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Originally published In Press as doi:10.1074/jbc.M207657200 on August 8, 2002

J. Biol. Chem., Vol. 277, Issue 42, 39401-39408, October 18, 2002
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The N-terminal SH2 Domains of Syk and ZAP-70 Mediate Phosphotyrosine-independent Binding to Integrin beta  Cytoplasmic Domains*

Darren G. WoodsideDagger, Achim Obergfell, Anupam Talapatra, David A. Calderwood, Sanford J. Shattil, and Mark H. Ginsberg§

From the Division of Vascular Biology, Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037

Received for publication, July 29, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Syk and ZAP-70 form a subfamily of nonreceptor tyrosine kinases that contain tandem SH2 domains at their N termini. Engagement of these SH2 domains by tyrosine-phosphorylated immunoreceptor tyrosine-based activation motifs leads to kinase activation and downstream signaling. These kinases are also regulated by beta 3 integrin-dependent cell adhesion via a phosphorylation-independent interaction with the beta 3 integrin cytoplasmic domain. Here, we report that the interaction of integrins with Syk and ZAP-70 depends on the N-terminal SH2 domain and the interdomain A region of the kinase. The N-terminal SH2 domain alone is sufficient for weak binding, and this interaction is independent of tyrosine phosphorylation of the integrin tail. Indeed, phosphorylation of tyrosines within the two conserved NXXY motifs in the integrin beta 3 cytoplasmic domain blocks Syk binding. The tandem SH2 domains of these kinases bind to multiple integrin beta  cytoplasmic domains with varying affinities (beta 3 (Kd = 24 nM) > beta 2 (Kd = 38 nM) > beta 1 (Kd = 71 nM)) as judged by both affinity chromatography and surface plasmon resonance. Thus, the binding of Syk and ZAP-70 to integrin beta  cytoplasmic domains represents a novel phosphotyrosine-independent interaction mediated by their N-terminal SH2 domains.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Integrin adhesion receptors bind components of the extracellular matrix or cell surface molecules and transmit signals that regulate processes such as cell proliferation, differentiation, migration, and death (reviewed in Refs. 1 and 2). Integrin signaling is initiated by ligand binding, which is thought to involve conformational changes in integrins that are propagated to their intracellular, cytoplasmic domains. The ability of integrins to function as signaling receptors is dependent on these cytoplasmic domains, which are typically short (13-70 amino acid residues in length) and lack known catalytic activity. Thus, integrins rely on either direct or indirect associations of their cytoplasmic domains with signaling and/or adaptor molecules to initiate signal transduction cascades.

An early event in integrin signaling in certain cells involves activation of the nonreceptor tyrosine kinase Syk (3, 4). In platelets, integrin alpha IIbbeta 3 engagement (3) or clustering (5) rapidly activates Syk in a manner independent of an intact actin cytoskeleton (4, 6), differentiating integrin activation of Syk from that of another tyrosine kinase, FAK. Integrin alpha IIbbeta 3-dependent regulation of Syk depends on the direct interaction of Syk with the beta 3 cytoplasmic domain (7). Integrins of other classes including the beta 1 (4) and beta 2 (8) integrins regulate Syk activity. Syk is essential for integrin beta 2-dependent morphological changes and respiratory burst in neutrophils (9, 10). However, the spectrum of integrin beta  cytoplasmic domains that bind Syk has yet to be assessed.

The Syk family of kinases (Syk and Zap-70) are essential for normal development and function of the immune system (reviewed in Refs. 11 and 12), and Syk is required for the maintenance of vascular integrity (13, 14). These kinases are structurally distinct in that they contain tandem N-terminal SH2 domains followed by a C-terminal kinase domain. A helical Y-shaped linker region termed "interdomain A" joins the tandem SH2 domains. Kinase activity and subcellular localization of this kinase family within immune cells can be controlled by binding of its tandem SH2 domains to a doubly phosphorylated tyrosine ligand in immune response receptors (immunoreceptor tyrosine-based activation motif (ITAM))1 (11). Linking the tandem SH2 domains with the kinase domain is the "interdomain B" region. This region contains a number of tyrosines that are phosphorylated in vivo and can recruit other signaling/adaptor molecules such as Src family members (15), Vav-1 (16), and Cbl (17).

Residues 6-370 in Syk and 1-337 in Zap-70, which contain the tandem SH2 domains and part of the interdomain B region, directly bind the integrin beta 3 cytoplasmic domain (7). Furthermore, Syk(1-330) acts as a dominant interfering mutant with respect to integrin alpha IIbbeta 3-dependent regulation of Syk (7). Integrin beta 3 cytoplasmic domain phosphorylation leads to binding of the adapter, Shc (18). However, the interaction between Syk and integrin beta 3 tails does not require tyrosine phosphorylation of the beta 3 tail or intact phosphotyrosine-binding sites in the Syk tandem SH2 domains (7). We now report that Syk and ZAP-70 can directly interact with integrin beta 1, beta 2, and beta 3 cytoplasmic domains with high affinity. Furthermore, the interaction involves the N-terminal SH2 domain of both kinases and is enhanced by the presence of the interdomain A region. Thus, the Syk/ZAP-70 integrin interaction is mediated by a phosphorylation-independent interaction of an SH2 domain.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cells, Reagents, Antibodies, and cDNAs-- The CHO cell line A5 stably expressing integrin alpha IIbbeta 3 (19) and was maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, L-glutamine, penicillin/streptomycin, and 0.5 mg/ml G418 (Invitrogen). The cells were grown at 37 °C in 6% CO2.

Anti-Syk Polyclonal antibody 0134, anti-beta 3 polyclonal antibody number 8053, and mAb 15 specific for beta 3 integrin have been described (7). Anti-Syk mAb 4D10, anti-GST mAb, and polyclonal anti-His were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated secondary goat anti-mouse and goat anti-rabbit F(ab')2 were purchased from BIOSOURCE International (Camarillo, CA).

Expression vectors encoding GST-Syk(6-370), GST-Syk(6-270), and GST-Syk(163-270) have been described (20). GST-Syk(6-109) was produced by inserting a stop codon at residue 109 of Syk by QuikChangeTM (Stratagene) site-directed mutagenesis. Construction of pGEX/GST-Syk(1-370,Delta 6-109) and the expression vector EMCV/myc-sykDelta (6-109) was performed by first introducing a blunt end StuI site at position 109 in both vectors with the QuikChange system. pGEX/GST-Syk(6-370) was cut with StuI and NheI to remove the N-terminal SH2 domain of Syk. Gel-purified vector was ligated to the annealed oligonucleotides 5'-gatccgccagcagcggc and 3'-gcggtcgtcgccg encoding amino acid residues 2-5 of Syk. For EMCV/myc-Syk, vector was digested with StuI and BamHI, gel-purified, and ligated with the annealed oligonucleotides 5'-ctagcagcggc and 3'-gtcgccg encoding amino acids 2-5 of Syk. All site-directed mutagenesis and deletion mutations were verified by sequence analysis. GST-Zap-70(1-337), GST-Zap-70(1-103), and GST-Zap-70(163-254) were generated by PCR amplification of wild type Zap70 cDNA (pBS-Zap-70) and cloned into pGEX-2T.

Coprecipitation-- A5 CHO cells transiently transfected with full-length Syk or Syk with residues 6-109 deleted were plated on bovine serum albumin- or fibrinogen-coated bacteriological dishes, precoated as described (21). After a 1-h incubation at 37 °C and 6% CO2, the cells were lysed in 50 mM Tris (pH 7.4) containing 0.5% Nonidet P-40, 50 mM NaCl, and a protease inhibitor mixture (Roche Molecular Biochemicals). After clarification at 12,000 rpm for 20 min, 500-750 µg of lysate was incubated with primary antibody (polyclonal antibody 8053) overnight at 4 °C. After incubation with primary antibody, protein A beads (10 µl packed; Amersham Biosciences) were then added and rotated for 2 h at 4 °C. After washing beads in lysis buffer, the immune complexes were separated by SDS-PAGE, transferred to nitrocellulose filters, and Western blotted for the indicated proteins.

Purification of GST Fusion Proteins-- GST fusion proteins were produced as described (20). Briefly, exponential growth phase bacteria were induced with 0.1 mM isopropyl-1-thio-beta -D-galactopyranoside for 4 h at 37 °C. The cells were then pelleted, resuspended in lysis buffer (phosphate-buffered saline with 0.5% Triton X-100, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitors (Roche Molecular Biochemicals)), and sonicated at 4 °C. The lysates were clarified at 20,000 × g for 30 min, and supernatant was incubated with glutathione-SepharoseTM 4B (Amersham Biosciences) pre-equilibrated in lysis buffer. The mixture was incubated overnight at 4 °C and then washed four times in elution buffer (100 mM Tris, pH 8.0, 100 mM NaCl, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride) without GSH. Final elution was carried out in elution buffer containing 20 mM glutathione. For BIAcore analysis of Syk binding to integrin cytoplasmic domains, Syk(6-370) was cleaved from GST-Syk(6-370)-coated glutathione-SepharoseTM 4B by thrombin (1 unit/mg protein) overnight at 4 °C.

Integrin Cytoplasmic Domain Model Proteins-- Model protein mimics of integrin cytoplasmic domains alpha IIb, beta 1A, and beta 3 have been described previously (22). The beta 2 integrin cytoplasmic domain model protein was developed by PCR amplification of beta 2 cDNA using 5'-ccaagcttctgatccacctgagcgacctccgg and 3'-ttggggttcaaacgactctcaatcatccctagggg primers. This primer set introduced a 5' HindIII restriction site into the N-terminal region of the beta 2 cytoplasmic domain, resulting in the mutation A725L. The 3' primer contained a BamHI restriction site immediately downstream of dual stop codons. The PCR product was cloned into pCR2.1 as described in the TA® cloning system (Invitrogen). After sequencing, pCR2.1-beta 2cyt was digested with BamHI and HindIII and subcloned into the modified bacterial expression plasmid pET15bm (22) (lacking the HindIII restriction site). All of the model proteins were verified by sequence analysis, expressed, high pressure liquid chromatography-purified, and verified by electrospray ionization mass spectroscopy as described (22).

Direct Binding Assays-- The model peptides (1 mg) were dissolved in 1 ml of buffer containing 20 mM Tris, pH 7.9, 500 mM NaCl, and 6 M urea. To this, 50 µl of packed His-Bind® resin (Novagen, Madison, WI) was added, and the mixture was rotated for 1 h at room temperature. Immobilized peptides were washed five times in the above buffer without urea and resuspended in 500 µl of buffer containing 20 mM PIPES, pH 6.8, 50 mM NaCl, 3 mM MgCl2, 150 mM sucrose, 50 mM NaF, 40 mM Na4P2O7·10H20 (buffer A) supplemented with 0.1% Triton X-100 and 3 mM MgCl2. Purified GST fusion proteins were added to 0.5 ml of buffer A supplemented with 0.1% Triton X-100 and 3 mM MgCl2. Five µl of total packed resin loaded with various integrin cytoplasmic domain model proteins was added to the GST fusion protein. The mixture was incubated for 40 min at room temperature with continuous rotation. The beads were then washed five times in buffer A with 0.1% Triton X-100 and 3 mM MgCl2. The bound proteins were eluted by boiling in reducing sample buffer, fractionated by SDS-PAGE, and immunoblotted.

For peptide competition assays, the peptides were preincubated with Syk(6-370) for 1 h before direct binding assays were performed. The peptides were used at a concentration of 1 × 10-4 M, and Syk(6-370) was used at a concentration of 2 × 10-9 M. Bound Syk was detected as described above. The concentration of integrin cytoplasmic tail model protein estimated in the direct binding assay is 1.7 × 10-5 M.

Surface Plasmon Resonance-- BIAcore 3000 (BIAcore, Uppsala, Sweden) was used for real time kinetic analysis of the Syk/integrin cytoplasmic domain interactions. The experimental procedures, integrin cytoplasmic domain modifications, and data analysis were performed as described (23).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The N-terminal SH2 Domain of Syk Mediates Binding to the Integrin beta 3 Cytoplasmic Tail-- The N terminus of Syk tyrosine kinase (Syk(6-370)) interacts with integrin alpha IIbbeta 3 by binding to the beta 3 cytoplasmic domain (7). Syk(6-370) contains two N-terminal tandem SH2 domains separated by a linker sequence termed interdomain A. Following the tandem SH2 domains is another linker region termed interdomain B, which contains a number of tyrosine residues that are phosphorylated in vivo and regulate Syk and Zap-70 function (reviewed in Ref. 11). The C terminus of Syk (residues 370-635) contains its kinase domain (Fig. 1A). To further map the integrin binding site within Syk, we tested a number of Syk constructs represented schematically in Fig. 1A. In direct binding assays, the interdomain B (Syk(270-370)) and C-terminal SH2 domain of Syk (Syk(163-270)) were dispensable for binding (Fig. 1B, middle panels). In contrast, the N-terminal SH2 domain (Syk(6-109)) alone was sufficient for interaction with the beta 3 tail (Fig. 1B, right panel). To determine whether the N-terminal SH2 domain was necessary for the interaction of Syk(6-370) with beta 3, it was removed (Syk(Delta 6-109)). This deletion markedly reduced binding to the beta 3 tail (Fig. 2). Thus, the N-terminal SH2 domain of Syk is sufficient for interaction with the integrin beta 3 cytoplasmic tail (Fig. 1) and is necessary for strong binding.


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  		Fig. 1.   Direct binding of the N-terminal SH2 domain of Syk to the integrin beta 3 cytoplasmic domain. A, schematic representation of the GST-Syk fusion proteins used. B, direct binding of various GST-Syk constructs to Ni2+-charged resin containing the integrin beta 3 or alpha IIb cytoplasmic domain. Bound protein was eluted in reducing sample buffer, separated by SDS-PAGE (4-20% gradient gels), and visualized by immunoblotting with anti-GST mAb. The doublet in the left panel is a C-terminal cleavage product of GST-Syk (6-370) that copurifies with intact GST-Syk (6-370). Equal loading of cytoplasmic domain peptides was confirmed by Coomassie staining (not shown). One of four representative experiments is shown. MW, molecular mass; WB, Western blot; Rec Prt, recombinant protein.


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  		Fig. 2.   Removal of the N-terminal SH2 domain from Syk(6-370) decreases its direct binding to the integrin beta 3 tail. A, indicated concentrations of Syk(6-370) and SykDelta (6-109) were tested for binding to integrin beta 3 or alpha IIb cytoplasmic domain coated Ni2+-charged resin. Bound protein was eluted by boiling in reducing sample buffer, separated by SDS-PAGE (4-20% gradient gels), and visualized by immunoblotting with anti-Syk mAb 4D10. One-third of the GST-Syk starting material (Rec Prt) is shown in the left panel (for the 2.5 nM test). One of three representative experiments is shown. B, densitometric analysis of the amount of Syk bound to the integrin beta 3 cytoplasmic domain in A was performed with NIH Image software. MW, molecular mass; WB, Western blot.

The N-terminal SH2 Domain of Syk Is Necessary for Adhesion-dependent Association of Syk and Integrin alpha IIbbeta 3-- Syk physically associates with integrin alpha IIbbeta 3 (7) following adhesion of alpha IIbbeta 3-expressing cells to fibrinogen. To test whether deletion of the N-terminal SH2 domain would affect integrin association with Syk in vivo, CHO cells stably expressing integrin alpha IIbbeta 3 were transfected with full-length Syk or Syk lacking the N-terminal SH2 domain (SykDelta (6-109)). When these cells were plated on fibrinogen, no association between alpha IIbbeta 3 and SykDelta (6-109) was observed (Fig. 3). In contrast, wild type Syk manifested adhesion-dependent association with alpha IIbbeta 3 (Fig. 3). The recombinant Syk proteins were expressed at similar levels (Fig. 3, bottom panel), and equal amounts of the beta 3 subunit were immunoprecipitated (middle panel). The N-terminal SH2 domain (residues 6-109) is thus necessary for strong binding to the integrin beta 3 tail (Fig. 2) and for the physical association of Syk with integrin alpha IIbbeta 3.


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  		Fig. 3.   Deletion of the N-terminal SH2 domain from full-length Syk prevents its adhesion-dependent association with integrin alpha IIbbeta 3 in CHO cells. CHO cells stably expressing integrin alpha IIbbeta 3 were transfected with intact Syk (Syk(wt)) or Syk with the N-terminal SH2 domain removed (SykDelta (6-109)). Integrin alpha IIbbeta 3 was precipitated from CHO cells either in suspension (Sus) or adherent to fibrinogen (Fib). The immunoprecipitates (IP) were boiled in reducing sample buffer, separated by SDS-PAGE (4-20% gradient gels), and visualized by immunoblotting with anti-Syk mAb 4D10. Equal precipitation of integrin beta 3 was verified by immunoblotting with mAb15 (middle panel), and equal Syk expression was verified from immunoblots of whole cell lysate (bottom panel, WCL). One of four representative experiments is shown. WB, Western blot

Binding of the Syk Paralogue, ZAP-70, Involves Its N-terminal SH2 Domain-- Zap-70 is a paralogue of Syk that binds directly to integrin beta  cytoplasmic domains (reviewed in Ref. 11). It shares a similar domain structure with Syk (see schematic in Fig. 4A) and is essential for T cell development and function (24, 25). To determine whether the N-terminal SH2 domain of Zap-70 could interact with the beta 3 integrin tail, we measured the binding of recombinant purified ZAP-70(1-103) to the beta 3 cytoplasmic domain. The N-terminal SH2 domain of Zap-70 (residues 1-103) directly bound the integrin beta 3 tail (Fig. 4B, right panel). Binding of the C-terminal SH2 domain of Zap-70 (residues 163-254) was not detected (Fig. 4B, middle panel). Thus, in both ZAP-70 and Syk, binding to the integrin beta 3 cytoplasmic domain involves the N-terminal SH2 domain.


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  		Fig. 4.   Direct binding of the N-terminal SH2 domain of Zap-70 to the integrin beta 3 cytoplasmic domain. A, schematic representation of the GST-Zap-70 fusion proteins used in this study. B, direct binding of various GST-Zap-70 constructs to Ni2+-charged resin containing the integrin beta 3 or alpha IIb cytoplasmic domains. Bound protein was eluted in reducing sample buffer, separated by SDS-PAGE (4-20% gradient gels), and visualized by immunoblotting with anti-GST mAb. Equal loading of cytoplasmic domain peptides was confirmed by Coomassie staining (not shown). One of three representative experiments is shown. MW, molecular mass; WB, Western blot; Rec Prt, recombinant protein.

The Syk and Zap-70 Interdomain A Is Involved in Binding to the Integrin beta 3 Cytoplasmic Domain-- In direct binding assays, the interaction between Syk and Zap-70 N-terminal SH2 domains generally appear to be of lower affinity than the tandem SH2 domains together (data not shown and Fig. 5C). Also, removal of the N-terminal SH2 domain of Syk decreases but does not prevent binding of Syk to the integrin beta 3 cytoplasmic domain (Fig. 2). These results suggest that regions in addition to the N-terminal SH2 domains of Syk and Zap-70 may be involved in binding to the integrin beta 3 tail. Because the C-terminal SH2 domain of Syk and Zap-70 failed to bind the integrin beta 3 cytoplasmic domain, we examined the interdomain A region further. We expressed the interdomain A of Syk as a GST fusion protein to determine whether it could directly interact with the cytoplasmic domain of beta 3. No binding was detected (Fig. 5A). The orientation of the SH2 domains of Syk and Zap-70 are such that the phosphotyrosine-binding domains bind to dually phosphorylated ITAM sequences (YXX(I/L)X6-8YXX(I/L)) in a reverse colinear fashion (26, 27). The tandem SH2 domains of SHP-2 are oriented differently from those of Syk and Zap-70. The regions involved in phosphotyrosine binding are widely separated and in opposite orientation (28). When tested in a direct binding assay, the tandem SH2 domains of SHP-2 did not bind the beta 3 tails (Fig. 5B). Thus, interaction with the integrin beta 3 cytoplasmic tail is not a general property of tandem SH2 domain-containing proteins. When the Syk interdomain A was inserted into the interdomain region of SHP-2, SHP-2 now bound to the integrin beta 3 cytoplasmic domain (Fig. 5B). To examine the role of the interdomain A region of Zap-70, a series of truncation mutants were tested for binding to the integrin beta 3 cytoplasmic domain. The N-terminal SH2 domain of Zap-70, when expressed in conjunction with its intact IA domain (Zap-70(1-162)), bound beta 3 to a similar extent as the tandem SH2 domains (Fig. 5C). However, removal of the C-terminal half of interdomain A (residues Leu133-Pro162, Zap-70(1-132)) resulted in a decrease in binding similar to levels of the N-terminal SH2 domain alone (Fig. 5C). Thus, the interdomain A is necessary for optimal binding of Syk and Zap-70 to the beta 3 integrin cytoplasmic domain and confers binding to the SHP-2 tandem SH2 domains.


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  		Fig. 5.   The interdomain A region of Syk and Zap-70 is involved in binding the integrin beta 3 cytoplasmic domain. Syk interdomain A (Syk(IA)) and Syk(6-370) (A), Syk(6-370), SHP2(1-219), and SHP-2 with the intervening sequence between its tandem SH2 domains replaced with the interdomain A region of Syk (SHP-2/Syk(IA)) (B), and various truncations of GST-Zap-70 (C) were tested for direct binding to Ni2+-charged resin containing the integrin beta 3 or alpha IIb cytoplasmic domain. Bound protein was eluted in reducing sample buffer, separated by SDS-PAGE (4-20% gradient gels), and visualized by immunoblotting with anti-GST mAb. The doublet in Syk(6-370) preparations is a C-terminal cleavage product of GST-Syk(6-370) that copurifies with intact GST-Syk (6-370). Equal loading of cytoplasmic domain peptides was confirmed by Coomassie staining (not shown). Each experiment was performed at least three times. MW, molecular mass; WB, Western blot; Rec Prt, recombinant protein.

Tyrosine Phosphorylation of the beta 3 Cytoplasmic Domain Inhibits Binding to Syk-- The binding of Syk to beta 3 involves the N-terminal SH2 domain and does not require phosphorylation of the two tyrosines contained in the beta 3 tail (7). However, mutation of these two tyrosine residues to phenylalanine (beta 3(Y747F,Y759F)) within the beta 3 cytoplasmic domain can alter alpha IIbbeta 3-dependent functions (29). We therefore tested the effect of the beta 3(Y747F,Y759F) mutation on Syk or Zap-70 binding to the beta 3 tail. The beta 3(Y747F,Y759F) mutations had no effect on the binding of Syk or Zap-70 to the beta 3 cytoplasmic domain (Fig. 6A). Phosphorylation of the beta 3 integrin cytoplasmic domain occurs as a consequence of integrin engagement (30). To determine whether phosphorylation of the beta 3 cytoplasmic domain could affect Syk binding, competition assays were performed using tyrosine-phosphorylated or nonphosphorylated peptides in the context of the last 23 amino acids of the beta 3 cytoplasmic domain. Peptides were used at a concentration 1 × 10-4 M, and Syk was used at a concentration of 5 × 10-9 M. The nonphosphorylated peptide competed for integrin beta 3 tail binding to Syk (Fig. 6B). In three experiments, there was a 52 ± 3.4% decrease in Syk binding to the beta 3 tail model proteins in the presence of nonphosphorylated beta 3C-23 peptide as compared with the phosphorylated peptide (beta 3C-23P). The phosphorylated peptide did not detectably compete (Fig. 6B), although this peptide preparation is active in binding Shc (18). Thus, substitution of Phe residues at beta 3 Tyr747 and Tyr759 does not disrupt Syk binding, confirming the lack of requirement for phosphorylation. Furthermore, phosphorylation of beta 3(Y747F,Y759F) reduces its capacity to compete for Syk binding to the nonphosphorylated beta 3 tail.


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  		Fig. 6.   Effects of mutation or phosphorylation of beta 3 Tyr747 and Tyr759 on the direct binding of Syk and Zap-70. A, GST-Syk or GST-Zap-70 was incubated with Ni2+-charged resin coated with integrin alpha IIb, beta 3, or mutant beta 3(Y747F,Y759F) cytoplasmic domains. Equal loading of cytoplasmic domain peptides was confirmed by Coomassie staining (not shown). Depicted is one representative experiment of three performed. B, competition assays were performed with peptides based on the C-terminal 23 amino acid residues of the beta 3 cytoplasmic domain. GST-Syk(6-370) (2 nM) was preincubated with beta 3-C23 (100 µM) or beta 3 C-23P (100 µM) and then added to Ni2+-charged resin coated with alpha IIb or beta 3 cytoplasmic domains. Bound protein was eluted in reducing sample buffer, separated by SDS-PAGE (4-20% gradient gels), and visualized by immunoblotting with anti-Syk mAb 4D10. Starting material (Rec Prt) represents one-third of the original amount of GST-Syk. Equal cytoplasmic domain loading was confirmed by Coomassie staining (Loading, bottom panel). One of three representative experiments is shown. WT, wild type; WB, Western blot.

Syk Interacts with Multiple Integrin beta  Cytoplasmic Domains-- Syk binds to the beta 3 integrin cytoplasmic domain. Because Syk activation occurs after engagement of integrins that contain other beta  subunits (4, 8, 9, 31, 32), we tested the interaction of Syk with other integrin beta  cytoplasmic tails (Fig. 7A). The integrin beta 2 cytoplasmic domain bound Syk (6-370) to nearly the same extent as beta 3. Binding to beta 1A was detectable but modest relative to beta 3. No binding of GST was observed to any of the cytoplasmic domain constructs, establishing specificity. Scanning densitometry was used to quantify Syk binding relative to beta 3; beta 2 bound approximately half as much Syk as beta 3, and beta 1A bound approximately one-twentieth as much (Fig. 7B). The integrin cytoplasmic domains were in excess (i.e. the binding of Syk(6-370) was not saturating), and only a fraction (less than 20%; data not shown) of Syk(6-370) bound the integrin beta  cytoplasmic domains. Thus, Syk binding was limited primarily by the affinity of the interaction rather than depletion of the interacting proteins. Consequently, the apparent affinity of Syk for integrin beta  tails is beta 3 > beta 2 > beta 1A.


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  		Fig. 7.   Integrin class-specific binding to Syk. Ni2+-charged resin was loaded with the indicated integrin cytoplasmic domain model proteins and then incubated with Syk(6-370) or GST. Both Syk(6-370) and GST were used at a concentration of 5 nM. Bound fractions were solubilized in reducing SDS-PAGE sample buffer. After separation on 4-20% gels, the proteins were transferred to nitrocellulose membranes and blotted with anti-GST mAb. Depicted in A is one of three representative experiments. Coomassie staining of integrin cytoplasmic domains was used to monitor loading of the affinity matrix (Loading). B, data were analyzed by scanning densitometry (NIH Image software), and the means ± S.D. from four experiments are shown. WB, Western blot; Rec Prt, recombinant protein.

High Affinity Interactions between Syk and Integrin beta  Cytoplasmic Domains-- To determine the affinity of the interaction between various integrin cytoplasmic domains and Syk, binding parameters were measured by surface plasmon resonance. GST-Syk (6-370) was cleaved by thrombin to remove the GST and then dialyzed in BIAcore analysis buffer. This cleavage product retained the ability to specifically bind the integrin beta 3 cytoplasmic domain (Fig. 8A). Integrin alpha IIb, beta 1A, beta 2, and beta 3 cytoplasmic domain peptides were biotinylated and immobilized (23) onto avidin-coated BIAcore sensor chips at levels limiting mass-transport effects. The analyte, Syk, was used at four different concentrations, and the binding parameters for Syk interaction with various integrin cytoplasmic domains were calculated as described (23). The results are summarized in Table I. In sum, the cytoplasmic domain of beta 3 bound Syk with highest affinity (Kd = 24 nM). Similar results were obtained when the integrin beta 3 cytoplasmic domain was captured with immobilized anti-His antibodies (data not shown). The binding of Syk to the integrin beta 2 cytoplasmic domain was of slightly lower affinity (Kd = 38 nM), whereas the binding of the beta 1A cytoplasmic domain to Syk was of the lowest affinity (Kd = 71 nM).


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  		Fig. 8.   Surface plasmon resonance analysis of the binding kinetics between integrin beta 3, beta 2, and beta 1A cytoplasmic domains and Syk. A, GST was removed from Syk by thrombin cleavage, and Syk was added to Ni2+-charged resin coated with indicated integrin cytoplasmic domains. Bound protein was eluted in reducing sample buffer, separated by SDS-PAGE (4-20% gradient gels), and visualized by Coomassie staining. Equal loading of integrin cytoplasmic domains was confirmed by Coomassie staining (Loading). Starting material (Rec Prt) represents one-fourth of the original amount of Syk used. B, Syk (25, 50, 100, and 200 starting from the upper trace) was injected onto avidin-coated CM5 chips bound with biotin-maleimide beta 3, beta 2, beta 1A, or alpha IIb cytoplasmic domains as described (23). No binding was detected to immobilized integrin alpha IIb cytoplasmic domains (bottom trace, top panel) using an analyte concentration of 200 nM.

                              
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Table I
Surface plasmon resonance analysis of the binding kinetics between Syk and the integrin beta 1, beta 2, and beta 3 cytoplasmic domains


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Syk and ZAP-70 form a subfamily of nonreceptor tyrosine kinases that contain tandem SH2 domains at their N terminus. The binding of Syk to the beta 3 integrin tail leads to its physical association with integrin alpha IIbbeta 3 and to Syk activation (7). Here, we report that the interaction of integrins with these kinases depends on the N-terminal SH2 domain and the interdomain A region of the kinase. The N-terminal SH2 domain alone is sufficient for weak binding, and this interaction is independent of tyrosine phosphorylation of the integrin tail. Indeed, phosphorylation of tyrosines within the two conserved NXXY motifs in the integrin beta 3 cytoplasmic domain blocks Syk binding. The tandem SH2 domains of these kinases bind to multiple integrin beta  cytoplasmic domains with varying affinities (beta 3 (Kd = 24 nM) > beta 2 (Kd = 38 nM) > beta 1 (Kd = 71 nM)) as judged by both affinity chromatography and surface plasmon resonance. Thus, the binding of Syk and ZAP-70 to integrin beta  cytoplasmic domains represents a novel phosphotyrosine-independent interaction mediated by their N-terminal SH2 domains.

The requirement of the Syk N-terminal SH2 domain for its binding to integrin beta  cytoplasmic domains is based on three observations. First, the N-terminal SH2 domain of Syk and Zap-70, but not their C-terminal SH2 domains, are sufficient for binding to integrin beta  cytoplasmic domains. Second, deletion of this SH2 domain decreases binding to integrin cytoplasmic domains in vitro. Third, deletion of the N-terminal SH2 domain of Syk prevents adhesion-dependent association of Syk with integrin alpha IIbbeta 3 in cells. In hematopoietic cells, the tandem SH2 domains of Syk and Zap-70 interact in an antiparallel colinear fashion with dually phosphorylated tyrosines of ITAMs (26, 27). Although the N-terminal SH2 domains of Syk and Zap-70 function cooperatively to enhance binding of the C-terminal SH2 domain to pITAMs (20, 33) and promote specificity with respect to pITAM binding (34), this domain does not bind phosphorylated ITAM motifs when in isolation (20, 33). Indeed, to our knowledge, the ligands of the isolated N-terminal SH2 domain of Syk or Zap-70 have not been identified. Thus, phosphotyrosine-independent binding to integrin beta  cytoplasmic domains is a novel function of the N-terminal SH2 domains of Syk and Zap-70.

The tandem SH2 domains of Syk and Zap-70 are joined by an intervening sequence (the interdomain A region) that is involved in binding integrin cytoplasmic domains. Even after deletion of the N-terminal SH2 domain, Syk specifically bound to the integrin beta 3 cytoplasmic domain, albeit with reduced affinity. Furthermore, binding of the isolated N-terminal SH2 domains of Syk and Zap-70 to integrin cytoplasmic domains was weaker than the tandem SH2 domains. These two results suggest the participation of another region of Syk in the interaction. Interdomain A was implicated as the additional site because the addition of interdomain A to the Zap-70 N-terminal SH2 domain markedly increased binding to the beta 3 integrin cytoplasmic domain. Additional mapping implicated Leu133-Pro162 within the interdomain A region in increased binding. This region of Zap-70 forms a helix-turn-helix motif in very close proximity to the N-terminal SH2 domain (27). Interestingly, the corresponding sequence in Syk (Leu138-Pro167) has a similar structure (26) and shares 97% sequence similarity and 83% amino acid identity. Furthermore, the tandem SH2 domains of SHP-2 do not interact with the integrin beta 3 cytoplasmic domain. Replacing the interdomain sequence of the SHP-2 tandem SH2 domains with the Syk interdomain A region results in binding to the integrin beta  cytoplasmic domain. Thus, in addition to the N-terminal SH2 domain, the interdomain A region of Syk and Zap-70 is involved in their binding to integrin beta  cytoplasmic domains.

The interaction of Syk family kinases with integrin cytoplasmic domains is an example of phosphotyrosine-independent interaction of an SH2 domain. The Syk and Zap-70 tandem SH2 domains interact with pITAMs containing the phosphorylated YXX(I/L)X8-10YXX(I/L) motifs. The beta 3 cytoplasmic domain contains two conserved NXXY motifs that are spaced similarly to those in ITAM motifs. However, the integrin cytoplasmic domains bound Syk and ZAP-70 and lacked post-translational modification, as judged by mass spectroscopy. SH2 domains can interact with nonphosphorylated peptides. For instance, the interaction between the cytoplasmic domain of the signaling lymphocyte-activation molecule (SLAM) and SLAM-associated protein (SAP, also called SH2D1A and DSHP) can occur in the absence of tyrosine phosphorylation (35-38), even though SAP is comprised of one SH2 domain. SAP binds peptides with the consensus sequence (T/S)XXXX(V/I) (38), which is also present in the beta 1A, beta 2, and beta 3 integrin cytoplasmic domains. However, the SLAM/SAP interaction is reported to require an intact phosphotyrosine-binding site in SAP (35). Integrin/Syk interactions do not require intact phosphotyrosine-binding sites (5, 7), and Syk tandem SH2 domains can interact with the integrin beta 3 cytoplasmic domain in the presence of excess pITAM peptide. Together with the observations that the isolated N-terminal SH2 domain of Syk can directly bind integrin cytoplasmic domains, these results show that the beta  cytoplasmic domains of integrins interact with Syk kinase SH2 domains with a novel specificity. The studies reported here will enable high resolution structural analysis of this interaction.

Tyrosine phosphorylation of the integrin beta 3 cytoplasmic domain reduces its association with Syk. In peptide competition assays, a peptide containing the C-terminal 23 amino acid residues (including the two conserved NXXY motifs) competed for the beta 3 cytoplasmic tail binding to Syk(6-370). When residues Tyr747 and Tyr759 were phosphorylated, no competition was detected. Integrin beta  tail NXXY motifs can become phosphorylated in vivo (30). Replacement of the two NXXY tyrosines with nonphosphorylatable phenylalanines still permits Syk binding and should therefore oppose phosphorylation induced Syk dissociation from integrin beta  tails, thus prolonging Syk activation following integrin engagement. The beta 2 cytoplasmic domain naturally contains Phe residues in the NXXY motifs, suggesting that beta 2 integrins may promote prolonged activation of Syk and ZAP-70. Mice expressing mutant integrin alpha IIbbeta 3(Y747F,Y759F) manifest mild bleeding accompanied by unstable platelet aggregates and reduced clot retraction (29). These defects have been ascribed to inhibition of binding of Tyr(P)-dependent ligands such as myosin (39) or Shc (18) to the beta 3 tail (40). Our results raise the alternative possibility that interrupting tyrosine phosphorylation of the beta 3 cytoplasmic domain may perturb platelet function by prolonging association of Syk with the beta 3 tail, leading to prolonged signaling. Syk signaling is known to result in elevated cytoplasmic Ca2+ (41), which could promote calpain-dependent cleavage of cytoskeletal proteins (42) and thus block clot retraction (43). Further analysis of the signaling properties of these mutant platelets and the effect of combining this mutation with Syk deficiency should permit an evaluation of this potential mechanism of platelet dysfunction.

The integrin beta 3, beta 2, and beta 1A cytoplasmic domains directly bind Syk with relatively high affinity, and this interaction is likely to account for integrin beta 1 (4), beta 2 (8), and beta 3 (3) regulation of Syk function. As measured by surface plasmon resonance, the affinity of the integrin beta 3 cytoplasmic domain for Syk was 24 × 10-9 nM in contrast to the 10-fold higher affinity of Syk for the dually phosphorylated Fcepsilon RIgamma -ITAM, 2.6 × 10-9 M (34). However, integrins are far more abundant than ITAM containing receptor complexes on most cells. For example, Jurkat T cells express ~80,000 copies of alpha 4beta 1 (44) and only ~12,000 copies of the T cell receptor (TCR) (45) on their cell surface. Syk colocalizes with alpha IIbbeta 3 integrins in lamellipodia (6). Integrin-dependent recruitment of this kinase family to lamellipodia may contribute to the mechanism whereby polarized migrating lymphocytes are more sensitive to antigenic stimulation at their leading edge (46). pITAM binding to Syk directly regulates its functions (47). Thus, the interaction with integrin cytoplasmic domains could serve to modulate or focus the regulation of Syk and ZAP-70 by immune response receptors.

    ACKNOWLEDGEMENTS

We gratefully acknowledge the advice and critical input from Joan Brugge and the gift of the di-phosphorylated beta 3 peptide by David Phillips and Debbie Law. Brian Yaspan and Jessica Torruella provided expert technical support.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants HL 48728, HL 59007, and HL 31950.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Recipient of a post-doctoral fellowship from the Arthritis Foundation.

§ To whom correspondence should be addressed: Div. of Vascular Biology, Dept. of Cell Biology, Scripps Research Inst., 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-784-7124; Fax: 858-784-7343; E-mail: ginsberg@scripps.edu.

Published, JBC Papers in Press, August 8, 2002, DOI 10.1074/jbc.M207657200

    ABBREVIATIONS

The abbreviations used are: ITAM, immunoreceptor tyrosine-based activation motif; CHO, Chinese hamster ovary; mAb, monoclonal antibody; GST, glutathione S-transferase; PIPES, 1,4-piperazinediethanesulfonic acid; SLAM, signaling lymphocyte-activation molecule; SAP, SLAM-associated protein.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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