Time-resolved fluorescence resonance energy transfer kinase assays using physiological protein substrates: Applications of terbium–fluorescein and terbium–green fluorescent protein fluorescence resonance energy transfer pairs

ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 356 (2006) 108–116 www.elsevier.com/locate/yabio

Time-resolved fluorescence resonance energy transfer kinase assays using physiological protein substrates: Applications of terbium–fluorescein and terbium–green fluorescent protein fluorescence resonance energy transfer pairs Steven M. Riddle 1, Kevin L. Vedvik 1, George T. Hanson, Kurt W. Vogel

*

Invitrogen Corp., 501 Charmany Dr., Madison, WI 53719, USA Received 13 March 2006 Available online 9 June 2006

Abstract Fluorescence-based kinase assays using peptide substrates are an established format for high-throughput screening and profiling of kinases. Among fluorescence-based formats, time-resolved fluorescence resonance energy transfer (TR-FRET) using a lanthanide donor species has advantages over other fluorescent formats in being resistant to many types of optical interference such as autofluorescent compounds, scattered light from precipitated compounds, or colored compounds that absorb excitation or emission radiation (‘‘color quenchers’’). By taking advantage of the fact that acceptors such as fluorescein or green fluorescent protein (GFP) can be paired with a terbium donor in a TR-FRET assay, we have developed TR-FRET kinase assays that use physiologically relevant native protein substrates, either labeled with fluorescein or expressed as GFP fusions. Phosphorylation of the labeled protein substrate results in an increase in TR-FRET when incubated with a terbium-labeled antibody that specifically recognizes the phosphorylated product. Thus, a strategy of using terbium-based TR-FRET can be applied to develop kinase assays, and the unique properties of terbium lead to a high degree of flexibility with regard to specific assay design. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Green fluorescent protein; Kinase assay; TR-FRET

The importance of amino acid sequence proximal to the site of phosphorylation has long been recognized as a method for imparting specificity to a kinase for its substrate [1,2]. In addition to such recognition motifs, additional levels of regulation such as modulation of subcellular localization [3,4] or association with substrates (either directly or through scaffolding proteins) [5,6] have been described. Synthetic peptides containing appropriate recognition motifs have proven to be useful tools for the in vitro assay of kinases in a variety of homogenous fluorescent formats [7–12]. Although many tyrosine kinases will readily accept random copolymers of glutamate, *

1

Corresponding author. Fax: +1 608 204 5100. E-mail address: [email protected] (K.W. Vogel). These authors contributed equally to this work.

0003-2697/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2006.05.017

alanine, and tyrosine as a substrate [13,14], such a generic substrate does not exist for serine/threonine kinases, and considerable effort is often required to identify a suitable peptide-based substrate [15]. However, even when suitable peptide substrates are identified they can suffer from poor kinetics relative to those of native protein substrates [16]. Additionally, when attempting to mimic complex biological processes in vitro, the use of physiological protein substrates rather than peptide surrogates is often preferred. The application of time-resolved fluorescence (or Fo¨rster) resonance energy transfer (TR-FRET)2 to assay kinase activity was first described by Mathis [17]. In a 2

Abbreviations used: TR-FRET, time-resolved fluorescence resonance energy transfer; APC, allophycocyanin; GFP, green fluorescent protein; HTS, high-throughput screening; JNK, c-Jun N-terminal kinase.

Kinase assays with physiological substrates / S.M. Riddle et al. / Anal. Biochem. 356 (2006) 108–116

TR-FRET kinase assay, a long-lifetime lanthanide donor species is conjugated to an antibody that specifically binds to a phosphorylated product of a kinase reaction that is labeled with a suitable acceptor fluorophore. This antibody-mediated interaction brings the lanthanide donor and the acceptor into proximity such that resonance energy transfer can take place, resulting in a detectible increase in FRET. Inhibition of the kinase can be detected by a decrease in the FRET signal. Because the excited state lifetime of a lanthanide donor can be on the order of hundreds of microseconds to several milliseconds, FRET can be measured in a gated detection mode following a suitable delay during which interfering background signals completely decay. In this manner, TR-FRET assays are remarkably resistant to matrix components such as autofluorescent compounds or scattered light from precipitated compounds, both of which generate interfering signals with lifetimes several orders of magnitude shorter than that of the lanthanide-specific signal. Additionally, in FRET-based assays the acceptor signal can be ratioed relative to the donor signal [18,19], which provides a robust internal stan-

Excitation / Emission

D A

300

350

400

450

500

550

600

650

Wavelength (nm) Fig. 1. Spectra of terbium chelate excitation (- - -) and emission (—) and fluorescein (orange) or emerald GFP (green) emission. The terbium chelate excitation spectrum follows the absorbance of the CS124 sensitizer that is attached to the chelate structure. The first emission peak of terbium (centered at approximately 490 nm) overlaps with the excitation spectrum of fluorescein or GFP (not shown for clarity). This donor signal, indicated by D, is used as an internal reference for the emission from the acceptor that is measured in the silent region between the first two emission peaks of terbium (indicated by A).

109

dard to compensate for optical effects due to the presence of colored compounds or slight variations in assay volume. Because of these properties, TR-FRET kinase assays have found wide application in the field of high-throughput screening. Initial studies using TR-FRET to assay kinase activity used europium as the donor species and the red fluorescent algae protein allophycocyanin (APC) as the acceptor [17]. Because APC is a multimeric complex that is prone to dissociation at low concentrations, it must be chemically cross-linked to maintain integrity under assay conditions. Additionally, because it is cumbersome to produce substrate-specific APC conjugates, streptavidin-conjugated APC is typically prepared in bulk, which is then used to indirectly label assay-specific biotinylated substrate peptides through interaction with streptavidin. In contrast to europium, the spectral properties of terbium are such that common fluorophores such as fluorescein can be used as the acceptor species [20], facilitating the use of directly fluorescein-labeled substrate peptides in kinase assays (Fig. 1). In addition, many GFP variants will serve as acceptors for terbium, opening the possibility of performing TR-FRET kinase assays on GFP fusions of native, physiologically relevant substrates. In this paper, we report the use of terbium–fluorescein or terbium–GFP FRET pairs to develop TR-FRET kinase assays using physiologically relevant proteins or protein domains (Fig. 2). The method is quite flexible, showing good performance with either fluorescein or GFP as the acceptor, and works with phosphospecific antibodies either directly conjugated with terbium or indirectly labeled through terbium-conjugated species-specific anti-IgG antibodies. Using directly labeled antibodies, we have applied this method to monitor JNK activity with GFP fusions of cJun or ATF2 and p38 kinase activity with a GFP fusion of ATF2, and demonstrated isoform-specific inhibition using a reference inhibitor. We have also quantitatively compared the performance of directly or indirectly labeled antibodies in this format by measuring cRAF activity against a fluorescein-labeled MEK substrate. Finally, we have demonstrated the insensitivity of the assay format to common forms of optical interference that are encountered in HTS assays.

Fig. 2. Schematic of a TR-FRET kinase assay using a GFP fusion of a protein substrate. Association of a terbium-labeled phosphospecific antibody with GFP- (or fluorescein-) labeled, phosphorylated kinase substrate results in resonance energy transfer between terbium and fluorescent label.

110

Kinase assays with physiological substrates / S.M. Riddle et al. / Anal. Biochem. 356 (2006) 108–116

Purified recombinant kinases and terbium chelate labeling reagents were from Invitrogen Corp. (Carlsbad, CA). Affinity-purified, polyclonal, phosphospecific antibodies and SB202190 were from Biosource (Camarillo, CA). SP600125 (JNK inhibitor II) was from Calbiochem (San Diego, CA). Reactive fluorescein label was from Molecular Probes (Eugene, OR). Kinase assays were performed in black low-volume 384-well plates (Part No. 3676 from Corning, Corning, NY) and read on a Tecan Ultra plate reader (Tecan, Durham, NC). TR-FRET values were taken as the ratio of raw acceptor to donor intensities measured over a 200-ls signal integration time following a 100-ls postexcitation delay, averaged over 10 excitations (flashes) per well. No background subtraction or cross-talk correction was required. Acceptor signal was measured using a 520-nm filter with a 25-nm bandwidth, and donor signal was measured using a 495-nm filter with a 10-nm bandwidth (filter set PV003 from Chroma Technology Corp., Rockingham, VT). Both signals were measured using the stock ‘‘fluorescein’’ dichroic mirror supplied by Tecan. Data analysis was performed with GraphPad Prism software (GraphPad Software, Inc., San Diego CA).

mended conditions. The amine-reactive Tb chelate is based upon a TTHA core structure that is terminally functionalized with CS124 on one end and a linker attached to an amine-reactive aromatic isothiocyanate on the other [21,22]. Briefly, the carrier-protein-free antibody is dialyzed against HBS and then concentrated to 5 mg/mL using a Centricon concentrator (Millipore, Billerica, MA). Separately, amine-reactive chelate is dissolved to a concentration of 5 mg/mL in 1.0 M sodium bicarbonate, pH 9.4. The dissolved chelate is then immediately added to the antibody at a 1:10 (vol:vol) ratio. After 4 h at room temperature the antibody is dialyzed twice against HBS to remove unreacted and hydrolyzed label, and the resulting labeled antibody is then stored at 4 °C. We have observed no degradation in performance of antibodies prepared and stored under these conditions for at least 6 months. The labeling efficiency (chelates per antibody) is calculated from the absorbance of the chelate at 343 nm (e343 = 12,570 M1 cm1) and the absorbance of the antibody (e280 = 210,000 M1 cm1) after subtracting out the absorbance of the chelate at 280 nm (1.1 times its absorbance at 343 nm). Under these conditions a labeling efficiency of four to nine chelates per antibody is typical.

Preparation of GFP fusion protein substrates

Kinase reactions and inhibitor assays

Escherichia coli expression plasmids for GFP fusions of kinase substrates cJun (residues 1–179) or ATF2 (residues 19–96) were constructed using the pRSET(B) vector (Invitrogen Corp.) such that the fusion proteins encoded were comprised of (N-terminal to C-terminal) a His-tag, EmGFP, and the kinase substrate. After purification on nitrilotriacetic acid–agarose, purified GFP-tagged substrates were quantified using the empirically determined extinction coefficient for GFP of 40,000 M1 cm1 at 480 nm and stored at 80 °C until use in the kinase assay.

All kinase reactions were performed at room temperature in 50 mM Hepes, pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, and 1 mM EGTA in 384-well low-volume plates. Reactions were allowed to proceed 1 h at room temperature with substrate and ATP in the presence or absence of inhibitor, after which EDTA and terbium-labeled phosphospecific antibody or unlabeled phosphospecific antibody and terbium-labeled species-specific IgG were added. The antibodies and EDTA solutions contained 20 mM Tris, pH 7.5, and 0.01% Nonidet P40. After a brief (10- to 60-min) incubation at room temperature the assay plate was read as described above. Specific assay conditions were as follows: p38a (MAPK14), b (MAPK11), d (MAPK13), c (MAPK12). In duplicate 10-lL assay reactions, a dilution series of p38 MAP kinases was assayed against 400 nM GFP–ATF2 in the presence of 10 lM ATP, which is at or below the Km, app for ATP for these kinases. After 1 h, a 10-lL solution of terbium-labeled anti-p-ATF2 (pThr 71) and EDTA was added to each well, for a final concentration of 2.5 nM antibody and 10 mM EDTA. After a 1 h incubation the plate was read and TR-FRET values were calculated. From this initial activity assay, EC80 concentrations of each kinase (MAPK11 = 1.3 lg/mL, MAPK12 = 1.6 lg/mL, MAPK13 = 250 lg/mL, MAPK14 = 5.2 lg/ mL) were determined for subsequent inhibition studies using SB202190. Inhibition studies were performed in a similar manner against a half-log dilution series of SB202190 and IC50 values determined from the resulting inhibition curves.

Materials and methods

Preparation of fluorescein-labeled MEK1 A fluorescein–MEK1 conjugate was prepared from fulllength inactive wild-type MEK1. After dialysis against HBS (137 mM NaCl, 2.7 mM KCl, and 10 mM Hepes, pH 7.5) the protein was labeled using a 10-fold molar excess of 5-iodoacetaminofluorescein and TCEP at a MEK1 concentration of 6.2 lM. After a 2-h reaction at room temperature, the excess dye was removed by desalting the MEK1 samples over a NAP-5 column into HBS. The labeled MEK1 preparations were stored frozen at 80 °C until used in the assay. Preparation of terbium-labeled antibodies Terbium-labeled antibodies are produced by labeling of phosphospecific antibodies with amine-reactive Tb chelate (Invitrogen) following the manufacturer’s recom-

Kinase assays with physiological substrates / S.M. Riddle et al. / Anal. Biochem. 356 (2006) 108–116

JNK. In triplicate 10-lL assay reactions, a dilution series of JNK1 or JNK2 was assayed against 400 nM GFP– cJun (1–179) in the presence of 100 lM ATP. After 1 h, a 10-lL solution of terbium-labeled anti-phospho-cJun (pSer 73) and EDTA was added to each well, for a final concentration of 2 nM antibody and 10 mM EDTA. After a 1 h incubation the plate was read and TR-FRET values were calculated. For assay robustness (Z 0 ) and interfering compound experiments, the concentration of JNK1 required to effect an approximate 80% change in the TR-FRET value between nonphosphorylated and fully phosphorylated product was used. Control wells containing five-times this concentration of kinase were also measured to verify that the experiments were performed near the EC80 for kinase. For Z 0 experiments, 48 positive control wells and 48 negative control (no ATP) wells were measured, and Z 0 was calculated according to the equation [23] Z 0 ¼ 1  ½ð3rcþ þ 3rc Þ=ðlcþ  lc Þ; where rc+ and rc- are the standard deviations of the positive and negative control wells on the assay plate, respectively, and lc+ and lc- are average values for the positive and negative control wells on the assay plate, respectively. To approximate Z 0 values at lower assay volumes, 4-lL aliquots were removed from the control wells, placed into empty wells, and read following re adjustment of the instrument’s Z axis focal height. Interfering compound experiments were performed by measuring six positive and six negative control wells in the presence of interferant that was added subsequent to the kinase assay. NADPH, tartrazine, and allura red were added to a final concentration of 5 lM, coumarin and fluorescein to a final concentration of 100 nM, and nondairy creamer to a final concentration of 0.5 mg/mL. For JNK inhibition studies using GFP–ATF2 as the substrate, activity assays were performed against a dilution series of JNK1 or JNK2, using 200 nM GFP–ATF2 as the substrate and 2 lM ATP, which was determined to be the concentration of ATP that gave half maximal assay response when the assay was performed against a dilution series of ATP (the ATP Km, app). From the initial activity assay, EC80 concentrations of kinase were determined to be 350 and 650 ng/mL for JNK1 and JNK2, respectively, and these concentrations were used for subsequent inhibition studies using SP600125. Inhibition studies were performed against a threefold dilution series of inhibitor and IC50 values determined from the resulting inhibition curves. cRAF cRAF was assayed in triplicate reaction wells using 100 lM ATP and 400 nM fluorescein–MEK substrate. After 1 h, EDTA and antibodies were added to a final volume of 20 lL. The final concentration of EDTA was 10 mM, the final concentration of Tb-labeled (or unlabeled) anti-p-Mek (Ser 222) was 2 nM, and the reactions developed using unlabeled primary antibody contained 5 nM Tb-labeled anti-rabbit antibody.

111

Results and discussion Assay of kinase activity and inhibition c-Jun N-terminal kinases are members of the MAP kinase family that specifically phosphorylate the AP-1 component c-Jun at Ser-63 and Ser-73 or the leucine zipper transcription factor ATF2 at Thr-69 and Thr-71, following UV irradiation or other stress stimuli. The phosphorylation of cJun by JNK is dependant on a ‘‘docking’’ event mediated by residues 30–60 of the c-Jun substrate [24] and residues within different domains near the JNK active site [25,26]. Similarly, ATF2 contains a docking site for JNKs that shares minimal amino acid identity with that of cJun and that is required for efficient phosphorylation by JNK [27,28]. We evaluated the ability to detect phosphorylation of GFP fusions of c-Jun or ATF2 by titrating JNK1 and JNK2 against either substrate in the presence of ATP and Mg2+, followed by the addition of terbium-labeled phosphospecific antibodies (Fig. 3). Under the conditions tested, the c-Jun fusion was more efficiently phosphorylated, requiring approximately 100-fold less kinase to effect a half-maximal TR-FRET value than was required for the ATF2 substrate. Additionally, the relative size of the assay window was larger for the c-Jun substrate. However, for either substrate the error associated with each measurement was extremely small. SP600125 is a recently described inhibitor of JNK1 and JNK2 [29], and was discovered using a high-throughput screen using gluthione-S-transferase-tagged ATF2 [30]. The previously described high-throughput assay involves JNK-mediated phosphorylation of immobilized ATF2 that was then followed by a plate-washing step, addition of europium-labeled antibody, another plate-washing step, and finally liberation and development of the europium label. In this format, an IC50 value of 40 nM was determined for inhibition of both JNK1 and JNK2. When tested in our format the IC50 values were approximately threefold higher, with IC50 values of 143 nM for JNK1 and 120 nM for JNK2 (Fig. 3B). These relatively minor differences are likely results of differences in assay conditions and format. Like the JNKs, the p38 family of MAP kinases are involved in stress-mediated signal transduction and as such are a potential target for therapeutic intervention in inflammatory and other disease states. Using GFP–ATF2 as a substrate, p38a, p38b, p38c, and p38d were evaluated against the inhibitor SB202190. SB202190 is reported to be a potent and selective p38 MAP kinase inhibitor, inhibiting the p38a and p38b isoforms but not the p38c or p38d isoforms, ERK2, other members of the MAP kinase family, or their upstream activators. This selectivity makes SB202190 a useful tool for dissecting the role of p38 in signaling pathways. SB202190 is reported to have IC50 values of 50 nM for p38a and 100 nM for p38b and negligible activity against the d or c isoforms when measured in a radiometric assay [31]. When SB202190 was tested as an inhibitor in the TR-FRET format using GFP–ATF2 as

Kinase assays with physiological substrates / S.M. Riddle et al. / Anal. Biochem. 356 (2006) 108–116

A

B Emission Ratio

1.25 1.00 0.75 0.50 0.25

C

Normalized Emission Ratio

0.001 0.01 0.1 1 10 100 100010000 Kinase (ng/mL)

Normalized Emission Ratio

112

1.00 0.75 0.50 0.25 0.00 0.1

1

10 100 1000 10000 [SP600125] (nM)

1.00 0.75 0.50 0.25 0.00 0.01 0.1

1 10 100 100010000 [SB202190] (nM)

Fig. 3. Kinase inhibition measured using GFP–fusion protein substrates. (A) Assay of JNK1 on GFP–cJun (s) or GFP–ATF2 (h) and JNK2 on GFP– cJun (d) or GFP–ATF2 (j). (B) JNK1 (h) and JNK2 (j) assayed against a dilution series of SP600125 using GFP–ATF2 as the substrate. (C) p38 family kinases MAPK11/p38b (m), MAPK12/p38c (n), MAPK13/p38d (), and MAPK14/p38a () assayed against a dilution series of SB202190 using GFP– ATF2 as the substrate.

Comparison of directly or indirectly labeled antibody performance To evaluate the performance of our assay format using either a directly labeled phosphospecific antibody or an indirectly labeled antibody, we measured cRaf kinase activity against a fluorescein-labeled conjugate of its downstream target, MEK, in both formats. The indirectly labeled format used the same phosphospecific antibody as that used in the directly labeled format and a terbium-labeled anti-IgG secondary antibody that binds to the phosphospecific antibody. In each experiment, 2 nM phosphospecific antibody was used, and in the experiment using indirectly labeled antibody, 5 nM Tb-labeled antirabbit antibody was used. There was negligible difference in the EC50 for the detection (directly labeled antibody EC50 = 42 ng/mL, indirectly labeled antibody EC50 = 30 ng/mL) (Fig. 4). It is interesting to note that in the assay using an indirectly labeled antibody, the response window was slightly larger than that in the assay using directly labeled antibody. In practice, this minor improvement will have negligible effect on the quality of the assay, and benefits may be offset by additional reagent costs. However,

1.0

Emission Ratio

the substrate, p38a was inhibited with an IC50 of 39 nM and p38b with an IC50 of 53 nM (Fig. 3C). In accord with previously published results, no inhibition was seen on the p38c or p38d isoforms up to the highest concentration of inhibitor tested, 20 lM.

0.9 0.8 0.7 0.6 0.5 0.4 0.01

0.1

1

10

100

1000 10000

cRaf (ng/mL) Fig. 4. Comparison of detection of cRAF activity using anti-p-Mek antibody that was either directly (s) labeled with terbium or indirectly (d) labeled with terbium via association with terbium-labeled anti-rabbit antibody.

terbium-labeled secondary antibodies are useful reagents for rapidly determining the suitability of a given primary antibody for use in an assay. In a FRET-based assay, the efficiency of energy transfer is inversely proportional to the sixth power of the distance between the donor and the acceptor fluorophore. For the terbium–GFP pair, the distance at which energy transfer is half-maximal (the Fo¨rster radius) has been calculated ˚ [20]. However, in a FRET-based to be approximately 43 A assay using a long-lifetime donor fluorophore, the magnitude of the Fo¨rster radius can give an incomplete indication of the suitability of a given pair of fluorophores for

Kinase assays with physiological substrates / S.M. Riddle et al. / Anal. Biochem. 356 (2006) 108–116

providing a robust assay signal if there is suitable flexibility in the system, as is the case in antibody-based assays [32]. Because conformational flexibility can allow the donor to sample a large volume of space during the excited state lifetime and because the energy transfer dependence on the distance weights the efficiency of energy transfer toward the distance of closest approach between donor and acceptor partners during the excited state lifetime, the efficiency of energy transfer can be greatly enhanced relative to systems built around standard fluorophores possessing nanosecond-duration excited state lifetimes [33,34]. In the example that we have shown, there is no decrease in assay sensitivity when using a primary antibody that is indirectly labeled via association with Tb-labeled secondary antibody relative to when the assay is performed using a directly labeled primary antibody. In the former case, the average donor–acceptor distance would be larger, but, because of the conformational flexibility of the antibodies, there is ample opportunity for energy transfer to take place during the extended (200 ls) detection window of the measurement. Assay robustness and resistance to interfering compounds In assessing the quality of a ratiometric assay and its ability to reliably identify compounds that have biological activity, it can be tempting (but misleading) to look at the fold change between maximal and minimal assay values. In practice, the robustness of a ratiometric assay is determined not by the relative difference in these values but by the magnitude of the absolute difference in these values relative to the magnitude of the errors associated with these values [23]. With TR-FRET assays in particular, the magnitude of these errors can be quite small relative to the separation between maximal and minimal TR-FRET values, and, as a result, a large window is not necessary for the assay to be robust [14,35]. Competitive equilibrium binding assays are typically performed at concentrations of tracer and receptor that

provide a signal that is 80% between that of the fully bound and that of the fully competed tracer [36]. This provides a balance between the magnitude of the signal change and the ability of the assay to report changes in analyte concentration, which decreases as the initial concentration of complex in the uncompeted state increases. Similarly, TR-FRET kinase assays are often run at or near the EC80 concentration of the kinase (under a given set of substrate and ATP concentrations), so that small changes in the amount of active kinase present will result in appreciable changes in the TR-FRET value while maintaining a suitable separation between the readouts of active and those of inactive kinase. Z 0 is a statistical parameter used to describe the suitability of an assay for correctly identifying active compounds in an HTS setting and is a measure of the robustness of an assay as defined by the difference in maximal and minimal assay response (the assay window) relative to the error associated with those responses. An assay with a Z 0 of >0.5 is typically taken to be suitable for HTS [23]. To determine Z 0 values for an assay of JNK1 activity using GFP–cJun (1–179) as the substrate, an EC80 concentration of JNK1 was used to perform the assay in the presence or absence of ATP. In a 20-lL final assay volume, a Z 0 of 0.93 was determined using 48 positive and negative control wells in a low-volume 384-well plate (Fig. 5). To simulate conditions for an ultraminiaturized assay (in the absence of liquid handling capacity to carry out such an assay), 4 lL of each control well was transferred to an empty well and the Z 0 determined to be 0.88. Based upon these results, we expect that the assay could be readily miniaturized below a 10-lL reaction/20-lL final assay volume, given proper liquid handling abilities. In addition to the Z 0 value, fluorescence-based HTS assays must be resistant to optical interference from the high concentrations of library compounds that are present in HTS. Three common sources of interference are ‘‘color quenchers’’ (compounds that cause inner-filter effects by absorbing either excitation or emission light), autofluores-

B

C

1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0

1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0

10

20

Well #

30

40

50

1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0

10

20

Well #

30

40

50

In No te rf er an t N A D Ta PH rt ra zi ne A llu ra R e C ou d m ar Fl in uo re sc ei Sc n at te ra nt

Emission Ratio

A

113

Fig. 5. Assay robustness and resistance to interference. (A) TR-FRET values for 20-lL assay wells containing 125 ng/mL JNK1(j) or 25 ng/mL JNK1 in the presence (d) or absence (s) of ATP. (B) Same as (A), but 4-lL final well volume. Dotted lines above and below the control wells indicate ±3 standard deviations for the positive and negative control data. (C) TR-FRET values for positive and negative control reactions measured in the presence of interfering compounds. NADPH, tartrazine, and allura red absorb light at the wavelengths used to excite or read the assay wells, and coumarin, fluorescein, or scatterant cause interference on time scales that do not affect the TR-FRET readout.

114

Kinase assays with physiological substrates / S.M. Riddle et al. / Anal. Biochem. 356 (2006) 108–116

cent compounds, and light scatter from precipitated compounds. To demonstrate the resistance of terbium-based TR-FRET assays to these common interferences, positiveand negative-control wells were spiked with interfering compounds prior to being read. Color quenchers (NADPH, tartrazine, and allura red) were present at a concentration of 5 lM, to mimic a concentration of 10 lM in a kinase assay. Tartrazine and allura red are the major chromophores in the food dies FD& C Yellow 5 and FD& C Red 40, respectively. NADPH absorbs strongly in the UV region in which the terbium chelate is excited (kmax = 340 nm), tartrazine absorbs strongly in the region between terbium excitation and emission (kmax = 425 nm), and allura red absorbs strongly in the region of fluorescein emission (kmax = 524 nm). Highly fluorescent compounds coumarin and fluorescein were present at 100 nM, representing an assay concentration of 200 nM. This concentration of fluorescein represents 10 times the highest fluorescence intensity of any compound in the LOPAC1280 library (Sigma) at 10 lM when read with a fluorescein filter set. Finally, nondairy coffee creamer was used at 0.5 mg/ mL as a light-scattering agent as has been described by others [37]. At this concentration, the solution is visibly turbid. In all cases (Fig. 5C), negligible effect was seen on the ratiometric assay readout. In the raw donor and acceptor intensity data (not shown), only the wells containing allura red showed a noticeable (30% decrease) effect of interfering compound in the raw donor and acceptor signals. However, the magnitude of this affect was similar in both data channels and was corrected by ratioing the data. Because the magnitude of the effect was similar in the spectrally adjacent channels, the ratio of donor:acceptor signals are unchanged relative to that of the appropriate control wells, despite the effect on the underlying donor and acceptor signals. Interference from fluorescent or light-scattering compounds was avoided by the time-resolved nature of the readout: any interference had decayed to background levels long before the measurements were made. Additionally, other common assay components such as Mn2+ or EDTA (to stop the kinase reaction by chelating divalent metals) do not affect the signal (although Mn2+ will decrease the assay signal in the absence of EDTA by slowly displacing Tb from the chelate complex). In fact, terbium-labeled antibody and EDTA can be premixed several hours in advance prior to the assay, to stop the reaction and begin the detection with a single reagent addition step. This is consistent with previously reported experiments in which similar (DTPA-based) Tb complexes were seen to be stable to large excesses of EDTA, which has a high affinity for Tb (Kb 6 1017 M1) [38]. The advantages of long-lifetime donor fluorophores in TR-FRET applications extend beyond the resistance to optical interference and the increased distances over which biomolecular interactions can be detected relative to those of conventional fluorophores. Unlike standard FRETbased assays that use short-lifetime donor fluorophores, TR-FRET-based assays are relatively insensitive to the

presence of excess donor or acceptor fluorophore that might be present in the system [20,34,39,40]. In a standard FRETbased assay, excess acceptor can lead to increased background due to off-peak excitation (excitation of the acceptor fluorophore at the wavelengths used to excite the donor), and excess donor can lead to increased background due to bleed through of the donor emission into wavelengths used to measure the acceptor signal. In contrast, the effects of offpeak excitation in TR-FRET-based assays are minimized by both the large difference in wavelength between donor excitation and acceptor emission (approximately 200 nm for the Tb–GFP or fluorescein pair) and the fact that any acceptor emission due to direct off-peak excitation completely decays prior to the gated intensity measurement used in a TR-FRET assay. Background effects due to excess donor are also minimized in a Tb-based TR-FRET assay due to the sharp emission peaks of terbium, in which minimal emission of the Tb donor is present in the spectral area used to measure acceptor emission. Additionally, although there is some bleed-through of fluorescein or GFP emission into the Tb (donor) signal, the magnitude of this signal is small relative to that of the total donor signal and therefore does not affect assay quality. Conclusions The flexibility of the terbium-based TR-FRET assay system was demonstrated in several configurations, using either directly or indirectly labeled antibodies and fluorescein-labeled or GFP fusions of physiologically relevant proteins as substrates. Although the use of terbium-labeled primary antibodies simplifies assay configuration and optimization, the use of indirectly labeled antibodies (via terbium-labeled secondary antibodies) allows for the facile evaluation of different primary antibodies for a particular application, while using very small amounts of primary antibody. Similarly, the use of GFP fusion substrates simplifies reagent production and assures lot-to-lot consistency, which can vary when the labeling is via multiple surface-accessible amine or thiol groups on the target protein. However, if the unlabeled substrate protein is already in hand, it may be simpler to label the existing protein using a small-molecule fluorophore rather than produce a GFP fusion of the target. Although this study took advantage of the fact that MEK contains several surface-accessible thiol groups to which an acceptor fluorophore could be attached via an iodoacetamide-functionalized fluorescein derivative, amine-reactive isothiocyanate or activated ester derivatives of suitable fluorophores are equally appropriate. Additionally, although fluorescein was used in this study, other fluorophores with similar spectra (such as BODIPY-FL, Oregon green, or Alexa Fluor-488) would be equally suitable, and red shifted fluorophores may also be used by employing alternative filter sets. Although europium has been the most commonly used donor in TR-FRET applications, the use of terbium rather than europium maintains the advantages of a time-resolved

Kinase assays with physiological substrates / S.M. Riddle et al. / Anal. Biochem. 356 (2006) 108–116

ratiometric format but adds flexibility with regard to the types of acceptors that may be used. In addition to simplifying TR-FRET kinase assays by allowing the use of substrates that are directly labeled with fluorescein (rather than relying on biotinylated substrates that must then be developed by an addition of streptavidin–APC), physiologically relevant protein substrates may be prepared as GFP fusions, with GFP serving as the FRET acceptor for terbium. This strategy provides for a facile method to assay kinases that require a docking site on the substrate for efficient phosphorylation to take place, as is the case with many MAP kinase pathway members such as p38a or RAF. Labeling kinase substrate as a GFP fusion has other practical advantages, such as batch-to-batch consistency that is not possible when a substrate protein is randomly labeled through accessible amino groups and lower cost when compared to using an acceptor-labeled antibody. Acknowledgment We thank Beth Strachan for expert molecular biology assistance. References [1] B.E. Kemp, D.B. Bylund, T.S. Huang, E.G. Krebs, Substrate specificity of the cyclic AMP-dependent protein kinase, Proc. Natl. Acad. Sci. USA 72 (1975) 3448–3452. [2] P. Cohen, D.C. Watson, G.H. Dixon, The hormonal control of activity of skeletal muscle phosphorylase kinase. Amino-acid sequences at the two sites of action of adenosine-3 0 :5 0 -monophosphate-dependent protein kinase, Eur. J. Biochem. 51 (1975) 79–92. [3] T.K. Sato, M. Overduin, S.D. Emr, Location, location, location: membrane targeting directed by PX domains, Science 294 (2001) 1881–1885. [4] J.D. Bjorge, A. Jakymiw, D.J. Fujita, Selected glimpses into the activation and function of Src kinase, Oncogene 19 (2000) 5620–5635. [5] T.M. Vondriska, J.M. Pass, P. Ping, Scaffold proteins and assembly of multiprotein signaling complexes, J. Mol. Cell. Cardiol. 37 (2004) 391–397. [6] A.S. Dhillon, W. Kolch, Untying the regulation of the Raf-1 kinase, Arch. Biochem. Biophys. 404 (2002) 3–9. [7] G.J. Parker, T.L. Law, F.J. Lenoch, R.E. Bolger, Development of high throughput screening assays using fluorescence polarization: nuclear receptor-ligand-binding and kinase/phosphatase assays, J. Biomol. Screen. 5 (2000) 77–88. [8] A.G. Morgan, T.J. McCauley, M.L. Stanaitis, M. Mathrubutham, S.Z. Millis, Development and validation of a fluorescence technology for both primary and secondary screening of kinases that facilitates compound selectivity and site-specific inhibitor determination, Assay Drug Dev. Technol. 2 (2004) 171–181. [9] W. Xia, F. Rininsland, S.K. Wittenburg, X. Shi, K.E. Achyuthan, D.W. McBranch, D.G. Whitten, Applications of fluorescent polymer superquenching to high throughput screening assays for protein kinases, Assay Drug Dev. Technol. 2 (2004) 183–192. [10] E.E. Loomans, A.M. van Doornmalen, J.W. Wat, G.J. Zaman, Highthroughput screening with immobilized metal ion affinity-based fluorescence polarization detection, a homogeneous assay for protein kinases, Assay Drug Dev. Technol. 1 (2003) 445–453. [11] S.M. Rodems, B.D. Hamman, C. Lin, J. Zhao, S. Shah, D. Heidary, L. Makings, J.H. Stack, B.A. Pollok, A FRET-based assay platform for ultra-high density drug screening of protein kinases and phosphatases, Assay Drug Dev. Technol. 1 (2002) 9–19.

115

[12] M.D. Shults, K.A. Janes, D.A. Lauffenburger, B. Imperiali, A multiplexed homogeneous fluorescence-based assay for protein kinase activity in cell lysates, Nat. Methods 2 (2005) 277–283. [13] S. Braun, W.E. Raymond, E. Racker, Synthetic tyrosine polymers as substrates and inhibitors of tyrosine-specific protein kinases, J. Biol. Chem. 259 (1984) 2051–2054. [14] A.J. Kolb, P.V. Kaplita, D.J. Hayes, Y.W. Park, C. Pernelle, J.S. Major, G. Mathis, Tyrosine kinase assays adapted to homogenous time-resolved fluorescence, Drug Disc. Today 3 (1998) 333–342. [15] S. Panse, L. Dong, A. Burian, R. Carus, M. Schutkowski, U. Reimer, J. Schneider-Mergener, Profiling of generic anti-phosphopeptide antibodies and kinases with peptide microarrays using radioactive and fluorescence-based assays, Mol. Divers 8 (2004) 291–299. [16] B.E. Kemp, R.B. Pearson, Design and use of peptide substrates for protein kinases, Methods Enzymol. 200 (1991) 121–134. [17] G. Mathis, Probing molecular interactions with homogeneous techniques based on rare earth cryptates and fluorescence energy transfer, Clin. Chem. 41 (1995) 1391–1397. [18] S.M. Fernandez, R.D. Berlin, Cell surface distribution of lectin receptors determined by resonance energy transfer, Nature 264 (1976) 411–415. [19] M.E. Astill, L.R. Johnson, G.H. Thorne, G.H. Krauth, R.E. Smith, R.W. Smith, T.R. Witty, Dual fluorometric/colorimetric detection system for an automated random-access instrument utilizing standard polystyrene test tubes as precision cuvettes, Clin. Chem. 33 (1987) 1554–1557. [20] P.R. Selvin, Principles and biophysical applications of lanthanidebased probes, Annu. Rev. Biophys. Biomol. Struct. 31 (2002) 275– 302. [21] M. Li, P.R. Selvin, Amine-reactive forms of a luminescent diethylenetriaminepentaacetic acid chelate of terbium and europium: attachment to DNA and energy transfer measurements, Bioconjug. Chem. 8 (1997) 127–132. [22] M. Li, P.R. Selvin, Luminescent Polyaminocarboxylate Chelates of Terbium and Europium: The Effect of Chelate Structure, J. Am. Chem. Soc. 117 (1995) 8131–8138. [23] J.H. Zhang, T.D. Chung, K.R. Oldenberg, A simple statistical parameter for use in evaluation and validation of high throughput screening assays, J. Biomol. Screen. 4 (1999) 67–73. [24] M. Hibi, A. Lin, T. Smeal, A. Minden, M. Karin, Identification of an oncoprotein- and UV-responsive protein kinase that binds and potentiates the c-Jun activation domain, Genes Dev. 7 (1993) 2135– 2148. [25] T. Kallunki, B. Su, I. Tsigelny, H.K. Sluss, B. Derijard, G. Moore, R. Davis, M. Karin, JNK2 contains a specificity-determining region responsible for efficient c-Jun binding and phosphorylation, Genes Dev. 8 (1994) 2996–3007. [26] L.M. Mooney, A.J. Whitmarsh, Docking interactions in the c-Jun Nterminal kinase pathway, J. Biol. Chem. 279 (2004) 11843–11852. [27] S. Gupta, D. Campbell, B. Derijard, R.J. Davis, Transcription factor ATF2 regulation by the JNK signal transduction pathway, Science 267 (1995) 389–393. [28] C. Livingstone, G. Patel, N. Jones, ATF-2 contains a phosphorylation-dependent transcriptional activation domain, EMBO J. 14 (1995) 1785–1797. [29] B.L. Bennett, D.T. Sasaki, B.W. Murray, E.C. O’Leary, S.T. Sakata, W. Xu, J.C. Leisten, A. Motiwala, S. Pierce, Y. Satoh, S.S. Bhagwat, A.M. Manning, D.W. Anderson, SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase, Proc. Natl. Acad. Sci. USA. 98 (2001) 13681–13686. [30] W. Gaarde, T. Hunter, H. Brady, B. Murray, M. Goldman, Development of a Nonradioactive, Time-resolved Fluorescence Assay for the Measurement of Jun N-Terminal Kinase Activity, J. Biomol. Screen. 2 (1997) 213–223. [31] S.P. Davies, H. Reddy, M. Caivano, P. Cohen, Specificity and mechanism of action of some commonly used protein kinase inhibitors, Biochem. J. 351 (2000) 95–105.

116

Kinase assays with physiological substrates / S.M. Riddle et al. / Anal. Biochem. 356 (2006) 108–116

[32] L.J. Harris, S.B. Larson, K.W. Hasel, J. Day, A. Greenwood, A. McPherson, The three-dimensional structure of an intact monoclonal antibody for canine lymphoma, Nature 360 (1992) 369–372. [33] D. Thomas, W. Carlsen, L. Stryer, Fluorescence Energy Transfer at the rapid-diffusion limit, Proc. Natl. Acad. Sci. USA. 75 (1978) 5746– 5750. [34] A. Cha, G.E. Snyder, P.R. Selvin, F. Bezanilla, Atomic scale movement of the voltage-sensing region in a potassium channel measured via spectroscopy, Nature 402 (1999) 809–813. [35] R.T. Cummings, H.M. McGovern, S. Zheng, Y.W. Park, J.D. Hermes, Use of a phosphotyrosine-antibody pair as a general detection method in homogeneous time-resolved fluorescence: application to human immunodeficiency viral protease, Anal. Biochem. 269 (1999) 79–93.

[36] X. Huang, Fluorescence polarization competition assay: the range of resolvable inhibitor potency is limited by the affinity of the fluorescent ligand, J. Biomol. Screen. 8 (2003) 34–38. [37] P. Rai, T.D. Cole, E. Thompson, D.P. Millar, S. Linn, Steady-state and time-resolved fluorescence studies indicate an unusual conformation of 2-aminopurine within ATAT and TATA duplex DNA sequences, Nucleic Acids Res. 31 (2003) 2323–2332. [38] P.R. Selvin, J.E. Hearst, Luminescence energy transfer using a terbium chelate: improvements on fluorescence energy transfer, Proc. Natl. Acad. Sci. USA. 91 (1994) 10024–10028. [39] P.R. Selvin, The renaissance of fluorescence resonance energy transfer, Nat. Struct. Biol. 7 (2000) 730–734. [40] P. Selvin, T. Rana, J. Hearst, Luminescence Resonance Energy Transfer, J. Am. Chem. Soc. 116 (1994) 6029–6030.