Tyr'd and True: Immunochemical reagents and kits for studying tyrosine phosphorylation

Tyrosine Phosphorylantion State-Sensitive Antibodies Protein tyrosine kinases (PTKs) are a diverse lot, including both transmembrane and soluble cytoplasmic enzymes. Roughly 10 percent of a cell's proteins are subject to covalent modification via phosphorylation, but tyrosine (Tyr) residues are modified in only about one out of every 100 protein phosphorylation events. When Tyr-phosphorylation occurs, cells pay heed. PTKs play vital roles in a wide variety of cellular processes, including growth, differentiation, and metabolism. Immunohistochemical staining with mAb 4G10. Image provided by Upstate Biotechnology. If you are in the business of detecting, visualizing, or purifying Tyr-phosphorylated proteins, then antiphosphotyrosine antibodies [ anti-(P)Tyrs] will definitely be on your shopping list. Several types of anti-(P)Tyrs are commercially available. Polyclonal preparations are sold by various vendors, and a number of different monoclonal anti (P)Tyrs are also on the market. Most of the currently available anti-(P)Tyrs bind free (P)Tyr quite well--with affinities in the µM range or better. Cross-reaction with phosphoserine and phosphothreonine [(P)Ser and (P)Thr] is not generally a problem with these immunoreagents, but monoclonal anti- bodies generated in some of the early efforts can bind to nucleotides and (P)His. For a review of commercially available anti-(P)Tyr reagents and kits that contain them, read on. In the 1980s, different research groups were trying out various antigens for generating anti-(P)Tyrs. The first-reported anti-(P)Tyr was not generated directly against an immunogen bearing the phosphorylated amino acid residue but rather against a carrier protein coupled to p-azobenzyl phosphonate (ABP). The nonhydrolyzable moiety ABP was substituted for (P)Tyr because of fears that intracellular phosphatases might degrade (P)Tyr before an immune response could be mounted. "One ABP-induced mAb, 2G8, was extremely useful for characterizing and purifying a variety of phosphotyrosyl proteins," comments A. Raymond Frackelton, Jr., an associate professor at Brown University (Providence, R.I.). Clone 2G8 is still in use, and can be purchased from Covance Research Products, Inc. (Richmond, Calif.). However, Frackelton is quick to point out that this mAb has a couple of drawbacks. First, its binding affinity for phosphoproteins is relatively low. Because of this low binding affinity, clone 2G8 is most effectively used in applications in which it is coupled with a matrix such as agarose beads. Second, clone 2G8, like a number of other anti-ABPs, cross-reacts with mononucleotides and phosphohistidine. Measuring PTK activity with an ELISA-based kit. Data provided by Sigma. Immunogens composed of carrier proteins coupled with other substitutes for (P)Tyr were also evaluated, and some of the resultant antibodies are commercially available. For example, clone 4G10 was generated against a protein coupled with the nonhydrolyzable analog phosphotyramine, which may be purchased from Upstate Biotechnology (Lake Placid, N.Y.). In a different effort, mice were immunized with carrier proteins coupled with (P)Tyr itself, then boosted with phosphotyramine-containing conjugates. One mAb generated using this protocol, clone 1G2, is still widely used by different research groups and is marketed by a number of different companies. Oncogene Research Products (Cambridge, Mass.) includes clone 1G2 in its Immunohistochemistry System, a kit with reagents and controls for immunostaining tissue sections. Other researchers have found that (P)Tyr coupled with a carrier protein is a good immunogen for generating antibody reagents. In the late 1980s and early 1990s, research groups led by John R. Glenney, Jr. generated, then carefully characterized, a panel of eight such anti-(P)Tyrs. These were certainly not the first reports of mAbs of this type. But the high-quality work described in them resulted in a standout group of mAbs, several of which are still used in many laboratories. These mAb clones are easy to find in catalogs--they all have names with the prefix "PY." Antibody PY69 is of the IgG2a subtype, and PY20 is an IgG2b. The rest are IgG1. It is important to know the subtype of the mAb you are using because this will greatly influence your choice of antibody-binding reagents. For example, IgG2a/b mAbs bind well to Protein A, but mouse IgG1 does not. (P)Tyr binding of each mAb in this panel is inhibited by high salt concentrations as well as by divalent cations. The inhibitory salt effects are not particularly surprising. It seems reasonable to imagine that electrostatic interactions, which are inhibited by ionic salts, could play a role in the binding of some (P)Tyr-specific antibodies to the charged residue. The mAbs should be used with buffers that contain less than 150 mM NaCl, and divalent cation concentrations of no greater than 1 mM. Numerous vendors carry mAbs from this series, including PY20, PY54, PY69, and PY72. PY20 is something of a celebrity, and is marketed by almost every company that dabbles in anti-(P)Tyrs. Its (P)Tyr binding affinity is about three to four times greater than the others', and it is compatible with Protein A-based systems. A recombinant form of PY20, designated RC20, is available from Signal Transduction Laboratories (Lexington, Ky.). RC20 is basically an altered form of the antigen-binding portions of PY20. Mutations resulting in a 10-fold increase in RC20's affinity for (P)Tyr were introduced to improve its binding to the phosphorylated residue. RC20 lacks immunoglobulin constant regions, which do not contribute to antigen binding. Removing the antibody's constant regions decreases background problems associated with non-antigen-directed binding. However, it also takes away RC20's ability to bind Protein A or secondary antibodies specific for antibody constant regions. RC20 is sold as conjugates of biotin, horseradish peroxidase (HRP), or alkaline phosphatase (AP). These conjugates are useful for immunoprecipitation and immunoblotting applications. Measuring PTK activity with an HTRF-based assay. Image provided by Packard Instruments. Quite a few anti-(P)Tyr mAbs generated against (P)Tyr-carrier protein conjugates have been generated since those reported by Glenney's group, and many of them are commercially available. Most of the currently sold anti-(P)Tyr rabbit polyclonal preparations were also raised against this type of immunogen. Monoclonal offerings include clone B120.1, from Biomeda (Foster City, Calif.), and clone anti-(P)Tyr sold by PanVera Corp. (Madison, Wis.). Several companies marketing clone PT-66, which was generated against (P)Tyr coupled to KLH, include Sigma (St. Louis, Mo.) and RBI (Natick, Pa.). Oncogene Research Products offers three anti-(P)Tyr mAbs generated at Cold Spring Harbor (clones 2-104, 2-105, and 2-153). Clone 3B12, sold by Alexis Corp. (San Diego, Calif.), is noted for its preferential binding to the phosphorylated EGF receptor. Carrier proteins coupled with (P)Tyr or nonhydrolyzable substitutes were used to generate the mAbs discussed in the preceding sections. Luckily, reagents generated using these rationales cross-reacted quite well with a host of (P)Tyr-containing proteins. But do these mAbs bind equally well to all Tyr-phosphorylated proteins? Do the affinities of anti-(P)Tyrs for particular phosphoproteins vary in the context of the whole-protein environment? These and similar questions have prompted some research groups to try different strategies for generating anti-(P)Tyr mAbs. In one scheme, antibodies are first generated against a Tyr-phosphorylated protein or even a mixture of phosphoproteins. Resultant clones are then screened for the ability to bind (P)Tyr specifically. In 1990, several (P)Tyr-specific mAbs obtained after immunizing mice with different Tyr-phosphoproteins from chicken embryo fibroblasts were described by Steven B. Kanner and colleagues. One of these, clone 6G9, can be purchased from Covance Research Products or Life Technologies (Rockville, Md.). Several additional monoclonal preparations generated against purified (P)Tyr proteins, including clones Z-PY1, PY-7E1, and PY-1B2, are available from Zymed Laboratories, Inc. (San Francisco, .), and New England Biolabs (Beverly, Mass.) offers P-Tyr-100, a high-affinity mAb with (P)Tyr binding that is largely independent of the local amino acid sequence. Synthetic phosphopeptides coupled to carrier proteins have been used to raise antibodies that recognize (P)Tyr in the context of flanking amino acids. Alexis Corp. offers two mAbs of this type. Clone 9H8 was generated against a phosphopeptide containing the sequence Arg-Gly-(P)Tyr-Val-Pro. This mAb is capable of cross-reacting with proteins containing basic residues that are amino-terminal to (P)Tyr. The synthetic peptide portion of the immunogen used to generate clone 16F4 was Asp-Ile-(P)Tyr-Ala-Glu. Clone 16F4 tolerates hydrophobic amino acids directly neighboring the (P)Tyr. Phosphopeptide-carrier protein conjugates are also being used to generate a growing number of immunoreagents specific for the phosphorylated (or nonphosphorylated) forms of a given protein. Some mAbs of this type are available, but most of these reagents are polyclonal preparations. Among these reagents are those sensitive to the phosphorylation state of proteins that undergo Tyr-phosphorylation (see table). These antibodies are extremely useful for studying many different proteins regulated via phosphorylation. "Pan" antibodies that don't distinguish between different phosphorylation states enable estimations of a given protein's total levels. Phosphorylation state-specific antibodies allow one to access the fraction of particular proteins that have been called into service (or shut down) by phosphorylation. Most of the antibodies currently available bind (P)Tyr, but it is likely that more reagents targeting nonphosphorylated antigens will be developed. Phosphorylation-state-specific antibodies are offered by a growing number of companies. New England Biolabs offers a broad selection of individual phosphospecific antibodies, many of which are also available as components of the company's PhosphoPlus® kits. These kits include control antibodies, control proteins or cell extracts, and even detection reagents and molecular weight markers for chemiluminescent Western blot analyses. For example, some of these reagents have been used to study kinases important in different MAPK (mitogen-activated protein kinase)-type signal transduction pathways, including p38 MAPK, SAPK1, and p44,42 MAPK. These enzymes have activation loops containing Thr-X-Tyr motifs, and are fully activated only when diphosphorylated. Antibodies that bind only the dually phosphorylated forms of these important signal transduction proteins enable one to monitor the levels of activated enzymes. The signal transduction enzyme p38 MAPK is active when phosphorylated at residues Thr180 and Tyr182. New England Biolabs offers a nonradioactive immunoprecipitation-based kit for the detection of p38 MAPK activity. This kit uses two different phosphospecific antibodies: one that selectively precipitates active (diphosphorylated) p38 MAPK, and one that detects p38 MAPK-catalyzed phosphorylation of its substrate ATF-2. An immobilized mouse mAb specific for the dually phosphorylated form of p38 MAPK is used to immunoprecipitate the active fraction of the enzyme from cell lysates. The immunoprecipitate is then incubated with an ATF-2 fusion protein in a reaction buffer. Active p38 MAPK phosphorylates ATF-2 at Thr71. The reaction is monitored via Western blot analyses utilizing a rabbit polyclonal antibody specific for phosphorylated ATF-2. The kit includes reagents for the HRP-based chemiluminescent detection of rabbit antibodies on immunoblots. Along the same lines is a MAPK activity kit containing an immobilized mAb for the selective precipitation of activated MAPK, which is phosphorylated at Thr202 and Tyr204. MAPK catalyzes the phosphorylation of Elk-1 at Ser383. A phosphospecific polyclonal antibody is used to follow the conversion of an Elk-1 fusion protein. In a similar kit for monitoring MEK1,2 activity, active MEK1,2 is immunoprecipitated using an immobilized rabbit polyclonal antibody. The antibody binds specifically to MEK1,2 dually phosphorylated at Ser217 and Ser221. The substrate in this case is p42 MAPK. Activated MEK1,2 phosphorylates and activates p42 MAPK at Thr202 and Tyr204. The accumulation of dually phosphorylated p42 MAPK is monitored via Western blot analyses with a phosphospecific mouse mAb. Reagents for the chemiluminescent detection of the mAb are included. Antibodies specific for (P)Tyr have also been harnessed for use in PTK activity assays. For example, an ELISA-based kit offered by Sigma on microtiter plates employs a random polymer (PGT-poly-Glu,Tyr) that can be phosphorylated by a wide range of PTKs. This synthetic polypeptide contains multiple Tyr residues but is devoid of Ser and Thr residues. PTK-containing samples are diluted into a reaction buffer containing ATP and requisite cations, then added to the plate wells. Reactions are stopped by simply removing the mixtures, then washing the plates. Tyr-phosphorylated substrate is probed using HRP-conjugated to an anti-(P)Tyr mAb (clone PT-66). Conversion of the chromogenic HRP substrate o-phenylenediamine dihydrochloride is monitored using an ELISA plate reader. Using mAB PY20 to immunoprecipitate Tyr-phosphorylated paxillin from carbachol-treated cells. Figure adapted from B. E. Slack (1998) PNAS 95:7281-7286. Copyright (1998) National Academy of Sciences, U.S.A. Similar principles are employed in the PTK assay kit offered by Oncogene Research Products, but Oncogene's kit detects phosphorylated substrate using PY20. This kit also uses a different HRP substrate, tetramethylbenzidine (TMB). Three different ELISA-based kits offered by Pierce (Rockford, Ill.) also employ HRP-labeled PY20 for the detection of phosphorylated PTK substrates. In these kits, any of three different biotin-labeled peptide substrates are immobilized to NeutrAvidin™-coated microplate wells. The biotinylated PTK substrates correspond to portions of cdc2, src, and human gastrin peptide. PanVera offers a unique PTK homogeneous assay kit that uses fluorescence polarization (FP) as the detection method for a competitive binding assay. A fluorescent phosphopeptide tracer competes with nonfluorescent phosphopeptides generated during a PTK assay for binding to an anti-(P)Tyr. In FP, polarized light is used to excite fluorescent molecules in solution. Fluorescent probes that are free in solution rotate and tumble, causing the emitted light to become depolarized. When bound to a larger molecule, such as anti-(P)Tyr, the tumbling rates of the fluorescent molecules are greatly decreased, and the emitted light remains highly polarized. If there are no PTK reaction products present, most of the phosphopeptide tracer binds to anti-(P)Tyr and the polarization value is high. As a PTK phosphorylates any of a number of different peptide and protein substrates, these products compete with the tracer for anti-(P)Tyr binding. As the reaction progresses, an increasing fraction of the fluorescent tracer is displaced, resulting in a proportional decrease in polarization that is easily quantified. PanVera's PTK assay can also be used in kinetic studies to monitor phosphorylation events in real time. As the reaction proceeds and phosphotyrosines are generated, the polarization of the sample decreases. The assay is formatted for use in single tubes and microplates. A PTK assay based on homogenous time-resolved fluorescence (HTRF) is available from Packard Instrument Co. (Meriden, Conn.). This assay exploits fluorescence resonance energy transfer (FRET) between europium cryptate and XL665, a modified allophycocyanin. Europium cryptate becomes excited when bombarded with light at 337 nm. If left to its own devices, europium cryptate then fluoresces at 620 nm. But if this fluorophore is close to XL665, FRET occurs, resulting in a shift of the output signal to 665 nm. The PTK substrate in the Packard Instrument kit is a synthetic, biotin-labeled, Tyr-containing polymer. After the kinase reaction, europium cryptate-labeled anti-(P)Tyr conjugate is added along with streptavidin-XL665. The biotinylated peptide substrate binds streptavidin-XL665. Europium-antibody conjugate that is free in solution emits light at 620 nm after laser-induced excitation. The immunoconjugate then binds the fraction of biotinylated substrate that has become Tyr-phosphorylated. When this happens, the europium cryptate-labeled antibody is brought into close proximity with streptavidin-XL665 and FRET occurs, resulting in a long-lived signal at 665 nm. This assay is particularly useful for microplate-based high-throughput screening applications. Many other useful products are available for studying PTKs and (P)Tyr-proteins. If you are interested in generating and purifying large quantities of a protein modified by (P)Tyr, check out the TKPhos Express™ kit from Stratagene (La Jolla, Calif.). This product enables the generation of Tyr-phosphorylated fusion proteins in E. coli. The phosphoprotein of interest is cloned into a vector encoding a calmodulin-binding peptide (CBP) fusion protein, then transfected into competent cells bearing PTK-expressing plasmids. Expressed proteins are purified via binding to a calmodulin resin, followed by immunoaffinity chromatography using immobilized PY20. The kit also contains free PY20 for use in Western blot analyses and immunoassays. Various companies also market kits for radioactive PTK assays. Some of these assays now exploit biotin/avidin interactions for capturing the radiolabeled substrates. One example of a system employing this technology is the AUSA™ (Affinity Ultrafiltration Separation Assay) PTK assay, from Transbio Corp. (Baltimore, Md.). The reaction mix includes the PTK, along with a biotinylated peptide substrate and gamma-[32P]ATP. After the reaction, avidin, which binds the biotinylated peptides, is added to the mix. The mixture is then subjected to a centrifugal ultrafiltration through a membrane. The membrane retains the biotinylated peptide/avidin complexes, and free gamma-[32P]ATP passes through. Evaluating cdc-2 phosphorylation status using a PhosphoPlus® kit. Image provided by New England Biolabs. The SignaTECT™ PTK assay system from Promega (Madison, Wis.) provides another example of the adaptation of biotin/avidin interactions for kinase assays. Biotinylated peptide substrates are also used in this kit. After terminating the kinase reactions, aliquots of the mixtures are spotted onto SAM2™ Biotin Capture Membranes, which bind the biotinylated substrates, but not free gamma-[32P]ATP. A plethora of PTK inhibitors are also available from different commercial sources. Where there are kinases, there are phosphatases, and a number of different assay kits for Tyr phosphatases are also on the market. Of course, many cellular events are modulated via the covalent modification of other residues, particularly Ser and Thr. Phosphospecific antibodies for many proteins regulated by covalent modification of Ser and/or Thr (but not Tyr) are available from the same companies listed in the table. In fact, for practically every type of (P)Tyr-centered product mentioned in this article, parallel versions specific for (P)Ser and (P)Thr exist. Happy hunting. Tyrosine Phosphorylantion State-Sensitive Antibodies A.R. Frackelton Jr. et al., "Characterization and use of monoclonal antibodies for isolation of phosphotyrosyl proteins from retrovirus-transformed cells and growth factor-stimulated cells," Molecular and Cellular Biology, 3:1343-52, 1983. A.R. Frackelton Jr. et al., "Characterization and use of monoclonal antibodies for isolation of phosphotyrosyl proteins from retrovirus-transformed cells and growth factor-stimulated cells. The generation of monoclonal antibodies against phosphotyrosine and their use for affinity-purification of phosphotyrosine-containing proteins," Methods in Enzymology, 201:79-92, 1991. J.R. Glenney et al., "Monoclonal antibodies to phosphotyrosine," Journal of Immunological Methods, 109:277-85, 1988. R.D. Huhn et al., "Cell lines and peripheral blood leukocytes derived from individuals with chronic myelogenous leukemia display virtually identical proteins phosphorylated on tyrosine residues," Proceedings of the National Academy of Sciences (PNAS), 84(13):4408-12. S.B. Kanner et al., "Monoclonal antibodies to individual tyrosine-phosphorylated protein substrates of oncogene-encoded tyrosine kinases," PNAS, 87:3328-32, 1990. M. Ohtsuka et al., "Preparation and characterization of antibodies to o-phosphotyrosine and their use for identification of phosphotyrosine-containing proteins," International Journal of Cancer, 34:855-61, 1984. A.H. Ross et al., "Phosphotyrosine-containing proteins isolated by affinity chromatography with antibodies to a synthetic hapten," Nature, 294:654-6, 1981. S. Ruff-Jamison et al., "Heavy and light chain variable region sequences and antibody properties of anti-phosphotyrosine antibodies reveal both common and distinct features," Journal of Biological Chemistry, 266:6607-13, 1991. Sengupta et al., "Identification and subcellular localization of proteins that are rapidly phosphorylated in tyrosine in response to colony-stimulating factor 1," PNAS, 85[21]:8062-6, 1988. J.Y.J. Wang, "Isolation of antibodies for phosphotyrosine by immunization with a v-abl oncogene-encoded protein," Molecular and Cellular Biology, 5:3640-3, 1985. The author, Deborah Wilkinson, can be reached at deborahwilkinson@sprynet.com.

Tyr'd and True: Immunochemical reagents and kits for studying tyrosine phosphorylation

Interested in reading more?

Become a Member of