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Cyclic-AMP-dependent protein kinase (PKA) activity assay based on FRET between cationic conjugated polymer and chromophore-labeled peptide† Shiyun Tang, Yufang Hu, Qinpeng Shen, Heting Fang, Wang Li,* Zhou Nie and Shouzhuo Yao A sensitive fluorescence turn-on biosensing platform for protein kinase activity assay has been developed based on fluorescence resonance energy transfer (FRET) between a fluorophore labeled peptide and a water soluble cationic conjugated polymer (CCP). The CCP-based assay is based on the electrostatic interaction between the peptide and the CCP. The FRET efficiency will change with the changing charges around the peptide after phosphorylation. The feasibility of this method has been demonstrated by sensitive measurement of the activity of cAMP-dependent protein kinase (PKA) with a low detection limit (0.3 mU mL1). Based on its simple mechanism, this assay is also sensitive and robust enough to be
Received 7th May 2014 Accepted 19th June 2014
applied to the evaluation of PKA inhibitor H-89. The IC50 value, the half maximal inhibitory concentration, was 40 nM. Furthermore, our method has excellent selectivity. CCP-based assay is
DOI: 10.1039/c4an00814f
sensitive, versatile, cost-effective and easy to operate, so, this method is a promising candidate for
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kinase activity assay and inhibitor screening.
1. Introduction Protein phosphorylation by kinases plays a vital role in many cellular processes, including metabolism, transcription, cell cycle progression, cytoskeletal rearrangement, apoptosis, and differentiation.1–3 The human genome encodes more than 500 kinds of protein kinases, exerting tight and reversible control on protein phosphorylation.4 Aberrant phosphorylation and overexpression of protein kinases are closely associated with the pathogenesis of many human diseases,2 such as cancer,5 Parkinson's disease,6 and Alzheimer's disease.7 In order to understand the pathogenesis of carcinogenesis and discover proteinkinase-targeted drug, it is very important to detect kinase activities and screen their potential inhibitors.8 Therefore, developing accurate and sensitive methods for kinase assays and potential inhibitor screening is of great signicance for biochemical investigation and drug discovery.9–11 Even though there are a number of methods to investigate protein kinase phosphorylation events, potential pitfalls exist in these methods. Traditional methods for assessing protein kinase activities are radiometric assays.12,13 Because the radioactive g-phosphoryl moiety is hazardous and has a short State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, P. R. China. E-mail: [email protected]; Fax: +86-731-88821848; Tel: +86-731-88821626 † Electronic supplementary information (ESI) available: Fig. S1–S6. See DOI: 10.1039/c4an00814f
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half-life, immunoassay by using specialized biological reagents such as phosphopeptide-recognized antibody has been developed as an alternative to the radiometric method.14–16 These methods, however, are costly and suffer from inconsistent batch-to-batch quality of these proteins. As a consequence, signicant efforts have been devoted to the development of nonradioactive and antibody-free assays for detecting protein kinase activity with various analytical methods, such as colorimetry,17,18 electro-generated chemiluminescence,19,20 electrochemical,21,22 mass spectrometry,23,24 surface-enhanced Raman spectroscopy (SERS),25 and quartz-crystal microbalance.26 However, a majority of these methods require a surfaceconned process, multistep washing, sophisticated preparation, or expensive instrumentation. Among these existing protein kinase assays, the inherent advantages of the uorescence-based methods, such as homogeneous detection, high sensitivity, easy readout and simple operation, make them very attractive for detection of protein kinase activities. Therefore, many kinds of uorescence assays for detection of protein kinase activities have been developed based on electrophoresis,27 microarrays,28 magnetic microspheres,29,30 quantum dots,31,32 and uorescent proteins.33,34 Although these methods are novel and offer great potential to be applied to detection of protein kinase activity, there are still some limitations, such as time-consuming, labor-intense process, complicated preparation and costly materials.
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Over the past several years, water soluble conjugated polymers (WCPs) have gained much attention in applications for highly sensitive detection of biomacromolecules.35,36 These materials are characterized by backbones with p-delocalized electronic structures and pendant substituents with ionic functionalities.37 In contrast to bioassays with small-molecule counterparts, the excitation energy along the whole backbone of the WCP transferring to lower energy chromophore acceptor sites over long distances results in the amplication of uorescence signals.38 So, WCPs can be used for the function of very sensitive optical biosensors.39 D. Whitten et al. developed a sensitive, homogeneous and versatile method for detection of kinase activity based on quenching of the uorescence of anionic uorescent-conjugated polymers.40 However, the uorescence quenching-based methods oen suffer from the disadvantage that they may be difficult to interpret whether a reduction in uorescence intensity is caused by poor assay performance or the presence of target kinases. To overcome this limitation, L. F. Hancock, Z. Li and co-workers developed homogeneous approaches based on anionic uorescent-conjugated polymers coupled with the metal ion-mediated FRET.41,42 Nevertheless, these uorescence assays based on metal ion complexing actions are subjected to the weakness that the phosphate coordinating metal ions are easily hydrolysed to hydroxide and adenosine triphosphate (ATP) will oen interfere with the assay process. From the above, it still remains a great challenge to develop homogeneous, sensitive, reliable, and simple protein kinase assays for detection of protein kinase activities and for high-throughput screening of kinase inhibitors. Here, we report a sensitive, selective and simple method based on the FRET between a water soluble cationic conjugated polymer (CCP) and a uorophore labeled peptide. In this method, a CCP was introduced in a protein kinase detection and kinase inhibition study, which expands the application range of the CCP. Compared with the previous reports, this CCP-based assay offers a mix-and-detect assay format based on electrostatic interactions, avoiding the weakness caused by metal ion complexing actions. Because the CCP-based assay is sensitive, versatile, cost-effective and easy to operate, it has good potential to be used for the detection of protein kinase activity and for the screening of protein kinase inhibitors as potential drugs.
2. 2.1
Experimental details Materials
The water soluble CCP, poly(9,9-bis(60 -N,N,N-trimethylammonium)hexyl)uorenylene phenylene (PF), was synthesized according to a reported method.43 A uorophore labeled substrate peptide was purchased from GL Biochem Ltd. (Shanghai, China). Cyclic adenosine 30 ,50 -monophosphatedependent protein kinase (PKA, catalytic subunit) and casein kinase II (CK2) were obtained from New England Biolabs Inc. (Beverly, MA). N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) was obtained from EMD Biosciences (Calbiochem-Novabiochem. La Jolla, CA). Thrombin was
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obtained from Sigma-Aldrich (St. Louis, MO). A recombinant GST-puried p300 HAT (histone acetyltransferase) domain was purchased from Signal-Chem (USA). All solutions were prepared using ultrapure water (18.25 MU cm) from a Millipore Milli-Q system. All samples were illuminated at an excitation wavelength of 384 nm and the uorescence emission spectra were scanned from 400 to 600 nm at room temperature. Fluorescence measurements were performed in Tris–HCl (50 mM, pH 7.5). Fluorescence spectra were recorded by using a QuantaMaster™4 uorescence spectrometer (PTI). 2.2
Detection of activity and inhibition of PKA
The PKA storing solutions were composed of 20 mM Tris–HCl buffer (pH 7.5, 25 C), 50 mM NaCl, 1 mM EDTA, 2 mM DTT, and 50% glycerol. The PKA reaction solutions were composed of PKA (from 0 mU mL1 to 150 mU mL1), peptide (600 nM), MgCl2 (10 mM), ATP (4 mM), and 50 mM Tris–HCl buffer (pH 7.5, 25 C). Aer incubation at 30 C for PKA-catalyzed reaction, the resulting solution (10 mL) was added to 90 mL of CCP (the nal concentration of the CCP was 2.95 106 M in repeat units (RUs)). Aer incubation at room temperature for 1 min, uorescence spectra were recorded at room temperature. For PKA inhibition assay, the experiments were carried out using a similar procedure as those for PKA assay stated above, except for mixing PKA (50 mU mL1) with different concentrations of H-89 (from 0 nM to 2000 nM) before the addition of the substrate peptide.
3.
Results and discussion
3.1 Detection mechanism of the CCP-based biosensor for kinase activity assay As reported, the electrostatic interactions between the CCP and target molecules can be used for probing biomolecules.36 PF has abundant pendant substituents with ionic functionalities and was employed as a CCP in this study. It can offer a large number of opportunities to couple analytic receptor interactions, as well as nonspecic interactions, into detectable responses.39 Fluorescein isothiocyanate (FITC), with absorption spectra roughly from 430 nm to 510 nm and emission spectra approximately from 500 nm to 550 nm, was chosen since its absorption spectra overlap with the emission spectra of the CCP with the photoluminescence spectra approximately from 400 nm to 500 nm (as shown in Fig. S1 in the ESI†). As a consequence, a CCP and FITC were chosen to meet the requirement conditions for efficient FRET.36 Irradiation at 384 nm selectively excites the CCP, and FRET generated from the CCP (donor) to FITC (acceptor) was favored (as shown in Fig. 1). The uorescence intensity of the CCP was quenched efficiently by FITC at approximately 419 nm and the uorescence intensity of FITC was enhanced greatly at about 520 nm through the transfer of uorescence resonance energy. Based on the above, a sensitive method for kinase activity assay could be developed based on FRET. Kemptide, Leu-Arg-Arg-Ala-Ser-Leu-Gly (LRRASLG) containing the phosphorylation site Ser, is the substrate peptide for PKA, which has a high specicity for PKA.2 Therefore, the
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Fluorescence emission spectra of the FRET generated from the CCP to free FITC. Conditions: 2.95 106 M (in repeat units (RUs)) CCP. Fig. 1
kemptide labeled with FITC was chosen as the substrate peptide for PKA in our method. Conventional methods for detecting protein kinase are based on the measurement of kinase-catalyzed phosphorylation and involve two steps, a phosphorylation step and a signal report step. We investigated the FRET generated from the CCP to the FITC labeled substrate peptide (F-Speptide, FITC-LRRASLG) or the phosphorylated peptide (F-Ppeptide, FITC-LRRApSLG) since the F-S-peptide and the F-Ppeptide are the reactant and product of PKA-catalyzed phosphorylation. The mechanism for the PKA activity biosensor is shown in Scheme 1 based on the idea that the F-S-peptide is phosphorylated by PKA and generates an F-P-peptide, then, the F-P-peptide was mixed with the CCP and the uorescence spectrum curve was recorded and output as a uorescence signal. Since one arginine residue possesses a positive charge (+1) and one carboxyl group possesses a negative charge (1) at the carboxyl end of the peptide, the PKA-specic F-S-peptide (FITC-LRRASLG) would have a positive charge (+1). Because of one phosphate group with two negative charges, the net charge of its counterpart phosphorylated peptide will have one negative charge (1) aer introducing a phosphate group from ATP.32 The distance between the CCP and the peptide is affected by the charge around the peptide and the distance will inuence the efficiency of FRET generated from the CCP to the FITC
Scheme 1 Schematic representation of the strategy of PKA activity assay based on the FRET between the FITC labeled peptide and the CCP.
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labeled at the substrate peptide. The electrostatic repulsion of the F-S-peptide with the CCP keeps the F-S-peptide far away from the CCP, while the electrostatic attraction of the F-Ppeptide phosphorylated by PKA with the CCP keeps the F-Ppeptide close to the CCP, resulting in a signicant FRET from the CCP to the F-P-peptide. In comparison with the uorescence quenching-based assay, simultaneous measurement of uorescence intensity of the CCP and FITC is credible for detection of PKA activity. For the purpose of visualizing the results, the FRET ratio I is dened as I ¼ I520 nm/I419 nm (I520 nm and I419 nm are dened as the uorescence intensities at 520 nm and 419 nm respectively) and the signal to noise ratio S/N is dened as S/N ¼ I/I0 (I0 is the FRET ratio of the control experiment). The use of the signal to noise ratio of this PKA biosensor system is feasible for the detection of PKA activity. In order to exhibit our results clearly, the uorescence intensities at 419 nm were all normalized to 1. A phenomenon was found that we could get high FRET efficiency upon addition of the CCP to solutions of the F-Speptide only in the presence of ATP and PKA (see Fig. 2A). As shown in Fig. 2B, when PKA or ATP was absent, the value of I/I0 has no obvious difference with the blank experiment (without PKA and ATP). The value of I/I0 of the experiment only in the presence of ATP and PKA was clearly different from that of other experiments, suggesting that the F-S-peptide was phosphorylated by PKA and generated the F-P-peptide. This also indicates that CCP-based assay has good potential to be used for the detection of protein kinase activity. 3.2
Optimization of the experimental conditions
Since protein kinase requires ATP as the cofactor, which may induce undesirable inuence on the uorescence intensity of the CCP, its effect on the CCP was investigated carefully in our experiments. A gradual decline was discovered in the uorescence intensity of CCP solution applying to different concentrations of ATP from 2 mM to 50 mM (Fig. S2†). However, there was little inuence on the uorescence intensity of the CCP suspension when the ATP concentration was 4 mM. Since we
Fig. 2 (A) Normalized fluorescence emission spectra of the effect of PKA and ATP on CPP-based assay. (B) The signal to noise ratio of the effect of PKA and ATP on CPP-based assay. (a) 600 nM F-S-peptide was phosphorylated by 50 mU mL1 PKA in the presence of 4 mM ATP; (b) without PKA; (c) without ATP; (d) without PKA and ATP.
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employed 4 mM ATP for each enzyme reaction and also for each control experiment, the effect of ATP on the CCP uorescence intensity was negligible. Since PKA may induce undesirable inuence on the uorescence intensity of the CCP, we also studied the inuence of PKA on the uorescence intensity of the CCP in our experiments. Fig. S3† shows the uorescence intensity of the CCP as a function of the concentration of PKA. When the concentration of PKA was lower than 200 mU mL1, no signicant variation at 419 nm of the CCP uorescence intensity was observed. To obtain the best sensing performance of the PKA biosensor system, the concentration of F-S-peptides was also optimized. The effect of F-S-peptide concentration (from 100 nM to 1400 nM) on the value of I/I0 of the PKA biosensor system was tested with a xed concentration of the CCP (the nal concentration of the CCP was 2.95 106 M (in RUs)), and the results are shown in Fig. S4 in the ESI.† The best concentration of the F-S-peptide for the PKA biosensor system was found to be 600 nM with a constant concentration of the CCP. To conrm that the FRET change was really caused by phosphorylation, the effect of PKA on the FRET between the F-S-peptide and the CCP was studied. Here, the uorescence intensity was measured immediately aer mixing PKA and the F-S-peptide under similar conditions using the experiments of PKA activity detection without any incubation at 30 C. As shown in Fig. S5,† when the concentration of PKA was lower than 150 mU mL1, there was not signicant inuence upon FRET from the CCP to the F-Speptide. Therefore, the CCP-based method is feasible to detect the activity of PKA. With the purpose of getting the best incubation time between the F-P-peptide and the CCP, the impact of incubation time on the FRET between the F-P-peptide and the CCP was studied. Fig. S6† shows that the electrostatic interaction between the CCP and the F-P-peptide was very rapid and the numerical value of I/I0 has little change from 1 min to 10 min (start time aer mixing the F-P-peptide and CCP). There is no signicant change of I/I0 with the extended reaction time from 10 min to 40 min. For the sake of improving the measurement efficiency and shortening the time of measurement, 1 min was employed as the incubation time between the F-P-peptide and the CCP. The F-P-peptide was obtained by mixing the F-S-peptide with PKA and incubation at 30 C for 60 min. This phosphorylation process by PKA is necessary for the general uorescence detection method.31,42 However, our method can detect the uorescence signals without separating the substrate peptides and phosphorylated peptides. Furthermore, our method is quicker and simpler than those assays based on anionic uorescent-conjugated polymers since no chelate metal ions are needed.
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peptide and CCP at room temperature, the uorescence signal of the CCP and F-S-peptide was recorded. So, our method possessed simple operation and easy readout. As shown in Fig. 3A, the FRET efficiency signicantly increased with increasing concentration of PKA, indicating that the efficient FRET was generated from the CCP to the F-P-peptide. Furthermore, the quantitative assessment of the FRET-based assay for PKA activity detection was investigated by monitoring the dependence of the numerical value of I/I0 on the concentrations of PKA. As shown in Fig. 3B, when the numerical value of I/ I0 versus the concentration of PKA was plotted, a sigmoidal prole was obtained. The numerical value of I/I0 increased with increasing concentration of PKA within certain limits, indicating that a higher degree of phosphorylation induced efficient FRET. Meanwhile, control experiments were also carried out in the absence of PKA. By measuring the value of I/I0, PKA activity could be quantitatively detected over a wide concentration range from 0.3 mU mL1 to 150 mU mL1. The detection limit was 0.3 mU mL1. This demonstrates that the proposed uorescence sensor can be employed for highly sensitive protein kinase detection over a wide concentration range. Detection sensitivity is one of the most important aspects of bioassay development. The detection limits of the most
Detection of the activity of PKA
In order to detect the activity of PKA, a series of F-S-peptide mixtures with various concentrations of PKA were tested. The reaction solutions (10 mL) were incubated for 60 min at 30 C for phosphorylation under the optimized experimental conditions, and the phosphate groups were transferred from ATP to the F-Speptide to generate an F-P-peptide. Then, aer mixing the F-P-
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Fig. 3 (A) Normalized fluorescence emission spectra of the CCPbased FRET assay in response to different concentrations of PKA. PKA concentrations from bottom to top: 0 (control), 0.3, 0.94, 3.1, 6.25, 12.5, 18.75, 31.25, 37.5, 50, 68.5, 81.25, 100, and 150 mU mL1. (B) The signal to noise ratio of the CCP-based FRET assay as a function of the concentration of PKA.
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sensitive assays for PKA activity detection recently reported, including metal ion mediated FRET using an anionic conjugated polymer,42 magnetic microsphere-based assay,29,30 the DNA-based electrochemical strategy21 and unmodied quantum dot-based enzyme activity assay,31 are in the range from 0.1 mU mL1 to 0.5 mU mL1. Therefore, the CCP-based method is comparable to the known highly sensitive assays. Moreover, the CCP is easily synthesized with low cost and the CCP-based method offers a mix-and-detect assay format with signicantly improved detection speed. The CCP-based PKA biosensor system based on electrostatic interactions does not need the action of metal ion complexation. These uorescencebased methods based on anionic uorescent-conjugated polymers coupled with the metal ion-mediated FRET oen suffer from some disadvantages, such as the metal ions are easily hydrolysed and ATP will frequently interfere with metal ion complexing actions. Therefore, compared with methods based on anionic uorescent-conjugated polymers coupled with metal ion-mediated FRET, the CCP-based method is rapid, straightforward and could avoid the weakness caused by metal ions. Furthermore, the CCP-based method provides a platform for kinase detection, which accelerates the development of derivative sensors based on the CCP. 3.4
Potential applications for inhibitor screening
To further demonstrate its potential applications in other molecular assays, the CCP-based method was applied to the inhibition assay. H-89, a potent and cell-permeable inhibitor of PKA, was chosen for this study. The experiments were performed at a constant PKA concentration (50 mU mL1) in the presence of H-89 with different concentrations (from 0 nM to 2000 nM). As can be seen in Fig. 4A, the FRET efficiency signicantly decreased with increasing concentration of H-89. When the value of I/I0 versus the concentration of H-89 was plotted, a sigmoidal prole was obtained (Fig. 4B). In Fig. 4B, on increasing the concentration of H-89, the numerical value of I/I0 decreased gradually, which revealed the inhibition of PKA and low levels of F-S-peptide phosphorylation. The IC50 value, the half maximal inhibitory concentration, is 40 nM, which is comparable with that reported in the literature.13,30 Taking advantage of rapidness, simplicity, low cost, and high sensitivity, the CCP-based method supports that the proposed method has great potential in high-throughput screening of protein kinase inhibitors and for drug development. 3.5
Fig. 4 (A) Normalized fluorescence emission spectra of the CCPbased FRET assay in the presence of different concentrations of H-89. H-89 concentrations from top to bottom: 0 (control), 1, 2.5, 4, 25, 50, 100, 200, 400, 800, 1000, and 2000 nM. (B) The signal to noise ratio of the CCP-based FRET assay as a function of the concentration of PKA inhibitor H-89. IC50 ¼ 40 nM. Conditions: 2.95 106 M (in repeat units (RUs)) CCP, 4 mM ATP.
pH 7.5, 10 mM MgCl2). The numerical values of I/I0 are shown in Fig. 5A. The results clearly indicate that the F-S-peptide could only be phosphorylated by PKA. The values of I/I0 of the other proteins are almost the same as the blank, which clearly suggests that they did not interfere with our assay. Furthermore,
Selectivity
To assess the selectivity of our protein kinase assay, control experiments were performed. Bovine serum albumin (BSA), lysozyme, thrombin, histone acetyltransferase (HAT) and casein kinase II (CK2) were introduced as interference proteins. In order to compare with other proteins, we adopt the amount-ofsubstance concentration for calculation. The concentration of PKA was 50 mU mL1 (about 0.26 nM) and the concentration of CK2 was 100 mU mL1 (about 0.83 nM). The concentration of all other proteins was 0.83 nM. The samples were incubated at 30 C for 60 min in PKA reaction buffer (50 mM Tris–HCl,
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The signal to noise ratio of experiments in the presence of different proteins. Conditions: 2.95 106 M (in repeat units (RUs)) CCP, 4 mM ATP. Fig. 5
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the activity of PKA was also studied in the presence of other proteins. As shown in Fig. S7,† PKA also has a good activity in presence of other proteins. So, our method has excellent selectivity.
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4. Conclusions In this work, a simply operating, sensitive uorescent peptide/ CCP system for probing the activity and inhibition of protein kinases has been developed. In this method, kinase-catalyzed peptide phosphorylation was monitored via a simple mechanism based on the FRET between the CCP and the FITC labeled peptide by changing the charge of the substrate peptide, which provides a new and attractive alternative to the commonly used mechanisms. This strategy does not require expensive equipment or complicated protein recognition treatment. Moreover, the introduction of peptide/CCP provides a diversied platform for kinase activity assay, which facilitates the development of derivative assays based on various CCP-based techniques. Furthermore, the method developed here shows potential applications in kinase inhibitor screening and kinase-related drug discovery. Given its sensitivity, selectivity, simplicity, and convenience, this method can be extended to other biological molecular assays and high-throughput screening of protease inhibitors.
Acknowledgements This work was nancially supported by the National Natural Science Foundation of China (nos 21305037, 21235002 and 21175036) and the Fundamental Research Funds for the Central Universities.
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Cyclic-AMP-dependent protein kinase (PKA) activity assay based on FRET between cationic conjugated polymer and chromophore-labeled peptide† Shiyun Tang, Yufang Hu, Qinpeng Shen, Heting Fang, Wang Li,* Zhou Nie and Shouzhuo Yao A sensitive fluorescence turn-on biosensing platform for protein kinase activity assay has been developed based on fluorescence resonance energy transfer (FRET) between a fluorophore labeled peptide and a water soluble cationic conjugated polymer (CCP). The CCP-based assay is based on the electrostatic interaction between the peptide and the CCP. The FRET efficiency will change with the changing charges around the peptide after phosphorylation. The feasibility of this method has been demonstrated by sensitive measurement of the activity of cAMP-dependent protein kinase (PKA) with a low detection limit (0.3 mU mL1). Based on its simple mechanism, this assay is also sensitive and robust enough to be
Received 7th May 2014 Accepted 19th June 2014
applied to the evaluation of PKA inhibitor H-89. The IC50 value, the half maximal inhibitory concentration, was 40 nM. Furthermore, our method has excellent selectivity. CCP-based assay is
DOI: 10.1039/c4an00814f
sensitive, versatile, cost-effective and easy to operate, so, this method is a promising candidate for
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kinase activity assay and inhibitor screening.
1. Introduction Protein phosphorylation by kinases plays a vital role in many cellular processes, including metabolism, transcription, cell cycle progression, cytoskeletal rearrangement, apoptosis, and differentiation.1–3 The human genome encodes more than 500 kinds of protein kinases, exerting tight and reversible control on protein phosphorylation.4 Aberrant phosphorylation and overexpression of protein kinases are closely associated with the pathogenesis of many human diseases,2 such as cancer,5 Parkinson's disease,6 and Alzheimer's disease.7 In order to understand the pathogenesis of carcinogenesis and discover proteinkinase-targeted drug, it is very important to detect kinase activities and screen their potential inhibitors.8 Therefore, developing accurate and sensitive methods for kinase assays and potential inhibitor screening is of great signicance for biochemical investigation and drug discovery.9–11 Even though there are a number of methods to investigate protein kinase phosphorylation events, potential pitfalls exist in these methods. Traditional methods for assessing protein kinase activities are radiometric assays.12,13 Because the radioactive g-phosphoryl moiety is hazardous and has a short State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, P. R. China. E-mail: [email protected]; Fax: +86-731-88821848; Tel: +86-731-88821626 † Electronic supplementary information (ESI) available: Fig. S1–S6. See DOI: 10.1039/c4an00814f
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half-life, immunoassay by using specialized biological reagents such as phosphopeptide-recognized antibody has been developed as an alternative to the radiometric method.14–16 These methods, however, are costly and suffer from inconsistent batch-to-batch quality of these proteins. As a consequence, signicant efforts have been devoted to the development of nonradioactive and antibody-free assays for detecting protein kinase activity with various analytical methods, such as colorimetry,17,18 electro-generated chemiluminescence,19,20 electrochemical,21,22 mass spectrometry,23,24 surface-enhanced Raman spectroscopy (SERS),25 and quartz-crystal microbalance.26 However, a majority of these methods require a surfaceconned process, multistep washing, sophisticated preparation, or expensive instrumentation. Among these existing protein kinase assays, the inherent advantages of the uorescence-based methods, such as homogeneous detection, high sensitivity, easy readout and simple operation, make them very attractive for detection of protein kinase activities. Therefore, many kinds of uorescence assays for detection of protein kinase activities have been developed based on electrophoresis,27 microarrays,28 magnetic microspheres,29,30 quantum dots,31,32 and uorescent proteins.33,34 Although these methods are novel and offer great potential to be applied to detection of protein kinase activity, there are still some limitations, such as time-consuming, labor-intense process, complicated preparation and costly materials.
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Over the past several years, water soluble conjugated polymers (WCPs) have gained much attention in applications for highly sensitive detection of biomacromolecules.35,36 These materials are characterized by backbones with p-delocalized electronic structures and pendant substituents with ionic functionalities.37 In contrast to bioassays with small-molecule counterparts, the excitation energy along the whole backbone of the WCP transferring to lower energy chromophore acceptor sites over long distances results in the amplication of uorescence signals.38 So, WCPs can be used for the function of very sensitive optical biosensors.39 D. Whitten et al. developed a sensitive, homogeneous and versatile method for detection of kinase activity based on quenching of the uorescence of anionic uorescent-conjugated polymers.40 However, the uorescence quenching-based methods oen suffer from the disadvantage that they may be difficult to interpret whether a reduction in uorescence intensity is caused by poor assay performance or the presence of target kinases. To overcome this limitation, L. F. Hancock, Z. Li and co-workers developed homogeneous approaches based on anionic uorescent-conjugated polymers coupled with the metal ion-mediated FRET.41,42 Nevertheless, these uorescence assays based on metal ion complexing actions are subjected to the weakness that the phosphate coordinating metal ions are easily hydrolysed to hydroxide and adenosine triphosphate (ATP) will oen interfere with the assay process. From the above, it still remains a great challenge to develop homogeneous, sensitive, reliable, and simple protein kinase assays for detection of protein kinase activities and for high-throughput screening of kinase inhibitors. Here, we report a sensitive, selective and simple method based on the FRET between a water soluble cationic conjugated polymer (CCP) and a uorophore labeled peptide. In this method, a CCP was introduced in a protein kinase detection and kinase inhibition study, which expands the application range of the CCP. Compared with the previous reports, this CCP-based assay offers a mix-and-detect assay format based on electrostatic interactions, avoiding the weakness caused by metal ion complexing actions. Because the CCP-based assay is sensitive, versatile, cost-effective and easy to operate, it has good potential to be used for the detection of protein kinase activity and for the screening of protein kinase inhibitors as potential drugs.
2. 2.1
Experimental details Materials
The water soluble CCP, poly(9,9-bis(60 -N,N,N-trimethylammonium)hexyl)uorenylene phenylene (PF), was synthesized according to a reported method.43 A uorophore labeled substrate peptide was purchased from GL Biochem Ltd. (Shanghai, China). Cyclic adenosine 30 ,50 -monophosphatedependent protein kinase (PKA, catalytic subunit) and casein kinase II (CK2) were obtained from New England Biolabs Inc. (Beverly, MA). N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) was obtained from EMD Biosciences (Calbiochem-Novabiochem. La Jolla, CA). Thrombin was
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obtained from Sigma-Aldrich (St. Louis, MO). A recombinant GST-puried p300 HAT (histone acetyltransferase) domain was purchased from Signal-Chem (USA). All solutions were prepared using ultrapure water (18.25 MU cm) from a Millipore Milli-Q system. All samples were illuminated at an excitation wavelength of 384 nm and the uorescence emission spectra were scanned from 400 to 600 nm at room temperature. Fluorescence measurements were performed in Tris–HCl (50 mM, pH 7.5). Fluorescence spectra were recorded by using a QuantaMaster™4 uorescence spectrometer (PTI). 2.2
Detection of activity and inhibition of PKA
The PKA storing solutions were composed of 20 mM Tris–HCl buffer (pH 7.5, 25 C), 50 mM NaCl, 1 mM EDTA, 2 mM DTT, and 50% glycerol. The PKA reaction solutions were composed of PKA (from 0 mU mL1 to 150 mU mL1), peptide (600 nM), MgCl2 (10 mM), ATP (4 mM), and 50 mM Tris–HCl buffer (pH 7.5, 25 C). Aer incubation at 30 C for PKA-catalyzed reaction, the resulting solution (10 mL) was added to 90 mL of CCP (the nal concentration of the CCP was 2.95 106 M in repeat units (RUs)). Aer incubation at room temperature for 1 min, uorescence spectra were recorded at room temperature. For PKA inhibition assay, the experiments were carried out using a similar procedure as those for PKA assay stated above, except for mixing PKA (50 mU mL1) with different concentrations of H-89 (from 0 nM to 2000 nM) before the addition of the substrate peptide.
3.
Results and discussion
3.1 Detection mechanism of the CCP-based biosensor for kinase activity assay As reported, the electrostatic interactions between the CCP and target molecules can be used for probing biomolecules.36 PF has abundant pendant substituents with ionic functionalities and was employed as a CCP in this study. It can offer a large number of opportunities to couple analytic receptor interactions, as well as nonspecic interactions, into detectable responses.39 Fluorescein isothiocyanate (FITC), with absorption spectra roughly from 430 nm to 510 nm and emission spectra approximately from 500 nm to 550 nm, was chosen since its absorption spectra overlap with the emission spectra of the CCP with the photoluminescence spectra approximately from 400 nm to 500 nm (as shown in Fig. S1 in the ESI†). As a consequence, a CCP and FITC were chosen to meet the requirement conditions for efficient FRET.36 Irradiation at 384 nm selectively excites the CCP, and FRET generated from the CCP (donor) to FITC (acceptor) was favored (as shown in Fig. 1). The uorescence intensity of the CCP was quenched efficiently by FITC at approximately 419 nm and the uorescence intensity of FITC was enhanced greatly at about 520 nm through the transfer of uorescence resonance energy. Based on the above, a sensitive method for kinase activity assay could be developed based on FRET. Kemptide, Leu-Arg-Arg-Ala-Ser-Leu-Gly (LRRASLG) containing the phosphorylation site Ser, is the substrate peptide for PKA, which has a high specicity for PKA.2 Therefore, the
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Fluorescence emission spectra of the FRET generated from the CCP to free FITC. Conditions: 2.95 106 M (in repeat units (RUs)) CCP. Fig. 1
kemptide labeled with FITC was chosen as the substrate peptide for PKA in our method. Conventional methods for detecting protein kinase are based on the measurement of kinase-catalyzed phosphorylation and involve two steps, a phosphorylation step and a signal report step. We investigated the FRET generated from the CCP to the FITC labeled substrate peptide (F-Speptide, FITC-LRRASLG) or the phosphorylated peptide (F-Ppeptide, FITC-LRRApSLG) since the F-S-peptide and the F-Ppeptide are the reactant and product of PKA-catalyzed phosphorylation. The mechanism for the PKA activity biosensor is shown in Scheme 1 based on the idea that the F-S-peptide is phosphorylated by PKA and generates an F-P-peptide, then, the F-P-peptide was mixed with the CCP and the uorescence spectrum curve was recorded and output as a uorescence signal. Since one arginine residue possesses a positive charge (+1) and one carboxyl group possesses a negative charge (1) at the carboxyl end of the peptide, the PKA-specic F-S-peptide (FITC-LRRASLG) would have a positive charge (+1). Because of one phosphate group with two negative charges, the net charge of its counterpart phosphorylated peptide will have one negative charge (1) aer introducing a phosphate group from ATP.32 The distance between the CCP and the peptide is affected by the charge around the peptide and the distance will inuence the efficiency of FRET generated from the CCP to the FITC
Scheme 1 Schematic representation of the strategy of PKA activity assay based on the FRET between the FITC labeled peptide and the CCP.
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labeled at the substrate peptide. The electrostatic repulsion of the F-S-peptide with the CCP keeps the F-S-peptide far away from the CCP, while the electrostatic attraction of the F-Ppeptide phosphorylated by PKA with the CCP keeps the F-Ppeptide close to the CCP, resulting in a signicant FRET from the CCP to the F-P-peptide. In comparison with the uorescence quenching-based assay, simultaneous measurement of uorescence intensity of the CCP and FITC is credible for detection of PKA activity. For the purpose of visualizing the results, the FRET ratio I is dened as I ¼ I520 nm/I419 nm (I520 nm and I419 nm are dened as the uorescence intensities at 520 nm and 419 nm respectively) and the signal to noise ratio S/N is dened as S/N ¼ I/I0 (I0 is the FRET ratio of the control experiment). The use of the signal to noise ratio of this PKA biosensor system is feasible for the detection of PKA activity. In order to exhibit our results clearly, the uorescence intensities at 419 nm were all normalized to 1. A phenomenon was found that we could get high FRET efficiency upon addition of the CCP to solutions of the F-Speptide only in the presence of ATP and PKA (see Fig. 2A). As shown in Fig. 2B, when PKA or ATP was absent, the value of I/I0 has no obvious difference with the blank experiment (without PKA and ATP). The value of I/I0 of the experiment only in the presence of ATP and PKA was clearly different from that of other experiments, suggesting that the F-S-peptide was phosphorylated by PKA and generated the F-P-peptide. This also indicates that CCP-based assay has good potential to be used for the detection of protein kinase activity. 3.2
Optimization of the experimental conditions
Since protein kinase requires ATP as the cofactor, which may induce undesirable inuence on the uorescence intensity of the CCP, its effect on the CCP was investigated carefully in our experiments. A gradual decline was discovered in the uorescence intensity of CCP solution applying to different concentrations of ATP from 2 mM to 50 mM (Fig. S2†). However, there was little inuence on the uorescence intensity of the CCP suspension when the ATP concentration was 4 mM. Since we
Fig. 2 (A) Normalized fluorescence emission spectra of the effect of PKA and ATP on CPP-based assay. (B) The signal to noise ratio of the effect of PKA and ATP on CPP-based assay. (a) 600 nM F-S-peptide was phosphorylated by 50 mU mL1 PKA in the presence of 4 mM ATP; (b) without PKA; (c) without ATP; (d) without PKA and ATP.
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employed 4 mM ATP for each enzyme reaction and also for each control experiment, the effect of ATP on the CCP uorescence intensity was negligible. Since PKA may induce undesirable inuence on the uorescence intensity of the CCP, we also studied the inuence of PKA on the uorescence intensity of the CCP in our experiments. Fig. S3† shows the uorescence intensity of the CCP as a function of the concentration of PKA. When the concentration of PKA was lower than 200 mU mL1, no signicant variation at 419 nm of the CCP uorescence intensity was observed. To obtain the best sensing performance of the PKA biosensor system, the concentration of F-S-peptides was also optimized. The effect of F-S-peptide concentration (from 100 nM to 1400 nM) on the value of I/I0 of the PKA biosensor system was tested with a xed concentration of the CCP (the nal concentration of the CCP was 2.95 106 M (in RUs)), and the results are shown in Fig. S4 in the ESI.† The best concentration of the F-S-peptide for the PKA biosensor system was found to be 600 nM with a constant concentration of the CCP. To conrm that the FRET change was really caused by phosphorylation, the effect of PKA on the FRET between the F-S-peptide and the CCP was studied. Here, the uorescence intensity was measured immediately aer mixing PKA and the F-S-peptide under similar conditions using the experiments of PKA activity detection without any incubation at 30 C. As shown in Fig. S5,† when the concentration of PKA was lower than 150 mU mL1, there was not signicant inuence upon FRET from the CCP to the F-Speptide. Therefore, the CCP-based method is feasible to detect the activity of PKA. With the purpose of getting the best incubation time between the F-P-peptide and the CCP, the impact of incubation time on the FRET between the F-P-peptide and the CCP was studied. Fig. S6† shows that the electrostatic interaction between the CCP and the F-P-peptide was very rapid and the numerical value of I/I0 has little change from 1 min to 10 min (start time aer mixing the F-P-peptide and CCP). There is no signicant change of I/I0 with the extended reaction time from 10 min to 40 min. For the sake of improving the measurement efficiency and shortening the time of measurement, 1 min was employed as the incubation time between the F-P-peptide and the CCP. The F-P-peptide was obtained by mixing the F-S-peptide with PKA and incubation at 30 C for 60 min. This phosphorylation process by PKA is necessary for the general uorescence detection method.31,42 However, our method can detect the uorescence signals without separating the substrate peptides and phosphorylated peptides. Furthermore, our method is quicker and simpler than those assays based on anionic uorescent-conjugated polymers since no chelate metal ions are needed.
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peptide and CCP at room temperature, the uorescence signal of the CCP and F-S-peptide was recorded. So, our method possessed simple operation and easy readout. As shown in Fig. 3A, the FRET efficiency signicantly increased with increasing concentration of PKA, indicating that the efficient FRET was generated from the CCP to the F-P-peptide. Furthermore, the quantitative assessment of the FRET-based assay for PKA activity detection was investigated by monitoring the dependence of the numerical value of I/I0 on the concentrations of PKA. As shown in Fig. 3B, when the numerical value of I/ I0 versus the concentration of PKA was plotted, a sigmoidal prole was obtained. The numerical value of I/I0 increased with increasing concentration of PKA within certain limits, indicating that a higher degree of phosphorylation induced efficient FRET. Meanwhile, control experiments were also carried out in the absence of PKA. By measuring the value of I/I0, PKA activity could be quantitatively detected over a wide concentration range from 0.3 mU mL1 to 150 mU mL1. The detection limit was 0.3 mU mL1. This demonstrates that the proposed uorescence sensor can be employed for highly sensitive protein kinase detection over a wide concentration range. Detection sensitivity is one of the most important aspects of bioassay development. The detection limits of the most
Detection of the activity of PKA
In order to detect the activity of PKA, a series of F-S-peptide mixtures with various concentrations of PKA were tested. The reaction solutions (10 mL) were incubated for 60 min at 30 C for phosphorylation under the optimized experimental conditions, and the phosphate groups were transferred from ATP to the F-Speptide to generate an F-P-peptide. Then, aer mixing the F-P-
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Fig. 3 (A) Normalized fluorescence emission spectra of the CCPbased FRET assay in response to different concentrations of PKA. PKA concentrations from bottom to top: 0 (control), 0.3, 0.94, 3.1, 6.25, 12.5, 18.75, 31.25, 37.5, 50, 68.5, 81.25, 100, and 150 mU mL1. (B) The signal to noise ratio of the CCP-based FRET assay as a function of the concentration of PKA.
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sensitive assays for PKA activity detection recently reported, including metal ion mediated FRET using an anionic conjugated polymer,42 magnetic microsphere-based assay,29,30 the DNA-based electrochemical strategy21 and unmodied quantum dot-based enzyme activity assay,31 are in the range from 0.1 mU mL1 to 0.5 mU mL1. Therefore, the CCP-based method is comparable to the known highly sensitive assays. Moreover, the CCP is easily synthesized with low cost and the CCP-based method offers a mix-and-detect assay format with signicantly improved detection speed. The CCP-based PKA biosensor system based on electrostatic interactions does not need the action of metal ion complexation. These uorescencebased methods based on anionic uorescent-conjugated polymers coupled with the metal ion-mediated FRET oen suffer from some disadvantages, such as the metal ions are easily hydrolysed and ATP will frequently interfere with metal ion complexing actions. Therefore, compared with methods based on anionic uorescent-conjugated polymers coupled with metal ion-mediated FRET, the CCP-based method is rapid, straightforward and could avoid the weakness caused by metal ions. Furthermore, the CCP-based method provides a platform for kinase detection, which accelerates the development of derivative sensors based on the CCP. 3.4
Potential applications for inhibitor screening
To further demonstrate its potential applications in other molecular assays, the CCP-based method was applied to the inhibition assay. H-89, a potent and cell-permeable inhibitor of PKA, was chosen for this study. The experiments were performed at a constant PKA concentration (50 mU mL1) in the presence of H-89 with different concentrations (from 0 nM to 2000 nM). As can be seen in Fig. 4A, the FRET efficiency signicantly decreased with increasing concentration of H-89. When the value of I/I0 versus the concentration of H-89 was plotted, a sigmoidal prole was obtained (Fig. 4B). In Fig. 4B, on increasing the concentration of H-89, the numerical value of I/I0 decreased gradually, which revealed the inhibition of PKA and low levels of F-S-peptide phosphorylation. The IC50 value, the half maximal inhibitory concentration, is 40 nM, which is comparable with that reported in the literature.13,30 Taking advantage of rapidness, simplicity, low cost, and high sensitivity, the CCP-based method supports that the proposed method has great potential in high-throughput screening of protein kinase inhibitors and for drug development. 3.5
Fig. 4 (A) Normalized fluorescence emission spectra of the CCPbased FRET assay in the presence of different concentrations of H-89. H-89 concentrations from top to bottom: 0 (control), 1, 2.5, 4, 25, 50, 100, 200, 400, 800, 1000, and 2000 nM. (B) The signal to noise ratio of the CCP-based FRET assay as a function of the concentration of PKA inhibitor H-89. IC50 ¼ 40 nM. Conditions: 2.95 106 M (in repeat units (RUs)) CCP, 4 mM ATP.
pH 7.5, 10 mM MgCl2). The numerical values of I/I0 are shown in Fig. 5A. The results clearly indicate that the F-S-peptide could only be phosphorylated by PKA. The values of I/I0 of the other proteins are almost the same as the blank, which clearly suggests that they did not interfere with our assay. Furthermore,
Selectivity
To assess the selectivity of our protein kinase assay, control experiments were performed. Bovine serum albumin (BSA), lysozyme, thrombin, histone acetyltransferase (HAT) and casein kinase II (CK2) were introduced as interference proteins. In order to compare with other proteins, we adopt the amount-ofsubstance concentration for calculation. The concentration of PKA was 50 mU mL1 (about 0.26 nM) and the concentration of CK2 was 100 mU mL1 (about 0.83 nM). The concentration of all other proteins was 0.83 nM. The samples were incubated at 30 C for 60 min in PKA reaction buffer (50 mM Tris–HCl,
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The signal to noise ratio of experiments in the presence of different proteins. Conditions: 2.95 106 M (in repeat units (RUs)) CCP, 4 mM ATP. Fig. 5
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the activity of PKA was also studied in the presence of other proteins. As shown in Fig. S7,† PKA also has a good activity in presence of other proteins. So, our method has excellent selectivity.
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4. Conclusions In this work, a simply operating, sensitive uorescent peptide/ CCP system for probing the activity and inhibition of protein kinases has been developed. In this method, kinase-catalyzed peptide phosphorylation was monitored via a simple mechanism based on the FRET between the CCP and the FITC labeled peptide by changing the charge of the substrate peptide, which provides a new and attractive alternative to the commonly used mechanisms. This strategy does not require expensive equipment or complicated protein recognition treatment. Moreover, the introduction of peptide/CCP provides a diversied platform for kinase activity assay, which facilitates the development of derivative assays based on various CCP-based techniques. Furthermore, the method developed here shows potential applications in kinase inhibitor screening and kinase-related drug discovery. Given its sensitivity, selectivity, simplicity, and convenience, this method can be extended to other biological molecular assays and high-throughput screening of protease inhibitors.
Acknowledgements This work was nancially supported by the National Natural Science Foundation of China (nos 21305037, 21235002 and 21175036) and the Fundamental Research Funds for the Central Universities.
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