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In vitro protein kinase activity measurement by flow cytometry
Analytical Biochemistry 383 (2008) 180–185
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
In vitro protein kinase activity measurement by flow cytometry Donald J. Bernsteel a, David L. Roman a, Richard R. Neubig a,b,* a b
Department of Pharmacology, University of Michigan, 1301 MSRB III, Ann Arbor, MI 48109, USA Department of Internal Medicine (Cardiovascular Medicine), University of Michigan, Ann Arbor, MI 48109, USA
a r t i c l e
i n f o
Article history: Received 6 May 2008 Available online 3 September 2008 Keywords: Akt Protein kinase A Protein kinase C Flow cytometry Kinase Phosphorylation Detection
a b s t r a c t Protein kinases are important drug targets, and a wide variety of methods have been developed for assessing their activity. A key element in developing selective kinase inhibitors is the ability to rapidly compare the effects of an inhibitor on several related or unrelated kinases. We describe a simple, nonradioactive, bead-based method for detecting kinase activity in vitro. Biotinylated peptide substrates are immobilized on beads and phosphorylation is detected with anti-phosphopeptide antibodies with no separation steps required. Phosphorylation is dependent on the amount of kinase in the assay and can be inhibited by known kinase inhibitors in a concentration-dependent manner. Using Luminex technology, we measured the activity of three kinases (PKA, PKC-l, and Akt) on multiple substrates simultaneously. We also discuss conditions necessary to optimize measurement of the activity of several kinases in a single sample. Ó 2008 Elsevier Inc. All rights reserved.
Protein kinases play a major role in both signal transduction and oncogenesis [1,2]. Given the importance of protein kinases as therapeutic drug targets [3], a large number of methods have been developed to assess protein kinase activity. In addition to biochemical measures of phosphate incorporation using [32P]ATP as a substrate, a number of high-throughput methods generally using antibody detection of phosphopeptide products have been perfected [4]. Most of these either use washing steps (enzyme-linked immunosorbent assay [ELISA])1 or require advanced plate reader capabilities such as time-resolved fluorescence resonance energy transfer (TR–FRET) or AlphaScreen technology. Early efforts in kinase inhibitor development aimed at complete specificity for only the target kinase. Recently, interest has shifted to inhibitors with an appropriate pattern of kinase inhibition that includes several kinases as the target [3]. Regardless of the drug development philosophy, understanding the actions of kinase inhibitors at a number of protein kinases is essential to fully evaluate the nature of the compound. Toward that goal, a number of groups have proposed approaches to multiplex the measurements of protein kinases and the assessment of their inhibitors [5–7].
* Corresponding author. Address: Department of Pharmacology, University of Michigan, Ann Arbor, MI 48109, USA. Fax: +1 734 763 4450. E-mail address: [email protected] (R.R. Neubig). 1 Abbreviations used: ELISA, enzyme-linked immunosorbent assay; TR–FRET, timeresolved fluorescence resonance energy transfer; PE, phycoerythrin; IgG, immunoglobulin G; PMA, phorbol-12-myristate-13-acetate; BCB, bead-coupling buffer; BSA, bovine serum albumin; RT, room temperature; EDTA, ethylenediaminetetraacetic acid; EGTA, ethyleneglycoltetraacetic acid; TPA, 12-O-tetradecanoyl-phorbol-13acetate. 0003-2697/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2008.08.026
In this article, we describe a simple method for measuring the activity of several protein kinases using a ‘‘mix and read” assay that uses flow cytometry to detect the production of phosphopeptides. The ability of flow cytometry to detect the accumulation of a fluorescent probe on a bead without physically separating bound and free probes provides a powerful approach to detecting molecular interactions (Fig. 1) [8]. Furthermore, advances in sample handling in flow cytometry now provide access to 96- and 384-well plate formats [9], and a novel high-throughput flow cytometry system was also reported recently [10]. The Luminex platform for multiplexed immunochemical analysis is one such 96-well cytometry system for analysis of bead-based antigens. We use that system to efficiently detect the in vitro phosphorylation of biotinylated substrate peptides in both single- and multiplexed samples. We describe the use of this method with three kinases (PKA, PKC-l, and Akt) and demonstrate kinase-dependent phosphorylation and its inhibition by a known kinase inhibitor. A preliminary report of this method with ROCK2 kinase was included in the online supplement of a recent article [11]. Conditions to permit true multiplexing of multiple kinases with this method are discussed.
Materials and methods Materials Avidin-coated microspheres (LumAvidin beads) were purchased from Luminex (Austin, TX, USA). PKA catalytic subunit from bovine heart was purchased from Sigma–Aldrich (cat. no. P2645, St. Louis, MO, USA). PKC-l and AKT1 enzymes were obtained from
Protein kinase activity measurement by flow cytometry / D.J. Bernsteel et al. / Anal. Biochem. 383 (2008) 180–185
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PKA assay The reaction buffer consisted of 20 mM Hepes (pH 7.4), 2 mM MgCl2, 0.5 mM ATP, 100 mM KCl, and 0.2 mM dithiothreitol. PKA catalytic subunit was resuspended in 25 mM Tris–HCl (pH 7.4), 0.5 mM ethylenediaminetetraacetic acid (EDTA), 0.5 mM ethyleneglycoltetraacetic acid (EGTA), 150 mM NaCl, 10 mM b-mercaptoethanol, and Complete Mini protease inhibitor (Roche Diagnostics, Mannheim, Germany) prior to initiation of the kinase reaction.
Fig. 1. Illustration of the kinase assay detection scheme. A biotinylated peptide is coupled to a LumAvidin microsphere through the high-affinity biotin–avidin interaction. The microspheres are then incubated with kinase, and the phosphopeptide is recognized by an anti-phospho-Ser primary antibody. A secondary antibody conjugated to PE is then added, and the sample is aspirated into a Luminex 200, where bead-associated fluorescence is measured.
Cell Signaling Technology (cat. nos. 7602 and 7500, respectively, Boston, MA, USA). Rabbit phospho-(Ser/Thr) PKA substrate antibody was purchased from Cell Signaling Technology (cat. no. 9621). R-PE (phycoerythrin) goat anti-rabbit immunoglobulin G (IgG) was obtained from Invitrogen (cat. no. P2771MP, Carlsbad, CA, USA). Biotinylated substrate for PKA (biot-AGAALRRASLAG) and prephosphorylated PKA substrate (biot-AGAALRRA[pSer]LAG) were prepared by custom synthesis from Sigma–Genosys (The Woodlands, TX, USA). Biotinylated substrates for PKC-l (biotAALVRGMSVAFFFK) and Akt (biot-ARKRNERTYSFGHHA) were purchased from Anaspec (cat. nos. 29927 and 29945, respectively, San Jose, CA, USA). Peptide substrates for PKC-e, PKC-f, and PKC-h were obtained from Quality Controlled Biochemicals (Hopkinton, MA, USA). Staurosporine was obtained from Sigma–Aldrich. Phorbol12-myristate-13-acetate (PMA) was obtained from Calbiochem (San Diego, CA, USA). Preparing peptide-bound beads Beads were labeled using a protocol similar to that of Roman and coworkers [12]. Luminex LumAvidin beads ( 105 to provide 1000/well) were briefly vortexed and then added to 1 ml of bead-coupling buffer (BCB: 154 mM NaCl, 2.97 mM Na2HPO47H2O, and 1 mM KH2PO4 [pH 7.4] containing 1% bovine serum albumin [BSA]). Beads were then pelleted in a microcentrifuge (2 min at 11,000g), the supernatant was decanted, and beads were resuspended in 1 ml of BCB. This process was repeated three times. For the third spin, the beads were reconstituted in a total volume providing 40 ll of BCB for each assay well, and peptide substrate was added at 100 or 20 nM to produce a final concentration of 50 or 10 nM peptide during kinase incubations. Peptides and beads were incubated for 30 min at room temperature (RT). Following the incubation, the beads were again washed three times. In the case of multiplex kinase assays, each group of beads was washed two times separately and then combined in BCB for the final spin. Beads were then resuspended in the specific buffer depending on the kinase being measured. Kinase assays General protocol All reactions were carried out in Axygen PCR plates (cat. no. PCR-96-FS-C, Union City, CA, USA) in 80 ll with gentle shaking. PKA and PKC incubations were for 10 min at 30 °C, whereas Akt assays were for 30 min at RT. Reactions were initiated by adding 40 ll of bead–peptide mixture in reaction buffer to the wells containing 40 ll of kinase in resuspension buffer.
PKC assay The reaction buffer contained 50 mM Hepes (pH 7.0), 0.5 mM EDTA, 0.0375 (v/v) b-mercaptoethanol, 2.5 mM MgCl2, 0.25% Triton X-100, 2.5 ng/ll PMA/TPA (12-O-tetradecanoyl-phorbol-13-acetate), and 125 lM ATP. PKC-l was resuspended in reaction buffer. Akt assay The reaction buffer contained 25 mM Tris (pH 7.4), 10 mM MgCl2, 5 mM b-glycerophosphate, 2 mM dithiothreitol, 200 lM ATP, and 0.1 mM sodium orthovanadate. Akt was resuspended in reaction buffer. Staurosporine inhibition For measurements of the staurosporine effect, PKA was preincubated for 20 min at RT with the indicated concentration of inhibitor in suspension buffer before initiating the reaction with the reaction buffer and 50 nM PKA substrate on beads. Detection of phosphorylation Phosphorylation of peptides on beads was detected with the anti-phospho-(Ser/Thr) PKA substrate antibody, which recognizes phosphorylated serine or threonine in a sequence such as RXXT or RXXS. Primary antibody was diluted 1:400 in BCB, and then 10 ll was added to each well (to produce a 1:4000 final dilution). Samples were then incubated for 60 min at RT with shaking at 440 rpm. Then secondary antibody, R-PE goat anti-rabbit IgG, was added in 10 ll to provide a final dilution of 1:2000. Samples were then incubated for 30 min at RT with shaking at 440 rpm. The 96-well plates were placed in a Luminex 200 and set to read 100 events per bead region per well. Median fluorescence intensity was used to determine the amount of phosphopeptide on the beads. Data analysis Median fluorescence reported from the Luminex instrument was obtained from at least three experiments performed with duplicate samples. Enzyme concentration curves were fitted to a hyperbolic equation, B = B0 + Bmax[E/(E + E50)], using nonlinear least squares fitting in GraphPad Prism software (version 5, San Diego, CA, USA). Inhibition data were fitted using Prism to a competition equation with a variable Hill slope B = B0 + (Bmax – B0)/ (1 + 10[(LogIC50 – LogI)*nH]), where B0 and Bmax are the blank signal with no kinase and the maximum phosphorylation detected, respectively. Also, E is the enzyme concentration, E50 is the concentration that gives half-maximal phosphorylation, LogIC50 is the logarithm of the concentration of inhibitor giving half-maximal inhibition, LogI is the logarithm of the inhibitor concentration, and nH is the Hill slope of the inhibition curve. Results and discussion To establish antibody dilutions and ensure low nonspecific binding of antibody reagents to beads, we tested different dilutions of the primary anti-phospho-PKA substrate antibody against a
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prephosphorylated PKA substrate peptide. With the BCB that contains 1% BSA and a 1:2000 dilution of the PE-labeled goat anti-rabbit secondary antibody, we obtained excellent specificity of detection of phospho-PKA substrate peptide compared with unphosphorylated peptide or blank beads (Fig. 2). Background signal was determined by using unphosphorylated peptides in the absence of kinase but in the presence of all other reaction components. The intermediate dilution (1:4000) was used for all subsequent studies. The sequence specificity of this antibody is such that it detects phosphorylated peptides with RXXpS/pT sequence. Interestingly, it also is quite efficient at detecting phosphorylated substrates for PKC-l and Akt despite the fact that neither is a good substrate for PKA (see below). Surprisingly, several commercially available phospho-Ser antibodies were unable to detect the phosphorylated PKA substrate peptide. Although this would have facilitated multiplexing of a wider range of kinases, the fact that other kinase substrates contain the same sequence motif suggested that the phospho-PKA substrate antibody could possibly be used to assay other kinases. We then tested phosphorylation of the bead-bound PKA peptide substrate by incubation with purified PKA catalytic subunit. The background staining of the unphosphorylated peptide is very low (Fig. 3; see also Fig. 2). The addition of nanogram quantities of the PKA catalytic subunit in a 10-min incubation stimulated the antibody signal from phosphorylation of the PKA substrate peptide nearly 100-fold (Fig. 3A). The enzyme concentration dependence was linear to approximately 10 ng (12 times background) and then showed a simple hyperbolic increase as expected for an endpoint assay. Studies assessing the time dependence of the phosphorylation were complicated by the long antibody incubations after the kinase reaction. Preliminary attempts to stop the reaction at the end of the enzyme incubation with acidic pH buffers were unsuccessful due to loss of phosphate from the serine in those quenching buffers (data not shown). Subsequent studies made no attempts to quench the reaction. It is possible, however, that Mg2+ chelation or a broad-spectrum kinase inhibitor such as staurosporine could be used to permit time course studies. The generality of this method for other kinases was tested next. Due to the lack of effectiveness of the anti-phospho-Ser antibodies, we limited our peptide substrate choice to those containing the RXXS motif that, on phosphorylation, should be able to bind the
Fig. 3. Generality of method and concentration dependence of kinase phosphorylation. Because the general PKA substrate motif (RXXS/T) is present in substrates for several kinases, we tested PKA, PKC-l, and Akt substrate peptides for detection by this method. Reactions were carried out as described in Materials and Methods using the indicated amounts of PKA on PKA substrate (10 nM) (A), PKC-l on PKC-l substrate (10 nM) (B), and Akt on Akt substrate (50 nM) (C). Antibody concentrations were held constant at 1:4000 for primary antibody and 1:2000 for secondary antibody. MFI, median fluorescence intensity. Values are means ± standard errors of three experiments with duplicate determinations.
Fig. 2. Detection of phosphorylated peptide. Primary antibody against the phosphorylated PKA substrate sequence was tested for signal with prephosphorylated PKA substrate (PKA–PS) compared with unphosphorylated PKA substrate (PKA) and beads with no peptide bound. The secondary antibody R-PE-goat anti-rabbit (Invitrogen) was held constant at a 1:2000 dilution. MFI, median fluorescence intensity. Values are means ± standard errors of three experiments with duplicate determinations.
phospho-PKA substrate antibody. Both PKC and Akt show efficient phosphorylation of those peptides as detected by the antibody. Fig. 3B and C show efficient phosphorylation of cognate substrates by PKC-l and Akt1, with reactions linear to 5 and 10 ng and five and four times background, respectively. As with PKA, a hyperbolic enzyme concentration dependence was seen at higher levels for both kinases. The signal/background was not quite as high for these substrates as for the PKA substrate peptide, but very reasonable signals were obtained for both PKC-l and Akt1. A goal of this project was to evaluate this method for potential multiplex kinase assays in which multiple kinases are mea-
Protein kinase activity measurement by flow cytometry / D.J. Bernsteel et al. / Anal. Biochem. 383 (2008) 180–185
sured simultaneously. By use of the uniquely tagged avidincoated LumAvidin beads, we tested the activity of each of the three kinases on all three substrates simultaneously. To do this, each peptide was bound to an identifiable bead, and after two washes to eliminate free peptide the three bead sets (each with a different peptide substrate) were combined. In separate incubations, each kinase was incubated with the mixture of the three substrate beads. As shown in Fig. 4, PKA showed a very high degree of substrate specificity, with only the PKA substrate showing any detectable signal. In contrast, PKC-l and Akt1 showed phosphorylation of the PKA substrate peptide that was as good as, or better than, that for their own substrates. The strong sig-
Fig. 4. Multiplex analysis of substrate specificity. Each enzyme—PKA (50 ng/ reaction) (A), PKC-l (20 ng/reaction) (B), and AKT (20 ng/reaction) (C)—was incubated with the three substrates simultaneously on distinguishable beads to rapidly assess the substrate specificity of the kinases. Phosphorylation of each substrate was determined separately by reading distinct bead regions in the Luminex cytometer. Blanks are similar to singleplex blanks. Once again, 1:4000 primary and 1:2000 secondary antibody concentrations were used. MFI, median fluorescence intensity. Values are means ± standard errors of three experiments with duplicate determinations.
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nal for the PKA substrate with PKC-l may be due in part to better detection of the phosphorylated PKA substrate peptide by the antibody. However, a significant signal is detected for each of the peptides with Akt1 and especially PKC-l. The fluorescence seen for the different substrates is not due to leaching of the PKA peptide from its bead to the other beads given that the PKA enzyme gives a very clear and specific phosphorylation of the PKA substrate without any detectable increase with the other substrate peptides. Furthermore, the PKC-l and Akt are definitely able to give a detectable signal with their own substrate peptides, as shown in Fig. 3. It may be possible to improve the specificity of PKC and Akt for their cognate substrate by reducing the amount of enzyme, but multiplexing of kinases with more different substrate specificities would likely be even more effective (as discussed below). Thus, we are able to assess phosphorylation of several substrate peptides by a panel of kinases (each introduced individually) using this multiplexed in vitro kinase assay. One key use of kinase assays is to assess the activity of kinase inhibitors. To determine whether our method can be used for that purpose, we measured the IC50 of the general kinase inhibitor staurosporine on the activity of PKA. Fig. 5 shows the inhibition of PKA activity by staurosporine. The data were fit well to a curve with an IC50 of 0.85 nM and an nH of 0.5. Thus, kinase inhibitors can be easily assessed with this method. In a recent online supplement, we also recently showed inhibition of ROCK2 kinase by the known ROCK inhibitor Y-27632 [11]. To create a true multiplexed kinase assay, one would need to be able to generate a specific signal on each peptide by its kinase and only its kinase. Given our current requirement for the RXXS sequence driven by the detection antibody, it is not surprising that there was overlap in the activity of the different kinases on some substrates (i.e., PKC-l and Akt on the PKA substrate and PKC-l on the Akt substrate and vice versa). This problem could be ameliorated by using a mixture of different antibodies that are selective for individual substrate peptides. This would permit the choice of more orthogonal peptide substrates and distinct kinases that would not have significant activity at the noncognate substrate. A somewhat similar approach was published recently by Shults and coworkers [7] using an Illumina detection system to explore activity at 900 different
Fig. 5. Staurosporine inhibition of PKA. Varying concentrations of staurosporine were incubated with PKA (10 ng/reaction) for 20 min in kinase incubation buffer prior to initiating the kinase reaction with PKA substrate (10.5 nM) in PKA reaction buffer. Antibody concentrations were the same as in Figs. 3 and 4. Values are means ± standard errors of three experiments with duplicate determinations. The curve is a nonlinear least squares fit to a competitive inhibition curve with a variable Hill slope. MFI, median fluorescence intensity.
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substrates. They showed sets of 2- and 4-enzyme multiplex analyses in that system. Using the substrates and kinases described in their report, a similar multiplex analysis could be set up with the more accessible flow cytometry multiplexing assay that we describe here. The Luminex system can easily multiplex 10 to perhaps 30 substrates. Using a standard flow cytometer with bead sets having different intensities of labeling (e.g., Spherotech Blue Pack), up to 5 or perhaps 10 different substrates could be analyzed in multiplex. In addition to identifying appropriate sets of kinases and substrates to permit specificity of the reactions, one would also need to optimize assay conditions to ensure good activity of each kinase in the set. Work on that problem has already been done in other studies [7]. This use of nucleotidetagged substrates with Illumina detection provides high-level multiplexing but not high-throughput sample handling. Several other ‘‘multiplexed” kinase assays use parallel nondifferentiable readouts (e.g., [32P] incorporation or a single fluorophore) that cannot separately detect multiple kinases in a single incubation sample [5,6]. Comparison of the current approach with the many other available kinase assays is warranted. The ‘‘gold standard” method is to measure incorporation of [32P]phosphate into proteins from a gamma [32P]ATP substrate donor. This requires physical separation of labeled substrate from unlabeled substrate and some form of radioactive detection such as autoradiography or scintillation counting. In general, these are not high-throughput but can be used with scintillation proximity detection for highthroughput studies. Common immunochemical methods include the use of Western blot or ELISA to detect phosphorylated substrates. The former is optimal with large protein substrates, whereas the latter is applicable for both protein and peptide substrates. Many of the considerations for those two standard approaches are also evident in our current method. One needs to pick an appropriate substrate/antibody combination and kinase incubation conditions. They differ in their need for separation and washing steps, adding significantly to the complexity of the method. Our bead-based approach did not require any separation steps and yielded (at least for the optimized PKA substrate) outstanding signal/background. Furthermore, one could easily immobilize complex substrates on beads either with biochemically purified and biotin-tagged proteins or by capturing the substrate protein on the bead from complex mixtures by the use of a specific antibody. Many true high-throughput methods have also been developed, including LANCE, which is a TR–FRET method using a Eu-chelate donor anti-phospho-antibody and an APC-labeled acceptor peptide. Similarly, AlphaScreen uses the proximity of donor and acceptor beads that are conjugated to either a substrate peptide or an anti-phospho-antibody. Both of these methods use the same principle as our flow cytometry approach—detecting the accumulation of a complex of phosphorylated peptide substrate and an antiphospho-antibody. Both also require complex plate reader optics permitting time-resolved reading or 620-nm excitation and 520nm emission from the O2-sensitized acceptor bead. Any application developed for those methods could be adapted to our flow cytometry detection. Given the increasing availability of flow cytometry and plate sampling front-end systems (including Luminex, BD, and Guava), the flow cytometry kinase method could be simply implemented outside of screening centers. More recently, a number of methods have been developed using kinase- and substrate-dependent ATP depletion with luciferase detection of ATP (e.g., Kinase-Glo) or production of ADP (e.g., ADPHunter). None of the methods described here, however, is easily adapted to multiplex measurements, as is our detection method based on beads and flow cytometry.
The quality of signal, in terms of both magnitude and wellto-well consistency, is important when considering an assay for a high-throughput screen. The standard measure of assay quality is the Z factor, a measure calculated from the following rpþrnÞ , where r denotes the standard deviaequation: Z ¼ 1 3ð jlplnj tions of the positive and negative controls (rp and rn, respectively) and l indicates the mean values of the positive and negative controls. Using control values from our PKA experiments as shown in Fig. 5 (100 nM staurosporine and no staurosporine) from four individual dose–response experiments with values determined in triplicate yields a range of Z factors (calculated individually) of 0.7–0.8, comfortably within the range of 0.5–1 that indicates an excellent assay where positive and negative control values are well separated (good signal strength) and highly reproducible (low standard deviation values). Therefore, this assay paradigm should be suitable for high-throughput screening application. Finally, the question of throughput is important. In general, flow cytometry is not considered to be a high-throughput method. However, the advent of plate reading cytometers is changing that view. The Luminex 200 can read a 96-well plate in 30 min; this permits analysis of more than 1000 samples per day and could yield 5000 data points per day if a 5-kinase multiplex analysis were implemented. Using the HyperCyt high-throughput flow cytometry front-end (IntelliCyt, Albuquerque, NM, USA), which can run a 384-well plate in 10–15 min, more than 10,000 samples per day should be possible—with additional information obtained by multiplexing. In summary, we have described a simple bead-based flow cytometry method to assess in vitro activity of several protein kinases. The method is easily generalizable, and optimization that has been done for other methods based on substrate peptides and antibodies (e.g., ELISA, LANCE, AlphaScreen) could be applied with this detection methodology as well. A key advantage of the method is the potential for multiplexing that has been shown clearly in the immunoassay field with the use of Luminex and other flow cytometry bead detection systems. Acknowledgments This research was supported by the Biomedical Interactions Technology Center (University of New Hampshire and National Science Foundation [NSF]) and a gift of equipment and supplies from Luminex Corporation. The authors thank Susan Wade for assistance with the manuscript. References [1] P. Blume-Jensen, T. Hunter, Oncogenic kinase signaling, Nature 411 (2001) 355–365. [2] G. Manning, D.B. Whyte, R. Martinez, T. Hunter, S. Sudarsanam, The protein kinase complement of the human genome, Science 298 (2002) 1912–1934. [3] J. Dancey, E.A. Sausville, Issues and progress with protein kinase inhibitors for cancer treatment, Nat. Rev. Drug Discov. 2 (2003) 296–313. [4] L.E. DeForge, A.G. Cochran, S.H. Yeh, B.S. Robinson, K.L. Billeci, W.L. Wong, Substrate capacity considerations in developing kinase assays, Assay Drug Dev. Technol. 2 (2004) 131–140. [5] K.A. Janes, J.G. Albeck, L.X. Peng, P.K. Sorger, D.A. Lauffenburger, M.B. Yaffe, A high-throughput quantitative multiplex kinase assay for monitoring information flow in signaling networks: application to sepsis–apoptosis, Mol. Cell. Proteomics 2 (2003) 463–473. [6] M.D. Shults, K.A. Janes, D.A. Lauffenburger, B. Imperiali, A multiplexed homogeneous fluorescence-based assay for protein kinase activity in cell lysates, Nat. Methods 2 (2005) 277–283. [7] M.D. Shults, I.A. Kozlov, N. Nelson, B.G. Kermani, P.C. Melnyk, V. Shevchenko, A. Srinivasan, J. Musmacker, J.P. Hachmann, D.L. Barker, M. Lebl, C. Zhao, A multiplexed protein kinase assay, ChemBioChem 8 (2007) 933–942. [8] L.A. Sklar, B.S. Edwards, S.W. Graves, J.P. Nolan, E.R. Prossnitz, Flow cytometric analysis of ligand–receptor interactions and molecular assemblies, Annu. Rev. Biophys. Biomol. Struct. 31 (2002) 97–119.
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[11] C.R. Evelyn, S.M. Wade, Q. Wang, M. Wu, J.A. Iniguez-Lluhi, S.D. Merajver, R.R. Neubig, CCG-1423: a small-molecule inhibitor of RhoA transcriptional signaling, Mol. Cancer Ther. 6 (2007) 2249–2260. [12] D.L. Roman, J.N. Talbot, R.A. Roof, R.K. Sunahara, J.R. Traynor, R.R. Neubig, Identification of small-molecule inhibitors of RGS4 using a high-throughput flow cytometry protein interaction assay, Mol. Pharmacol. 71 (2007) 169–175.
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
In vitro protein kinase activity measurement by flow cytometry Donald J. Bernsteel a, David L. Roman a, Richard R. Neubig a,b,* a b
Department of Pharmacology, University of Michigan, 1301 MSRB III, Ann Arbor, MI 48109, USA Department of Internal Medicine (Cardiovascular Medicine), University of Michigan, Ann Arbor, MI 48109, USA
a r t i c l e
i n f o
Article history: Received 6 May 2008 Available online 3 September 2008 Keywords: Akt Protein kinase A Protein kinase C Flow cytometry Kinase Phosphorylation Detection
a b s t r a c t Protein kinases are important drug targets, and a wide variety of methods have been developed for assessing their activity. A key element in developing selective kinase inhibitors is the ability to rapidly compare the effects of an inhibitor on several related or unrelated kinases. We describe a simple, nonradioactive, bead-based method for detecting kinase activity in vitro. Biotinylated peptide substrates are immobilized on beads and phosphorylation is detected with anti-phosphopeptide antibodies with no separation steps required. Phosphorylation is dependent on the amount of kinase in the assay and can be inhibited by known kinase inhibitors in a concentration-dependent manner. Using Luminex technology, we measured the activity of three kinases (PKA, PKC-l, and Akt) on multiple substrates simultaneously. We also discuss conditions necessary to optimize measurement of the activity of several kinases in a single sample. Ó 2008 Elsevier Inc. All rights reserved.
Protein kinases play a major role in both signal transduction and oncogenesis [1,2]. Given the importance of protein kinases as therapeutic drug targets [3], a large number of methods have been developed to assess protein kinase activity. In addition to biochemical measures of phosphate incorporation using [32P]ATP as a substrate, a number of high-throughput methods generally using antibody detection of phosphopeptide products have been perfected [4]. Most of these either use washing steps (enzyme-linked immunosorbent assay [ELISA])1 or require advanced plate reader capabilities such as time-resolved fluorescence resonance energy transfer (TR–FRET) or AlphaScreen technology. Early efforts in kinase inhibitor development aimed at complete specificity for only the target kinase. Recently, interest has shifted to inhibitors with an appropriate pattern of kinase inhibition that includes several kinases as the target [3]. Regardless of the drug development philosophy, understanding the actions of kinase inhibitors at a number of protein kinases is essential to fully evaluate the nature of the compound. Toward that goal, a number of groups have proposed approaches to multiplex the measurements of protein kinases and the assessment of their inhibitors [5–7].
* Corresponding author. Address: Department of Pharmacology, University of Michigan, Ann Arbor, MI 48109, USA. Fax: +1 734 763 4450. E-mail address: [email protected] (R.R. Neubig). 1 Abbreviations used: ELISA, enzyme-linked immunosorbent assay; TR–FRET, timeresolved fluorescence resonance energy transfer; PE, phycoerythrin; IgG, immunoglobulin G; PMA, phorbol-12-myristate-13-acetate; BCB, bead-coupling buffer; BSA, bovine serum albumin; RT, room temperature; EDTA, ethylenediaminetetraacetic acid; EGTA, ethyleneglycoltetraacetic acid; TPA, 12-O-tetradecanoyl-phorbol-13acetate. 0003-2697/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2008.08.026
In this article, we describe a simple method for measuring the activity of several protein kinases using a ‘‘mix and read” assay that uses flow cytometry to detect the production of phosphopeptides. The ability of flow cytometry to detect the accumulation of a fluorescent probe on a bead without physically separating bound and free probes provides a powerful approach to detecting molecular interactions (Fig. 1) [8]. Furthermore, advances in sample handling in flow cytometry now provide access to 96- and 384-well plate formats [9], and a novel high-throughput flow cytometry system was also reported recently [10]. The Luminex platform for multiplexed immunochemical analysis is one such 96-well cytometry system for analysis of bead-based antigens. We use that system to efficiently detect the in vitro phosphorylation of biotinylated substrate peptides in both single- and multiplexed samples. We describe the use of this method with three kinases (PKA, PKC-l, and Akt) and demonstrate kinase-dependent phosphorylation and its inhibition by a known kinase inhibitor. A preliminary report of this method with ROCK2 kinase was included in the online supplement of a recent article [11]. Conditions to permit true multiplexing of multiple kinases with this method are discussed.
Materials and methods Materials Avidin-coated microspheres (LumAvidin beads) were purchased from Luminex (Austin, TX, USA). PKA catalytic subunit from bovine heart was purchased from Sigma–Aldrich (cat. no. P2645, St. Louis, MO, USA). PKC-l and AKT1 enzymes were obtained from
Protein kinase activity measurement by flow cytometry / D.J. Bernsteel et al. / Anal. Biochem. 383 (2008) 180–185
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PKA assay The reaction buffer consisted of 20 mM Hepes (pH 7.4), 2 mM MgCl2, 0.5 mM ATP, 100 mM KCl, and 0.2 mM dithiothreitol. PKA catalytic subunit was resuspended in 25 mM Tris–HCl (pH 7.4), 0.5 mM ethylenediaminetetraacetic acid (EDTA), 0.5 mM ethyleneglycoltetraacetic acid (EGTA), 150 mM NaCl, 10 mM b-mercaptoethanol, and Complete Mini protease inhibitor (Roche Diagnostics, Mannheim, Germany) prior to initiation of the kinase reaction.
Fig. 1. Illustration of the kinase assay detection scheme. A biotinylated peptide is coupled to a LumAvidin microsphere through the high-affinity biotin–avidin interaction. The microspheres are then incubated with kinase, and the phosphopeptide is recognized by an anti-phospho-Ser primary antibody. A secondary antibody conjugated to PE is then added, and the sample is aspirated into a Luminex 200, where bead-associated fluorescence is measured.
Cell Signaling Technology (cat. nos. 7602 and 7500, respectively, Boston, MA, USA). Rabbit phospho-(Ser/Thr) PKA substrate antibody was purchased from Cell Signaling Technology (cat. no. 9621). R-PE (phycoerythrin) goat anti-rabbit immunoglobulin G (IgG) was obtained from Invitrogen (cat. no. P2771MP, Carlsbad, CA, USA). Biotinylated substrate for PKA (biot-AGAALRRASLAG) and prephosphorylated PKA substrate (biot-AGAALRRA[pSer]LAG) were prepared by custom synthesis from Sigma–Genosys (The Woodlands, TX, USA). Biotinylated substrates for PKC-l (biotAALVRGMSVAFFFK) and Akt (biot-ARKRNERTYSFGHHA) were purchased from Anaspec (cat. nos. 29927 and 29945, respectively, San Jose, CA, USA). Peptide substrates for PKC-e, PKC-f, and PKC-h were obtained from Quality Controlled Biochemicals (Hopkinton, MA, USA). Staurosporine was obtained from Sigma–Aldrich. Phorbol12-myristate-13-acetate (PMA) was obtained from Calbiochem (San Diego, CA, USA). Preparing peptide-bound beads Beads were labeled using a protocol similar to that of Roman and coworkers [12]. Luminex LumAvidin beads ( 105 to provide 1000/well) were briefly vortexed and then added to 1 ml of bead-coupling buffer (BCB: 154 mM NaCl, 2.97 mM Na2HPO47H2O, and 1 mM KH2PO4 [pH 7.4] containing 1% bovine serum albumin [BSA]). Beads were then pelleted in a microcentrifuge (2 min at 11,000g), the supernatant was decanted, and beads were resuspended in 1 ml of BCB. This process was repeated three times. For the third spin, the beads were reconstituted in a total volume providing 40 ll of BCB for each assay well, and peptide substrate was added at 100 or 20 nM to produce a final concentration of 50 or 10 nM peptide during kinase incubations. Peptides and beads were incubated for 30 min at room temperature (RT). Following the incubation, the beads were again washed three times. In the case of multiplex kinase assays, each group of beads was washed two times separately and then combined in BCB for the final spin. Beads were then resuspended in the specific buffer depending on the kinase being measured. Kinase assays General protocol All reactions were carried out in Axygen PCR plates (cat. no. PCR-96-FS-C, Union City, CA, USA) in 80 ll with gentle shaking. PKA and PKC incubations were for 10 min at 30 °C, whereas Akt assays were for 30 min at RT. Reactions were initiated by adding 40 ll of bead–peptide mixture in reaction buffer to the wells containing 40 ll of kinase in resuspension buffer.
PKC assay The reaction buffer contained 50 mM Hepes (pH 7.0), 0.5 mM EDTA, 0.0375 (v/v) b-mercaptoethanol, 2.5 mM MgCl2, 0.25% Triton X-100, 2.5 ng/ll PMA/TPA (12-O-tetradecanoyl-phorbol-13-acetate), and 125 lM ATP. PKC-l was resuspended in reaction buffer. Akt assay The reaction buffer contained 25 mM Tris (pH 7.4), 10 mM MgCl2, 5 mM b-glycerophosphate, 2 mM dithiothreitol, 200 lM ATP, and 0.1 mM sodium orthovanadate. Akt was resuspended in reaction buffer. Staurosporine inhibition For measurements of the staurosporine effect, PKA was preincubated for 20 min at RT with the indicated concentration of inhibitor in suspension buffer before initiating the reaction with the reaction buffer and 50 nM PKA substrate on beads. Detection of phosphorylation Phosphorylation of peptides on beads was detected with the anti-phospho-(Ser/Thr) PKA substrate antibody, which recognizes phosphorylated serine or threonine in a sequence such as RXXT or RXXS. Primary antibody was diluted 1:400 in BCB, and then 10 ll was added to each well (to produce a 1:4000 final dilution). Samples were then incubated for 60 min at RT with shaking at 440 rpm. Then secondary antibody, R-PE goat anti-rabbit IgG, was added in 10 ll to provide a final dilution of 1:2000. Samples were then incubated for 30 min at RT with shaking at 440 rpm. The 96-well plates were placed in a Luminex 200 and set to read 100 events per bead region per well. Median fluorescence intensity was used to determine the amount of phosphopeptide on the beads. Data analysis Median fluorescence reported from the Luminex instrument was obtained from at least three experiments performed with duplicate samples. Enzyme concentration curves were fitted to a hyperbolic equation, B = B0 + Bmax[E/(E + E50)], using nonlinear least squares fitting in GraphPad Prism software (version 5, San Diego, CA, USA). Inhibition data were fitted using Prism to a competition equation with a variable Hill slope B = B0 + (Bmax – B0)/ (1 + 10[(LogIC50 – LogI)*nH]), where B0 and Bmax are the blank signal with no kinase and the maximum phosphorylation detected, respectively. Also, E is the enzyme concentration, E50 is the concentration that gives half-maximal phosphorylation, LogIC50 is the logarithm of the concentration of inhibitor giving half-maximal inhibition, LogI is the logarithm of the inhibitor concentration, and nH is the Hill slope of the inhibition curve. Results and discussion To establish antibody dilutions and ensure low nonspecific binding of antibody reagents to beads, we tested different dilutions of the primary anti-phospho-PKA substrate antibody against a
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prephosphorylated PKA substrate peptide. With the BCB that contains 1% BSA and a 1:2000 dilution of the PE-labeled goat anti-rabbit secondary antibody, we obtained excellent specificity of detection of phospho-PKA substrate peptide compared with unphosphorylated peptide or blank beads (Fig. 2). Background signal was determined by using unphosphorylated peptides in the absence of kinase but in the presence of all other reaction components. The intermediate dilution (1:4000) was used for all subsequent studies. The sequence specificity of this antibody is such that it detects phosphorylated peptides with RXXpS/pT sequence. Interestingly, it also is quite efficient at detecting phosphorylated substrates for PKC-l and Akt despite the fact that neither is a good substrate for PKA (see below). Surprisingly, several commercially available phospho-Ser antibodies were unable to detect the phosphorylated PKA substrate peptide. Although this would have facilitated multiplexing of a wider range of kinases, the fact that other kinase substrates contain the same sequence motif suggested that the phospho-PKA substrate antibody could possibly be used to assay other kinases. We then tested phosphorylation of the bead-bound PKA peptide substrate by incubation with purified PKA catalytic subunit. The background staining of the unphosphorylated peptide is very low (Fig. 3; see also Fig. 2). The addition of nanogram quantities of the PKA catalytic subunit in a 10-min incubation stimulated the antibody signal from phosphorylation of the PKA substrate peptide nearly 100-fold (Fig. 3A). The enzyme concentration dependence was linear to approximately 10 ng (12 times background) and then showed a simple hyperbolic increase as expected for an endpoint assay. Studies assessing the time dependence of the phosphorylation were complicated by the long antibody incubations after the kinase reaction. Preliminary attempts to stop the reaction at the end of the enzyme incubation with acidic pH buffers were unsuccessful due to loss of phosphate from the serine in those quenching buffers (data not shown). Subsequent studies made no attempts to quench the reaction. It is possible, however, that Mg2+ chelation or a broad-spectrum kinase inhibitor such as staurosporine could be used to permit time course studies. The generality of this method for other kinases was tested next. Due to the lack of effectiveness of the anti-phospho-Ser antibodies, we limited our peptide substrate choice to those containing the RXXS motif that, on phosphorylation, should be able to bind the
Fig. 3. Generality of method and concentration dependence of kinase phosphorylation. Because the general PKA substrate motif (RXXS/T) is present in substrates for several kinases, we tested PKA, PKC-l, and Akt substrate peptides for detection by this method. Reactions were carried out as described in Materials and Methods using the indicated amounts of PKA on PKA substrate (10 nM) (A), PKC-l on PKC-l substrate (10 nM) (B), and Akt on Akt substrate (50 nM) (C). Antibody concentrations were held constant at 1:4000 for primary antibody and 1:2000 for secondary antibody. MFI, median fluorescence intensity. Values are means ± standard errors of three experiments with duplicate determinations.
Fig. 2. Detection of phosphorylated peptide. Primary antibody against the phosphorylated PKA substrate sequence was tested for signal with prephosphorylated PKA substrate (PKA–PS) compared with unphosphorylated PKA substrate (PKA) and beads with no peptide bound. The secondary antibody R-PE-goat anti-rabbit (Invitrogen) was held constant at a 1:2000 dilution. MFI, median fluorescence intensity. Values are means ± standard errors of three experiments with duplicate determinations.
phospho-PKA substrate antibody. Both PKC and Akt show efficient phosphorylation of those peptides as detected by the antibody. Fig. 3B and C show efficient phosphorylation of cognate substrates by PKC-l and Akt1, with reactions linear to 5 and 10 ng and five and four times background, respectively. As with PKA, a hyperbolic enzyme concentration dependence was seen at higher levels for both kinases. The signal/background was not quite as high for these substrates as for the PKA substrate peptide, but very reasonable signals were obtained for both PKC-l and Akt1. A goal of this project was to evaluate this method for potential multiplex kinase assays in which multiple kinases are mea-
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sured simultaneously. By use of the uniquely tagged avidincoated LumAvidin beads, we tested the activity of each of the three kinases on all three substrates simultaneously. To do this, each peptide was bound to an identifiable bead, and after two washes to eliminate free peptide the three bead sets (each with a different peptide substrate) were combined. In separate incubations, each kinase was incubated with the mixture of the three substrate beads. As shown in Fig. 4, PKA showed a very high degree of substrate specificity, with only the PKA substrate showing any detectable signal. In contrast, PKC-l and Akt1 showed phosphorylation of the PKA substrate peptide that was as good as, or better than, that for their own substrates. The strong sig-
Fig. 4. Multiplex analysis of substrate specificity. Each enzyme—PKA (50 ng/ reaction) (A), PKC-l (20 ng/reaction) (B), and AKT (20 ng/reaction) (C)—was incubated with the three substrates simultaneously on distinguishable beads to rapidly assess the substrate specificity of the kinases. Phosphorylation of each substrate was determined separately by reading distinct bead regions in the Luminex cytometer. Blanks are similar to singleplex blanks. Once again, 1:4000 primary and 1:2000 secondary antibody concentrations were used. MFI, median fluorescence intensity. Values are means ± standard errors of three experiments with duplicate determinations.
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nal for the PKA substrate with PKC-l may be due in part to better detection of the phosphorylated PKA substrate peptide by the antibody. However, a significant signal is detected for each of the peptides with Akt1 and especially PKC-l. The fluorescence seen for the different substrates is not due to leaching of the PKA peptide from its bead to the other beads given that the PKA enzyme gives a very clear and specific phosphorylation of the PKA substrate without any detectable increase with the other substrate peptides. Furthermore, the PKC-l and Akt are definitely able to give a detectable signal with their own substrate peptides, as shown in Fig. 3. It may be possible to improve the specificity of PKC and Akt for their cognate substrate by reducing the amount of enzyme, but multiplexing of kinases with more different substrate specificities would likely be even more effective (as discussed below). Thus, we are able to assess phosphorylation of several substrate peptides by a panel of kinases (each introduced individually) using this multiplexed in vitro kinase assay. One key use of kinase assays is to assess the activity of kinase inhibitors. To determine whether our method can be used for that purpose, we measured the IC50 of the general kinase inhibitor staurosporine on the activity of PKA. Fig. 5 shows the inhibition of PKA activity by staurosporine. The data were fit well to a curve with an IC50 of 0.85 nM and an nH of 0.5. Thus, kinase inhibitors can be easily assessed with this method. In a recent online supplement, we also recently showed inhibition of ROCK2 kinase by the known ROCK inhibitor Y-27632 [11]. To create a true multiplexed kinase assay, one would need to be able to generate a specific signal on each peptide by its kinase and only its kinase. Given our current requirement for the RXXS sequence driven by the detection antibody, it is not surprising that there was overlap in the activity of the different kinases on some substrates (i.e., PKC-l and Akt on the PKA substrate and PKC-l on the Akt substrate and vice versa). This problem could be ameliorated by using a mixture of different antibodies that are selective for individual substrate peptides. This would permit the choice of more orthogonal peptide substrates and distinct kinases that would not have significant activity at the noncognate substrate. A somewhat similar approach was published recently by Shults and coworkers [7] using an Illumina detection system to explore activity at 900 different
Fig. 5. Staurosporine inhibition of PKA. Varying concentrations of staurosporine were incubated with PKA (10 ng/reaction) for 20 min in kinase incubation buffer prior to initiating the kinase reaction with PKA substrate (10.5 nM) in PKA reaction buffer. Antibody concentrations were the same as in Figs. 3 and 4. Values are means ± standard errors of three experiments with duplicate determinations. The curve is a nonlinear least squares fit to a competitive inhibition curve with a variable Hill slope. MFI, median fluorescence intensity.
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substrates. They showed sets of 2- and 4-enzyme multiplex analyses in that system. Using the substrates and kinases described in their report, a similar multiplex analysis could be set up with the more accessible flow cytometry multiplexing assay that we describe here. The Luminex system can easily multiplex 10 to perhaps 30 substrates. Using a standard flow cytometer with bead sets having different intensities of labeling (e.g., Spherotech Blue Pack), up to 5 or perhaps 10 different substrates could be analyzed in multiplex. In addition to identifying appropriate sets of kinases and substrates to permit specificity of the reactions, one would also need to optimize assay conditions to ensure good activity of each kinase in the set. Work on that problem has already been done in other studies [7]. This use of nucleotidetagged substrates with Illumina detection provides high-level multiplexing but not high-throughput sample handling. Several other ‘‘multiplexed” kinase assays use parallel nondifferentiable readouts (e.g., [32P] incorporation or a single fluorophore) that cannot separately detect multiple kinases in a single incubation sample [5,6]. Comparison of the current approach with the many other available kinase assays is warranted. The ‘‘gold standard” method is to measure incorporation of [32P]phosphate into proteins from a gamma [32P]ATP substrate donor. This requires physical separation of labeled substrate from unlabeled substrate and some form of radioactive detection such as autoradiography or scintillation counting. In general, these are not high-throughput but can be used with scintillation proximity detection for highthroughput studies. Common immunochemical methods include the use of Western blot or ELISA to detect phosphorylated substrates. The former is optimal with large protein substrates, whereas the latter is applicable for both protein and peptide substrates. Many of the considerations for those two standard approaches are also evident in our current method. One needs to pick an appropriate substrate/antibody combination and kinase incubation conditions. They differ in their need for separation and washing steps, adding significantly to the complexity of the method. Our bead-based approach did not require any separation steps and yielded (at least for the optimized PKA substrate) outstanding signal/background. Furthermore, one could easily immobilize complex substrates on beads either with biochemically purified and biotin-tagged proteins or by capturing the substrate protein on the bead from complex mixtures by the use of a specific antibody. Many true high-throughput methods have also been developed, including LANCE, which is a TR–FRET method using a Eu-chelate donor anti-phospho-antibody and an APC-labeled acceptor peptide. Similarly, AlphaScreen uses the proximity of donor and acceptor beads that are conjugated to either a substrate peptide or an anti-phospho-antibody. Both of these methods use the same principle as our flow cytometry approach—detecting the accumulation of a complex of phosphorylated peptide substrate and an antiphospho-antibody. Both also require complex plate reader optics permitting time-resolved reading or 620-nm excitation and 520nm emission from the O2-sensitized acceptor bead. Any application developed for those methods could be adapted to our flow cytometry detection. Given the increasing availability of flow cytometry and plate sampling front-end systems (including Luminex, BD, and Guava), the flow cytometry kinase method could be simply implemented outside of screening centers. More recently, a number of methods have been developed using kinase- and substrate-dependent ATP depletion with luciferase detection of ATP (e.g., Kinase-Glo) or production of ADP (e.g., ADPHunter). None of the methods described here, however, is easily adapted to multiplex measurements, as is our detection method based on beads and flow cytometry.
The quality of signal, in terms of both magnitude and wellto-well consistency, is important when considering an assay for a high-throughput screen. The standard measure of assay quality is the Z factor, a measure calculated from the following rpþrnÞ , where r denotes the standard deviaequation: Z ¼ 1 3ð jlplnj tions of the positive and negative controls (rp and rn, respectively) and l indicates the mean values of the positive and negative controls. Using control values from our PKA experiments as shown in Fig. 5 (100 nM staurosporine and no staurosporine) from four individual dose–response experiments with values determined in triplicate yields a range of Z factors (calculated individually) of 0.7–0.8, comfortably within the range of 0.5–1 that indicates an excellent assay where positive and negative control values are well separated (good signal strength) and highly reproducible (low standard deviation values). Therefore, this assay paradigm should be suitable for high-throughput screening application. Finally, the question of throughput is important. In general, flow cytometry is not considered to be a high-throughput method. However, the advent of plate reading cytometers is changing that view. The Luminex 200 can read a 96-well plate in 30 min; this permits analysis of more than 1000 samples per day and could yield 5000 data points per day if a 5-kinase multiplex analysis were implemented. Using the HyperCyt high-throughput flow cytometry front-end (IntelliCyt, Albuquerque, NM, USA), which can run a 384-well plate in 10–15 min, more than 10,000 samples per day should be possible—with additional information obtained by multiplexing. In summary, we have described a simple bead-based flow cytometry method to assess in vitro activity of several protein kinases. The method is easily generalizable, and optimization that has been done for other methods based on substrate peptides and antibodies (e.g., ELISA, LANCE, AlphaScreen) could be applied with this detection methodology as well. A key advantage of the method is the potential for multiplexing that has been shown clearly in the immunoassay field with the use of Luminex and other flow cytometry bead detection systems. Acknowledgments This research was supported by the Biomedical Interactions Technology Center (University of New Hampshire and National Science Foundation [NSF]) and a gift of equipment and supplies from Luminex Corporation. The authors thank Susan Wade for assistance with the manuscript. References [1] P. Blume-Jensen, T. Hunter, Oncogenic kinase signaling, Nature 411 (2001) 355–365. [2] G. Manning, D.B. Whyte, R. Martinez, T. Hunter, S. Sudarsanam, The protein kinase complement of the human genome, Science 298 (2002) 1912–1934. [3] J. Dancey, E.A. Sausville, Issues and progress with protein kinase inhibitors for cancer treatment, Nat. Rev. Drug Discov. 2 (2003) 296–313. [4] L.E. DeForge, A.G. Cochran, S.H. Yeh, B.S. Robinson, K.L. Billeci, W.L. Wong, Substrate capacity considerations in developing kinase assays, Assay Drug Dev. Technol. 2 (2004) 131–140. [5] K.A. Janes, J.G. Albeck, L.X. Peng, P.K. Sorger, D.A. Lauffenburger, M.B. Yaffe, A high-throughput quantitative multiplex kinase assay for monitoring information flow in signaling networks: application to sepsis–apoptosis, Mol. Cell. Proteomics 2 (2003) 463–473. [6] M.D. Shults, K.A. Janes, D.A. Lauffenburger, B. Imperiali, A multiplexed homogeneous fluorescence-based assay for protein kinase activity in cell lysates, Nat. Methods 2 (2005) 277–283. [7] M.D. Shults, I.A. Kozlov, N. Nelson, B.G. Kermani, P.C. Melnyk, V. Shevchenko, A. Srinivasan, J. Musmacker, J.P. Hachmann, D.L. Barker, M. Lebl, C. Zhao, A multiplexed protein kinase assay, ChemBioChem 8 (2007) 933–942. [8] L.A. Sklar, B.S. Edwards, S.W. Graves, J.P. Nolan, E.R. Prossnitz, Flow cytometric analysis of ligand–receptor interactions and molecular assemblies, Annu. Rev. Biophys. Biomol. Struct. 31 (2002) 97–119.
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[11] C.R. Evelyn, S.M. Wade, Q. Wang, M. Wu, J.A. Iniguez-Lluhi, S.D. Merajver, R.R. Neubig, CCG-1423: a small-molecule inhibitor of RhoA transcriptional signaling, Mol. Cancer Ther. 6 (2007) 2249–2260. [12] D.L. Roman, J.N. Talbot, R.A. Roof, R.K. Sunahara, J.R. Traynor, R.R. Neubig, Identification of small-molecule inhibitors of RGS4 using a high-throughput flow cytometry protein interaction assay, Mol. Pharmacol. 71 (2007) 169–175.