Modulation of Lck Function through Multisite Docking to T Cell-specific Adapter Protein*

T cell-specific adapter protein (TSAd), encoded by the SH2D2A gene, interacts with Lck through its C terminus and thus modulates Lck activity. Here we mapped Lck phosphorylation and interaction sites on TSAd and evaluated their functional importance. The three C-terminal TSAd tyrosines Tyr280, Tyr290, and Tyr305 were phosphorylated by Lck and functioned as docking sites for the Lck Src homology 2 (SH2) domain. Binding affinities of the TSAd Tyr(P)280 and Tyr(P)290 phosphopeptides to the isolated Lck SH2 domain were similar to that observed for the Lck Tyr(P)505 phosphopeptide, whereas the TSAd Tyr(P)305 peptide displayed a 10-fold higher affinity. The proline-rich Lck SH3-binding site on TSAd as well as the Lck SH2 domain were required for efficient tyrosine phosphorylation of TSAd by Lck. Interaction sites on TSAd for both Lck SH2 and Lck SH3 were necessary for TSAd-mediated modulation of proximal TCR signaling events. We found that 20–30% of TSAd molecules are phosphorylated in activated T cells and that the proportion of TSAd to Lck molecules in such cells is ∼1:1. Therefore, in activated T cells, a considerable number of Lck molecules may potentially be engaged by TSAd. In conclusion, Lck binds to TSAd prolines and phosphorylates and interacts with the three C-terminal TSAd tyrosines. We propose that through multivalent interactions with Lck, TSAd diverts Lck from phosphorylating other substrates, thus modulating its functional activity through substrate competition.

T cell-specific adapter protein (TSAd), encoded by the SH2D2A gene, interacts with Lck through its C terminus and thus modulates Lck activity. Here we mapped Lck phosphorylation and interaction sites on TSAd and evaluated their functional importance. The three C-terminal TSAd tyrosines Tyr 280 , Tyr 290 , and Tyr 305 were phosphorylated by Lck and functioned as docking sites for the Lck Src homology 2 (SH2) domain. Binding affinities of the TSAd Tyr(P) 280 and Tyr(P) 290 phosphopeptides to the isolated Lck SH2 domain were similar to that observed for the Lck Tyr(P) 505 phosphopeptide, whereas the TSAd Tyr(P) 305 peptide displayed a 10-fold higher affinity. The proline-rich Lck SH3-binding site on TSAd as well as the Lck SH2 domain were required for efficient tyrosine phosphorylation of TSAd by Lck. Interaction sites on TSAd for both Lck SH2 and Lck SH3 were necessary for TSAd-mediated modulation of proximal TCR signaling events. We found that 20 -30% of TSAd molecules are phosphorylated in activated T cells and that the proportion of TSAd to Lck molecules in such cells is ϳ1:1. Therefore, in activated T cells, a considerable number of Lck molecules may potentially be engaged by TSAd. In conclusion, Lck binds to TSAd prolines and phosphorylates and interacts with the three C-terminal TSAd tyrosines. We propose that through multivalent interactions with Lck, TSAd diverts Lck from phosphorylating other substrates, thus modulating its functional activity through substrate competition.
Activation of T cells through T cell receptor (TCR) 2 triggering is tightly controlled by transient changes in protein conformation, phosphorylation status, and catalytic activities of signaling molecules. These events may either be promoted or inhibited by adapter proteins, which lack catalytic activity, but serve as molecular bridges mediating protein-protein or protein-lipid interactions (1). Adapter proteins contain multiple protein interaction motifs and domains that allow these molecules to interact with several proteins simultaneously. They are therefore often referred to as scaffolding proteins. The picture is, however, becoming increasingly complex, since some signaling molecules that possess enzymatic activity also fulfill adapter or scaffolding characteristics.
The majority of intracellular molecules known to be involved in the early stages of T cell activation are already present in the T cell prior to T cell triggering; TCR stimulation results in Lck (lymphoid cell kinase)-mediated phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 chains, which form the TCR-CD3 complex together with the TCR ␣ and ␤ chains. Phosphorylated ITAMs are docking sites for the tandem SH2 domains of the Zap-70 (-associated protein of 70 kDa) kinase (2,3). ITAM-bound Zap-70 becomes catalytically active when phosphorylated by Lck on residue Tyr 494 in its catalytic domain (4), as well as on the residue Tyr 319 in the interdomain (5)(6)(7). Activated Zap-70 phosphorylates the adapter proteins SLP-76 (leukocyte-specific protein of 76 kDa) and LAT (linker of activated T cells) (8 -10), which serve as scaffolding proteins for other molecules crucial for signaling downstream of the TCR (11).
Lck is regulated by phosphorylation of its Tyr 394 and Tyr 505 tyrosines (26). Csk-mediated phosphorylation of Lck Tyr 505 results in an intramolecular interaction between phosphorylated Tyr 505 and its SH2 domain (27)(28)(29), whereby Lck is kept in an inactive conformation due to blockade of its substrate binding site. Moreover, the SH3 domain of Lck interacts with the linker between the SH2 and kinase domain, which further blocks the kinase activity (30,31). Dephosphorylation of Tyr(P) 505 by the CD45 phosphatase (32) and Lck autophosphorylation of Tyr 394 activates Lck (33)(34)(35). The intramolecular interactions controlling Lck activity are of low affinity (36), allowing specific ligands with stronger affinity for the Lck SH2 or SH3 domains to compete out the intramolecular interactions and thus activate Lck (37).
Here we dissected the TSAd-Lck interaction by mutational analysis in transfected cell lines as well as by chemical analysis using isothermal calorimetry (ITC) and in vitro kinase assays. We show that Lck phosphorylates and interacts with all three C-terminal tyrosines of TSAd. There is a hierarchy of affinities for the phosphorylated TSAd peptides to the Lck SH2 domain, where the lowest affinity observed was in the same range as the Lck-Tyr 505 peptide, and the highest was 10-fold higher. The tyrosine phosphorylation level of TSAd in vivo is dependent on the Lck SH3 interaction site in TSAd (aa 239 -256) as well as a functional Lck SH2 domain. Moreover, the TSAd-mediated modulation of Lck activity is dependent on both the Lck SH2 and SH3 ligands on TSAd. Our data suggest that multisite docking of Lck to TSAd leads to efficient phosphorylation of TSAd and concomitant modulation of Lck activity toward other substrates.
Expression and Purification of GST Fusion Proteins-GST fusion proteins of TSAd-aa 236 -312 as well as the Lck SH2 domain and the Lck SH3 domain were produced in BL21 Codon plus bacteria (Stratagene), and purified on glutathione-Sepharose beads (GE Healthcare) according to the instructions of the manufacturer. For calorimetric analysis of peptide binding to the Lck SH2 domain, isolated Lck SH2 domain was also excised from the glutathione-Sepharose beads using thrombin.
Cell Cultures and Transfections-293T cells and Jurkat E6.1 (American Type Culture Collection) or TAg cells (38) were cultured in RPMI 1640, 5-10% fetal calf serum supplemented with 1 mM sodium pyruvate, 1 mM nonessential amino acids (all from GIBCOBRL, Invitrogen, The Netherlands), and antibiotics. Transfections of 5-20 ϫ 10 6 Jurkat T cells in RPMI 1640 with 5% fetal calf serum with 5-30 g of plasmid DNA were performed using a BTX electroporator (Genetronix, San Diego, CA) at 200 V and 70 ms or the Amaxa nucleofector with the cell line Nucleofector TM kit (catalog number VCA-1003), using either program S18 or I-10. Transient transfectants were cultured for 16 -48 h. 293T cells (2 ϫ 10 6 ) were washed with Opti-MEM I medium (GIBCOBRL) before transfection with a mixture of 0.5-3 g of DNA and 25 l of Lipofectin in Opti-MEM I medium. Transfections were terminated after 5-8 h by the addition of 1:1 ml of RPMI 1640 with 20% fetal calf serum. The cells were further propagated for 16 -24 h. Human peripheral blood CD4 ϩ T cells were positively or negatively selected from healthy blood donors as previously described (15) and cultured in either RPMI 1640 or hTC culture medium (Amaxa Biosystems, Cologne, Germany) with 5-10% fetal calf serum.
Cell Stimulation, Lysis, Immunoprecipitation, and Western Blot-TSAd protein expression was induced in Jurkat T cells and in peripheral CD4 ϩ T cells by stimulation for various times with OKT3 (5 g/ml)-coated wells or in peripheral CD4 ϩ T cells by stimulation with anti-CD3/CD28 Dynabeads (1 bead/ cell). Jurkat cells were washed with phosphate-buffered saline, resuspended, and stimulated with 5-10 g/ml anti-CD3 (OKT3) or 1:200 acites anti-TCR (C305) monoclonal antibodies for specific times. Cells were lysed by the addition of an equal volume of 2ϫ lysis buffer (1ϫ lysis buffer: 20 mM Tris (pH 7.5), 100 mM NaCl, 50 mM NaF, 1 mM Na 3 VO 4 , 0.25-1% Igepal, 12.5-50 mM n-octyl-␤-D-glucoside, and a 10 g/ml concentration of the protease inhibitors leupeptin, pepstatin A, chymostatin, and antipain (all from Sigma). 293T cells were washed with phosphate-buffered saline and lysed by the addition of 1ϫ lysis buffer. Lysates were precleared 2-3 times for 45 min with protein A/G-Sepharose TM (Amersham Biosciences) or Dynabeads protein G (Invitrogen), followed by incubation for 1 h with anti-TSAd or normal rabbit serum followed by a 1-h incubation with protein A/G-Sepharose TM or for 1 h with protein G Dynabeads precoated with anti-Tyr(P) antibodies as described by the manufacturer. Pull-down experiments with GST-Lck SH2 or GST-Lck SH3 were performed in precleared lysates as described (18). After immunoprecipitation or pulldown, beads were washed three times in 1ϫ lysis buffer, and isolated proteins were separated by 7.5-12.5% SDS-PAGE. Gels were blotted onto a polyvinylidine difluoride membrane (Bio-Rad) or directly stained in Coomassie Blue solution. Blots were probed with the indicated antibodies in Tris-buffered saline (pH 7.4) with 0.1% Tween (Sigma) plus 3% bovine serum albumin (Biotest, Dreieich, Germany) or 3% skimmed milk (Sigma). Bound antibodies were visualized by incubation with secondary horseradish peroxidase-labeled antibodies and Super Signal West Pico stable peroxide solution (Pierce) (18). In some experiments, the intensities of the bands of interest were quantitated by the use of an Eastman Kodak Co. Image Station 2000R.
ITC-ITC was performed using the VP-ITC MicroCal instrument (MicroCal, Northampton, MA) (39). Recombinant Lck SH2 domain was made as described, and the concentration was determined using the A 280 value, with extinction coefficient 9650 M Ϫ1 cm Ϫ1 . The concentration of Lck SH2 domain in the reaction cell typically was 10 mM, and the concentrations of the phosphotyrosine peptides in the ITC syringe were 0.5 mM, all dissolved in a modified MBS buffer (50 mM MOPS, 50 mM NaCl, 2 mM dithiothreitol, pH 6.8) (36). All experiments were performed at 25°C. The initiation delay was set to 60 s, and aliquots of 5 l of peptide solution were injected into the reaction cell at 120-s intervals with a stirring speed of 260 rpm. Typically, 20 -25 injections were used to complete the titration. ITC data were collected automatically using the ITC Origin version 7.0 software (MicroCal) accompanying the VP-ITC system. The data were fitted to a single binding site mechanism using the ITC fitting algorithm included with the instrument after correcting for the heat of dilution. All data from the ITC binding reactions fitted well to a single-site binding model, yielding the stoichiometry (n), equilibrium binding association constant (K a ), and enthalpy change (⌬H) of the reaction. The value of n was found to be between 0.9 and 1.1 for all reactions. The changes in reaction free energy (⌬G) and entropy (⌬S) as well as the dissociation constant (K d ) were calculated using the relation ⌬G ϭ ⌬H Ϫ T⌬S ϭ ϪRTlnK a ϭ RTlnK d . Errors in ⌬H r and K d were obtained as S.D. values of three or more experiments.
Mass Spectrometry-Recombinant TSAd proteins were subjected to in-solution tryptic digestion as indicated under "Results." Therefore, 0.5 g of recombinant proteins were digested by 25 ng of trypsin in 50 mM NH 4 HCO 3 (37°C, 3 h). The kinase buffer was not removed prior to digestion. Approximately 75 ng of digested recombinant protein were desalted by using C18-stop and go extraction tips (C18-stage tips) and analyzed by matrix-assisted laser desorption/ionization-time-offlight mass spectrometry in the positive and reflector mode (Ultraflex II; Bruker Daltonics). The observed masses were compared with an artificial tryptic digest of the recombinant TSAd proteins. Tandem mass spectrometry experiments were performed to confirm the peptide sequences and to verify the phosphorylation sites. To further confirm the identity of the detected phosphopeptides, an aliquot of the tryptic digest (150 ng of recombinant TSAd protein) was treated with alkaline phosphatase (2 units) in 50 mM NH 4 HCO 3 (37°C, 30 min). Half of the sample was desalted and analyzed as described. Since alkaline phosphatase-mediated dephosphorylation results in a mass shift of Ϫ80 Da per phosphorylation site, this treatment proves the presence and number of phosphogroups in the given peptides.
Kinase Assay-In vitro kinase assays were performed using various GST-TSAd constructs diluted in kinase buffer containing 50 mM Tris-HCl, pH 7.5, 0.1 mM EGTA, 75 mM MgCl 2 , 15 mM dithiothreitol, and 1-2 M ATP before recombinant Lck was mixed into the samples. The samples were then immediately put on a heating block prewarmed to 30°C, aliquots were withdrawn at specific time points, and the kinase assays were stopped by adding SDS-PAGE loading buffer and incubation at 100°C for 5 min. Purified, recombinant GST-TSAd constructs were used at an estimated final concentration in the range of 0.175-20 M, whereas concentrations between 7.5 and 200 nM full-length, active Lck have been used per kinase assay reaction.

TSAd Is Tyrosine-phosphorylated by Lck on Several
Tyrosines-TSAd has 10 potential tyrosine phosphorylation sites ( Fig. 1A and supplemental Table 1). We previously reported that TSAd is tyrosine-phosphorylated in stimulated peripheral blood mononuclear cells (12) and Jurkat T cells (17). Co-expression with Lck is required for phosphorylation of TSAd upon tyrosine in 293T cells (17,18) and To determine which of the TSAd tyrosines could be phosphorylated, we made single Tyr to Phe TSAd mutants for all 10 tyrosines and expressed them in 293T cells together with Lck. Immunoprecipitates (IPs) of TSAd from these cells were examined for phosphorylation by immunoblotting using anti-Tyr(P) antibody. Two distinct bands corresponding to phosphorylated TSAd were detected when TSAd IPs were resolved by SDS-PAGE. As shown in Fig. 1C, all 10 TSAd tyrosine to phenylalanine mutants were tyrosine-phosphorylated in the presence of Lck, showing that more than one tyrosine was phosphorylated. A slower migrating band appeared only in the presence of Tyr 290 (Fig. 1C, lane 11). All single Tyr to Phe mutants interacted with Lck SH2 in a GST pull-down assay using the same lysates as in Fig. 1C (Fig. 1D). These data indicate that Lck is able to phosphorylate and interact via its SH2 domain with more than one of the TSAd tyrosines.
Lck Phosphorylates and Interacts with the Highly Conserved Tyr 280 , Tyr 290 , and Tyr 305 of TSAd-Murine TSAd displays 68% sequence identity to human TSAd, with the SH2 domain showing the highest degree of homology (19). However, also in the C terminus, there are highly conserved elements. The region encompassing aa 236 -312 has a proline-rich motif, as well as the three C-terminal tyrosines of TSAd, all of which have essentially the same flanking sequences in humans, chimpanzees, cows, mice, and rats ( Fig. 2A). The only other tyrosine in the TSAd sequence that has equally well conserved flanking sequence is Tyr 117 , which is part of the conserved phosphotyrosine binding pocket of the SH2 domain (supplemental Table  1). This degree of conservation of the aa 236 -312 in TSAd strongly indicates that this region has functional importance. Accordingly, we and others previously showed that TSAd mutated for its four or three C-terminal tyrosines is not phosphorylated by Lck (18,24).
Since single mutations of any of the three C-terminal tyrosines were not sufficient to abolish Lck-mediated TSAd phosphorylation or Lck SH2 domain binding to TSAd, we made double or triple Tyr to Phe mutants of the four C-terminal tyrosines. All double Tyr to Phe mutants were both phosphorylated in the presence of Lck and interacted with Lck SH2 (lanes 9 -11). As shown in Fig. 2B 5-7). Taken together, these data indicate that Lck phospho-rylates and may interact through its SH2 domain with all three C-terminal tyrosines of TSAd.
None of the 10 tyrosines in TSAd (supplemental Table 1) are preferred Lck substrates. Only at a low stringency search using the Scansite algorithm (40) are the Tyr 280 and Tyr 305 identified as potential Lck phosphorylation sites. To confirm that Lck is able to phosphorylate the three C-terminal tyrosines in TSAd, we therefore expressed the conserved C-terminal region (aa 236 -312) of human TSAd (depicted in Fig. 2A) fused to GST (hereafter referred to as TSAd-PY-YYY; Fig. 2C). The intact protein TSAd-PY-YYY includes the Lck SH3 recognition site (aa 239 -256; i.e. P in the abbreviation PY-YYY) (18) and the four C-terminal tyrosines of TSAd (i.e. Y-YYY). In the mutated TSAd-PY-FFF protein, the three C-terminal tyrosines had been mutated to phenylalanine (Fig. 2C). These two recombinant GST-TSAd proteins were subjected to an in vitro kinase assay using a 1:10 molar ratio of recombinant active human Lck to TSAd. Mass spectrometry analysis of tryptic peptide digests of the reaction mixtures revealed that all four TSAd tyrosines were phosphorylated (Fig. 2, D and E, and Table 1).
Although Tyr 260 was phosphorylated by Lck in vitro, Tyr 260 seems to be neither a major Lck tyrosine phosphorylation site in vivo in 293T cells nor an interaction site for the Lck SH2 domain (Fig. 2B). Moreover, the sequence flanking the Tyr 260 is less well conserved in evolution than the three C-terminal tyrosines ( Fig. 2A and supplemental Table 1). Therefore, we chose to focus primarily on elucidating the interaction of Lck with the three C-terminal tyrosines.
A Hierarchy of Binding Affinities for TSAd Phosphopeptides to the Lck SH2 Domain-Interactions of SH2 domains with their phosphorylated tyrosine ligands are generally weak (36). The specificity and affinity of a particular SH2 domain for its ligands are known to be dependent on the amino acids immediately C-terminal to the tyrosine (41). TSAd Tyr 305 and TSAd Tyr 280 were identified as putative Lck SH2 domain ligands through high or medium stringency motif scanning, respectively, using Scansite (40). In contrast, TSAd Tyr 290 was not predicted to be an Lck SH2 ligand, even at low stringency search. To further probe the significance of the results obtained by the Lck SH2 pull-down analysis (Fig. 2B), we determined the binding affinities of the three C-terminal TSAd tyrosines to the Lck SH2 domain. Hexameric polypeptides representing each of the three phosphorylated TSAd tyrosines were synthesized, and their binding affinities for isolated Lck SH2 domain were assessed by ITC. In accordance with previous studies (36), the high affinity ligand HMT Tyr(P) 324 peptide displayed a 100-fold higher affinity than the Lck Tyr(P) 505 peptide for the isolated Lck SH2 domain (Table 2), providing a quality control for our assay. The TSAd phosphopeptides Tyr(P) 280 and Tyr(P) 290 had affinities for Lck SH2 in the same range as that observed for Lck Tyr(P) 505 , whereas the TSAd phosphopeptide Tyr(P) 305 bound to Lck SH2 with ϳ10-fold higher affinity (Table 2). This result indicates that all three TSAd phosphopeptides, and in particular the TSAd Tyr(P) 305 peptide, might be able to compete out the Lck Tyr(P) 505 intramolecular binding to the Lck SH2 domain and thus unlock the inhibitory conformation of Lck.   TSAd Association with Lck Requires Only Lck SH2 or SH3 Interaction Sites on TSAd-We have previously shown that TSAd interacts with Lck in primary T cells as well as in Jurkat T cells transfected with TSAd (17,18). In order to assess whether TSAd interacts with Lck also in the absence of either the Lck SH2 or Lck SH3 interaction sites, we performed co-immunoprecipitation experiments in Jurkat cells transfected with intact TSAd or TSAd Y280F/Y290F/Y305F or TSAd ⌬239 -256. In all cases, Lck co-precipitated with TSAd; however, the amount of associated Lck was clearly reduced in the absence of either the  SH2 or the SH3 interaction sites (Fig. 3A). This result shows that interaction of TSAd with Lck through either the Lck SH3 or SH2 domain is sufficient for stable association of TSAd with Lck and that both interactions occur in vivo. TSAd Is Phosphorylated by Lck in a Processive Manner-Multisite phosphorylation by a single kinase may occur in a distributive or processive manner. Distributive phosphorylation implies that phosphorylation of substrates with multiple sites requires multiple hits between the kinase and its substrate. By contrast, processive phosphorylation involves only one hit between the kinase and the substrate for phosphorylation of multiple sites (42). Nonreceptor tyrosine kinases typically contain both SH2 and SH3 domains. Binding of the kinase to the substrate via the kinase SH3 domain may allow for processive phosphorylation of the substrate (43,44). Moreover, SH2 domains of nonreceptor tyrosine kinases typically have specificity for tyrosine motifs phosphorylated by the same kinase, and once phosphorylated these sites may contribute to phosphorylation of the substrate through binding to the SH2 domain of the kinase (44,45).
Since TSAd includes multiple SH2 interaction sites as well as an SH3 interaction site for Lck, it is possible that Lck phosphorylates TSAd through a processive mechanism. We tested this hypothesis by monitoring phosphorylation of different mutated and truncated versions of the TSAd-PY-YYY constructs (Fig. 2C) in our in vitro Lck kinase assay. Phosphotyrosine levels of recombinant TSAd proteins were assessed by SDS-PAGE followed by immunoblotting with anti-Tyr(P) and anti-GST antibodies.
For substrates phosphorylated by a distributive mode, the level of phosphorylation will be dependent on the kinase concentration in the reaction mixture. For substrates that are phosphorylated by a processive mode, the concentration of the kinase is less important once the kinase has bound to the substrate. Substrates that are phosphorylated through a processive mechanism should therefore be less sensitive to changes in substrate and kinase concentrations after the initial encounter between the kinase and the substrate. In agreement with this, 2-fold dilutions of Lck and TSAd-PY-YYY after initiation of the kinase assay did not affect the level of TSAd phosphorylation (Fig.  3B). In contrast, the TSAd-dY-YYY protein lacking the Lck SH3 interaction site (aa 239 -256), was less tyrosine-phosphorylated at low Lck concentrations, indicating a distributive mode of TSAd phosphorylation in the absence of its Lck SH3 ligand (Fig. 3B).
Since processive phosphorylation of multiple tyrosines in a substrate only requires one hit between the substrate and the kinase, the kinetics of phosphorylation should be more rapid than for distributive phosphorylation of multiple sites, which requires repeated hits between the substrate and the kinase (42). We therefore monitored the level of tyrosine-phosphorylated GST-TSAd protein in the in vitro Lck kinase assay over time. As seen in Fig. 3C, phosphorylation of GST-TSAd-PY-   YYY could be detected at an earlier time point than the GST-TSAd-dY-YYY, where the phosphorylation was clearly delayed. This suggests that interaction of the Lck SH3 domain with TSAd contributes to phosphorylation efficiency and thereby gives further support to the notion that Lck phosphorylates TSAd in a processive manner.
Because all three C-terminal tyrosines of TSAd may bind to Lck SH2, interaction of Lck SH2 to any of the TSAd phosphotyrosines could contribute to the processive phosphorylation of the other tyrosines. In order to address whether the Lck SH2 domain contributes to processive phosphorylation of TSAd, we took advantage of our observation that TSAd Tyr 260 is phosphorylated by Lck in vitro (Table 1 and Fig. 2E) but does not interact with Lck SH2 (Fig. 2B). Compared with the GST-TSAd-PY-YYY, GST-TSAd-PY-FFF displayed a clearly delayed kinetics (Fig. 3D, compare lanes 1 and 2).
If Lck SH2 domain docking onto one of the TSAd phosphotyrosines promotes phosphorylation of the other tyrosines, recombinant TSAd proteins carrying Tyr 260 combined with any of the three C-terminal tyrosines should display a more rapid rate of tyrosine phosphorylation than GST-TSAd-PY-FFF carrying Tyr 260 alone. In agreement with this notion, we found that the recombinant protein including both Tyr 260 and either Tyr 280 or Tyr 305 displayed clearly a higher level of tyrosine phosphorylation than the protein including Tyr 260 only (Fig. 3D, compare lanes 2, 3, and 5). By contrast, inclusion of Tyr 290 showed only a weak increase in the overall rate of phosphorylation compared with the GST-TSAd Tyr 260 construct (Fig. 3D, compare lanes 2 and 4). Since Tyr(P) 280 and Tyr(P) 290 have essentially the same affinity for Lck SH2 (Table 2), the difference in phosphorylation efficiency conferred by the two tyrosines could be due to differences in Lck kinase preference, since residues Tyr 280 and Tyr 305 but not Tyr 290 were preferred Lck substrates, as predicted by Scansite (40) (Fig. 3D). This notion was supported by the observation that the phosphotyrosine peptide representing TSAd Tyr(P) 305 promoted accelerated activation of Lck, as indicated by the level of Tyr 394 phosphorylation after a 1-min co-incubation of Lck with the phosphopeptide in kinase buffer (Fig. 3E). By contrast, the addition of either the TSAd Tyr(P) 280 or the TSAd Tyr(P) 290 peptide to the kinase buffer did not significantly alter the kinetics of Lck Tyr 394 autophosphorylation, showing that TSAd Tyr(P) 280 is not more potent than TSAd Tyr(P) 290 in promoting Lck tyrosine phosphorylation.
Taken together, the in vitro kinase experiments indicate that interaction with the Lck SH3 domain contributes to processive phosphorylation of TSAd. However, to what extent the Lck SH2 domain also contributes to processive phosphorylation of TSAd could not be determined. We therefore asked whether interaction of Lck with TSAd through the Lck SH2 and/or the Lck SH3 domains determines the extent of TSAd tyrosine phosphorylation also in vivo.
Maximal Tyrosine Phosphorylation of TSAd Is Dependent on both Lck SH2 and SH3 Domain Interactions-TSAd or TSAd lacking the Lck SH3 interaction site (aa 239 -256) was expressed in 293T cells in the presence of Lck or Lck mutated at arginine 154 (R154K), which disrupts the normal binding function of the Lck SH2 domain (46). TSAd IPs from these cells were assessed for level of tyrosine phosphorylation by immunoblotting (Fig. 4A) and quantitation of chemiluminesence signals (Fig. 4B). When co-expressed together with Lck, TSAd was found to be highly tyrosine-phosphorylated (here referred to as 100% tyrosine phosphorylation). Disruption of the Lck SH3 interaction site on TSAd resulted in a 40% reduction of TSAd tyrosine phosphorylation. A 60% reduction in tyrosine-phosphorylated TSAd was evident when intact TSAd was expressed in the presence of Lck lacking a functional SH2 domain (LckR154K). TSAd molecules lacking the Lck SH3 ligand coexpressed with Lck lacking a functional SH2 domain displayed less than 10% of the TSAd tyrosine phosphorylation level observed when co-expressing full-length TSAd and Lck. This result shows that interaction of TSAd with both the Lck SH3 and SH2 domains is necessary for maximal tyrosine phosphorylation of TSAd and indirectly indicates that the Lck SH2 domain also contributes to processive phosphorylation of TSAd.
TSAd-dependent Modulation of Lck Activity Is Dependent on Intact SH2 and SH3 Ligands on TSAd-We have previously shown that TSAd modulates multiple proximal signaling events in T cells (14). Upon TCR stimulation, the Src kinase family members Lck and Fyn are the major protein-tyrosine kinases to become activated and initiate intracellular signaling events (47). Reconstitution studies of the Lck-deficient T cell line (JCaM1) with either Lck or Fyn reveal that only Lck fully restores tyrosine phosphorylation and activation of Zap-70 (48). Moreover, the Zap-70 Tyr 319 is phosphorylated by Lck upon TCR triggering (5)(6)(7). When Lck activity is abolished by siRNA knock down (49) or by the Src family kinase inhibitor PP2 (Fig. 5A) and the TSAd Y280F/Y290F/Y305F mutants displayed a level of Zap-70 Tyr(P) 319 similar to that observed in control cells transfected with empty vector () (Fig. 5B). This effect was not due to altered phosphorylation kinetics, since expression of intact TSAd reduced the level of phosphorylation of Zap-70 Tyr 319 after 1, 2.5, and 5 min of stimulation (Fig. 5B). We previously reported that phosphorylation of both Lck Tyr 394 and Tyr 505 is increased in Jurkat T cells stably expressing TSAd (17). This is in accordance with Marti et al. (24), who showed that intact TSAd promotes phosphorylation of Lck Tyr 394 in primary mouse CD4 ϩ T cells. Here we found that the level of Lck Tyr(P) 394 in Jurkat T cells transiently transfected with intact or mutated TSAd was similar to that observed in Jurkat T cells transfected with empty vector (Fig. 5C). In sum, these results indicate that attenuation of Zap-70 Tyr(P) 319 levels in T cells expressing intact TSAd cannot be explained by altered activation status of Lck as evidenced by the level of Tyr(P) 394 autophosphorylation. Our data thus suggest that tyrosine phosphorylation of TSAd is a prerequisite for modulation of Lck. TSAd is phosphorylated both in resting and activated Jurkat T cells transiently expressing TSAd (17,18) and in phytohemagglutinin-stimulated peripheral blood mononuclear cells (12).
To explore to what extent TSAd is tyrosine-phosphorylated under physiological conditions, we immunoprecipitated TSAd from human CD4 ϩ T cells stimulated for 72 h with anti-CD3/ CD28-coated Dynabeads to induce TSAd expression (14). In these activated CD4 ϩ T cells, TSAd was already tyrosine-phosphorylated, and restimulation of the cells resulted in a minor increase in phosphotyrosine level (Fig. 5D). To estimate the proportion of tyrosine-phosphorylated TSAd molecules in activated T cells, we then induced endogenous TSAd expression in Jurkat T cells by anti-CD3 stimulation for 16 h and performed immunodepletion of tyrosine-phosphorylated proteins using an anti-phosphotyrosine antibody.
Immunoblotting of lysates from resting or CD3-stimulated cells before and after phosphotyrosine immunodepletion revealed that tyrosine-phosphorylated TSAd constitutes ϳ20% of the total amount of TSAd expressed in CD3-stimulated Jurkat T cells (Fig. 5E). In cells transiently expressing TSAd, the tyrosine-phosphorylated pool of TSAd was somewhat higher (30%), whereas the fraction of tyrosine-phosphorylated TSAd mutated for the three C-terminal tyrosines or the proline-rich region 239 -256 was virtually zero (Fig. 5F, i and ii). Fig. 5F, iii, shows that there was no gross difference in overall level of protein-tyrosine phosphorylation in the lysates from the transfected cells before and after phosphotyrosine immunodepletion, which could not explain this result. Thus, also in T cells, both the Lck SH3 and Lck SH2 ligands are necessary for TSAd to be strongly tyrosine-phosphorylated. Taken together, these results indicate that 20 -30% of TSAd is phosphorylated in vivo.
In order to put our results into a physiological context, we enumerated TSAd and Lck molecules in activated CD4 ϩ T cells. To this end, we first determined that the polyclonal anti-TSAd antibody 1715T (15) contains two major epitopes, one of which accounted for 30% of the anti-TSAd reactivity and was contained in the GST-TSAd-PY-YYY protein (supplemental Fig. 1). As a result, the signal from intact TSAd in cell lysates was 10:3 (i.e. 3.3)-fold stronger per molecule than the signal obtained from the recombinant TSAd-PY-YYY protein. By comparing the immunoblot signals from known amounts of recombinant Lck and GST-TSAd-PY-YYY proteins with the immunoblot signals of lysates from a defined number of cells, we found that peripheral CD4 ϩ T cells activated with anti-CD3 antibodies for 24 h express 20,000 -50,000 TSAd and 50,000 Lck molecules per cell. In comparison, anti-CD3-stimulated Jurkat T cells express in the order of 200,000 Lck and 200,000 TSAd molecules per cell (Fig. 5G). Hence, in activated T cells expressing TSAd, the relative amount of TSAd to Lck is in the order of 1:1. Taken together, our data suggest that a considerable number of Lck molecules may at any given time be engaged by TSAd in activated T cells.
We therefore propose a model whereby TSAd, through multivalent interactions with Lck, diverts Lck kinase activity away from other substrates (Fig. 6A). When bound to TSAd, Lck may eventually phosphorylate other proteins that are brought into the vicinity of the TSAd-Lck complex (Fig. 6B), as we recently have demonstrated for Itk. 3

DISCUSSION
The main conclusion from this study is that TSAd phosphorylation is dependent on its interaction with both Lck SH3 and SH2 domains and that it proceeds in a processive rather than a distributive manner. We propose that through multisite interactions with Lck, TSAd modulates Lck activity by sequestering Lck from some of its substrates (i.e. Zap-70).
Displacement of the intramolecular binding of Lck SH3 to prolines in the linker between the SH2 and the kinase domains is known to partially activate Src family kinases (SFKs) (31). TSAd molecules lacking the Lck SH3 ligand (aa 239 -256) have reduced levels of tyrosine phosphorylation compared with intact TSAd both in vitro and in vivo. Thus, the initial event during TSAd phosphorylation is probably the interaction between Lck SH3 and the proline-rich region of TSAd.
Our model for how TSAd interacts with and becomes phosphorylated by Lck may explain how TSAd can mediate modulation of proximal tyrosine phosphorylation events upon TCR triggering. The multivalent interaction between Lck and TSAd, where TSAd remains in complex with Lck through multiple rounds of phosphorylation, sequesters Lck away from other substrates. Recently, a crucial role for TSAd in activating Lck was reported (24). Marti et al. (24) showed that both the Lck SH3 interaction site and the three C-terminal tyrosines of murine TSAd were required for TSAd to activate Lck in an in vitro kinase assay. Our data are in agreement with theirs; however, our interpretations of the data differ. It is well established that SH3 or SH2 ligands of SFK increase the activity of the kinase (31,37). Thus, when recombinant TSAd molecules are added to Lck in vitro, the net result may be increased activity of  Lck (24). Also, if TSAd is a general and crucial activator of Lck, we would expect that transient expression of TSAd in Jurkat T cell lines would lead to increased amounts of tyrosine-phosphorylated proteins similar to what is observed for instance in Jurkat cells expressing hyperactive Lck mutated for the regulatory Tyr 505 . However, this is not the case. On the contrary, we regularly observe reduced tyrosine phosphorylation of Lck-dependent substrates when TSAd is overexpressed in Jurkat cells (14,17,18). Moreover, some proteins display increased tyrosine phosphorylation level when TSAd is expressed in Jurkat T cells (e.g. see Fig. 5F, iii). Upon disruption of either the TSAd Lck SH3 or SH2 domain interaction sites, TSAd is no longer able to inhibit phosphorylation of Zap-70 Tyr 319 (Fig. 5B). Both of these sites were also necessary for the reported activation of Lck (24). Collectively, this indicates that the Lck SH3 and SH2 interactions with TSAd work in synergy to exert the modulatory effect TSAd has on Lck activity in vivo.
The phenotype of mice lacking TSAd implies that TSAd is required for proper activation of T cells. TSAd is expressed only at low levels in resting naive T cells but is induced during the first few hours after triggering of the T cell (15). This suggests that TSAd is not essential for the initial triggering of the T cells, but plays a role later during the activation. The exact role of TSAd in T cell activation is yet poorly defined. However, we recently found that TSAd promotes Lck-mediated phosphorylation of Itk. 3 Moreover, a number of other interaction partners, including VCP (23), MEKK2 (50), and Grb2 (22), have been reported, indicating that TSAd may participate in several signaling pathways.
The presence of three combined Lck tyrosine phosphorylation sites and Lck SH2 ligands on TSAd within a distance of only 25 residues is analogous to the ITAMs of the T cell receptor complex, where tyrosine motifs typically spaced 10 -12 amino acid residues from each other are phosphorylated by Lck and serve as docking sites for Zap-70 SH2 domains (51). The conserved nature of these three C-terminal tyrosines strongly indicate that these tyrosines are of importance for the function of TSAd. It is therefore highly likely that one or several of the C-terminal tyrosines serve as docking sites for other signaling molecules that, when bound to TSAd, may eventually become phosphorylated by Lck.
Our observations of the Lck-TSAd interaction may have a biological significance extending above and beyond that related to the role of TSAd in T cell activation. Although processive phosphorylation as a phenomenon is described in the literature, the concept and its possible consequences are not often alluded to. Many SFK substrates also harbor interaction sites for the SH2 and SH3 domains of the kinase. Indeed, the adapter protein Cas (Crk-associated substrate) has been shown to be phosphorylated in a processive manner by Src (52). The Cas family member Sin was recently reported to modulate activation of T cells through regulation of Fyn availability. In resting cells, Sin is constitutively phosphorylated by Fyn, which remains in complex with Sin until the latter becomes transiently dephosphorylated upon TCR triggering (53). Processive phosphorylation of SFK substrates may be an important mechanism not only to ensure proper phosphorylation of a given substrate but also to ensure that the kinase activity is contained within a certain molecular microenvironment. In the absence of its SH2 or SH3 domain, SFK may become hyperactive and oncogenic (54), which can be explained based on lack of intramolecular regulatory interactions. But another not mutually exclusive consequence of lacking SFK SH2 or SH3 domains could be inappropriate tyrosine phosphorylation of substrates that normally are not tyrosine-phosphorylated by SFKs.
In conclusion, we have found that the three C-terminal tyrosines of TSAd are phosphorylated by Lck through a processive mechanism, involving TSAd interactions with both the Lck SH2 and SH3 domains. The multivalent interactions between TSAd and Lck are also necessary for TSAd-mediated modulation of proximal signaling observed in TSAd-expressing T cell lines. We thus propose that processive phosphorylation, where the kinase docks onto the substrate through protein interaction domains, is an important mechanism not only to ensure proper phosphorylation of kinase substrates with multiple phosphorylation sites but also to keep kinase activity confined to particular molecular microenvironments within the cell.