Homogeneous Single-Label Biochemical Ras Activation Assay Using

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Homogeneous Single-Label Biochemical Ras Activation Assay Using Time-Resolved Luminescence Eija Martikkala,*,† Stefan Veltel,‡ Jonna Kirjavainen,† Anita Rozwandowicz-Jansen,† Urpo Lamminm€aki,§ Pekka H€anninen,† and Harri H€arm€a† †

Laboratory of Biophysics and Medicity, University of Turku, FI-20520 Turku, Finland VTT Medical Biotechnology, FI-20520 Turku, Finland § Department of Biotechnology, University of Turku, FI-20520 Turku, Finland ‡

ABSTRACT:

Mutations of the small GTP-binding protein Ras have been commonly found in tumors, and Ras oncogenes have been established to be involved in the early steps of cancerogenesis. The detection of Ras activity is critical in the determination of the cell signaling events controlling cell growth and differentiation. Therefore, development of improved methods for primary screening of novel potential drugs that target small GTPase or their regulators and their signaling pathways is important. Several assays have been developed for small GTPases studies, but all these methods have limitations for a high-throughput screening (HTS) use. Multiple steps including separation, use of radioactive labels or time-consuming immunoblotting, and a need of large quantities of purified proteins are decreasing the user-friendliness of these methods. Here, we have developed a homogeneous H-Ras activity assay based on a single-label utilizing the homogeneous quenching resonance energy transfer technique (QRET). In the QRET method, the binding of a terbium-labeled GTP (Tb-GTP) to small GTPase protein H-Ras protects the signal of the label from quenching, whereas the signal of the nonbound fraction of Tb-GTP is quenched by a soluble quencher. This enables a rapid determination of the changes in the activity status of Ras. The assay optimization showed that only 60 nM concentration of purified H-Ras protein was needed. The functionality of the assay was proved by detecting the effect of H-Ras guanine nucleotide exchange factor, Son of Sevenless. The signal-to-background ratio up to 7.7 was achieved with an average assay coefficient of variation of 9.1%. The use of a low concentration of purified protein is desirable and the signal-to-background ratio of 3.4 was achieved in the assay at a concentration of 60 nM for H-Ras and SOS proteins. The need of only one labeled molecule and the ability to decrease the quantities of purified proteins used in the experiments are valuable qualities in HTS showing the potential of the QRET method.

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as proteins have shown to be strongly involved in cancer as 30% of all human cancers include mutated Ras by stabilizing Ras to a constitutively active conformation.1,2 This has made the development of the Ras activity measurements an interesting research field. Ras proteins are small (∼21 kDa) monomeric guanosinetriphosphate (GTP) hydrolyzing proteins (GTPases) which have essential roles in many cellular processes. They regulate major cellular functions like cell growth, proliferation, differentiation, and apoptosis.3 Ras cycles between its inactive guanosinediphosphate (GDP)-bound form and active guanosinetriphosphate (GTP)-bound form.4 The conversion from the active GTP-bound form into the inactive GDP-bound form is the result of an intrinsic GTPase activity of Ras, catalyzed by GTPase activating proteins (GAPs). Activation of Ras is r 2011 American Chemical Society

catalyzed by guanine nucleotide exchange factors (GEFs) which accelerate the exchange of GDP to GTP. Guanine nucleotide dissociation inhibitors can also regulate the small GTPases by preventing nucleotide exchange and thereby blocking downstream signaling. Immunopreciptation of the GTPase followed by a thin-layer chromatography separation of bound 32P-radiolabeled GDP from GTP was the only method to measure the active GTPbound form of small GTPases in cell based assays for decades.5,6 To avoid the use of radioactive labels, nonradioactive pull-down Received: October 14, 2011 Accepted: November 1, 2011 Published: November 18, 2011 9230

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Analytical Chemistry

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assays using Ras-binding domains, which preferentially bind the GTP-form of Ras, were developed.7 In these assays, the need of Western blotting after precipitation is, however, time-consuming and limits the use of the method. Many biochemical methods have also been developed to measure small GTPases activity with purified proteins but the methods rely on the use of radiolabeled nucleotides.8 Recently, novel methods based on NMR and fluorescence have been discovered.9
11 Fluorescence based methods use for example BODIPY- or Mant-labeled nucleotides to detect small GTPases activation.10,11 Since these methods are based on conventional fluorescence, fluorescence background may cause significant problems especially in large scale compound library studies. NMR methods have been used successfully in high-throughput secondary screening. The high cost, the need of large quantities of protein, and the limitations in the number and the type of compounds applicable in testing are limiting the use of heterogeneous NMR methods in primary screenings.12 Despite their advantages, most of the current methods require large quantities of purified protein and are thus not suitable to detect the small GTPase activity in primary screenings. The need for new compounds in cancer treatment implies that novel HTS ready assay formats are required. Therefore, we have developed a homogeneous single-label quenching resonance energy transfer technique (QRET) using time-resolved luminescence detection to measure activity of the Ras protein.13
17 The Son of Sevenless (SOS) protein, an established guanine nucleotide exchange factor for Ras, activated the nucleotide exchange in these experiments.

’ EXPERIMENTAL SECTION Materials. Tris(hydroxymethyl)aminomethane and triton X-100 were from Fluka (Buchs, Switzerland). γ-Globulins from bovine blood were purchased from Sigma-Aldrich (St. Louis, MO). Magnesium chloride hexahydrate was from Merck (Darmstadt, Germany). GDP-loaded H-Ras protein and His-SOS protein (catalytic domain of SOS (REM+GEF) aminoacids 564-1049) were from VTT (Turku, Finland). Terbium-labeled GTP (Tb-GTP) (20 /30 -O-(2-aminoethyl-carbamoyl)-guanosine-50 triphosphate (EDA-GTP) labeled with {2,20 ,200 ,2000 -{{6,60 -{400 -[2-(4-isothiocyanatophenyl)ethyl]pyrazole-100 ,300 -diyl}bis(pyridine)-2,20 -diyl}bis(methylenenitrilo)}tetrakis(acetato)}terbium(III)) was from the Department of Biotechnology, University of Turku.18 Microtiterplates OptiPlate384F were obtained from PerkinElmer (Turku, Finland). The soluble quencher Quench II was obtained from QRET Technologies (Turku, Finland). Detection of Effect of Ras Protein Concentration. For detection of the effect of Ras protein concentration, various concentrations of H-Ras (0
600 nM) were tested by activating the exchange of guanine nucleotide with His-SOS. A volume of 100 μL of reaction buffer (10 mM MgCl2, 3 mg/mL Triton X-100, 0.1 mg/mL γ-globulins in 50 mM Tris buffer pH 8.0), 39.5 μL of H-Ras solution, and 3 μL of Tb-GTP dilution in the reaction buffer (final concentration 10 nM) were added to the microcentrifuge tube and mixed. His-SOS in 7.5 μL of the reaction buffer solution (final concentration 250 nM) or reaction buffer (control reactions without SOS) was then added to the reaction. Reactions were incubated for 20 min at room temperature in the dark, and 40 μL of the sample were transferred to OptiPlate384F plates. Thereafter, 20 μL of the soluble quencher solution at 47 μM concentration was added to each well, and the reactions

Figure 1. (A) Structure of the terbium-labeled GTP. (B) Principle of the Ras activation QRET assay. Activation of Ras and thus binding of the terbium-labeled GTP are induced by the nucleotide exchange factor SOS. The luminescence signal of the free labeled GTP is quenched in solution whereas the bound fraction remains luminescent.

were further incubated for 30 min at room temperature in the dark. The time-gated luminescence signal was measured at 545 nm (bandwidth 6
8 nm) using a 320 nm (bandwidth 90 nm) excitation wavelength, 400 μs delay, and 400 μs decay times with Victor2 1420 multilabel counter (PerkinElmer, Turku, Finland). Detection of Effect of SOS Protein Concentration. The effect of SOS protein concentration from 0 to 2000 nM was tested to activate the exchange of guanine nucleotide in Ras. The assay was performed as above using 60 nM concentration of Ras. Control reactions were performed without Ras protein. The detection limit was calculated with equation xL = xbi + ksbi, where xbi is the mean of the blank measures, sbi is the root-meansquare of the blank measures, and k is 3.

’ RESULTS A homogeneous in vitro H-Ras protein activity assay was developed using the time-resolved luminescence single-label approach of the QRET technique.13
17 The structure of the labeled GTP is shown and the principle of the H-Ras protein activity QRET assay is demonstrated in Figure 1. The guanine 9231

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Analytical Chemistry

Figure 2. Optimization of the Ras protein concentration. The effect of protein concentration was observed by testing Ras concentration as it varied from 0 to 600 nM with 200 nM SOS (9) and without (b) SOS. The final concentration of Tb-GTP used in the experiment was 10 nM. The signal-to-background ratio over 2 was detected with 60
600 nM Ras concentration. However with a 600 nM Ras protein concentration, the background signal increased lowering the signal-to-background ratio (signal of Ras with SOS divided by the signal of Ras without SOS). A 60 nM protein concentration was decided to be used in all subsequent experiments.

exchange factor of H-Ras protein, Son of Sevenless (SOS), catalyzes the exchange of guanine nucleotide in H-Ras. Unlabeled GDP is dissociated from H-Ras and replaced by terbium labeled GTP. The luminescence of the bound labeled GTP is protected from quenching of the soluble quencher, and a high luminescence signal is obtained. As the signal of the nonbound labeled GTP is quenched in solution, the SOS-mediated activation of H-Ras can be detected as an increase in luminescence. Optimization of the H-Ras concentration and testing of the functionality were studied to prove the potential of the assay as a primary screening method. Optimization of H-Ras Assay Protein Concentration. The effect of the H-Ras protein concentration was investigated using a varied concentration of H-Ras (0
600 nM) in a measurement where 200 nM of His-SOS activated the exchange of guanine nucleotide (Figure 2). The control reactions were carried out without SOS, and their signals were used as background signals in the signal-to-background ratio calculations. An increasing signalto-background ratio was observed up to 200 nM of H-Ras. At higher H-Ras concentrations, the increased background luminescence signal decreased the signal-to-background ratio. Although the highest signal-to-background ratio of 2.9 was found at 200 nM concentration of H-Ras, we chose to employ a 60 nM protein concentration (signal-to-background ratio 2.4) as the use of low protein concentration is desirable for cost reasons. The coefficient of variation of the assays varied from 2 to 18%. Functionality of the Developed Assay. The functionality of the developed H-Ras protein activity assay was proved by testing the effect of various concentrations of guanine exchange factor His-SOS (0
2000 nM, Figure 3). The reactions without H-Ras were measured as control reactions, and their signals were used as the background signals in the signal-to background ratio calculations. An increased luminescence signal was observed as higher His-SOS concentrations were applied. This was a result of the higher binding rate of the Tb-labeled GTP to H-Ras protein. The

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Figure 3. Functionality of the Ras activation assay. Varied concentrations of SOS (0
2000 nM) were tested to prove the functionality of the assay. Two curves with Ras (9) and the control reactions carried out without Ras (b) are shown. At a high SOS concentration, an increased binding of the labeled GTP to Ras protein was observed. The signal-tobackground ratio (signal of SOS with Ras divided by the signal of SOS without Ras) up to 7.7 was measured.

signal-to-background ratio increased up to 7.7 using the His-SOS concentration of 2000 nM. However, a lower protein concentration of 60 nM with a signal-to-background ratio of 3.4 is considered sufficient for a successful H-Ras activation assay. The detection limit of the assay was 6 nM of His-SOS. The coefficient of variation (CV) of the assays varied between 0.5 and 17%.

’ DISCUSSION Here we have developed a single-label time-resolved luminescence assay to detect the activation of small GTPases. The activation of H-Ras protein by nucleotide exchange factor was monitored by binding of the terbium labeled GTP. Binding of the Tb-GTP to H-Ras protects the fluorophore from the soluble quencher leading to increased time-resolved luminescence. The signal-to-background ratio of the developed assay gradually increased as the concentration of H-Ras and SOS were increased. A signal-to-background ratio up to 7.7 was achieved which is significantly higher than typically measured for the widely used Mant-GDP assay, which are typically in the range of 2 or 3.19,20 This is associated with an efficient protection of bound Tblabeled GTP and quenching of the nonbound labeled GTP. In addition the highly sensitive time-resolved luminescence detection allows the use of a low concentration of assay components in comparison to conventional fluorescence methods such as Mant-, BODIPY-, and Tamra-GDP/GTP methods.19,21
24 Although the highest signal-to-background ratio 7.7 was achieved with the highest SOS concentration used in these experiments, a higher signal-to-background ratio may be achieved at higher concentrations of H-Ras and His-SOS. Then, the concentration level of the proteins may be very high but still comparable to the conventional fluorescence based methods. The terbium-labeled GTP has been previously applied to the guanine nucleotide exchange assay. The assay was based on the F€orster resonance energy transfer (FRET) method where TbGTP transferred energy to green fluorescent protein (GFP) fused with small GTPase Rab21.18 The use of the FRET method is clearly more limited for assay purposes as the preparation and 9232

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Analytical Chemistry purification of GFP fused protein is required, and sterical hindrances might occur when large target proteins are involved such as guanine nucleotide exchange factors or GTPase activating proteins. The developed QRET method is not limited only to small GTPase H-Ras. We have successfully obtained preliminary data on Rab21 and Rab5 showing the potential of the assay to other small GTPases (data not shown). The amount of purified protein needed in biochemical small GTPases binding and dissociation assays varies significantly. Typically the purified protein concentration ranges from low micromolar to 100 μM.19,22,25,26 The developed method required only 60 nM concentration of purified proteins decreasing the need for highly concentrated protein solutions. Many assays use also nucleotide free proteins.18,19,25 Here assays were done with protein, which was not purified free of nucleotide. Use of nucleotide containing protein is desirable as a nucleotide binding pocket of GTPases is nearly always occupied by guanine nucleotide27
30and extra effort is needed to purify protein nucleotide free. To develop an assay with a high signal-tobackground ratio, an increased amount of SOS can be employed. The highest SOS concentration in this study was 2 μM, which is similar to the concentrations typically applied to GEF experiments using fluorescence-based methods.19,25

’ CONCLUSIONS GTPases have a crucial role in the regulation of multiple signaling events in cells, and their role in cancer is major. The methods for effective primary screening of novel potential drugs that target GTPases are valuable. Our homogeneous single-label biochemical Ras activation assay is simple and fast to use with high signal-to-background ratios. The need for a significantly lower quantity of purified proteins compared to many existing methods brings cost-savings to screenings. These qualities indicate the HTS-compatible potential of the method for screening compounds targeting the GTPases or their activators. ’ AUTHOR INFORMATION Corresponding Author

*E-mail: eija.martikkala@utu.fi. Notes

Harri H€arm€a has commercial interest to the Quench II molecule through QRET Technologies.

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’ ACKNOWLEDGMENT This work was generously supported by the Emil Aaltonen Foundation, Turku University Foundation, the Graduate School of Chemical Sensors and Microanalytical Systems, and Academy of Finland (Projects 138584 and 125777). The synthesis of TbGTP was performed in the projects supported by the Academy of Finland (Grants 114903 and 119497). ’ REFERENCES (1) Oxford, G.; Theodorescu, D Cancer Lett. 2003, 189, 117–128. (2) Bos, J. Cancer Res. 1989, 49, 4682–4689. (3) Takai, Y; Sasaki, T.; Matozaki, T. Physiol. Rev. 2001, 81, 153–208. (4) Vetter, I. R.; Wittinghofer, A. Science 2001, 294, 1299–304. (5) Gibbs, J.; Schaber, M.; Marshall, M.; Scolnick, E.; Sigal, I J. Biol. Chem. 1987, 262, 10426–10429. 9233

dx.doi.org/10.1021/ac202723h |Anal. Chem. 2011, 83, 9230–9233