The N-terminal SH2 Domains of Syk and ZAP-70 Mediate Phosphotyrosine-independent Binding to Integrin β Cytoplasmic Domains*

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 β3 integrin-dependent cell adhesion via a phosphorylation-independent interaction with the β3 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 β3 cytoplasmic domain blocks Syk binding. The tandem SH2 domains of these kinases bind to multiple integrin β cytoplasmic domains with varying affinities (β3 (K d = 24 nm) > β2 (K d = 38 nm) > β1 (K d = 71 nm)) as judged by both affinity chromatography and surface plasmon resonance. Thus, the binding of Syk and ZAP-70 to integrin β cytoplasmic domains represents a novel phosphotyrosine-independent interaction mediated by their N-terminal SH2 domains.

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  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 ␣ IIb ␤ 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 ␣ IIb ␤ 3 -dependent regulation of Syk depends on the direct interaction of Syk with the ␤ 3 cytoplasmic domain (7). Integrins of other classes including the ␤ 1 (4) and ␤ 2 (8) integrins regulate Syk activity. Syk is essential for integrin ␤ 2 -dependent morphological changes and respiratory burst in neutrophils (9,10). However, the spectrum of integrin ␤ 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 ␤ 3 cytoplasmic domain (7). Furthermore, Syk(1-330) acts as a dominant interfering mutant with respect to integrin ␣ IIb ␤ 3 -dependent regulation of Syk (7). Integrin ␤ 3 cytoplasmic domain phosphorylation leads to binding of the adapter, Shc (18). However, the interaction between Syk and integrin ␤ 3 tails does not require tyrosine phosphorylation of the ␤ 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 ␤ 1 , ␤ 2 , and ␤ 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.
Coprecipitation-A5 CHO cells transiently transfected with fulllength 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% CO 2 , 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 g of lysate was incubated with primary antibody (polyclonal antibody 8053) overnight at 4°C. After incubation with primary antibody, protein A beads (10 l 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.
Integrin Cytoplasmic Domain Model Proteins-Model protein mimics of integrin cytoplasmic domains ␣ IIb , ␤ 1A , and ␤ 3 have been described previously (22). The ␤ 2 integrin cytoplasmic domain model protein was developed by PCR amplification of ␤2 cDNA using 5Ј-ccaagcttctgatccacctgagcgacctccgg and 3Ј-ttggggttcaaacgactctcaatcatccctagggg primers. This primer set introduced a 5Ј HindIII restriction site into the Nterminal region of the ␤ 2 cytoplasmic domain, resulting in the mutation A725L. The 3Ј 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-␤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 l 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 l of buffer containing 20 mM PIPES, pH 6.8, 50 mM NaCl, 3 mM MgCl 2 , 150 mM sucrose, 50 mM NaF, 40 mM Na 4 P 2 O 7 ⅐10H 2 0 (buffer A) supplemented with 0.1% Triton X-100 and 3 mM MgCl 2 . Purified GST fusion proteins were added to 0.5 ml of buffer A supplemented with 0.1% Triton X-100 and 3 mM MgCl 2 . Five l 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 MgCl 2 . 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 ϫ 10 Ϫ4 M, and Syk(6 -370) was used at a concentration of 2 ϫ 10 Ϫ9 M. Bound Syk was detected as

FIG. 1. Direct binding of the N-terminal SH2 domain of Syk to the integrin ␤ 3 cytoplasmic domain.
A, schematic representation of the GST-Syk fusion proteins used. B, direct binding of various GST-Syk constructs to Ni 2ϩcharged resin containing the integrin ␤ 3 or ␣ 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.
described above. The concentration of integrin cytoplasmic tail model protein estimated in the direct binding assay is 1.7 ϫ 10 Ϫ5 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
The N-terminal SH2 Domain of Syk Mediates Binding to the Integrin ␤ 3 Cytoplasmic Tail-The N terminus of Syk tyrosine kinase (Syk(6 -370)) interacts with integrin ␣ IIb ␤ 3 by binding to the ␤ 3 cytoplasmic domain (7). Syk(6 -370) contains two Nterminal 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 ␤ 3 tail (Fig. 1B,  right panel). To determine whether the N-terminal SH2 domain was necessary for the interaction of Syk(6 -370) with ␤ 3 , it was removed (Syk(⌬6 -109)). This deletion markedly reduced binding to the ␤ 3 tail (Fig. 2). Thus, the N-terminal SH2 domain of Syk is sufficient for interaction with the integrin ␤ 3 cytoplasmic tail (Fig. 1)

FIG. 3. Deletion of the N-terminal SH2 domain from full-length
Syk prevents its adhesion-dependent association with integrin ␣ IIb␤ 3 in CHO cells. CHO cells stably expressing integrin ␣ IIb ␤ 3 were transfected with intact Syk (Syk(wt)) or Syk with the N-terminal SH2 domain removed (Syk⌬ (6 -109)). Integrin ␣ IIb ␤ 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 ␤ 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 were transfected with full-length Syk or Syk lacking the Nterminal SH2 domain (Syk⌬(6 -109)). When these cells were plated on fibrinogen, no association between ␣ IIb ␤ 3 and Syk⌬(6 -109) was observed (Fig. 3). In contrast, wild type Syk manifested adhesion-dependent association with ␣ IIb ␤ 3 (Fig. 3). The recombinant Syk proteins were expressed at similar levels (Fig. 3, bottom panel), and equal amounts of the ␤ 3 subunit were immunoprecipitated (middle panel). The N-terminal SH2 domain (residues 6 -109) is thus necessary for strong binding to the integrin ␤ 3 tail (Fig. 2) and for the physical association of Syk with integrin ␣ IIb ␤ 3 .
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 ␤ 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 ␤ 3 integrin tail, we measured the binding of recombinant purified ZAP-70(1-103) to the ␤ 3 cytoplasmic domain. The N-terminal SH2 domain of Zap-70 (residues 1-103) directly bound the integrin ␤ 3 tail (Fig.  4B, right panel). Binding of the C-terminal SH2 domain of Zap-70 (residues 163-254) was not detected (Fig. 4B,
the Integrin ␤ 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 ␤ 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 ␤ 3 tail. Because the Cterminal SH2 domain of Syk and Zap-70 failed to bind the integrin ␤ 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 ␤ 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)X 6 -8 YXX(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 ␤ 3 tails (Fig. 5B). Thus, interaction with the integrin ␤ 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 ␤ 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 ␤ 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 ␤ 3 to a similar extent as the tandem SH2 domains (Fig. 5C). However, removal of the C-terminal half of interdomain A (residues Leu 133 -Pro 162 , Zap-70(1-132)) resulted in a decrease in binding similar to levels of the N-terminal SH2 domain alone (Fig. 5C). Thus, the interdo- main A is necessary for optimal binding of Syk and Zap-70 to the ␤ 3 integrin cytoplasmic domain and confers binding to the SHP-2 tandem SH2 domains.
Tyrosine Phosphorylation of the ␤ 3 Cytoplasmic Domain Inhibits Binding to Syk-The binding of Syk to ␤ 3 involves the N-terminal SH2 domain and does not require phosphorylation of the two tyrosines contained in the ␤3 tail (7). However, mutation of these two tyrosine residues to phenylalanine (␤ 3 (Y747F,Y759F)) within the ␤ 3 cytoplasmic domain can alter ␣ IIb ␤ 3 -dependent functions (29). We therefore tested the effect of the ␤ 3 (Y747F,Y759F) mutation on Syk or Zap-70 binding to the ␤ 3 tail. The ␤ 3 (Y747F,Y759F) mutations had no effect on the binding of Syk or Zap-70 to the ␤ 3 cytoplasmic domain (Fig.  6A). Phosphorylation of the ␤ 3 integrin cytoplasmic domain occurs as a consequence of integrin engagement (30). To determine whether phosphorylation of the ␤ 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 ␤ 3 cytoplasmic domain. Peptides were used at a concentration 1 ϫ 10 Ϫ4 M, and Syk was used at a concentration of 5 ϫ 10 Ϫ9 M. The nonphosphorylated peptide competed for integrin ␤ 3 tail binding to Syk (Fig. 6B). In three experiments, there was a 52 Ϯ 3.4% decrease in Syk binding to the ␤ 3 tail model proteins in the presence of nonphosphorylated ␤ 3 C-23 peptide as compared with the phosphorylated peptide (␤ 3 C-23P). The phosphorylated peptide did not detectably compete (Fig. 6B), although this peptide prepa-ration is active in binding Shc (18). Thus, substitution of Phe residues at ␤ 3 Tyr 747 and Tyr 759 does not disrupt Syk binding, confirming the lack of requirement for phosphorylation. Furthermore, phosphorylation of ␤ 3 (Y747F,Y759F) reduces its capacity to compete for Syk binding to the nonphosphorylated ␤ 3 tail.
Syk Interacts with Multiple Integrin ␤ Cytoplasmic Domains-Syk binds to the ␤ 3 integrin cytoplasmic domain. Because Syk activation occurs after engagement of integrins that contain other ␤ subunits (4,8,9,31,32), we tested the interaction of Syk with other integrin ␤ cytoplasmic tails (Fig. 7A). The integrin ␤ 2 cytoplasmic domain bound Syk (6 -370) to nearly the same extent as ␤ 3 . Binding to ␤ 1A was detectable but modest relative to ␤ 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 ␤ 3 ; ␤ 2 bound approximately half as much Syk as ␤ 3 , and ␤ 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 ␤ 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 ␤ tails is High Affinity Interactions between Syk and Integrin ␤ 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 ␤ 3 cytoplasmic domain (Fig. 8A). Integrin ␣ IIb , ␤ 1A , ␤ 2 , and ␤ 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 ␤ 3 bound Syk with highest affinity (K d ϭ 24 nM). Similar results were obtained when the integrin ␤ 3 cytoplasmic domain was captured with immobilized anti-His antibodies (data not shown). The binding of Syk to the integrin ␤ 2 cytoplasmic domain was of slightly lower affinity (K d ϭ 38 nM), whereas the binding of the ␤ 1A cytoplasmic domain to Syk was of the lowest affinity (K d ϭ 71 nM).

DISCUSSION
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 ␤ 3 integrin tail leads to its physical association with integrin ␣ IIb ␤ 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 ␤ 3 cytoplasmic domain blocks Syk binding. The tandem SH2 domains of these kinases bind to multiple integrin ␤ cytoplasmic domains with varying affinities (␤ 3 (K d ϭ 24 nM) Ͼ ␤ 2 (K d ϭ 38 nM) Ͼ ␤ 1 (K d ϭ 71 nM)) as judged by both affinity chromatography and surface plasmon resonance. Thus, the binding of Syk and ZAP-70 to integrin ␤ 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 ␤ 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 ␤ 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 ␣ IIb ␤ 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 ␤ 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 ␤ 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 ␤ 3 integrin cytoplasmic domain. Additional mapping implicated Leu 133 -Pro 162 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 (Leu 138 -Pro 167 ) 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 ␤ 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 ␤ 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 ␤ 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)X 8 -10 YXX(I/L) motifs. The ␤ 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)(36)(37)(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 ␤ 1A , ␤ 2 , and ␤ 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 FIG. 8. Surface plasmon resonance analysis of the binding kinetics between integrin ␤ 3 , ␤ 2 , and ␤ 1A cytoplasmic domains and Syk. A, GST was removed from Syk by thrombin cleavage, and Syk was added to Ni 2ϩ -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 avidincoated CM5 chips bound with biotin-maleimide ␤ 3 , ␤ 2 , ␤ 1A , or ␣ IIb cytoplasmic domains as described (23). No binding was detected to immobilized integrin ␣ IIb cytoplasmic domains (bottom trace, top panel) using an analyte concentration of 200 nM. The studies reported here will enable high resolution structural analysis of this interaction. Tyrosine phosphorylation of the integrin ␤ 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 ␤ 3 cytoplasmic tail binding to Syk(6 -370). When residues Tyr 747 and Tyr 759 were phosphorylated, no competition was detected. Integrin ␤ 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 ␤ tails, thus prolonging Syk activation following integrin engagement. The ␤ 2 cytoplasmic domain naturally contains Phe residues in the NXXY motifs, suggesting that ␤ 2 integrins may promote prolonged activation of Syk and ZAP-70. Mice expressing mutant integrin ␣ IIb ␤ 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 ␤ 3 tail (40). Our results raise the alternative possibility that interrupting tyrosine phosphorylation of the ␤ 3 cytoplasmic domain may perturb platelet function by prolonging association of Syk with the ␤ 3 tail, leading to prolonged signaling. Syk signaling is known to result in elevated cytoplasmic Ca 2ϩ (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 ␤ 3 , ␤ 2 , and ␤ 1A cytoplasmic domains directly bind Syk with relatively high affinity, and this interaction is likely to account for integrin ␤ 1 (4), ␤ 2 (8), and ␤ 3 (3) regulation of Syk function. As measured by surface plasmon resonance, the affinity of the integrin ␤ 3 cytoplasmic domain for Syk was 24 ϫ 10 Ϫ9 nM in contrast to the 10-fold higher affinity of Syk for the dually phosphorylated Fc⑀RI␥-ITAM, 2.6 ϫ 10 Ϫ9 M (34). However, integrins are far more abundant than ITAM containing receptor complexes on most cells. For example, Jurkat T cells express ϳ80,000 copies of ␣ 4 ␤ 1 (44) and only ϳ12,000 copies of the T cell receptor (TCR) (45) on their cell surface. Syk colocalizes with ␣ IIb ␤ 3 integrins in lamellipodia (6). Integrindependent 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.