The Utility of FK506-Binding Protein as a Fusion Partner in Scintillation Proximity Assays: Application to SH2 Domains

ANALYTICAL BIOCHEMISTRY ARTICLE NO.

240, 289–297 (1996)

0359

The Utility of FK506-Binding Protein as a Fusion Partner in Scintillation Proximity Assays: Application to SH2 Domains Lisa M. Sonatore, Doug Wisniewski, Lori J. Frank, Patricia M. Cameron, Jeffrey D. Hermes, Alice I. Marcy, and Scott P. Salowe1 Department of Molecular Design and Diversity, Merck Research Laboratories, P.O. Box 2000, Rahway, New Jersey 07065

Received May 1, 1996

Methodology has been developed which gives a specific measure of the interaction of an SH2 domain with a phosphopeptide ligand using scintillation proximity assay (SPA) technology. Recombinant SH2 domains were expressed from a T7 RNA polymerase-based vector in Escherichia coli as fusions to the C-terminus of the FK506-binding protein (FKBP) and purified from freeze-thaw lysates in high yield by affinity chromatography using immobilized phosphopeptides. For binding assays the phosphopeptide ligands were synthesized with a biotin tag and the FKBP fusion proteins were noncovalently radiolabeled with commercially available [3H]dihydroFK506. Complexes of tritiated SH2 fusion protein and biotinyl-phosphopeptide were then captured on streptavidin-coated SPA beads and counted. The modular protocol is an equilibrium technique that does not employ washing steps or specialized radiochemical syntheses required in other binding assays. The utility of the assay has been demonstrated in an examination of the ligand specificity of the SH2 domains of the tyrosine kinases ZAP70, Syk, and Lck. The methodology is potentially generalizable to any receptor–ligand interaction in which one component can be expressed as a fusion partner with FKBP and the other component can be captured on a SPA bead. q 1996 Academic Press, Inc.

It has become increasingly clear that many cellular processes are initiated and/or controlled by signal transduction pathways that employ protein–protein interactions as key elements in the transmission of information. Critical to such processes are modular binding domains within signal transduction proteins that confer both recognition and specificity (1). The best 1 To whom correspondence and reprint requests should be addressed. Fax: (908) 594-6100. E-mail: [email protected].

characterized of these modular domains is the Src Homology 2 (SH2)2 domain (2). SH2 domains comprise approximately 100 amino acids and are found in both tyrosine kinases and phosphatases as well as adaptor proteins with no catalytic function. These independently folded domains share the common property of recognizing phosphorylated tyrosine residues in specific peptide contexts. As many as 100 SH2 domains have been discovered, and in many cases the ligand specificity and/or functional role have been defined. An example of an important signal transduction process involving SH2 domains is T-cell activation. Engagement of the extracellular domains of the T-cell receptor (TCR) triggers rapid induction of intracellular tyrosine phosphorylation (3, 4). While the cytoplasmic domains of the TCR do not have intrinsic kinase activity, they do have specific sequences critical to signalling referred to as immunoreceptor tyrosine-based activation motifs (ITAM). Phosphorylation of both tyrosines in the minimal ITAM consensus sequence YxxL(x)7-8 YxxL enables recruitment of the tyrosine kinase ZAP70 via a specific interaction with the tandem SH2 domains present in that protein. A number of biochemical and genetic studies implicate an essential role for ZAP70 in the propagation of signals which lead to T-cell activation (5–7). Given the important role that SH2 domains play in signal transduction, it would be expected that compounds which inhibit specific SH2-ligand associations could have utility in the elucidation of signalling pathways and in the modulation of pathological cellular processes. The investigation of SH2 domain function, as well as the search for compounds which interrupt 2 Abbreviations used: SH2, Src homology 2; TCR, T cell receptor; ITAM, immunoreceptor tyrosine-based activation motif; SPA, scintillation proximity assay; FKBP, FK506-binding protein; GST, glutathione S-transferase; DTT, dithiothreitol; PLCg1, phospholipase Cg1.

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0003-2697/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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that function, requires a reliable biochemical assay. Since SH2 domains have no catalytic activity, a sensitive and quantitative binding assay is required, preferably one that is adaptable to automation to facilitate high throughput. There are significant disadvantages to existing procedures, particularly when applied to screening. We have employed scintillation proximity assay (SPA) technology (8) to develop a modular binding assay that uses the Mr 12,000 FK506-binding protein (FKBP) as a fusion partner for the SH2 domain of interest. The assay is simple to implement and readily expandable for high volume work. The methodology is potentially generalizable to any receptor–ligand interaction in which one component can be expressed as a fusion partner with FKBP. MATERIALS AND METHODS

[3H]dihydroFK506, Ç90 Ci/mmol, was obtained from DuPont-NEN. Biotinyl-e-aminocaproyl-EPQpYEEIPIYL was purchased from Bachem Bioscience; all other phosphopeptides were synthesized by California Peptide Research (Napa, CA). The concentrations of phosphopeptide stock solution were determined spectrophotometrically using e267 Å 500 M01 per pY residue (9). Streptavidin-coated SPA beads (typical capacity 100 pmol biotin/mg) were purchased from Amersham. White microplates (Optiplates) were obtained from Packard. Flag epitope-tagged full-length ZAP70 was obtained from insect cells by methods to be described elsewhere. Glutathione S-transferase (GST) fusions to various SH2 domains were purchased from Santa Cruz Biotechnology; protein concentrations given by the supplier were used in calculations. Preparation of the FKBP Fusion Cloning Vector Sequences for a 3*- altered human FKBP fragment that contained a glycine codon (GGT) in place of the stop (TGA) codon followed by a sequence encoding a thrombin site (Leu–Val–Pro–Arg) and BamHI restriction site (GAATTC) were amplified using PCR. The PCR reaction contained the following primers: 5*-GATCGCCATGGGAGTGCAGGTGGAAACCATCTCCCCA3* and 5*-TACGAATTCTGGCGTGGATCCACGCGGAACCAGACCTTCCAGTTTTAG-3* and a plasmid containing human FKBP as the template. The resulting 367-base pair amplification product was ligated into the vector pCRII (Invitrogen) and the ligation mixture was transformed into competent Escherichia coli cells. Clones containing an insert were identified using PCR with flanking vector primers. Dideoxy DNA sequencing confirmed the nucleotide sequence of one positive isolate. The altered 338-base pair FKBP fragment was excised from the pCRII plasmid using NcoI and BamHI and ligated into NcoI- and BamHI-digested pET9d (Novagen) plasmid. Competent E. coli were transformed

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with the ligation mixture, and colonies containing the insert were identified using PCR with primers encoding for flanking vector sequences. The FKBP fusion cloning vector is called pET9dFKBPt. Preparation of the ZAP(SH2), FKBP-ZAP(SH2), FKBP-SYK(SH2), and FKBP-LCK(SH2) Expression Vectors A DNA fragment encoding for the tandem SH2 domains of ZAP70 (amino acids 1–263) was prepared by PCR to contain a BamHI site at the 5*-end such that the reading frame was conserved with that of FKBP in the fusion vector. At the 3*-end, the fragment also incorporated a stop codon followed by a BamHI site. The PCR reaction contained Molt-4 cDNA (Clontech) and the following primers: 5*-ATTAGGATCCATGCCAGATCCTGCAGCTCACCTGCCCT-3* and 5*-ATATGGATCCTTACCAGAGGCGTTGCT-3*. The fragment was cloned, sequenced, digested with BamHI, and the insert containing the SH2 domains was ligated to BamHI-treated pET9dFKBPt and transformed into E. coli. Clones containing inserts in the correct orientation were identified by PCR or restriction enzyme analysis. The unfused ZAP70 tandem SH2 domains were cloned using PCR with a reaction containing the above FKBP-ZAP(SH2) fusion plasmid DNA and the following primers: 5*-ATTACATATGCCAGATCCTGCAGCTCACCTGC-3* and 5*-ATATGGATCCTTAAGAGGCGTTGCT-3*. The PCR product was cloned, sequenced, and digested with NdeI and BamHI and inserted into NdeI/BamHI-digested pET9a. The expression vector for the tandem SH2 domains of human Syk (amino acids 1–269) fused to FKBP was prepared as above for FKBP-ZAP(SH2) except that the PCR reaction contained Raji cell cDNA (Clontech) and the following primers: 5*-CAATAGGATCCATGGCCAGCAGCGGCATGGCTGA-3* and 5*-GACCTAGGATCCCTAATTAACATTTCCCTGTGTGCCGAT-3*. The expression vector for the SH2 domain of human Lck (amino acids 119–226) fused to FKBP was prepared as above for FKBP-ZAP(SH2) except that the PCR reaction contained Molt-4 cDNA (Clontech) and the following primers: 5*-ATATGGATCCATGGCGAACAGCCTGGAGCCCGAACCCT-3* and 5*-ATTAGGATCCTTAGGTCTGGCAGGGGCGGCTCAACCGTGTGCA-3*. Expression and Purification of SH2 Domains E. coli BL21 (DE3) cells containing an SH2 expression plasmid were grown in Luria–Bertani media containing 50 mg/ml kanamycin at 377C until the optical density measured at 600 nm was 0.5–1.0. Protein expression was induced with 0.1 mM isopropyl b-thiogalactopyranoside and the cells were grown for another 3–5 h at 307C. They were pelleted at 4400g for 10 min

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at 47C and resuspended in 2% of the original culture volume with 100 mM Tris, pH 8.0, containing 1 mg/ml each aprotinin, pepstatin, leupeptin, and bestatin. The resuspended pellet was frozen at 0207C until further purification. The affinity matrix for purification of FKBPZAP(SH2), FKBP-SYK(SH2), and unfused ZAP70 SH2 domains was prepared by combining agarose-immobilized avidin with excess biotinylated phosphopeptide derived from the zeta 1 ITAM sequence of the human T-cell receptor, biotinyl-GSNQLpYNELNLGRREEpYDVLDK, and washing out unbound peptide. The affinity matrix for purification of FKBP-LCK(SH2) was prepared in similar fashion substituting the peptide biotinyl-e-aminocaproyl-EPQpYEEIPIYL. In all cases, frozen cells containing the SH2 domain-containing protein were thawed in warm water, refrozen on dry ice for about 25 min, then thawed again. After the addition of 0.1% octyl glucoside, 1 mM dithiothreitol (DTT), and 500 mM NaCl, the extract was centrifuged at 35,000g for approximately 30 min. The supernatant was loaded onto the phosphopeptide affinity column at 47C and washed with phosphate-buffered saline containing 1 mM DTT and 0.1% octyl glucoside. The SH2 domain was eluted with 200 mM phenyl phosphate in the same buffer at 377C. The protein pool was concentrated by ultrafiltration and the phenyl phosphate removed on a desalting column. The purified protein was stored at 0207C in 10 mM Hepes/150 mM NaCl/1 mM DTT/0.1 mM EDTA/10% glycerol. Protein concentrations were determined from A280 using molar extinction coefficients calculated from the amino acid content (10).

Assay The following components were added sequentially to the wells of a white microplate to a final volume of 150 ml: biotinylated phosphopeptide ligand in assay buffer, test compound, or protein (when present) and FKBP-SH2 fusion protein preloaded with [3H]dihydroFK506. After 30 min at room temperature, 50 ml of a 4 mg/ml suspension of streptavidin-coated SPA beads in assay buffer was dispensed into the wells and the plate was sealed and shaken for 30 s. The final concentrations of the assay components were 25 nM biotinyl-phosphopeptide, 25 nM FKBP-SH2 fusion protein, 10 nM [3H]dihydroFK506 (0.2 mCi), 1 mg/ml streptavidin-coated SPA beads, 50 mM Hepes (pH 7.0), 10 mM DTT, 0.01% Tween-20. Bead-bound radioactivity was measured in a Packard Topcount microplate scintillation counter after an additional 4-h incubation at room temperature. While the plate could be read immediately, the results were more precise and reproducible after the beads settled.

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RESULTS

The Mr 12,000 FKBP is an abundant cytosolic protein that was first identified as the receptor for the immunosuppressive drug FK506 (11, 12). Since FKBP domains have also been found within larger proteins, it was expected that FKBP would be a versatile expression partner. To express FKBP fusion proteins, a T7 RNA polymerase-based bacterial expression vector containing sequences coding for a modified version of human FKBP was constructed. The polymerase chain reaction was used to replace the stop codon of FKBP with GGT (encodes glycine), add sequences encoding for a thrombin cleavage site and incorporate sequences for a BamHI restriction site. This 345-bp fragment was cloned into Nco- and BamHI-digested pET9d to generate plasmid pET9dFKBPt (Fig. 1). Genes of interest were cloned at the BamHI site, transformed into E. coli strain BL21 (DE3), and protein expression was appropriately induced. Following a period of induction, the cells were pelleted and resuspended in a suitable buffer. Although FKBP lacks sequences that specifically direct it to the periplasm, FKBP fusions were primarily located there and could be released by a straightforward freeze/thaw treatment of the cell pellet. Following centrifugation, the resulting supernatant contained ú80% pure FKBP fusion, which if desired, could be additionally purified. We found affinity chromatography to be a rapid and efficient method of purifying our SH2 constructs. The affinity matrices were formed by coupling an appropriate biotinylated phosphopeptide ligand for the SH2 domain of interest to agarose-immobilized streptavidin. After loading the extract and washing the column free of nonspecifically bound materials, the SH2 domain was specifically eluted with phenyl phosphate. The resulting protein was at least 95% pure as judged by SDS–PAGE, analytical HPLC, and ES-MS. The principle of the binding assay for a generic SH2 domain and its phosphopeptide ligand is illustrated in Fig. 2. The biotinylated phosphopeptide is mixed with the FKBP fusion protein that has been preloaded with [3H]dihydroFK506; the tritiated drug binds with high affinity (Kd 0.4 nM) (13) to the FKBP portion of the fusion and serves as a noncovalent radiolabel for the SH2 domain. After a suitable incubation period to allow complex formation to occur, streptavidin-coated SPA beads are added to capture the biotinylated ligand and any bound fusion protein. After the capture is completed, the assay sample is counted in a scintillation counter without any separation step. Screening for inhibitors of the interaction is carried out by performing the initial incubation of protein and biotinylated ligand in the presence of a test compound prior to the capture step with SPA beads. If the test compound binds the

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FIG. 1. (A) Vector for the expression of FKBP fusion proteins in E. coli. (B) The gene for the protein of interest is cloned into the BamHI site as illustrated for the SH2 domains of ZAP70.

target protein competitively with the biotinylated ligand, the detected signal will be reduced by that portion of the complex displaced from the SPA bead. It is also possible to test other non-FKBP-fused SH2 domains which bind the same ligand as competitors against the FKBP-fused target; this could be useful in identifying or confirming the suspected specificity of a newly identified domain. The implementation of assays for the tandem SH2 domains found in the tyrosine kinases ZAP70 and Syk, as well as the single domain in the tyrosine kinase Lck, are demonstrated in Fig. 3. Two biotinylated phosphopeptides were used: the zeta 1 ITAM of the T-cell receptor, a known high affinity ligand for ZAP70, and a hamster polyoma middle T antigen sequence (YEEI), a known high affinity ligand for Lck. Titration of the biotinylated ITAM into a fixed concentration of either FKBP-ZAP(SH2) or FKBP-SYK(SH2) (Fig. 3A) gave a steadily increasing signal until a plateau was reached due to saturation of the available protein. The signalto-noise ratio at the plateau was ú50:1. The sharp breaks in the titration curves indicated that in both cases the Kd for the ITAM was !25 nM, the protein concentration used in the experiment. In contrast, titration of the YEEI peptide into FKBP-ZAP(SH2) resulted in only a very weak signal (21 background); Kd

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was estimated as ú1 mM. FKBP-SYK(SH2) was captured significantly better by this ligand, with an estimated Kd of Ç50 nM. Ligand specificity was reversed for the FKBPLCK(SH2) protein where YEEI was the preferred peptide (Fig. 3B). The titration curve indicated that Kd was õ25 nM, although the more gradual curvature suggested a lower affinity for this protein/ligand pair than for either FKBP-ZAP(SH2) or FKBP-SYK(SH2) with the ITAM. FKBP-LCK(SH2) also had significant, albeit weaker, affinity for the ITAM ligand. If it is assumed that each phosphotyrosine in the ITAM can independently bind an Lck SH2 domain (vide infra), a Kd of Ç60 nM may be estimated. All three SH2 domain fusion proteins examined in this work exhibited weaker affinity for their preferred ligand as the ionic strength was increased through a physiologically relevant range. The apparent IC50 for NaCl against the FKBP-LCK(SH2) complex with YEEI was 70 mM, while the IC50s against the FKBPSYK(SH2) and FKBP-ZAP(SH2) ITAM complexes were 100 and 400 mM, respectively. Peptides and compounds with the potential to bind to a target SH2 domain may be screened in the assay via competition against a biotinylated ligand. This is demonstrated in Fig. 4 and summarized in Table 1,

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FIG. 2. Schematic diagram of the assay principle. A biotinylated phosphopeptide ligand is mixed with a FKBP fusion protein. Tritiated FK506 binds tightly to the FKBP fusion partner and serves as a noncovalent radiolabel for the SH2 domain. The complex is captured on streptavidin-coated SPA beads for detection by scintillation counting. The signal is reduced by compounds which bind the SH2 domain competitively with the biotinylated ligand or by other SH2 domains which bind the biotinylated ligand competitively with the FKBP fusion protein.

where various phosphorylated ITAMs from the cytoplasmic chains of the T-cell receptor are compared as inhibitors of the interaction of the zeta 1 ITAM with FKBP-ZAP(SH2) or FKBP-SYK(SH2). There was little difference between the two proteins or between the six ITAM sequences tested, with the possible exception of

the weaker CD3e inhibition of FKBP-SYK(SH2). It should be noted that the IC50s for the nonbiotinylated zeta 1 ITAM were 40 and 47 nM (Table 1), very similar to the concentration of the biotinylated zeta 1 captured on the bead. In a separate experiment with FKBPLCK(SH2), the IC50 for nonbiotinylated YEEI was also

FIG. 3. Titration of phosphopeptide ligands into FKBP–SH2 fusion proteins. Either the ITAM (biotinyl-GSNQLpYNELNLGRREEpYDVLDK; open symbols) or YEEI (biotinyl-e-aminocaproyl-EPQpYEEIPIYL; closed symbols) phosphopeptide was added in increasing amounts to an assay containing 25 nM of the FKBP-ZAP(SH2) (circles), FKBP-SYK(SH2) (squares), or FKBP-LCK(SH2) (triangles).

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FIG. 4. Competition binding by a T-cell receptor ITAM to FKBPZAP(SH2). Varying amounts of nonbiotinylated zeta 1 ITAM were added to the standard assay containing biotinylated zeta 1 ITAM and FKBP-ZAP(SH2).

nearly equal to the concentration of the biotinylated YEEI used for capture (data not shown). These important results indicate that the affinities of SH2 domains for their ligands are essentially unaltered by the immobilization of the phosphopeptide on the bead. The results for several other zeta 1 ITAM-derived peptides containing a single phosphotyrosine, as well as the phosphotyrosine mimic phenyl phosphate, are also summarized in Table 1. The IC50s for these peptides with FKBP-ZAP(SH2) and FKBP-SYK(SH2) were

at least three orders of magnitude higher than the doubly phosphorylated parent sequence, confirming the expected requirement for two phosphotyrosines for productive engagement of the tandem SH2 domains in these proteins. Several of these peptides were also tested against FKBP-LCK(SH2) since the results of Fig. 3B indicated that this SH2 domain could bind the ITAM sequence. Inhibition by the singly and doubly phosphorylated ITAM-derived peptides was weak but measurable versus the strong YEEI ligand (Table 1). There was apparently little preference for a particular phosphotyrosine site in the zeta 1 ITAM. As noted earlier, the assay format also allows nonFKBP-fused SH2 domains to be tested, via competition against an FKBP fusion, for binding to a target phosphopeptide ligand. This is illustrated in Fig. 5 and Table 2 for several ZAP70 constructs as well as a variety of commercially available GST fusions to other SH2 domains. Figure 5A shows that the ZAP70 SH2 domains, whether unfused or present in the full-length protein, compete nearly equivalently (IC50s 69 and 51 nM, respectively) with 25 nM FKBP-ZAP(SH2) fusion protein for the ITAM sequence. This result indicates that the affinity of the tandem SH2 domains for the ITAM is essentially independent of the overall protein context. A variety of other GST-SH2 domains, including the tandem domains present in phospholipase Cg1 (PLCg1) and the tyrosine phosphatase SHPTP2 failed to effectively compete against FKBP-ZAP(SH2) for ITAM binding, even when present in large excess (Table 2). This is consistent with the high affinity and specificity that has been observed for the ZAP70/ITAM interaction (7, 14).

TABLE 1

Inhibition of Phosphopeptide Binding to FKBP-SH2 Fusion Proteins by ITAM-Derived Peptides IC50 (mM) Competitor

FKBP-ZAP (SH2)a

FKBP-SYK (SH2)a

FKBP-LCK (SH2)b

0.040 0.041 0.066 0.033 0.060 0.084 ú25 ú25 ú25 ú25 ú25 ú25 ú25 5800

0.047 0.022 0.024 0.022 0.029 0.183 ND ú25 ú25 ú25 ú25 ND ú25 5600

2.6 NDc ND ND ND ND ND ND ND Ç10 Ç20 ND ND 960

NQLpYNELNLGRREEpYDVLDK (zeta 1) EGLpYNELQKDKMAEApYSEIGM (zeta 2) DGLpYQGLSTATKDTpYDALHM (zeta 3) DQLpYQPLKDREDDQpYSHLQG (CD3g) DQVpYQPLRDRDDAQpYSHLGG (CD3d) NPDpYEPIRKGQRDLpYSGLNQ (CD3e) NQL YNELNLGRREE YDVLDK NQLpYNELNLGRREE YDVLDK Ac-NQL YNELNLGRREEpYDVLDK NQLpYNELNL REEpYDVLDK NQLpYNELNL / REEpYDVLDK (1:1 mixture) Ac-LNLGRREEpY Phenyl phosphate a

Conducted in the standard assay using biotinylated zeta 1 ITAM as ligand. Conducted in the standard assay using biotinylated YEEI as ligand. c ND, not determined. b

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FIG. 5. Competition binding by SH2 domains. (A) Varying amounts of ZAP(SH2) (filled circles) or Flag-ZAP70 (open circles) were added to the standard assay containing FKBP-ZAP(SH2) and biotinylated ITAM ligand. (B) Varying amounts of GST-LCK (filled circles), GSTFyn (open circles), GST-PLCg1 (closed squares), GST-PI3K (open squares), or GST-GRB2 (triangles) were added to the standard assay containing FKBP-LCK(SH2) and biotinylated YEEI ligand.

Competition experiments were also conducted against the FKBP fusion to the single SH2 domain of Lck. In Figure 5B it can be seen that GST-Lck competes well (IC50 7 nM), confirming that this SH2 domain also behaves independently of its fusion partner. The SH2 domain of Fyn, a closely related Src family kinase, also competed well (IC50 17 nM). Other domains from more distantly related proteins inhibited weakly or not at all in the concentration range tested (Fig. 5B and Table 2). The exception was the PLCg1 tandem SH2 construct (IC50 58 nM). This observation prompted a follow

TABLE 2

Inhibition of Phosphopeptide Binding to FKBP-SH2 Fusion Proteins by Other SH2 Domains IC50 (mM) Competitor

FKBP-ZAP (SH2)a

FKBP-LCK (SH2)b

Flag-ZAP70 ZAP (SH2) GST-Lck (120-226) GST-Fyn (145-247) GST-PI3K (333-430) GST-GRB2 (54-164) GST-SHPTP2 (6-213) GST-PLCg1 (548-760) GST-PLCg1 (548-659) GST-PLCg1 (663-760)

0.051 0.069 ú3 ú3 ú3 ú3 ú3 ú3 ND ND

NDc ú3 0.008 0.017 Ç2 Ç3 ú3 0.058 1.8 0.064

a

Conducted in the standard assay using biotinylated zeta 1 ITAM as ligand. b Conducted in the standard assay using biotinylated YEEI as ligand. c ND, not determined.

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up experiment with the individual domains which demonstrated that the inhibition was attributable to the C-terminal domain (Table 2). DISCUSSION

A variety of methods have been applied to assay the binding of ligands to SH2 domains. Biospecific interaction analysis using surface plasmon resonance has been frequently used (15–19); less commonly seen are isothermal titration calorimetry (20, 21) and NMR (22). Each of these techniques requires specialized instrumentation (and in the latter two cases large quantities of protein) that is not amenable to high throughput screening. Other methods rely on the expression of SH2 domains as GST fusion proteins; ligand binding can be assayed by incubating a GST–SH2 fusion with a radiolabeled phosphopeptide and determining bound radioactivity after precipitation of the complex with glutathione-agarose (23–25). There are several disadvantages to this procedure, particularly when applied to high-throughput screening. First, radiolabeling of the peptide with either [125I]Bolton–Hunter reagent or [32P]ATP and a kinase uses short-lived isotopes that require frequent preparation of material. Furthermore, in the case of enzymatic phosphorylation a kinase with the appropriate specificity must be available in sufficient quantity to generate enough material for screening purposes. Second, dissociation of the bound ligand can occur during washing of the glutathione-agarose beads to separate bound complex from free phosphopeptide, lowering the signal-to-noise of the assay and the accuracy of the results. Third, the manipulations required for centrifugations and filtrations are tedious

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when conducted manually and not readily adaptable to automation to increase throughput. In this paper we present a new binding assay for SH2 domains fused to FKBP. We have found that FKBPcontaining fusion proteins are expressed at high levels in E. coli (5–10 mg/liter culture) and are easily purified from the periplasmic fraction. Furthermore, the FKBP fusion partner provides straightforward means of making use of SPA technology for the functional assay of phosphopeptide binding. Our methodology has several advantages over assays employing GST fusions. The phosphopeptide ligand is modified by chemically stable biotinylation, eliminating the need for repeated preparation. The FKBP–SH2 fusion is noncovalently radiolabeled as needed with commercially available [3H]dihydroFK506, removing any requirement for specialized radiochemical synthesis to implement the assay. Since the SPA methodology is an equilibrium technique that does not require the separation of complex from free protein or ligand, there are no washing steps that might introduce errors due to rapid ligand dissociation. Finally, the protocol is mechanically simple, requiring only that reagents be added together in a suitable vessel for scintillation counting. The method is thus readily adaptable to 96-well microplates and automated pipetting stations for high volume work. While we have focussed on the use of SPA beads for the detection of radioactivity, we have also found that a streptavidin-coated microscintillation plate (e.g., Flashplate) may be employed (unpublished results). Several technical aspects need to be kept in mind during the design of a new assay using this methodology. Since the radiolabel is noncovalent, the concentrations of FKBP fusion and [3H]dihydroFK506 are best set above their Kd (Ç0.4 nM) to maximize the signal and minimize the noise from nonspecific binding of the drug to the beads. Such conditions also minimize false positives during screening from compounds which might bind FKBP competitively with the radiolabeled drug. False positives, which also include agents which compete with the biotinylated ligand during the streptavidin capture step, can be readily identified in a secondary assay which uses [3H]dihydroFK506 with biotinylated, unfused FKBP (unpublished results). We chose to illustrate the new assay methodology by establishing assays for the SH2 domains of tyrosine kinases that are important in the signal transduction pathways of hematopoietic cells. The ITAM sequence proved to be an excellent capture ligand for the tandem domains of ZAP70 and Syk for both protein purification and the binding assay. As expected, the high affinity interaction required both ITAM tyrosines to be phosphorylated. The affinity of other T-cell receptor ITAMs for FKBP-ZAP(SH2) were examined through competition with biotinylated zeta 1. Despite the limited sequence homology of the ITAM sequences, no great dif-

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ferences in affinity were observed between them, consistent with literature reports using other methods (17, 24). The affinity and specificity of the Syk SH2 domains for TCR ITAMs has not previously been examined; the competition experiments described in this paper indicate that FKBP-SYK(SH2) behaves very much like the analogous ZAP70 construct. Under physiologic conditions the ZAP70 SH2 domains may have a competitive advantage with respect to ITAM binding because of the greater sensitivity of the Syk SH2 domains to ionic strength. It remains a challenging task to find compounds that demonstrate selectivity between the SH2 domains of ZAP70 and Syk. Among the few differences observed between these two proteins was the recognition of the YEEI peptide (Fig. 3A). Previous work with phosphotyrosine-containing peptide libraries has led to classification of SH2 domains based upon their selection of ligands (26, 27), e.g., ‘‘Group 1’’ which select for pYhydrophilic-hydrophilic-hydrophobic residues. Each of the two SH2 domains present in ZAP70 and Syk have been classified as Group 1B based upon the key protein residues that are predicted to interact with ligands. Only the Syk C-terminal domain has been experimentally tested and was found to select pY-Q/T/E-E/Q-L/I, with a strong preference for L at the pY / 3 position. This is consistent with the preferential binding of Syk to the pY-X-X-L signature sequence of the zeta 1 ITAM over YEEI. The existing predictive models are insufficient, however, to explain the differences between the homologous ZAP70 and Syk SH2 domains with respect to YEEI binding. The limitations of peptide libraries are also evident when considering the interesting new findings of substantial affinity between the Lck SH2 domain and the ITAM (Fig. 3B) and between the PLCg1 C-terminal SH2 domain and the YEEI sequence (Table 2). The Lck SH2 domain is Group 1A which selects most strongly for pY-E/T-E/D-I/V/M (27). While the high affinity for the YEEI peptide is thus correctly predicted, the substantial affinity for the pYNEL and pYDVL sequences in the zeta 1 ITAM is not. It remains to be determined whether ITAM-bound Lck is significant in the process of lymphocyte activation. The PLCg1 C-terminal SH2 domain is Group 3 which selects for pY-V/I/L-I/L-P/ V/I (27); the affinity for the YEEI peptide was thus unexpected. Residues /4, /5, and /6 carboxyl to the phosphotyrosine have been shown to make important binding interactions with this SH2 domain (28, 29), and their contributions must override the less favorable interactions of the glutamates at /1 and /2. A similar result has been seen before in the context of a high affinity peptide sequence from the platelet-derived growth factor receptor (30). The application of the procedures described in this work should not be limited to SH2 domain–phospho-

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peptide interactions. In principle many protein–protein or protein–small molecule ligand systems could be adapted. The main requirements are that one component can be expressed as a fusion partner with FKBP while the other component can be captured on a SPA bead. While the biotin-streptavidin pair is likely to be the most robust combination for capture, an epitope tag may also be used in concert with an appropriate antibody and anti-IgG- or protein A-coated beads (8). A variety of available formats, modular components, and easy implementation should make the methodology described in this paper an attractive alternative for consideration when designing new assays in both basic research and compound screening applications. REFERENCES 1. Cohen, G. B., Ren, R., and Baltimore, D. (1995) Cell 80, 237– 248. 2. Schaffhausen, B. (1995) Biochim. Biophys. Acta 1242, 61–75. 3. Bolen, J. B. (1995) Curr. Opin. Immunol. 7, 306–311. 4. Weiss, A., and Littman, D. R. (1994) Cell 76, 263–274. 5. Chan, A. C., Kadlecek, T. A., Elder, M. E., Filipovich, A. H., Kuo, W. L., Iwashima, M., Parslow, T. G., and Weiss, A. (1994) Science 264, 1599–1601. 6. Elder, M. E., Lin, D., Clever, J., Chan, A. C., Hope, T. J., Weiss, A., and Parslow, T. G. (1994) Science 264, 1596–1599. 7. Iwashima, M., Irving, B. A., Vanoers, N. S. C., Chan, A. C., and Weiss, A. (1994) Science 263, 1136–1139. 8. Skinner, R. H., Picardo, M., Gane, N. M., Cook, N. D., Morgan, L., Rowedder, J., and Lowe, P. N. (1994) Anal. Biochem. 223, 259–265. 9. Cooper, J. A., Sefton, B. M., and Hunter, T. (1983) in Methods in Enzymology (Corbin, J. D., and Hardman, J. G., Eds.), Vol. 99, pp. 387–402, Academic Press, San Diego, CA. 10. Mach, H., Middaugh, C. R., and Lewis, R. V. (1992) Anal. Biochem. 200, 74–80. 11. Trandinh, C. C., Pao, G. M., and Saier, M. H. (1992) FASEB J. 6, 3410–3420. 12. Wiederrecht, G., and Etzkorn, F. (1994) Perspect. Drug Discovery Des. 2, 57–84. 13. Siekierka, J. J., Hung, S. H. Y., Poe, M., Lin, C. S., and Sigal, N. H. (1989) Nature 341, 755–757.

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