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Recent Advances in Technologies for Analyzing Protein Kinases
Journal of Pharmacological Sciences
J Pharmacol Sci 103, 5 – 11 (2007)
©2007 The Japanese Pharmacological Society
Current Perspective: New Technique
Recent Advances in Technologies for Analyzing Protein Kinases Atsuhiko Ishida1, Isamu Kameshita2, Noriyuki Sueyoshi2, Takanobu Taniguchi1, and Yasushi Shigeri3,* 1
Department of Biochemistry, Asahikawa Medical College, Asahikawa 078-8510, Japan Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan 3 National Institute of Advanced Industrial Science and Technology (AIST), Ikeda, Osaka 563-8577, Japan 2
Received September 7, 2006; Accepted November 24, 2006
Abstract. Most cellular events are regulated by protein phosphorylation mediated by protein kinases, whose malfunction is involved in the etiology of various disorders. The elucidation of the biochemical properties of the protein phosphorylation reaction will lead not only to a better understanding of the signal transduction mechanism, but also to developing new therapeutic agents. In this review, we briefly summarize the technologies to detect or characterize protein kinases with special emphasis on recently developed and / or commercially available techniques. Keywords: protein kinase, assay, method, high-throughput screening, proteomic analysis
potent kinase inhibitors or to assess their inhibitory potency toward a particular protein kinase. Therefore, biochemical studies on protein kinases are important not only for basic biology to clarify molecular mechanisms of signal transduction, but also for clinical pharmacology to develop novel anticancer agents. In general, researchers in this field use two different approaches to study protein kinases. One is to characterize an individual protein kinase in detail by measuring its kinase activity. Protein kinase activities are conventionally measured by, for example, filterbinding procedures using radioactive ATP. In this assay, substrate proteins or peptides phosphorylated by protein kinases are adsorbed onto filter papers, followed by liquid scintillation counting (3). In addition, a variety of useful assay techniques to detect protein kinase activities with high sensitivity have recently been developed (see below). The second approach is to comprehensively analyze the extent of phosphorylation of substrate proteins or expression profiles of protein kinases themselves under various conditions. These include differential display or proteomic analyses focused on phosphoproteins or protein kinases. In this review, we will briefly discuss the methodology for these two different kinds of approaches, focusing on novel techniques to analyze protein kinases.
Introduction A variety of biological processes, including cell growth, differentiation, and death, are regulated by protein phosphorylation. It is catalyzed by protein kinases, which are encoded by more than 500 different genes in the human genome (1). Since many of the oncogenes encode protein kinases, they are potential targets for cancer chemotherapy. A variety of protein kinase inhibitors have been developed to date, and some of them are now clinically used as cancer therapeutics (2). Imatinib mesylate (Gleevec®; Novartis, Basel, Switzerland) and gefitinib (Iressa®; AstraZeneca, London, UK) are typical examples of such drugs. The former has been designed to inhibit Bcr / Abl tyrosine kinase, which is generated by a chimeric gene resulting from a chromosomal translocation characteristic of chronic myelogenous leukemia. The latter has been developed as a potent inhibitor of epidermal growth factor (EGF) receptor tyrosine kinase, and it selectively inhibits EGF-stimulated tumor cell growth. Imatinib mesylate and gefitinib are now employed as effective anticancer drugs for the treatment of chronic myelogenous leukemia and non-small cell lung carcinoma, respectively. To develop useful drugs like them, it is essential to screen a variety of chemical libraries for *Corresponding author. [email protected] Published online in J-STAGE: January 1, 2007 doi: 10.1254/jphs.CP0060026
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Various techniques to detect activities of protein kinases Until recently, radioactive compounds including [γ-32P]ATP and [32P]inorganic phosphate were widely employed to detect protein phosphorylation by protein kinases (3). Initially, some abundant proteins, such as histone or casein, were used as substrates for protein kinases. Thereafter, various synthetic peptides were developed as substrates for each protein kinase. Most of them are now commercially available and widely used for detection and characterization of various individual protein kinases (3). The in-gel protein kinase assay is a useful technique for detecting proteins with protein kinase activity in crude cell extracts (4). To simultaneously detect many more protein kinases, including protein kinase A, calmodulin (CaM)-kinase II, protein kinase C, and mitogen-activated protein (MAP) kinases by the in-gel kinase assay, a synthetic substrate peptide designated as a Multide with multiple phosphorylation sites was developed (5). These conventional techniques are simple, economical, and suitable for detection of protein kinase activity with high sensitivity. However, they are not suitable for high-throughput screening aimed at the development of novel drugs because they require radioactive isotopes (RI) for detection and thus require inconvenient washing steps. To avoid the utilization of RI, non-RI protein kinase assays using matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry were developed (6). Following on from the use of anti-phospho-Tyr antibodies for the study of tyrosine phosphorylation, a number of phospho-specific antibodies have been raised against phosphoproteins, phosphopeptides, and phosphoamino acids (7). Using these antibodies, it is now possible to detect protein phosphorylation with high sensitivity without RI (8). However, these techniques are not applicable to high-throughput screening. Recently, various novel techniques suitable for highthroughput screening have been developed to detect protein kinase activities more efficiently. Some of them are shown in Table 1. Fluorometry is the most promising technique to monitor phosphorylation reactions with high sensitivity as an alternative to radiometric assay. Lanthanoid fluorescent labels with long decay times have been employed in the DELFIA® assay (dissociation-enhanced lanthanide fluoroimmunoassay) (Perkin Elmer, Wellesley, MA, USA) (2). In this system, a lanthanide-labeled secondary antibody is employed instead of the enzyme-labeled antibody in an enzymelinked immunosorbent assay (ELISA). Time-resolved fluorometry of the enhanced fluorescence of the lanthanide, which is dissociated by an enhancement
solution, is carried out to reduce non-specific fluorescent background. The HTScanTM kinase assay kit (Cell Signaling Technology, Danvers, MA, USA) is equipped with the protein kinase of interest, its substrate peptide, and the first antibody to detect phosphorylation, and therefore can be readily used for DELFIA® assay. Fluorescence resonance energy transfer (FRET) technology is widely used for intracellular imaging and various fluorescence-based assays (9). ELISA assay formats using phospho-specific antibodies also have been combined with FRET or time-resolved FRET (TR-FRET) technology to develop novel assay systems for protein kinases (10). These include the Z'-LYTETM kinase assay platform (Invitrogen, Carlsbad, CA, USA) (11), LanthaScreen TR-FRET (Invitrogen), IMAP TRFRET (Molecular Devices, Sunnyvale, CA, USA), HTRF TR-FRET (Cisbio International, Bedford, MA, USA) (12), LANCE assay (Perkin Elmer), and AlphaScreen assay (Perkin Elmer) (2). Fluorescence polarization is a simple and convenient homogenous assay format not requiring a separation procedure of phosphorylated peptides or proteins from the reaction mixture (2). It is based on the principle that fluorophores with a low molecular weight have low polarization values, while those with a high molecular weight have higher polarization values. Non-RI protein kinase assays based on fluorescence polarization, such as the Far-Red PolarScreenTM fluorescence polarization kinase assay kit (Invitrogen) and KinEASE (Upstate Cell Signaling Solutions, Charlottesville, VA, USA), have been developed. A highly sensitive fluorescence polarization assay combined with bead technology has also been reported (IMAP-FP assay) (Molecular Devices, Sunnyvale, CA, USA) (13). Detecting the changes in ATP levels associated with the kinase reaction is another way to monitor kinase activity. Kinase-Glo® luminescent kinase assay (Promega, Madison, WI, USA) was developed to assess protein kinase activity by a luciferase-based ATP assay (14). The increase in fluorescence of a fluorescently labeled phosphopeptide by digestion with an appropriate protease was applied to a protein kinase assay to develop the ProFluorTM kinase assay (Promega) (15). Antibody BeaconTM assay (Invitrogen) was developed based on a detection complex comprising a small-molecule tracer ligand that exhibits quenched fluorescence when bound to anti-phospho-Tyr antibodies. In the presence of phospho-Tyr containing peptides, the ligand is rapidly displaced and the fluorescence increases (2). Quenching of fluorescence upon binding of a Fe3+- or Ga3+-containing compound to a phosphate group in a fluorescently labeled phosphopeptide can be applicable to protein kinase assays. The IQ kinase assay (Pierce, Rockford,
Technologies for Analyzing Protein Kinases
IL, USA) (16) and QTL LightspeedTM assay (QTL Biosystems, Santa Fe, NM, USA) (17) were designed on the basis of such methodology. Recently, the TruLightTM kinase assay (Merck Biosciences, San Diego, CA, USA) was developed. Compared to FRET assays or conventional fluorescence quenching assays, it exploits a 50fold increase in quenching magnitude, resulting in 10 times more sensitivity than FRET-based assays. Although the scintillation proximity assay (GE Healthcare Biosciences, Piscataway, NJ, USA) requires an RI such as [γ-33P]ATP, it is applicable to highthroughput assays because there is no need for separation of phosphorylated peptides from the reaction mixture (18). Besides these, some other techniques such as PepTag® assay (Promega), SignaTECT® protein kinase assay systems (Promega) (19), SignalScoutTM kinase profiling system (Stratagene, La Jolla, CA, USA), and AUSA® universal protein kinase assay kit (Transbio Corporation, Baltimore, MD, USA) are also commercially available for protein kinase assay. Some of the assay formats mentioned above are discussed in detail by Olive (2). Proteomic analysis in protein kinase research Proteomics is an emerging area of research in the post-genome era that deals with global analysis of gene expression. Application of proteomic analysis in protein kinase research may lead to a breakthrough in this field. Although proteomic analysis focused on phosphoproteins (phosphoproteomics) is one of the important applications, proteomic analysis focused on protein kinases themselves has not yet been established due to lack of specific probes to comprehensively detect or trap multiple protein kinases. We recently developed novel antibodies that could detect multiple protein kinases. In this section, we will discuss some techniques for phosphoproteomics together with the usefulness of the novel antibodies. Useful techniques for phosphoproteomics: For the study of protein kinases, it is very important to evaluate what kinds of phosphoproteins exist in cells and to what extent these proteins are phosphorylated. Recent advances in the techniques for proteomic analysis enable us to analyze comprehensively phosphoproteins in cells. Phosphoproteomics, proteomic analysis focused on phosphoproteins, is a powerful approach to obtain a variety of important information as to signaling pathways in which protein kinases are involved. We briefly summarize some of the commercially available techniques that are useful for phosphoproteomics in Table 2. Electrospray ionization (ESI) and MALDI-TOF mass spectrometry are the most sensitive techniques to
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detect phosphoproteins or phosphopeptides without RI (20), and thus are indispensable for phosphoproteomics. To increase the sensitivity and accuracy in mass spectrometry, phosphoproteins must be enriched and separated from nonphosphorylated proteins prior to isolation by two-dimensional electrophoresis and identification by mass spectrometry. Immunoprecipitation using phospho-specific antibodies is a conventional, but powerful technique for the enrichment. However, some antibodies are not suitable for immunoprecipitation, and even though the antibodies can be used for immunoprecipitation, these phospho-specific antibodies are occasionally too expensive. Recently, immobilized metal-ion affinity chromatography, which is based on specific interactions between the phosphate group in phosphopeptides or phosphoproteins and Fe3+ or Ga3+ on the metal affinity support, has been developed; and convenient kits, such as Phosphopeptide enrichment spin columns (Clontech, Mountain View, CA, USA), Phosphoprotein enrichment kit (Clontech), Phosphoprotein purification kit (QIAGEN, Hilden, Germany), and Phosphopeptide isolation kit (Pierce), are now commercially available (21). On the other hand, a chemical reaction method to substitute the phosphate group is efficient for the enrichment of phosphoproteins and phosphopeptides. In this protocol, the phosphate group in phosphoproteins or phosphopeptides can be replaced by several functional groups, such as a biotincontaining tag, and subsequently purified by affinity chromatography based on the avidin-biotin interaction (22). Phosphoprotein-specific fluorescence staining using ProQ diamond phosphoprotein stain (Invitrogen) (23) is a simple and useful way for the comprehensive detection of phosphoproteins displayed on SDS-polyacrylamide gels, but care should be taken to avoid nonspecific staining of proteins. To detect phosphopeptides or phosphoproteins on blot membranes, ELISA plates, or tissue slices by fluorescence staining, Phosphoprotein detection reagent and kit (Pierce) can be used. Besides these techniques, a novel approach for proteomic analysis of phosphoproteins using antibodies broadly reactive against the consensus motif of phosphorylation sites has been developed to identify substrate proteins for the protein kinase of interest (24). In some cases, microarray technology can be utilized for screening and identification of kinase substrates (ProtoarrayTM kinase substrate identification kit, Invitrogen). For phosphoproteomic analysis, it is often important to estimate the phosphorylation level. Malachite Green phosphate detection kit (R & D systems, Minneapolis, MN, USA) and Phosphoprotein phosphate estimation assay kit (Pierce) (25) are used to determine the phosphorylation level of substrate proteins without using RIs.
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Some commercially available techniques to detect protein kinase activities
Key technique
Tool
Summary
Company and References
Time-resolved fluorometry
DELFIA® assay
Time-resolved fluorometry in ELISA format using lanthanide labeled phosphospecific antibody, biotin labeled phosphopeptide, and streptavidin-coated microwell plate. An enhancement solution of low pH is used to dissociate the lanthanide, which forms a stable fluorescent chelate. Low background due to long fluorescent decay times of the lanthanide.
Perkin Elmer (Wellesley, MA, USA) (www.perkinelmer.com) Ref. 2
HTScanTM kinase assay kit
A kit containing biotinylated substrate peptide, anti-phosphopeptide antibody and the Cell Signaling Technology protein kinase of interest. Readily compatible with DELFIA® assay. Europium labeled (Danvers, MA, USA) secondary antibody and DELFIA® enhancement solution are recommended. (www.cellsignal.com)
FRET
Z'-LYTETM kinase assay
FRET using substrate peptide with donor (coumarin) and acceptor (fluorescein). The differential protease sensitivity of phosphorylated versus non-phosphorylated substrate peptide is utilized. Unnecessary for specific antibody but difficulty in substrate peptide design.
TR-FRET
LanthaScreenTM TR-FRET assay
TR-FRET using terbium-conjugated phosphospecific antibody and fluorescein-labeled Invitrogen substrate peptide. Low background due to long fluorescent decay times of the lanthanide. (Carlsbad, CA, USA) (www.invitrogen.com)
IMAP (immobilized metal assay for phosphochemicals) TR-FRET
Both terbium-labeled TR-FRET donor and fluorescence labeled phosphopeptide bind to Molecular Devices nanoparticles through immobilized trivalent metals resulting in TR-FRET. No requirement (Sunnyvale, CA, USA) for antibody. (www.moleculardevices.com)
HTRF® (homogeneous time resolved fluorescence) TR-FRET
TR-FRET using Eu3+ cryptate-conjugated phosphospecific antibody and streptavidin Cisbio International coupled to allophycocyanin (XL665). Biotinylated peptide is phosphorylated by the (Bedford, MA, USA) kinase. Low background due to long fluorescent decay times of the lanthanide. (www.htrf-assays.com) Ref. 12
LANCE assay
TR-FRET between Eu3+-labeled phosphospecific antibody and streptavidin-coated Perkin Elmer fluorescent bead bound to biotin-labeled phosphopeptide. Low background due to long (Wellesley, MA, USA) fluorescent decay times of the lanthanide. (www.perkinelmer.com) Ref. 2
AlphaScreen assay
TR-FRET between a streptavidin-coated fluorescent dye-containing bead, which is bound to biotinylated phosphopeptide and acts as donor, and a second fluorescent dye-containing bead coated with a phosphospecific antibody binding to biotinylated phosphopeptide. The signal is significantly amplified by the singlet oxygen. Highly sensitive, greater dynamic range along with good assay reproducibility.
Far-Red PolarScreenTM fluorescence polarization kinase assay kit
Fluorescence polarization derived from binding of fluorescence-labeled phosphopeptide to Invitrogen phosphospecific antibody is detected. Far-red fluorescence polarization reduces back- (Carlsbad, CA, USA) ground. (www.invitrogen.com)
KinEASE
Fluorescence polarization derived from binding of fluorescence-labeled phosphopeptide to phosphospecific antibody is detected. Far-red fluorescence polarization at 645 nm to reduce background.
Upstate Cell Signaling Solutions (Charlottesville, VA, USA) (www.upstate.com)
IMAP-FP
Fluorescence labeled phosphopeptide, captured on modified nanoparticles through interactions with immobilized trivalent metals, results in high fluorescence polarization. Insensitivity to the sequence of flanking amino acids. Change in polarization derived from kinase reaction is largely enhanced.
Molecular Devices (Sunnyvale, CA, USA) (www.moleculardevices.com) Ref. 13
Luciferase-based ATP assay detected by luminescence
Kinase-Glo® luminescent kinase assay
Amount of ATP remaining in solution after termination of kinase reaction is quantified by Promega luminescence. No requirement for antibody, RI, specially designed peptide substrates, or (Madison, WI, USA) long reading time. (www.promega.com) Ref. 14
Fluorescence quenching assay
ProFluorTM kinase assay
Non-phosphorylated bisamide rhodamine substrate peptide is digested by protease, Promega resulting in the increase in fluorescence of rhodamine. Fluorescence signal is very stable. (Madison, WI, USA) (www.promega.com) Ref. 15
Antibody BeaconTM tyrosine kinase assay kit
Fluorescence quenching assay employing a small molecule tracer ligand labeled by Oregon Green 488. The tracer ligand exhibits quenched fluorescence when bound to antiphosphoTyr antibody. In the presence of phosphoTyr-containing peptides, the ligand is rapidly displaced, resulting in an increase in fluorescence. Real-time monitoring of kinase activity.
IQ kinase assay
Binding of Fe3+-containing compound to a phosphate group on fluorescence labeled Pierce phosphopeptide results in the quenching of fluorescence. Tolerant to both high and low (Rockford, IL, USA) concentrations of ATP and substrate. (www.piercenet.com) Ref. 16
QTL lightspeedTM kinase activity assays
Rhodamine-labeled phosphopeptide bound to Ga3+ metal ion on the polymer results in quench of polymer fluorescence. Wide dynamic range. Unnecessary for specific antibody.
TruLightTM kinase assays
Utilization of peptide substrates labeled with a quencher and microsphere coated with Merck Biosciences fluorescent polymers and a Ga3+. The phosphorylated peptides bind to microsphere, (San Diego, CA, USA) resulting in fluorescence quenching. Ten times more sensitivity than FRET-based assays. (www.merckbiosciences.com)
Fluorescence polarization
Invitrogen (Carlsbad, CA, USA) (www.invitrogen.com) Ref. 11
Perkin Elmer (Wellesley, MA, USA) (www.perkinelmer.com), Ref. 2
Invitrogen (Carlsbad, CA, USA) (www.invitrogen.com) Ref. 2
QTL Biosystems (Santa Fe, NM, USA) (www.qtlbio.com) Ref. 17
Technologies for Analyzing Protein Kinases
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SPA beads
Scintillation proximity assay (SPA)
Phosphorylation of biotinylated protein substrate with [γ-33P]ATP and capture of radiolabeled product on streptavidin-coated SPA beads. Light is emitted and detected by scintillation counter or CCD imager. Radiometric assay. Unnecessary for separation step and available for microplate scintillation counter.
Fluorometry, Agarose electrophoresis
PepTag® assay for non-radioactive detection of protein kinases
Phosphorylated and non-phosphorylated substrate peptides labeled by fluorescence are Promega separated by agarose electrophoresis due to the change in peptide’s net charge. Quantifica- (Madison, WI, USA) tion of kinase activity can be determined by fluorometric densitometer or spectro- (www.promega.com) fluorometer.
Streptavidin membrane
SignaTECT® protein kinase assay systems
Biotinylated substrate peptide is phosphorylated by kinase and [γ-32P]ATP, and then Promega captured by membrane with a high density of streptavidin. Low level of non-specific (Madison, WI, USA) substrate peptide binding results in low background. (www.promega.com) Ref. 19
Substrate beads
SignalScoutTM kinase profiling system
Kinase substrate proteins immobilized on beads. Radioactive, colorimetric and Stratagene fluorometric detection can be used. (La Jolla, CA, USA) (www.stratagene.com)
Biotin-avidin interaction, Ultrafiltration
AUSA® universal protein kinase assay kit
Binding of biotinylated substrate peptide to avidin is separated by ultrafiltration. Ultrafil- Transbio Corporation tration technique and radioactive detection. (Baltimore, MD, USA) (www.transbiocorp.com)
Table 2.
GE Healthcare Biosciences (Piscataway, NJ, USA) (www. gehealthcare.com) Ref. 18
Some commercially available techniques useful for phosphoproteomics
Key technique
Tool
Summary
Company and References
Metal affinity chromatography resin
Phosphopeptide enrichment spin columns
Spin columns contain an immobilized metal affinity chromatography resin that binds to phosphopeptides. Enrichment of phosphopeptides by spin column.
Clontech (Mountain View, CA, USA) (www.clontech.com) Ref. 21
Phosphoprotein enrichment kit
Immobilized affinity chromatography resin that binds to phosphoproteins or phospho- Clontech peptides. Enrichment of phosphoproteins by affinity column. (Mountain View, CA, USA) (www.clontech.com) Ref. 21
Phosphoprotein purification kit
Immobilized affinity chromatography resin that binds to phosphoproteins or phospho- QIAGEN peptides. Eluate from the affinity column is further concentrated and desalted by ultrafil- (Hilden, Germany) tration. (www1.qiagen.com) Ref. 21
Phosphopeptide isolation kit
Ga3+-chelated iminodiacetic acid (IDA)-based resin specifically traps phosphopeptides. Pierce (Rockford, IL, USA) Ga3+-based resin shows more specific binding than iron-chelated resin. (www.piercenet.com) Ref. 21
Fluorescence dye
ProQ diamond phosphoprotein stain
Fluorescent dye for detecting phosphoprotein displayed on SDS-PAGE and protein Invitrogen microarray. Small molecular fluorophore recognizes phosphoamino acids. (Carlsbad, CA, USA) (www.invitrogen.com) Ref. 23
Phosphoprotein detection
Phosphoprotein detection reagent and kit
Fe3+-activated derivatives of horseradish peroxidase can detect phosphorylated molecules Pierce by ELISA, immunohistochemistry, and Western blotting. Applicable to ELISA, immuno- (Rockford, IL, USA) histochemistry, and Western blotting. (www.piercenet.com) Ref. 21
Microarray
ProtoarrayTM kinase substrate identification kit
Kinase substrate protein microarray. Identification of kinase substrate using [γ-33P]ATP and autoradiography.
Colorimetric assay by Malachite Green
Malachite Green phosphate detection kit
Based on the Malachite Green-molybdate binding reaction. Phosphate from alkaline R & D systems hydrolysis of phosphoprotein is quantified by Malachite Green. (Minneapolis, MN, USA) (www.rndsystems.com) Ref. 25
Phosphoprotein phosphate estimation assay kit
Based on the Malachite Green-molybdate binding reaction. Phosphate from alkaline Pierce hydrolysis of phosphoprotein is quantified by Malachite Green. (Rockford, IL, USA) (www.piercenet.com) Ref. 25
Antibodies to detect multiple protein kinases simultaneously: Although hundreds of protein kinases have been documented to date, how many and what kind of protein kinases are expressed in cells and tissues under varying situations or in response to various stimuli are
Invitrogen (Carlsbad, CA, USA) (www.invitrogen.com)
still unknown. Therefore, it would be of great help if we could detect a variety of protein kinases expressed in cells or tissues with a simple procedure. For this purpose, we developed unique antibodies directed towards a highly conserved region of protein kinases.
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Monoclonal antibodies as well as polyclonal antibodies broadly reactive against serine / threonine kinases were obtained by immunization of a synthetic peptide (CVVHRDLKPENLLLAS), which was derived from a highly conserved subdomain VIB of serine / threonine kinases, as an anitigen (26, 27). Since these antibodies could recognize various serine / threonine kinases including CaM-kinases I, II, IV, CaM-kinase kinase, protein kinase A, MAP-kinase (ERK), and MAP-kinase kinase (MEK), we called them “Multi-PK antibodies”. By screening with the monoclonal antibodies, cDNA clones for various known and novel serine / threonine kinases, but not tyrosine kinases, have been isolated (27, 28). Moreover, these antibodies could immunoprecipitate various protein kinases from crude cell extracts. This implies that protein kinases can be immunoprecipitated with the “Multi-PK antibody” and analyzed for their activation states in response to various extracellular stimuli by an in-gel protein kinase assay (27). The “Multi-PK antibody” is also applicable to plant molecular biology because the subdomain VIB of protein kinases is conserved from plant to mammals. When a cDNA library of Lotus japonicus was screened with the “Multi-PK antibodies”, 178 positive clones were obtained. Among them, 164 clones (92%) were found to encode putative serine / threonine protein kinases and kinase-like proteins (29). We also prepared monoclonal antibodies with broad reactivity to tyrosine kinases using a conserved amino acid sequence (CYVHRDLRAAN) in the subdomain VIB of Src kinase as an immunogen (30). A monoclonal antibody, designated as YK34, reacted with not only Src kinase, but also nine different Src mutants with various amino acid substitutions in the subdomain VIB, which were designed so as to correspond to those of many other tyrosine kinases in humans. Since YK34 appeared to react with various tyrosine kinases, but not with serine / threonine kinases, it seems to be a “Multi-PK antibody” for tyrosine kinases. We observed significant changes in immunoreactive bands with YK34 in HL-60 cell extracts along with changes in cell morphology induced by TPA treatment (30). Thus, the “Multi-PK antibodies” can be powerful tools for proteomic analysis focused on serine / threonine and / or tyrosine kinases. Conclusion In this review, we discussed recent advances in technologies useful for protein kinase research. At first, we summarized recently developed and commercially available techniques to characterize activities of protein kinases as well as conventional protein kinase assays. Most of the former are rapid and highly sensitive
techniques suitable for high-throughput screening. Although we surveyed and listed representative techniques, modified, improved, or combined techniques will appear soon. Next, we discussed recently developed techniques useful for phosphoproteomics. Finally, we introduced a novel approach for detection and characterization of protein kinases using “Multi-PK antibodies”. The techniques described here will provide us with powerful tools to explore the protein kinase world, leading to clarification of molecular mechanisms of signal transduction, and to establishment of novel therapeutic approaches for human disorders including cancer. Acknowledgments We thank Prof. Leslie Sargent Jones (University of South Carolina) and Prof. Kenneth Takeda (University of Louis Pasteur, Strasbourg) for critical reading of this manuscript. This work was supported, in part, by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan, the Smoking Research Foundation of Japan, the Akiyama Foundation, the Osaka Medical Research Foundation for Incurable Diseases, the Naito Foundation, and also by an AIST Research Grant. References 1 Manning G, Plowman GD, Hunter T, Sudarsanam S. Evolution of protein kinase signaling from yeast to man. Trends Biochem Sci. 2002;27:514–520. 2 Olive DM. Quantitative methods for the analysis of protein phosphorylation in drug development. Expert Rev Proteomics. 2004;1:327–341. 3 Hardie DG, editor. Protein phosphorylation: a practical approach. 2nd ed. New York: Oxford University Press; 1999. 4 Bischoff KM, Liang S, Kennelly PJ. The detection of enzyme activity following sodium dodecyl sulfate polyacrylamide gel electrophoresis. Anal Biochem. 1998;260:1–17. 5 Kameshita I, Taketani S, Ishida A, Fujisawa H. Detection of a variety of Ser/Thr protein kinases using a synthetic peptide with multiple phosphorylation sites. J Biochem. 1999;126:991–995. 6 Matsumoto H, Kahn ES, Komori N. Nonradioactive phosphopeptide assay by matrix-assisted laser desorption ionization time-of-flight mass spectrometry: application to calcium / calmodulin-dependent protein kinase II. Anal Biochem. 1998;260:188–194. 7 Ross AH, Baltimore D, Eisen HN. Phosphotyrosine-containing proteins isolated by affinity chromatography with antibodies to a synthetic hapten. Nature. 1981;294:654–656. 8 Rijksen G, Van Oirschot BA, Staal GE. Nonradioactive assays of protein-tyrosine kinase activity using anti-phosphotyrosine antibodies. Methods Enzymol. 1991;200:98–107. 9 Miyawaki A, Tsien RY. Monitoring protein conformations and interactions by fluorescence resonance energy transfer between
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mutants of green fluorescent protein. Methods Enzymol. 2000;327:472–500. Han S, Zhou V, Pan S, Liu Y, Hornsby M, McMullan D, et al. Identification of coumarin derivatives as a novel class of allosteric MEK1 inhibitors. Bioorg Med Chem Lett. 2005;15: 5467–5473. Rodems SM, Hamman BD, Lin C, Zhao J, Shah S, Heidary D, et al. A FRET-based assay platform for ultra-high density drug screening of protein kinases and phosphatases. Assay Drug Dev Technol. 2002;1:9–19. Mathis, G. HTRF technology. J Biomol Screen. 1999;4:309– 314. Sportsman JR, Gaudet EA, Boge A. Immobilized metal ion affinity-based fluorescence polarization (IMAP): advances in kinase screening. Assay Drug Dev Technol. 2004;2:205–214. Koresawa M, Okabe T. High-throughput screening with quantitation of ATP consumption: a universal non-radioisotope, homogeneous assay for protein kinase. Assay Drug Dev Technol. 2004;2:153–160. Leytus SP, Melhado LL, Mangel WF. Rhodamine-based compounds as fluorogenic substrates for serine proteinases. Biochem J. 1983;209:299–307. Morgan AG, McCauley TJ, Stanaitis ML, Mathrubutham M, Millis SZ. Development and validation of a fluorescence technology for both primary and secondary screening of kinases that facilitates compound selectivity and site-specific inhibitor determination. Assay Drug Dev Technol. 2004;2:171–181. Rininsland F, Xia W, Wittenburg S, Shi X, Stankewicz C, Achyuthan K, et al. Metal ion-mediated polymer superquenching for highly sensitive detection of kinase and phosphatase activities. Proc Natl Acad Sci U S A. 2004;101:15295– 15300. Beveridge M, Park YW, Hermes J, Marenghi A, Brophy G, Santos A. Detection of p56(lck) kinase activity using scintillation proximity assay in 384-well format and imaging proximity assay in 384- and 1536-well format. J Biomol Screen. 2000;5:205–212. Xuei X, David CA, Middleton TR, Lim B, Pithawalla R, Chen CM, et al. Use of SAM2® biotin capture membrane in microarrayed compound screening (µARCS) format for nucleic acid polymerization assays. J Biomol Screen. 2003;8:273–282.
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20 Reinders J, Sickmann A. State-of-the-art in phosphoproteomics. Proteomics. 2005;5:4052–4061. 21 Andersson L, Porath J. Isolation of phosphoproteins by immobilized metal (Fe3+) affinity chromatography. Anal Biochem. 1986;154:250–254. 22 Sechi S, Oda Y. Quantitative proteomics using mass spectrometry. Curr Opin in Chem Biol. 2003;7:70–77. 23 Schulenberg B, Aggeler R, Beechem JM, Capaldi RA, Patton WF. Analysis of steady-state protein phosphorylation in mitochondria using a novel fluorescent phosphosensor dye. J Biol Chem. 2003;278:27251–27255. 24 Zhang H, Zha X, Tan Y, Hornbeck PV, Mastrangelo AJ, Alessi DR, et al. Phosphoprotein analysis using antibodies broadly reactive against phosphorylated motifs. J Biol Chem. 2002;277:39379–39387. 25 Harder KW, Owen P, Wong L, Aebersold R, Clark-Lewis I, Jirik FR. Characterization and kinetic analysis of the intracellular domain of human protein tyrosine phosphatase beta (HPTP beta) using synthetic phophopeptides. Biochem J. 1994;298:395–401. 26 Kameshita I, Kinoshita S, Shigeri Y, Tatsu Y, Yumoto N, Ishida A. Generation of a polyclonal antibody that simultaneously detects multiple Ser /Thr protein kinases. J Biochem Biophys Methods. 2004;60:13–22. 27 Kameshita I, Tsuge T, Kinashi T, Kinoshita S, Sueyoshi N, Ishida A, et al. A new approach for the detection of multiple protein kinases using monoclonal antibodies directed to the highly conserved region of protein kinases. Anal Biochem. 2003;322:215–224. 28 Kinoshita S, Sueyoshi N, Suetake I, Nakamura M, Tajima S, Kameshita I. Cloning and characterization of a novel Ca2+ /calmodulin-dependent protein kinase I homologue in Xenopus laevis. J Biochem. 2004;135:619–630. 29 Kameshita I, Nishida T, Nakamura S, Sugiyama Y, Sueyoshi N, Umehara Y, et al. Expression cloning of a variety of novel protein kinases in Lotus japonicus. J Biochem. 2005;137:33–39. 30 Sugiyama Y, Sueyoshi N, Shigeri Y, Tatsu Y, Yumoto N, Ishida A, et al. Generation and application of a monoclonal antibody that detects a wide variety of protein kinases. Anal Biochem. 2005;347:112–120.
J Pharmacol Sci 103, 5 – 11 (2007)
©2007 The Japanese Pharmacological Society
Current Perspective: New Technique
Recent Advances in Technologies for Analyzing Protein Kinases Atsuhiko Ishida1, Isamu Kameshita2, Noriyuki Sueyoshi2, Takanobu Taniguchi1, and Yasushi Shigeri3,* 1
Department of Biochemistry, Asahikawa Medical College, Asahikawa 078-8510, Japan Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan 3 National Institute of Advanced Industrial Science and Technology (AIST), Ikeda, Osaka 563-8577, Japan 2
Received September 7, 2006; Accepted November 24, 2006
Abstract. Most cellular events are regulated by protein phosphorylation mediated by protein kinases, whose malfunction is involved in the etiology of various disorders. The elucidation of the biochemical properties of the protein phosphorylation reaction will lead not only to a better understanding of the signal transduction mechanism, but also to developing new therapeutic agents. In this review, we briefly summarize the technologies to detect or characterize protein kinases with special emphasis on recently developed and / or commercially available techniques. Keywords: protein kinase, assay, method, high-throughput screening, proteomic analysis
potent kinase inhibitors or to assess their inhibitory potency toward a particular protein kinase. Therefore, biochemical studies on protein kinases are important not only for basic biology to clarify molecular mechanisms of signal transduction, but also for clinical pharmacology to develop novel anticancer agents. In general, researchers in this field use two different approaches to study protein kinases. One is to characterize an individual protein kinase in detail by measuring its kinase activity. Protein kinase activities are conventionally measured by, for example, filterbinding procedures using radioactive ATP. In this assay, substrate proteins or peptides phosphorylated by protein kinases are adsorbed onto filter papers, followed by liquid scintillation counting (3). In addition, a variety of useful assay techniques to detect protein kinase activities with high sensitivity have recently been developed (see below). The second approach is to comprehensively analyze the extent of phosphorylation of substrate proteins or expression profiles of protein kinases themselves under various conditions. These include differential display or proteomic analyses focused on phosphoproteins or protein kinases. In this review, we will briefly discuss the methodology for these two different kinds of approaches, focusing on novel techniques to analyze protein kinases.
Introduction A variety of biological processes, including cell growth, differentiation, and death, are regulated by protein phosphorylation. It is catalyzed by protein kinases, which are encoded by more than 500 different genes in the human genome (1). Since many of the oncogenes encode protein kinases, they are potential targets for cancer chemotherapy. A variety of protein kinase inhibitors have been developed to date, and some of them are now clinically used as cancer therapeutics (2). Imatinib mesylate (Gleevec®; Novartis, Basel, Switzerland) and gefitinib (Iressa®; AstraZeneca, London, UK) are typical examples of such drugs. The former has been designed to inhibit Bcr / Abl tyrosine kinase, which is generated by a chimeric gene resulting from a chromosomal translocation characteristic of chronic myelogenous leukemia. The latter has been developed as a potent inhibitor of epidermal growth factor (EGF) receptor tyrosine kinase, and it selectively inhibits EGF-stimulated tumor cell growth. Imatinib mesylate and gefitinib are now employed as effective anticancer drugs for the treatment of chronic myelogenous leukemia and non-small cell lung carcinoma, respectively. To develop useful drugs like them, it is essential to screen a variety of chemical libraries for *Corresponding author. [email protected] Published online in J-STAGE: January 1, 2007 doi: 10.1254/jphs.CP0060026
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Various techniques to detect activities of protein kinases Until recently, radioactive compounds including [γ-32P]ATP and [32P]inorganic phosphate were widely employed to detect protein phosphorylation by protein kinases (3). Initially, some abundant proteins, such as histone or casein, were used as substrates for protein kinases. Thereafter, various synthetic peptides were developed as substrates for each protein kinase. Most of them are now commercially available and widely used for detection and characterization of various individual protein kinases (3). The in-gel protein kinase assay is a useful technique for detecting proteins with protein kinase activity in crude cell extracts (4). To simultaneously detect many more protein kinases, including protein kinase A, calmodulin (CaM)-kinase II, protein kinase C, and mitogen-activated protein (MAP) kinases by the in-gel kinase assay, a synthetic substrate peptide designated as a Multide with multiple phosphorylation sites was developed (5). These conventional techniques are simple, economical, and suitable for detection of protein kinase activity with high sensitivity. However, they are not suitable for high-throughput screening aimed at the development of novel drugs because they require radioactive isotopes (RI) for detection and thus require inconvenient washing steps. To avoid the utilization of RI, non-RI protein kinase assays using matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry were developed (6). Following on from the use of anti-phospho-Tyr antibodies for the study of tyrosine phosphorylation, a number of phospho-specific antibodies have been raised against phosphoproteins, phosphopeptides, and phosphoamino acids (7). Using these antibodies, it is now possible to detect protein phosphorylation with high sensitivity without RI (8). However, these techniques are not applicable to high-throughput screening. Recently, various novel techniques suitable for highthroughput screening have been developed to detect protein kinase activities more efficiently. Some of them are shown in Table 1. Fluorometry is the most promising technique to monitor phosphorylation reactions with high sensitivity as an alternative to radiometric assay. Lanthanoid fluorescent labels with long decay times have been employed in the DELFIA® assay (dissociation-enhanced lanthanide fluoroimmunoassay) (Perkin Elmer, Wellesley, MA, USA) (2). In this system, a lanthanide-labeled secondary antibody is employed instead of the enzyme-labeled antibody in an enzymelinked immunosorbent assay (ELISA). Time-resolved fluorometry of the enhanced fluorescence of the lanthanide, which is dissociated by an enhancement
solution, is carried out to reduce non-specific fluorescent background. The HTScanTM kinase assay kit (Cell Signaling Technology, Danvers, MA, USA) is equipped with the protein kinase of interest, its substrate peptide, and the first antibody to detect phosphorylation, and therefore can be readily used for DELFIA® assay. Fluorescence resonance energy transfer (FRET) technology is widely used for intracellular imaging and various fluorescence-based assays (9). ELISA assay formats using phospho-specific antibodies also have been combined with FRET or time-resolved FRET (TR-FRET) technology to develop novel assay systems for protein kinases (10). These include the Z'-LYTETM kinase assay platform (Invitrogen, Carlsbad, CA, USA) (11), LanthaScreen TR-FRET (Invitrogen), IMAP TRFRET (Molecular Devices, Sunnyvale, CA, USA), HTRF TR-FRET (Cisbio International, Bedford, MA, USA) (12), LANCE assay (Perkin Elmer), and AlphaScreen assay (Perkin Elmer) (2). Fluorescence polarization is a simple and convenient homogenous assay format not requiring a separation procedure of phosphorylated peptides or proteins from the reaction mixture (2). It is based on the principle that fluorophores with a low molecular weight have low polarization values, while those with a high molecular weight have higher polarization values. Non-RI protein kinase assays based on fluorescence polarization, such as the Far-Red PolarScreenTM fluorescence polarization kinase assay kit (Invitrogen) and KinEASE (Upstate Cell Signaling Solutions, Charlottesville, VA, USA), have been developed. A highly sensitive fluorescence polarization assay combined with bead technology has also been reported (IMAP-FP assay) (Molecular Devices, Sunnyvale, CA, USA) (13). Detecting the changes in ATP levels associated with the kinase reaction is another way to monitor kinase activity. Kinase-Glo® luminescent kinase assay (Promega, Madison, WI, USA) was developed to assess protein kinase activity by a luciferase-based ATP assay (14). The increase in fluorescence of a fluorescently labeled phosphopeptide by digestion with an appropriate protease was applied to a protein kinase assay to develop the ProFluorTM kinase assay (Promega) (15). Antibody BeaconTM assay (Invitrogen) was developed based on a detection complex comprising a small-molecule tracer ligand that exhibits quenched fluorescence when bound to anti-phospho-Tyr antibodies. In the presence of phospho-Tyr containing peptides, the ligand is rapidly displaced and the fluorescence increases (2). Quenching of fluorescence upon binding of a Fe3+- or Ga3+-containing compound to a phosphate group in a fluorescently labeled phosphopeptide can be applicable to protein kinase assays. The IQ kinase assay (Pierce, Rockford,
Technologies for Analyzing Protein Kinases
IL, USA) (16) and QTL LightspeedTM assay (QTL Biosystems, Santa Fe, NM, USA) (17) were designed on the basis of such methodology. Recently, the TruLightTM kinase assay (Merck Biosciences, San Diego, CA, USA) was developed. Compared to FRET assays or conventional fluorescence quenching assays, it exploits a 50fold increase in quenching magnitude, resulting in 10 times more sensitivity than FRET-based assays. Although the scintillation proximity assay (GE Healthcare Biosciences, Piscataway, NJ, USA) requires an RI such as [γ-33P]ATP, it is applicable to highthroughput assays because there is no need for separation of phosphorylated peptides from the reaction mixture (18). Besides these, some other techniques such as PepTag® assay (Promega), SignaTECT® protein kinase assay systems (Promega) (19), SignalScoutTM kinase profiling system (Stratagene, La Jolla, CA, USA), and AUSA® universal protein kinase assay kit (Transbio Corporation, Baltimore, MD, USA) are also commercially available for protein kinase assay. Some of the assay formats mentioned above are discussed in detail by Olive (2). Proteomic analysis in protein kinase research Proteomics is an emerging area of research in the post-genome era that deals with global analysis of gene expression. Application of proteomic analysis in protein kinase research may lead to a breakthrough in this field. Although proteomic analysis focused on phosphoproteins (phosphoproteomics) is one of the important applications, proteomic analysis focused on protein kinases themselves has not yet been established due to lack of specific probes to comprehensively detect or trap multiple protein kinases. We recently developed novel antibodies that could detect multiple protein kinases. In this section, we will discuss some techniques for phosphoproteomics together with the usefulness of the novel antibodies. Useful techniques for phosphoproteomics: For the study of protein kinases, it is very important to evaluate what kinds of phosphoproteins exist in cells and to what extent these proteins are phosphorylated. Recent advances in the techniques for proteomic analysis enable us to analyze comprehensively phosphoproteins in cells. Phosphoproteomics, proteomic analysis focused on phosphoproteins, is a powerful approach to obtain a variety of important information as to signaling pathways in which protein kinases are involved. We briefly summarize some of the commercially available techniques that are useful for phosphoproteomics in Table 2. Electrospray ionization (ESI) and MALDI-TOF mass spectrometry are the most sensitive techniques to
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detect phosphoproteins or phosphopeptides without RI (20), and thus are indispensable for phosphoproteomics. To increase the sensitivity and accuracy in mass spectrometry, phosphoproteins must be enriched and separated from nonphosphorylated proteins prior to isolation by two-dimensional electrophoresis and identification by mass spectrometry. Immunoprecipitation using phospho-specific antibodies is a conventional, but powerful technique for the enrichment. However, some antibodies are not suitable for immunoprecipitation, and even though the antibodies can be used for immunoprecipitation, these phospho-specific antibodies are occasionally too expensive. Recently, immobilized metal-ion affinity chromatography, which is based on specific interactions between the phosphate group in phosphopeptides or phosphoproteins and Fe3+ or Ga3+ on the metal affinity support, has been developed; and convenient kits, such as Phosphopeptide enrichment spin columns (Clontech, Mountain View, CA, USA), Phosphoprotein enrichment kit (Clontech), Phosphoprotein purification kit (QIAGEN, Hilden, Germany), and Phosphopeptide isolation kit (Pierce), are now commercially available (21). On the other hand, a chemical reaction method to substitute the phosphate group is efficient for the enrichment of phosphoproteins and phosphopeptides. In this protocol, the phosphate group in phosphoproteins or phosphopeptides can be replaced by several functional groups, such as a biotincontaining tag, and subsequently purified by affinity chromatography based on the avidin-biotin interaction (22). Phosphoprotein-specific fluorescence staining using ProQ diamond phosphoprotein stain (Invitrogen) (23) is a simple and useful way for the comprehensive detection of phosphoproteins displayed on SDS-polyacrylamide gels, but care should be taken to avoid nonspecific staining of proteins. To detect phosphopeptides or phosphoproteins on blot membranes, ELISA plates, or tissue slices by fluorescence staining, Phosphoprotein detection reagent and kit (Pierce) can be used. Besides these techniques, a novel approach for proteomic analysis of phosphoproteins using antibodies broadly reactive against the consensus motif of phosphorylation sites has been developed to identify substrate proteins for the protein kinase of interest (24). In some cases, microarray technology can be utilized for screening and identification of kinase substrates (ProtoarrayTM kinase substrate identification kit, Invitrogen). For phosphoproteomic analysis, it is often important to estimate the phosphorylation level. Malachite Green phosphate detection kit (R & D systems, Minneapolis, MN, USA) and Phosphoprotein phosphate estimation assay kit (Pierce) (25) are used to determine the phosphorylation level of substrate proteins without using RIs.
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Some commercially available techniques to detect protein kinase activities
Key technique
Tool
Summary
Company and References
Time-resolved fluorometry
DELFIA® assay
Time-resolved fluorometry in ELISA format using lanthanide labeled phosphospecific antibody, biotin labeled phosphopeptide, and streptavidin-coated microwell plate. An enhancement solution of low pH is used to dissociate the lanthanide, which forms a stable fluorescent chelate. Low background due to long fluorescent decay times of the lanthanide.
Perkin Elmer (Wellesley, MA, USA) (www.perkinelmer.com) Ref. 2
HTScanTM kinase assay kit
A kit containing biotinylated substrate peptide, anti-phosphopeptide antibody and the Cell Signaling Technology protein kinase of interest. Readily compatible with DELFIA® assay. Europium labeled (Danvers, MA, USA) secondary antibody and DELFIA® enhancement solution are recommended. (www.cellsignal.com)
FRET
Z'-LYTETM kinase assay
FRET using substrate peptide with donor (coumarin) and acceptor (fluorescein). The differential protease sensitivity of phosphorylated versus non-phosphorylated substrate peptide is utilized. Unnecessary for specific antibody but difficulty in substrate peptide design.
TR-FRET
LanthaScreenTM TR-FRET assay
TR-FRET using terbium-conjugated phosphospecific antibody and fluorescein-labeled Invitrogen substrate peptide. Low background due to long fluorescent decay times of the lanthanide. (Carlsbad, CA, USA) (www.invitrogen.com)
IMAP (immobilized metal assay for phosphochemicals) TR-FRET
Both terbium-labeled TR-FRET donor and fluorescence labeled phosphopeptide bind to Molecular Devices nanoparticles through immobilized trivalent metals resulting in TR-FRET. No requirement (Sunnyvale, CA, USA) for antibody. (www.moleculardevices.com)
HTRF® (homogeneous time resolved fluorescence) TR-FRET
TR-FRET using Eu3+ cryptate-conjugated phosphospecific antibody and streptavidin Cisbio International coupled to allophycocyanin (XL665). Biotinylated peptide is phosphorylated by the (Bedford, MA, USA) kinase. Low background due to long fluorescent decay times of the lanthanide. (www.htrf-assays.com) Ref. 12
LANCE assay
TR-FRET between Eu3+-labeled phosphospecific antibody and streptavidin-coated Perkin Elmer fluorescent bead bound to biotin-labeled phosphopeptide. Low background due to long (Wellesley, MA, USA) fluorescent decay times of the lanthanide. (www.perkinelmer.com) Ref. 2
AlphaScreen assay
TR-FRET between a streptavidin-coated fluorescent dye-containing bead, which is bound to biotinylated phosphopeptide and acts as donor, and a second fluorescent dye-containing bead coated with a phosphospecific antibody binding to biotinylated phosphopeptide. The signal is significantly amplified by the singlet oxygen. Highly sensitive, greater dynamic range along with good assay reproducibility.
Far-Red PolarScreenTM fluorescence polarization kinase assay kit
Fluorescence polarization derived from binding of fluorescence-labeled phosphopeptide to Invitrogen phosphospecific antibody is detected. Far-red fluorescence polarization reduces back- (Carlsbad, CA, USA) ground. (www.invitrogen.com)
KinEASE
Fluorescence polarization derived from binding of fluorescence-labeled phosphopeptide to phosphospecific antibody is detected. Far-red fluorescence polarization at 645 nm to reduce background.
Upstate Cell Signaling Solutions (Charlottesville, VA, USA) (www.upstate.com)
IMAP-FP
Fluorescence labeled phosphopeptide, captured on modified nanoparticles through interactions with immobilized trivalent metals, results in high fluorescence polarization. Insensitivity to the sequence of flanking amino acids. Change in polarization derived from kinase reaction is largely enhanced.
Molecular Devices (Sunnyvale, CA, USA) (www.moleculardevices.com) Ref. 13
Luciferase-based ATP assay detected by luminescence
Kinase-Glo® luminescent kinase assay
Amount of ATP remaining in solution after termination of kinase reaction is quantified by Promega luminescence. No requirement for antibody, RI, specially designed peptide substrates, or (Madison, WI, USA) long reading time. (www.promega.com) Ref. 14
Fluorescence quenching assay
ProFluorTM kinase assay
Non-phosphorylated bisamide rhodamine substrate peptide is digested by protease, Promega resulting in the increase in fluorescence of rhodamine. Fluorescence signal is very stable. (Madison, WI, USA) (www.promega.com) Ref. 15
Antibody BeaconTM tyrosine kinase assay kit
Fluorescence quenching assay employing a small molecule tracer ligand labeled by Oregon Green 488. The tracer ligand exhibits quenched fluorescence when bound to antiphosphoTyr antibody. In the presence of phosphoTyr-containing peptides, the ligand is rapidly displaced, resulting in an increase in fluorescence. Real-time monitoring of kinase activity.
IQ kinase assay
Binding of Fe3+-containing compound to a phosphate group on fluorescence labeled Pierce phosphopeptide results in the quenching of fluorescence. Tolerant to both high and low (Rockford, IL, USA) concentrations of ATP and substrate. (www.piercenet.com) Ref. 16
QTL lightspeedTM kinase activity assays
Rhodamine-labeled phosphopeptide bound to Ga3+ metal ion on the polymer results in quench of polymer fluorescence. Wide dynamic range. Unnecessary for specific antibody.
TruLightTM kinase assays
Utilization of peptide substrates labeled with a quencher and microsphere coated with Merck Biosciences fluorescent polymers and a Ga3+. The phosphorylated peptides bind to microsphere, (San Diego, CA, USA) resulting in fluorescence quenching. Ten times more sensitivity than FRET-based assays. (www.merckbiosciences.com)
Fluorescence polarization
Invitrogen (Carlsbad, CA, USA) (www.invitrogen.com) Ref. 11
Perkin Elmer (Wellesley, MA, USA) (www.perkinelmer.com), Ref. 2
Invitrogen (Carlsbad, CA, USA) (www.invitrogen.com) Ref. 2
QTL Biosystems (Santa Fe, NM, USA) (www.qtlbio.com) Ref. 17
Technologies for Analyzing Protein Kinases
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SPA beads
Scintillation proximity assay (SPA)
Phosphorylation of biotinylated protein substrate with [γ-33P]ATP and capture of radiolabeled product on streptavidin-coated SPA beads. Light is emitted and detected by scintillation counter or CCD imager. Radiometric assay. Unnecessary for separation step and available for microplate scintillation counter.
Fluorometry, Agarose electrophoresis
PepTag® assay for non-radioactive detection of protein kinases
Phosphorylated and non-phosphorylated substrate peptides labeled by fluorescence are Promega separated by agarose electrophoresis due to the change in peptide’s net charge. Quantifica- (Madison, WI, USA) tion of kinase activity can be determined by fluorometric densitometer or spectro- (www.promega.com) fluorometer.
Streptavidin membrane
SignaTECT® protein kinase assay systems
Biotinylated substrate peptide is phosphorylated by kinase and [γ-32P]ATP, and then Promega captured by membrane with a high density of streptavidin. Low level of non-specific (Madison, WI, USA) substrate peptide binding results in low background. (www.promega.com) Ref. 19
Substrate beads
SignalScoutTM kinase profiling system
Kinase substrate proteins immobilized on beads. Radioactive, colorimetric and Stratagene fluorometric detection can be used. (La Jolla, CA, USA) (www.stratagene.com)
Biotin-avidin interaction, Ultrafiltration
AUSA® universal protein kinase assay kit
Binding of biotinylated substrate peptide to avidin is separated by ultrafiltration. Ultrafil- Transbio Corporation tration technique and radioactive detection. (Baltimore, MD, USA) (www.transbiocorp.com)
Table 2.
GE Healthcare Biosciences (Piscataway, NJ, USA) (www. gehealthcare.com) Ref. 18
Some commercially available techniques useful for phosphoproteomics
Key technique
Tool
Summary
Company and References
Metal affinity chromatography resin
Phosphopeptide enrichment spin columns
Spin columns contain an immobilized metal affinity chromatography resin that binds to phosphopeptides. Enrichment of phosphopeptides by spin column.
Clontech (Mountain View, CA, USA) (www.clontech.com) Ref. 21
Phosphoprotein enrichment kit
Immobilized affinity chromatography resin that binds to phosphoproteins or phospho- Clontech peptides. Enrichment of phosphoproteins by affinity column. (Mountain View, CA, USA) (www.clontech.com) Ref. 21
Phosphoprotein purification kit
Immobilized affinity chromatography resin that binds to phosphoproteins or phospho- QIAGEN peptides. Eluate from the affinity column is further concentrated and desalted by ultrafil- (Hilden, Germany) tration. (www1.qiagen.com) Ref. 21
Phosphopeptide isolation kit
Ga3+-chelated iminodiacetic acid (IDA)-based resin specifically traps phosphopeptides. Pierce (Rockford, IL, USA) Ga3+-based resin shows more specific binding than iron-chelated resin. (www.piercenet.com) Ref. 21
Fluorescence dye
ProQ diamond phosphoprotein stain
Fluorescent dye for detecting phosphoprotein displayed on SDS-PAGE and protein Invitrogen microarray. Small molecular fluorophore recognizes phosphoamino acids. (Carlsbad, CA, USA) (www.invitrogen.com) Ref. 23
Phosphoprotein detection
Phosphoprotein detection reagent and kit
Fe3+-activated derivatives of horseradish peroxidase can detect phosphorylated molecules Pierce by ELISA, immunohistochemistry, and Western blotting. Applicable to ELISA, immuno- (Rockford, IL, USA) histochemistry, and Western blotting. (www.piercenet.com) Ref. 21
Microarray
ProtoarrayTM kinase substrate identification kit
Kinase substrate protein microarray. Identification of kinase substrate using [γ-33P]ATP and autoradiography.
Colorimetric assay by Malachite Green
Malachite Green phosphate detection kit
Based on the Malachite Green-molybdate binding reaction. Phosphate from alkaline R & D systems hydrolysis of phosphoprotein is quantified by Malachite Green. (Minneapolis, MN, USA) (www.rndsystems.com) Ref. 25
Phosphoprotein phosphate estimation assay kit
Based on the Malachite Green-molybdate binding reaction. Phosphate from alkaline Pierce hydrolysis of phosphoprotein is quantified by Malachite Green. (Rockford, IL, USA) (www.piercenet.com) Ref. 25
Antibodies to detect multiple protein kinases simultaneously: Although hundreds of protein kinases have been documented to date, how many and what kind of protein kinases are expressed in cells and tissues under varying situations or in response to various stimuli are
Invitrogen (Carlsbad, CA, USA) (www.invitrogen.com)
still unknown. Therefore, it would be of great help if we could detect a variety of protein kinases expressed in cells or tissues with a simple procedure. For this purpose, we developed unique antibodies directed towards a highly conserved region of protein kinases.
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Monoclonal antibodies as well as polyclonal antibodies broadly reactive against serine / threonine kinases were obtained by immunization of a synthetic peptide (CVVHRDLKPENLLLAS), which was derived from a highly conserved subdomain VIB of serine / threonine kinases, as an anitigen (26, 27). Since these antibodies could recognize various serine / threonine kinases including CaM-kinases I, II, IV, CaM-kinase kinase, protein kinase A, MAP-kinase (ERK), and MAP-kinase kinase (MEK), we called them “Multi-PK antibodies”. By screening with the monoclonal antibodies, cDNA clones for various known and novel serine / threonine kinases, but not tyrosine kinases, have been isolated (27, 28). Moreover, these antibodies could immunoprecipitate various protein kinases from crude cell extracts. This implies that protein kinases can be immunoprecipitated with the “Multi-PK antibody” and analyzed for their activation states in response to various extracellular stimuli by an in-gel protein kinase assay (27). The “Multi-PK antibody” is also applicable to plant molecular biology because the subdomain VIB of protein kinases is conserved from plant to mammals. When a cDNA library of Lotus japonicus was screened with the “Multi-PK antibodies”, 178 positive clones were obtained. Among them, 164 clones (92%) were found to encode putative serine / threonine protein kinases and kinase-like proteins (29). We also prepared monoclonal antibodies with broad reactivity to tyrosine kinases using a conserved amino acid sequence (CYVHRDLRAAN) in the subdomain VIB of Src kinase as an immunogen (30). A monoclonal antibody, designated as YK34, reacted with not only Src kinase, but also nine different Src mutants with various amino acid substitutions in the subdomain VIB, which were designed so as to correspond to those of many other tyrosine kinases in humans. Since YK34 appeared to react with various tyrosine kinases, but not with serine / threonine kinases, it seems to be a “Multi-PK antibody” for tyrosine kinases. We observed significant changes in immunoreactive bands with YK34 in HL-60 cell extracts along with changes in cell morphology induced by TPA treatment (30). Thus, the “Multi-PK antibodies” can be powerful tools for proteomic analysis focused on serine / threonine and / or tyrosine kinases. Conclusion In this review, we discussed recent advances in technologies useful for protein kinase research. At first, we summarized recently developed and commercially available techniques to characterize activities of protein kinases as well as conventional protein kinase assays. Most of the former are rapid and highly sensitive
techniques suitable for high-throughput screening. Although we surveyed and listed representative techniques, modified, improved, or combined techniques will appear soon. Next, we discussed recently developed techniques useful for phosphoproteomics. Finally, we introduced a novel approach for detection and characterization of protein kinases using “Multi-PK antibodies”. The techniques described here will provide us with powerful tools to explore the protein kinase world, leading to clarification of molecular mechanisms of signal transduction, and to establishment of novel therapeutic approaches for human disorders including cancer. Acknowledgments We thank Prof. Leslie Sargent Jones (University of South Carolina) and Prof. Kenneth Takeda (University of Louis Pasteur, Strasbourg) for critical reading of this manuscript. This work was supported, in part, by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan, the Smoking Research Foundation of Japan, the Akiyama Foundation, the Osaka Medical Research Foundation for Incurable Diseases, the Naito Foundation, and also by an AIST Research Grant. References 1 Manning G, Plowman GD, Hunter T, Sudarsanam S. Evolution of protein kinase signaling from yeast to man. Trends Biochem Sci. 2002;27:514–520. 2 Olive DM. Quantitative methods for the analysis of protein phosphorylation in drug development. Expert Rev Proteomics. 2004;1:327–341. 3 Hardie DG, editor. Protein phosphorylation: a practical approach. 2nd ed. New York: Oxford University Press; 1999. 4 Bischoff KM, Liang S, Kennelly PJ. The detection of enzyme activity following sodium dodecyl sulfate polyacrylamide gel electrophoresis. Anal Biochem. 1998;260:1–17. 5 Kameshita I, Taketani S, Ishida A, Fujisawa H. Detection of a variety of Ser/Thr protein kinases using a synthetic peptide with multiple phosphorylation sites. J Biochem. 1999;126:991–995. 6 Matsumoto H, Kahn ES, Komori N. Nonradioactive phosphopeptide assay by matrix-assisted laser desorption ionization time-of-flight mass spectrometry: application to calcium / calmodulin-dependent protein kinase II. Anal Biochem. 1998;260:188–194. 7 Ross AH, Baltimore D, Eisen HN. Phosphotyrosine-containing proteins isolated by affinity chromatography with antibodies to a synthetic hapten. Nature. 1981;294:654–656. 8 Rijksen G, Van Oirschot BA, Staal GE. Nonradioactive assays of protein-tyrosine kinase activity using anti-phosphotyrosine antibodies. Methods Enzymol. 1991;200:98–107. 9 Miyawaki A, Tsien RY. Monitoring protein conformations and interactions by fluorescence resonance energy transfer between
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mutants of green fluorescent protein. Methods Enzymol. 2000;327:472–500. Han S, Zhou V, Pan S, Liu Y, Hornsby M, McMullan D, et al. Identification of coumarin derivatives as a novel class of allosteric MEK1 inhibitors. Bioorg Med Chem Lett. 2005;15: 5467–5473. Rodems SM, Hamman BD, Lin C, Zhao J, Shah S, Heidary D, et al. A FRET-based assay platform for ultra-high density drug screening of protein kinases and phosphatases. Assay Drug Dev Technol. 2002;1:9–19. Mathis, G. HTRF technology. J Biomol Screen. 1999;4:309– 314. Sportsman JR, Gaudet EA, Boge A. Immobilized metal ion affinity-based fluorescence polarization (IMAP): advances in kinase screening. Assay Drug Dev Technol. 2004;2:205–214. Koresawa M, Okabe T. High-throughput screening with quantitation of ATP consumption: a universal non-radioisotope, homogeneous assay for protein kinase. Assay Drug Dev Technol. 2004;2:153–160. Leytus SP, Melhado LL, Mangel WF. Rhodamine-based compounds as fluorogenic substrates for serine proteinases. Biochem J. 1983;209:299–307. Morgan AG, McCauley TJ, Stanaitis ML, Mathrubutham M, Millis SZ. Development and validation of a fluorescence technology for both primary and secondary screening of kinases that facilitates compound selectivity and site-specific inhibitor determination. Assay Drug Dev Technol. 2004;2:171–181. Rininsland F, Xia W, Wittenburg S, Shi X, Stankewicz C, Achyuthan K, et al. Metal ion-mediated polymer superquenching for highly sensitive detection of kinase and phosphatase activities. Proc Natl Acad Sci U S A. 2004;101:15295– 15300. Beveridge M, Park YW, Hermes J, Marenghi A, Brophy G, Santos A. Detection of p56(lck) kinase activity using scintillation proximity assay in 384-well format and imaging proximity assay in 384- and 1536-well format. J Biomol Screen. 2000;5:205–212. Xuei X, David CA, Middleton TR, Lim B, Pithawalla R, Chen CM, et al. Use of SAM2® biotin capture membrane in microarrayed compound screening (µARCS) format for nucleic acid polymerization assays. J Biomol Screen. 2003;8:273–282.
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20 Reinders J, Sickmann A. State-of-the-art in phosphoproteomics. Proteomics. 2005;5:4052–4061. 21 Andersson L, Porath J. Isolation of phosphoproteins by immobilized metal (Fe3+) affinity chromatography. Anal Biochem. 1986;154:250–254. 22 Sechi S, Oda Y. Quantitative proteomics using mass spectrometry. Curr Opin in Chem Biol. 2003;7:70–77. 23 Schulenberg B, Aggeler R, Beechem JM, Capaldi RA, Patton WF. Analysis of steady-state protein phosphorylation in mitochondria using a novel fluorescent phosphosensor dye. J Biol Chem. 2003;278:27251–27255. 24 Zhang H, Zha X, Tan Y, Hornbeck PV, Mastrangelo AJ, Alessi DR, et al. Phosphoprotein analysis using antibodies broadly reactive against phosphorylated motifs. J Biol Chem. 2002;277:39379–39387. 25 Harder KW, Owen P, Wong L, Aebersold R, Clark-Lewis I, Jirik FR. Characterization and kinetic analysis of the intracellular domain of human protein tyrosine phosphatase beta (HPTP beta) using synthetic phophopeptides. Biochem J. 1994;298:395–401. 26 Kameshita I, Kinoshita S, Shigeri Y, Tatsu Y, Yumoto N, Ishida A. Generation of a polyclonal antibody that simultaneously detects multiple Ser /Thr protein kinases. J Biochem Biophys Methods. 2004;60:13–22. 27 Kameshita I, Tsuge T, Kinashi T, Kinoshita S, Sueyoshi N, Ishida A, et al. A new approach for the detection of multiple protein kinases using monoclonal antibodies directed to the highly conserved region of protein kinases. Anal Biochem. 2003;322:215–224. 28 Kinoshita S, Sueyoshi N, Suetake I, Nakamura M, Tajima S, Kameshita I. Cloning and characterization of a novel Ca2+ /calmodulin-dependent protein kinase I homologue in Xenopus laevis. J Biochem. 2004;135:619–630. 29 Kameshita I, Nishida T, Nakamura S, Sugiyama Y, Sueyoshi N, Umehara Y, et al. Expression cloning of a variety of novel protein kinases in Lotus japonicus. J Biochem. 2005;137:33–39. 30 Sugiyama Y, Sueyoshi N, Shigeri Y, Tatsu Y, Yumoto N, Ishida A, et al. Generation and application of a monoclonal antibody that detects a wide variety of protein kinases. Anal Biochem. 2005;347:112–120.