-COR, Lincoln, NE) for 1 h, the membranes were incubated with primary antibodies diluted in Odyssey blocking buffer, followed by secondary antibodies diluted in Odyssey blocking buffer. Proteins were detected by an Odyssey Infrared scanner (LI-COR, Lincoln, NE) and analyzed by using the Odyssey imaging software.

Results

Design of Eevee-iAkt

Eevee-iAkt is a genetically encoded intramolecular FRET biosensor designed to monitor Akt activity in living cells. An intramolecular FRET biosensor of a protein kinase typically comprises a phosphopeptide-binding domain (PBD), linker, and substrate sequence concatenated between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) serving as FRET donor and acceptor, respectively (Ni et al., 2006). Phosphorylation of the substrate sequence causes a conformational change due to binding of the PBD to the phosphorylated substrate, leading to an increase in the FRET efficiency (Fig. 1A). All Eevee-iAkt constructs developed in this study were based on the optimized Eevee backbone (Komatsu et al., 2011), which consisted of the optimized fluorescent protein pair, YPet and ECFP, and the long flexible EV-linker, which rendered the biosensor “distance-dependent.” We used an Akt substrate sequence of GSK3β, SGRPRTTTFADSCKP, as the substrate, in which the phosphorylation site Ser was replaced by a Thr (underlined) and the +3 residue from the said Thr was changed to an Asp to increase affinity to the PBD, FHA1. For the analysis of subcellular Akt activity, various localization sequences were added at the C-termini (Fig. 1B): The nuclear export sequence (NES) targets the biosensor to the cytosol and the nuclear localization signal (NLS) targets the biosensor to the nucleus. The CAAX sequence along with a polybasic motif from KRas provides anchoring to the non-raft plasma membrane, whereas the CAAX sequence with the adjacent palmitoylation sites from HRas and the N-terminal portion of Lyn kinase provide localization to raft regions of the plasma membrane (Prior et al., 2001; Zacharias et al., 2002). The HRas CAAX box additionally targets the sensor to the Golgi apparatus (Rocks et al., 2005). For targeting to the mitochondrial outer membrane, the mitochondrial localization sequence of human phospholipase D6 was added (Choi et al., 2006). To confirm their specific targeting, HeLa cells expressing the differently localized biosensors were imaged using confocal microscopy. The images indicated the correct localization of each biosensor in HeLa cells (Fig. 1C). Eevee-iAkt was found at the cytosol. Eevee-iAkt-nls was restricted to the nucleus. Eevee-iAkt-pm (KRas), Eevee-iAkt-pm (HRas), and Eevee-iAkt-pm (Lyn) were localized at the plasma membrane. In addition, we detected Eevee-iAkt-pm (HRas) at a location in the center of the cell, most likely the Golgi. Finally, Eevee-iAkt-mito was localized to a tubular network, which was typical for mitochondria.

Fig. 1

Structure of Eevee-iAkt FRET biosensors. (A) Mode of action of an intramolecular FRET biosensor for Akt, Eevee-iAkt. Phosphorylation of the substrate sequence by the kinase induces binding of the PBD to the phosphorylated sequence, resulting in a conformational change and concomitant increase in FRET efficiency from the FRET donor CFP to the acceptor YFP. (B) Structure of the Akt biosensor Eevee-iAkt with different localization signals. Eevee-iAkt comprises YPet (acceptor), an FHA1 phosphopeptide-binding domain, an EV Linker, an Akt substrate sequence derived from GSK3β, ECFP (donor), and the N- or C-terminal localization signals NES, NLS, KRas CAAX, HRas CAAX, Lyn, and mito. (C) HeLa cells expressing Eevee-iAkt, -nls, -pm (KRas), -pm (HRas), -pm (Lyn), and -mito, respectively (from top to bottom). The localization of the biosensors in HeLa cells was determined by confocal microscopy of CFP. Scale bar=10 μm.

Comparison of Eevee-iAkt with previously-developed Akt biosensors

We compared the sensitivity and specificity of Eevee-iAkt with those of previously-developed Akt biosensors: Eevee-Akt-cyt, Aktus, and BKAR are structurally very similar to iAkt, whereas Akind comprises Akt and fluorescent proteins designed to monitor the conformational change of Akt (Fig. 2A). Eevee-iAkt differs from Eevee-Akt-cyt only in the substrate sequence (Fig. 2B and 2D), while Aktus and BKAR consist of different fluorescent protein pairs, substrate sequences, PBDs, and linkers (Fig. 2F and 2H). Eevee-Akt-cyt and BKAR share the Akt phosphorylation consensus sequence as substrate peptide (Fig. 2D and 2H). It should be noted that BKAR exhibits a decrease in FRET efficiency. Therefore, the CFP/FRET ratio was used to show the phosphorylation of the substrate sequence. Akind is composed of the PH domain of Akt, Venus, the catalytic and regulatory domains of Akt, and CFP, and therefore mimicked Akt. Phosphorylation of the corresponding Thr308 and Ser473 induces a conformational change which results in an increase in the FRET/CFP ratio (Fig. 2J).

Fig. 2

Comparison of Eevee-iAkt with previously developed Akt sensors. (A) HeLa cells transiently expressing Eevee-iAkt, Eevee-Akt, BKAR, Aktus, and Akind, respectively, were serum-starved for at least 3 h and stimulated with 10 ng/ml EGF. The CFP and FRET intensities of each cell were monitored by time-lapse epifluorescence microscopy. The FRET/CFP ratio was normalized by the averaged FRET/CFP value before stimulation. The mean normalized FRET/CFP ratios (except for BKAR, for which CFP/FRET was used) and SD (Eevee-iAkt, n=40; Eevee-Akt, n=40; BKAR, n=40; Aktus, n=40; Akind, n=20) are shown. (B) Mode of action of Eevee-iAkt. (C) HeLa cells expressing Eevee-iAkt were serum-starved for at least 3 h, pre-treated with 10 μM PI-103 and 10 μM LY294002, or DMSO as a control, and stimulated with 10 ng/ml EGF. The mean normalized FRET/CFP ratios are shown with the SD (DMSO, n=20; PI-103, n=10; LY294002, n=20). (D) Mode of action of Eevee-Akt-cyt. (E) HeLa cells transiently expressing Eevee-Akt-cyt were serum-starved for at least 3 h, pre-treated with 10 μM PI-103, 20 μM LY294002, or DMSO as a control, and stimulated with 10 ng/ml EGF. The mean normalized FRET/CFP ratios and SD (DMSO, n=40; PI-103, n=40; LY294002, n=40) are shown. (F) Mode of action of Aktus. (G) HeLa cells transiently expressing Aktus were serum-starved for at least 3 h, pre-treated with 10 μM PI-103 and 20 μM LY294002, or DMSO as a control, and stimulated with 10 ng/ml EGF. The mean normalized FRET/CFP ratios are shown with the SD (DMSO, n=20; PI-103, n=20; LY294002, n=20). (H) Mode of action of BKAR. (I) HeLa cells transiently expressing BKAR were serum-starved for at least 3 h, pre-treated with 10 μM PI-103, 20 μM LY294002, or DMSO as a control, and stimulated with 10 ng/ml EGF. The mean normalized CFP/FRET ratios and SD (DMSO, n=20; PI-103, n=20; LY294002, n=20) are shown. (B) Mode of action of Akind. (K) HeLa cells transiently expressing Akind were serum-starved for at least 3 h, pre-treated with 10 μM PI-103 and 20 μM LY294002, or DMSO as a control, and stimulated with 10 ng/ml EGF. The mean normalized FRET/CFP ratios are shown with the SD (DMSO, n=20; PI-103, n=20; LY294002, n=20).

For comparison of the sensitivity of all biosensors, HeLa cells transiently expressing Eevee-iAkt, Eevee-Akt-cyt, BKAR, Aktus or Akind were serum-starved and stimulated with EGF (Fig. 2A). Eevee-iAkt responded immediately to EGF stimulation, as evidenced by the increase in the FRET/CFP ratio, reaching its maximum of ∼8% 3 min after EGF treatment, followed by the gradual decrease of the emission ratio, demonstrating its reversibility (Fig. 2A). The increase in the FRET/CFP ratio of Eevee-iAkt was greater than those of BKAR and Akind, indicating higher sensitivity. Under the same conditions, a slow and gradual increase of the FRET/CFP ratio was observed for Aktus, which did not necessarily reflect the kinetics of Akt activation (Yoshizaki et al., 2007). Eevee-Akt-cyt exhibited the largest increase of the FRET/CFP ratio (13%) among the biosensors.

We next examined the specificity by PI3K inhibitors, PI-103 and LY294002. Surprisingly, except for Eevee-iAkt and Akind, none of the biosensors could be inhibited significantly by the PI3K inhibitors in the EGF-stimulated HeLa cells (Fig. 2B–K). These data raised strong doubts as to the specificity of Eevee-Akt-cyt, BKAR and Aktus.

Specificity in Eevee-iAkt

We further characterized the specificity and dynamic range of Eevee-iAkt. In EGF-treated HeLa cells, Eevee-iAkt showed a rapid and transient increase in FRET/CFP ratio (approximately 8% at the peak) (Fig. 3A, blue line). Under the same condition, Eevee-iAkt-T/A, in which the threonine at the phosphorylation site was mutated to an alanine, did not respond to EGF stimulation (Fig. 3A, red line). Thus, the observed change in the FRET/CFP ratio of Eevee-iAkt was attributed to phosphorylation of the designed substrate site. The suppression of Ser/Thr phosphatase activities by calyculin A treatment robustly enhanced the increase in the EGF-stimulated response of Eevee-iAkt (Fig. 3B). Furthermore, calyculin A treatment allowed us to estimate the dynamic range of Eevee-iAkt as at least 30% (Fig. 3B). We then examined whether protein kinase A (PKA) and protein kinase C (PKC), which, like Akt, belonged to the AGC-family of kinases, could phosphorylate Eevee-iAkt. Neither dbcAMP nor TPA, activators of PKA and PKC, respectively, induced any remarkable change in the FRET/CFP ratio of Eevee-iAkt (Fig. 3C), excluding the cross-reactivity to PKA and PKC.

Fig. 3

Characterization of Eevee-iAkt. (A) HeLa cells stably expressing Eevee-iAkt or its T/A mutant Eevee-iAkt-TA (transient) were serum-starved for 3 h and stimulated with 10 ng/ml EGF. The mean normalized FRET/CFP ratios are shown with the SD (Eevee-iAkt n=36, Eevee-iAkt-TA n=76). (B) HeLa cells expressing Eevee-iAkt were serum-starved for 3 h, stimulated with 10 ng/ml EGF and subsequently treated with 50 nM calyculin A. The mean normalized FRET/CFP ratios are shown with the SD (n=20). (C) HeLa cells expressing Eevee-iAkt were serum-starved for at least 3 h, and stimulated with 10 ng/ml EGF, 1 mM dbcAMP, and 1 μM TPA, respectively. The mean normalized FRET/CFP ratios are shown with the SD (EGF n=40, dbcAMP n=40, TPA n=40). (D) Scheme of the PI3K-Akt-mTOR pathway with inhibitors. (E) HeLa cells expressing Eevee-iAkt were serum-starved for at least 3 h, stimulated with10 ng/ml EGF, and then treated with 10 μM PI-103, 10 uM LY294002, or DMSO as a control. The mean normalized FRET/CFP ratios are shown with the SD (DMSO, n=20; PI103, n=15; LY294002, n=20). (F) HeLa cells expressing Eevee-iAkt were serum-starved for at least 3 h, pretreated with 5 μM rapamycin, 1 μM Torin1, or DMSO as a control, and stimulated with 10 ng/ml EGF. The mean normalized FRET/CFP ratios are shown with the SD (DMSO, n=20; rapamycin, n=20; Torin1, n=20). (G) HeLa cells expressing Eevee-iAkt were serum-starved for at least 3 h, stimulated with 10 ng/ml EGF, and then treated with 5 μM rapamycin, 1 μM Torin1, or DMSO as control. The mean normalized FRET/CFP ratios are shown with the SD (DMSO, n=20; rapamycin, n=20; Torin1, n=20).

We further investigated the specificity of Eevee-iAkt by inhibitors targeting kinases upstream or downstream of Akt (Fig. 3D). Treatment with the PI3K inhibitors PI-103 and LY294002 completely prevented the EGF-stimulated increase in the FRET/CFP ratio of Eevee-iAkt, as shown in Fig. 2C. The PI3K inhibitor treatment after EGF stimulation resulted in a rapid drop of the FRET/CFP ratio (Fig. 3E). Analogous experiments were conducted with the mTORC1-specific inhibitor, rapamycin, and mTORC1/mTORC2 inhibitor, Torin1 (Fig. 3D). As expected, rapamycin treatment before or after EGF stimulation yielded no differences in comparison to the DMSO control, whereas Torin1 treatment resulted in the inhibition of the EGF-stimulated increase in FRET/CFP ratio and rapid decrease after EGF stimulation (Fig. 3F and 3G).

To further confirm that Eevee-iAkt monitored PI3Kdependent Akt activation, we applied the rapamycin-inducible PI3K activation system to Eevee-iAkt (Suh et al., 2006). This system employs two fusion proteins, the rapamycin-binding domain of the FK506-binding protein (FKBP) fused to the inter-Src homology 2 domain (iSH2) of the regulatory PI3K subunit p85 (FKBP-iSH2) and the FK506-rapamycin-binding (FRB) domain of mTOR fused to the myristoylation signal of Lyn (Lyn11-FRB) (Suh et al., 2006). Stimulation with rapamycin or its analog, iRAP, leads to rapid translocation of FKBP-iSH2 to the membrane-anchored Lyn11-FRB via hetero-dimerization of FKBP and FRB. According to the translocation, the catalytic subunit of PI3K, p110, is recruited to the plasma membrane where it converts PI(4,5)P₂ to PI(3,4,5)P₃ (Fig. 4A). Therefore, this system activates the PI3K pathway more specifically than does EGF stimulation. As expected, the FRET/CFP ratio of Eevee-iAkt rapidly increased upon stimulation with iRAP, indicating that the response of Eevee-iAkt was dependent on the PI3K pathway (Fig. 4B). Consistently, subsequent addition of PI-103 and LY294002 led to a prompt decline of the FRET/CFP ratio (Fig. 4B). The slower inhibition by LY294002 than by PI-103 (Fig. 4B) may be attributable to the higher IC50 value of LY294002 (Chaussade et al., 2007) and/or the difference of those membrane permeability. In addition to these results, Torin1, but not rapamycin, inhibited the increase in the FRET/CFP ratio induced by the rapamycin-inducible PI3K activation system (Fig. 4C). Taken together, these results indicated that Eevee-iAkt specifically senses Akt activity.

Fig. 4

Eevee-iAkt is specific for Akt. (A) Schematic representation of the rapamycin-inducible PI3K activation system. (B) HeLa cells expressing Eevee-iAkt and the two fusion proteins Lyn11-FKB and FRBP-iSH2 were serum-starved for at least 3 h, incubated with 5 μM iRAP for 20 min, and then treated with 10 μM PI-103, 10 μM LY294002, or DMSO as a control. The mean normalized FRET/CFP ratios are shown with the SD (DMSO, n=20; PI-103, n=10; LY294002, n=20). (C) HeLa cells expressing Eevee-iAkt and the two fusion proteins Lyn11-FKB and FRBP-iSH2 were serum-starved for at least 3 h, incubated with 5 μM iRAP for 20 min, and then treated with 5 μM rapamycin, 1 μM Torin1, or DMSO as a control. The mean normalized FRET/CFP ratios are shown with the SD (DMSO, n=20; rapamycin, n=20; Torin1, n=20).

Subcellular Akt activity in HeLa and Cos7 cells

Akt is mainly activated at the plasma membrane, from where the activated Akt is propagated to the cytosol and other subcellular locations. Using the Eevee-iAkt constructs with different subcellular localization signals (Fig. 1B), we aimed to examine the difference in Akt activation among distinct plasma membrane microdomains and among subcellular locations in EGF-stimulated HeLa or Cos7 cells. In HeLa cells, Eevee-iAkt localized in the cytosol exhibited a rapid and transient increase in the FRET/CFP ratio of ∼8%, which reached a maximum value 3 min after EGF stimulation (Fig. 5A and 5B). Similarly to the cytosolic Eevee-iAkt, all three plasma membrane-localized biosensors Eevee-iAkt-pm (KRas), Eevee-iAkt-pm (HRas), and Eevee-iAkt-pm (Lyn) demonstrated a comparable increase in the FRET/CFP ratio of ∼8% (Fig. 5A and 5B). No distinct difference in the spatial Akt activation pattern could be detected between the non-raft region as monitored by Eevee-iAkt-pm (KRas) and the raft region as monitored by Eevee-iAkt-pm (HRas) and Eevee-iAkt-pm (Lyn). Thus, we concluded that the activity of Akt at the plasma membrane does not show significant heterogeneity. The time-course and the range of the EGF-induced FRET/CFP ratio change of Eevee-iAkt-mito upon EGF stimulation were almost identical between Eevee-iAkt-mito and the Eevee-iAkt (Fig. 5A and 5B). HeLa cells expressing Eevee-iAkt-nls apparently did not show any response to EGF stimulation. Thus, in EGF-stimulated HeLa cells, Akt showed similar activation patterns in terms of amplitude and subcellular location, except for the nucleus.

Fig. 5

Subcellular Akt activity in HeLa and Cos7 cells. HeLa cells (A, B) and Cos7 cells (C, D) expressing the indicated Eevee-iAkt forms were serum-starved for at least 3 h and stimulated with 10 ng/ml EGF and 50 ng/ml EGF, respectively. The CFP and FRET intensities of each cell were monitored by time-lapse epifluorescence microscopy. Representative FRET/CFP ratio images at the indicated time points are shown in the intensity-modulated display mode. Scale bar=20 μm (A, C). The FRET/CFP ratio of each cell was normalized by the averaged FRET/CFP value before stimulation. The mean normalized FRET/CFP ratios are shown with the SD (HeLa, n>35; Cos7, n>13) (B, D).

In contrast, we observed a different activation pattern of Akt in EGF-stimulated Cos7 cells. The cytoplasmic Eevee-iAkt responded to EGF stimulation with a smaller increase in the FRET/CFP ratio of 2–3% in Cos7 cells than in HeLa cells (Fig. 5C and D). The non-raft-targeted Eevee-iAkt-pm (KRas) detected a rapid and sustained increase of ∼13% upon EGF stimulation diffusely in the plasma membrane (Fig. 5C and 5D). Meanwhile, the raft-targeted biosensors Eevee-iAkt-pm (HRas) and Eevee-iAkt-pm (Lyn) showed smaller increases in the FRET/CFP ratio (increases of 8% and 6%, respectively) upon EGF stimulation than did Eevee-iAkt-pm (KRas). Neither the nuclear-localized Eevee-iAkt-nls nor the mitochondrial-localized Eevee-iAkt-mito responded to EGF stimulation in Cos7 cells (Fig. 5C and 5D). Taken together, these results indicated that Cos7 cells exhibited rapid and sustained Akt activation at the plasma membrane, to a larger extent in non-raft regions of the plasma membrane than in raft regions, but much less in the cytosol and none in the nucleus or mitochondria.

Finally, we confirmed the temporal activation patterns of Akt by immunoblotting. Immunoblotting for the Ser473 phosphorylation of endogenous Akt revealed a transient increase in such phosphorylation in HeLa cells (Fig. 6A) and a more sustained increase in Cos7 cells (Fig. 6B). The temporal dynamics of Ser473 phosphorylation was almost comparable to that observed in Eevee-iAkt, supporting the specificity and validity of Eevee-iAkt.

Fig. 6

Time course of endogenous Akt phosphorylation upon EGF stimulation in HeLa cells and Cos7 cells. HeLa cells (A) and Cos7 cells (B) were serum-starved for at least 3 h and stimulated with 10 ng/ml EGF and 50 ng/ml EGF, respectively. The total amount and phosphorylation of endogenous Akt on Ser473 were analyzed by immunoblotting (top) and quantified (bottom). The Ser473 phosphorylation was normalized by the maximum value (n=3).

Discussion

In this study, we developed Eevee-iAkt, a new FRET-based biosensor monitoring Akt activity in living cells. Eevee-iAkt was highly specific for Akt phosphorylation, reversible, and applicable for monitoring Akt activity in different subcellular compartments. By taking full advantage of Eevee-iAkt, we found the different spatial and temporal dynamics of Akt activity in HeLa and Cos7 cells upon EGF stimulation (Fig. 7).

Fig. 7

Model of Akt signaling in HeLa and Cos7 cells. (A) Upon EGF stimulation, Akt is activated at raft and non-raft regions of the plasma membrane, and subsequently translocates to the cytosol and mitochondria in HeLa cells. The amplitudes of Akt signals are comparable among the plasma membrane, cytosol, and mitochondria. (B) In Cos7 cells, Akt shows higher activation at non-raft regions than raft regions upon EGF stimulation. The Akt activity in the cytosol is significantly low and active Akt does not reach mitochondria and the nucleus.

When HeLa cells were used for the assay, the substrate-type Akt biosensors that have been hitherto reported responded not only to Akt but also other EGF-stimulated kinase(s) (Fig. 2). Most of the previous studies have utilized cells stimulated with PDGF and insulin during the development of the biosensors (Gao and Zhang, 2008; Kunkel et al., 2005; Yoshizaki et al., 2007). PDGF and insulin seem to stimulate the PI3K pathway selectively; therefore, the specificity of the biosensors has not necessarily been fully characterized. Meanwhile, the EGF stimulation used in the present study activates a wide range of kinases, such as PKA, PKC, CaMK, RSK, MAP kinases, and so on (Olsen et al., 2006), one of which might phosphorylate the substrate sequences used in the previous studies. Akt belongs to the AGC family of kinases, which preferentially phosphorylate a common consensus motif comprised of basic amino acids around the phospho-acceptor Ser or Thr (Hornbeck et al., 2012). CaMK family kinases also phosphorylate a similar consensus phosphorylation motif (Hornbeck et al., 2012). Therefore, it is plausible that AGC- and/or CaMK-family kinases phosphorylate the substrate peptides applied in the previous Akt biosensor development.

Our present results revealed a significant subcellular difference in Akt activation between HeLa cells and Cos7 cells. Akt was activated at similar levels in the raft regions, non-raft regions, cytoplasm, and mitochondria in HeLa cells, whereas Akt activation was limited mostly to the plasma membrane in Cos7 cells (Fig. 7). The uneven distribution of Akt activity in Cos7 cells likely arose from the uneven distribution of either active Akt or active phosphatases. Little is known about the dephosphorylation of the FRET biosensors. The substrate specificity and the subcellular localization of serine/threonine phosphatases such as PP1 and PP2 show broad spectra in many cases (Millward et al., 1999). Thus, it is unlikely that uneven phosphatase activities caused the uneven Akt activity distribution in Cos7 cells. Therefore, the spatial gradient might have been due to the high phosphatase activity toward Akt in the cytosol in Cos7 cells. As shown theoretically by Brown and Kholodenko (Brown and Kholodenko, 1999), the distance over which the spatial gradient in the concentration of the phosphorylated form of the substrate decays, L_gradient, is given by   

$$L_{\mathit{gradient}}=\sqrt{\frac{D}{k_p}}$$

where k_p is the first-order phosphatase reaction rate constant and D is the diffusion coefficient for phosphorylated substrate. Thus, in HeLa cells, we suggest that a small amount of phosphatase activity, i.e., small k_p, and thereby phosphorylated Akt can reach the cytoplasm and mitochondria (large L_gradient). On the other hand, given the higher phosphatase activity (large k_p value) in Cos7 cells, phosphorylated Akt is rapidly dephosphorylated and is incapable of reaching the mitochondria (small L_gradient). With respect to the central role of mitochondria in apoptosis, growth factor-induced accumulation of active Akt in mitochondria has been observed in cardiomyocytes and the neuroblastoma cell line SH-SY5Y, where it protects cells from apoptosis (Bijur and Jope, 2003; Miyamoto et al., 2008; Mookherjee et al., 2007; Su et al., 2012). A future study will be needed to evaluate the functional relation between Akt activation and its anti-apoptotic role in living cells.

Torin1, an inhibitor for both mTORC1 and mTORC2 activities, but not Rapamycin, an mTORC1 specific inhibitor, abrogated the EGF-stimulated phosphorylation of Eevee-iAkt (Fig. 3G and 3F), demonstrating that mTORC2 activity is crucial for Akt activation. An earlier study with Akt mutants, in which Thr 308 and/or Ser 473 were replaced with alanine, showed that phosphorylation of both residues is required for maximum Akt activity (Alessi et al., 1997). Later studies showed that the phosphorylation of Ser 473 precedes the phosphorylation of Thr 308 by PDK1 and that phospho-Ser 473 is required for the PDK1’s recognition and activation of Akt/PKB (Scheid et al., 2002; Yang et al., 2002). In 2005, mTORC2 was found to phosphorylate Ser 473 of Akt (Hresko and Mueckler, 2005; Sarbassov et al., 2005). Following in vitro studies confirmed that mTORC2-mediated Ser 473 phosphorylation is necessary for the full activation of Akt (Jacinto et al., 2006; Sarbassov et al., 2005). Consistent with these findings, we observed that inhibition of mTORC2 with Torin1 almost completely impaired phosphorylation of Eevee-iAkt in living cells, emphasizing the critical role of Ser 473 phosphorylation for Akt activation. Seemingly in contrast to these observations, studies with mTORC2-deficient mice showed that, despite defective Ser 473 phosphorylation of Akt, some of Akt targets such as GSK3b, TSC2 and S6K are still phosphorylated (Jacinto et al., 2006; Shiota et al., 2006), suggesting that Ser 473 phosphorylation may rather determines substrate specificity than catalytic activity of Akt. Eevee-iAkt may be useful to dissect the role of Ser 473 phosphorylation in Akt activation.

Eevee-iAkt-nls failed to monitor any marked increase of Akt activity in HeLa and Cos7 cells upon growth factor stimulation. Previous studies have showed phosphorylated Akt in the nucleus of several cell lines, such as PC12 and MCF7E, upon growth factor stimulation by cell fractionation and immunofluorescence (Borgatti et al., 2003; Wang and Brattain, 2006; Xuan Nguyen et al., 2006). In the light of previous studies, our data suggest that nuclear Akt activity is strongly cell type- and/or stimulant-dependent. However, we cannot exclude the possibility that the sensitivity of Eevee-iAkt-nls may be insufficient for detecting nuclear Akt activity in HeLa and Cos7 cells. Future work will be needed to assess the role of Akt in the nucleus.

In conclusion, we described a novel genetically-encoded FRET biosensor that is highly specific to Akt activity.

Acknowledgments

We thank the members of the Matsuda Laboratory for their helpful discussions. We are also grateful to Y. Umezasa and A.C. Newton for the plasmids expressing Aktus and BKAR. KA and MM were supported by the Research Program of Innovative Cell Biology by Innovative Technology (Cell Innovation) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. KA was supported by the JST PRESTO program and by a JSPS KAKENHI (23136504, 25136706). HM, MM and KA designed the project. HM carried out experiments. HM, MM and KA wrote the manuscript. The authors declare that they have no conflict of interest.

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