Escaping the Heat

Nonradioactive Kinase Assay Kits Safety concerns and economic considerations have fueled a growing trend in the biomedical sciences: to shun the use of radioactivity when practical. Nonradioactive options for numerous applications have become widely available, including a number of nonradioactive kinase assay kits. Assays from different manufacturers employ a wide range of strategies. Most of these kits utilize antibodies, but two nonimmunochemical approaches use fluorescently tagged substrates and employ different means of separating phosphorylated and nonphosphorylated substrate fractions. Kits using immunochemical reagents employ either "general use" antiphosphotyrosine preparations or phosphospecific antibodies that bind target antigens phosphorylated at specific serine (Ser), threonine (Thr) and/or tyrosine (Tyr) residues. Substrates may be random polymers, peptide substrates based on the phosphorylation sites of different proteins, phosphorylatable domains, or full-length substrate proteins. Some kits can be used for the assay of a broad group of enzymes within a given kinase family; others are tailor-made for a particular kinase. The types of experimental techniques employed in these kits also vary widely. The equipment needed for kits that require electrophoresis, centrifugation, ELISA, or immunoprecipitation, can likely be found in most of today's basic research laboratories. Kits that employ fluorescence polarization, a fluorescent microsphere reporting system, homogenous time-resolved fluorescence, or amplified luminescent proximity homogeneous assays require more sophisticated instrumentation but are amenable to high-throughput (HT) analysis. Some of the key features of nonradioactive kinase kits on the market are outlined below. Upstate Biotechnology's BeadLyte kits uses color-coded microspheres. Playing Tag Promega of Madison, Wis., offers two PepTag® assays, one for protein kinase A (PKA), and one for protein kinase C (PKC). These unique assays utilize positively charged, fluorescently tagged peptide substrates; a fluorescently labeled Kemptide1 substrate peptide is used in the PKA kit, whereas the PKC assay uses an analog of the amino-terminal portion of glycogen synthase.1 Phosphorylation of either peptide substrate alters its net charge from +1 to -1 allowing the phosphorylated and nonphosphorylated substrate fractions to be separated by gel electrophoresis. Spectrophotometry, densitometry, or spectrofluorometry can be used to quantify the peptides. According to Promega's product literature, PepTag assays employing fluorescence detection can be used to detect subnanomolar levels of PKC or PKA. After demonstrating that residue Ser177 is the major site of rat prolactin phosphorylation, a research group at the University of California, Riverside, used the PepTag system to verify that a peptide substrate corresponding to the relevant portion of rat prolactin (residues 161-180) competes with the fluorescently labeled PepTag substrate in PKA assays.2 The SpinZyme™ colorimetric assays from Pierce of Rockford, Ill., also employ fluorescently labeled peptide substrates for the assay of PKA or PKC; however, these kits utilize affinity membrane separation units to separate phosphorylated and nonphosphorylated substrate fractions. The amino acid portion of the PKA substrate corresponds to the Kemptide1 substrate. Pierce offers four different SpinZyme kits for PKC assays, each containing a different peptide substrate. These substrates include a Ser-substituted analog of a PKC pseudosubstrate sequence1 as well as peptides based on phosphorylation sites of glycogen synthase1, myelin basic protein (MBP)3, or neurogranin.4 After the PKA or PKC reaction has been performed, an aliquot is applied to the metal chelate affinity membrane of a SpinZyme separation unit. The membrane is washed, removing nonphosphorylated substrate. The phosphorylated product is then eluted and quantified via spectrophotometry or spectrofluorometry. The SpinZyme devices are also sold separately for researchers who want to design their own assays. Wishing Wells A number of companies offer ELISA-based kinase assays. A versatile kit from Calbiochem® of San Diego can be used to selectively measure the activity of either PKC or PKA. The kit contains 12 eight-well microstrips coated with an immobilized peptide based on a pseudosubstrate sequence1 along with the reagents needed for both types of kinase reactions and subsequent ELISA. PKC or PKA reactions are performed in the microstrip wells and kinase activity is detected via subsequent incubations with a biotinylated antibody specific for the phosphorylated pseudosubstrate and streptavidin-horseradish peroxidase (HRP). Oxidation of the chromogenic HRP substrate o-phenylenediamine is monitored using an ELISA plate reader. "This kit is suitable for various applications, such as screening inhibitors or activators of PKA and/or PKC, monitoring the purification of either of these kinases, and evaluating the effects of pharmacological agents on either enzyme activity," comments Suzan Oberle, product manager, Immunochemicals, Calbiochem. However, Oberle adds, "We don't recommend using this kit for determining the calcium or phospholipid dependencies of PKC because certain isoforms of PKC (such as zeta) phosphorylate the pseudosubstrate in a calcium- and phospholipid-independent manner." Oncogene Research Products of Boston manufactures an ELISA-based PTK assay that employs a random polymer substrate. This polymer contains multiple Tyr residues but is devoid of Ser and Thr residues, and can be phosphorylated by a wide range of PTKs. Reactions are performed in microplate wells (12 strips of eight) coated with the polymer substrate. A HRP-conjugated anti- phosphotyrosine antibody (mAb PY20)5 is used to detect Tyr-phosphorylated substrate. Conversion of the chromogenic HRP substrate 3,3',5,5'-tetramethylbenzidine (TMB) is monitored using an ELISA plate reader. This kit is also available (with the same catalog number and pricing) from Calbiochem, a "sister company" of Oncogene Research Products. Similar principles are employed in the PTK assay kit offered by Sigma of St. Louis, but Sigma's kit employs a different anti-phosphotyrosine mAb (mAb PT-66) and uses o-phenylenediamine instead of TMB. Upstate Biotechnology of Lake Placid, N.Y. offers two versions of an ELISA-based kit that can be used for the assay of numerous Ser/Thr kinases. This ELISA-based kinase assay differs from those currently available from other companies in that it employs a chemiluminescent detection system. Kits contain a 96- or 384-well microplate coated with the substrate MBP. Immunodetection of phosphorylated MBP is via subsequent incubations with two antiphosphoserine/ phosphothreonine mouse mAbs and an HRP-labeled secondary antibody. The chemiluminescent DuoLuX™ substrate from Vector Laboratories Inc. of Burlingame, Calif., is supplied in the kits. "Chemiluminescent detection systems for microplate assays ... will perhaps replace traditional colorimetric ELISA detection methods in a few years, at least among industrial scientists. [They] are more sensitive, more time efficient, and less prone to variability than are their colorimetric counterparts," comments Greg Moore, drug discovery product manager, Upstate Biotechnology. The ELISA-based PTK assay from Roche Molecular Biochemicals of Indianapolis employs a somewhat different tactic in that biotinylated substrate is captured in streptavidin-coated wells. Two biotinylated peptide substrates supplied with the kit; one is based on the amino-terminal region of cdc2,6 and the other on a portion of gastrin.7 After the PTK reaction has been performed and quenched, the unfractionated biotinylated substrate (phosphorylated plus nonphosphorylated forms) is immobilized to streptavidin-coated wells. Phosphorylated substrate is immunodetected via binding to a HRP-conjugated anti-phosphotyrosine (mAb 3-365-10).8 This kit employs the HRP substrate azino-diethylbenzotyazol-sulfonate (ABTS) and contains 12 eight-well streptavidin-coated microtiter plate modules. Pierce's three ELISA-based PTK assay kits utilize a similar strategy. Each kit features a different biotinylated peptide substrate, based on phosphorylation sites of cdc2,6 gastrin,7 or c-Src,9 immobilized to NeutrAvidin™-coated microplate wells. After the PTK reaction is performed, phosphorylated substrate is detected via use of an HRP-labeled anti-phosphotyrosine (mAb PY20). The kit includes Pierce's ready-to-use 1-Step™ Turbo TMB HRP substrate for plate development. Packard Instrument's HTRF PTK assay (right) takes advantage of FRET to detect enzymatic activity. Come on Down! Cell Signaling Technology Inc. (CST) of Beverly, Mass., offers kits for the assay of Akt kinase, SAPK/JNK, p38 MAP kinase or p44/42 MAP kinase. The kinase is first selectively precipitated via use of an immobilized antibody or substrate. Kinase activity is then quantified in western blot assays using phosphospecific antibodies. These assay kits include reagents and membranes for the HRP-based chemiluminescent detection of the secondary antibody in immunoblot analyses. In the Akt kinase assay kit, a mouse anti-Akt mAb agarose conjugate is used to immunoprecipitate the serine kinase from cell lysates. The immunoprecipitate is then incubated with a GSK-3 fusion protein, which is phosphorylated by Akt at Ser9 and Ser21. The reaction is monitored using a rabbit polyclonal antibody specific for phosphorylated GSK-3-*/ß. The kits for measuring the activities of the mitogen-activated protein (MAP) kinases employ two phospho-specific antibodies each. The signal transduction enzyme p38 MAP kinase is active when phosphorylated at residues Thr180 and Tyr182. An immobilized mouse mAb specific for the dually phosphorylated form of p38 MAP kinase is used to precipitate the active fraction of the enzyme from cell lysates. Enzymatic activity is monitored via phosphorylation of an ATF-2 fusion protein at Thr71. Similarly, the kit for assaying p44/42 MAP kinase employs an immobilized phosphospecific mAb for the selective precipitation of activated p44/42 MAP kinase, which is phosphorylated at both Thr202 and Tyr204. The p44/42 MAP kinase catalyzes the phosphorylation of Elk-1 at Ser383, which is detected using a phosphospecific polyclonal antibody. The SAPK/JNK assay kit is different from the others in that the selective precipitation step is effected via use of an immobilized fusion protein corresponding to the amino-terminal 89 amino acid residues of c-Jun. This protein then serves as the substrate in the subsequent kinase reaction. SAPK/JNK binds the c-Jun fusion protein immediately amino-terminal to its phosphorylation sites at Ser63 and Ser73. SAPK/JNK activity is measured via use of an antibody specific for phosphorylation at Ser63. "CST also offers PhosphoPlus® Antibody Kits, which were developed to allow qualitative determination of the phosphorylation status of a specific substrate protein. Although these kits do not fall into the 'classic' category of kinase assays, they do allow the researcher to determine upstream kinase activity," explains Liisa Eisenlohr, marketing manager, CST. Fourteen different PhosphoPlus kits are currently available. Each kit contains one or two phosphospecific antibodies for the detection of the following targets: Bad (Ser112 and Ser136); CREB (Ser133); Elk-1 (Ser383); I*B-* (Ser32); c-Jun (Ser63 and Ser73); MEK-1/2 (Ser217/221); MKK3/MKK6 (Ser189/207); ATF-2 (Thr71); p70 S6 kinase (Thr389 and Thr421/Ser424); cdc2 (Tyr15); TrkA (Tyr490 and Tyr674/675); p38 MAP kinase (Thr180/Tyr182); p44/42 MAP kinase (Thr202/Tyr204); or SAPK/JNK (Thr183/Tyr185). These kits also contain "panning" antibodies for assessing total antigen levels (phosphorylated plus nonphosphorylated forms), along with the cell extract controls, reagents and membranes needed for western blot analyses. Upstate Biotechnology also vends several kinase assay kits that employ the "immunoprecipitation followed by western blot analysis" strategy. Kits for the assay of Akt, mitogen-activated protein kinase activated protein (MAPKAP) kinase-2, or MAP kinase activities are currently available. In the Akt assay kit, the kinase is precipitated and monitored by phosphorylation of a Bad fusion protein at Ser136. In the MAPKAP kinase-2 assay kit, the enzyme is immunoprecipitated and subsequent phosphorylation of recombinant HSP27 at Ser78 is detected via immunoblot analysis. In the kit for the assay of MAP kinase, the enzyme is precipitated with an immobilized anti-peptide rabbit antibody; its activity is monitored by use of a mouse mAb specific for myelin basic protein (MBP) phosphorylated at Thr98. Upstate Biotechnology's less expensive MAP Kinase Assay Kit contains the MBP substrate and reagents needed for performing the MAP kinase reaction as well as the phosphospecific antibody required for subsequent immunoblot analysis. However, this kit does not contain reagents for the selective precipitation of the kinase; thus, this kit is most useful for monitoring the activity of MAP kinase that has already been partially purified, perhaps by chromatography or via precipitation with the researcher's own immobilized antibody. Though the product insert indicates that this kit can also be used for monitoring MAP kinase activities of crude cell lysates, it issues a couple of well-founded cautions. Notably, although the kit includes an inhibitor cocktail that targets other kinases known to phosphorylate MBP, one cannot rule out the possibility of contributions from unknown kinases in crude lysates. Further, components of cell lysis buffers may inhibit MAP kinase. High Tech Solutions PanVera Corp. of Madison, Wis., offers homogeneous fluorescence polarization (FP)-based assays for the detection of PKC or PTK activities. PanVera's product literature indicates that the PKC assay kit can detect the alpha, beta I, beta II, gamma, delta, epsilon or zeta isoforms of PKC. Recent results show that researchers can also use the kit to detect the activity of PKA or GSK-3-ß. This kit can be used with a random polymer substrate (poly-Glu,Tyr), or with any of a wide variety of other peptide substrates. The company does not include substrates in these kits because they can each be used for the assay of a wide variety of different kinases. PanVera sells a pseudosubstrate-based peptide1 for use in PKC assays separately; researchers will need to obtain alternative PKC substrates and substrates for PTK assays elsewhere. The FP assays exploit the differential rotation rates of free versus antibody-bound fluorescent molecules. In these assays, proprietary fluorescent phosphopeptide tracers compete with nonfluorescent phosphopeptides generated during kinase reactions for binding to phosphospecific antibodies. Each kit includes a relevant phosphopeptide competitor that can be used for assay validation. After the kinase reactions have been quenched, fluorescent phosphopeptide tracer/antiphosphopeptide complexes are added to each sample. If no kinase reaction products are present in a given sample, most of the fluorescent tracer will remain bound to the antibody. If a kinase reaction occurred, the resultant unlabeled phosphopeptide products will compete with the tracer for binding to the antibody. After equilibrium is reached, plane-polarized light is used to excite the fluorescent tracers. This is the step at which differential tumbling rates come into play. If the excited molecule remains stationary during the period of excitation (4 nanoseconds for fluorescein), it will re-emit highly polarized light. However, if the molecule rotates or tumbles during this period, it will emit light in different directions as it moves about, and the light will be depolarized. In the kinase assays, the displaced fluorescent phosphopeptide tracers, unfettered by antibodies, will emit depolarized light, while the tumbling rates of the antibody-bound fluorescent molecules will be greatly decreased, resulting in highly polarized emission. As the amount of polarization depends upon the ratio of bound and free fluorescent molecules, the measured polarization value is an indicator of enzymatic activity. Researchers can measure fluorescence polarization via HT systems (described in a recent report from the scientific staff of PanVera10) or PanVera's single-tube Beacon® 2000 Fluorescence Polarization Instrument. Nanomolar concentrations of reaction products can be detected in assays employing the FP-based kits for PKC or PTKs. The relatively fast "mix-and-read" format of these assays makes them particularly useful for signal transduction research and HT screening drug discovery applications. Luminex Corp. of Austin, Texas, has developed the laboratory multianalyte profiling (LabMAP™) system, which employs internally color-coded microspheres and laser detection methodology for the simultaneous measurement of up to 100 analytes in a single reaction vessel. In September of this year, Upstate Biotechnology launched a homogenous fluorescent Ser/Thr kinase assay kit based on this detection technology using the trade name Beadlyte™. This kit can be used for the assay of a wide variety of Ser/Thr kinases. The substrate MBP is covalently linked to the surfaces of fluorescently labeled microspheres. After the kinase reaction, phosphorylated MBP is detected by adding a mixture of two antiphosphoserine/ phosphothreonine mouse mAbs, followed by a biotinylated secondary antibody and a streptavidin-phycoerythrin conjugate. Thus, there are two fluorescent species in this assay: the substrate-conjugated fluorescent microsphere, and the streptavidin-phycoerythrin "reporter" reagent. Quantification is via the Luminex 100™ instrument, which uses microfluidics to align the microspheres in single file and employs two lasers, one for the detection of the fluorescent microsphere itself, and the other for the reporter reagent. The color signals are captured by an optics system and translated into binding data via digital signal processing. Packard Instrument Co. of Meriden, Conn., offers homogenous time-resolved fluorescence (HTRF) PTK assays. HTRF assays exploit fluorescence resonance energy transfer (FRET) between europium cryptate and XL665, a modified allophycocyanin. Europium cryptate becomes excited when bombarded with light at 337 nm, and normally fluoresces at 620 nm. However, if this fluorophore is proximal to XL665, FRET occurs, and the output signal is shifted to 665 nm. Researchers may choose between kits containing either of two synthetic, biotin-labeled, Tyr-containing polypeptide substrates, or supply their own biotinylated PTK substrates. After the kinase reaction, a europium cryptate-labeled antiphosphotyrosine (mAb PY20 or mAb PT-66) conjugate is added along with a streptavidin-XL665 conjugate. The europium-antibody conjugate binds the fraction of biotinylated substrate that has become Tyr- phosphorylated and the biotinylated peptide substrate binds streptavidin-XL665. This brings the europium cryptate-labeled antibody into close proximity with streptavidin-XL665, which results in a long-lived signal at 665 nm via FRET. This assay can be performed in 96-, 384-, or 1536-well microplates, and is particularly useful for HT screening applications. Packard Instrument has also recently developed AlphaScreen™ (Amplified Luminescent Proximity Homogeneous Assay Screen) assays. The company offers an AlphaScreen kit for the assay of c-Src; reagents that may be used for the assay of ERK1 MAP kinase are sold separately. AlphaScreen assays utilize "donor" and "acceptor" beads. Upon irradiation with laser light (680 nm), a photosensitizer in the donor bead converts ambient oxygen to singlet-state oxygen. The excited singlet-state oxygen molecules normally diffuse approximately 250 nm (one bead diameter) and decay. However, if the donor bead is in close proximity to an acceptor bead, the singlet-state oxygen molecules react with chemiluminescent groups in the acceptor beads, transferring energy to fluorescent acceptors in the same bead, and shifting the emission wavelength to 520-620 nm. Beads are brought into proximity by biological interactions between proteins bound to the surfaces of the beads. For example, the AlphaScreen c-Src tyrosine kinase assay kit employs a biotinylated peptide substrate based on a region of cdc2.6 After the kinase reaction, streptavidin-labeled donor beads are added along with an anti-phosphotyrosine (mAb PT-66) conjugated to acceptor beads. The streptavidin-labeled donor beads bind the biotinylated substrate. Anti-phosphotyrosine/ acceptor bead conjugates bind the phosphorylated substrate fraction, thus bringing acceptor beads into close contact with donor beads. The MAP kinase assay employs a slightly different substrate capture strategy than does the c-Src assay. In this case, two different antibodies are used to capture the phosphorylated peptides onto acceptor beads: digoxigenin-labeled antiphosphothreonine, and antidigoxin immobilized onto acceptor beads. This "antibody sandwich" approach enables substitution with a wide range of substrate/antibody pairs. Packard Instruments supplies all of the reagents needed for the ERK1 MAP kinase assay except for digoxigenin-labeled antiphosphothreonine. Both AlphaScreen assays can be performed in standard 96-well microplates, and have also been miniaturized and fully automated in the 384- and 1536- well formats. Something for Everyone A wide variety of nonradioactive kinase assays have been developed, and some of the features of commercially available options have been discussed above. See the accompanying table for summary information regarding various kinase assay kits. Each assay has its own set of advantages and limitations, and the appropriateness of a given approach depends entirely on the nature and scale of the application at hand. In some cases, a conventional radioactive approach may still be the most viable option. Not surprisingly, nonradioactive phosphatase assays have also been developed and are available from various companies, including some of those featured in this article. S Deborah A. Fitzgerald can be contacted at df@sciwriters.com. References 1. B.E. Kemp, R.B. Pearson, "Design and use of peptide substrates for protein kinases," Methods in Enzymology, 200:121-34, 1991. 2. Y.-F. Wang et al., "Identification of the major site of rat prolactin phosphorylation as serine 177," Journal of Biological Chemistry, 271:2462-9, 1996. 3. I. Yasuda et al., "A synthetic peptide substrate for selective assay of protein kinase C." Biochemical and Biophysical Research Communications, 166:1220-7, 1990. 4. A. Gonzalez et al., "Use of the synthetic peptide neurogranin(28-43) as a selective protein kinase C substrate in assays of tissue homogenates," Analytical Biochemistry, 215:184-9, 1993. 5. J.R. Glenney Jr. et al., "Monoclonal antibodies to phosphotyrosine," Journal of Immunological Methods, 109:277-85, 1988. 6. H.C. Cheng et al., "A synthetic peptide derived from p34cdc2 is a specific and efficient substrate of src-family tyrosine kinases," Journal of Biological Chemistry, 267:9248-56, 1992. 7. G.S. Baldwin et al., "Phosphorylation of gastrin-17 by epidermal growth factor-stimulated tyrosine kinase," Nature, 301:435-7, 1983. 8. G. Steinhilber et al., "Highly specific characterization of tyrosine phosphoproteins in tumor cells based on monoclonal antibodies defined by conjugated phosphotyramine," Anticancer Research, 10:907-11, 1990. 9. J.E. Casnellie et al., "Phosphorylation of synthetic peptides by a tyrosine protein kinase from the particulate fraction of a lymphoma cell line," Proceedings of the National Academy of Sciences, 79:282-6, 1982. 10. G.J. Parker et al., "Development of high throughput screening assays using fluorescence polarization: nuclear receptor-ligand-binding and kinase/phosphatase assays," Journal of Biomolecular Screening, 5:77-88, April 2000.

Escaping the Heat

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