[8]

79

M O N O C L O N A L ANTIBODIES TO PHOSPHOTYROSINE

Assay of Phosphatidylinositol 3-Kinase 1. Add 10/xl of 100 mM MgCI2 and 10/xl of sonicated lipid (20/zg/assay point). Initiate the phosphorylation reaction by adding 10/xl of 440/zM ATP containing 30 ~Ci of [3Ep]ATP and 10 mM MgC12. Incubate the mixture for 10 min at 22° with occasional agitation. Stop the reaction by adding 20/zl of 8 N HCI. 2. Extract the PI from the reaction mixture with 160 /xl of chloroform : methanol (1 : 1). Vortex the mixture and separate the phases by centrifugation for 2-3 min in a microfuge. Remove 50/zl of the lower organic layer and apply it to a TLC plate next to PI-4-P and PI-3,4-P 2 standards. Develop the plates in CHC13 : CH3OH : H20 : NH4OH (60 : 47 : 11.3 : 2). Adequate separation can be obtained with a 10-cm plate. 3. Allow the plates to dry and identify the lipid products by autoradiography using Kodak X-Omat film and an intensifying screen (Fig. 4). Determine the position of lipid standards by iodine staining. Radioactivity in the lipid products can be quantified by cutting or scraping the TLC plate and Cerenkov counting (Fig. 4). Acknowledgments This work has been supported in part by NationalInstitutesof Health Grants DK38712 (M.F.W.) and a Diabetes and EndocrinologyResearchCenter Grant DK36836. J.M.B. is a recipient of a NationalResearch Serviceaward, DK08126;M.F.W. is a scholarof the PEW Foundation, Philadelphia.

[8] G e n e r a t i o n o f M o n o c l o n a l A n t i b o d i e s a g a i n s t P h o s p h o t y r o s i n e a n d T h e i r U s e for A f f i n i t y P u r i f i c a t i o n o f Phosphotyrosine-Containing Proteins

By A.

RAYMOND

J R . , M. P O S N E R , B. and F. MERMELSTEIN

FRACKELTON,

KANNAN,

The first polyclonal and monoclonal antibodies reactive with phosphotyrosine (PY)-containing proteins were generated by immunizing animals with p-aminobenzylphosphonic acid (ABP) diazotized to carrier protein. 1,2 This analog of phosphotyrosine was chosen over phosphotyrosine itself as the haptenic group because the phosphonate group, in contrast to the 1 A. H. Ross, D. Baltimore, and H. N. Eisen, Nature (London) 294, 654 (1981). 2 A. R. Frackelton, Jr., A. H. Ross, and H. N. Eisen, Mol. Cell. Biol. 3, 1343 (1983).

METHODS IN ENZYMOLOGY, VOL. 201

Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

80

ANALYSIS OF PROTEIN PHOSPHORYLATION

[8]

phosphate group of PY, was resistant to enzymatic hydrolysis, and thus might be a more persistent and efficient immunogen. Polyclonal and monoclonal antibodies to ABP, however, frequently cross-reacted with a number of other phosphate-containing compounds (for example, nucleotides). 2'3 One ABP-induced monoclonal antibody, 2G8, had been extremely useful for characterizing and purifying a variety o f p h o s p h o t y r o syl proteins. 2'4-8 It had, however, two limitations: (1) it had a relatively low binding constant for phosphotyrosyl proteins and therefore could be used effectively only when coupled at high density to an activated matrix such as C N B r - S e p h a r o s e ; (2) it cross-reacted with mononucleotides and phosphohistidine. 2 Because of these limitations, a substantial and successful effort was made to produce a panel of monoclonal antibodies that has higher affinity and greater specificity for phosphotyrosyl proteins. To accomplish this goal, we tested a variety of phosphotyrosine-analog conjugates, immunizing protocols, and screening procedures. The most successful immunogens were conjugates of phosphotyrosine or phosphotyramine (PYA) coupled via their free amino groups to keyhole limpet hemocyanin ( K L H ) using either the heterobifunctional cross-linking agent succinimidyl-4-(p-maleimidophenyl)butyrate (SMBP) or the homobifunctional cross-linker glutaraldehyde. We describe here methods for preparing the P Y - p r o t e i n and P Y A - p r o t e i n conjugates, immunizing mice, and screening hybridoma culture fluid for specific antibodies. We compare and contrast the affinities and fine specificities of these antibodies with those of the earlier anti-ABP monoclonal antibody (2G8), and provide detailed methods for using the most avid and specific monoclonal antibody (1G2) for purifying PY-containing proteins from cells. Preparation of Immunogens and Antigens for ELISA Preliminary studies demonstrated that the immune system of mice frequently recognizes the immunizing PY or PYA haptenic group in the 3 p. M. Comoglio, M. R. Di Renzo, G. Tarone, F. G. Giancotti, L. Naldini, and P. C. Marchisio, EMBO J. 3, 483 (1984). 4 A. R. Frackelton, Jr., P. M. Tremble, and L. T. Williams,J. Biol. Chem. 259, 7909(1984). 5 B. Friedman, A. R. Frackelton, Jr., A. H. Ross, J. M. Conners, H. Fujiki, T. Sugimura, and M. R. Rosner, Proc. Natl. Acad. Sci. U.S.A. 81, 3034 (1984). 6 j. G. Foulkes, M. Chow, C. Gorka, A. R. Frackelton, Jr., and D. Baltimore, J. Biol. Chem. 260, 8070 (1985). 7 T. O. Daniel, P. M. Tremble, A. R. Frackelton, Jr., and L. T. Williams, Proc. Natl. Acad. Sci. U.S.A. 82, 2684 (1985). 8 y. Yarden, J. A. Escobedo, W. J. Kuang, T. L. Yang-Feng, T. O. Daniel, P. M. Tremble, E. Y. Chen, M. E. Ando, R. N. Harkins, U. Francke, V. A. Fried, A. Ullrich, and L. T. Williams, Nature (London) 323, 226 (1986).

[8]

M O N O C L O N AANTIBODIES L TO PHOSPHOTYROSINE

81

context of the bifunctional cross-linking reagent. 9 To minimize clonal dominance by responses to PY (or PYA) cross-linker, we found it essential to use generically different chemical cross-linking agents for the original immunizations and the final antigenic challenge. P Y or P YA Conjugates with Keyhole Limpet Hemocyanin Using SMBP SMBP is a heterobifunctional agent that reacts with sulfhydryl groups via a maleimido group and with amino groups via a succinimidyl group. 10 PY (or PYA) is first linked via its free amino group to SMBP. Meanwhile, disulfide bonds of KLH are reduced with sodium borohydride. The PY-maleimide conjugate is then reacted with the free sulfhydryl groups of KLH. Materials for SMBP Conjugation SMBP (Pierce, Rockford, IL), 20 mM (7.3 mg/ml) in tetrahydrofuran Keyhole limpet hemocyanin (KLH) (Calbiochem, San Diego, CA), 50 /zM (5 mg/ml, assuming 105 g/mol), thoroughly dialyzed against 0.1 M EDTA and 6 M urea O-Phospho-L-tyrosine (Sigma, St. Louis, MO), 10 mM (2.61 mg/ml) in 0.05 M sodium phosphate buffer, pH 7.0 O-Phospho-DL-tyramine, prepared as described by Ross et al.,t 10 mM (2.17 mg/ml) in 0.05 M sodium phosphate buffer, pH 7.0 Dichloromethane (30 ml) Sodium borohydride (20 mg) Butanol (0.2 ml) NaHzPO 4 (1 ml), 0.1 M Acetone (0.4 ml) Nitrogen gas Procedure 1. Preparation of PY (or PYA)-Maleimide Conjugate: Combine 1 ml of 20 mM SMBP in tetrahydrofuran with 2 ml of 10 mM PY (or PYA) in 0.05 M sodium phosphate buffer, pH 7.0. Incubate at 30° for 30 min with occasional stirring. Remove tetrahydrofuran by gently bubbling nitrogen through the solution, and then extract excess SMBP with three 10-ml aliquots of dichloromethane, saving the aqueous phase for subsequent reaction with reduced KLH. 2. Reduction of KLH Disulfide Bonds: To 10.2 mg of KLH in 2 ml of 6 M urea, 0.1 M EDTA, alternately add small portions of sodium borohydride and n-butanol, totaling 20 mg and 0.2 ml, respectively, taking care 9 A. R. Frackelton, Jr., unpublished observations (1984). 10 T. Kitagawa, T. Kawasaki, and H. Munechika, J. Biochem. (Tokyo) 92, 585 (1982).

82

ANALYSIS OF PROTEIN PHOSPHORYLATION

[8]

that effervescence does not cause the reaction to spill over the tube. Incubate the reaction for 30 min at 30° (durinz which time considerable aggregation of K L H will be observed). Excess sodium borohydride is then decomposed by the addition of 1 ml of 0. I M NaH2PO4 and 0.4 ml of acetone. 3. Reaction of P Y (or P YA)-Maleimide Conjugate with Reduced KLH: One-half of the aqueous phase containing PY (or PYA)-maleimide is combined with the reduced K L H and incubated at 25° for 2 hr. Protein aggregates are then dispersed by homogenization, and the whole reaction mixture is exhaustively dialyzed against phosphate-buffered saline (PBS). These two conjugates contained 8 PY and 20 PYA haptenic groups per K L H 100,000 daltons, respectively, determined by inorganic phosphate analysis. 11

P Y-Protein or P YA-Protein Conjugates Using Glutaraldehyde Glutaraldehyde is a homobifunctional cross-linking agent reacting with amino groups. It is quite different structurally from the SMBP agent, and for this reason was used to produce K L H conjugates for the final immunogenic challenge, as well as to produce bovine serum albumin (BSA) conjugates for use in enzyme-linked immunosorbent assays (ELISA). The procedure detailed below is adapted from Reichlin 12 and Konopka et al.13

Materials for Glutaraldehyde Conjugation Keyhole limpet hemocyanin, 0.15 mM (15 mg/ml in PBS, pH 7.2) Bovine serum albumin [radioimmunoassay (RIA) grade; Sigma], 0.46 mM (30 mg/ml) in buffer A (0.15 M NaC1 and 0.1 M sodium phosphate, pH 7.2), and dialyzed against this buffer O-Phospho-L-tyrosine, 9 mM (2.35 mg/ml) in buffer A O-Phospho-DL-tyramine, 9 mM (1.95 mg/ml) in buffer A Glutaraldehyde (Sigma), 5 M in sealed ampoule. Prepare 20 mM glutaraldehyde in 0.15 M NaCl and 0.1 M sodium phosphate, pH 7.2

Procedure 1. Combine 15 mg of protein with 1-5/xmol of PY or PYA (0.1 ml to 0.5 ml of stock solution, above; the amount of hapten added will affect the haptenic valence of the conjugate, see below). II j. E. Buss and J. T. Stull, this series, Vol. 99, p. 7. 12 M. Reichlin, this series, Vol. 70, p. 159. t3 j. B. Konopka, R. L. Davis, S. M. Watanabe, A. S. Ponticelli, L. Schiff-Maker, N. Rosenberg, and O. N. Witte, J. Virol. 51, 223 (1984).

[8]

MONOCLONAL ANTIBODIES TO PHOSPHOTYROSINE

83

2. Add 1 ml of 20 mM glutaraldehyde and allow to react for 5 hr at room temperature. 3. Dialyze the reaction mixture against PBS containing 0.05% (w/v) sodium azide as a preservative. These haptenated proteins can be stored at 4° for >5 years. By quantitative phosphate analysis, the PY-glutaraldehyde-KLH conjugate prepared with 5/.~mol PY contained 1.5 PY groups/KLH (100,000 Da) whereas PYA-glutaraldehyde-BSA conjugates prepared with 1 and 5 /xmol PYA contained 1.5 and 7 PYA groups, respectively, per molecule of BSA. Immunization Protocol Of several immunization protocols tested, the most successful involves immunizing mice (BALB/c, female) with an intraperitoneal injection of 50/xg of PY8-SMBP-KLH, emulsified in Freund's complete adjuvant, challenging 2 months later with 50/zg of PYA20-SMBP-KLH in Freund's complete adjuvant, and finally challenging another 2 months later with PY15-glutaraldehyde-KLH, just 3 days before taking spleen cells (fusing as described previously with P3U1 myeloma-derived fusing partnersZ'14). Serum from the immunized mice is screened for antibody activity in a solid-phase enzyme-linked immunosorbent assay (see method below) using PYA linked at low valency via glutaraldehyde to BSA (1.5 haptenic groups/molecule of BSA). The use of low-valency antigen and low antigen concentration in the ELISA favors the detection of only high-affinity antibodies. T M All of the immunized mice had high titers of antibodies reactive with PYA in this assay. However, when these antibodies were screened using a much more demanding criterion--the ability to bind [32p]phosphotyrosyl epidermal growth factor (EGF) receptor in a solidphase assay (see method below and Fig. 1 legend)--antibodies from only those mice immunized by the protocol described above performed well. Fusion of the spleen cells from one of these mice with the P3U1 fusion partner resulted in approximately 3000 hybridomas, hundreds of which produced antibody to PY as judged by the ELISA. Of the 30 most reactive by ELISA, 4 hybridoma culture fluids showed strong reactivity in the EGF 14 D. E. Yelton, B. A. Diamond, S. P. Kwan, and M. D. Scharff, in "Current Topics in Microbiology and Immunology" (F. Melchers, M. Potter, and N. L. Warner, eds.), p. 81. Springer-Verlag, New York, 1978. 15 T. T. Tsu and L. A. Herzenberg, in "Selected Methods in Cellular Immunology" (B. B. Mishell and S. M. Shiigi, eds.), p. 373. Freeman, San Francisco, California, 1980. 16 A. Nieto, A. Gaya, M. Jansa, C. Moreno, and J. Vives, Mol. Immunol. 21, 537 (1984).

84

kDa

ANALYSIS OF PROTEIN PHOSPHORYLATION

[8]

1

6

8

10

G

H

F

F

2

9

1

7

2

G 8D

FIG. 1. Screening hybridoma fluid for ability of their antibodies to bind [32p]Tyr-EGF receptor. Microtiter wells were coated with rabbit anti-mouse Ig (50/A of 50 /~g/ml) as described in the text. Hybridoma culture fluids (70 t-*l) were incubated in the wells for 4 hr, after which the wells were rinsed several times with PBS. Meanwhile, a partially purified preparation of phosphotyrosyl EGF receptors was made as described in the text. Aliquots of the crude EGF receptors were dispensed into each microtiter well. After 14 hr at 4°, the wells were washed extensively and eluted with hot Laemmli SDS gel sample buffer. The eluates were boiled, resolved by SDS-polyacrylamide gel electrophoresis, and 32p-labeled proteins were visualized by overnight autoradiography. Lanes 1G2, 6H9, 8F1, and 10F7 contain the 170-kDa EGF receptor purified by antibodies from this fusion. Lane D and the next lane contain EGF receptor purified by 1 and 10/~g, respectively, of affinity-purified PY antibody 2D2 from a different fusion. Lane 2G8 shows the inability of the earlier ABP antibody (10 ~g of affinity-pure 2G8) to purify EGF receptors. Lane R shows the EGF receptor purified by EGF receptor antibody.

receptor-binding assay: hybrids IG2, 6H9, 10F7, and 8Fl (Fig. 1). In contrast the 2G8 monoclonal antibody that had been so useful in the past was completely ineffectual. Each of these four hybrids were passed as an ascites in mice, and the monoclonal antibodies were affinity purified from the ascites fluid by phosphotyramyl-Sepharose affinity chromatography, specifically eluting with the phosphotyrosine analog, phenyl phosphate, as described previously. 2 ELISA and RIA Protocols of P Y Antibodies 1. Dilute PYAl.5-glutaraldehyde-BSA to 100 ng/50/~1 with PBS. Immediately dispense 50/~1 into each well of an Immulon (Dynatech, Alexandria, VA) or Corning (Corning, NY) 96-well microtiter plate. Incubate overnight at 4°. 2. Flick out the contents of the wells and wash once with PBS using a squirt bottle.

R

[8]

MONOCLONAL ANTIBODIES TO PHOSPHOTYROSINE

85

3. Completely fill the wells with 5% (w/v) Milkman nonfat milk in PBS-azide. Incubate for 1 hr at 37°. 4. Wash two times with PBST [PBS containing 0.1% (v/v) Tween 20]. 5. Dispense antibody samples (or 10 ~1 of hybridoma fluid) into wells (in 50/xl of PBST). Incubate at 37 ° for 1-2 hr. 6. Flick out the solution and wash the wells three times with PBST. 7. RIA: For RIA, add sheep (Fab'2) anti-mouse 125I-labeled Ig (-105 cpm/well in 50/zl of PBST). Incubate 1-2 hr at 37°. Wash four times with PBST. Count wells. 7. ELISA: For ELISA, add 50 p.1 of a 2000-fold dilution of sheep anti-mouse Ig (peroxidase labeled; Jackson ImmunoResearch Labs, West Grove, PA) in PBST. Note: Be certain that no azide is used from here on as it inhibits peroxidase. Incubate 1-2 hr at 37°. 8. Wash four times with PBST. 9. Add 100/xl of substrate solution (10 ml of 0.1 M citrate buffer, pH 4.5, containing 4/zl of 30% (w/w) H202 and 10 mg of o-phenylenediamine. Prepare just before use and protect from light). Color develops in 2-15 min at room temperature. Preparation of P Y Proteins from A431 Cells Stimulated with EGF 1. Seed A431 cells 16-20 hr before experiment at 1-2 × 105 cells in 2 ml culture medium [with 10% fetal calf serum (FCS)] in 35-mm culture wells. 2. The next day, rinse wells two or three times with 2 ml of phosphatefree Dulbecco's minimal essential medium (DMEM), then add 1 ml of phosphate-free DMEM containing 1 mM sodium L-pyruvate, 2 mM Lglutamine, nonessential amino acids, 0.1% (w/v) BSA, and 1 mCi of 32p. Incubate at 37 ° in 5% CO2 for 3-4 hr. 3. Add 100 ng EGF/weI1. Mix and incubate at 37° for 5 min. 4. Transfer the plate quickly to ice, aspirate the culture fluid to radioactive waste, draining the fluid thoroughly. 5. Add phenylmethylsulfonyl fluoride (PMSF) (from 100 mM roomtemperature stock) to an aliquot of ice-cold extraction buffer (see recipe below); mix it quickly and then dispense 0.4 ml onto A431 cell monolayer. 6. Dislodge monolayer with a cell scraper (or rubber policeman). Transfer to a 1.4-ml conical plastic centrifuge tube. 7. Rinse the well with another 0.4-ml aliquot of extraction buffer, transferring this rinse into the same tube. 8. Incubate at 0 ° for 10-20 min, with intermittent vigorous vortexing. 9. Centrifuge mixture at 8000 g for 15 min. Phosphotyrosyl proteins are purified from the supernatant as described below. (For use in screening hybridoma culture fluids for antibodies to PY proteins, we enrich this

86

ANALYSIS OF PROTEIN PHOSPHORYLATION

[8]

extract for EGF receptors, taking advantage of their ability to bind to wheat germ agglutinin. The extract is passed over a small affinity matrix bearing wheat germ agglutinin (Pharmacia, Piscataway, N J), and partially pure EGF receptors are eluted with 0.3 M N-acetylglucosamine.) Antigenic Fine Specificity of Monoclonal Antibodies In order to begin the process of selecting the best antibody for further use, one can determine first the apparent binding constants of each antibody for PYA1.5-BSA and for a number of potentially cross-reacting molecules. To evaluate the hybrids described above, for example, microtiter plate wells are coated with PYA1.5-BSA and then incubated sequentially with monoclonal antibody and sheep anti-mouse 125I-labeled immunoglobulin (one can also use the ELISA assay described above). The concentration dependence of binding (Fig. 2) provides estimates of the apparent binding constant of each monoclonal antibody, ranked in order of decreasing affinity: 1G2 > 6H9 = 2G8 > 10F7 > 1H9 -> 2D2, with 1G2 having an apparent K d of about 4 x 10-10 M, nearly 10-fold better than our original anti-ABP antibody (2G8). The specificity of the most avid antibody, 1G2, was tested by determining the ability of various phosphate-containing compounds to inhibit its binding to P Y A - B S A in the radioimmunoassays just described above. 10,000

8,0006,000 O "O C

4,000

t~ 2,000

010~'~"" 161°

1(~9

1l~a

107

16 s

Antibody [M] FIG. 2. Concentration dependence of monoclonal antibody binding to PYA-BSA. Microtiter plate wells were coated with 25 ng of PYAl-glutaraldehyde-BSA, adsorptive sites were blocked with milk, and the wells were incubated sequentially with monoclonal antibody and sheep anti-mouse t2SI-labeled Ig as described in the text. II, 1G2; &, 2G8; [], 6H9; 0 , 10F7; O, IH9; A, 2D2.

[8]

MONOCLONAL ANTIBODIES TO PHOSPHOTYROSINE

87

o~ 1.0J •"O -¢ E C

0.8 ~

m

._E O.6X

'~

0.4-

C

.2

0.2-

0,0

_

010 1 0 9

16 + 11) ? l d 6 10 s 1 6 4 Hapten [M]

11} 3

FZG. 3. Inhibition of ]251-labeled IG2 binding to PYA-BSA by phosphotyrosine and its analogs. Microtiter plate wells were coated with antigen and then blocked with milk as described in the text. The various phosphotyrosine analogs and other phosphate-containing molecules were diluted to twice the indicated molarity in 25 txl PBST and added to the wells. Then 125I-labeled 1G2 (50,000 cpm in a total volume of 25/~1 of PBST) was added to each well. After 3 hr at room temperature, the wells were rinsed quickly with PBST four times and radioactivity remaining bound was quantitated. [], PYA-BSA; II, PY-BSA; O, PYA; A, PY; A, phenyl phosphate; O, others (5'-adenosine monophosphate, ATP, phosphoserine, phosphothreonine, ribose 5'-phosphate, ribose 1-phosphate, sodium pyrophosphate, and phosphoseryl-B SA).

The 1G2 antibody was inhibited only by phosphotyrosine analogs (Fig. 3), with an apparent Ki for PYA-BSA of 3 x 10 -9 M , compared to I × 10 -7 M for our original antibody (2G8, data not shown). Ribose phosphates, phosphoserine, phosphothreonine, phosphoseryl-BSA, and a variety of nucleotides (e.g., AMP, ATP) failed to inhibit. Similar analyses of the other monoclonal antibodies revealed that 1H9 and 2G8 cross-react with mononucleotides, while 2D2 cross-reacts with ATP and pyrophosphate. Antibodies 6H9 and 10F7, like 1G2, reacted only with close phosphotyrosine analogs (e.g., phenyl phosphate, p-nitrophenyl phosphate, and phosphotyramine). Thus, the new monoclonal antibodies generally lack the cross-reactivities that 2G8 and other antibodies to ABP exhibited for mononucleotides and certain other phosphate-containing compoundsY Notice that of this panel of monoclonal antibodies, only 1H9 and 2D2 share this cross-reactivity. It is interesting to contrast the fine specificity of the 1G2 antibody with another monoclonal antibody, PY20, produced by immunizing mice with conjugates of PY to KLH and ovalbumin, using 1-ethyl-3-(3-dimethyl-

88

ANALYSIS OF PROTEIN PHOSPHORYLATION

[8]

aminopropyl)-carbodiimide (EDAC) as a bifunctional cross-linking agent (see [9] in this volume). 17The IG2 antibody binds free phenyl phosphate (apparent Kd of 7 x 10 -6 M ) , about 30-fold stronger than free PY, and about 15-fold stronger than free PYA, suggesting that both the free amino group and carboxyl group on PY interfere with antibody binding. Of course, the PY amino and carboxyl groups would be absent in a tyrosinephosphorylated protein, being involved in peptide bonds. The PY20 antibody, on the other hand, binds free phenyl phosphate slightly less well than PY, with half-maximal inhibition in a similar ELISA occurring at about 5 x 10 -4 M phenyl phosphate. 17 Use of 1G2 MAb for Purifying PY Proteins Phosphotyrosine-containing proteins can be purified on a small scale for analytical purposes, from about I x 106 cells, typically labeled in vivo with [32p]Pi, or on a large scale for preparative purposes, using 1 x 109 cells or more. For both of these purposes we favor using the antibody as a solid-phase immunosorbent. This not only allows virtually unlimited reuse of the antibody, but also takes advantage of the "persistent hapten" effect, thereby increasing the apparent avidity of the antibody for any unusual PY protein that may bind weakly to the antibody. 2 For the purposes of illustration, we will describe the steps to purify PY proteins from EGF-stimulated cells on an analytical scale, and from chronic myelogenous leukemia cells on a preparative scale.

Microbatch Purification Extraction Buffer Triton X-100 (1%) SDS (0.1%, w/v) (optional) Tris base (10 mM) EDTA (5 mM) NaC1 (50 mM) Sodium pyrophosphate (30 mM) Sodium fluoride (50 mM) Sodium orthovanadate (100/zM) BSA, crystallized (0.1%, w/v) (Sigma, optional) Phenylmethylsulfonyl fluoride (PMSF; 1 mM) Adjust pH to 7.6 at 4 ° with HC1 PMSF (from a 100 mM stock in 2-propanol or absolute ethanol) must be added to the buffer immediately before it is used to lyse cells. The extraction buffer (lacking PMSF) stores many weeks at 4°. 17j. R. Glenney, Jr., L. Zokas, and M. J. Kamps, J. Imrnunol. Methods 109, 277 (1988).

[8]

MONOCLONAL ANTIBODIES TO PHOSPHOTYROSINE

89

Eluting Buffer Eluting buffer: Same as the extraction buffer, except that the 0.1% BSA is omitted, and 1.0 mM phenyl phosphate is included as a competing hapten to displace PY proteins. Adjust pH to 7.6 with HCI. Store aliquots at - 2 0 °

Affinity Purification of P Y Proteins 1. After the detergent extract (see above) has been clarified by centrifugation, the extract is transferred to a 1.5-ml tube containing 10 /xl of anti-phosphotyrosine-Sepharose beads (15 mg of PY antibody covalently linked to CNBr-activated Sepharose 4B; Pharmacia). This suspension is mixed end over end for 1-3 hr at 0°. 2. After this incubation, the anti-phosphotyrosine beads are sedimented (microcentrifuge, about 7 sec), and the supernatant fluid is carefully aspirated (leaving about 10-20/zl of fluid above the beads). 3. The beads are washed by resuspending them in 1 ml of extraction buffer lacking BSA, sedimenting the beads, and aspirating the supernatant fluid. This wash is repeated three times, with the last allowed to mix for 10 min or more (this helps to remove proteins that are nonspecifically adsorbed to the beads). 4. Phosphotyrosyl proteins that have bound to the anti-phosphotyrosine beads are specifically and gently eluted with the PY analog, phenyl phosphate. Add 30/~1 of eluting buffer to the washed beads. Incubate 5-10 min, occasionally resuspending the beads. 5. Prepare a hemostat with a 25-gauge needle protruding five- to sixhundredths of an inch from the side of the jaws. With another needle make a pinhole in the cap of the tube that contains the beads and eluting buffer. Wipe dry the bottom of the tube and puncture a single, straight hole in the bottom using the 25-gauge needle-hemostat. Place this tube piggyback onto an open 1.5-ml tube, and then insert them together into an appropriate carrier. 6. Centrifuge at 1500 g for 1 min. The eluate (45/xl) will collect in the lower tube, free of any Sepharose beads. The PY proteins can then be analyzed further by, e.g., immunoprecipitation with antibodies to specific proteins and SDS-PAGE.

Notes 1. When pipetting beads, cut off about 1/4-in. of pipette tip to afford a wider bore. 2. To regenerate used anti-phosphotyrosine beads: Wash the beads several times with a solution of one part extraction buffer and one part 2 M NaC1. Beads have been successfully regenerated and used through 100 cycles.

90

ANALYSIS OF PROTEIN PHOSPHORYLATION

[8]

3. To minimize artifactual dephosphorylation, do not exceed 2 × 106 cells/ml of extraction buffer unless 0.1% SDS is included in the buffer. We have used the microbatch procedure to isolate PY proteins from retrovirally transformed cells, from cells stimulated with EGF, transforming growth factor a (TGF-a), PDGF, fibroblast growth factor (FGF), insulin, colony-stimulating factor 1 (CSF-I), and interleukin 3 (IL-3), and from many other normal and transformed cells, including human chronic myelogenous leukemia cells expressing the aberrant p210 °.... b~ tyrosine kinase.Z'4-s'18-22 Antibody specificity has been evaluated by phosphoamino acid analysis of purified proteins,19,2° as well as by phenyl phosphate inhibition of binding to the antibody (Fig. 4). Binding of the abl p210 was inhibited >95% by as little as 0.04 mM phenyl phosphate, and 0.3 mM phenyl phosphate quantitatively eluted PY proteins.

Large-Scale Isolation of P Y Proteins Extraction Buffer Extraction buffer: As in the microbatch procedure, but eliminating BSA

Eluting Buffer Eluting buffer: As in the microbatch procedure, but using 40 mM phenyl phosphate. If ultrafiltration steps will be used to subsequently concentrate the PY proteins, substitute 0.7% (w/v)/3-octylglucoside for Triton X-100.

Extraction o f Nonadherent Cells 1. Nonadherent cells growing in spinner culture are harvested by centrifugation, and the cell pellets are treated with extraction buffer (all subsequent steps at 4°): 2 × 10 9 cells are resuspended in 20 ml of ice-cold PBS and vigorously vortexed while adding 1 liter of extraction buffer. 2. The extract is clarified by centrifugation at 1500 g for 30 min and subsequent filtration through fluted Whatman (Clifton, N J) # 1 filter paper. 18 j. C. Bell, L. C. Mahadevan, W. H. Colledge, A. R. Frackelton, Jr., M. G. Sargent, and J. G. Foulkes, Nature (London) 325, 552 (1987). 19R. D. Huhn, M. R. Posner, J. G. Foulkes, S. Rayter, and A. R. Frackelton, Jr., Proc. Natl. Acad. Sci. U.S.A. 84, 4408 (1987). 20 R. D. Huhn, M. E. Cicione, and A. R. Frackelton, Jr., J. Cell. Biochem. 39, 129 (1989). eJ R. Isfort, R. D. Huhn, A. R. Frackelton, Jr., and J. N. Ihle, J. Biol. Chem. 263, 19203 (1988). 22 A. Sengupta, W.-K. Liu, Y.-G. Yeung, D. Yeung, A. R. Frackelton, Jr., and E. R. Stanley, Proc. Natl. Acad. Sci. U.S.A. 83, 8062 (1988).

[8]

91

M O N O C L O N AANTIBODIES L TO PHOSPHOTYROSINE

A

B

0

c~

0

d

d

,~

o

~. c° ~ ~ 0 o

o

•

0

o~ ,-

kDa -205-

-116 -93-

-67-

-43-

-29FXG. 4. Isolation of phosphotyrosyl proteins is blocked by competing hapten. Chronic myelogenous leukemia cells (RWLeu4), which express an aberrant, 210-kDa abl protein with constitutive tyrosine kinase activity, were labeled and extracted as indicated in the text. (A) Phenyl phosphate was added to the cell extracts at 0.04, 0.2, or 1.0 mM as indicated. (B) Phosphotyrosyl proteins were purified as usual, except that the indicated concentration (raM) of phenyl phosphate was used for specific elution.

3. Gently stir the clarified extract overnight with 6 ml of the 1G2-Sepharose 4B immunosorbent. 4. Collect the immunosorbent beads on a sintered glass funnel (coarse), and wash with 100 ml of extraction buffer. 5. Transfer the immunosorbent into a 50-ml polypropylene culture tube, and wash three times for 10 min each with 50 ml of filtered extraction buffer (filtered through 0.45-/zm nitrocellulose to remove the ubiquitous keratins and any other trace contaminant proteins). 6. Transfer the beads into a small open-bed column, and wash with 12 ml of filtered extraction buffer. 7. Specifically elute PY proteins from the immunosorbent using 12 ml of the phenyl phosphate-containing elution buffer. These proteins can be concentrated by ultrafiltration, and further puri-

92

ANALYSIS OF PROTEIN PHOSPHORYLATION

[9]

tied by standard techniques including fast protein liquid chromatography (FPLC), glycerol gradient ultracentrifugation, and one- and two-dimensional SDS-PAGE. The eluted PY proteins are typically nondenatured, and thus can be analyzed for a variety of functional activities. These proteins can be stored at - 7 0 ° for >1 year with no obvious changes in their SDS electrophoretic properties. We have used this procedure with good success for isolating PY proteins from chronic myelogenous leukemia cells and, with minor modifications, for isolating PY proteins from growth factor-stimulated adherent cells. Greater than 90% of the protein phosphotyrosine in EGF-stimulated cells bound to the 1G2 immunosorbent (data not shown). The 1G2 hybridoma cells can be obtained from the ATCC (Rockville, MD) Safe Deposit collection, and the antibody itself is commercially available. Acknowledgment This w o r k was s u p p o r t e d in part by Grants R01-CA39235 and 5P30-CA13943 from the N a t i o n a l C a n c e r Institute.

[9] I s o l a t i o n o f T y r o s i n e - P h o s p h o r y l a t e d P r o t e i n s a n d Generation of Monoclonal Antibodies

By

JOHN R. GLENNEY

Introduction The mechanism by which tyrosine kinases exert their effects is not well understood. Although tyrosine-specific protein kinase activity was described more than 10 years ago, the relevant substrates that mediate the effects of tyrosine phosphorylation have been elusive. One approach to identification of tyrosine kinase substrates has been to analyze the phosphoamino acid content of proteins that are thought to play a role in the process affected by the tyrosine kinase. This approach assumes that we can deduce the identity of the relevant substrates and simply test them for the presence of phosphotyrosine. It is quite possible that some mediators of the tyrosine kinase signal are novel proteins that have not been previously studied. To gain insight into the function and regulation of such substrates we have taken the approach of (I) isolation of tyrosine-phosphorylated proteins using immunoaffinity chromatography with a highaffinity monoclonal anti-phosphotyrosine antibody and (2) generation of METHODS IN ENZYMOLOGY, VOL. 201

Copyright © 1991by AcademicPress, Inc. All rights of reproductionin any form reserved.