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required for the preparation of monoclonal antibodies. The starting material, goat immune serum, is plentiful and only the purified isozymes are required for the isolation of the specific antibodies. We found that these purified antibodies have a broader range of applications than the monoclonal antibodies we prepared.
[39] G e n e r a t i o n a n d Use of A n t i - p e p t i d e Antibodies D i r e c t e d against C a t a l y t i c D o m a i n of P r o t e i n Kinases
By GAIL M. CLINTON and NANCY A. BROWN The molecular architecture of the protein kinases includes regulatory regions which are diverse in their amino acid sequence and a catalytic domain which is composed of sequences that are conserved because they are required for catalysis. The 250 to 300 amino acid residues of the catalytic domain can be divided into highly conserved subdomains that are interspersed with relatively divergent sequences.l The highly conserved subdomains contain amino acid residues which are likely to participate directly in catalysis or to provide structural constraints that are necessary to form the active site pocket. Although it has been shown that an invariant lysine residue in the catalytic domain is involved in ATP binding2-4 (Lys-72 in the cAMP-dependent protein kinase 1) the functions of amino acid residues in other conserved subdomains are unknown _/---.- It Eemains to be determined which sequences within the eatal~,Iic domain form the active site pocket, which may be blocked or buried by regulatory sequences and thus are inaccessible to substrates, and which subdomains may be altered in conformation by binding of ligands and other effectors. Antibodies against selected amino acid sequences in proteins have been found to be valuable reagents for examining the location of these sequences within a native polypeptide. Immunoprecipitation with antipeptide antibodies has been used to determine whether the cognate peptide sequence is exposed or buried, and whether its conformation changes following binding of an effector. Ligand-activated autophosphorylation of the platelet-derived growth factor (PDGF) receptor results in enhanced accessibility of sites in the C-terminal autophosphorylation domain to 1 S. K. Hanks, A. M. Quinn, and T. Hunter, Science 241, 42 (1988). 2 M. J. Zoller, N. C. Nelson, and S. S. Taylor, J. Biol. Chem. 256, 10837 (1981). 3 M. D. Kamps, S. S. Taylor, and B. M. Sefton, Nature (London) 310, 589 (1984). 4 M. W. Russo, T. J. Lukes, S. Cohen, and J. V. Staros, J. Biol. Chem. 260, 5205 (1985).
METHODS IN ENZYMOLOGY,VOL. 200
Copyright © 1991by AcademicPress, Inc. All rightsof reproductionin any form reserved.
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binding with anti-peptide antibodies.5,6 Autophosphorylation of the insulin receptor exposes the previously inaccesible autophosphorylation sites in the catalytic domain and allows immunoprecipitation with anti-peptide antibodies. 7,8 The catalytic domain sequence, HRDLAARN [residues 811-8188a in the human epidermal growth factor (EGF) receptor], binds anti-peptide antibody only when the protein structure has been opened by an elevation in temperature or by mild denaturation,9 indicating that this sequence is buried in the native EGF receptor. Anti-peptide antibodies have also supplied information on the involvement in catalysis of selected sequences within protein kinases. If the antibody binds to a site directly involved in catalysis, it may block interaction of that site with substrates. Antibody directed against a v-abl tyrosine kinase sequence which includes the Gly at the C-terminal end of the nucleotide binding motif, GIy-X-GIy-X-X-Gly, inhibits both autophosphorylation and phosphorylation of exogenous substrates. 10This inhibition is likely due to interference with ATP binding. The kinase activities of the EGF receptor, the insulin receptor, and the IGF-I receptor are neutralized when site-directed antibodies bind to autophosphorylation sites (corresponding to Tyr-416 in Src) within the catalytic domain. 11A site near the C terminus of the catalytic domain, which contains an invariant Arg (residue 280 in the cAMP kinase), also appears critical for catalysis since antipeptide antibodies against a sequence within this site (residues 498-512 in Src) neutralize the kinase activity of Src.12 Kinase activity is not always neutralized, however, by binding of antibodies to sites in the catalytic domain. For example, the EGF receptor retains autophosphorylation activity when complexed to antibodies against either of two highly conserved sequences with the catalytic domain (residues 852-861 and 871-878). 9 Furthermore, there is no apparent effect on kinase activity when anti-
M. T. Keating, J. A. Escobedo, and L. T. Williams, J. Biol. Chem. 263, 12805 (1988). 6 S. Bishayee, S. Majumdar, C. D. Scher, and S. Khan, Mol. Cell. Biol. 8, 3696 (1988). 7 R. Perlman, D. P. Bottaro, M. F. White, and R. Kahn, J. Biol. Chem. 264, 8946 (1989). s R. Herrera and O. M. Rosen, J. Biol. Chem. 261, 1980. Sa Single-letter abbreviations are used for amino acids: A, alanine; D, aspartic acid; H, histidine; L, leucine; N, asparagine; R, arginine. 9 N. Brown, L. A. Compton, and G. M. Clinton, unpublished observations (1990). 10j. 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). I1 T. Izume, S. Yoshiyuki, Y. Akanuma, J. Takaku, and M. Kasuga, J. Biol. Chem. 263, 10386 (1988). 12 L. E. Gentry, L. R. Rohrschneider, J. E. Casnellie, and E. G. Krebs, J. Biol. Chem. 258, 11219 (1983).
[39]
ANTI-PEPTIDE Abs AGAINSTPK CATALYTICDOMAIN
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peptide antibodies are complexed to sites in the C-terminal domain of Src, 12 the PDGF receptor, 6 insulin receptor, 11,13and EGF receptor. 9:1,14 The interaction of antibodies with specific sites outside of the catalytic domain may be used to examine potential regulatory roles of these sites. When an antibody interacts with one site outside the insulin-binding site in the extracellular domain of the insulin receptor/3 subunit, autophosphorylation and kinase activity are inhibited.15 Similarly, binding of an antipeptide antibody to a region in the insulin receptor on the cytoplasmic side of and directly adjacent to the transmembrane domain inhibits kinase activity.13 Binding of an anti-peptide antibody to the pseudosubstrate sequence in the regulatory domain of protein kinase C has been found to activate this kinase in the absence of calcium and phospholipids, possibly by relieving the inhibitory effects of this sequence. 16 Generation of Anti-Peptide Antibodies to Sites in Catalytic Domain There have been several publications describing the generation and use of anti-peptide antibodies. 17-~9 The procedures described here are targeted toward the generation of antibodies against conserved sequences in the catalytic domain of protein kinases and the special considerations that may be required in using these antibodies. The methods presented are synthesized from our experience with anti-peptide antibodies directed against four highly conserved sites within the catalytic domain and two nonconserved regions from outside the catalytic domain of the EGF receptor (EGFR). Choosing A m i n o A c i d S e q u e n c e s f r o m Kinase Domain f o r Synthesis o f Peptide Antigen
When selecting the amino acid sequence from the catalytic domain to which the anti-peptide antibody will be directed, it is important to consider whether the epitopes will be collinear with the primary sequence. In the absence of information on the three-dimensional structure of the catalytic ~3R. Herrera, L. Petruzzelli,N. Thomas, H. N. Bramson,E. T. Kaiser,and O. M. Rosen, Proc. Natl. Acad. Sci. U.S.A. 82, 7899 (1985). 14W. J. Gullick, J. Downward, and M. D. Waterfield,EMBO J. 4, 2869 (1985). ~5R. Gherzi, G. Sesti, G. Andraghetti,R. De Pirro, R. Lauro, L. Adezanti,and R. Condera, J. Biol. Chem. 264, 8627 (1989). 16M. Makowskeand O. M. Rosen, J. Biol. Chem. 264, 16155(1989). 17R. A. Lerner, Nature (London) 299, 592 (1982). 18G. Walter and R. F. Doolittle, Genet. Eng. 5, 61 (1983). 19G. Walter, J. lmmunol. Methods 88, 149 (1986).
466
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domain, whether the sequence is conserved may be considered. Patterns of amino acid sequence conservation can most easily be assessed by consulting the publication of Hanks, Quinn, and Hunter, ~in which these authors aligned the sequences within the catalytic domains of 65 different protein kinases. Attention should be directed not only to the 11 conserved subdomains (numbered I through XI) but also to the patterns of conservation within and outside of these subdomains. For example, within subdomain VI, the sequence HRDLRAAN (HRD) for the Src family (residues 384-391) is one of the most highly conserved segments in the tyrosine kinase family and may, therefore, represent a site that is structurally or functionally important. Other considerations when choosing the amino acid sequence, including the length and the chemical properties of the amino acid side chains, have been discussed. 17-2°
Conjugation of Synthetic Peptide There are several procedures for conjugating the peptide to a carrier protein. We have found that cross-linking synthetic peptides with carbodiimide by the following procedure has yielded antibodies which are reactive with highly conserved sequences in the interior of the EGFR. 1. Dissolve 2.5 mg of the synthetic peptide in 1 ml water. Determine absorbance at 214 nm or at the wavelength of maximum absorbance for the particular peptide. 2. Add 7.5 mg hemocyanin (thyroglobulin works also) in 100/zl of water containing 0.5 mg/ml cross-linker 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (Sigma, St. Louis, MO). Vortex and let stand at room temperature for 30 min. 3. Dilute to 3 ml with water and put into dialysis tubing with a pore size that will allow free peptide to diffuse out. Dialyze against I00 ml of water overnight. 4. Determine absorbance of dialysate and calculate the amount of peptide in the dialysate to determine the efficiencyof conjugation. Alternatively, spot an aliquot of dialysate onto paper and react with ninhydrin to estimate amount of free peptide. We have found that 40 to 50% of the peptide is usually conjugated. We have also assessed the immunogenicity of peptide conjugated to hemocyanin using glutaraldehyde as the cross-linking reagent. Glutaraldehyde efficiently conjugated the peptide to the carrier protein and the conjugated peptide generated antibody reactive to the immobilized peptide in an enzyme-linked immunosorbent assay (ELISA). However, we have 2o T. P. Hopp and K. R. Woods, Proc. Natl. Acad. Sci. U.S.A. 78, 3824 (1981).
[39]
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found that these antibodies did not immunoprecipitate the EGFR if the cognate sequence was in the interior of the protein. The conformation of the peptide that is cross-linked with glutaraldehyde may be different than the conformation of the same sequence when it is located at an internal site in the protein. Anti-peptide antibodies to sites in the flexible C-terminal domain, on the other hand, bound to the EGFR whether glutaraldehyde or carbodiimide was used to prepare the immunogen. The other procedure we have evaluated for producing immunogen is the synthesis of a heptalysine c o r e to which peptide monomers were attached according to the method of Tam. 21,22 Peptides attached to the polylysine core have been found to be immunogenic, thereby eliminating the need for a carder protein. However, because the synthesis of the peptide on the polylysine core was more difficult and there did not appear to be improvement in the titer or in the proportion of immunized rabbits that produced antibodies, there seems little reason to use this procedure.
Immunization New Zealand White rabbits of 2.5-3.0 kg are bled for preimmune sera and then injected subcutaneously with conjugated peptide emulsified at a ratio of 1 to 1.2 with Freund's complete adjuvant for the first and last injections, and Freund's incomplete adjuvant for all other injections. The rabbits are given subcutaneous booster injections at 2-week intervals and weekly test bleeds are begun 2-3 weeks after the initial injection. The amount of peptide used for immunization varies from 50 to 500/zg/injection. Antibodies of highest titer are produced in animals immunized with 50 to 100/.~g peptide conjugated to hemocyanin using EDC. Our experience is that the titer and the time of production of antibody are more affected by differences in individual rabbits rather than by the injection scheme used.
Monitoring Anti-Peptide Antibody Production Production of polyclonal antisera which bind to conserved, internal sites in the catalytic domain needs to be monitored by immunoprecipitation or by Western blotting rather than by ELISA using immobilized, synthetic peptide. A discrepancy between the titer of the antibody that binds to the peptide versus the antibody that binds to the cognate sequence in the whole protein has previously been observed 7A° and may be caused by differences in how the antigenic determinants are displayed. In several 21 D. N. Posnett, H. McGrath, and J. P. Tam, J. Biol. Chem. 263, 1719 (1988). 22 j. p. Tam, Proc. Natl. Acad. Sci. U.S.A. 85, 5409 (1988).
468
ANTIBODIES AGAINST PROTEIN KINASES 50,
~
[39] .30
40 n-
,20
mo
"r"
s ~
oo Q.O
20
o~ ,E~
,lo
_1 .i
10,
0
I--
2
4
~
~
1'o
12
o
Weeks after Initial Injection FIG. 1. Production of anti-peptide antibodies to a conserved sequence from the catalytic domain of the EGF receptor (EGFR). Aliquots (50 /zl) of sera from rabbits immunized with HRDLAARN conjugated to hemocyanin were tested for the presence of antibody by immunoprecipitation of 32p-labeled EGF receptor. Parallel immunoprecipitations were conducted with a control polyclonal antiserum to the extracellular domain of the EGF receptor under conditions of antibody excess where 95% of the EGF receptor was immunoprecipitated. The serum from each test bleed was also tested by ELISA using the peptide immobilized on microtiter plates. The ELISA results are expressed relative to an ELISA using a control anti-peptide antibody against a peptide containing a sequence from the carboxy terminus of the EGF receptor.
cases, we have also found that the antibody reactive with the entire protein was produced transiently and early compared to antibody that reacted with the isolated peptide. In fact, when maximum ELISA titer was attained, the antibody that immunoprecipitated the entire protein was often no longer detectable. Eight different rabbits immunized with conjugated HRDLAARN (residues 811-818 of the EGFR) have displayed this pattern and Fig. I illustrates the profile of antibody production in one of these
[39]
ANTI-PEPTIDEAbs AGAINSTPK CATALYTICDOMAIN
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TABLE I BUFFERS USED IN IMMUNOPRECIPITATION a Concentration (%) Buffer
NaC1 (mM)
Deoxycholate
SDS
Glycerol
Kinase activity b
A B C
-150 150
--1
-0.05 0.1
10 10 --
+ + -
All buffers contain 20 m M Tris, p H 7.6, 1% Triton X-100, 1% aprotinin, 1 m M P M S F , 2 m M s o d i u m vanadate. b T h e a u t o p h o s p h o r y l a t i o n activity of the E G F receptor was m e a s u r e d in each of the buffers.
rabbits. Thus for rabbits immunized with peptides containing highly conserved sequences from the catalytic domain of protein kinases, we recommend that antibody production be monitored on a weekly basis by immunoprecipitation of the target protein kinase and that monitoring begin following the first booster injection. The most convenient and sensitive method to monitor production of antibodies that immunoprecipitate a protein kinase capable of autophosphorylation is to radiolabel the protein with [y)2p]ATP in an in vitro kinase reaction. Because the antibodies to highly conserved sites may recognize several protein kinases, it may be necessary to have a partially purified preparation or a concentrated source of the specific target protein kinase. For a source of concentrated EGFR, we use membrane vesicles prepared from A431 cells by a previously described procedure.23 The immunoprecipitation protocol follows: 1. The protein kinase is labeled by autophosphorylation in a reaction containing [y-32p]ATP. The components in the reaction and their concentration depend on the particular protein kinase to be investigated. 2. The 32p-labeled protein kinase is denatured in buffer C (Table I) to expose sites that may react with the antibody. 3. The denatured protein kinase is mixed with 50/zl antiserum from a test bleed and the volume is adjusted to 200 /.d with buffer C (50 /zl antiserum is the maximum amount that can be used before overloading the SDS gel with IgG). 4. The sera from the test bleed and the radiolabeled protein kinase are incubated for 1 hr at 4° with continuous rotation. 23 S. C o h e n , in " M e t h o d s in E n z y m o l o g y " (J. D. Corbin and J. D. H a r d m a n , eds.), Vol. 99, p. 379. A c a d e m i c Press, N e w York, 1983.
470
ANTIBODIES AGAINST PROTEIN KINASES
[39]
5. Protein A-Sepharose is added at a ratio of one bed volume to one volume of sera and the mixture is incubated with shaking at 4° for 30 min. 6. The immune complex is washed four times with 1 ml of buffer C. 7. The immune complex is suspended into 100/zl of sample buffer containing 63 mM Tris-HC1, pH 6.8, 10% (v/v) glycerol, 2% (w/v) SDS, 5% (v/v) 2-mercaptoethanol, and 0.002% (w/v) Bromphenol Blue and incubated in a boiling water bath for 2 min. The released proteins are analyzed by electrophoresis in an SDS-containing gel of 1.5 mm thickness with a well size that holds 200/xl of sample volume.
Tests of Specificity of Anti-Peptide Antibodies Binding of the anti-peptide antibody to a specific sequence in the target protein is best demonstrated by showing that immunoprecipitation is blocked by preincubation of the antibody with its cognate peptide. To conduct peptide-blocking experiments, the antiserum is incubated without or with different concentrations of peptide for 1 hr at 4° with continuous shaking prior to addition of the radiolabeled antigen. The remainder of the immunoprecipitation protocol is conducted as described above. We find that the concentration of peptide is sometimes important in determining the extent to which a specific antibody is blocked by its cognate peptide. For antibodies prepared to the HRDLAARN sequence, we have found that a peptide concentration of about 15/zg/ml is most effective at blocking immunoprecipitation of the EGFR by 250 ng of peptide affinity purified antibody.
Antibody Storage We find that anti-peptide antisera raised against some sequences from the catalytic domain in the EGFR, such as HRDLAARN, are unstable when stored outside of the rabbit. This is true only for the antibody that reacts with the entire EGFR, not for that which binds the immobilized peptide in an ELISA. The sera are therefore stored at 4° or at room temperature with the addition of 0.02% (w/v) sodium azide to prevent bacterial growth. Under these conditions, the activity is retained for 1-3 weeks. In contrast, antisera against nonconserved sequences within the EGFR are stable when stored at - 7 0 °. Unstable antibodies such as anti-HRD retain their activity best when bound to a peptide affinity column. The column is prepared using a 20-ml bed volume of Affi-Gel 10 (Bio-Rad, Richmond, CA) coupled to 15 mg of peptide using the procedure recommended by the manufacturer. We bind antibodies to their respective peptide affinity columns using the following procedure:
[39]
ANTI-PEPTIDE Abs AGAINSTPK CATALYTICDOMAIN
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1. Precipitate the IgG-containing fraction from 15 ml of antiserum by adding a saturated ammonium sulfate solution to a final concentration of 40% of saturation. 2. Centrifuge at 10,000 g for 30 min at 4 ° and resuspend the pellet in 3 ml of 20 mM HEPES, 150 mM NaC1, pH 7.5. Dialyze against the same buffer overnight at 4 °. 3. Slowly add the dialyzed IgG to the peptide affinity column and incubate at 4 ° for 1 hr. 4. Wash the column with equilibration buffer until the absorbance at 280 nm of the eluate is reduced to background levels. 5. Store the column at 4 ° in buffer containing 0.02% sodium azide. Antibodies appear to be stable while bound to the peptide affinity column and can be used after elution from the column. However, they lose activity with time following elution from the column. Elution is accomplished as follows: 1. Add 60 ml of 0.1 M sodium acetate, 0.5 M NaCI, pH 2 to the column. Collect 0.5-ml fractions directly into 0.5 ml of 0.1 M glycine, pH 12, to neutralize each fraction. 2. Monitor the protein content of the fractions by absorbance at 280 nm. 3. The fractions with maximum absorbance may be used directly. Otherwise, combine these fractions and precipitate the IgG using 40% (v/v) ammonium sulfate. 4. Centrifuge and resuspend the IgG pellet in water. Dialyze against 20 mM HEPES, pH 7.5 at 4 °. The protease inhibitors aprotinin (0.5%), leupeptin (4/zM), and phenylmethylsulfonyl fluoride (PMSF) (2 mM) (all from Sigma, St. Louis, MO) are added to the purified antibody if it is not used immediately. Use of Anti-Peptide Antibodies in Studies of Topology and Function of Sites in Catalytic Domain
Determination of Topology To determine the topology of a specific sequence in the native protein kinase, immunoprecipitation of the radiolabeled protein by the anti-peptide antibody is conducted in buffer A (Table I) at 4 ° under conditions of antibody excess. Buffer A is relatively nondenaturing and, in the case of EGFR, stabilizes the kinase activity. Subsequent steps in the immunoprecipitation are carded out as described above. It is critical that a control
472
ANTIBODIES AGAINST PROTEIN KINASES
[39]
antibody which is known to immunoprecipitate the protein kinase in its native state be used here as a standard. In addition, the proportion of the protein kinase immunoprecipitated under the nondenaturing conditions of buffer A should be compared with the amount immunoprecipitated in parallel under the denaturing conditions of buffer C. Such a comparison addresses the question of whether conformation has an effect on the efficiency of antibody binding to its cognate sequence. The radiolabeled protein kinase in the immune complex can be most accurately quantitated by SDS gel electrophoresis and scintillation counting of the radiolabeled bands excised from the gel. It may also be of interest to determine whether partial "opening" of the protein structure is sufficient to expose a particular site in the protein or whether the protein needs to be more thoroughly denatured. This can best be determined by immunoprecipitation of the radiolabeled protein kinase in buffer B (Table I). When buffer B with 0.05% SDS is used for immunoprecipitation the protein kinase activity in the immune complex is about 80% of that found when buffer A is used. We have further found that sites that are inaccessible to antibody in buffer A may be exposed in buffer B. Another way to achieve partial opening of the protein structure is to incubate the radiolabeled protein kinase together with the anti-peptide antibody in either buffer A or buffer B at 34° for 5 rain-before incubation at 4° for 1 hr. The effects of modulators of kinase activity on the topology of specific sites in the catalytic domain of protein kinases can be examined by binding of anti-peptide antibodies. For example, one can ask whether the exposure of the sites in the kinase domain is altered by ligand binding or by autophosphorylation of the protein kinase. This approach has been described in more detail for the PDGF receptor5'6 and the insulin receptor7'8 tyrosine kinases.
Identification of Sites Criticalfor Catalysis The following protocol can be used to determine whether binding of anti-peptide antibody to specific sites in the catalytic domain neutralizes kinase activity. It is critical that a control antibody that efficiently immunoprecipitates the protein kinase but does not inhibit kinase activity be included in order to determine the amount of kinase activity that is lost during the immunoprecipitation procedure. 1. Mix 1 vol of anti-peptide antisera with one bed volume of protein A-Sepharose and incubate at 4 ° for 30 min on a shaking platform. 2. Wash the IgG-protein A complex three times with 10 to 20 vol of buffer A.
[39]
ANTI-PEPTIDE Abs AGAINSTPK CATALYTICDOMAIN
473
3. Mix the protein kinase with the anti-peptide IgG that is bound to protein A-Sepharose in buffer A or buffer B at 4° for 1 hr with shaking. 4. Wash the antigen-antibody complex with about 1 ml of buffer B once and buffer A three times. Care should be taken to work quickly and keep the protein kinase on ice to preserve kinase activity. 5. To test for autophosphorylation suspend the immune complex in about 100/zl of a kinase reaction mixture containing [~/-nP]ATP. Incubate under conditions that achieve maximum levels of autophosphorylation for the particular protein kinase. (Because autophosphorylation occurs very quickly, it is not feasible to analyze the kinetics of autophosphorylation. Thus the overall level rather than the rate of autophosphorylation is determined.) 6. Wash the immune complex three times with buffer C to remove the unincorporated 32p and analyze the proteins in the immune complex by SDS gel electrophoresis. To test for kinase activity toward exogenous protein substrate: 1. Suspend the washed immune complex from step (4) above in a kinase reaction mixture containing [y-32p]ATP and an exogenous substrate such as enolase or angiotensin for tyrosine kinases such as the EGFR. Incubate the kinase reaction at a temperature where a linear rate of substrate phosphorylation can be maintained. 2. Remove aliquots at different times of incubation, plunge into a boiling water bath to stop the kinase reaction, and separate the soluble substrate from the immune complex by centrifugation. 3. Phosphorylation of the exogenous substrate is determined following separation of the substrate from the other reaction components by techniques such as SDS gel electrophoresis. The rate of phosphorylation of the exogenous substrate can then be determined by quantitation of the 32p incorporated with time. The rate of substrate phosphorylation will reveal whether the kinase activity is inhibited by binding of anti-peptide antibody to a specific sequence. In some cases it may be desirable to determine the effects of the antibody on the kinase activity in a soluble reaction where the kinase has not been immunoprecipitated. The advantage is that the effects of antibody on the entire population of kinase molecules rather than on those that are immunoprecipitated can be evaluated. This approach, however, requires that the antibody be of high titer, in excess of the protein kinase, and be highly purified, for example by peptide affinity chromatography. To test for antibody inhibition of kinase activity in a soluble reaction, add the purified anti-peptide IgG to the protein kinase and incubate the
474
ANTIBODIES AGAINST PROTEIN KINASES
[39]
mixture at 4° with shaking for 1 hr. Add the components of the kinase reaction, including [y-32p]ATP. Remove aliquots at increasing times to determine the level of substrate phosphorylation by the amount of 32p that is incorporated. Use peptide blocking (discussed above) of the purified IgG to establish that inhibition of kinase activity is caused by antibody binding to a specific sequence.
ANTI-PEPTIDE Abs AGAINSTPK CATALYTICDOMAIN
463
required for the preparation of monoclonal antibodies. The starting material, goat immune serum, is plentiful and only the purified isozymes are required for the isolation of the specific antibodies. We found that these purified antibodies have a broader range of applications than the monoclonal antibodies we prepared.
[39] G e n e r a t i o n a n d Use of A n t i - p e p t i d e Antibodies D i r e c t e d against C a t a l y t i c D o m a i n of P r o t e i n Kinases
By GAIL M. CLINTON and NANCY A. BROWN The molecular architecture of the protein kinases includes regulatory regions which are diverse in their amino acid sequence and a catalytic domain which is composed of sequences that are conserved because they are required for catalysis. The 250 to 300 amino acid residues of the catalytic domain can be divided into highly conserved subdomains that are interspersed with relatively divergent sequences.l The highly conserved subdomains contain amino acid residues which are likely to participate directly in catalysis or to provide structural constraints that are necessary to form the active site pocket. Although it has been shown that an invariant lysine residue in the catalytic domain is involved in ATP binding2-4 (Lys-72 in the cAMP-dependent protein kinase 1) the functions of amino acid residues in other conserved subdomains are unknown _/---.- It Eemains to be determined which sequences within the eatal~,Iic domain form the active site pocket, which may be blocked or buried by regulatory sequences and thus are inaccessible to substrates, and which subdomains may be altered in conformation by binding of ligands and other effectors. Antibodies against selected amino acid sequences in proteins have been found to be valuable reagents for examining the location of these sequences within a native polypeptide. Immunoprecipitation with antipeptide antibodies has been used to determine whether the cognate peptide sequence is exposed or buried, and whether its conformation changes following binding of an effector. Ligand-activated autophosphorylation of the platelet-derived growth factor (PDGF) receptor results in enhanced accessibility of sites in the C-terminal autophosphorylation domain to 1 S. K. Hanks, A. M. Quinn, and T. Hunter, Science 241, 42 (1988). 2 M. J. Zoller, N. C. Nelson, and S. S. Taylor, J. Biol. Chem. 256, 10837 (1981). 3 M. D. Kamps, S. S. Taylor, and B. M. Sefton, Nature (London) 310, 589 (1984). 4 M. W. Russo, T. J. Lukes, S. Cohen, and J. V. Staros, J. Biol. Chem. 260, 5205 (1985).
METHODS IN ENZYMOLOGY,VOL. 200
Copyright © 1991by AcademicPress, Inc. All rightsof reproductionin any form reserved.
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binding with anti-peptide antibodies.5,6 Autophosphorylation of the insulin receptor exposes the previously inaccesible autophosphorylation sites in the catalytic domain and allows immunoprecipitation with anti-peptide antibodies. 7,8 The catalytic domain sequence, HRDLAARN [residues 811-8188a in the human epidermal growth factor (EGF) receptor], binds anti-peptide antibody only when the protein structure has been opened by an elevation in temperature or by mild denaturation,9 indicating that this sequence is buried in the native EGF receptor. Anti-peptide antibodies have also supplied information on the involvement in catalysis of selected sequences within protein kinases. If the antibody binds to a site directly involved in catalysis, it may block interaction of that site with substrates. Antibody directed against a v-abl tyrosine kinase sequence which includes the Gly at the C-terminal end of the nucleotide binding motif, GIy-X-GIy-X-X-Gly, inhibits both autophosphorylation and phosphorylation of exogenous substrates. 10This inhibition is likely due to interference with ATP binding. The kinase activities of the EGF receptor, the insulin receptor, and the IGF-I receptor are neutralized when site-directed antibodies bind to autophosphorylation sites (corresponding to Tyr-416 in Src) within the catalytic domain. 11A site near the C terminus of the catalytic domain, which contains an invariant Arg (residue 280 in the cAMP kinase), also appears critical for catalysis since antipeptide antibodies against a sequence within this site (residues 498-512 in Src) neutralize the kinase activity of Src.12 Kinase activity is not always neutralized, however, by binding of antibodies to sites in the catalytic domain. For example, the EGF receptor retains autophosphorylation activity when complexed to antibodies against either of two highly conserved sequences with the catalytic domain (residues 852-861 and 871-878). 9 Furthermore, there is no apparent effect on kinase activity when anti-
M. T. Keating, J. A. Escobedo, and L. T. Williams, J. Biol. Chem. 263, 12805 (1988). 6 S. Bishayee, S. Majumdar, C. D. Scher, and S. Khan, Mol. Cell. Biol. 8, 3696 (1988). 7 R. Perlman, D. P. Bottaro, M. F. White, and R. Kahn, J. Biol. Chem. 264, 8946 (1989). s R. Herrera and O. M. Rosen, J. Biol. Chem. 261, 1980. Sa Single-letter abbreviations are used for amino acids: A, alanine; D, aspartic acid; H, histidine; L, leucine; N, asparagine; R, arginine. 9 N. Brown, L. A. Compton, and G. M. Clinton, unpublished observations (1990). 10j. 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). I1 T. Izume, S. Yoshiyuki, Y. Akanuma, J. Takaku, and M. Kasuga, J. Biol. Chem. 263, 10386 (1988). 12 L. E. Gentry, L. R. Rohrschneider, J. E. Casnellie, and E. G. Krebs, J. Biol. Chem. 258, 11219 (1983).
[39]
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peptide antibodies are complexed to sites in the C-terminal domain of Src, 12 the PDGF receptor, 6 insulin receptor, 11,13and EGF receptor. 9:1,14 The interaction of antibodies with specific sites outside of the catalytic domain may be used to examine potential regulatory roles of these sites. When an antibody interacts with one site outside the insulin-binding site in the extracellular domain of the insulin receptor/3 subunit, autophosphorylation and kinase activity are inhibited.15 Similarly, binding of an antipeptide antibody to a region in the insulin receptor on the cytoplasmic side of and directly adjacent to the transmembrane domain inhibits kinase activity.13 Binding of an anti-peptide antibody to the pseudosubstrate sequence in the regulatory domain of protein kinase C has been found to activate this kinase in the absence of calcium and phospholipids, possibly by relieving the inhibitory effects of this sequence. 16 Generation of Anti-Peptide Antibodies to Sites in Catalytic Domain There have been several publications describing the generation and use of anti-peptide antibodies. 17-~9 The procedures described here are targeted toward the generation of antibodies against conserved sequences in the catalytic domain of protein kinases and the special considerations that may be required in using these antibodies. The methods presented are synthesized from our experience with anti-peptide antibodies directed against four highly conserved sites within the catalytic domain and two nonconserved regions from outside the catalytic domain of the EGF receptor (EGFR). Choosing A m i n o A c i d S e q u e n c e s f r o m Kinase Domain f o r Synthesis o f Peptide Antigen
When selecting the amino acid sequence from the catalytic domain to which the anti-peptide antibody will be directed, it is important to consider whether the epitopes will be collinear with the primary sequence. In the absence of information on the three-dimensional structure of the catalytic ~3R. Herrera, L. Petruzzelli,N. Thomas, H. N. Bramson,E. T. Kaiser,and O. M. Rosen, Proc. Natl. Acad. Sci. U.S.A. 82, 7899 (1985). 14W. J. Gullick, J. Downward, and M. D. Waterfield,EMBO J. 4, 2869 (1985). ~5R. Gherzi, G. Sesti, G. Andraghetti,R. De Pirro, R. Lauro, L. Adezanti,and R. Condera, J. Biol. Chem. 264, 8627 (1989). 16M. Makowskeand O. M. Rosen, J. Biol. Chem. 264, 16155(1989). 17R. A. Lerner, Nature (London) 299, 592 (1982). 18G. Walter and R. F. Doolittle, Genet. Eng. 5, 61 (1983). 19G. Walter, J. lmmunol. Methods 88, 149 (1986).
466
ANTIBODIES AGAINST PROTEIN KINASES
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domain, whether the sequence is conserved may be considered. Patterns of amino acid sequence conservation can most easily be assessed by consulting the publication of Hanks, Quinn, and Hunter, ~in which these authors aligned the sequences within the catalytic domains of 65 different protein kinases. Attention should be directed not only to the 11 conserved subdomains (numbered I through XI) but also to the patterns of conservation within and outside of these subdomains. For example, within subdomain VI, the sequence HRDLRAAN (HRD) for the Src family (residues 384-391) is one of the most highly conserved segments in the tyrosine kinase family and may, therefore, represent a site that is structurally or functionally important. Other considerations when choosing the amino acid sequence, including the length and the chemical properties of the amino acid side chains, have been discussed. 17-2°
Conjugation of Synthetic Peptide There are several procedures for conjugating the peptide to a carrier protein. We have found that cross-linking synthetic peptides with carbodiimide by the following procedure has yielded antibodies which are reactive with highly conserved sequences in the interior of the EGFR. 1. Dissolve 2.5 mg of the synthetic peptide in 1 ml water. Determine absorbance at 214 nm or at the wavelength of maximum absorbance for the particular peptide. 2. Add 7.5 mg hemocyanin (thyroglobulin works also) in 100/zl of water containing 0.5 mg/ml cross-linker 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (Sigma, St. Louis, MO). Vortex and let stand at room temperature for 30 min. 3. Dilute to 3 ml with water and put into dialysis tubing with a pore size that will allow free peptide to diffuse out. Dialyze against I00 ml of water overnight. 4. Determine absorbance of dialysate and calculate the amount of peptide in the dialysate to determine the efficiencyof conjugation. Alternatively, spot an aliquot of dialysate onto paper and react with ninhydrin to estimate amount of free peptide. We have found that 40 to 50% of the peptide is usually conjugated. We have also assessed the immunogenicity of peptide conjugated to hemocyanin using glutaraldehyde as the cross-linking reagent. Glutaraldehyde efficiently conjugated the peptide to the carrier protein and the conjugated peptide generated antibody reactive to the immobilized peptide in an enzyme-linked immunosorbent assay (ELISA). However, we have 2o T. P. Hopp and K. R. Woods, Proc. Natl. Acad. Sci. U.S.A. 78, 3824 (1981).
[39]
ANTI-PEPTIDE Abs AGAINSTPK CATALYTICDOMAIN
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found that these antibodies did not immunoprecipitate the EGFR if the cognate sequence was in the interior of the protein. The conformation of the peptide that is cross-linked with glutaraldehyde may be different than the conformation of the same sequence when it is located at an internal site in the protein. Anti-peptide antibodies to sites in the flexible C-terminal domain, on the other hand, bound to the EGFR whether glutaraldehyde or carbodiimide was used to prepare the immunogen. The other procedure we have evaluated for producing immunogen is the synthesis of a heptalysine c o r e to which peptide monomers were attached according to the method of Tam. 21,22 Peptides attached to the polylysine core have been found to be immunogenic, thereby eliminating the need for a carder protein. However, because the synthesis of the peptide on the polylysine core was more difficult and there did not appear to be improvement in the titer or in the proportion of immunized rabbits that produced antibodies, there seems little reason to use this procedure.
Immunization New Zealand White rabbits of 2.5-3.0 kg are bled for preimmune sera and then injected subcutaneously with conjugated peptide emulsified at a ratio of 1 to 1.2 with Freund's complete adjuvant for the first and last injections, and Freund's incomplete adjuvant for all other injections. The rabbits are given subcutaneous booster injections at 2-week intervals and weekly test bleeds are begun 2-3 weeks after the initial injection. The amount of peptide used for immunization varies from 50 to 500/zg/injection. Antibodies of highest titer are produced in animals immunized with 50 to 100/.~g peptide conjugated to hemocyanin using EDC. Our experience is that the titer and the time of production of antibody are more affected by differences in individual rabbits rather than by the injection scheme used.
Monitoring Anti-Peptide Antibody Production Production of polyclonal antisera which bind to conserved, internal sites in the catalytic domain needs to be monitored by immunoprecipitation or by Western blotting rather than by ELISA using immobilized, synthetic peptide. A discrepancy between the titer of the antibody that binds to the peptide versus the antibody that binds to the cognate sequence in the whole protein has previously been observed 7A° and may be caused by differences in how the antigenic determinants are displayed. In several 21 D. N. Posnett, H. McGrath, and J. P. Tam, J. Biol. Chem. 263, 1719 (1988). 22 j. p. Tam, Proc. Natl. Acad. Sci. U.S.A. 85, 5409 (1988).
468
ANTIBODIES AGAINST PROTEIN KINASES 50,
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Weeks after Initial Injection FIG. 1. Production of anti-peptide antibodies to a conserved sequence from the catalytic domain of the EGF receptor (EGFR). Aliquots (50 /zl) of sera from rabbits immunized with HRDLAARN conjugated to hemocyanin were tested for the presence of antibody by immunoprecipitation of 32p-labeled EGF receptor. Parallel immunoprecipitations were conducted with a control polyclonal antiserum to the extracellular domain of the EGF receptor under conditions of antibody excess where 95% of the EGF receptor was immunoprecipitated. The serum from each test bleed was also tested by ELISA using the peptide immobilized on microtiter plates. The ELISA results are expressed relative to an ELISA using a control anti-peptide antibody against a peptide containing a sequence from the carboxy terminus of the EGF receptor.
cases, we have also found that the antibody reactive with the entire protein was produced transiently and early compared to antibody that reacted with the isolated peptide. In fact, when maximum ELISA titer was attained, the antibody that immunoprecipitated the entire protein was often no longer detectable. Eight different rabbits immunized with conjugated HRDLAARN (residues 811-818 of the EGFR) have displayed this pattern and Fig. I illustrates the profile of antibody production in one of these
[39]
ANTI-PEPTIDEAbs AGAINSTPK CATALYTICDOMAIN
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TABLE I BUFFERS USED IN IMMUNOPRECIPITATION a Concentration (%) Buffer
NaC1 (mM)
Deoxycholate
SDS
Glycerol
Kinase activity b
A B C
-150 150
--1
-0.05 0.1
10 10 --
+ + -
All buffers contain 20 m M Tris, p H 7.6, 1% Triton X-100, 1% aprotinin, 1 m M P M S F , 2 m M s o d i u m vanadate. b T h e a u t o p h o s p h o r y l a t i o n activity of the E G F receptor was m e a s u r e d in each of the buffers.
rabbits. Thus for rabbits immunized with peptides containing highly conserved sequences from the catalytic domain of protein kinases, we recommend that antibody production be monitored on a weekly basis by immunoprecipitation of the target protein kinase and that monitoring begin following the first booster injection. The most convenient and sensitive method to monitor production of antibodies that immunoprecipitate a protein kinase capable of autophosphorylation is to radiolabel the protein with [y)2p]ATP in an in vitro kinase reaction. Because the antibodies to highly conserved sites may recognize several protein kinases, it may be necessary to have a partially purified preparation or a concentrated source of the specific target protein kinase. For a source of concentrated EGFR, we use membrane vesicles prepared from A431 cells by a previously described procedure.23 The immunoprecipitation protocol follows: 1. The protein kinase is labeled by autophosphorylation in a reaction containing [y-32p]ATP. The components in the reaction and their concentration depend on the particular protein kinase to be investigated. 2. The 32p-labeled protein kinase is denatured in buffer C (Table I) to expose sites that may react with the antibody. 3. The denatured protein kinase is mixed with 50/zl antiserum from a test bleed and the volume is adjusted to 200 /.d with buffer C (50 /zl antiserum is the maximum amount that can be used before overloading the SDS gel with IgG). 4. The sera from the test bleed and the radiolabeled protein kinase are incubated for 1 hr at 4° with continuous rotation. 23 S. C o h e n , in " M e t h o d s in E n z y m o l o g y " (J. D. Corbin and J. D. H a r d m a n , eds.), Vol. 99, p. 379. A c a d e m i c Press, N e w York, 1983.
470
ANTIBODIES AGAINST PROTEIN KINASES
[39]
5. Protein A-Sepharose is added at a ratio of one bed volume to one volume of sera and the mixture is incubated with shaking at 4° for 30 min. 6. The immune complex is washed four times with 1 ml of buffer C. 7. The immune complex is suspended into 100/zl of sample buffer containing 63 mM Tris-HC1, pH 6.8, 10% (v/v) glycerol, 2% (w/v) SDS, 5% (v/v) 2-mercaptoethanol, and 0.002% (w/v) Bromphenol Blue and incubated in a boiling water bath for 2 min. The released proteins are analyzed by electrophoresis in an SDS-containing gel of 1.5 mm thickness with a well size that holds 200/xl of sample volume.
Tests of Specificity of Anti-Peptide Antibodies Binding of the anti-peptide antibody to a specific sequence in the target protein is best demonstrated by showing that immunoprecipitation is blocked by preincubation of the antibody with its cognate peptide. To conduct peptide-blocking experiments, the antiserum is incubated without or with different concentrations of peptide for 1 hr at 4° with continuous shaking prior to addition of the radiolabeled antigen. The remainder of the immunoprecipitation protocol is conducted as described above. We find that the concentration of peptide is sometimes important in determining the extent to which a specific antibody is blocked by its cognate peptide. For antibodies prepared to the HRDLAARN sequence, we have found that a peptide concentration of about 15/zg/ml is most effective at blocking immunoprecipitation of the EGFR by 250 ng of peptide affinity purified antibody.
Antibody Storage We find that anti-peptide antisera raised against some sequences from the catalytic domain in the EGFR, such as HRDLAARN, are unstable when stored outside of the rabbit. This is true only for the antibody that reacts with the entire EGFR, not for that which binds the immobilized peptide in an ELISA. The sera are therefore stored at 4° or at room temperature with the addition of 0.02% (w/v) sodium azide to prevent bacterial growth. Under these conditions, the activity is retained for 1-3 weeks. In contrast, antisera against nonconserved sequences within the EGFR are stable when stored at - 7 0 °. Unstable antibodies such as anti-HRD retain their activity best when bound to a peptide affinity column. The column is prepared using a 20-ml bed volume of Affi-Gel 10 (Bio-Rad, Richmond, CA) coupled to 15 mg of peptide using the procedure recommended by the manufacturer. We bind antibodies to their respective peptide affinity columns using the following procedure:
[39]
ANTI-PEPTIDE Abs AGAINSTPK CATALYTICDOMAIN
471
1. Precipitate the IgG-containing fraction from 15 ml of antiserum by adding a saturated ammonium sulfate solution to a final concentration of 40% of saturation. 2. Centrifuge at 10,000 g for 30 min at 4 ° and resuspend the pellet in 3 ml of 20 mM HEPES, 150 mM NaC1, pH 7.5. Dialyze against the same buffer overnight at 4 °. 3. Slowly add the dialyzed IgG to the peptide affinity column and incubate at 4 ° for 1 hr. 4. Wash the column with equilibration buffer until the absorbance at 280 nm of the eluate is reduced to background levels. 5. Store the column at 4 ° in buffer containing 0.02% sodium azide. Antibodies appear to be stable while bound to the peptide affinity column and can be used after elution from the column. However, they lose activity with time following elution from the column. Elution is accomplished as follows: 1. Add 60 ml of 0.1 M sodium acetate, 0.5 M NaCI, pH 2 to the column. Collect 0.5-ml fractions directly into 0.5 ml of 0.1 M glycine, pH 12, to neutralize each fraction. 2. Monitor the protein content of the fractions by absorbance at 280 nm. 3. The fractions with maximum absorbance may be used directly. Otherwise, combine these fractions and precipitate the IgG using 40% (v/v) ammonium sulfate. 4. Centrifuge and resuspend the IgG pellet in water. Dialyze against 20 mM HEPES, pH 7.5 at 4 °. The protease inhibitors aprotinin (0.5%), leupeptin (4/zM), and phenylmethylsulfonyl fluoride (PMSF) (2 mM) (all from Sigma, St. Louis, MO) are added to the purified antibody if it is not used immediately. Use of Anti-Peptide Antibodies in Studies of Topology and Function of Sites in Catalytic Domain
Determination of Topology To determine the topology of a specific sequence in the native protein kinase, immunoprecipitation of the radiolabeled protein by the anti-peptide antibody is conducted in buffer A (Table I) at 4 ° under conditions of antibody excess. Buffer A is relatively nondenaturing and, in the case of EGFR, stabilizes the kinase activity. Subsequent steps in the immunoprecipitation are carded out as described above. It is critical that a control
472
ANTIBODIES AGAINST PROTEIN KINASES
[39]
antibody which is known to immunoprecipitate the protein kinase in its native state be used here as a standard. In addition, the proportion of the protein kinase immunoprecipitated under the nondenaturing conditions of buffer A should be compared with the amount immunoprecipitated in parallel under the denaturing conditions of buffer C. Such a comparison addresses the question of whether conformation has an effect on the efficiency of antibody binding to its cognate sequence. The radiolabeled protein kinase in the immune complex can be most accurately quantitated by SDS gel electrophoresis and scintillation counting of the radiolabeled bands excised from the gel. It may also be of interest to determine whether partial "opening" of the protein structure is sufficient to expose a particular site in the protein or whether the protein needs to be more thoroughly denatured. This can best be determined by immunoprecipitation of the radiolabeled protein kinase in buffer B (Table I). When buffer B with 0.05% SDS is used for immunoprecipitation the protein kinase activity in the immune complex is about 80% of that found when buffer A is used. We have further found that sites that are inaccessible to antibody in buffer A may be exposed in buffer B. Another way to achieve partial opening of the protein structure is to incubate the radiolabeled protein kinase together with the anti-peptide antibody in either buffer A or buffer B at 34° for 5 rain-before incubation at 4° for 1 hr. The effects of modulators of kinase activity on the topology of specific sites in the catalytic domain of protein kinases can be examined by binding of anti-peptide antibodies. For example, one can ask whether the exposure of the sites in the kinase domain is altered by ligand binding or by autophosphorylation of the protein kinase. This approach has been described in more detail for the PDGF receptor5'6 and the insulin receptor7'8 tyrosine kinases.
Identification of Sites Criticalfor Catalysis The following protocol can be used to determine whether binding of anti-peptide antibody to specific sites in the catalytic domain neutralizes kinase activity. It is critical that a control antibody that efficiently immunoprecipitates the protein kinase but does not inhibit kinase activity be included in order to determine the amount of kinase activity that is lost during the immunoprecipitation procedure. 1. Mix 1 vol of anti-peptide antisera with one bed volume of protein A-Sepharose and incubate at 4 ° for 30 min on a shaking platform. 2. Wash the IgG-protein A complex three times with 10 to 20 vol of buffer A.
[39]
ANTI-PEPTIDE Abs AGAINSTPK CATALYTICDOMAIN
473
3. Mix the protein kinase with the anti-peptide IgG that is bound to protein A-Sepharose in buffer A or buffer B at 4° for 1 hr with shaking. 4. Wash the antigen-antibody complex with about 1 ml of buffer B once and buffer A three times. Care should be taken to work quickly and keep the protein kinase on ice to preserve kinase activity. 5. To test for autophosphorylation suspend the immune complex in about 100/zl of a kinase reaction mixture containing [~/-nP]ATP. Incubate under conditions that achieve maximum levels of autophosphorylation for the particular protein kinase. (Because autophosphorylation occurs very quickly, it is not feasible to analyze the kinetics of autophosphorylation. Thus the overall level rather than the rate of autophosphorylation is determined.) 6. Wash the immune complex three times with buffer C to remove the unincorporated 32p and analyze the proteins in the immune complex by SDS gel electrophoresis. To test for kinase activity toward exogenous protein substrate: 1. Suspend the washed immune complex from step (4) above in a kinase reaction mixture containing [y-32p]ATP and an exogenous substrate such as enolase or angiotensin for tyrosine kinases such as the EGFR. Incubate the kinase reaction at a temperature where a linear rate of substrate phosphorylation can be maintained. 2. Remove aliquots at different times of incubation, plunge into a boiling water bath to stop the kinase reaction, and separate the soluble substrate from the immune complex by centrifugation. 3. Phosphorylation of the exogenous substrate is determined following separation of the substrate from the other reaction components by techniques such as SDS gel electrophoresis. The rate of phosphorylation of the exogenous substrate can then be determined by quantitation of the 32p incorporated with time. The rate of substrate phosphorylation will reveal whether the kinase activity is inhibited by binding of anti-peptide antibody to a specific sequence. In some cases it may be desirable to determine the effects of the antibody on the kinase activity in a soluble reaction where the kinase has not been immunoprecipitated. The advantage is that the effects of antibody on the entire population of kinase molecules rather than on those that are immunoprecipitated can be evaluated. This approach, however, requires that the antibody be of high titer, in excess of the protein kinase, and be highly purified, for example by peptide affinity chromatography. To test for antibody inhibition of kinase activity in a soluble reaction, add the purified anti-peptide IgG to the protein kinase and incubate the
474
ANTIBODIES AGAINST PROTEIN KINASES
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mixture at 4° with shaking for 1 hr. Add the components of the kinase reaction, including [y-32p]ATP. Remove aliquots at increasing times to determine the level of substrate phosphorylation by the amount of 32p that is incorporated. Use peptide blocking (discussed above) of the purified IgG to establish that inhibition of kinase activity is caused by antibody binding to a specific sequence.
