- Email: [email protected]
A homogeneous assay of kinase activity that detects phosphopeptide using fluorescence polarization and zinc
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 316 (2003) 82–91 www.elsevier.com/locate/yabio
A homogeneous assay of kinase activity that detects phosphopeptide using fluorescence polarization and zinc John E. Scott* and John W. Carpenter Eli Lilly & Company, Sphinx Laboratories, 20 TW Alexander Drive, Research Triangle Park, NC, 27709, USA Received 28 October 2002
Abstract Homogeneous antibody-free assays of protein kinase activity have great utility in high-throughput screening in support of drug discovery. In an effort to develop such an assay, we have used a pair of fluorescein-labeled peptides of identical amino acid sequence with and without phosphorylation on serine to mimic the substrate and product, respectively, of a kinase. Using fluorescence polarization (FP), we have demonstrated that a mixture of zinc sulfate, phosphate-buffered saline, and bovine serum albumin added to the peptides dramatically and differentially increased the fluorescence polarization of the phosphorylated peptide over its nonphosphorylated derivative. A similar FP differential was observed using different peptide pairs, though the magnitude varied. The FP values obtained using this method were directly proportional to the fraction of phosphopeptide present. Therefore, an FP assay was developed using a proprietary kinase. Using this FP method, linear reaction kinetics were obtained in enzyme titration and reaction time course experiments. The IC50 values for a panel of inhibitors of kinase activity were determined using this FP method and a scintillation proximity assay. The IC50 values were comparable between the two methods, suggesting that the zinc FP assay may be useful as an inexpensive high-throughput assay for identifying inhibitors of kinase activity. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Kinase; Fluorescence polarization; Zinc
Protein phosphorylation is a frequently used mechanism in nature for regulating the activity of a host of enzymes, transcription factors, receptors, etc. There are estimated to be over 500 different protein kinases in the human proteome that catalyze the transfer of phosphate from ATP to serine, threonine, and tyrosine [1]. Protein kinases can be divided into two broad classes, serine/ threonine and tyrosine kinases, although a few kinases are able to phosphorylate both types of amino acid residues. Kinases play critical roles in many fundamental cell-signaling pathways including those that lead to growth, differentiation, motility, apoptosis, and survival. Not surprisingly, the dysregulation of protein kinase activity has been associated with numerous diseases including cancer, diabetes, and inflammation. As such, they are the focus of much interest for the development of compounds to selectively regulate the activity of disease-related kinases. The most common method to * Corresponding author. Fax: +919-314-4174. E-mail address: [email protected] (J.E. Scott).
discover selective novel inhibitors of protein kinase activity has been to screen large libraries of compounds for inhibitors of a biochemical assay designed to measure the phosphorylating activity of the kinase of interest. Numerous methods have been designed to measure protein kinase activity. Early methods require physical separation of substrate protein or peptide from its phosphorylated product. One method is to use radiolabeled ATP that allows the incorporated phosphate to be detected after the capture of the protein/peptide substrate by phosphocellulose membrane [2]. This type of assay of protein kinase activity requires multiple steps and the use of radioactivity with its accompanying hazards and costs. There are also ELISA1-based assays 1
Abbreviations used: ELISA, enzyme-linked immunosorbent assay; FP, fluorescence polarization; mP, millipolarization units; DTT, dithiothreitol; PBS, phosphate-buffered saline; BSA, bovine serum albumin; IMAC, immobilized metal ion affinity chromatography; SPA, scintillation proximity assay; FRET, fluorescence resonance energy transfer, TMR, tetramethylrhodamine; DMSO, dimethyl sulfoxide; SAP, shrimp alkaline phosphatase.
0003-2697/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0003-2697(03)00036-8
J.E. Scott, J.W. Carpenter / Analytical Biochemistry 316 (2003) 82–91
of kinase activity, but these also require multiple steps in addition to being dependent on availability of a phospho-specific antibody to capture product [3]. The scintillation proximity assay (SPA) has been used as a high-throughput homogeneous assay of protein kinase activity that does not require an antibody [4,5], but still has all the disadvantages inherent in the use of radioactivity. Homogeneous nonradioactive assay formats measuring protein kinase activity that include fluorescence polarization (FP) and time-resolved fluorescence resonance energy transfer (FRET) have been developed to eliminate the disadvantages of radioactivity and washing steps [6–8]. Fluorescence polarization is a ratiometric technique that reflects the rotational motion of a fluorescent molecule in solution with the rotational velocity dependent on the apparent molecular size [9]. A major disadvantage of both FP and FRET assays is that they are dependent on the availability of a phospho-specific antibody. Another drawback to the use of antibodies is that their high specificity for a given phosphopeptide often prevents the same antibody from being used with different substrates. Anti-phosphotyrosine-specific antibodies are broadly reactive since they are relatively insensitive to the surrounding amino acid context. In contrast, phosphoserine-/phosphothreoninespecific antibodies are highly dependent on the amino acid sequence surrounding the phosphorylated residue and thus tend to only bind a limited diversity of phosphorylated sites [10,11]. In general, a new antibody must be generated for each different substrate, which can be both time consuming and expensive. Several antibody-free nonradioactive assays of protein kinase activity have been reported. One such assay method replaces ATP in the reaction with ATPcS leading to the formation of a thiophosphorylated peptide product [12]. This product is then chemically biotinylated by a sulfur-specific biotinylation reagent. Streptavidin is then added to the fluorescently tagged peptide and an FP measurement is taken to measure the amount of product formed. The potential disadvantages of this assay include long incubation times and altered kinetics for some kinases due to the ATPcS. Another antibody-free FP assay of protein kinase activity that has been reported uses polyarginine to discriminate fluorescently labeled phosphorylated and nonphosphorylated peptides based on charge interaction of the phosphate group and the positively charged high molecular weight polyarginine [13,14]. Another potential antibody-free assay of protein kinase activity is the socalled IMAP assay that employs trivalent metal ions immobilized on nanoparticles to bind phosphate groups on molecules and thus increase their FP values [15]. This approach is similar in concept to immobilized metal affinity chromatography (IMAC) used to specifically isolate phosphopeptides from mixtures containing both phosphorylated and nonphosphorylated peptides
83
[16,17]. Finally, the development of microchip instruments has led to an additional approach to measuring the activity of kinases [18]. In the microfluidic assay of protein kinase activity, fluorophore-labeled peptide substrate and product are separated based on a difference in their charge-to-mass ratio. Separation of product and substrate is achieved by electrophoresis through an electric field, followed by quantitation of the individual fluorescent peaks. We have explored the use of zinc ions in solution to discriminate between phosphorylated and nonphosphorylated fluorescently labeled peptides. In this article, we report a simple homogeneous nonradioactive FP-based assay of protein kinase activity using commonly available reagents and fluorescently-tagged peptides to quantitate relative phosphopeptide levels. Our assay does not require an antibody and is very inexpensive. Experiments using a serine/threonine kinase and alkaline phosphatase show that this zinc FP assay represents a novel inexpensive alternative for measuring kinase and phosphatase activity.
Materials and methods General All peptides were obtained from Anaspec Inc. (San Jose, CA). All general biochemical reagents were obtained from Sigma (St. Louis, MO). DulbeccoÕs phosphate-buffered saline without calcium or magnesium (PBS, Catalog No. 17-512Q) was obtained as a liquid stock from BioWhittaker (Walkersville, MD). Bovine serum albumin (BSA) was obtained from Sigma (Catalog No. A7906 or A6918). ATP was purchased from Roche (Nutley, NJ). All assays were done in Corning/ Costar (Acton, MA) 384-well flat-bottom non-treated black polystyrene plates (No. 3710). Compounds for IC50 (inhibitor concentration that results in 50% inhibition of activity) value comparison experiments were obtained from Calbiochem (San Diego, CA). Fluorescence polarization measurements Fluorescence polarization measurements were performed on an Analyst HT instrument (Molecular Devices, Sunnyvale, CA). Fluorescence intensity measurements were taken in black 384-well microtiter plates where both the excitation and the emission occur from the top of the wells. In the Analyst HT, a xenon arc lamp provides excitation light that passes through an excitation filter and then a polarizer filter. A beamsplitter filter directs the polarized excitation light into the well and emitted fluorescence transmits back through the same beam-splitter filter, through a polarizer filter, then through the emission filter for detection.
84
J.E. Scott, J.W. Carpenter / Analytical Biochemistry 316 (2003) 82–91
Read time was 0.1 s per well. The excitation polarizer filter was fixed in the parallel position, while the emission polarizer filter was changed for measuring parallel and perpendicular emission fluorescence intensity. All polarization values are expressed in millipolarization units (mP). The mP values were calculated using the equation mP ¼ 1000 ½ðIS IP Þ=ðIS þ IP Þ, where IS is the parallel emission intensity measurement and IP is the perpendicular emission intensity measurement. For fluorescein-labeled peptides, measurements were made with excitation at 485 nm (25-nm bandwidth) and emission at 530 nm (25-nm bandwidth) using a 505-nm beam splitter. For the tetramethylrhodamine (TMR)labeled peptide, measurements were made with excitation at 530 nm (25-nm bandwidth) and emission at 580 nm (10-nm bandwidth) using a 561-nm beam splitter. Peptide experiments The sequences of the peptides used are shown in Table 1. All reagents and experiments were done at room temperature. Except where indicated, standard non-enzymatic peptide experiments were performed by diluting fluorescently labeled peptide to 0.5 lM in water and adding 30 ll of this peptide solution into the well of an assay plate. The zinc reagent, ZnSO4 /PBS/BSA, was prepared by gently mixing equal volumes of 1% (weight/ volume) BSA in PBS (PBS/BSA) and 60 mM ZnSO4 . Mixing was accomplished through gentle inversion. The zinc reagent is stable for at least 1 h. To 30 ll of peptide solution, 60 ll of zinc reagent was added. After approximately 10 min, the fluorescence polarization measurements were taken. Assay of protein kinase activity All assays of protein kinase activity were performed using a proprietary recombinant serine/threonine kinase prepared at Eli Lilly & Co. (Indianapolis, IN). The FP assay of protein kinase activity was performed as follows. The FP assay buffer used was 10 mM Tris, pH 8.0,
Table 1 Sequences of fluorescently labeled peptidesa No.
Sequence
1 2 3 4 5 6 7 8
Fl-GPLGRHGpSIRQKKEEV Fl-GPLGRHGSIRQKKEEV Fl-RFARKGpSLRQKNV Fl-RFARKGSLRQKNV Fl-LC-LRRApSLG Fl-LC-LRRASLG TMR-KVEKIGEGTpYGVVYK TMR-KVEKIGEGTYGVVYK
a Abbreviations used: pS, phosphoserine; pY, phosphotyrosine; Fl, fluorescein; TMR, tetramethylrhodamine; LC, linker chain.
10 mM MgCl2 , and 0.01% Triton X-100. The concentration of ATP used was 5 lM and the nonphosphorylated fluorescein-labeled peptide 2 was used at 0.5 lM (or 1 lM where indicated). The reaction volume was 30 ll and the amount of kinase used varied from 160 to 600 ng per reaction as indicated for each experiment. The assays were set up by the addition of 10 ll of 3% DMSO in water (with or without compound) to the assay plate followed by 10 ll kinase and 10 ll ATP/ peptide mixture, each diluted with 1.5X FP assay buffer. The reactions were incubated at room temperature, or 30 °C where indicated, for 3–4 h or 18–20 h, depending on the kinase concentration used and the activity of the particular kinase preparation. The preparation of zinc reagent and its use in developing the assay was done as described above. For the IC50 comparison experiments, 10- to 12-point concentration responses were generated starting at 100 lM (or 20 lM for staurosporine). Each concentration of compound was tested in triplicate (quadruplicate for staurosporine) per individual IC50 determination. Reactions were incubated at room temperature for 18–20 h with 160 ng kinase per reaction, except for the staurosporine IC50 determination experiments where 200–400 ng/well kinase and 1 or 0.5 lM peptide substrate was used in 3-h reactions. For the scintillation proximity assay, the final assay conditions were 50 mM Tris, pH 8.0, 10 mM MgCl2 , 0.01% Triton X-100, 1 mM DTT, 1 or 3.75 lM ATP, 0.375 lM peptide substrate (identical to peptide 2 except biotin replaced the fluorescein), 160 ng/reaction kinase enzyme, and 0.125 lCi/reaction of [33 P]ATP from Perkin Elmer (Boston, MA). The 40-ll reactions were incubated at room temperature for 4 h. A stop reagent (40 ll per well) containing 0.2 mg streptavidin-coated SPA beads (Amersham, Piscataway, NJ), 40 mM EDTA, and 74% CsCl was added to the reaction. After sealing the plates and incubating for 2 h at room temperature, the plates were read on a TopCount (Perkin Elmer) scintillation counter. IC50 values for compounds were generated as described above. Assay of phosphatase activity The assay of phosphatase activity used shrimp alkaline phosphatase (SAP) from Roche. The assay buffer used was the same as for the FP assay of protein kinase activity. SAP and the phosphopeptides 1 and 5 were diluted into assay buffer with the final assay concentrations of 0.1 lM peptide and 15 mU (peptide 1) or 0.25 mU (peptide 5) per reaction SAP. The reactions were started by the addition of 15 ll of SAP to 15 ll of phosphopeptide at various times. SAP was heat-inactivated for the control reactions by heating at 65 °C for 20 min. Nonphosphorylated peptide 2 along with SAP was also used as a reference for 100% dephosphorylation. The reactions were terminated with 60 ll zinc
J.E. Scott, J.W. Carpenter / Analytical Biochemistry 316 (2003) 82–91
85
reagent and the FP measurements taken as described for the FP assay of protein kinase activity.
Results In an effort to develop a homogeneous nonradioactive assay of protein kinase activity, we had synthesized a pair of fluorescein-labeled peptides (Table 1, peptides 1 and 2) with identical amino acid sequence with (peptide 1) and without (peptide 2) a phosphate on the serine residue to mimic the substrate and product, respectively, of a kinase. We tested several metal ions, including Zn2þ , Fe3þ , Ca2þ , Mn2þ , and Mg2þ , for their ability to preferentially aggregate the phosphopeptide such that the phosphorylated and nonphosphorylated peptide could be differentiated by their fluorescence polarization values. Aggregates/large complexes preferentially containing the phosphopeptide would have an increased apparent molecular size. These large aggregates would have a slower rate of rotation compared to nonphosphorylated peptide free in solution and hence the FP values would be higher for the phosphorylated peptide. Zinc sulfate produced the most dramatic FP differential observed for this set of peptides diluted into PBS and BSA solution (data not shown). In contrast, Mn2þ and Mg2þ ions, common cofactors for kinases and phosphatases, did not generate any significant difference in FP values for these peptides (data not shown). In further optimization experiments using zinc, peptides 1 and 2 were diluted to 0.5 lM in water. Varying concentrations of zinc sulfate solution were added to an equal volume of PBS containing 1% BSA (PBS/BSA). Sixty microliters of this ZnSO4 /PBS/BSA mixture (zinc reagent) was added to 30 ll of peptide and fluorescence polarization values were measured (Fig. 1). At final zinc sulfate concentrations of 2 mM or above, a dramatic difference in mP values was observed for the phosphorylated versus nonphosphorylated peptides, with the phosphorylated version of the peptide displaying FP values of 170–230 mP higher than its nonphosphorylated analog. We observed that 16–32 mM zinc sulfate generated the maximum fluorescence polarization difference between these two peptides, approximately 230 mP. We chose to use 20 mM final zinc sulfate concentration (or 60 mM zinc sulfate solution before dilution) for all subsequent experiments. To further explore this phenomenon, we determined some of the reagent requirements for generating this fluorescence polarization differential between the two peptides. We tested different combinations of zinc sulfate, PBS, and BSA along with water-solubilized peptide (data not shown). Neither zinc sulfate, PBS, nor BSA alone produced a significant FP differential between the two peptides. Generation of the observed differential polarization values for these peptides required both zinc sulfate and
Fig. 1. Effect of zinc sulfate concentration on the fluorescence polarization of phosphorylated and nonphosphorylated fluorescein-labeled peptides. Varying concentrations of aqueous zinc sulfate were added to an equal volume of PBS/1% BSA solution. The ZnSO4 /PBS/BSA solutions (60 ll) were added to 30 ll of 0.5 lM phosphorylated (peptide 1, N) and nonphosphorylated (peptide 2, j) versions of the same fluorescein-labeled peptide diluted with water. The fluorescence polarization (mP) of the resulting final solution was measured. The final concentration of zinc sulfate after addition to peptide is indicated. Each data point is an average of triplicate determinations and the error bars indicate standard deviation. Data are representative of three independent experiments.
PBS. While the presence of BSA was not required, BSA did enhance the polarization value difference between the phosphorylated and nonphosphorylated peptide. The inclusion of BSA increased the average FP differential from 183 to 242 mP, enhancing the FP differential by 59 mP. Therefore, all subsequent experiments had the final concentrations (after addition to the peptide solution) of a 1:3 dilution of PBS:BSA and 20 mM zinc sulfate. This was obtained by mixing 1 vol of PBS/BSA with an equal volume of 60 mM zinc sulfate and then two volumes of this zinc reagent added to one volume of peptide solution. In general, equivalent results were obtained by either adding the PBS/BSA and zinc sulfate separately (30 ll peptide plus 30 ll PBS/BSA, then 30 ll zinc sulfate) or gently premixing the two reagents before addition to peptide (30 ll peptide plus 60 ll of zinc reagent) (data not shown). All experiments shown in this report were performed using the premixed zinc reagent. To determine whether the increased mP observed for the phosphorylated peptide could be used to quantitate the percentage of phosphorylated peptide in a mixture, we used peptides 1 and 2 to mimic protein kinase activity. We mixed solutions of phosphorylated peptide 1 and nonphosphorylated peptide 2 such that the concentration of total labeled peptide was constant at 0.5 lM, but the percentage of phosphorylated peptide varied 0–100% of total peptide (Fig. 2). Zinc reagent was added to these defined peptide mixtures and the mP values were determined. The results demonstrated that the increase in polarization values observed for phosphorylated
86
J.E. Scott, J.W. Carpenter / Analytical Biochemistry 316 (2003) 82–91
Fig. 2. Fluorescence polarization of defined mixtures of a phosphorylated fluorescein-labeled peptide and its nonphosphorylated derivative. Phosphorylated peptide (peptide 1) and its nonphosphorylated peptide derivative (peptide 2) were mixed together in water to generate the indicated percentages of phosphorylated peptide compared to the concentration of total peptide. The concentration of total peptide remained constant at 0.5 lM. To 30 ll of the peptide mixtures, 60 ll of zinc reagent was added and the fluorescence polarization measured. Each data point is an average of triplicate determinations and the error bars indicate standard deviation. Data are representative of three independent experiments.
peptide was linear and directly proportional to the percentage of phosphorylated peptide in the mixture. These data suggested that this method might be used to quantitate the catalytic activity of a kinase using a fluorescein-labeled peptide as a substrate. We used a proprietary serine/threonine kinase and peptide 2 to test the utility of this system as a viable assay of protein kinase activity. This zinc FP assay was found to be sensitive to high concentrations of Tris buffer, which caused a decrease in the difference in mP between peptides 1 and 2. For the volumes used in this assay, 10 mM Tris, pH 8.0, buffer did not significantly affect the FP differential between peptides 1 and 2 and therefore was used for the reaction buffer (data not shown). An enzyme titration experiment with peptide 2 as substrate demonstrated a linear increase in fluorescence polarization values with increasing amount of kinase used in the reaction (Fig. 3). Also, this increase in FP values was ATP dependent (data not shown). If the change in FP values observed using this zinc FP assay was related to kinase activity, the mP values obtained would be expected to be directly proportional to the reaction incubation time. Kinase was incubated with peptide 2 and ATP for varying times, zinc reagent was added, and fluorescence polarization values were determined (Fig. 4). These data indicated a positive linear relationship between incubation time and mP values. Based on previous data with the model peptides 1 and 2, the 4-h incubation time with kinase resulted in 110 mP increase over background or approximately 50% of peptide 2 converted to phosphorylated product. We compared this zinc FP assay to a scintillation proximity
Fig. 3. Dependence of fluorescence polarization signal on kinase concentration. Reactions were performed with the indicated amount of kinase per 30 ll reaction, 0.5 lM peptide 2 (nonphosphorylated fluorescein-labeled peptide), and 5 lM ATP at 30 °C. Reaction time was 4 h. Reactions were terminated with 60 ll zinc reagent and fluorescence polarization measurements taken. Each data point is an average of triplicate determinations and the error bars indicate standard deviation. Data are representative of three independent experiments.
Fig. 4. Dependence of fluorescence polarization signal on reaction time. Reactions were performed for the indicated length of time using 600 ng/well kinase, 0.5 lM peptide 2 (nonphosphorylated fluoresceinlabeled peptide), and 5 lM ATP at 30 °C. The reactions were started at various times with the addition of enzyme and all reactions terminated together and the fluorescence polarization values determined. Each data point is an average of triplicate determinations and the error bars indicate standard deviation. Data are representative of three independent experiments.
assay of protein kinase activity with respect to compound potency determination using a panel of seven commercially available inhibitors of kinase activity (Table 2). The IC50 value for staurosporine generated using the zinc FP assay was 12 nM, which is comparable to 4 nM obtained using the SPA format. The zinc FP assay produced average IC50 values for six of the seven inhibitors that were within three-fold of the SPA format. One of these inhibitors, bisindolylmaleimide V, is a negative control for the bisindolylmaleimide series of
J.E. Scott, J.W. Carpenter / Analytical Biochemistry 316 (2003) 82–91
87
Table 2 Comparison of IC50 values for compounds using the zinc FP and SPA formatsa Compound
FP IC50 (lM)
SPA IC50 (lM)
Bisindolylmaleimide III Bisindolylmaleimide V H-9 Calmidazolium H-89 G€ o 7879 Staurosporineb
13 1:4 >100 2:2 0:4 3:5 0:2 0:62 0:18 1:5 0:4 0:012 0:0003
5:1 1:8 >100 0:55 0:23 8:9 5:3 0:24 0:13 0:98 0:45 0:004 0:001
a Reactions were performed as described under Materials and methods. Ten-point dose response curves were generated starting at a high concentration of 100 lM. Each compound concentration was tested in triplicate per IC50 determination. IC50 values are the mean of three independent determinations. Errors reported are standard deviations. b Staurosporine dose response curves were generated starting at a high concentration of 20 lM. Each compound concentration was tested in quadruplicate (FP assay) or triplicate (SPA) per IC50 determination. IC50 values are the mean of two independent determinations.
PKC inhibitors and also served as a negative control for our kinase, generating >100 lM IC50 value in both assay formats. The average IC50 value for H-9 in the zinc assay format was four-fold higher than in the SPA format. Approximately 25–50% of peptide substrate was converted to product in these zinc FP IC50 assays while in the SPA it was <10%. Overall, these data indicated that the zinc FP assay was comparable to the SPA in sensitivity to both potent and relatively weak inhibitors of kinase activity. In addition, this IC50 data demonstrated that the zinc FP assay tolerated these compounds at high concentration without effect on the assay. In order to determine how broadly applicable this zinc FP phosphopeptide detection method was in detecting the phosphorylation state of peptides, we obtained three other pairs of peptides, identical in sequence except that one residue was phosphorylated. Peptide pairs 3 and 4 (PKC pseudosubstrate analogs) and 5 and 6 (Kemptide) are fluoresceinated derivatives of commonly used substrates for kinases [19,20]. Peptide pair 7 and 8 is unique in this panel of peptides in that it is tagged with tetramethylrhodamine instead of fluorescein. Peptide 8 is a substrate for the tyrosine kinase cSrc and therefore, the phosphopeptide version (peptide 7) was synthesized with a phosphotyrosine residue. All four peptide pairs were tested with the zinc FP method as before (Fig. 5). The net differential polarization was determined for each pair of peptides; i.e., the mP value obtained for the nonphosphorylated peptide was subtracted from the phosphopeptide mP value. All four pairs of peptides in this panel displayed significantly higher mP values for the phosphorylated peptides with differences ranging from 38 to 235 mP. At 50% phosphopeptide concentration, three of the four peptide pairs would produce at least 50 mP of signal over
Fig. 5. Differential FP values for a panel of paired phosphorylated and nonphosphorylated fluorescently labeled peptides. A panel of four pairs of fluorescently labeled peptides was assembled where a pair has the same amino acid sequence, but one is phosphorylated. Each peptide was diluted to 0.5 lM in 30 ll water and 60 ll zinc reagent added followed by fluorescence polarization measurements. The difference in FP values (indicated above the bars) between phosphorylated and nonphosphorylated matched peptide pairs as indicated was plotted. The data shown are the average difference obtained in three independent experiments where each data point was an average of triplicate determinations. The error bars indicate standard deviation for the results of the three experiments.
background. These data suggest that the zinc FP assay has general utility in phosphopeptide detection. However, the magnitude of polarization differential observed between phosphorylated and nonphosphorylated peptides in this assay was clearly peptide sequence and/or net charge dependent. To determine if this zinc FP method of phosphopeptide detection could be used for phosphatases, we performed phosphatase activity time courses with phosphopeptides 1 and 5 as substrates for shrimp alkaline phosphatase (Fig. 6). Controls were included that corresponded to a 100% phosphopeptide level using heat-inactivated phosphatase and phosphopeptide. A control representing 0% phosphopeptide used active enzyme and the nonphosphorylated matched peptide. Both peptides 1 and 5 demonstrated a time-dependent decrease in polarization values. Peptide 1 showed a linear decrease in mP values for the first 30 min of incubation time (Fig. 6A, inset). Based on the mP values obtained with the 0 and 100% phosphopeptide controls, this linear range corresponded to between 100% (at 0 min) and approximately 50% (at 30 min) phosphopeptide content. The mP values for the phosphopeptides exposed to active phosphatase, but not inactivated phosphatase, closely approach the mP values of the nonphosphorylated control peptide after 2 h of reaction time. These data provided proof-of-concept that this assay method could be used to measure phosphatase activity using a fluorescein-labeled phosphopeptide as substrate. Moreover, the concentration of peptide substrate in this assay was only 100 nM, demonstrating that the zinc FP phosphopeptide detection assay can be employed as a very sensitive antibody-free assay of
88
J.E. Scott, J.W. Carpenter / Analytical Biochemistry 316 (2003) 82–91
Fig. 6. Phosphatase activity measured using FP assay. Shrimp alkaline phosphatase was incubated with 0.1 lM fluorescein-labeled phosphopeptide 1 (A) or 5 (B) for the indicated times. The reactions were started at various times with the addition of phosphatase and terminated at one time with zinc reagent. The phosphopeptides were incubated with either active phosphatase (d) or heat-inactivated phosphatase (N) as a control. The nonphosphorylated derivative of each peptide was also included as a control and incubated at the same times with active phosphatase (j). Each data point is an average of triplicate determinations and the error bars indicate standard deviation. Data are representative of three independent experiments.
phosphatase activity. In addition, these data further validated the idea that the mP values determined with the zinc FP method accurately reflect the phosphorylation state of the labeled peptide.
Discussion In this article, we have described a novel, inexpensive, nonradioactive, homogeneous fluorescence polarization assay that can be used to detect and quantitate relative phosphopeptide levels in a sample. Unlike traditional radiometric approaches, fluorescence polarization has the advantage of being a homogeneous method that does not require separation of product and substrate. Most commercially available FP-based assays of protein kinase activity require a phospho-specific antibody. There are several disadvantages to the use of antibodies in FP assays. Although phosphotyrosine-specific antibodies are common and relatively amino acid context
independent, they are expensive. Phosphoserine- or phosphothreonine-specific antibodies can be difficult and costly to produce and are sensitive to amino acid sequence surrounding the phosphorylation site and hence cannot be used for all serine/threonine peptide substrates. Unlike most other homogeneous fluorescent assays of protein kinase activity, reagents required for the zinc FP assay are simply a fluorescein-labeled peptide and common, inexpensive laboratory reagents, with no antibody required. Peptide pairs that are identical in amino acid sequence and fluorophore, but one modified with a phosphate group, were used as models of substrates and products of a kinase. A solution of zinc sulfate, PBS, and BSA was found to differentially effect the fluorescence polarization of phosphopeptides compared to nonphosphorylated analogs. The phosphorylated peptides generated significantly higher mP values in response to exposure to the zinc reagent. The difference in fluorescence polarization values between the fluorescein-
J.E. Scott, J.W. Carpenter / Analytical Biochemistry 316 (2003) 82–91
labeled phosphorylated and nonphosphorylated peptides within a matched pair ranged from 94 to 235 mP for the three sets we tested. The magnitude of this observed differential appears to depend on the amino acid sequence and/or net charge of the peptide. Of potential significance, the two fluorescein-labeled peptides that gave the highest FP differential (peptides 2 and 6) had a net charge of )1 in the nonphosphorylated state that changed to a net charge of )3 in the phosphorylated state. The other fluorescein-labeled peptide (peptide 4) had a net charge of +2 that became neutral upon addition of the phosphate group. Although the TMR-labeled peptides had a net neutral charge, it produced a much lower differential mP value. It is unknown whether this is due to the different fluorophore, as TMR has a positive and negative charge (net neutral), or because the peptide has a phosphotyrosine whereas all the other phosphopeptides tested were phosphoserines. Since we only tested four peptide pairs with this FP assay and they varied significantly in amino acid sequence, net charge, and fluorophore, it is not possible at this point to correlate peptide structure with performance in this assay format. In general, it appears that this differential FP observed for phosphopeptides in the presence of zinc reagent is broadly applicable and not the consequence of isolated unusual peptide sequences, although the magnitude of the FP differential varies with the peptide sequence and/or net charge. We have shown that this change in fluorescence polarization absolutely requires both zinc sulfate and PBS, while BSA just enhances the observed differential. While initially the PBS/BSA and the zinc sulfate reagents were added separately, it was more convenient to premix the two solutions so that only one addition to the assay was required. We demonstrated that the mP values obtained in the zinc FP assay were directly proportional to the percentage of phosphopeptide in mixtures of phosphorylated and nonphosphorylated peptides designed to mimic kinase activity. These data suggest that this method could be applied to assaying protein kinase activity. In order to measure kinase activity in the current configuration, the substrate needs to be a relatively short peptide labeled with a fluorophore. In addition, completely phosphorylated and nonphosphorylated versions of the peptide are required to establish the mP values for 0 and 100% phosphopeptide content. Therefore, this assay is not readily amenable to measuring kinase activity using a peptide or protein of unknown sequence. The assay of protein kinase activity in this report was designed using a proprietary serine/threonine kinase and a nonphosphorylated fluorescein-labeled peptide as substrate. The mP values obtained in enzyme titration experiments increased in direct proportion to the amount of kinase added to the reaction, as would be expected if the zinc FP method measured the fraction of phosphopeptide present. Likewise, a time course of the reaction
89
demonstrated that the mP values increased in direct proportion to the reaction time, which is consistent with the zinc FP method measuring kinase activity. Furthermore, we generated IC50 values for a panel of seven commercially available compounds using the zinc FP and SPA formats. The IC50 values for the two assay formats were within 1.6- to 4-fold of each other or an average of 2.4-fold difference. Taking the SPA data as the standard, both potent and weakly inhibitory compounds were detected with the zinc FP assay. Although the SPA and FP formats were set up with different concentrations of ATP (1–5 lM), these concentrations are well below the Km for ATP of 20 lM determined previously for this kinase (data not shown). In theory, the difference in ATP concentration between the two assay formats would not be expected to make a difference in sensitivity to ATP competitors. Overall, the IC50 comparison data validated the use of the zinc FP phosphopeptide detection method to determine IC50 values for inhibitors of kinase activity. The zinc FP method has the potential to be used as an inexpensive, automated, homogeneous, high-throughput assay for the discovery of inhibitors of kinase activity from compound collections. This assay uses high concentrations of fluorophore that produces very high fluorescence intensities leading to highly accurate FP measurements. In addition, the high fluorescence intensity readings would make the assay more resistant to interference by weakly fluorescent and quenching compounds. Another simple and inexpensive assay of protein kinase activity previously reported used fluoresceinlabeled peptides, polyarginine, and FP to measure kinase activity [13]. One advantage of our zinc FP assay over the polyarginine assay is that the signal observed for some peptides is larger allowing a smaller fraction of substrate-to-product conversion to generate a suitable assay signal. For instance, a nearly identical peptide to one used for the polyarginine study was used in our studies and had a differential of 110 mP for the polyarginine assay, but 175 mP for the zinc FP assay. Using the polyarginine method with peptides 1 and 2 produced a maximal 110 mP differential in our hands (data not shown). Using the zinc FP assay, the same peptides demonstrated a 235 mP differential—double the differential compared to polyarginine. This large differential enabled us to perform assays with peptide 2 where only 25–50% of substrate was converted to product, while maintaining a robust 50–100 mP signal. For optimal enzyme kinetic experiments, it is usually desired to convert less than 10% of the substrate to product. However, in practice, there is typically only a small or insignificant difference between the IC50 values for ATP competitive inhibitors generated by assays converting <10% and those converting 50% (or higher) of the peptide substrate to product, provided the rate of product generation in the assay remains linear with re-
90
J.E. Scott, J.W. Carpenter / Analytical Biochemistry 316 (2003) 82–91
spect to time ([13] and data not shown). The IC50 values presented in Table 2 also confirmed this observation since comparable results were observed even though substrate conversion for the FP assay and the SPA was quite different, at 25–50% and <10% substrate conversion, respectively. These data suggested that the sensitivity of the zinc FP assay to inhibitors was not significantly compromised by the relatively high amount of peptide substrate converted to product. One major disadvantage to the current version of the zinc FP phosphopeptide assay is that some buffer components interfere with achieving the maximal difference in mP values between phosphorylated and nonphosphorylated peptides. For the standard volumes used in the described version of the zinc FP assay, it was determined that concentrations of Tris in the assay buffer above 15 mM (before zinc reagent was added) negatively impacted the FP differential observed (data not shown). This was avoided simply by lowering the Tris concentration in the assay buffer to 10 mM. Preliminary data suggested that lowering the pH of the Tris buffer would permit a higher concentration of Tris to be used (data not shown). It would be interesting to determine if altering the pH after termination of the reaction would enhance the differential FP for different peptide pairs, perhaps by altering the net charge of the peptide and/or buffering agents. The zinc FP assay was also found to be negatively affected by 1 mM DTT, and this reagent was omitted from the reaction since it was not needed for kinase activity (data not shown). However, many kinases require DTT and negative effects on the FP assay signal could possibly be minimized by finding the lowest concentration of DTT required for acceptable enzyme activity, diluting the reaction further with zinc reagent, or using a higher concentration of zinc. Another potential way to avoid interference from assay buffer components would be to transfer a fraction of the stopped reaction into a relatively large volume of premixed zinc reagent; e.g., transfer 10 ll of reaction into 240 ll of zinc reagent and then take FP measurements. In addition to assaying protein kinase activity, another potential use for this zinc phosphopeptide detection technique is to quantify phosphatase activity. Our experiments using shrimp alkaline phosphatase and phosphopeptides provide proof-of-concept that one could use this zinc FP assay as an assay of phosphatase activity. We observed a direct linear relationship between the reaction time and the decrease in mP values up to approximately 50% substrate conversion, at which point the data curve to reach nearly the same mP values as nonphosphorylated peptide. These data also further validated that the zinc FP assay quantifies the fraction of phosphopeptide in solution. Assaying phosphatase activity at low concentration of substrate can be important if the Km for substrate is very low and one needs to design a screening assay that is sensitive to substrate
competitors. Malachite green is a frequently used reagent to assay phosphatase activity by detection of free phosphate, but it is not very sensitive, requiring micromolar levels of phosphate. Due to the concentrationindependent ratiometric nature of FP, our data show that the zinc FP assay can be used to measure phosphatase activity at very low (0.1 lM and probably lower) substrate concentrations. Conceivably, this FP assay could also be used to detect and quantify protease activity by creating a peptide substrate with a fluorophore at one end, a phosphorylated residue on the other end, and the protease recognition sequence in the middle. This substrate would have a high mP value in the presence of the zinc reagent. Protease cleavage of the substrate would lower the FP values since the fluor and the phosphate would be separated, allowing the fluorophore-containing fragment to rotate freely while the phosphate-containing fragment remains bound. Currently, the mechanism of this FP assay is unknown. One possibility is that zinc phosphate is highly insoluble and may precipitate when zinc sulfate and PBS are mixed. Our observations support this idea because when the zinc/PBS solution is stirred, a white precipitate is eventually observed. Gentle mixing of the two solutions results in only a slightly hazy solution, possibly reflecting very small aggregate formation (data not shown). Immobilized metal ion affinity chromatography employing such metals as iron, gallium, and aluminum has been used to selectively isolate phosphopeptides from mixtures of phosphorylated and nonphosphorylated peptides. IMAC exploits the affinity of these transition state metal ions for phosphate. Similar to IMAC, these putative small zinc phosphate aggregates may preferentially capture the phosphorylated peptide due to the affinity of the zinc metal ion for phosphate. Fluorescein-labeled phosphopeptides preferentially bound to relatively large aggregates would rotate slower and have higher polarization values than nonphosphorylated peptides free in solution. BSA may enhance the differential polarization observed by enlarging the size of the aggregates. If this theory is correct, this assay could be based on a similar concept to a recent publication describing the use of nanoparticles derivatized with trivalent metal ions in a homogeneous FP assay designed to quantitate AMP or GMP, molecules with a phosphate group [15]. Our assay may have the cost advantage of making metal ion affinity particles in situ, without the cost of purchasing commercial nanoparticles. Alternatively, the phosphopeptide, via the phosphate group, may mimic free phosphate in that it forms an insoluble zinc salt that coaggregates with the more abundant zinc phosphate into large aggregates. These large zinc phosphate/phosphopeptide aggregates would then produce the higher polarization values observed for phosphopeptides.
J.E. Scott, J.W. Carpenter / Analytical Biochemistry 316 (2003) 82–91
Acknowledgments The authors thank Dr. Xiang Ye for providing purified kinase. Additionally, discussions of this data with Dr. Louis Stancato, Dr. Keith Houck, and Dr. Patricia Johnston are gratefully acknowledged.
References [1] M. Kostich, J. English, V. Madison, F. Gheyas, L. Wang, P. Qiu, J. Greene, T.M. Laz, Human members of the eukaryotic protein kinase family, Genome Biol. 3 (2002) RESEARCH0043.1–0043.12. [2] J.J. Witt, R. Roskoski, Rapid protein kinase assay using phosphocelluolose-paper absorption, Anal. Biochem. 66 (1975) 253–258. [3] T.S. Angeles, C. Steffler, B.A. Bartlett, R.L. Hudkins, R.M. Stephens, D.R. Kaplan, C.A. Dionne, Enzyme-linked immunosrbent assay for trkA tyrosine kinase activity, Anal. Biochem. 236 (1996) 49–55. [4] A.F. Braunwalder, D.R. Yarwood, T. Hall, M. Missbach, K.E. Lipson, M.A. Sills, A solid phase assay for the determination of protein tyrosine kinase activity of c-src using scintillating microtitration plates, Anal. Biochem. 234 (1996) 23–26. [5] B. Antonsson, C.J. Marshall, S. Montessuit, S. Arkinstall, An in vitro 96-well plate assay of the mitogen-activated protein kinase cascade, Anal. Biochem. 267 (1999) 294–299. [6] R. Seetha, R. Menzel, A homogeneous, fluorescence polarization assay for src-family tyrosine kinases, Anal. Biochem. 253 (1997) 210–218. [7] R. Seetha, R. Menzel, A fluorescence polarization competition assay for tyrosine kinases, Anal. Biochem. 255 (1998) 257–262. [8] Y.-W. Park, R.T. Cummings, L. Wu, S. Zheng, P.M. Cameron, A. Woods, D.M. Zaller, A.I. Marcy, J.D. Hermes, Homogeneous proximity tyrosine kinase assays: scintillation proximity assay versus homogeneous time-resolved fluorescence, Anal. Biochem. 289 (1999) 94–104. [9] A.J. Pope, U.M. Haupts, K.J. Moore, Homogeneous fluorescence readouts for miniaturized high-throughput screening: theory and practice, Drug Discovery Today 4 (1999) 350–362.
91
[10] T.C. Turek, E.C. Small, R.W. Bryant, W.A.G. Hill, Development and validation of a competitive AKT serine/threonine kinase fluorescence polarization assay using a product-specific anti-phospho-serine antibody, Anal. Biochem. 299 (2001) 45–53. [11] J.J. Wu, D.R. Yarwood, Q. Pham, M.A. Sills, Identification of a high-affinity anti-phosphoserine antibody for development of a homogeneous fluorescence polarization assay for protein kinase C, J. Biomol. Screen. 5 (2000) 23–30. [12] S. Jeong, T.T. Nikiforov, A kinase assay based on thiophosphorylation and biotinylation, Biotechniques 27 (1999) 1232– 1238. [13] J. Coffin, M. Latev, X. Bi, T.T. Nikiforov, Detection of phosphopeptides by fluorescence polarization in the presence of cationic polyamino acids: application to kinase assays, Anal. Biochem. 278 (2000) 206–212. [14] A. Simeonov, X. Bi, T.T. Nikiforov, Enzyme assays by fluorescence polarization in the presence of polyarginine: study for kinase, phosphatase, and protease reactions, Anal. Biochem. 304 (2002) 193–199. [15] W. Huang, Y. Zhang, J.R. Sportsman, A fluorescence polarization assay for cyclic nucleotide phosphodiesterases, J. Biomol. Screen. 7 (2002) 215–222. [16] L. Andersson, J. Porath, Isolation of phosphoproteins by immobilized metal (Fe3þ ) affinity chromatography, Anal. Biochem. 154 (1986) 250–254. [17] S. Li, C. Dass, Iron(III)-immobilized metal ion affinity chromatography and mass spectrometry for the purification and characterization of synthetic phosphopeptides, Anal. Biochem. 270 (1999) 9–14. [18] C.B. Cohen, E. Chin-Dixon, S. Jeong, T.T. Niliforov, A microchip-based enzyme assay for protein kinase A, Anal. Biochem. 273 (1999) 89–97. [19] C. House, B.E. Kemp, Protein kinase C contains a pseudosubstrate prototype in its regulatory domain, Science 238 (1987) 1726–1728. [20] B.E. Kemp, M.G. Clark, Adrenergic control of the cyclic AMP-dependent protein kinase and pyruvate kinase in isolated hepatocytes. Application of a synthetic peptide substrate for measuring protein kinase activity, J. Biol. Chem. 253 (1978) 5147–5154.
A homogeneous assay of kinase activity that detects phosphopeptide using fluorescence polarization and zinc John E. Scott* and John W. Carpenter Eli Lilly & Company, Sphinx Laboratories, 20 TW Alexander Drive, Research Triangle Park, NC, 27709, USA Received 28 October 2002
Abstract Homogeneous antibody-free assays of protein kinase activity have great utility in high-throughput screening in support of drug discovery. In an effort to develop such an assay, we have used a pair of fluorescein-labeled peptides of identical amino acid sequence with and without phosphorylation on serine to mimic the substrate and product, respectively, of a kinase. Using fluorescence polarization (FP), we have demonstrated that a mixture of zinc sulfate, phosphate-buffered saline, and bovine serum albumin added to the peptides dramatically and differentially increased the fluorescence polarization of the phosphorylated peptide over its nonphosphorylated derivative. A similar FP differential was observed using different peptide pairs, though the magnitude varied. The FP values obtained using this method were directly proportional to the fraction of phosphopeptide present. Therefore, an FP assay was developed using a proprietary kinase. Using this FP method, linear reaction kinetics were obtained in enzyme titration and reaction time course experiments. The IC50 values for a panel of inhibitors of kinase activity were determined using this FP method and a scintillation proximity assay. The IC50 values were comparable between the two methods, suggesting that the zinc FP assay may be useful as an inexpensive high-throughput assay for identifying inhibitors of kinase activity. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Kinase; Fluorescence polarization; Zinc
Protein phosphorylation is a frequently used mechanism in nature for regulating the activity of a host of enzymes, transcription factors, receptors, etc. There are estimated to be over 500 different protein kinases in the human proteome that catalyze the transfer of phosphate from ATP to serine, threonine, and tyrosine [1]. Protein kinases can be divided into two broad classes, serine/ threonine and tyrosine kinases, although a few kinases are able to phosphorylate both types of amino acid residues. Kinases play critical roles in many fundamental cell-signaling pathways including those that lead to growth, differentiation, motility, apoptosis, and survival. Not surprisingly, the dysregulation of protein kinase activity has been associated with numerous diseases including cancer, diabetes, and inflammation. As such, they are the focus of much interest for the development of compounds to selectively regulate the activity of disease-related kinases. The most common method to * Corresponding author. Fax: +919-314-4174. E-mail address: [email protected] (J.E. Scott).
discover selective novel inhibitors of protein kinase activity has been to screen large libraries of compounds for inhibitors of a biochemical assay designed to measure the phosphorylating activity of the kinase of interest. Numerous methods have been designed to measure protein kinase activity. Early methods require physical separation of substrate protein or peptide from its phosphorylated product. One method is to use radiolabeled ATP that allows the incorporated phosphate to be detected after the capture of the protein/peptide substrate by phosphocellulose membrane [2]. This type of assay of protein kinase activity requires multiple steps and the use of radioactivity with its accompanying hazards and costs. There are also ELISA1-based assays 1
Abbreviations used: ELISA, enzyme-linked immunosorbent assay; FP, fluorescence polarization; mP, millipolarization units; DTT, dithiothreitol; PBS, phosphate-buffered saline; BSA, bovine serum albumin; IMAC, immobilized metal ion affinity chromatography; SPA, scintillation proximity assay; FRET, fluorescence resonance energy transfer, TMR, tetramethylrhodamine; DMSO, dimethyl sulfoxide; SAP, shrimp alkaline phosphatase.
0003-2697/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0003-2697(03)00036-8
J.E. Scott, J.W. Carpenter / Analytical Biochemistry 316 (2003) 82–91
of kinase activity, but these also require multiple steps in addition to being dependent on availability of a phospho-specific antibody to capture product [3]. The scintillation proximity assay (SPA) has been used as a high-throughput homogeneous assay of protein kinase activity that does not require an antibody [4,5], but still has all the disadvantages inherent in the use of radioactivity. Homogeneous nonradioactive assay formats measuring protein kinase activity that include fluorescence polarization (FP) and time-resolved fluorescence resonance energy transfer (FRET) have been developed to eliminate the disadvantages of radioactivity and washing steps [6–8]. Fluorescence polarization is a ratiometric technique that reflects the rotational motion of a fluorescent molecule in solution with the rotational velocity dependent on the apparent molecular size [9]. A major disadvantage of both FP and FRET assays is that they are dependent on the availability of a phospho-specific antibody. Another drawback to the use of antibodies is that their high specificity for a given phosphopeptide often prevents the same antibody from being used with different substrates. Anti-phosphotyrosine-specific antibodies are broadly reactive since they are relatively insensitive to the surrounding amino acid context. In contrast, phosphoserine-/phosphothreoninespecific antibodies are highly dependent on the amino acid sequence surrounding the phosphorylated residue and thus tend to only bind a limited diversity of phosphorylated sites [10,11]. In general, a new antibody must be generated for each different substrate, which can be both time consuming and expensive. Several antibody-free nonradioactive assays of protein kinase activity have been reported. One such assay method replaces ATP in the reaction with ATPcS leading to the formation of a thiophosphorylated peptide product [12]. This product is then chemically biotinylated by a sulfur-specific biotinylation reagent. Streptavidin is then added to the fluorescently tagged peptide and an FP measurement is taken to measure the amount of product formed. The potential disadvantages of this assay include long incubation times and altered kinetics for some kinases due to the ATPcS. Another antibody-free FP assay of protein kinase activity that has been reported uses polyarginine to discriminate fluorescently labeled phosphorylated and nonphosphorylated peptides based on charge interaction of the phosphate group and the positively charged high molecular weight polyarginine [13,14]. Another potential antibody-free assay of protein kinase activity is the socalled IMAP assay that employs trivalent metal ions immobilized on nanoparticles to bind phosphate groups on molecules and thus increase their FP values [15]. This approach is similar in concept to immobilized metal affinity chromatography (IMAC) used to specifically isolate phosphopeptides from mixtures containing both phosphorylated and nonphosphorylated peptides
83
[16,17]. Finally, the development of microchip instruments has led to an additional approach to measuring the activity of kinases [18]. In the microfluidic assay of protein kinase activity, fluorophore-labeled peptide substrate and product are separated based on a difference in their charge-to-mass ratio. Separation of product and substrate is achieved by electrophoresis through an electric field, followed by quantitation of the individual fluorescent peaks. We have explored the use of zinc ions in solution to discriminate between phosphorylated and nonphosphorylated fluorescently labeled peptides. In this article, we report a simple homogeneous nonradioactive FP-based assay of protein kinase activity using commonly available reagents and fluorescently-tagged peptides to quantitate relative phosphopeptide levels. Our assay does not require an antibody and is very inexpensive. Experiments using a serine/threonine kinase and alkaline phosphatase show that this zinc FP assay represents a novel inexpensive alternative for measuring kinase and phosphatase activity.
Materials and methods General All peptides were obtained from Anaspec Inc. (San Jose, CA). All general biochemical reagents were obtained from Sigma (St. Louis, MO). DulbeccoÕs phosphate-buffered saline without calcium or magnesium (PBS, Catalog No. 17-512Q) was obtained as a liquid stock from BioWhittaker (Walkersville, MD). Bovine serum albumin (BSA) was obtained from Sigma (Catalog No. A7906 or A6918). ATP was purchased from Roche (Nutley, NJ). All assays were done in Corning/ Costar (Acton, MA) 384-well flat-bottom non-treated black polystyrene plates (No. 3710). Compounds for IC50 (inhibitor concentration that results in 50% inhibition of activity) value comparison experiments were obtained from Calbiochem (San Diego, CA). Fluorescence polarization measurements Fluorescence polarization measurements were performed on an Analyst HT instrument (Molecular Devices, Sunnyvale, CA). Fluorescence intensity measurements were taken in black 384-well microtiter plates where both the excitation and the emission occur from the top of the wells. In the Analyst HT, a xenon arc lamp provides excitation light that passes through an excitation filter and then a polarizer filter. A beamsplitter filter directs the polarized excitation light into the well and emitted fluorescence transmits back through the same beam-splitter filter, through a polarizer filter, then through the emission filter for detection.
84
J.E. Scott, J.W. Carpenter / Analytical Biochemistry 316 (2003) 82–91
Read time was 0.1 s per well. The excitation polarizer filter was fixed in the parallel position, while the emission polarizer filter was changed for measuring parallel and perpendicular emission fluorescence intensity. All polarization values are expressed in millipolarization units (mP). The mP values were calculated using the equation mP ¼ 1000 ½ðIS IP Þ=ðIS þ IP Þ, where IS is the parallel emission intensity measurement and IP is the perpendicular emission intensity measurement. For fluorescein-labeled peptides, measurements were made with excitation at 485 nm (25-nm bandwidth) and emission at 530 nm (25-nm bandwidth) using a 505-nm beam splitter. For the tetramethylrhodamine (TMR)labeled peptide, measurements were made with excitation at 530 nm (25-nm bandwidth) and emission at 580 nm (10-nm bandwidth) using a 561-nm beam splitter. Peptide experiments The sequences of the peptides used are shown in Table 1. All reagents and experiments were done at room temperature. Except where indicated, standard non-enzymatic peptide experiments were performed by diluting fluorescently labeled peptide to 0.5 lM in water and adding 30 ll of this peptide solution into the well of an assay plate. The zinc reagent, ZnSO4 /PBS/BSA, was prepared by gently mixing equal volumes of 1% (weight/ volume) BSA in PBS (PBS/BSA) and 60 mM ZnSO4 . Mixing was accomplished through gentle inversion. The zinc reagent is stable for at least 1 h. To 30 ll of peptide solution, 60 ll of zinc reagent was added. After approximately 10 min, the fluorescence polarization measurements were taken. Assay of protein kinase activity All assays of protein kinase activity were performed using a proprietary recombinant serine/threonine kinase prepared at Eli Lilly & Co. (Indianapolis, IN). The FP assay of protein kinase activity was performed as follows. The FP assay buffer used was 10 mM Tris, pH 8.0,
Table 1 Sequences of fluorescently labeled peptidesa No.
Sequence
1 2 3 4 5 6 7 8
Fl-GPLGRHGpSIRQKKEEV Fl-GPLGRHGSIRQKKEEV Fl-RFARKGpSLRQKNV Fl-RFARKGSLRQKNV Fl-LC-LRRApSLG Fl-LC-LRRASLG TMR-KVEKIGEGTpYGVVYK TMR-KVEKIGEGTYGVVYK
a Abbreviations used: pS, phosphoserine; pY, phosphotyrosine; Fl, fluorescein; TMR, tetramethylrhodamine; LC, linker chain.
10 mM MgCl2 , and 0.01% Triton X-100. The concentration of ATP used was 5 lM and the nonphosphorylated fluorescein-labeled peptide 2 was used at 0.5 lM (or 1 lM where indicated). The reaction volume was 30 ll and the amount of kinase used varied from 160 to 600 ng per reaction as indicated for each experiment. The assays were set up by the addition of 10 ll of 3% DMSO in water (with or without compound) to the assay plate followed by 10 ll kinase and 10 ll ATP/ peptide mixture, each diluted with 1.5X FP assay buffer. The reactions were incubated at room temperature, or 30 °C where indicated, for 3–4 h or 18–20 h, depending on the kinase concentration used and the activity of the particular kinase preparation. The preparation of zinc reagent and its use in developing the assay was done as described above. For the IC50 comparison experiments, 10- to 12-point concentration responses were generated starting at 100 lM (or 20 lM for staurosporine). Each concentration of compound was tested in triplicate (quadruplicate for staurosporine) per individual IC50 determination. Reactions were incubated at room temperature for 18–20 h with 160 ng kinase per reaction, except for the staurosporine IC50 determination experiments where 200–400 ng/well kinase and 1 or 0.5 lM peptide substrate was used in 3-h reactions. For the scintillation proximity assay, the final assay conditions were 50 mM Tris, pH 8.0, 10 mM MgCl2 , 0.01% Triton X-100, 1 mM DTT, 1 or 3.75 lM ATP, 0.375 lM peptide substrate (identical to peptide 2 except biotin replaced the fluorescein), 160 ng/reaction kinase enzyme, and 0.125 lCi/reaction of [33 P]ATP from Perkin Elmer (Boston, MA). The 40-ll reactions were incubated at room temperature for 4 h. A stop reagent (40 ll per well) containing 0.2 mg streptavidin-coated SPA beads (Amersham, Piscataway, NJ), 40 mM EDTA, and 74% CsCl was added to the reaction. After sealing the plates and incubating for 2 h at room temperature, the plates were read on a TopCount (Perkin Elmer) scintillation counter. IC50 values for compounds were generated as described above. Assay of phosphatase activity The assay of phosphatase activity used shrimp alkaline phosphatase (SAP) from Roche. The assay buffer used was the same as for the FP assay of protein kinase activity. SAP and the phosphopeptides 1 and 5 were diluted into assay buffer with the final assay concentrations of 0.1 lM peptide and 15 mU (peptide 1) or 0.25 mU (peptide 5) per reaction SAP. The reactions were started by the addition of 15 ll of SAP to 15 ll of phosphopeptide at various times. SAP was heat-inactivated for the control reactions by heating at 65 °C for 20 min. Nonphosphorylated peptide 2 along with SAP was also used as a reference for 100% dephosphorylation. The reactions were terminated with 60 ll zinc
J.E. Scott, J.W. Carpenter / Analytical Biochemistry 316 (2003) 82–91
85
reagent and the FP measurements taken as described for the FP assay of protein kinase activity.
Results In an effort to develop a homogeneous nonradioactive assay of protein kinase activity, we had synthesized a pair of fluorescein-labeled peptides (Table 1, peptides 1 and 2) with identical amino acid sequence with (peptide 1) and without (peptide 2) a phosphate on the serine residue to mimic the substrate and product, respectively, of a kinase. We tested several metal ions, including Zn2þ , Fe3þ , Ca2þ , Mn2þ , and Mg2þ , for their ability to preferentially aggregate the phosphopeptide such that the phosphorylated and nonphosphorylated peptide could be differentiated by their fluorescence polarization values. Aggregates/large complexes preferentially containing the phosphopeptide would have an increased apparent molecular size. These large aggregates would have a slower rate of rotation compared to nonphosphorylated peptide free in solution and hence the FP values would be higher for the phosphorylated peptide. Zinc sulfate produced the most dramatic FP differential observed for this set of peptides diluted into PBS and BSA solution (data not shown). In contrast, Mn2þ and Mg2þ ions, common cofactors for kinases and phosphatases, did not generate any significant difference in FP values for these peptides (data not shown). In further optimization experiments using zinc, peptides 1 and 2 were diluted to 0.5 lM in water. Varying concentrations of zinc sulfate solution were added to an equal volume of PBS containing 1% BSA (PBS/BSA). Sixty microliters of this ZnSO4 /PBS/BSA mixture (zinc reagent) was added to 30 ll of peptide and fluorescence polarization values were measured (Fig. 1). At final zinc sulfate concentrations of 2 mM or above, a dramatic difference in mP values was observed for the phosphorylated versus nonphosphorylated peptides, with the phosphorylated version of the peptide displaying FP values of 170–230 mP higher than its nonphosphorylated analog. We observed that 16–32 mM zinc sulfate generated the maximum fluorescence polarization difference between these two peptides, approximately 230 mP. We chose to use 20 mM final zinc sulfate concentration (or 60 mM zinc sulfate solution before dilution) for all subsequent experiments. To further explore this phenomenon, we determined some of the reagent requirements for generating this fluorescence polarization differential between the two peptides. We tested different combinations of zinc sulfate, PBS, and BSA along with water-solubilized peptide (data not shown). Neither zinc sulfate, PBS, nor BSA alone produced a significant FP differential between the two peptides. Generation of the observed differential polarization values for these peptides required both zinc sulfate and
Fig. 1. Effect of zinc sulfate concentration on the fluorescence polarization of phosphorylated and nonphosphorylated fluorescein-labeled peptides. Varying concentrations of aqueous zinc sulfate were added to an equal volume of PBS/1% BSA solution. The ZnSO4 /PBS/BSA solutions (60 ll) were added to 30 ll of 0.5 lM phosphorylated (peptide 1, N) and nonphosphorylated (peptide 2, j) versions of the same fluorescein-labeled peptide diluted with water. The fluorescence polarization (mP) of the resulting final solution was measured. The final concentration of zinc sulfate after addition to peptide is indicated. Each data point is an average of triplicate determinations and the error bars indicate standard deviation. Data are representative of three independent experiments.
PBS. While the presence of BSA was not required, BSA did enhance the polarization value difference between the phosphorylated and nonphosphorylated peptide. The inclusion of BSA increased the average FP differential from 183 to 242 mP, enhancing the FP differential by 59 mP. Therefore, all subsequent experiments had the final concentrations (after addition to the peptide solution) of a 1:3 dilution of PBS:BSA and 20 mM zinc sulfate. This was obtained by mixing 1 vol of PBS/BSA with an equal volume of 60 mM zinc sulfate and then two volumes of this zinc reagent added to one volume of peptide solution. In general, equivalent results were obtained by either adding the PBS/BSA and zinc sulfate separately (30 ll peptide plus 30 ll PBS/BSA, then 30 ll zinc sulfate) or gently premixing the two reagents before addition to peptide (30 ll peptide plus 60 ll of zinc reagent) (data not shown). All experiments shown in this report were performed using the premixed zinc reagent. To determine whether the increased mP observed for the phosphorylated peptide could be used to quantitate the percentage of phosphorylated peptide in a mixture, we used peptides 1 and 2 to mimic protein kinase activity. We mixed solutions of phosphorylated peptide 1 and nonphosphorylated peptide 2 such that the concentration of total labeled peptide was constant at 0.5 lM, but the percentage of phosphorylated peptide varied 0–100% of total peptide (Fig. 2). Zinc reagent was added to these defined peptide mixtures and the mP values were determined. The results demonstrated that the increase in polarization values observed for phosphorylated
86
J.E. Scott, J.W. Carpenter / Analytical Biochemistry 316 (2003) 82–91
Fig. 2. Fluorescence polarization of defined mixtures of a phosphorylated fluorescein-labeled peptide and its nonphosphorylated derivative. Phosphorylated peptide (peptide 1) and its nonphosphorylated peptide derivative (peptide 2) were mixed together in water to generate the indicated percentages of phosphorylated peptide compared to the concentration of total peptide. The concentration of total peptide remained constant at 0.5 lM. To 30 ll of the peptide mixtures, 60 ll of zinc reagent was added and the fluorescence polarization measured. Each data point is an average of triplicate determinations and the error bars indicate standard deviation. Data are representative of three independent experiments.
peptide was linear and directly proportional to the percentage of phosphorylated peptide in the mixture. These data suggested that this method might be used to quantitate the catalytic activity of a kinase using a fluorescein-labeled peptide as a substrate. We used a proprietary serine/threonine kinase and peptide 2 to test the utility of this system as a viable assay of protein kinase activity. This zinc FP assay was found to be sensitive to high concentrations of Tris buffer, which caused a decrease in the difference in mP between peptides 1 and 2. For the volumes used in this assay, 10 mM Tris, pH 8.0, buffer did not significantly affect the FP differential between peptides 1 and 2 and therefore was used for the reaction buffer (data not shown). An enzyme titration experiment with peptide 2 as substrate demonstrated a linear increase in fluorescence polarization values with increasing amount of kinase used in the reaction (Fig. 3). Also, this increase in FP values was ATP dependent (data not shown). If the change in FP values observed using this zinc FP assay was related to kinase activity, the mP values obtained would be expected to be directly proportional to the reaction incubation time. Kinase was incubated with peptide 2 and ATP for varying times, zinc reagent was added, and fluorescence polarization values were determined (Fig. 4). These data indicated a positive linear relationship between incubation time and mP values. Based on previous data with the model peptides 1 and 2, the 4-h incubation time with kinase resulted in 110 mP increase over background or approximately 50% of peptide 2 converted to phosphorylated product. We compared this zinc FP assay to a scintillation proximity
Fig. 3. Dependence of fluorescence polarization signal on kinase concentration. Reactions were performed with the indicated amount of kinase per 30 ll reaction, 0.5 lM peptide 2 (nonphosphorylated fluorescein-labeled peptide), and 5 lM ATP at 30 °C. Reaction time was 4 h. Reactions were terminated with 60 ll zinc reagent and fluorescence polarization measurements taken. Each data point is an average of triplicate determinations and the error bars indicate standard deviation. Data are representative of three independent experiments.
Fig. 4. Dependence of fluorescence polarization signal on reaction time. Reactions were performed for the indicated length of time using 600 ng/well kinase, 0.5 lM peptide 2 (nonphosphorylated fluoresceinlabeled peptide), and 5 lM ATP at 30 °C. The reactions were started at various times with the addition of enzyme and all reactions terminated together and the fluorescence polarization values determined. Each data point is an average of triplicate determinations and the error bars indicate standard deviation. Data are representative of three independent experiments.
assay of protein kinase activity with respect to compound potency determination using a panel of seven commercially available inhibitors of kinase activity (Table 2). The IC50 value for staurosporine generated using the zinc FP assay was 12 nM, which is comparable to 4 nM obtained using the SPA format. The zinc FP assay produced average IC50 values for six of the seven inhibitors that were within three-fold of the SPA format. One of these inhibitors, bisindolylmaleimide V, is a negative control for the bisindolylmaleimide series of
J.E. Scott, J.W. Carpenter / Analytical Biochemistry 316 (2003) 82–91
87
Table 2 Comparison of IC50 values for compounds using the zinc FP and SPA formatsa Compound
FP IC50 (lM)
SPA IC50 (lM)
Bisindolylmaleimide III Bisindolylmaleimide V H-9 Calmidazolium H-89 G€ o 7879 Staurosporineb
13 1:4 >100 2:2 0:4 3:5 0:2 0:62 0:18 1:5 0:4 0:012 0:0003
5:1 1:8 >100 0:55 0:23 8:9 5:3 0:24 0:13 0:98 0:45 0:004 0:001
a Reactions were performed as described under Materials and methods. Ten-point dose response curves were generated starting at a high concentration of 100 lM. Each compound concentration was tested in triplicate per IC50 determination. IC50 values are the mean of three independent determinations. Errors reported are standard deviations. b Staurosporine dose response curves were generated starting at a high concentration of 20 lM. Each compound concentration was tested in quadruplicate (FP assay) or triplicate (SPA) per IC50 determination. IC50 values are the mean of two independent determinations.
PKC inhibitors and also served as a negative control for our kinase, generating >100 lM IC50 value in both assay formats. The average IC50 value for H-9 in the zinc assay format was four-fold higher than in the SPA format. Approximately 25–50% of peptide substrate was converted to product in these zinc FP IC50 assays while in the SPA it was <10%. Overall, these data indicated that the zinc FP assay was comparable to the SPA in sensitivity to both potent and relatively weak inhibitors of kinase activity. In addition, this IC50 data demonstrated that the zinc FP assay tolerated these compounds at high concentration without effect on the assay. In order to determine how broadly applicable this zinc FP phosphopeptide detection method was in detecting the phosphorylation state of peptides, we obtained three other pairs of peptides, identical in sequence except that one residue was phosphorylated. Peptide pairs 3 and 4 (PKC pseudosubstrate analogs) and 5 and 6 (Kemptide) are fluoresceinated derivatives of commonly used substrates for kinases [19,20]. Peptide pair 7 and 8 is unique in this panel of peptides in that it is tagged with tetramethylrhodamine instead of fluorescein. Peptide 8 is a substrate for the tyrosine kinase cSrc and therefore, the phosphopeptide version (peptide 7) was synthesized with a phosphotyrosine residue. All four peptide pairs were tested with the zinc FP method as before (Fig. 5). The net differential polarization was determined for each pair of peptides; i.e., the mP value obtained for the nonphosphorylated peptide was subtracted from the phosphopeptide mP value. All four pairs of peptides in this panel displayed significantly higher mP values for the phosphorylated peptides with differences ranging from 38 to 235 mP. At 50% phosphopeptide concentration, three of the four peptide pairs would produce at least 50 mP of signal over
Fig. 5. Differential FP values for a panel of paired phosphorylated and nonphosphorylated fluorescently labeled peptides. A panel of four pairs of fluorescently labeled peptides was assembled where a pair has the same amino acid sequence, but one is phosphorylated. Each peptide was diluted to 0.5 lM in 30 ll water and 60 ll zinc reagent added followed by fluorescence polarization measurements. The difference in FP values (indicated above the bars) between phosphorylated and nonphosphorylated matched peptide pairs as indicated was plotted. The data shown are the average difference obtained in three independent experiments where each data point was an average of triplicate determinations. The error bars indicate standard deviation for the results of the three experiments.
background. These data suggest that the zinc FP assay has general utility in phosphopeptide detection. However, the magnitude of polarization differential observed between phosphorylated and nonphosphorylated peptides in this assay was clearly peptide sequence and/or net charge dependent. To determine if this zinc FP method of phosphopeptide detection could be used for phosphatases, we performed phosphatase activity time courses with phosphopeptides 1 and 5 as substrates for shrimp alkaline phosphatase (Fig. 6). Controls were included that corresponded to a 100% phosphopeptide level using heat-inactivated phosphatase and phosphopeptide. A control representing 0% phosphopeptide used active enzyme and the nonphosphorylated matched peptide. Both peptides 1 and 5 demonstrated a time-dependent decrease in polarization values. Peptide 1 showed a linear decrease in mP values for the first 30 min of incubation time (Fig. 6A, inset). Based on the mP values obtained with the 0 and 100% phosphopeptide controls, this linear range corresponded to between 100% (at 0 min) and approximately 50% (at 30 min) phosphopeptide content. The mP values for the phosphopeptides exposed to active phosphatase, but not inactivated phosphatase, closely approach the mP values of the nonphosphorylated control peptide after 2 h of reaction time. These data provided proof-of-concept that this assay method could be used to measure phosphatase activity using a fluorescein-labeled phosphopeptide as substrate. Moreover, the concentration of peptide substrate in this assay was only 100 nM, demonstrating that the zinc FP phosphopeptide detection assay can be employed as a very sensitive antibody-free assay of
88
J.E. Scott, J.W. Carpenter / Analytical Biochemistry 316 (2003) 82–91
Fig. 6. Phosphatase activity measured using FP assay. Shrimp alkaline phosphatase was incubated with 0.1 lM fluorescein-labeled phosphopeptide 1 (A) or 5 (B) for the indicated times. The reactions were started at various times with the addition of phosphatase and terminated at one time with zinc reagent. The phosphopeptides were incubated with either active phosphatase (d) or heat-inactivated phosphatase (N) as a control. The nonphosphorylated derivative of each peptide was also included as a control and incubated at the same times with active phosphatase (j). Each data point is an average of triplicate determinations and the error bars indicate standard deviation. Data are representative of three independent experiments.
phosphatase activity. In addition, these data further validated the idea that the mP values determined with the zinc FP method accurately reflect the phosphorylation state of the labeled peptide.
Discussion In this article, we have described a novel, inexpensive, nonradioactive, homogeneous fluorescence polarization assay that can be used to detect and quantitate relative phosphopeptide levels in a sample. Unlike traditional radiometric approaches, fluorescence polarization has the advantage of being a homogeneous method that does not require separation of product and substrate. Most commercially available FP-based assays of protein kinase activity require a phospho-specific antibody. There are several disadvantages to the use of antibodies in FP assays. Although phosphotyrosine-specific antibodies are common and relatively amino acid context
independent, they are expensive. Phosphoserine- or phosphothreonine-specific antibodies can be difficult and costly to produce and are sensitive to amino acid sequence surrounding the phosphorylation site and hence cannot be used for all serine/threonine peptide substrates. Unlike most other homogeneous fluorescent assays of protein kinase activity, reagents required for the zinc FP assay are simply a fluorescein-labeled peptide and common, inexpensive laboratory reagents, with no antibody required. Peptide pairs that are identical in amino acid sequence and fluorophore, but one modified with a phosphate group, were used as models of substrates and products of a kinase. A solution of zinc sulfate, PBS, and BSA was found to differentially effect the fluorescence polarization of phosphopeptides compared to nonphosphorylated analogs. The phosphorylated peptides generated significantly higher mP values in response to exposure to the zinc reagent. The difference in fluorescence polarization values between the fluorescein-
J.E. Scott, J.W. Carpenter / Analytical Biochemistry 316 (2003) 82–91
labeled phosphorylated and nonphosphorylated peptides within a matched pair ranged from 94 to 235 mP for the three sets we tested. The magnitude of this observed differential appears to depend on the amino acid sequence and/or net charge of the peptide. Of potential significance, the two fluorescein-labeled peptides that gave the highest FP differential (peptides 2 and 6) had a net charge of )1 in the nonphosphorylated state that changed to a net charge of )3 in the phosphorylated state. The other fluorescein-labeled peptide (peptide 4) had a net charge of +2 that became neutral upon addition of the phosphate group. Although the TMR-labeled peptides had a net neutral charge, it produced a much lower differential mP value. It is unknown whether this is due to the different fluorophore, as TMR has a positive and negative charge (net neutral), or because the peptide has a phosphotyrosine whereas all the other phosphopeptides tested were phosphoserines. Since we only tested four peptide pairs with this FP assay and they varied significantly in amino acid sequence, net charge, and fluorophore, it is not possible at this point to correlate peptide structure with performance in this assay format. In general, it appears that this differential FP observed for phosphopeptides in the presence of zinc reagent is broadly applicable and not the consequence of isolated unusual peptide sequences, although the magnitude of the FP differential varies with the peptide sequence and/or net charge. We have shown that this change in fluorescence polarization absolutely requires both zinc sulfate and PBS, while BSA just enhances the observed differential. While initially the PBS/BSA and the zinc sulfate reagents were added separately, it was more convenient to premix the two solutions so that only one addition to the assay was required. We demonstrated that the mP values obtained in the zinc FP assay were directly proportional to the percentage of phosphopeptide in mixtures of phosphorylated and nonphosphorylated peptides designed to mimic kinase activity. These data suggest that this method could be applied to assaying protein kinase activity. In order to measure kinase activity in the current configuration, the substrate needs to be a relatively short peptide labeled with a fluorophore. In addition, completely phosphorylated and nonphosphorylated versions of the peptide are required to establish the mP values for 0 and 100% phosphopeptide content. Therefore, this assay is not readily amenable to measuring kinase activity using a peptide or protein of unknown sequence. The assay of protein kinase activity in this report was designed using a proprietary serine/threonine kinase and a nonphosphorylated fluorescein-labeled peptide as substrate. The mP values obtained in enzyme titration experiments increased in direct proportion to the amount of kinase added to the reaction, as would be expected if the zinc FP method measured the fraction of phosphopeptide present. Likewise, a time course of the reaction
89
demonstrated that the mP values increased in direct proportion to the reaction time, which is consistent with the zinc FP method measuring kinase activity. Furthermore, we generated IC50 values for a panel of seven commercially available compounds using the zinc FP and SPA formats. The IC50 values for the two assay formats were within 1.6- to 4-fold of each other or an average of 2.4-fold difference. Taking the SPA data as the standard, both potent and weakly inhibitory compounds were detected with the zinc FP assay. Although the SPA and FP formats were set up with different concentrations of ATP (1–5 lM), these concentrations are well below the Km for ATP of 20 lM determined previously for this kinase (data not shown). In theory, the difference in ATP concentration between the two assay formats would not be expected to make a difference in sensitivity to ATP competitors. Overall, the IC50 comparison data validated the use of the zinc FP phosphopeptide detection method to determine IC50 values for inhibitors of kinase activity. The zinc FP method has the potential to be used as an inexpensive, automated, homogeneous, high-throughput assay for the discovery of inhibitors of kinase activity from compound collections. This assay uses high concentrations of fluorophore that produces very high fluorescence intensities leading to highly accurate FP measurements. In addition, the high fluorescence intensity readings would make the assay more resistant to interference by weakly fluorescent and quenching compounds. Another simple and inexpensive assay of protein kinase activity previously reported used fluoresceinlabeled peptides, polyarginine, and FP to measure kinase activity [13]. One advantage of our zinc FP assay over the polyarginine assay is that the signal observed for some peptides is larger allowing a smaller fraction of substrate-to-product conversion to generate a suitable assay signal. For instance, a nearly identical peptide to one used for the polyarginine study was used in our studies and had a differential of 110 mP for the polyarginine assay, but 175 mP for the zinc FP assay. Using the polyarginine method with peptides 1 and 2 produced a maximal 110 mP differential in our hands (data not shown). Using the zinc FP assay, the same peptides demonstrated a 235 mP differential—double the differential compared to polyarginine. This large differential enabled us to perform assays with peptide 2 where only 25–50% of substrate was converted to product, while maintaining a robust 50–100 mP signal. For optimal enzyme kinetic experiments, it is usually desired to convert less than 10% of the substrate to product. However, in practice, there is typically only a small or insignificant difference between the IC50 values for ATP competitive inhibitors generated by assays converting <10% and those converting 50% (or higher) of the peptide substrate to product, provided the rate of product generation in the assay remains linear with re-
90
J.E. Scott, J.W. Carpenter / Analytical Biochemistry 316 (2003) 82–91
spect to time ([13] and data not shown). The IC50 values presented in Table 2 also confirmed this observation since comparable results were observed even though substrate conversion for the FP assay and the SPA was quite different, at 25–50% and <10% substrate conversion, respectively. These data suggested that the sensitivity of the zinc FP assay to inhibitors was not significantly compromised by the relatively high amount of peptide substrate converted to product. One major disadvantage to the current version of the zinc FP phosphopeptide assay is that some buffer components interfere with achieving the maximal difference in mP values between phosphorylated and nonphosphorylated peptides. For the standard volumes used in the described version of the zinc FP assay, it was determined that concentrations of Tris in the assay buffer above 15 mM (before zinc reagent was added) negatively impacted the FP differential observed (data not shown). This was avoided simply by lowering the Tris concentration in the assay buffer to 10 mM. Preliminary data suggested that lowering the pH of the Tris buffer would permit a higher concentration of Tris to be used (data not shown). It would be interesting to determine if altering the pH after termination of the reaction would enhance the differential FP for different peptide pairs, perhaps by altering the net charge of the peptide and/or buffering agents. The zinc FP assay was also found to be negatively affected by 1 mM DTT, and this reagent was omitted from the reaction since it was not needed for kinase activity (data not shown). However, many kinases require DTT and negative effects on the FP assay signal could possibly be minimized by finding the lowest concentration of DTT required for acceptable enzyme activity, diluting the reaction further with zinc reagent, or using a higher concentration of zinc. Another potential way to avoid interference from assay buffer components would be to transfer a fraction of the stopped reaction into a relatively large volume of premixed zinc reagent; e.g., transfer 10 ll of reaction into 240 ll of zinc reagent and then take FP measurements. In addition to assaying protein kinase activity, another potential use for this zinc phosphopeptide detection technique is to quantify phosphatase activity. Our experiments using shrimp alkaline phosphatase and phosphopeptides provide proof-of-concept that one could use this zinc FP assay as an assay of phosphatase activity. We observed a direct linear relationship between the reaction time and the decrease in mP values up to approximately 50% substrate conversion, at which point the data curve to reach nearly the same mP values as nonphosphorylated peptide. These data also further validated that the zinc FP assay quantifies the fraction of phosphopeptide in solution. Assaying phosphatase activity at low concentration of substrate can be important if the Km for substrate is very low and one needs to design a screening assay that is sensitive to substrate
competitors. Malachite green is a frequently used reagent to assay phosphatase activity by detection of free phosphate, but it is not very sensitive, requiring micromolar levels of phosphate. Due to the concentrationindependent ratiometric nature of FP, our data show that the zinc FP assay can be used to measure phosphatase activity at very low (0.1 lM and probably lower) substrate concentrations. Conceivably, this FP assay could also be used to detect and quantify protease activity by creating a peptide substrate with a fluorophore at one end, a phosphorylated residue on the other end, and the protease recognition sequence in the middle. This substrate would have a high mP value in the presence of the zinc reagent. Protease cleavage of the substrate would lower the FP values since the fluor and the phosphate would be separated, allowing the fluorophore-containing fragment to rotate freely while the phosphate-containing fragment remains bound. Currently, the mechanism of this FP assay is unknown. One possibility is that zinc phosphate is highly insoluble and may precipitate when zinc sulfate and PBS are mixed. Our observations support this idea because when the zinc/PBS solution is stirred, a white precipitate is eventually observed. Gentle mixing of the two solutions results in only a slightly hazy solution, possibly reflecting very small aggregate formation (data not shown). Immobilized metal ion affinity chromatography employing such metals as iron, gallium, and aluminum has been used to selectively isolate phosphopeptides from mixtures of phosphorylated and nonphosphorylated peptides. IMAC exploits the affinity of these transition state metal ions for phosphate. Similar to IMAC, these putative small zinc phosphate aggregates may preferentially capture the phosphorylated peptide due to the affinity of the zinc metal ion for phosphate. Fluorescein-labeled phosphopeptides preferentially bound to relatively large aggregates would rotate slower and have higher polarization values than nonphosphorylated peptides free in solution. BSA may enhance the differential polarization observed by enlarging the size of the aggregates. If this theory is correct, this assay could be based on a similar concept to a recent publication describing the use of nanoparticles derivatized with trivalent metal ions in a homogeneous FP assay designed to quantitate AMP or GMP, molecules with a phosphate group [15]. Our assay may have the cost advantage of making metal ion affinity particles in situ, without the cost of purchasing commercial nanoparticles. Alternatively, the phosphopeptide, via the phosphate group, may mimic free phosphate in that it forms an insoluble zinc salt that coaggregates with the more abundant zinc phosphate into large aggregates. These large zinc phosphate/phosphopeptide aggregates would then produce the higher polarization values observed for phosphopeptides.
J.E. Scott, J.W. Carpenter / Analytical Biochemistry 316 (2003) 82–91
Acknowledgments The authors thank Dr. Xiang Ye for providing purified kinase. Additionally, discussions of this data with Dr. Louis Stancato, Dr. Keith Houck, and Dr. Patricia Johnston are gratefully acknowledged.
References [1] M. Kostich, J. English, V. Madison, F. Gheyas, L. Wang, P. Qiu, J. Greene, T.M. Laz, Human members of the eukaryotic protein kinase family, Genome Biol. 3 (2002) RESEARCH0043.1–0043.12. [2] J.J. Witt, R. Roskoski, Rapid protein kinase assay using phosphocelluolose-paper absorption, Anal. Biochem. 66 (1975) 253–258. [3] T.S. Angeles, C. Steffler, B.A. Bartlett, R.L. Hudkins, R.M. Stephens, D.R. Kaplan, C.A. Dionne, Enzyme-linked immunosrbent assay for trkA tyrosine kinase activity, Anal. Biochem. 236 (1996) 49–55. [4] A.F. Braunwalder, D.R. Yarwood, T. Hall, M. Missbach, K.E. Lipson, M.A. Sills, A solid phase assay for the determination of protein tyrosine kinase activity of c-src using scintillating microtitration plates, Anal. Biochem. 234 (1996) 23–26. [5] B. Antonsson, C.J. Marshall, S. Montessuit, S. Arkinstall, An in vitro 96-well plate assay of the mitogen-activated protein kinase cascade, Anal. Biochem. 267 (1999) 294–299. [6] R. Seetha, R. Menzel, A homogeneous, fluorescence polarization assay for src-family tyrosine kinases, Anal. Biochem. 253 (1997) 210–218. [7] R. Seetha, R. Menzel, A fluorescence polarization competition assay for tyrosine kinases, Anal. Biochem. 255 (1998) 257–262. [8] Y.-W. Park, R.T. Cummings, L. Wu, S. Zheng, P.M. Cameron, A. Woods, D.M. Zaller, A.I. Marcy, J.D. Hermes, Homogeneous proximity tyrosine kinase assays: scintillation proximity assay versus homogeneous time-resolved fluorescence, Anal. Biochem. 289 (1999) 94–104. [9] A.J. Pope, U.M. Haupts, K.J. Moore, Homogeneous fluorescence readouts for miniaturized high-throughput screening: theory and practice, Drug Discovery Today 4 (1999) 350–362.
91
[10] T.C. Turek, E.C. Small, R.W. Bryant, W.A.G. Hill, Development and validation of a competitive AKT serine/threonine kinase fluorescence polarization assay using a product-specific anti-phospho-serine antibody, Anal. Biochem. 299 (2001) 45–53. [11] J.J. Wu, D.R. Yarwood, Q. Pham, M.A. Sills, Identification of a high-affinity anti-phosphoserine antibody for development of a homogeneous fluorescence polarization assay for protein kinase C, J. Biomol. Screen. 5 (2000) 23–30. [12] S. Jeong, T.T. Nikiforov, A kinase assay based on thiophosphorylation and biotinylation, Biotechniques 27 (1999) 1232– 1238. [13] J. Coffin, M. Latev, X. Bi, T.T. Nikiforov, Detection of phosphopeptides by fluorescence polarization in the presence of cationic polyamino acids: application to kinase assays, Anal. Biochem. 278 (2000) 206–212. [14] A. Simeonov, X. Bi, T.T. Nikiforov, Enzyme assays by fluorescence polarization in the presence of polyarginine: study for kinase, phosphatase, and protease reactions, Anal. Biochem. 304 (2002) 193–199. [15] W. Huang, Y. Zhang, J.R. Sportsman, A fluorescence polarization assay for cyclic nucleotide phosphodiesterases, J. Biomol. Screen. 7 (2002) 215–222. [16] L. Andersson, J. Porath, Isolation of phosphoproteins by immobilized metal (Fe3þ ) affinity chromatography, Anal. Biochem. 154 (1986) 250–254. [17] S. Li, C. Dass, Iron(III)-immobilized metal ion affinity chromatography and mass spectrometry for the purification and characterization of synthetic phosphopeptides, Anal. Biochem. 270 (1999) 9–14. [18] C.B. Cohen, E. Chin-Dixon, S. Jeong, T.T. Niliforov, A microchip-based enzyme assay for protein kinase A, Anal. Biochem. 273 (1999) 89–97. [19] C. House, B.E. Kemp, Protein kinase C contains a pseudosubstrate prototype in its regulatory domain, Science 238 (1987) 1726–1728. [20] B.E. Kemp, M.G. Clark, Adrenergic control of the cyclic AMP-dependent protein kinase and pyruvate kinase in isolated hepatocytes. Application of a synthetic peptide substrate for measuring protein kinase activity, J. Biol. Chem. 253 (1978) 5147–5154.