Immunological methods for the detection of phosphotyrosine-containing proteins in neural tissues

NEUROPROTOCOLS: A Companion to Methods in Neurosciences Vol. 1, No. 3, December, pp. 177-184, 1992

Immunological Methods for the Detection of Phosphotyrosine-Containing Proteins in Neural Tissues Constance M. Ely, Sarah J. Parsons, and J. Thomas Parsons Department of Microbiology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908

The development, survival, and function of mature cells in the central and peripheral nervous systems are associated with changes in protein tyrosine phosphorylation, suggesting that this modification plays a central role in neural regulatory pathways. A major advance in the study of tyrosine kinases and their substrates in neural cells has been the development of antibodies that recognize tyrosine-phosphorylated proteins. This article describes methodologies involved in the generation of phosphotyrosine-specific antibodies and in the use of these antibodies to analyze and purify phosphotyrosine-containing proteins. Technical and theoretical considerations regarding these methods are discussed. o 1992 Academic press. I~C.

Phosphorylation of cellular proteins on tyrosine rep- resents a key regulator modification in the molecular pathways that control many diverse cellular functions, including cell proliferation, cell and tissue differentiation, metabolism, secretion, and oncogenic transformation (1, 2). Two classes of protein tyrosine kinases contribute sig- nificantly to the regulation of cell signaling pathways. Receptor tyrosine kinases trigger intracellular signals in response to binding of a specific ligand, usually a growth factor or hormone (2-4). These transmembrane receptors have a large extracellular ligand binding domain linked to a cytoplasmic tyrosine kinase domain. Ligand binding and subsequent receptor oligomerization induce the activation of the cytoplasmic tyrosine kinase domain, lead- ing to autophosphorylation of the receptor on tyrosine and tyrosine phosphorylation of numerous cellular targets. Nonreceptor protein tyrosine kinases are associated with cytoplasmic membranes, including vesicle and plasma membranes, as well as with proteins making up the cell cytoskeleton (5-7). Activation of these protein tyrosine kinases occurs in response to a variety of extracellular signals, including antibody-mediated aggregation of cell surface molecules (e.g., CD4, CD8, and the T-cell receptor (8, 9)), engagement of cell surface integrins with components of the extracellular matrix (10, ll), aggregation

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of surface immunoglobulins (In), and stimulation of cells with peptide growth factors (13-17) and agents that pro- mote the secretory process (18). The stable association of some nonreceptor tyrosine kinases with membrane- spanning cell surface molecules (e.g., the association of c-lck with the CD4 and CD8 molecules (19, 20) and the c-src, fyn, and c-yes proteins with the platelet-derived growth factor receptor (PDGFR) (21)) suggests that activation of these kinases occurs normally in response to engagement of cell surface molecules with both soluble and cell-associated ligands. As with the receptor family of protein tyrosine kinases, activation of the nonreceptor kinases leads to the tyrosine phosphorylation of many cellular proteins.

Interactions of neurotrophic factors such as nerve growth factor (NGF), neurotrophin, and brain-derived neurotrophic factor (BDNF) with specific receptors is crucial to the development of cells in the central and peripheral nervous systems (22). For example, NGF binding to the trk/NGF receptor family of receptors results in rapid changes in the extent of tyrosine phosphorylation of cellular proteins (23) and is essential to the initiation of intracellular responses which culminate in the development and survival of neurons (24). In addition, three members of the nonreceptor tyrosine kinase family are expressed at high levels in neural tissues, c-src, fyn, and c-yes (reviewed in (22)). The c-src protein is expressed in neural retina (25) and is enriched in nerve growth cones (26, 27). High levels of this protein are also detected in neurally derived chromaffin cells (7,28). c-yes expression shows considerable overlap with c-src and is found in high levels in brain (29, 30) and retina (3l), as well as in neurally derived adrenal medullary tissue (32). Like c-yes and c-src, c-fyn is found in the retinal neurons (33). The rapid induction of tyrosine-phosphorylated proteins observed upon in vitro stimulation of a variety of neural cells is consistent with tyrosine phosphorylation playing an im- portant role in neural signaling pathways.

Antibodies recognizing proteins containing phosphotyrosine have revolutionized the study of protein tyrosine

177

178 ELY, PARSONS, AND PARSONS -

kinases and their targets. In general, these antibodies are used for immunoprecipitation and Western immunoblotting. However, subcellular localization and tissue distri- bution of phosphotyrosine-containing proteins have also been analyzed using immunofluorescence techniques. Large-scale immunoaffinity purification of phosphotyrosine proteins and the subsequent generation of specific antibodies for individual substrates have also been achieved successfully by the use of phosphotyrosine antibodies.

The primary purpose of this brief review is to consider the methodologies employed in the generation of phosphotyrosine-specific antibodies and the use of these antibodies to analyze and purify phosphotyrosine-containing proteins. The methods outlined herein reflect the accrued experience of our laboratories.

GENERATION AND PURIFICATION OF PHOSPHOTYROSINE ANTIBODIES

Several forms of antigen have been used to generate antibodies against phosphotyrosine-containing proteins. These include: (i) recombinant v-&l protein (a tyrosine- phosphorylated, viral-transforming protein, which is itself a tyrosine kinase) (34); (ii) structural homologs of phosphotyrosine, such as phosphotyramine, p-aminobenzyl- phosphonic acid or arsanylic acid; and (iii) peptides containing phosphotyrosine, alanine, and threonine or phosphotyrosine, alanine, and glycine conjugated to key- hole limpet hemocyanin (KLH) using 1-ethyl-3-(3-di- methylaminopropyl) carbodiimide (EDAC) (35). Inter- estingly, when reactivity with cellular proteins was analyzed by Western immunoblotting, antibodies raised to each of these immunogens recognized a similar spectrum of tyrosine-phosphorylated cellular proteins (35).

It has previously been reported that the conjugation of KLH to mixtures of amino acids using EDAC is a simple, efficient, and reproducible method for generating high- titer anti-phosphotyrosine antibodies (36). We have uti- lized this method almost exclusively for the preparation of immunogen in the generation of rabbit polyclonal phosphotyrosine antibody.

Preparation of Antigen: Conjugation of Phosphotyrosine, Alanine, and Glycine to KLH The polymerization of phosphotyrosine, alanine, and

glycine and conjugation of the resulting mixed peptides to KLH using EDAC have been shown to maximize the numbers of phosphotyrosines attached to KLH (when compared to the direct polymerization of phosphotyrosine to KLH) and results in an immunogen that presents phosphotyrosines in the context of a polypeptide back- bone (35). This method requires little time and generates large quantities of antigen that are stable for several years

when stored at 4°C in the presence of 0.02% sodium azide. Antigen is prepared as follows:

1. L-Tyr (160 mg, 0.64 nM), L-Ala (56 mg, 0.64 nM), and L-Gly (47 mg, 0.65 nM) (Sigma) are added to 3 ml distilled water, and the pH is adjusted to 5.7 with 1 N NaOH.

2. Water is added to give a final volume of 4 ml. 3. KLH (10 mg; Calbiochem) is added to this mixture,

followed by EDAC (480 mg; Sigma) in 80-mg aliquots. 4. The pH of the solution is maintained using 1 N

NaOH or 1 N HCl and monitored for 2 h until it becomes stabilized at pH 6.0.

5. The resulting solution is transferred to dialysis tub- ing (10 mm, 12,000 molecular weight cutoff) and dialyzed for 4 days at 4°C against 150 vol of phosphate buffer (PBS) (150 mM NaCl, 15 mM sodium phosphate, pH 7.4). The buffer is changed daily. A solution of 5 mg of KLH is dialyzed in phosphate buffer as a control.

The efficiency of antigen conjugation to KLH is determined spectrophotometrically by comparing the optical densities of the dialyzed solutions containing either conjugated or unconjugated KLH. Formulas for these cal- culations are found in (37). Using this procedure, 40-100 phosphotyrosines are normally incorporated per 100 kDa of KLH.

Procedure for the Immunization and Bleeding of Rabbits The schedule for immunizations and bleeding of rabbits

is described below and involves primary and secondary injections of antigen emulsified in Freund’s adjuvant, followed by a third injection of antigen mixed with alum adjuvant. Maximum titers generally develop after the third injection and by the second or third bleed. Although individual rabbits produce antibodies that show differ- ential binding to specific proteins, the overall antibody titers between animals are similar and reproducible.

1. For the primary immunization, 200 pg KLH-peptide conjugate is emulsified in complete Freund’s adjuvant at a 1:l ratio of antigen:adjuvant and administered to the rabbits via 15-20 intradermal injections in the back using 50-100 ~1 per injection site.

2. On Day 14, the first boost is given, using 100 pg KLH-peptide conjugate emulsified 1:l in incomplete Freund’s adjuvant. Each rabbit receives two subcutaneous injections, 0.5 ml/site, on either side of the neck.

3. On Day 20, 10 ml blood is drawn (designated bleed l), and the serum is screened by direct Western immunoblotting of whole-cell extracts (described below).

4. The second boost is administered on Day 21, using 100 pg KLH-peptide conjugate mixed with alum (AlNH,(SO,),) adjuvant (made fresh for each boost) in one 0.5-ml intraperitoneal injection. The alum adjuvant is prepared as follows:

a. Alum (purchased in drug stores as powdered alum) is prepared by adding 1.25 g alum to 25 ml (total volume)

DETECTION OF PHOSPHOTYROSINE-CONTAINING PROTEINS 179

water in a 50-ml polypropylene tube followed by the addition of 12.5 ml 1 M NaOH.

b. This solution is mixed, resulting in the formation of a white A1(OH)B precipitate, which is collected by centrifugation in a clinical centrifuge for 2 min.

c. The precipitate is washed three times with 25-50 ml PBS and resuspended in 25 ml PBS.

d. The alum solution is adjusted to pH 7.4 with 1 N

HCl. This solution is centrifuged once again, and the final precipitate is resuspended in 25 ml PBS.

e. One milliliter of alum solution (containing 40 mg alum) is added to 100 pg conjugate and the solution is centrifuged, resulting in a antigen/alum pellet. The extent of adsorption of the KLH-conjugated antigen to the alum is determined by assaying residual antigen levels in the supernatant using a standard protein assay (BCA method) (Pierce, Rockford, IL). This measurement is compared to a positive control (the antigenic protein) and a negative control (buffer alone). The optimum ratio of alum to conjugate can be determined by titration, if necessary.

5. On Day 28, a 50-ml blood sample is collected, designated “bleed 2,” and tested by Western immunoblotting.

6. On Day 35, a third bleed is taken and assayed as before, followed by a 3-week waiting period before the boosting-bleeding cycle (Day 21, step 4) is repeated.

It is not known how long this boosting-bleeding schedule can be maintained. However, rabbits in our laboratories are still producing antibodies after a period of several years.

Purification of Phosphotyrosine Antibodies To eliminate antibodies in the phosphotyrosine antisera

that are directed to carrier epitopes or to phosphoserine and phosphothreonine, immunoglobulin is purified by affinity chromatography. Phosphotyrosine-linked Affigel is used as the affinity matrix, and elution is achieved by competition with phenyl phosphate (34-36).

I. Freshly drawn blood is allowed to coagulate at room temperature for l-2 h, the blood clot is “rimmed” to free it from the edges of the tube, and the tube is placed at 4°C overnight.

2. The following day the tube is centrifuged at 18OOg for 20 min at 4”C, and the serum is transferred to a fresh tube and frozen at -20°C.

3. Sera (typically 200 ml) are pooled from multiple bleeds that have tested positive by Western immunoblotting before the addition of solid (NH&SO4 (AS) to give a final concentration of 50%. AS is added gradually (over a 30-min period) to a glass beaker of serum stirring gently on ice.

4. The resulting precipitate is centrifuged at 1800g for 20 min at 4°C and the precipitate is resuspended in TN buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl) to l/4 of the original serum volume.

5. This solution is dialyzed against 4 liters of TN buffer at 4”C, with two changes of buffer over 48 h.

6. During the dialysis, phosphotyrosine is linked to Af- figel 15 (Sigma) for the subsequent affinity purification of the antibody. Coupling is accomplished in the following manner: 0.2 g phosphotyrosine (Sigma) is added to 1 ml 1 M Mops, pH 7.5, and 7 ml water. The pH of this solution is adjusted to 7.5 by adding 10 N NaOH dropwise, and the final volume is then brought to 10 ml with 100 mM

Mops buffer. 7. Five milliliters of this solution is added to 10 ml

Affige115 beads that had previously been washed with 5- 10 vol ice-cold deionized water and resuspended in 5 ml water. Affigel washes are performed in centrifuge tubes rather than on sintered glass filters to avoid drying and loss of beads.

8. Phosphotyrosine is allowed to couple to Affigel overnight at 4°C in a small polypropylene tube attached to an end-over-end tube rotator (Labquake shaker, Lab- oratory and Research Instruments, Berkeley CA).

9. The next day the beads are allowed to settle, and then 1 ml of the “postsupernatant” is removed and centrifuged in a microcentrifuge for 1 min. The coupling efficiency is determined spectrophotometrically (OD,,) by quantitating the amount of uncoupled phosphotyrosine in the supernatant.

10. To block unreacted sites on the Affigel 15 beads, l/10 volume of 1 M ethanolamine, pH 8.0, is added and reacted for 1 h at 4°C using the end-over-end tube rotator.

11. The Affigel is then washed with 40 ml TN buffer in a conical 50-ml centrifuge tube, the supernatant is removed, and the dialyzed IgG fraction (prepared from 200 ml of serum) is added to the Affigel beads and mixed at room temperature for 3 h by gentle rocking.

12. A column is made by removing the top of a lo-ml serological pipet and fixing glass wool into the tip as a barrier to the Affigel. This column is then attached to a peristaltic pump (Gilson Minipuls 2, Gilson Medical Electronics, Madison WI), and the beads are poured into the column with a pump setting of 40 (approximate flow rate of 100 ml/h).

13. The beads are washed once with 40 ml of TN buffer and once with 40 ml of buffer II (100 mM NaCl, 5 mM

NaPO,, pH 7.5,5 mM phosphoserine, 5 mM phosphothreonine).

14. When buffer II has moved into the column bed, the elution of antibodies is initiated by the addition of 100 ml buffer III (40 mM phenyl phosphate, 90 mM NaCl, 30 mM Tris-HCl, pH 7.5). Twelve l-ml fractions are collected, and the peak is pooled from fractions containing greater than 0.2 mg of protein, as measured by the BCA method.

15. Pooled antibody is dialyzed at 4°C for 14 days against TN containing 0.02% sodium azide, with buffer changes every 48 h.

16. Following dialysis, protein concentration is determined by the BCA method, and the antibody is tested,

180 ELY, PARSONS, AND PARSONS

typically by Western immunoblotting of whole-cell extracts.

Approximately 5-10 mg of purified IgG is recovered from 200 ml of serum.

rescence, and (iv) large-scale affinity purification of phosphotyrosine proteins. The methods described here are highly reproducible and sensitive, although there are many variations of these techniques described in the literature.

Production of Monoclonal Antibodies Recognizing Lysate Preparation Phosphotyrosine The patterns of reactivity of rabbit polyclonal phos-

photyrosine antibodies have been found to vary not only between different animals, but also between bleeds from the same animal. In an effort to develop a more homo- geneous and limitless set of reagents, monoclonal antibodies directed to phosphotyrosine have been generated.

The earliest reports of monoclonal antibodies specifi- cally recognizing phosphotyrosine-containing proteins were generated using KLH-coupled azylbenzyl phospho- nate as antigen (38). One such monoclonal antibody was capable of recognizing aminophenyl phosphate (a close analog of phosphotyrosine), as well as phosphotyrosine and phosphohistidine, the latter with low affinity. In a second approach, antigen was synthesized as described above for generation of rabbit polyclonal antibodies (phosphotyrosine polymerized with alanine and glycine and conjugated to KLH). However, to screen against antibodies specific for carrier epitopes, the mice were boosted with bovine serum albumin (BSA)-conjugated phosphotyrosine and then screened using ovalbumin (OVA)-conjugated phosphotyrosine (39). All of the antibodies derived using this procedure showed similar affinities and speci- ficities. Still other investigators have reported the use of KLH-conjugated phosphotyrosine or phosphotyramine, without prior polymerization with other amino acids, as a source of antigen in the generation of phosphotyrosine monoclonal antibodies (40). A final method used mixtures of immunoaffinity-purified tyrosine-phosphoproteins (isolated using rabbit phosphotyrosine antibody) as antigen. Although many of the antibodies generated in this study recognized epitopes that did not contain phosphotyrosine, a number of hybridomas produced antibody that was specific for phosphotyrosine residues (41). The ap- parent affinities of different monoclonal antibodies for phosphotyrosine have been found to vary as much as lOOO- fold, related in part to the type of antigen used for immunization (40).

Many investigations have demonstrated that phosphotyrosine groups on proteins are labile (e.g., susceptible to endogenous cellular phosphatases), making the study of rare proteins with this modification especially difficult. However, the inclusion of protease and phosphatase inhibitors and chelating agents during cell lysis, along with low temperatures and less concentrated extracts, has helped to maintain these phosphorylations, thereby in- creasing the sensitivity of detection (35,42-44). The following method affords a rapid and reliable procedure for the preparation of cell extracts with a minimum loss of phosphotyrosine.

1. Cell monolayers are washed twice with ice-cold PBS and then placed on a cold metal tray, on ice (0°C).

2. Ice-cold “ptyr RIPA” (150 mM NaCl, 50 mM Tris- HCl, pH 7.5,1% NP-40, and 0.5% sodium deoxycholate), supplemented with 1 mM sodium orthovanadate, 1 mM

phenylmethylsulfonyl fluoride, 0.5% aprotinin, 50 wg/ml leupeptin, 10 pg/ml a,-macroglobulin (Boehringer- Mannheim), and 2 mM EGTA, is added to each dish, 0.5 ml for 60-mm dishes and 1 ml for loo-mm dishes.

3. The plates are rocked for 5 min while remaining on the metal tray, and then the lysed cells are pipetted off the dish (or scraped with a rubber policeman) and transferred to 1.5-ml Eppendorf tubes.

4. The lysates are centrifuged for 5 min in a refrigerated microfuge and the protein concentrations of the supernatants determined by the BCA method.

5. If the lysates are not to be used immediately, they are frozen quickly by placing them into an ethanol-dry ice bath.

6. For direct Western immunoblotting, equal protein concentrations are mixed with l/10 vol of 10X gel electrophoresis sample buffer (0.5 M Tris, pH 6.8, 10% SDS,

Some commercially available monoclonal antibodies recognizing phosphotyrosine-containing proteins are listed in Table 1.

Commercially Available Phosphotyrosine Monoclonal Antibodies

ANALYSIS OF TYROSINE-PHOSPHORYLATED PROTEINS

Outlined below are procedures used for the detection of phosphotyrosine proteins in (i) immunoprecipitation assays, (ii) Western immunoblotting, (iii) immunofluo-

Company

Boehringer Mannheim Chemicon Gibco-BRL Oncogene Sciences Santa Cruz Biotechnology Sigma Upstate Biotechnology Zymed

TABLE 1

Name of monoclonal antibody

lG2 lG2, Py20, Py69 6G9 lG2 lG2 PT-66 4GlO Py20,2021

DETECTION OF PHOSPHOTYROSINE-CONTAINING PROTEINS 181

1.4 M fl-mercaptoethanol, 50% sucrose, and 0.02% brom- phenol blue).

7. Samples are boiled for 2-5 min and then resolved by electrophoresis on SDS-polyacrylamide gels.

8. For direct Western blotting, cells may also be lysed directly in sample buffer. In this case, cellular DNA is sheared by sonication using three l-s bursts or by passage several times through a small-gauge needle prior to boiling.

Extracts in ptyr RIPA buffer can be stored at -70°C for up to a year with little change in phosphotyrosine content. However, we recommend, after thawing, the addition of l/4 to l/3 volume fresh ptyr RIPA buffer containing protease and phosphatase inhibitors.

Comparative Western blot analyses of samples lysed directly in sample buffer or prepared in ptyr RIPA show no differences in the resolution or in the phosphotyrosine content of cellular proteins. However, scraping monolayers off dishes (using a rubber policeman) before the addition of buffers containing sodium orthovanadate causes a significant reduction in tyrosine phosphorylation of cellular proteins, presumably due to the activation of cellular phosphatases (45). Furthermore, it has previously been reported that the overall rate of protein dephosphorylation is more rapid in concentrated cell extracts (prepared at 2 X lo7 cells/ml) than in more dilute cell extracts (5 X lo6 cells/ml), although the rates of dephosphorylation for individual proteins are quite variable (35). We find that multiple freezings and thawings (up to three cycles) cause little change in the phosphotyrosine pattern of cellular proteins.

Immunoprecipitation Immunoprecipitation of cellular proteins with phos-

photyrosine antibodies provides a rapid and effective approach to the isolation and quantitation of phosphotyrosine-containing proteins (43, 46). However, it should be noted that protein-protein interactions involving phosphotyrosine (e.g., SHB-phosphotyrosine interactions) or protein folding may mask or decrease the efficiency of immunoprecipitation by phosphotyrosine antibodies. The following immunoprecipitation protocol has successfully been used in our laboratories.

All steps of the immunoprecipitation are performed at 0°C (on wet ice) and in the presence of protease and phosphatase inhibitors.

1. One to three micrograms of rabbit polyclonal or mouse monoclonal phosphotyrosine antibody is added to ptyr RIPA cell extracts and incubated at 0°C for 1 h.

2. Protein A-Sepharose (Sigma) or Pansorbin (Cal- biochem, La Jolla, CA) is washed once in ptyr RIPA containing protease and phosphatase inhibitors and then added to the above solution to bind antibody-antigen complexes. Incubations are carried out for 30 min or overnight at 4°C using an end-over-end tube rotator. In cases in which mouse monoclonal antibodies with isotypes

that bind weakly to protein A-Sepharose are used, secondary antibody (anti-mouse IgG) is added to the protein A-Sepharose beads before binding the antigen-antibody complexes. This is accomplished by:

a. Adding affinity-purified secondary antibody (rabbit anti-mouse IgG, Jackson Immuno Research Labora- tories Inc., West Grove, PA) to protein A-Sepharose beads at a ratio of 5 pg:50 ~1 packed beads.

b. Incubating this mixture for 1 h on ice. c. Washing protein A-Sepharose with bound sec-

ondary antibody once with TBS and then adding the “coated” protein A-Sepharose to the antigen-antibody complexes.

3. Immune complexes bound to protein A-Sepharose are washed three times in ptyr-RIPA, as follows: The samples are centrifuged in a refrigerated microcentrifuge at lO,OOOg, the supernatant is removed, and the pellet is thoroughly resuspended in 0.5 ml ptyr RIPA.

4. The final pellet is resuspended in 100 ~1 PBS, mixed with l/10 volume of 10X sample buffer, and boiled for 2 min.

5. Before polyacrylamide gels are loaded, samples are again centrifuged in the microcentrifuge for 2 min to remove Pansorbin or protein A-Sepharose.

6. After electrophoresis, SDS-polyacrylamide gels are transferred to nitrocellulose and subjected to Western immunoblotting (described below) using polyclonal or monoclonal antibodies as blotting reagents.

Typically, 200 pug extract per sample can be immuno- precipitated using l-3 pg purified antibody and 25 ~1 Pansorbin or 100 ~1 protein A-Sepharose (50 ~1 packed beads). However, we recommend that the optimum ratio of antiserum to antigen be determined for each antibody by titrating the antibody against 200 pg cell extract and monitoring the efficiency of immunoprecipitation by Western immunoblotting.

As mentioned above, not all phosphotyrosine-containing proteins are recognized in immunoprecipitation assays using phosphotyrosine antibodies. For example, calpactin 1 (~36) (47), extracellular signal regulated kinase-2 (ERK2) (48), and ~~60’.“‘” (5, 6), each a tyrosine-phosphorylated protein, are not recognized by phosphotyrosine antibodies with this method. Since the denatured forms of these proteins are detected by Western blotting with phosphotyrosine antibodies, it is likely that protein conformation restricts binding of the antibody to the phosphotyrosine epitope(s). Thus, the degree to which a specific protein is recognized using this technique can be influenced by intra- or interprotein complex formation or by the amino acid sequence surrounding the phosphotyrosine.

Western Transfer of Phosphotyrosine-Containing Proteins The most frequent application of phosphotyrosine an-

tibodies has been in the detection of phosphotyrosine-


containing proteins by Western immunoblotting. This is a rapid and specific method for analyzing both small and large numbers of samples simultaneously, with the added advantage of avoiding in vivo radiolabeling, techniques that are inherent in methods such as phosphoamino acid analysis and alkali hydrolysis. A sensitive and reproducible method for Western immunoblotting with phosphotyrosine antibodies (for which many variations have been reported elsewhere) (35,42-4549-56) is described below.

1. Following SDS-polyacrylamide gel electrophoresis, proteins are transferred to nitrocellulose or Immobilon membranes (Schleicher & Schuell, Keene, NH) at 0.5 A for 3 h or 0.12 A for 15 h at 4°C using the Bio-Rad Trans- blot Cell unit. Transfer buffer contains 25 mM Tris, 190 mM glycine, 0.08% SDS, 20% methanol, and 0.01% Na orthovanadate.

2. Following transfer, the filters are incubated in blocking buffer (50 mM Tris-HCl, pH 7.2, 0.15 M NaCl, 0.05% Tween 20, 4% BSA, 0.01% sodium azide) for 2 h at 37°C (or overnight at room temperature).

3. Phosphotyrosine antibody (approximately 20 pg/20 ml of blocking buffer) is added to the filters and rocked (we recommend using the Belly Dancer rocker, Stovall Life Science, Inc., Greensboro, NC) at room temperature for 3 h or at 4°C for 15 h.

4. The filters are washed five times (ca. 5 min each) with rinsing buffer (50 mM Tris-HCl, pH 7.2,0.15 M NaCl, 0.05% Tween 20, and 0.01% sodium azide) and then incubated for 1 h at room temperature with 1 pCi/ml lz51- protein A (Amersham Corp., Arlington Heights, IL) in fresh blocking buffer.

5. The filters are washed as before and then dried for 20 min on 3MM filter paper. Once dried, the filter is cov- ered with a piastic sheet protector and then exposed to Kodak X-AR or X-RP film with an intensifying screen at -70°C.

Note: In some cases in which the nonspecific “gray” background of the filter is high, exposures are performed at room temperature. This has been shown to reduce the nonspecific background while maintaining specific signals and is particularly useful when using whole serum as the Western blotting reagent instead of purified antibody.

It has previously been shown that 0.1% SDS in the rinsing buffer reduces the extent of binding of phosphotyrosine antibodies to specific cellular proteins (50-80%) as does Tween 20 at a concentration of 0.2% (20% reduction) (36) and NP-40 at 0.1% (for some but not all proteins) (57). In addition, blocking buffers containing nonfat dry milk cannot be used because of reactivity with the phosphotyrosine antibodies (36).

Enzyme-linked reagents are frequently employed in place of lz51-labeled reagents. However, we have found that lz51-labeled reagents are more readily quantitated. Also, when monoclonal antibodies are used as Western blotting reagents, lz51-labeled secondary antibody can be used in place of labeled protein A.

Comparison of different phosphotyrosine antisera using Western immunoblotting reveals considerable overlap in substrate recognition, although different antisera can display differences in the extent of binding to individual phosphotyrosine-containing proteins (57). In addition, immunoblotting appears to preferentially detect high- molecular-weight phosphotyrosine-containing proteins when compared to other methods, such as phosphoamino acid analysis of in vivo 32P-labeled proteins resolved by SDS-PAGE. Thus, while quantitative comparisons by Western immunoblotting using a single antibody preparation are highly reproducible, the spectrum of proteins recognized may be somewhat selective.

Subcellular Localization of Phosphotyrosine-Containing Proteins by Immunofluorescence

Phosphotyrosine antibodies have provided a powerful method for the localization of phosphotyrosine proteins in cells or tissues using indirect immunofluorescence and have been used to survey a wide range of tissues at different developmental stages (58, 59). The following method has been adopted in our laboratories to localize tyrosine-phosphorylated proteins in cells transformed by pp60’-“” and activated ~~60’.““, as well as in nontrans- formed cell types.

1. For the detection of phosphotyrosine proteins by immunofluorescence, cells are cultured at subconfluence on sterile coverslips (Corning) and placed in fresh tissue culture dishes at room temperature for the remaining steps of the procedure.

2. To remove excess medium components, the slides are washed three times with PBS and then fixed by incubation for 20 min in 3% paraformaldehyde (Sigma).

3. Following fixation, the slides are washed three more times in PBS before the cells are permeabilized by incubation for 3 min in 0.4% Triton X-100, diluted in PBS.

4. Detergent is removed, and the cells are washed three times in PBS. Phosphotyrosine antibody is then added at a concentration of 10 pg/ml for a l-h incubation.

5. To remove excess unbound IgG, the slides are washed two times with PBS and then fluorescently labeled (FITC or Texas red), and secondary antibody (5 pg/ml) (Jackson Immuno Research Laboratories Inc., West Grove, PA) is added and incubated with the cells for 1 h in the dark.

6. A final series of washes, two times with PBS, is performed before the slides are aspirated dry and mounted in 90% glycerol, 10% PBS, and 1% N-propylgalate (Sigma).

The intensity of fluorescence can be enhanced by using a “bridge” antibody, i.e., an unlabeled secondary antibody, before the sample is incubated with fluorescently labeled tertiary antibody. Background fluorescence due to debris can be decreased by centrifuging the fluorescent-conjugated antibody for 1 min in the microfuge before use.

DETECTION OF PHOSPHOTYROSINE-CONTAINING PROTEINS ~~~- ~ -_ _~ ~~~___~__ 183

Use of Phosphotyrosine Antibodies for Large-Scale Phosphoprotein Purification Pertinent to our understanding of the role of tyrosine

kinases are the identification, purification, and analysis of their relevant substrates. Phosphotyrosine antibodies have been used to purify phosphotyrosine-containing proteins for generation of specific monoclonal antibodies. This method is useful for the purification of native (60, 61) or denatured (61) proteins. The method described below has been used successfully to affinity-purity phosphotyrosine proteins in their native form from chicken embryo fibroblasts expressing activated pp60”+” (~~60~~~~) (60).

1. Cells are lysed (as described above) in ptyr RIPA with protease and phosphatase inhibitors.

2. Lysates are cleared by centrifugation at 100,OOOg and incubated for l-2 h at 0°C with phosphotyrosine antibodies, using 1 mg antibody for every 50 mg cellular protein.

3. Immune complexes are recovered by incubation with protein A-Sepharose (1 ml packed beads/2 mg antibody) for 1 h at 0°C.

4. Sepharose beads are washed three times with lysis buffer, once with phosphoserine/phosphothreonine buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5,5 mM phosphoserine, and 5 mM phosphothreonine) and twice in TN buffer.

5. The proteins are eluted with two successive incubations in 40 mM phenyl phosphate, 10 mM Tris-HCl, pH 7.5, and 20 mM NaCl at 0°C for 30 min using a 1:l ratio of elution buffer:beads.

6. For analysis of purified proteins, supernatants are mixed 1:l with 2X sample buffer and boiled for 5 min before SDS-PAGE.

7. Following electrophoresis, the gels are analyzed by silver staining or Western immunoblotting as described above.

Recovery of proteins from columns prepared with phosphotyrosine antibodies that have not been previously purified using a phosphotyrosine affinity matrix and elution with phenyl phosphate, i.e., some monoclonal phosphotyrosine antibodies, can be enhanced by washing the column with 1% SDS (following step 5) to elute tightly bound phosphoproteins.

Analysis of proteins using this procedure has demonstrated that this single-step immunoaffinity procedure generates large amounts of relatively pure proteins (approximately a 3000-fold enrichment) that retain their native conformation (60).

CONCLUDING REMARKS

This report describes methods involved in the generation and use of rabbit polyclonal and mouse monoclonal

antibodies directed to phosphotyrosine residues of proteins. These reagents have been useful in studying tyrosine phosphorylation, both extents of phosphorylation and localization during many cellular processes. However, these analyses have been limited by the nature of the specificity of these antibodies (recognizing only the tyrosine-phosphorylated form of each protein). Therefore, to facilitate the study of both tyrosine-phosphorylated and unphosphorylated forms of individual proteins, phosphotyrosine antibodies have been used to purify tyrosine protein kinase substrates with the aim of generating specific antibodies against individual proteins. These reagents, combined with molecular cloning techniques and mutational analysis, make it possible to address pertinent questions regarding the relevance of these proteins in cell processes.

ACKNOWLEDGMENTS

The authors acknowledge the contributions of our colleagues, A. Reynolds, M. Payne, T. Rossomando, S. Kanner, L. Kozma, A. Bouton, B. Cobb, and R. Vines, whose efforts have contributed to the formulation of the protocols described herein. We also thank Dr. M. Weber for his helpful discussions on the theoretical and technical aspects of these methods. This research was supported by DHHS Grants CA40042. CA39438, and CA29243 and American Cancer Society Grant MV 430.

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Immunological methods for the detection of phosphotyrosine-containing proteins in neural tissues