Regulation and Function of SKAP-55 Non-canonical Motif Binding to the SH3c Domain of Adhesion and Degranulation-promoting Adaptor Protein*

The immune cell adaptor adhesion and degranulation promoting adaptor protein (ADAP) and its binding to T-cell adaptor Src kinase-associated protein of 55 kDa (SKAP-55) play a key role in the modulation of T-cell adhesion. While primary binding occurs via SKAP-55 SH3 domain binding to a proline-rich region in ADAP, a second interaction occurs between the ADAP C-terminal SH3 domain (ADAP-SH3c) and a non-canonical RKXXY294XXY297 motif in SKAP-55. Increasing numbers of non-canonical SH3 domain binding motifs have been identified in a number of biological systems. The presence of tyrosine residues in the SKAP-55 RKXXY294XXY297 motif suggested that phosphorylation might influence this unusual SH3 domain interaction. Here, we show that the Src kinase p59fyn can induce the in vivo phosphorylation of the motif, and this event blocks ADAP-SH3c domain binding to the peptide motif. The importance of tyrosine phosphorylation was confirmed by plasmon resonance interaction analysis showing that phosphorylation of Tyr294 residue plays a central role in mediating dissociation, whereas phosphorylation of the second Tyr297 had no effect. Although loss of this secondary interaction did not result in the disruption of the complex, the Y294F mutation blocked T-cell receptor-induced up-regulation of lymphocyte function-associated antigen-1-mediated adhesion to intercellular adhesion molecule-1 and interleukin-2 promoter activity. Our findings identify a RKXXY294 motif in SKAP-55 that mediates unique ADAP SH3c domain binding and is needed for LFA-1-mediated adhesion and cytokine production.


The immune cell adaptor adhesion and degranulation promoting adaptor protein (ADAP) and its binding to T-cell adaptor Src kinaseassociated protein of 55 kDa (SKAP-55) play a key role in the modulation of T-cell adhesion. While primary binding occurs via SKAP-55 SH3 domain binding to a proline-rich region in ADAP, a second interaction occurs between the ADAP C-terminal SH3 domain (ADAP-SH3c) and a non-canonical RKXXY 294 XXY 297 motif in SKAP-55.
Increasing numbers of non-canonical SH3 domain binding motifs have been identified in a number of biological systems. The presence of tyrosine residues in the SKAP-55 RKXXY 294 XXY 297 motif suggested that phosphorylation might influence this unusual SH3 domain interaction. Here, we show that the Src kinase p59 fyn can induce the in vivo phosphorylation of the motif, and this event blocks ADAP-SH3c domain binding to the peptide motif. The importance of tyrosine phosphorylation was confirmed by plasmon resonance interaction analysis showing that phosphorylation of Tyr 294 residue plays a central role in mediating dissociation, whereas phosphorylation of the second Tyr 297 had no effect. Although loss of this secondary interaction did not result in the disruption of the complex, the Y294F mutation blocked T-cell receptor-induced up-regulation of lymphocyte function-associated antigen-1-mediated adhesion to intercellular adhesion molecule-1 and interleukin-2 promoter activity. Our findings identify a RKXXY 294 motif in SKAP-55 that mediates unique ADAP SH3c domain binding and is needed for LFA-1-mediated adhesion and cytokine production.
Modular domains in proteins such as protein-tyrosine kinases, phosphatases and adaptors play central roles in the generation of signals needed for mammalian cell function (1). Of these, Src homology domain 2 (SH2) 3 recognizes phosphotyrosine-based motifs, whereas Src homology domain 3 (SH3) domains recognize proline-based PXXP motifs (2). Since the description of Abelson SH3 domain recognition of the 3BP1 protein (3), numerous SH3 domain-mediated interactions have been documented. Examples include SH3-mediated interactions between the adaptor Grb-2 (growth factor receptor-bound protein-2) and Son-of-Sevenless, Src kinase SH3 domain binding to the p85 subunit of phosphatidylinositol 3-kinase, and Crk SH3 domain binding to Crk SH3 domain-binding guanine nucleotide-releasing factor, among others. SH3 domain binding is involved in subcellular localization, cytoskeletal organization, and signal transduction (4).
Structurally, SH3 domains are comprised of two anti-parallel ␤ sheets packed at right angles to one other (2,4,5). The core øPXøP motif (where ø represents a hydrophobic residue) interacts with SH3 through two defined consensus sequences: Class I (R/KXXPXXP) and Class II (PXXPXR) (6). Domains can bind ligands in either an N-to C-terminal or C-to N-terminal orientation due to the pseudosymmetrical nature of the polyproline class II helix that is stabilized primarily by hydrophobic and additional electrostatic interactions. Directionality is conferred by the interaction of the arginine or lysine residues with the charged outer face on the SH3 domain, while the tandem prolines bind to two hydrophobic pockets. Binding depends on the two SH3 variable loops, the RT and n-Src loops, that flank a ligand-binding region.
In addition to the binding to øPXøP-based motifs, an increasing number of studies have documented SH3 domain binding to non-canonical motifs (7). These include a PX(V/I)(D/N)RXXKP motif that is responsible for STAM2 SH3 domain binding to the deubiquitination enzyme ubiquitin isopeptidase Y/Usp8 (8,9) and Grb-2 related protein (GADS), SH3 domain binding to SH2-domain-containing leukocyte protein of 76 kDa (SLP-76) (10 -13). In the latter case, the interaction has a 10 -20 times higher affinity than interaction between SH3 domains and their øPXøP motifs. Other examples of non-canonical interactions include amphiphysin SH3 domain binding to dynamin (14), Eps8 SH3 domain binding to the PXXDY motif (15), and HPK1 SH3 domain binding to a RXXK motif (12). We recently identified a novel RKXXYXXY motif that is found in the T-cell adaptor Src kinase-associated protein of 55 kDa (SKAP-55) and that is recognized by the FYN-SH3 and ADAP-SH3c domains (16).
In the immune system, SH3 domains are found in Src-related kinases and immune specific adaptor proteins (17,18). Adaptors lack enzymatic activity and transcription binding domains, and instead are comprised of multiple binding domains and sites that facilitate protein-protein aggregation. This family of adaptors in T-cells includes linker for activation of T cells (LAT), lymphocyte cytosolic protein-2 (SLP-76), adaptor adhesion and degranulation promoting adaptor protein (ADAP, previously known as FYN T-binding protein/SLP-76-associated protein (FYB/SLAP)), and SKAP-55 (also known as SCAP1). ADAP and SKAP-55 are unique proteins with C-terminal SH3 domains and are selectively phosphorylated by the Src kinase FYN-T (19 -22). The SH3 domain of SKAP-55 can bind to a prolinerich region in ADAP (20,21), whereas the ADAP SH3c domain binds to the RKXXY 294 XXY 297 motif in SKAP-55 (16). Essentially all cellular SKAP-55 is complexed to ADAP (21), an interaction that protects free SKAP-55 from rapid degradation by proteolysis (23). Furthermore, both ADAP and SKAP-55 have been implicated in T-cell adhesion. Transfection and knockout models have shown that ADAP mediates inside-out signaling events that regulate LFA-1 clustering (24 -27). Retroviral expression of SKAP-55 also enhances LFA-1 clustering and adhesion (28,29), whereas the loss of SKAP-55 by small interference RNA blocks these events (30). Disruption of SLP-76-ADAP also interferes with peripheral supramolecular activation complex formation at the immunological synapse (28), whereas ADAP and SLP-76 can cooperatively up-regulate IL-2 transcription (22,31,32).
Unlike other non-canonical motifs, the SKAP-55 RKXXY 294 XXY 297 sequence incorporates two tyrosine residues that may serve as sites for phosphorylation, which could potentially regulate SH3 domain binding and influence the interaction with ADAP. Here, we show that the Src kinase p59 fyn can induce the in vivo phosphorylation of tyrosines within the SKAP-55 motif, and this event potently blocks ADAP-SH3c domain binding to the ligand. The importance of tyrosine phosphorylation was confirmed by plasmon resonance interaction analysis showing that phosphorylation of Tyr 294 residue plays a central role in mediating dissociation, whereas phosphorylation of the second Tyr 297 had no effect. Although loss of this secondary interaction did not result in the disruption of the complex and likely plays little role in complex structural integrity, the Y294F mutation blocked TcR-induced up-regulation of LFA-1-mediated adhesion to ICAM-1 and IL-2 promoter activity. Our findings thus identify a RKXXY 294 motif in SKAP-55 that interacts with ADAP SH3c domain and is required for LFA-1-mediated adhesion and cytokine production.

EXPERIMENTAL PROCEDURES
Reagents and Cell Culture-COS-1 cells were maintained in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (Intergen), 1% (w/v) penicillin and streptomycin (Invitrogen), and 1% (v/v) L-glutamine (Invitrogen). COS-1 cell transfection was conducted using standard protocols (32). Jurkat cells were maintained in the same medium and expressed SKAP-55 or appropriate mutants following transfection as previously described (28 -30). Anti-GST monoclonal antibody was purchased from Santa Cruz Biotechnology, and anti-HA monoclonal antibody was from Roche Biochemicals. Anti-phosphotyrosine monoclonal antibody 4G10 was kindly provided by Dr. Tom Roberts (Dana-Farber Cancer Institute, Boston, MA). Anti-FYN-T, LCK, and ZAP70 monoclonal antibodies were purchased from BD Transduction. Peptides were synthesized and high-performance liquid chromatography-purified by the Molecular Biology Core Facility (Dana-Farber Cancer Institute) with sequences as follows: unphosphorylated peptide TRRKGDY 294 ASY 297 Y 298 QG, the same peptide phosphorylated at Tyr 294 , phosphorylated at Tyr 297 , phosphorylated at Tyr 294 and Tyr 297 , and phosphorylated at Tyr 294 , Tyr 297 , and Tyr 298 ( Table 1). TTGVFVKMPPTE served as an irrelevant peptide.
Immunoprecipitation and Immunoblotting-Cell lysis, immunoprecipitation, and detection were performed as described previously (19). Briefly, 2 ϫ 10 5 COS-1 or Jurkat cells were transfected with cDNA using DEAE-dextran as described (30). After 2 days, cells were harvested and lysed with 200 l of lysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% (v/v) Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na 3 VO4, 1 mM NaF, 1 mM leupeptin, 1 mM pepstatin, and 1% aprotinin). Immunoprecipitation was carried out by incubation of the lysate with the antibody for 1 h at 4°C, followed by incubation with 50 l of gluta-thione-Sepharose beads (50% w/v) for 1 h at 4°C. For immunoblotting, the immunoprecipitates were separated by SDS-PAGE and transferred onto nitrocellulose filters (Schleicher and Schuell). Filters were blocked with 5% (w/v) skimmed milk for 1 h in Tris-buffered saline (Tris buffered saline) pH 8.0 and then probed with the indicated antibody or GST fusion proteins followed by binding with an anti-GST monoclonal antibody. Bound antibody was revealed with horseradish peroxidaseconjugated rabbit anti-mouse antibody using enhanced chemiluminescence (ECL, Amersham Biosciences). For the Far Western technique, ADAP-SH3c was expressed with a T7 tag, and binding to SDS-PAGEseparated and blotted GST fusion proteins was detected with anti-T7 antibody followed by chemiluminescence development.
Expression and Purification of GST Fusion Proteins-Plasmids were transformed into the DH5 strain of Escherichia coli and induced with isopropyl-␤-D-thiogalactopyranoside to produce GST fusion proteins as described (16).
Luciferase Assays for IL-2 Promoter Activity-A total of 5 ϫ 10 5 Jurkat cells transfected with the test SKAP-55 constructs, IL-2 promoter reporter vector, and control vector (Promega, Madison, WI) were stimulated with 1 g/ml anti-CD3 and 2 g/ml rabbit anti-mouse Ab at 37°C for 6 h and subsequently assayed for luciferase activity using a luminometer (MicroLumat, EG&G Berthold). Luciferase units of the experimental vector were normalized to the level of the control vector in each sample. Adhesion assays were conducted according to Jo et al. (30).
Measurement of Peptide Binding Using Surface Plasmon Resonance-All interactions were determined using a BIAcore 3000 and a fully upgraded BIAcore 1000 instrument. Various SH3 fusion proteins (10 g/ml in 100 mM sodium acetate, pH 4.5) were immobilized on CM5 Biosensor chips (BIAcore) through cross-linking of free amine groups to the N-hydroxysuccinimide/1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl-activated flow cell surface, followed by blocking of free succinimide ester with 1 M ethanolamine. After extensive washing of the surface with binding buffer (10 mM HEPES, 150 mM NaCl, pH 7.0), peptide binding was assessed by injecting the indicated concentrations in binding buffer over the flow cell surface. When required, the surface was regenerated using 100 mM glycine, pH 2.8. The data were analyzed using the BIAevaluation version 3.2 software and fitted to a 1:1 Langmuir binding model with separate k d and k a determination. The association constant (K a ) was determined as k a /k d , and the dissociation constant (K d ) was determined as 1/K a . (16) have demonstrated that the atypical ADAP SH3c domain binds to a non-canonical non-Tyrphosphorylated RKXXYXXY motif in the SK4 region of the SKAP-55 SK region (Fig. 1A). To assess whether the tyrosine residues in the motif could be phosphorylated and whether this affects ADAP SH3c domain binding, cells were co-transfected with GST-tagged peptide SK4, which carries the RKXXYXXY motif (residues 290 -297) and the protein-tyrosine kinase p59 fyn . Within the SK4 peptide, tyrosines are located at residues 294, 297, and 298. We had previously shown that SKAP-55 is phosphorylated by the Src kinase p59 fyn (19,22). Following cell lysis, the GST-tagged SK4 was precipitated using glutathione beads, and tyrosine phosphorylation was detected by anti-pTyr blotting. Co-expression of p59 fyn resulted in the phosphorylation of pEBG-expressed SK4 (Fig. 1B, left panel, lane 4 versus 3). As a control for nonspecific effects, coexpression of p59 fyn plus GST alone failed to result in GST phosphorylation (lane 2 versus 1). SK4 SKAP-55 peptide was preferentially phosphorylated by p59 fyn , because co-expression of another T-cell kinase ZAP-70 resulted in only a background phosphorylation (lane 5). Anti-GST blotting of cell lysates confirmed the expression of GST (lanes 1 and 2) and GST-SK4 (lanes 3-5) (Fig. 1B, right upper panel), whereas anti-p59 fyn and anti-ZAP70 blotting confirmed expression of the kinases (Fig. 1B, right middle and lower panels). These observations indicate that p59 fyn can phosphorylate the SK4 region of SKAP-55, the region that interacts with the C-terminal SH3 domain of ADAP.

Phosphorylation of the RKXXY 294 XXY 297 Motif Interferes with ADAP SH3c Domain Binding-Previous studies
To assess whether phosphorylation might interfere with ADAP SH3c domain binding, the same samples were blotted with ADAP-SH3c-T7 in a Far-Western protocol followed by detection with anti-T7 antibody (Fig. 1C). Under these conditions, the ADAP-SH3c domain could readily bind to GST-SKAP-55-SK4 on nitrocellulose (lane 3). This confirms by another technique the binding of the ADAP-SH3c domain with the SK4 peptide (16). As a control, no binding was observed between ADAP-SH3c and the GST control, verifying the specificity of the interaction between SKAP-55 and ADAP-SH3c. Significantly, in the presence of p59 fyn , where the SKAP-55 peptide was phosphorylated (Fig. 1B,   left panel, lane 4), the ADAP-SH3c domain was greatly impaired in binding to the SKAP-55 peptide (Fig. 1C, left panel, lane 4). As a further control, in the absence of SKAP-55 peptide but the presence of p59 fyn , no ADAP-SH3c was precipitated (Fig. 1C, left panel, lane 2). Co-expression of ZAP-70 partially inhibited the interaction between SKAP-55 peptide and ADAP-SH3c (Fig. 1C, left panel, lanes 5 versus 3), with little if any phosphorylation (Fig. 1B, left panel, lane 5), but it was significantly less than the phosphorylation and inhibition by p59 fyn (Fig. 1C, left  panel, lanes 4 versus 5). Overall, these observations show that phosphorylation of the SK4 peptide in vivo correlated with a major reduction in the ability of the ADAP-SH3c domain to interact with the RKXXYXXYcontaining peptide.
RKXXYXXY Phosphorylation Interferes with ADAP-SH3 Domain Binding as Confirmed by Surface Plasmon Resonance-The co-expression studies suggested that the ADAP-SH3c domain interaction with the RKXXYXXY motif can be regulated by a phosphorylation event. Nevertheless, the complexity of the interactions of kinases with sub-  MAY 12, 2006 • VOLUME 281 • NUMBER 19

SKAP-55 Motif Binding to ADAP SH3c
strates within the intact cell may introduce inadvertent interactions. For example, the presence of the SH3 domain within p59 fyn itself might compete with ADAP-SH3c in binding to the peptide. We therefore confirmed the SKAP-55 peptide-ADAP-SH3c interaction using the BIAcore surface plasmon resonance (SPR) technique. We had previously demonstrated that RKXXYXXY-containing peptides can bind to ADAP-and Fyn-SH3 domains using the same approach (16). Utilizing synthetically phosphorylated peptides of the SK4 region of SKAP-55, SPR analysis yields not only association/dissociation constants but also allows the mapping of the tyrosines within SKAP-55 SK4 domain that blocks the interaction with ADAP-SH3 domain. Five peptides were produced of the unmodified peptide and several permutations of phosphorylated tyrosines that might be expected following p59 fyn kinase activity (Table 1). Confirming our previous results and the results of Far Western blotting (Fig. 1C), a significant interaction between the control peptide and ADAP-SH3c was revealed by SPR interaction analysis ( Fig. 2A). This showed a dissociation constant (K D ) of ϳ30 M (Table 1 and Fig. 2  (F and G)). Significantly, modification at the Tyr 294 residue had a dramatic effect in reducing the association so that a binding constant could not be determined (Fig. 2D). Phosphorylation at Tyr 294 in the context of Tyr 297 phosphorylation with or without modification of Tyr 298 decreased the response further toward baseline (Fig. 2, B and C). By contrast, phosphorylation at Tyr 297 had minimal effect upon equilibrium binding to ADAP-SH3c, increasing the K D to ϳ40 M (Fig. 2E). In this context, it is unlikely that the inhibition of the association was a non-position-specific phosphate charge effect, because phosphorylation of the proximal Tyr 297 had little effect on binding. It is important to note here that the in vitro-expressed GST-SK4-peptide contains a long glutamic acid repeat (Fig. 1A) upstream of the synthetic peptides used for the BIAcore analysis. This charged region is irrelevant to the SKAP-55-ADAP interaction, because SKAP peptides SK1, SK2, and SK3 also harbor these glutamic acid repeats (Fig. 1A) but do not interact with ADAP (16). These observations confirmed the binding of the SK-4 peptide to the ADAP-SH3c domain and showed that phosphorylation of the Tyr 294 residue had a specific inhibitory effect on the interaction.
Inorganic Phosphate Interferes with the SKAP-55 SK4-ADAP-SH3c Interaction-Although confirming our finding that the SKAP-55 SK4 motif can interact with the Fyn-SH3 domain, a recent report had difficulty confirming the interaction with the ADAP-SH3c domain by nuclear magnetic resonance (34). This was surprising given the high reproducibility of our results in showing the interaction by co-expression studies (16), far-Western blotting, and BIAcore SPR (Figs. 1 and 2). Furthermore, we found that the SKAP-55 SK4 peptide interaction with the ADAP-SH3c domain was stronger than with the Fyn-SH3 domain (16). In comparing the composition of buffers used in the SPR versus NMR analysis, it was evident that the NMR buffer had 20 mM phosphate anion that was absent in the SPR buffer. Given that we were examining an event inhibited by phosphorylation, we reasoned that the presence of high buffer inorganic phosphate might be of particular concern given the lower avidity and charge-dependent interaction of SK4 with the ADAP SH3c domain. To assess this, we repeated the SPR analysis in the presence of the buffer used in the NMR analysis that included 20 mM phosphate. Whereas the ADAP-SH3c domain-SK4 interaction occurred in HEPES buffer in the absence of phosphate (Fig. 3A), no interaction of SKAP-55 SK4 peptide with ADAP-SH3c was detected in the presence of high levels of free inorganic phosphate anion (Fig. 3B) in agreement with Heuer and co-workers (34). This indicates that the presence of free inorganic phosphate anion can interfere with the binding of ADAP-SH3c domain to the SK4 peptide and offers an explanation for the differing result.
Mutation of Tyr 294 Does Not Disrupt SKAP-55⅐ADAP Complex Formation-In addition to ADAP-SH3c domain binding to SKAP-55, we and others have shown that a second interaction occurs between the SKAP-55 SH3 domain and proline residues in ADAP (21,22), where this latter interaction is generally accepted to be the major basis of binding between the two proteins.
Nevertheless, to assess whether the ADAP-SH3c-mediated interaction is necessary for complex formation, or whether it constitutes a secondary interaction, the Tyr 294 residue was mutated, tagged with GFP, and assessed for binding to ADAP via co-precipitation analysis (Fig. 4). Independent of whether the transfected cells were resting or activated by anti-CD3 cross-linking, both wild-type SKAP-55-GFP and mutant SKAP-55-Y294F-GFP could be detected as an 80-kDa band following ADAP immunoprecipitation (Fig. 4C). Detection of phosphotyrosine in the same co-precipitates revealed that SKAP-55 was phosphorylated in all instances (Fig. 4D). The lack of SKAP-55 in appropriate controls (Fig. 4, A and B) confirmed the specificity of the ADAP-mediated co-precipitation. These data indicate that the loss of the Tyr 294 site has no effect on ADAP⅐SKAP-55 complex formation in T-cells. The interaction must therefore be considered as a secondary interaction to SKAP-55 SH3 domain binding to ADAP (21,22).
Mutation of Tyr 294 Disrupts SKAP-55 Regulation of LFA-1-mediated Adhesion-We have shown previously that ADAP and SKAP-55 cooperate to provide the inside-out signals that induce LFA-1-mediated T cell adhesion to ICAM-1 (28 -30). Despite it playing a secondary role in complex formation, the ADAP-SH3c domain interaction with SKAP-55 could still influence the conformation and functionality of the ADAP⅐SKAP-55 complex. We therefore tested the effects of mutations at Tyr 294 and Tyr 294 /Tyr 297 (SKAP-55-Y294F and SKAP-55-Y294F/ Y297F, respectively) on TcR induction of adhesion and IL-2 transcription. Adhesion to ICAM-1 of cells expressing native or mutant SKAP-55 was assessed by binding of anti-CD3-stimulated Jurkat T cells to recombinant ICAM-1-coated plates as described previously (28 -30). As shown in Fig. 5, stimulation through CD3 of control vector-transfected cells increased adhesion 2-fold, and this could be increased a further 50% by introduction of exogenous full-length SKAP-55. Transfection with either SKAP-55-Y294F or SKAP-55-Y294F/Y297F abrogated completely the adhesion response, implying that the mutant SKAP-55 has a dominant negative effect in blocking the function of endogenous SKAP-55. The lower panels in Fig. 5 show representative light microscopy images of each condition, indicating the effects of each SKAP-55 variant and confirming that the SKAP-55 Tyr 294 residue is needed for TcR up-regulation of LFA-1 adhesion.
As shown in Fig. 6, transfection of wild-type SKAP-55 resulted in a 3-fold increase in IL-2 promoter activity over non-transfected control.
In contrast, expression of the SKAP-55-Y294F mutant blocked up-regulation of IL-2 promoter activity, as did expression of the SKAP-55-Y294F/Y297F double mutant. Overall, these data underscore the importance of the ADAP-SH3c binding motif in the TcR up-regulation of LFA-1 adhesion and IL-2 promoter activity.

DISCUSSION
The present study builds on our initial observation that the SH3c domain of ADAP (as well as Fyn and Lck SH3 domains) can bind to a novel motif in SKAP-55 by showing that phosphorylation of a core Tyr 294 in the motif disrupts binding, and that, although the interaction is not required for ADAP⅐SKAP-55 complex formation, loss of the key tyrosine abrogates TcR-driven LFA-1 adhesion and IL-2 gene transcription. The interaction must therefore be secondary to the interaction to SKAP-55 SH3 domain binding to ADAP (21,22), but is nevertheless important for functionality of the ADAP⅐SKAP-55 complex.
SH3 domains can recognize classic proline-based øPXøP motifs as well as non-canonical motifs (35). Atypical motifs include PX(V/I)(D/ N)RXXKP binding to the STAM2 and GADS SH3 domains (8 -13) and Eps8 SH3 domain binding to the PXXDY motif (15). The RKXXYXXY For all curves, the end of injection resulted in a large spike between the association and dissociation phases. For presentation purposes, the spike was removed and is represented by the linear region present in all curves. This region was not used for kinetic calculations of association and dissociation as presented in Table 1. F, equilibrium resonance units (R.Eq) obtained by BIAevaluation software for the control and Tyr 297 -phosphorylated peptide. G, equilibrium resonance unit values from panel F were used to calculate dissociation constants by Scatchard analysis for peptides binding to ADAP-SH3c (Table 1).
motif of ADAP-SH3c domain binding to SKAP-55 is unique in that it possesses bipartide tyrosine residues that could serve as sites for phosphorylation (16). This possibility was underscored by p59 fyn binding to ADAP and its phosphorylation of SKAP-55 (19,33). In agreement with this model, we showed that p59 fyn phosphorylation of RKXXY 294 XXY 297 blocked the binding to the ADAP SH3c domain (Fig. 1). Furthermore, plasmon resonance using peptides phosphorylated at Tyr 294 , Tyr 297 , or Tyr 294 /297 confirmed this finding and identified Tyr 294 as the major residue involved in ADAP SH3c domain binding (Fig. 2). Phosphorylation of this site completely prevented SH3c domain binding, a finding in keeping with the physical proximity of the Tyr 294 residue to the Arg 290 / Lys 291 basic residues we had previously shown to be essential for binding (16). By contrast, phosphorylation of residue Tyr 297 or the adjacent Tyr 298 residue had only a nominal effect on binding. Our findings therefore identify a role for protein-tyrosine phosphorylation in the negative regulation of an SH3 domain binding to ligand. Another example involves platelet-derived growth factor-stimulated phosphorylation of c-Src, where phosphorylation of Tyr 138 on the SH3 peptide-binding surface diminished ligand-binding ability (36). These cases may serve as prototypes for phosphoregulation of other SH3 domain interactions, including Eps8 SH3 domain binding to the PXXDY motif (15).
Other examples of non-canonical interactions include STAM2 and GADS SH3 domain binding to a PX(V/I)(D/N)RXXKP motif in ubiquitin isopeptidase Y/Usp8 and SLP-76, respectively (8 -13). In the latter case, the interaction has a 10 -20 times higher affinity (K D ϭ 0.24 M) than SH3 domain binding to øPXøP motifs. Another example is HPK1 SH3 domain binding to RXXK and a cluster of N-terminal prolines (12). In each of these cases, charged interactions between RXXK and the negatively charged surface of the SH3 domain play a central role in binding. In the case of the RKXXY 294 XXY 297 motif, we previously documented the key role played by electrostatic interactions between the RK residues and the negatively charged surface of the FYN SH3 domain (16). The binding site overlapped with that of the classic øPXøP motif and induced a chemical shift perturbation of the negatively charged FIGURE 3. Effect of free phosphate on the interaction between SKAP55 SK4 domain non-phosphorylated peptide and ADAP SH3 domain. A, binding of control peptide (TRRKGDYASYYQG) in HEPES-saline to immobilized ADAP-SH3c detected by surface plasmon resonance. B, binding of control peptide in phosphate-buffered saline to the same regenerated ADAP-SH3c surface detected by surface plasmon resonance. Regeneration (100 mM glycine, pH 2.8) had no effect in subsequent cycles on the capacity of immobilized ADAP-SH3c to bind control peptide in HEPES-saline. The dominant effect of Tyr 294 , but not Tyr 297 phosphorylation on binding supports the notion that the bipartide YXXY residues do not operate in the same manner as a bipartide PXXP motif. The tyrosinebased motif is unlikely to form a pseudosymmetric polyproline class II helix that has been observed with proline-based motifs (7). Furthermore, the ADAP SH3c domain is unusual in that it lacks the key residue Trp 119 that has been substituted by a lysine or tyrosine (16,19). Furthermore, the RKXXY 294 XXY 297 motif failed to induce a shift perturbation for the NH groups of Trp 119 and Tyr 137 in the FYN SH3 domain indicating that these residues are unlikely to contribute to the binding of the tyrosine-based motif (16).
Our findings also indicate that the Tyr 294 site is not needed for complex formation between ADAP and SKAP-55 (Fig. 4). This is consistent with previous observations by ourselves and others that the major interaction between ADAP and SKAP-55 relies on SKAP-55 SH3 domain binding to proline residues in ADAP (20,21). In this way, the ADAP-SH3c domain interaction is likely to act in a supplemental fashion to reinforce or alter the orientation of components within the ADAP⅐SKAP-55 complex. The slight increase in tyrosine phosphorylation of SKAP-55 in response to anti-CD3 is consistent with previous data showing that both ADAP and SKAP-55 undergo weak but extended levels of phosphorylation (19,22,32). Nevertheless, the loss of Tyr 294 had a major inhibitory effect on TcR-driven LFA-1 clustering and IL-2 promoter activity (Figs. 5 and 6). Both mutation and phosphorylation resulted in a loss of SH3c domain binding. The effect of the mutation 4 was observed with both reported isoforms of human SKAP-55 (20,33). The first, represented by gb:BC047870 and used here, has the motif RKXXY 294 XXY 297 , whereas the second variant represented by gb:NP_003717 has an additional residue generating the motif RKXXXY 295 XXY 298 . The ultimate basis for negative regulation of ADAP-SH3c domain binding and inhibition of adhesion and IL-2 transcription is unclear but may involve other as yet discovered receptors that target the Tyr 294 site in the down-regulation of the immune response. This would be consistent both with the relatively weak phosphorylation of the site by anti-CD3 ligation (Fig. 4) 4 as well as with our previous demonstration that peptides with the RKXXY 295 XXY 298 motif, which interfered with complex formation, also inhibited IL-2 production (16).
Initial attempts to purify the complex have been complicated by protein solubility issues. The only ADAP-SH3c domain purified for structural analysis to date failed to bind to any protein or peptide, raising the issues of correct folding or appropriateness of the conditions for measuring peptide binding (34). The absence of binding was surprising given our previous work showing binding by a combination of in vivo co-expression, protein-protein blotting, peptide precipitation, alanine-scanning mutagenesis, and surface plasmon resonance (16). Instead, non-selective binding to an array of polyvalent phosphoinositides was reported (37). Using far-Western blotting, we confirm ADAP-SH3c domain binding to RKXXY 294 XXY 297 motif (Fig. 1). The selective effect of phospho-Tyr 294 by SPR analysis underscored the specificity of the interaction (Fig. 2). To address the reported discrepancy, we noticed that the buffer conditions used in the studies by Heuer and coworkers (37) contained 20 mM free inorganic phosphate. Given the moderate affinity of the ADAP SH3c-SKAP-55 peptide interaction when isolated in vitro, high conductivity buffers incorporating phosphate may well mask interactions that can be detected by NMR with greater sensitivity in low conductivity buffers (38). Phosphate in NMR buffer can interfere with SH2/ SH3 domain-ligand interactions. 5 Consistent with this, SPR analysis using the NMR buffer containing 20 mM phosphate as reported failed to reveal significant ADAP-SH3c domain-RKXXY 294 XXY 297 binding (Fig.  3). From this, we conclude that the presence of inorganic phosphate interferes with ADAP-SH3c binding. Although the intracellular concentration of phosphate can rise to almost ϳ100 mM in resting cells, the majority is complexed to cellular intermediates and only a smaller fraction is available as free anion, predominantly in the form of inorganic pyrophosphate with a concentration in mammalian cells of ϳ1.5 mM, significantly less than the 20 mM representation in the NMR buffer (39). Whether FYN SH3 domain binding to the motif is also partially affected by the inorganic phosphate remains to be determined.
Increasing evidence points to the importance of multiple modular interactions in the formation of protein-protein aggregates in the generation of signals that regulate cell function (17,18). Examples include tandem SH2 domain binding for ZAP-70 and phosphatases such as SHP-2. Preliminary studies using gel filtration has indicated that the ADAP complex is greater than 10 6 Da (unpublished data). Biphasic high affinity and moderate affinity modes for ADAP⅐SKAP-55 binding could act to reinforce complex formation or alter the orientation of components within the ADAP⅐SKAP-55 complex. This may be of particular relevance given the importance of ADAP in stabilizing SKAP-55 expression in cells. In the absence of ADAP, the intracellular t1 ⁄ 2 of SKAP-55 is only 18 min, whereas complexing with ADAP enhances the t1 ⁄ 2 to 90 min (23). These differences in the rate of SKAP-55 degradation could have profound effects on the requirement for SKAP-55 in the transmission of inside-out signals needed for integrin-mediated T cell adhesion and up-regulation of IL-2 promoter activity (28 -30).