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A gold nanoparticles colorimetric assay for label-free detection of protein kinase activity based on phosphorylation protection against exopeptidase cleavage
Biosensors and Bioelectronics 53 (2014) 295–300
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A gold nanoparticles colorimetric assay for label-free detection of protein kinase activity based on phosphorylation protection against exopeptidase cleavage Jiang Zhou, Xiahong Xu, Xin Liu, Hao Li, Zhou Nie n, Meng Qing, Yan Huang, Shouzhuo Yao State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 23 July 2013 Received in revised form 17 September 2013 Accepted 26 September 2013 Available online 8 October 2013
Protein kinases are significant regulators in the cell signaling pathways, and it is still greatly desirable to achieve simple and quick kinase detection. Herein, we present a novel colorimetric gold nanoparticles (AuNPs)/peptide platform for probing the activity and inhibition of protein kinases based on phosphorylation-induced suppression of carboxypeptidase Y (CPY) cleavage. This AuNPs/peptide platform can easily monitor the kinase activity by a UV–vis spectrometer or even by the naked eye. The feasibility of the method has been demonstrated by sensitive measurement of the cAMP-dependent protein kinase (PKA) activity with a low detection limit of 0.232 mU/mL and assessment of kinase inhibition by H-89 with an IC50 value of 18.13 nM. The assay was also successfully put into practice for the detection of kinase activity in cell lysate. Because of its label-free, homogenous and colorimetric merits, the proposed assay presents great potential in high-throughput screening for kinase-targeted drug discovery. & 2013 Elsevier B.V. All rights reserved.
Keywords: Gold nanoparticles Protein kinase Protein phosphorylation Carboxypeptidase Y Cell lysate
1. Introduction Protein phosphorylation catalyzed by kinase is a universal regulatory mechanism in the cell signaling pathways, which plays a crucial role in various vital cellular physiological processes including cell growth, metabolism, differentiation, and apoptosis (Manning et al., 2002; Cohen, 2002a, 2002b; Kalume et al., 2003). The over-expression and aberrant activity of protein kinases are closely related to a number of severe diseases such as cancer, diabetes, inflammation, cardiac diseases, and Alzheimer's disease (Mark and Joseph, 2010; Cohen, 2002a, 2002b; Hanger et al., 2007). Therefore, the development of kinase assays capable of monitoring kinases activity, identifying their substrates and screening potential inhibitors is important for the research of fundamental biochemical pathways, clinical diagnosis and drug discovery (Cohen, 2002a, 2002b; Noble et al., 2004). Traditional method for assessing kinase activity relied on radioactive isotope-labeled ATP, which is general but hampered by the hazardous effect of radioactive materials (Turk et al., 2006; Hastie et al., 2006; Houseman et al., 2002). To overcome this shortcoming, various kinase assays, including electrochemical (Wieckowska et al., 2008; Ji et al., 2009; Xu et al., 2009; Kerman et al., 2008), surface-plasmon resonant(Yoshida et al., 2000;
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Corresponding author. Tel.: þ 86 731 88821626; fax: þ 86 731 88821848. E-mail address: [email protected] (Z. Nie).
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Viht et al., 2007), and mass spectroscopic techniques (Mann et al., 2002; Watts et al., 1994) have been developed as the alternatives of radioactive assay. Most of these techniques involve a surfaceconfined process of substrate peptide, heterogeneous enzymatic reaction at peptide-modified surface, and multistep-washing procedure. Compared with those heterogeneous kinase assays, the approaches for homogenous detection of kinase activity possess intrinsic merits such as the short detection time, facile detection without separation, and readiness for high-throughput screening (HTS). Great progress has been achieved on the design of homogenous fluorescence measurements of kinase-catalyzed phosphorylation (Sato et al., 1999; Sharma et al., 2007; Agnes et al., 2010; Xu et al., 2011; Bai et al., 2013; Zhou et al., 2013). Because the color change can be directly monitored by naked eye without sophisticated instruments, the colorimetric biosensing for protein phosphorylation has attracted much attention because of its low cost, simplicity, and practicality. Although it is a promising technique for developing homogenous kinase assay, the existing colorimetric assays for kinase activity are still scarce in comparison with welldeveloped fluorescent counterparts. Gold nanoparticles (AuNPs) represent a potent colorimetric probe mainly due to their unique optical properties, particularly their size-dependent surface plasmon resonance absorption (Lim et al., 2007; Mirkin et al., 1996; Sato et al., 2003). There are only a few AuNPs-based methods currently available for homogenous activity detection of protein kinase. Brust et al. developed a colorimetric kinase assay in which the biotin-labeled ATP was
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used to probe phosphorylation process on peptide-modified AuNPs, and consequently the phosphorylated AuNPs with biotin label reacted with avidin-capped AuNPs to generate cross-linking and corresponding color-change (Wang et al., 2006; Wang et al., 2005). Similar cross-linking strategy has been designed by Stevens' group using AuNPs conjugated with substrate peptide and phospho-specific antibody, respectively, as dual probes to assess the activity of kinase (Gupta et al., 2010). Recently, Katayama group reported an intriguing kinase assay using phosphorylationinduced peptide charge change to mediate AuNPs aggregation (Oishi et al., 2008). Among these methods, most of them relied on phosphorylation-induced interparticles cross-linking of AuNPs, which are effective but suffer from the complicated modification of AuNPs, the high cost of labeled substrate, and relative long responsive time for observing the aggregation-induced color change (Lévy et al., 2004). Hence, it is still desirable to develop new label-free strategy to fulfill the phosphorylation-responsive non-crosslinking AuNPs aggregation. In this paper, we present a novel colorimetric AuNPs/peptide platform for probing the activity and inhibition of protein kinase. This AuNPs/peptide platform is composed of the unmodified AuNPs and the rationally designed substrate peptide probe. The phosphorylation recognition is dependent on the phosphorylation protection against carboxypeptidase digestion. The carboxypeptidase refers to a kind of exopeptidase that hydrolyzes peptide bond from the carboxyl terminal (C-terminal) of a protein or peptide (Low and Yuan, 1996). It has been de monstrated that the activity of exopeptidase are susceptible to modification on the amino acid (Dass and Mahalakshmi, 1996). With the aid of carboxypeptidase, the phosphorylated peptide could be discriminated from unphosphorylated one by its resistance to enzymatic cleavage, leading to retain the peptide fragment to mediate the aggregation of AuNPs. The cAMP-dependent protein kinase (PKA) and carboxypeptidase Y (CPY) were used as typical models. Because of its label-free property and no modification required for AuNPs and peptides, this method represents a promising application of AuNPs in protein kinase analysis.
The UV–vis absorption spectra were recorded on a Beckman DU800 spectrophotometer in a wavelength range from 400 to 800 nm. The absorbance measurements of each sample were conducted in three independent times (n ¼3). High-resolution transmission electron microscopy (HRTEM) measurements were made on a JEOL JEC-3010 electron microscope. The samples of TEM were prepared by placing a drop of solution (10 μL) on carboncoated copper grid and drying at room temperature in drying cabinet. Cell-breaking was performed using a JY92-IIN ultrasonic cell disruption system (Scientz, Ningbo, China). The photographs were recorded with digital camera.
2.2. Synthesis of gold nanoparticles (AuNPs) AuNPs (13 nm) were synthesized according to the previously reported method (Jin et al., 2003). Generally, 38.8 mM sodium citrate solution (5 mL) was rapidly added into 1 mM boiled HAuCl4 solution (50 mL) under vigorous stirring in a 100 mL round flask. The mixture was maintained boiling for 10 min, and the corresponding color change from yellow to deep wine red. The obtained gold nanoparticles solution was stored at 4 1C. The final concentration of AuNPs was calculated to be 11 nM using the UV–vis absorption spectrum based on Lambert–Beer's law and extinction coefficients (ε) of 2.7 108 M 1 cm 1 at λ520 for 13 nm AuNPs. The so-prepared AuNPs dispersion was diluted 2.5 times by ultrapure water and used as stock solution in the subsequent absorbance experiments. The absorption ratio (Abs520/Abs620) of the AuNPs dispersion was chosen as the respond signal of the AuNPs dispersion stability.
2.3. Peptide-induced AuNPs aggregation A series of 40 mL S-pep samples at different concentrations (0, 0.001, 0.01, 0.1, 1, 2.5, 5, 10, 20 and 40 mM) were prepared in 10 mM Tris–HCl buffer (pH 7.5, 25 1C), then an equal volume of the unmodified AuNPs dispersion (4.4 nM) was added and mixed for 5 s. After mixing, UV–vis spectroscopic measurements at the 400–800 nm wavelength region were made at room temperature.
2. Experimental methods 2.1. Materials and measurements
2.4. Treatment of S-pep by carboxypeptidases Y (CPY)
Cyclic adenosine 3′,5′-monophosphate-dependent protein kinase (PKA, catalytic subunit) and Casein kinase II (CKII) were purchased from New England Biolabs (Beverly, MA, USA). Generally, PKA was diluted using the storing solutions (50 mM NaCl, 1 mM EDTA, 2 mM DTT, 50% glycerol in 20 mM Tris–HCl buffer (pH 7.5, 25 1C)) and stored in the refrigerator at 80 1C. Substrate peptide probe (S-pep, CGGRRGLRRASLG) for PKA was synthesized by GL Biochem (Shanghai, China). ATP was bought from Generay (Shanghai, China). HAuCl4 4H2O was supplied from Shanghai Reagent Company (Shanghai, China). H-89 was obtained from EMD Biosciences (Calbiochem-Novabiochem. La Jolla, CA, USA). Carboxypeptidase Y (CPY), forskolin and 3-isobutyl-1methylxantine (IBMX) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The improved Bradford protein assay dye reagent kit was purchased from Sangon (Shanghai, China). The rest of the chemical reagents including bovine serum albumin (BSA), Tris, glycerol, DTT, and EDTA used in this study were obtained from Bio Basic (Ontario, Canada). Human breast cancer cells (MCF-7) were bought from the Cell Bank of Xiangya Central Laboratory of Central South University (Changsha, China). The ultrapure water (18.3 MΩ cm) from the Millipore Milli-Q system was used in all experiments.
The S-pep (1 mM) was treated with the CPY (2.632 U/mL) for 45 min. The resulting solution (40 mL) was mixed with an equal volume of AuNPs dispersion (4.4 nM) and then measured by spectroscopy. A range of concentrations (0–4 U/mL) of CPY were exploited to optimize the quantity of CPY. For optimization of the digestion time, the same experiments were conducted incubating with 2.632 U/mL CPY for different incubation times (0—90 min).
2.5. Detection of activity and inhibition of PKA For PKA-catalyzed phosphorylation, 200 mL of the PKA reaction solutions composed of PKA (0–1 U/mL), S-pep (1 mM), MgCl2 (1 mM), ATP (0.1 mM) in 20 mM Tris–HCl buffer (pH 7.5, 25 1C) was incubated for 60 min at 30 1C, and the resulting phosphorylated solution was incubated with 2.632 U/mL CPY for 45 min at 25 1C, then 40 mL of the mixture was mixed with the AuNPs dispersion (40 mL, 4.4 nM) and measured by spectroscopy. For PKA inhibition assays, 250 mU/mL PKA and H-89 at different concentrations (0–0.5 mM) were added into the reaction solutions, and the experiment procedures were set under the above-mentioned conditions.
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2.6. Detection of PKA activity in MCF-7 cell lysate Ten percent fetal bovine serum, 0.1 mM of mimimum eagle's essential medium (MEM) nonessential amino acid solution, 1% insulin–transferrin–selenium A supplement, 100 U/mL of penicillin, 100 mg/mL of streptomycin, and 0.25 mg/mL amphotericin B composed the MCF-7 breast cancer cells (1 106 cells) supplement. The MCF-7 cells were incubated in a humidified atmosphere with 5% CO2 at 37 1C. After a 4 h incubation in the serum-free medium replacing the culture medium, then the forskolin and IBMX mixture diluted with dimethyl sulfoxide (DMSO) was added into the medium at various concentrations (the final concentrations of forskolin and IBMX are shown in the inset table of Fig. 5A) with the purpose of stimulating intracellular PKA activity. Equal volume of DMSO was added into the medium as the unstimulated control sample. After 30 min of stimulation, the cultured cells were removed by scraping and lysed in Dulbecco's phosphate-buffered saline (D-PBS) by sonication (200 W) 2 s for 60 times at a 3 s interval each time for protein extraction. The samples were clarified by centrifugation for 60 min at 41125 g, 4 1C and the extracted supernatants were stored in the freezing tubes at 20 1C and used in the following experiments. The Bradford method was used to determine the total protein content of cell lysate supernatants, and the protein concentration of each cell lysate samples was diluted to 12.5 μg/mL used for the experiments. For kinase activity assays in MCF-7 cell lysate, the same experiments were performed except that the above-prepared cell lysate samples instead of pure PKA were investigated.
3. Results and discussion 3.1. The principle of the colorimetric assay for kinase activity detection The kemptide, Leu–Arg–Arg–Ala–Ser–Leu–Gly (LRRASLG) containing the phosphorylation site Ser, is the substrate peptide for cAMP-dependent protein kinase (PKA), which has a high affinity for PKA with Km ¼ 5 μM (Cheng et al., 1986). The detection mechanism of the proposed assay is shown in the Scheme 1. An artificially designed peptide CGGRRGLRRASLGA, namely S-pep, is employed here for the assay. It is clearly observed that the S-pep is comprised of three parts, i.e., the kemptide motif (LRRASLG), two additional positively charged Arg residues (RR) to ensure the
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peptide net charge being positive both before and after phosphorylation, and the cysteine at N terminal for anchoring on the surface of citrate-capped AuNPs by Au–S bond. This rationally designed positively charged S-pep is capable of inducing AuNPs aggregation because it can readily bind to negatively charged AuNPs, neutralizing the AuNPs surface charges, and disrupt the stability of AuNPs. CPY, a representative exopeptidase, was applied to recognize the kinase catalysis event due to its activity susceptible to phosphorylated modification of amino acid (Dass and Mahalakshmi, 1996; Murray et al., 1996). When non-phosphorylated S-pep was treated with CPY, it is literally cleaved from C-terminal into the free amino acids (Low and Yuan, 1996). The full degradation of S-pep leads to only cysteine attached on the AuNPs surface, thus keeping dispersion of AuNPs and the color of the solution remaining red due to no disturbance of AuNPs surface charge (route a). However, after phosphorylation by kinase, the phosphorylated peptide (CGGRRGLRRApSLG) can effectively block CPY cleavage by the bulky phosphorylated serine sites (Kupcho et al., 2003; Zhou et al., 2013), and the CPY-treated S-pep fragment retaining two positive net charges still can cause AuNPs aggregation and corresponding red-to-blue color change (route b). Hence, we could successfully detect activity of PKA by the color change and the UV–vis spectra of unmodified AuNPs with the assistance of CPY.
3.2. Optimization of S-pep induced aggregation of AuNPs In order to obtain the best colorimetric performance, we investigated the effect of S-pep concentrations on the colorimetric response of the unmodified AuNPs dispersion, which is described by the Abs520/Abs620 ratio of AuNPs solutions. Fig. 1 shows the Abs520/Abs620 ratio and the color change of the AuNPs as a function of S-pep concentration (0, 0.001, 0.01, 0.1, 1, 2.5, 5, 10, 20 and 40 mM, respectively). It can be seen that, at the S-pep concentration ranging from 0 to 0.1 μM, the Abs520/Abs620 ratio keeps more than 5.78 and the solution color is red, while the Abs520/Abs620 ratio decreases sharply to 0.7 and the blue color is observed at the concentration reaching or beyond 1 μM, indicating the aggregation of AuNPs. Hence we could conclude that, in order to induce aggregation of AuNPs, S-pep concentration should be kept higher than 1 μM. Furthermore, the effect of S-pep in the presence and absence of CPY was further examined, and the results are shown in the inset of Fig. 1A. The most obvious response reflected by the largest ΔAbs520/Abs620 ratio was observed at 1 μM S-pep, and the corresponding UV–vis absorption spectra of the AuNPs dispersion
Scheme 1. The principle of colorimetric assay of PKA activity detection based on carboxypeptidase Y (CPY) and unmodified gold nanoparticles (AuNPs).
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are shown in Fig. S1 in Supporting Information (SI). Therefore, 1 μM substrate peptide was chosen as the optimal concentration for the following experiment. 3.3. Optimization of CPY treatment of S-pep At first, the S-pep degradation by CPY digestion was investigated, and the UV–vis absorption spectra and color changes (inset) of the AuNPs dispersion of the S-pep and S-pep/CPY samples are displayed in Fig. S2. When the AuNPs was mixed with the S-pep (1 mM), a distinct absorption peak at 628 nm, the symbol of aggregated AuNPs, was observed (spectrum a), and this absorption peak is much higher than that at 520 nm. The corresponding
images of solution color (a) also demonstrate the aggregation of AuNPs which is in accordance with the result of the UV–vis spectra. Nevertheless, the S-pep treated by CPY (5 U/mL) causes only one absorption peak at 520 nm, indicating good dispersion of AuNPs, which is confirmed by the red solution color (b). Then, to obtain the best hydrolysis performance of CPY, the concentration and reaction time of CPY were optimized. Various CPY concentrations (0–4 U/mL) and treating times (0–90 min) were investigated for optimization in the presence of S-pep (1 mM), and the results are shown in Fig. S3. The Abs520/Abs620 ratio of the AuNPs mixture increases with the CPY concentration increasing, until it reaches a plateau at 2.632 U/mL CPY. With the reaction time increasing, the Abs520/Abs620 ratio gradually increases and then turns to be a platform at reaction time 45 min. So 2.632 U/mL CPY and 45 min of reaction time were employed in the subsequent assays.
3.4. Detection of activity and inhibition of PKA
Fig. 1. The Abs520/Abs620 ratio (A) and the color change (B) of the AuNPs dispersion after addition of S-pep solutions (0, 0.001, 0.01, 0.1, 1, 2.5, 5, 10, 15, 20 and 40 mM) in 10 mM Tris–HCl buffer (pH 7.5). The concentrations of AuNPs dispersion used for absorbance measurement and colorimetric observation were 2.2 nM and 5.5 nM, respectively. The inset of (A) shows the bar chart of Abs520/Abs620 ratios of the AuNPs dispersion in the presence of S-pep sample (1–40 μM) before and after addition of 13.16 U/mL CPY. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Mg2 þ , ATP, and Tris–HCl buffer (pH 7.5) are required for protein kinase reaction, which may have undesirable influence on the stability of AuNPs, so their effects on AuNPs stability were investigated in our experiments. As shown in Fig. S4A, the Abs520/Abs620 ratio of the AuNPs dispersion remains unaffected when Mg2 þ concentration is lower than 1 mM. It can also be concluded that the ATP concentration (0–0.2 mM) has no significant influence on the stability of AuNPs (Fig. S4B). It was found that the Tris–HCl (pH 7.5) would not induce AuNPs aggregation unless the buffer concentration was higher than 20 mM (Fig. S4C). So we employed 1 mM Mg2 þ , 0.1 mM ATP, and 20 mM Tris–HCl (pH 7.5) for the kinase reaction. The UV–vis spectra, transmission electron microscope (TEM) images, and color changes of the AuNPs dispersion of the various samples were studied and the results are displayed in Fig. 2. The S-pep, after addition of CPY (5 U/mL), exhibits only one absorption peak at 520 nm (spectrum 1), and the corresponding TEM images (1) and red solution color (1) also are in accordance with the UV–vis spectrum result, indicating the S-pep was digested and the AuNPs were well-dispersed. However, two obvious absorption peaks of AuNPs at 520 nm and 620 nm can be observed in the AuNPs dispersion after addition of the phosphorylated peptide by PKA (250 mU/mL) in the presence of CPY (5 U/mL) (spectrum 4) and the solution color is blue (4) as expected. TEM image
Fig. 2. UV–vis absorption spectra (A), color changes (the inset of A), and transmission electron microscope (TEM) images (B) of the AuNPs dispersion in different conditions. The concentrations of AuNPs dispersion used for absorbance measurement and colorimetric observation were 2.2 nM and 5.5 nM, respectively. AuNPs dispersion mixed with (1) 1 mM S-pep treated by 2.632 U/mL CPY, (2) 1 mM S-pep with 250 mU/mL CKII and 0.1 mM ATP in the presence of 2.632 U/mLCPY, (3) 1 mM S-pep, 250 mU/mL PKA with addition of 2.632 U/mL CPY, and (4) (3) with 0.1 mM ATP. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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AuNPs when the H-89 concentration is lower than 0.5 μM, therefore, 0–0.5 μM H-89 is used for the following inhibition experiment. A sigmoidal profile of the Abs520/Abs620 ratio versus the H-89 concentration was obtained (Fig. 4). With the increase of the H-89 concentration, Abs520/Abs620 ratio of the AuNPs mixture increases until H-89 concentration reaches up to 250 nM, at which the corresponding Abs520/Abs620 ratio reaches a platform. The IC50 value (the corresponding inhibitor concentration required to reduce protein kinase activity by 50%) was found to be 23.09 nM, which is in accordance with the reported value in literature (Rodems et al., 2002). These results indicated that our method can be used to study the protein kinase inhibitor and is feasible for protein kinase inhibitors screening.
3.5. Protein kinase A activity detection in cell lysate
Fig. 3. Colorimetric assay of PKA activity based on the measurements of the Abs520/ Abs620 ratios (A) and the color changes (B) of AuNPs. The results were obtained from the mixtures of AuNPs dispersion and S-pep (1 mM) phosphorylated by PKA (0–1 U/mL) in the absence (black) and presence (gray) of 2.632 mU/mL CPY. The concentrations of AuNPs dispersion used for absorbance measurement and colorimetric observation were 2.2 nM and 5.5 nM, respectively. The inset shows the calibration curve of the Abs520/Abs620 ratio dependent on the PKA concentration.
(4) further demonstrated that AuNPs were aggregated. There is no obvious spectra shift in the sample without ATP (spectrum 3) and non-target kinase sample with CKII (250 mU/mL) (spectrum 2) compared with the S-pep/CPY sample, indicating that the signals of this sensor can specifically and selectively respond to the occurrence of phosphorylation. Therefore, it is feasible and convenient to monitor the PKA activity with the aid of CPY based on the change of color and absorption spectra of the AuNPs dispersion as the read-out. Quantitative detection of kinase activity was further studied under the pre-optimized conditions with different concentrations of PKA (0–1 U/mL). As depicted in Fig. 3, with the increase of PKA concentration, the Abs520/Abs620 ratios of phosphorylated peptide/ AuNPs sample in the presence of CPY significantly decreases while those ratios of the samples without CPY only slightly increases, indicating that the phosphorylation recognition in this sensor is CPY-dependent and a higher degree of phosphorylation can induce a greater blocking effect on CPY enzymatic hydrolysis. The calibration curve of Abs520/Abs620 ratios versus logarithm of the PKA concentration is plotted in the inset of Fig. 3. It is observed that the Abs520/Abs620 signal turns to a platform when PKA concentration reaches 250 mU/mL. The EC50 value (the corresponding protein kinase concentration required to phosphorylate 50% substrate) of PKA was estimated to be 41.58 mU/mL. The detection limit of PKA is estimated to be 0.232 mU/mL which is lower than the data reported previously (Ji et al., 2009;Xu et al., 2011; Oishi et al., 2007; Kerman et al., 2007; Miao et al., 2012). To investigate whether colorimetric assay could be further used to study the PKA inhibition, the experiments were performed as the same procedures in the presence of a well-known PKA inhibitor H-89 with different concentrations. H-89 is an isoquinoline derivative capable of inhibiting PKA activity by acting as the competitive antagonist of ATP at its binding site on the PKA catalytic subunit (Engh et al., 1996). Firstly, the influence of H-89 concentration on the stability of AuNPs was tested. As shown in Fig. S5, there is negligible effect on the Abs520/Abs620 ratio of
The activation of kinases in a cell caused by extracellular stimulation can trigger a series of important cellular processes including transcription, differentiation, and apoptosis (Godwin et al., 1993). Thus, whether the assays are competent to detect kinase activity in cell lysate is significant for the study of kinase regulation in the cell system. PKA in human cells could be activated using extracellular stimulation by forskolin, the adenylyl cyclase activator, and IBMX, a phosphodiesterase inhibitor. First of all, whether cell lysate samples could affect the stability of AuNPs was tested. As shown in Fig. S6, the cell lysate samples have little influence on the stability of AuNPs. Then, the detection of PKA activity in cell lysate was performed, and the samples were prepared by mixing the AuNPs dispersion with the S-pep phosphorylated by stimulated lysate samples (number 1–6) and untreated lysate sample (blank). The corresponding Abs520/Abs620 ratios of all the samples are depicted in Fig. 5. The bar graph of stimulant concentration-dependent Abs520/Abs620 ratio indicates that, with the increasing concentration of stimulants, the kinase activity in cell lysate gradually increases and then slightly decreases after reaching the maximum when the concentrations of forskolin and IBMX were 25 and 50 μM respectively. Therefore, the PKA activation in MCF-7 cells by the drugs is successfully detected through the Abs520/Abs620 ratios of the AuNPs dispersions as read-out. That is, this colorimetric assay is applicable for in vitro kinase assay in cell lysate.
Fig. 4. The Abs520/Abs620 signal of the AuNPs dispersion (2.2 nM) as a function of the concentration of the inhibitor H-89. The reaction mixture contained 1 mM S-pep, 250 mU/mL PKA, and 2.632 U/mL CPY.
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References
Fig. 5. The Abs520/Abs620 ratio as a function of PKA activity in the cell lysate stimulated by various concentrations of stimulants (forskolin/IBMX) shown in the inset table. The results were obtained from the mixtures of the AuNPs dispersion (2.2 nM) with S-pep phosphorylated by varying stimulated cell lysate samples (number1–6) and unstimulated lysate sample (blank). The total protein concentration of MCF-7 cell lysate (stimulated and unstimulated) was diluted to 12.5 mg/mL.
4. Conclusions In summary, we have developed a label-free colorimetric assay for detecting protein kinase activity and inhibition based on unmodified AuNPs. This kinase sensing platform is relied on the phosphorylation protection against CPY degradation and peptide-mediated non-crosslinking aggregation of AuNPs. Compared with the traditional methods using phosphorylation-specific antibodies and proteins, the CPYdependent phosphorylation recognition possesses the advantages of versatility, cost efficiency, and reliability due to its phosphorylationspecific inhibition and residue-nonspecific cleavage. Our assay is simple and homogenous without multistep washing process, sophistical labeling treatment of peptide, and the modification of AuNPs probe. Its colorimetric read-out could be easily observed by the naked eye without sophisticated instrument. Moreover, this colorimetric biosensing technique can readily expand to detect different protein kinase by simply changing specific positively charged substrate peptide sequences. Therefore, the proposed AuNPs/peptide kinase sensing platform shows great potential in fundamental research of protein kinase-related biochemistry and high-throughput screening for kinase-targeted drug discovery.
Acknowledgments This work was financially supported by the National Basic Research Program of China (973Program, Nos. 2009CB421601 and 2011CB911002), the Foundation for Innovative Research Groups of NSFC (No. 21221003), the National Natural Science Foundation of China (Nos. 21222507, 21175036, 21190044, and 21075031), the Program for New Century Excellent Talents in University (NCET-10-0366), and the Ph.D. Programs Foundation of the Ministry of Education of China (No. 20120161110025).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2013.09.070.
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A gold nanoparticles colorimetric assay for label-free detection of protein kinase activity based on phosphorylation protection against exopeptidase cleavage Jiang Zhou, Xiahong Xu, Xin Liu, Hao Li, Zhou Nie n, Meng Qing, Yan Huang, Shouzhuo Yao State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 23 July 2013 Received in revised form 17 September 2013 Accepted 26 September 2013 Available online 8 October 2013
Protein kinases are significant regulators in the cell signaling pathways, and it is still greatly desirable to achieve simple and quick kinase detection. Herein, we present a novel colorimetric gold nanoparticles (AuNPs)/peptide platform for probing the activity and inhibition of protein kinases based on phosphorylation-induced suppression of carboxypeptidase Y (CPY) cleavage. This AuNPs/peptide platform can easily monitor the kinase activity by a UV–vis spectrometer or even by the naked eye. The feasibility of the method has been demonstrated by sensitive measurement of the cAMP-dependent protein kinase (PKA) activity with a low detection limit of 0.232 mU/mL and assessment of kinase inhibition by H-89 with an IC50 value of 18.13 nM. The assay was also successfully put into practice for the detection of kinase activity in cell lysate. Because of its label-free, homogenous and colorimetric merits, the proposed assay presents great potential in high-throughput screening for kinase-targeted drug discovery. & 2013 Elsevier B.V. All rights reserved.
Keywords: Gold nanoparticles Protein kinase Protein phosphorylation Carboxypeptidase Y Cell lysate
1. Introduction Protein phosphorylation catalyzed by kinase is a universal regulatory mechanism in the cell signaling pathways, which plays a crucial role in various vital cellular physiological processes including cell growth, metabolism, differentiation, and apoptosis (Manning et al., 2002; Cohen, 2002a, 2002b; Kalume et al., 2003). The over-expression and aberrant activity of protein kinases are closely related to a number of severe diseases such as cancer, diabetes, inflammation, cardiac diseases, and Alzheimer's disease (Mark and Joseph, 2010; Cohen, 2002a, 2002b; Hanger et al., 2007). Therefore, the development of kinase assays capable of monitoring kinases activity, identifying their substrates and screening potential inhibitors is important for the research of fundamental biochemical pathways, clinical diagnosis and drug discovery (Cohen, 2002a, 2002b; Noble et al., 2004). Traditional method for assessing kinase activity relied on radioactive isotope-labeled ATP, which is general but hampered by the hazardous effect of radioactive materials (Turk et al., 2006; Hastie et al., 2006; Houseman et al., 2002). To overcome this shortcoming, various kinase assays, including electrochemical (Wieckowska et al., 2008; Ji et al., 2009; Xu et al., 2009; Kerman et al., 2008), surface-plasmon resonant(Yoshida et al., 2000;
n
Corresponding author. Tel.: þ 86 731 88821626; fax: þ 86 731 88821848. E-mail address: [email protected] (Z. Nie).
0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.09.070
Viht et al., 2007), and mass spectroscopic techniques (Mann et al., 2002; Watts et al., 1994) have been developed as the alternatives of radioactive assay. Most of these techniques involve a surfaceconfined process of substrate peptide, heterogeneous enzymatic reaction at peptide-modified surface, and multistep-washing procedure. Compared with those heterogeneous kinase assays, the approaches for homogenous detection of kinase activity possess intrinsic merits such as the short detection time, facile detection without separation, and readiness for high-throughput screening (HTS). Great progress has been achieved on the design of homogenous fluorescence measurements of kinase-catalyzed phosphorylation (Sato et al., 1999; Sharma et al., 2007; Agnes et al., 2010; Xu et al., 2011; Bai et al., 2013; Zhou et al., 2013). Because the color change can be directly monitored by naked eye without sophisticated instruments, the colorimetric biosensing for protein phosphorylation has attracted much attention because of its low cost, simplicity, and practicality. Although it is a promising technique for developing homogenous kinase assay, the existing colorimetric assays for kinase activity are still scarce in comparison with welldeveloped fluorescent counterparts. Gold nanoparticles (AuNPs) represent a potent colorimetric probe mainly due to their unique optical properties, particularly their size-dependent surface plasmon resonance absorption (Lim et al., 2007; Mirkin et al., 1996; Sato et al., 2003). There are only a few AuNPs-based methods currently available for homogenous activity detection of protein kinase. Brust et al. developed a colorimetric kinase assay in which the biotin-labeled ATP was
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used to probe phosphorylation process on peptide-modified AuNPs, and consequently the phosphorylated AuNPs with biotin label reacted with avidin-capped AuNPs to generate cross-linking and corresponding color-change (Wang et al., 2006; Wang et al., 2005). Similar cross-linking strategy has been designed by Stevens' group using AuNPs conjugated with substrate peptide and phospho-specific antibody, respectively, as dual probes to assess the activity of kinase (Gupta et al., 2010). Recently, Katayama group reported an intriguing kinase assay using phosphorylationinduced peptide charge change to mediate AuNPs aggregation (Oishi et al., 2008). Among these methods, most of them relied on phosphorylation-induced interparticles cross-linking of AuNPs, which are effective but suffer from the complicated modification of AuNPs, the high cost of labeled substrate, and relative long responsive time for observing the aggregation-induced color change (Lévy et al., 2004). Hence, it is still desirable to develop new label-free strategy to fulfill the phosphorylation-responsive non-crosslinking AuNPs aggregation. In this paper, we present a novel colorimetric AuNPs/peptide platform for probing the activity and inhibition of protein kinase. This AuNPs/peptide platform is composed of the unmodified AuNPs and the rationally designed substrate peptide probe. The phosphorylation recognition is dependent on the phosphorylation protection against carboxypeptidase digestion. The carboxypeptidase refers to a kind of exopeptidase that hydrolyzes peptide bond from the carboxyl terminal (C-terminal) of a protein or peptide (Low and Yuan, 1996). It has been de monstrated that the activity of exopeptidase are susceptible to modification on the amino acid (Dass and Mahalakshmi, 1996). With the aid of carboxypeptidase, the phosphorylated peptide could be discriminated from unphosphorylated one by its resistance to enzymatic cleavage, leading to retain the peptide fragment to mediate the aggregation of AuNPs. The cAMP-dependent protein kinase (PKA) and carboxypeptidase Y (CPY) were used as typical models. Because of its label-free property and no modification required for AuNPs and peptides, this method represents a promising application of AuNPs in protein kinase analysis.
The UV–vis absorption spectra were recorded on a Beckman DU800 spectrophotometer in a wavelength range from 400 to 800 nm. The absorbance measurements of each sample were conducted in three independent times (n ¼3). High-resolution transmission electron microscopy (HRTEM) measurements were made on a JEOL JEC-3010 electron microscope. The samples of TEM were prepared by placing a drop of solution (10 μL) on carboncoated copper grid and drying at room temperature in drying cabinet. Cell-breaking was performed using a JY92-IIN ultrasonic cell disruption system (Scientz, Ningbo, China). The photographs were recorded with digital camera.
2.2. Synthesis of gold nanoparticles (AuNPs) AuNPs (13 nm) were synthesized according to the previously reported method (Jin et al., 2003). Generally, 38.8 mM sodium citrate solution (5 mL) was rapidly added into 1 mM boiled HAuCl4 solution (50 mL) under vigorous stirring in a 100 mL round flask. The mixture was maintained boiling for 10 min, and the corresponding color change from yellow to deep wine red. The obtained gold nanoparticles solution was stored at 4 1C. The final concentration of AuNPs was calculated to be 11 nM using the UV–vis absorption spectrum based on Lambert–Beer's law and extinction coefficients (ε) of 2.7 108 M 1 cm 1 at λ520 for 13 nm AuNPs. The so-prepared AuNPs dispersion was diluted 2.5 times by ultrapure water and used as stock solution in the subsequent absorbance experiments. The absorption ratio (Abs520/Abs620) of the AuNPs dispersion was chosen as the respond signal of the AuNPs dispersion stability.
2.3. Peptide-induced AuNPs aggregation A series of 40 mL S-pep samples at different concentrations (0, 0.001, 0.01, 0.1, 1, 2.5, 5, 10, 20 and 40 mM) were prepared in 10 mM Tris–HCl buffer (pH 7.5, 25 1C), then an equal volume of the unmodified AuNPs dispersion (4.4 nM) was added and mixed for 5 s. After mixing, UV–vis spectroscopic measurements at the 400–800 nm wavelength region were made at room temperature.
2. Experimental methods 2.1. Materials and measurements
2.4. Treatment of S-pep by carboxypeptidases Y (CPY)
Cyclic adenosine 3′,5′-monophosphate-dependent protein kinase (PKA, catalytic subunit) and Casein kinase II (CKII) were purchased from New England Biolabs (Beverly, MA, USA). Generally, PKA was diluted using the storing solutions (50 mM NaCl, 1 mM EDTA, 2 mM DTT, 50% glycerol in 20 mM Tris–HCl buffer (pH 7.5, 25 1C)) and stored in the refrigerator at 80 1C. Substrate peptide probe (S-pep, CGGRRGLRRASLG) for PKA was synthesized by GL Biochem (Shanghai, China). ATP was bought from Generay (Shanghai, China). HAuCl4 4H2O was supplied from Shanghai Reagent Company (Shanghai, China). H-89 was obtained from EMD Biosciences (Calbiochem-Novabiochem. La Jolla, CA, USA). Carboxypeptidase Y (CPY), forskolin and 3-isobutyl-1methylxantine (IBMX) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The improved Bradford protein assay dye reagent kit was purchased from Sangon (Shanghai, China). The rest of the chemical reagents including bovine serum albumin (BSA), Tris, glycerol, DTT, and EDTA used in this study were obtained from Bio Basic (Ontario, Canada). Human breast cancer cells (MCF-7) were bought from the Cell Bank of Xiangya Central Laboratory of Central South University (Changsha, China). The ultrapure water (18.3 MΩ cm) from the Millipore Milli-Q system was used in all experiments.
The S-pep (1 mM) was treated with the CPY (2.632 U/mL) for 45 min. The resulting solution (40 mL) was mixed with an equal volume of AuNPs dispersion (4.4 nM) and then measured by spectroscopy. A range of concentrations (0–4 U/mL) of CPY were exploited to optimize the quantity of CPY. For optimization of the digestion time, the same experiments were conducted incubating with 2.632 U/mL CPY for different incubation times (0—90 min).
2.5. Detection of activity and inhibition of PKA For PKA-catalyzed phosphorylation, 200 mL of the PKA reaction solutions composed of PKA (0–1 U/mL), S-pep (1 mM), MgCl2 (1 mM), ATP (0.1 mM) in 20 mM Tris–HCl buffer (pH 7.5, 25 1C) was incubated for 60 min at 30 1C, and the resulting phosphorylated solution was incubated with 2.632 U/mL CPY for 45 min at 25 1C, then 40 mL of the mixture was mixed with the AuNPs dispersion (40 mL, 4.4 nM) and measured by spectroscopy. For PKA inhibition assays, 250 mU/mL PKA and H-89 at different concentrations (0–0.5 mM) were added into the reaction solutions, and the experiment procedures were set under the above-mentioned conditions.
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2.6. Detection of PKA activity in MCF-7 cell lysate Ten percent fetal bovine serum, 0.1 mM of mimimum eagle's essential medium (MEM) nonessential amino acid solution, 1% insulin–transferrin–selenium A supplement, 100 U/mL of penicillin, 100 mg/mL of streptomycin, and 0.25 mg/mL amphotericin B composed the MCF-7 breast cancer cells (1 106 cells) supplement. The MCF-7 cells were incubated in a humidified atmosphere with 5% CO2 at 37 1C. After a 4 h incubation in the serum-free medium replacing the culture medium, then the forskolin and IBMX mixture diluted with dimethyl sulfoxide (DMSO) was added into the medium at various concentrations (the final concentrations of forskolin and IBMX are shown in the inset table of Fig. 5A) with the purpose of stimulating intracellular PKA activity. Equal volume of DMSO was added into the medium as the unstimulated control sample. After 30 min of stimulation, the cultured cells were removed by scraping and lysed in Dulbecco's phosphate-buffered saline (D-PBS) by sonication (200 W) 2 s for 60 times at a 3 s interval each time for protein extraction. The samples were clarified by centrifugation for 60 min at 41125 g, 4 1C and the extracted supernatants were stored in the freezing tubes at 20 1C and used in the following experiments. The Bradford method was used to determine the total protein content of cell lysate supernatants, and the protein concentration of each cell lysate samples was diluted to 12.5 μg/mL used for the experiments. For kinase activity assays in MCF-7 cell lysate, the same experiments were performed except that the above-prepared cell lysate samples instead of pure PKA were investigated.
3. Results and discussion 3.1. The principle of the colorimetric assay for kinase activity detection The kemptide, Leu–Arg–Arg–Ala–Ser–Leu–Gly (LRRASLG) containing the phosphorylation site Ser, is the substrate peptide for cAMP-dependent protein kinase (PKA), which has a high affinity for PKA with Km ¼ 5 μM (Cheng et al., 1986). The detection mechanism of the proposed assay is shown in the Scheme 1. An artificially designed peptide CGGRRGLRRASLGA, namely S-pep, is employed here for the assay. It is clearly observed that the S-pep is comprised of three parts, i.e., the kemptide motif (LRRASLG), two additional positively charged Arg residues (RR) to ensure the
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peptide net charge being positive both before and after phosphorylation, and the cysteine at N terminal for anchoring on the surface of citrate-capped AuNPs by Au–S bond. This rationally designed positively charged S-pep is capable of inducing AuNPs aggregation because it can readily bind to negatively charged AuNPs, neutralizing the AuNPs surface charges, and disrupt the stability of AuNPs. CPY, a representative exopeptidase, was applied to recognize the kinase catalysis event due to its activity susceptible to phosphorylated modification of amino acid (Dass and Mahalakshmi, 1996; Murray et al., 1996). When non-phosphorylated S-pep was treated with CPY, it is literally cleaved from C-terminal into the free amino acids (Low and Yuan, 1996). The full degradation of S-pep leads to only cysteine attached on the AuNPs surface, thus keeping dispersion of AuNPs and the color of the solution remaining red due to no disturbance of AuNPs surface charge (route a). However, after phosphorylation by kinase, the phosphorylated peptide (CGGRRGLRRApSLG) can effectively block CPY cleavage by the bulky phosphorylated serine sites (Kupcho et al., 2003; Zhou et al., 2013), and the CPY-treated S-pep fragment retaining two positive net charges still can cause AuNPs aggregation and corresponding red-to-blue color change (route b). Hence, we could successfully detect activity of PKA by the color change and the UV–vis spectra of unmodified AuNPs with the assistance of CPY.
3.2. Optimization of S-pep induced aggregation of AuNPs In order to obtain the best colorimetric performance, we investigated the effect of S-pep concentrations on the colorimetric response of the unmodified AuNPs dispersion, which is described by the Abs520/Abs620 ratio of AuNPs solutions. Fig. 1 shows the Abs520/Abs620 ratio and the color change of the AuNPs as a function of S-pep concentration (0, 0.001, 0.01, 0.1, 1, 2.5, 5, 10, 20 and 40 mM, respectively). It can be seen that, at the S-pep concentration ranging from 0 to 0.1 μM, the Abs520/Abs620 ratio keeps more than 5.78 and the solution color is red, while the Abs520/Abs620 ratio decreases sharply to 0.7 and the blue color is observed at the concentration reaching or beyond 1 μM, indicating the aggregation of AuNPs. Hence we could conclude that, in order to induce aggregation of AuNPs, S-pep concentration should be kept higher than 1 μM. Furthermore, the effect of S-pep in the presence and absence of CPY was further examined, and the results are shown in the inset of Fig. 1A. The most obvious response reflected by the largest ΔAbs520/Abs620 ratio was observed at 1 μM S-pep, and the corresponding UV–vis absorption spectra of the AuNPs dispersion
Scheme 1. The principle of colorimetric assay of PKA activity detection based on carboxypeptidase Y (CPY) and unmodified gold nanoparticles (AuNPs).
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are shown in Fig. S1 in Supporting Information (SI). Therefore, 1 μM substrate peptide was chosen as the optimal concentration for the following experiment. 3.3. Optimization of CPY treatment of S-pep At first, the S-pep degradation by CPY digestion was investigated, and the UV–vis absorption spectra and color changes (inset) of the AuNPs dispersion of the S-pep and S-pep/CPY samples are displayed in Fig. S2. When the AuNPs was mixed with the S-pep (1 mM), a distinct absorption peak at 628 nm, the symbol of aggregated AuNPs, was observed (spectrum a), and this absorption peak is much higher than that at 520 nm. The corresponding
images of solution color (a) also demonstrate the aggregation of AuNPs which is in accordance with the result of the UV–vis spectra. Nevertheless, the S-pep treated by CPY (5 U/mL) causes only one absorption peak at 520 nm, indicating good dispersion of AuNPs, which is confirmed by the red solution color (b). Then, to obtain the best hydrolysis performance of CPY, the concentration and reaction time of CPY were optimized. Various CPY concentrations (0–4 U/mL) and treating times (0–90 min) were investigated for optimization in the presence of S-pep (1 mM), and the results are shown in Fig. S3. The Abs520/Abs620 ratio of the AuNPs mixture increases with the CPY concentration increasing, until it reaches a plateau at 2.632 U/mL CPY. With the reaction time increasing, the Abs520/Abs620 ratio gradually increases and then turns to be a platform at reaction time 45 min. So 2.632 U/mL CPY and 45 min of reaction time were employed in the subsequent assays.
3.4. Detection of activity and inhibition of PKA
Fig. 1. The Abs520/Abs620 ratio (A) and the color change (B) of the AuNPs dispersion after addition of S-pep solutions (0, 0.001, 0.01, 0.1, 1, 2.5, 5, 10, 15, 20 and 40 mM) in 10 mM Tris–HCl buffer (pH 7.5). The concentrations of AuNPs dispersion used for absorbance measurement and colorimetric observation were 2.2 nM and 5.5 nM, respectively. The inset of (A) shows the bar chart of Abs520/Abs620 ratios of the AuNPs dispersion in the presence of S-pep sample (1–40 μM) before and after addition of 13.16 U/mL CPY. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Mg2 þ , ATP, and Tris–HCl buffer (pH 7.5) are required for protein kinase reaction, which may have undesirable influence on the stability of AuNPs, so their effects on AuNPs stability were investigated in our experiments. As shown in Fig. S4A, the Abs520/Abs620 ratio of the AuNPs dispersion remains unaffected when Mg2 þ concentration is lower than 1 mM. It can also be concluded that the ATP concentration (0–0.2 mM) has no significant influence on the stability of AuNPs (Fig. S4B). It was found that the Tris–HCl (pH 7.5) would not induce AuNPs aggregation unless the buffer concentration was higher than 20 mM (Fig. S4C). So we employed 1 mM Mg2 þ , 0.1 mM ATP, and 20 mM Tris–HCl (pH 7.5) for the kinase reaction. The UV–vis spectra, transmission electron microscope (TEM) images, and color changes of the AuNPs dispersion of the various samples were studied and the results are displayed in Fig. 2. The S-pep, after addition of CPY (5 U/mL), exhibits only one absorption peak at 520 nm (spectrum 1), and the corresponding TEM images (1) and red solution color (1) also are in accordance with the UV–vis spectrum result, indicating the S-pep was digested and the AuNPs were well-dispersed. However, two obvious absorption peaks of AuNPs at 520 nm and 620 nm can be observed in the AuNPs dispersion after addition of the phosphorylated peptide by PKA (250 mU/mL) in the presence of CPY (5 U/mL) (spectrum 4) and the solution color is blue (4) as expected. TEM image
Fig. 2. UV–vis absorption spectra (A), color changes (the inset of A), and transmission electron microscope (TEM) images (B) of the AuNPs dispersion in different conditions. The concentrations of AuNPs dispersion used for absorbance measurement and colorimetric observation were 2.2 nM and 5.5 nM, respectively. AuNPs dispersion mixed with (1) 1 mM S-pep treated by 2.632 U/mL CPY, (2) 1 mM S-pep with 250 mU/mL CKII and 0.1 mM ATP in the presence of 2.632 U/mLCPY, (3) 1 mM S-pep, 250 mU/mL PKA with addition of 2.632 U/mL CPY, and (4) (3) with 0.1 mM ATP. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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AuNPs when the H-89 concentration is lower than 0.5 μM, therefore, 0–0.5 μM H-89 is used for the following inhibition experiment. A sigmoidal profile of the Abs520/Abs620 ratio versus the H-89 concentration was obtained (Fig. 4). With the increase of the H-89 concentration, Abs520/Abs620 ratio of the AuNPs mixture increases until H-89 concentration reaches up to 250 nM, at which the corresponding Abs520/Abs620 ratio reaches a platform. The IC50 value (the corresponding inhibitor concentration required to reduce protein kinase activity by 50%) was found to be 23.09 nM, which is in accordance with the reported value in literature (Rodems et al., 2002). These results indicated that our method can be used to study the protein kinase inhibitor and is feasible for protein kinase inhibitors screening.
3.5. Protein kinase A activity detection in cell lysate
Fig. 3. Colorimetric assay of PKA activity based on the measurements of the Abs520/ Abs620 ratios (A) and the color changes (B) of AuNPs. The results were obtained from the mixtures of AuNPs dispersion and S-pep (1 mM) phosphorylated by PKA (0–1 U/mL) in the absence (black) and presence (gray) of 2.632 mU/mL CPY. The concentrations of AuNPs dispersion used for absorbance measurement and colorimetric observation were 2.2 nM and 5.5 nM, respectively. The inset shows the calibration curve of the Abs520/Abs620 ratio dependent on the PKA concentration.
(4) further demonstrated that AuNPs were aggregated. There is no obvious spectra shift in the sample without ATP (spectrum 3) and non-target kinase sample with CKII (250 mU/mL) (spectrum 2) compared with the S-pep/CPY sample, indicating that the signals of this sensor can specifically and selectively respond to the occurrence of phosphorylation. Therefore, it is feasible and convenient to monitor the PKA activity with the aid of CPY based on the change of color and absorption spectra of the AuNPs dispersion as the read-out. Quantitative detection of kinase activity was further studied under the pre-optimized conditions with different concentrations of PKA (0–1 U/mL). As depicted in Fig. 3, with the increase of PKA concentration, the Abs520/Abs620 ratios of phosphorylated peptide/ AuNPs sample in the presence of CPY significantly decreases while those ratios of the samples without CPY only slightly increases, indicating that the phosphorylation recognition in this sensor is CPY-dependent and a higher degree of phosphorylation can induce a greater blocking effect on CPY enzymatic hydrolysis. The calibration curve of Abs520/Abs620 ratios versus logarithm of the PKA concentration is plotted in the inset of Fig. 3. It is observed that the Abs520/Abs620 signal turns to a platform when PKA concentration reaches 250 mU/mL. The EC50 value (the corresponding protein kinase concentration required to phosphorylate 50% substrate) of PKA was estimated to be 41.58 mU/mL. The detection limit of PKA is estimated to be 0.232 mU/mL which is lower than the data reported previously (Ji et al., 2009;Xu et al., 2011; Oishi et al., 2007; Kerman et al., 2007; Miao et al., 2012). To investigate whether colorimetric assay could be further used to study the PKA inhibition, the experiments were performed as the same procedures in the presence of a well-known PKA inhibitor H-89 with different concentrations. H-89 is an isoquinoline derivative capable of inhibiting PKA activity by acting as the competitive antagonist of ATP at its binding site on the PKA catalytic subunit (Engh et al., 1996). Firstly, the influence of H-89 concentration on the stability of AuNPs was tested. As shown in Fig. S5, there is negligible effect on the Abs520/Abs620 ratio of
The activation of kinases in a cell caused by extracellular stimulation can trigger a series of important cellular processes including transcription, differentiation, and apoptosis (Godwin et al., 1993). Thus, whether the assays are competent to detect kinase activity in cell lysate is significant for the study of kinase regulation in the cell system. PKA in human cells could be activated using extracellular stimulation by forskolin, the adenylyl cyclase activator, and IBMX, a phosphodiesterase inhibitor. First of all, whether cell lysate samples could affect the stability of AuNPs was tested. As shown in Fig. S6, the cell lysate samples have little influence on the stability of AuNPs. Then, the detection of PKA activity in cell lysate was performed, and the samples were prepared by mixing the AuNPs dispersion with the S-pep phosphorylated by stimulated lysate samples (number 1–6) and untreated lysate sample (blank). The corresponding Abs520/Abs620 ratios of all the samples are depicted in Fig. 5. The bar graph of stimulant concentration-dependent Abs520/Abs620 ratio indicates that, with the increasing concentration of stimulants, the kinase activity in cell lysate gradually increases and then slightly decreases after reaching the maximum when the concentrations of forskolin and IBMX were 25 and 50 μM respectively. Therefore, the PKA activation in MCF-7 cells by the drugs is successfully detected through the Abs520/Abs620 ratios of the AuNPs dispersions as read-out. That is, this colorimetric assay is applicable for in vitro kinase assay in cell lysate.
Fig. 4. The Abs520/Abs620 signal of the AuNPs dispersion (2.2 nM) as a function of the concentration of the inhibitor H-89. The reaction mixture contained 1 mM S-pep, 250 mU/mL PKA, and 2.632 U/mL CPY.
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References
Fig. 5. The Abs520/Abs620 ratio as a function of PKA activity in the cell lysate stimulated by various concentrations of stimulants (forskolin/IBMX) shown in the inset table. The results were obtained from the mixtures of the AuNPs dispersion (2.2 nM) with S-pep phosphorylated by varying stimulated cell lysate samples (number1–6) and unstimulated lysate sample (blank). The total protein concentration of MCF-7 cell lysate (stimulated and unstimulated) was diluted to 12.5 mg/mL.
4. Conclusions In summary, we have developed a label-free colorimetric assay for detecting protein kinase activity and inhibition based on unmodified AuNPs. This kinase sensing platform is relied on the phosphorylation protection against CPY degradation and peptide-mediated non-crosslinking aggregation of AuNPs. Compared with the traditional methods using phosphorylation-specific antibodies and proteins, the CPYdependent phosphorylation recognition possesses the advantages of versatility, cost efficiency, and reliability due to its phosphorylationspecific inhibition and residue-nonspecific cleavage. Our assay is simple and homogenous without multistep washing process, sophistical labeling treatment of peptide, and the modification of AuNPs probe. Its colorimetric read-out could be easily observed by the naked eye without sophisticated instrument. Moreover, this colorimetric biosensing technique can readily expand to detect different protein kinase by simply changing specific positively charged substrate peptide sequences. Therefore, the proposed AuNPs/peptide kinase sensing platform shows great potential in fundamental research of protein kinase-related biochemistry and high-throughput screening for kinase-targeted drug discovery.
Acknowledgments This work was financially supported by the National Basic Research Program of China (973Program, Nos. 2009CB421601 and 2011CB911002), the Foundation for Innovative Research Groups of NSFC (No. 21221003), the National Natural Science Foundation of China (Nos. 21222507, 21175036, 21190044, and 21075031), the Program for New Century Excellent Talents in University (NCET-10-0366), and the Ph.D. Programs Foundation of the Ministry of Education of China (No. 20120161110025).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2013.09.070.
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