Functional association between SLAP-130 and SLP-76 in Jurkat T cells.

T cell antigen receptor (TCR) engagement results in protein-tyrosine kinase activation which initiates signaling cascades leading to induction of the interleukin-2 gene. Previous studies identified two substrates of the TCR-induced protein-tyrosine kinases, SH2 domain-containing leukocyte specific protein of 76 kDa (SLP-76) and SLP-76-associated phosphoprotein of 130 kDa (SLAP-130). While SLP-76 appears to couple the TCR with downstream signals, SLAP-130 may play a negative regulatory role in T cell activation. In this study, we demonstrate that consistent with its ability to abrogate the SLP-76 augmentation of TCR-induced activation of the NFAT/AP1 region of the interleukin-2 promoter, overexpression of SLAP-130 also interferes with the rescue of signaling in SLP-76-deficient Jurkat cells in co-transfection experiments. The effect of SLAP-130 on SLP-76 function is specific for regulating TCR-induced ERK activation, but not phospholipase Cgamma 1 phosphorylation. By generating both deletion and point mutants of SLAP-130, we identified tyrosine 559 as critical for the interaction between SLP-76 and SLAP-130. We show that mutation of this residue in context of full-length SLAP-130 diminishes the ability of SLAP-130 to abrogate SLP-76 function. These data suggest that the SLAP-130/SLP-76 association is important for the negative regulatory role that SLAP-130 appears to play in T cell signaling.

T cell receptor (TCR) 1 engagement results in the activation of numerous signaling cascades leading ultimately to new gene transcription and cellular proliferation and differentiation (1,2). Work from various laboratories has revealed that the most proximal signaling event following TCR ligation is the activation of several protein-tyrosine kinases (3)(4)(5)(6). These include the Src family kinases Lck and Fyn (7)(8)(9)(10) as well as the Syk family kinase ZAP-70 (11,12). Following protein-tyrosine kinase activation, other signaling events including production of phosphoinositide-derived second messengers (13)(14)(15)(16)(17), in-creases in cytosolic calcium levels (4,18,19), protein kinase C activation (20,21), stimulation of lipid kinases (22), and Ras/ MAPK activation (23) have been shown to be important for T cell activation (24). One experimental strategy to understand better the coordinate regulation of these diverse signaling events is to identify and characterize substrates of the TCRactivated protein-tyrosine kinases. In addition to effector molecules, recent work has shown that adapter proteins such as Cbl (25,26), Shc (27), LAT (28), and SLP-76 (29), can serve as substrates of the TCR-activated protein-tyrosine kinases. In an attempt to determine how these adapter molecules regulate the signaling events involved in T cell activation, studies have been designed to identify molecules which associate with these proteins and to determine the functional significance of these interactions.
Recently, we and others have shown that the SLP-76 associated phosphoprotein of 130 kDa (30), SLAP-130 (also known as FYB for Fyn-binding protein (31)), becomes tyrosine phosphorylated and associates with SLP-76 in T cells following TCR engagement. Analysis of the primary sequence of SLAP-130 reveals no probable enzymatic function; however, it contains several domains that may mediate associations with other proteins. SLAP-130 contains 16 possible tyrosine phosphorylation sites, a central proline-rich region which has recently been identified as the SKAP-55 (another adapter molecule (32)) binding site (33), two possible nuclear localization sites, and an SH3-like domain (31). Although SLAP-130 inducibly binds to SLP-76, we were surprised to find that in our model system unlike SLP-76, overexpression of SLAP-130 does not augment TCR-induced activation of the nuclear factor of activated T cells (NFAT)/AP1 region of the IL-2 promoter in Jurkat T cells (30). More striking was our observation that co-overexpression of SLAP-130 and SLP-76 abrogates the ability of SLP-76 to augment this TCR-induced activation event suggesting that SLAP-130 may serve as a negative regulator of TCR mediated signaling. It should be noted, however, that controversy remains regarding the role of SLAP-130/FYB in the regulation of TCR signaling as others have shown (31,34) that transfection of SLAP-130/FYB can augment TCR-induced IL-2 gene activity.
In a series of experiments designed to investigate further the role of SLAP-130 in the regulation of SLP-76 function in T cell signaling, we found that in our model system overexpression of SLAP-130 along with SLP-76 inhibits SLP-76 mediated augmentation of the entire IL-2 promoter, as well as the NFAT/ AP1 region, following TCR ligation. Consistent with this observation, we show that co-transfection of SLAP-130 along with SLP-76 inhibits the ability of SLP-76 to rescue TCR-induced signaling in a SLP-76-deficient Jurkat variant. Next, we asked whether the SLAP-130/SLP-76 association is important for the effect of SLAP-130 on SLP-76 function by determining the region of SLAP-130 which is important for association with SLP-76 in activated T cells. In these experiments, we identified a tyrosine residue that serves as a phosphorylation site responsible for promoting the binding of SLAP-130 to the SLP-76 SH2 domain in Jurkat T cells. We found that the binding of SLAP-130 to SLP-76 is important for the inhibitory effect of SLAP-130 on the regulation of NFAT/AP1 promoter activity following TCR engagement.
cDNA Constructs-The cDNA for SLAP-130 was cloned as described previously (30). Briefly, the amino-terminal 1350 nucleotides of SLAP-130 were amplified from Jurkat RNA by reverse transcriptase-polymerase chain reaction using the GeneAmp kit (Perkin-Elmer), and were subcloned into pEF/Flag at the BamHI and XbaI sites in-frame with the Flag epitope to generate pEF/Flag/SLAP-130/1-460. The remaining 1000 base pairs were amplified from Jurkat RNA by overlap extension reverse transcriptase-polymerase chain reaction. The resulting fragment was ligated in-frame with the amino-terminal 1350 base pairs (pEF/Flag/SLAP-130/1-460) described above to create pEF/Flag/SLAP-130 or ligated into pEF/Flag at the XbaI site to generate pEF/Flag/ SLAP-130/460 -783. The pEF/HA/SLAP-130 truncation mutants were prepared by generating a series of polymerase chain reaction products using primers corresponding to the designated amino acids. Using a 5Ј sense primer (TCGGATCCGCGGCCCCTCTAGAT) for each of the three truncation mutants, we used the antisense primers TCGGGTACCGG-TATGACTTGAATAGGGCCTG for pEF/SLAP-130/460 -525, GGATCC-GCGGCCGCAGACTCTCTTGGTGC for pEF/SLAP-130/460 -595, and TCGGTACCTGGTTTGTATAACTTCTAGAGA for pEF/SLAP-130/460 -750, respectively. The pEF/Flag/SLAP-130 point mutations were generated by overlap extension of pEF/Flag/SLAP/460 -783 and were initially cloned into pEF/HA at the XbaI site. Using the same sense primer used for the truncation mutants in combination with primers derived from the SLP-76-binding domain and corresponding to a single base pair change of tyrosine (TAT) to phenylalanine (TTT) and an antisense primer for the 3Ј coding region (TCTAGAGGTACCGAC-TAGTCATTGTCATA), we generated the tyrosine point mutations. Both sense and antisense primers were designed corresponding to each point mutation (GGGGTTCATTTGGCTATATTAAAAC for pEF/SLAP-130/Y559F, GGGGTTCATATGGCTTTATTAAAAC for pEF/SLAP-130/Y559F, and GATTGACTTTGATTCTTTG for pEF/SLAP-130/ Y559F). For generation of the full-length SLAP-130 point mutants, the constructs containing the mutation were subcloned in-frame with pEF/ Flag/SLAP-130/1-460 at the XbaI site. All constructs generated using polymerase chain reaction were sequenced and the fidelity confirmed at the University of Iowa DNA Sequencing Core (Iowa City, IA).
Transfections and Luciferase Assays-Cells were washed in phosphate-buffered saline and suspended in cytomix (120 mM KCl, 0.15 mM CaCl 2 , 10 mM K 2 HPO 4 /KH 2 PO 4 , pH 7.6, 25 mM HEPES, pH 7.6, 2 mM EGTA, 5 mM MgCl 2 , pH adjusted with KOH) (39) at a concentration of 1.5 ϫ 10 7 cells/400 l of cytomix/cuvette. Cells were electroporated at 250 V, 960 microfarads using a Gene Pulser (Bio-Rad) with 25 g of pIL-2 or pIL2-NFAT/AP1-luciferase, 5 g of pCMV/␤-galactosidase, and 40 g of the SLAP-130 and SLP-76 expression vectors. The total amount of plasmid DNA was equilibrated to 100 g with the vector control pEF/HLA-A2. After 24 h, 5 ϫ 10 5 cells were stimulated in triplicate for 16 h with media, immobilized anti-TCR mAb C305 (ascites 1:1000), or 50 ng/ml phorbol ester (PMA) plus 1 M ionomycin (maximum response). Additionally, triplicate samples of 5 ϫ 10 5 unstimulated cells were assayed for ␤-galactosidase activity using the Galacto-Light Plus Reporter Gene Assay System (Tropix Inc., Bedford, MA). Luciferase activity was determined as described previously (37). Luciferase light units were normalized to ␤-galactosidase activity present in each transfectant to standardize for transfection efficiency. The luciferase activity is expressed as percentage of the maximal promoter activity which is induced by incubation of the transfected cells with PMA and ionomycin.
GST Fusion Protein Precipitations-Following transformation of DH5Ј␣ with pGEX/SLP-SH2, recombinant protein was purified from logarithmically growing cells (A 500 ϭ 0.6) that had been incubated for 2 h at 25°C with 1 mM isopropyl-1-thio-␤-D-galactopyranoside (Life Technologies, Inc., Grand Island, NY). The bacterial pellet was resuspended in ice-cold phosphate-buffered saline (50 mM sodium phosphate, pH 7.6, 100 mM KCl, 100 mM NaCl) and sonicated (Branson Sonifier 250; Bio-Rad) at 50% power and 20 duty cycles. Following incubation of the lysates with 1% Triton X-100 and 1 mM phenylmethylsulfonyl fluoride at 4°C for 10 min, the cell lysates were cleared at 10,000 ϫ g for 10 min at 4°C. The GST fusion protein was bound to glutathioneagarose beads (Sigma), and the complex was washed with phosphatebuffered saline and stored at 4°C in phosphate-buffered saline with 1 mM phenylmethylsulfonyl fluoride.
Immunoprecipitations--Transfected cells (2 ϫ 10 6 cells/sample) were left unstimulated or stimulated with C305 for 5 min and lysed in Nonidet P-40 lysis buffer including protease inhibitors and proteintyrosine phosphatase inhibitors (33). In experiments involving the detection of phosphorylated PLC␥1, cells stimulated with pervanadate were used to assess TCR-independent phosphorylation as a positive control (16). For immunoprecipitations, antibodies (2 g/immunoprecipitation) were conjugated to GammaBind Plus Sepharose (Amersham Pharmacia Biotech) for 2 h at 4°C. Lysates were subjected to precipitation with the indicated antibody-conjugated Sepharose beads for 2 h at 4°C. The immune complexes were washed 3 times with Nonidet P-40 lysis buffer with 500 mM NaCl, subjected to SDS-PAGE (10% polyacrylamide gels for separation of full-length SLAP-130 and SLP-76, 15% for separation of the SLAP-130 truncation mutants, and 8% for detection of PLC␥1), and transferred to nitrocellulose for immunoblot analysis as described previously.
Phosphopeptide Competition Assays-Jurkat cells transfected with pEF/Flag/SLAP-130 were stimulated with C305 for 5 min and lysed in Nonidet P-40 lysis buffer including protease inhibitors and proteintyrosine phosphatase inhibitors. The GST/SLP-76 SH2 domain agarose beads were preincubated with vehicle or 50 M of each peptide (see Fig.  6A, for peptide sequences) for 30 min at 4°C. Lysates were then subjected to precipitation with the GST/SLP-76 SH2 domain agarose beads in the presence of peptides for 2 h at 4°C. Precipitated complexes were washed 3 times in high salt lysis buffer and resolved by reducing SDS-PAGE followed by immunoblot analysis to detect Flag-tagged SLAP-130.

Regulation of SLP-76 Function by SLAP-130 -Stimulation
of the TCR on Jurkat T-cells results in the transactivation of the IL-2 gene which can be measured using a transfectable luciferase reporter construct encoding the full-length IL-2 promoter or multiple copies of the NFAT/AP1 region of this promoter (41,42). We and others have shown that overexpression of SLP-76 augments IL-2 promoter function in response to TCR ligation (43)(44)(45). Since the SLP-76 SH2 domain is required for augmentation of TCR signaling by SLP-76 (37, 40), we speculated that SLAP-130, which was initially isolated as a SLP-76 SH2 domain-binding protein, would also serve as a positive regulator of TCR signals. Surprisingly, we found that overexpression of SLAP-130 blocks the augmentation of TCR-induced NFAT/AP1 activity by overexpressed SLP-76 30) (Fig. 1A, left panel). As shown, SLAP-130 overexpression does not change TCR-stimulated NFAT/AP1 promoter activity compared with cells transfected with an unrelated cDNA (HLA-A2), while overexpression of SLP-76 dramatically augments NFAT/AP1 function. Expression of the Flag epitope-tagged SLAP-130 and SLP-76 was confirmed by immunoblot analysis of whole cell lysates (Fig. 1A, right panel).
To extend this finding to activity of the entire IL-2 promoter, we co-transfected Jurkat T cells with a full-length IL-2 promoter construct driving the luciferase gene along with expression vectors that encode for SLAP-130, SLP-76, or both. Fig. 1B (left panel) shows that again, in contrast to overexpression of SLP-76 alone, overexpressed SLAP-130 has little effect on TCR-induced IL-2 promoter function although equivalent levels of SLAP-130 and SLP-76 were expressed in the transfected cells (Fig. 1B, right panel). However, when overexpressed along with SLP-76, SLAP-130 inhibits the augmentation of TCRinducible promoter activity by SLP-76.  We extended these observations by studying the effect of transfecting SLAP-130 into J14-v-29, a SLP-76 deficient variant of Jurkat. As previously shown by others (36) and demonstrated in Fig. 2, TCR stimulation fails to activate the NFAT/ AP1 promoter in this mutant. Reconstitution of SLP-76 expression rescues this signaling defect (left panel). Consistent with the experiment shown in Fig. 1, co-transfection of SLAP-130 along with SLP-76 blocks reconstitution of TCR-induced NFAT/AP1 promoter activity.
To determine where in the TCR-regulated signaling cascade SLAP-130 interferes with SLP-76 function, we examined the effect of co-transfection of SLAP-130 and SLP-76 on TCR-induced second messenger activation. First, J14-v-29 cells were transfected with a control plasmid (HLA-A2), SLAP-130, SLP-76, or the combination of SLAP-130 plus SLP-76. Cells were left unstimulated or stimulated via the TCR or with pervanadate, then lysed and subjected to immunoprecipitation with antibody directed against PLC␥1. As shown in Fig. 3A, engagement of the TCR on J14-v-29 fails to induce tyrosine phosphorylation of PLC␥1, although this event is seen following pervanadate treatment. This signaling defect is rescued when cells are transfected with SLP-76. Co-transfection of SLAP-130 along with SLP-76 does not interfere with this readout of TCR ligation.
In contrast, when TCR-stimulated activation of ERK is studied, co-transfection of SLAP-130 with SLP-76 blocks rescue of J14-v-29. This is shown in Fig. 3, B-D. For the experiment depicted in Fig. 3B, J14-v-29 cells transfected with SLP-76, SLAP-130, or the combination of SLP-76 plus SLAP-130 were left unstimulated or treated with anti-TCR or PMA. Lysates were then prepared and analyzed for ERK activation by a gel shift assay. As shown, transfection of SLP-76, but not SLAP-130 results in TCR-induced ERK activation. Co-transfection of SLP-76 plus SLAP-130 prevents TCR-stimulated ERK function, but has no effect on the ability of ERK to be activated following PMA treatment. These findings are corroborated in the experiment shown in Fig. 3, C and D. In this study, J14v-29 cells were transfected with Myc-ERK along with control plasmid, SLP-76, SLAP-130, or the combination of SLP-76 plus SLAP-130. Cells were left unstimulated or stimulated via the TCR or with PMA. Lysates were prepared and subjected to immunoprecipitation with anti-Myc. Kinase assays using myelin basic protein as a substrate were performed on each immune complex and assayed by autoradiography (Fig. 3C) and quantitated by scanning densitometry of the autoradiogram (Fig. 3D). While SLP-76 rescues TCR-induced ERK activation in J14-v-29, the combination of SLAP-130 plus SLP-76 abrogates the rescue. Collectively, these results suggest that when SLAP-130 is overexpressed it is able to interfere with the ability of SLP-76 to promote TCR-induced ERK kinase activity, but not phosphorylation of PLC␥1.
Preliminary Mapping of the SLAP-130/SLP-76 Association-To define the SLP-76-binding site in SLAP-130 and to determine the functional significance of this intermolecular interaction, we created a series of truncation mutants of SLAP-130 (Fig. 4A). For our initial studies, SLAP-130 was divided into two regions, one containing the first 460 amino acids (SLAP-130/1-460) and the second including the carboxyl-terminal 323 amino acids with 14 of the 16 possible tyrosine- phosphorylation sites (SLAP-130/460 -783). Jurkat T cells were transfected with these constructs and were then either stimulated via their TCR or left unstimulated. Lysates were precipitated with a GST fusion protein containing the SLP-76 SH2 domain conjugated to glutathione-conjugated agarose beads. Fig. 4B (left panel) shows that full-length SLAP-130 inducibly associates with the SLP-76 SH2 domain in vitro as has been described previously (30). Analysis of the SLAP-130 truncation mutants reveals that SLAP-130/460 -783 contains the SLP-76 SH2 domain-binding site while SLAP-130/1-460 fails to bind. Note that the observed difference in binding was not due to differences in the amount of protein expressed as shown in the anti-Flag immunoblot analysis (Fig. 4B, right panel).
Next, we asked whether the in vitro SLAP-130/SLP-76 association also occurs in intact cells. For this study, we co-transfected Jurkat cells with an HA-tagged SLP-76 construct along with cDNAs encoding the Flag-tagged SLAP-130 truncation mutants. Transfected cells were left unstimulated or stimulated via the TCR. Lysates were then subjected to immunoprecipitation of the Flag-tagged molecules, followed by detection of SLP-76 binding by immunoblot analysis using an anti-HA antibody. As shown in Fig. 4C (left panel)

SLAP-130 Tyr-559 Is Important for Association with SLP-76
the TCR, and the HA-tagged fusion proteins were immunoprecipitated using an HA mAb. All three truncation mutants can be phosphorylated in stimulated Jurkat cells (data not shown); although, SLAP-130/460 -524 appears to be phosphorylated to a lesser extent than the longer SLAP-130 constructs. This is likely due to the fact that this truncation mutant retains only a single tyrosine residue. To address which truncation mutant could bind to SLP-76, we then co-transfected each construct into Jurkat T cells and performed binding experiments. First, we determined whether they could bind to the SLP-76 SH2 domain in vitro. As illustrated in Fig. 6B, although similar levels of each construct were expressed (right panel), two of the SLAP-130 mutants, SLAP-130/460 -595 and SLAP-130/460 -750, inducibly bind to the SLP-76 SH2 domain. In contrast, the shortest construct (SLAP-130/460 -524) fails to associate with the SLP-76 fusion protein in this in vitro experiment (left panel).
We next addressed further the requirements for the association of SLAP-130 and SLP-76 in intact T cells (Fig. 6C). In this experiment, we co-transfected Jurkat T cells with HA-tagged SLAP-130 constructs along with a Flag-tagged SLP-76 cDNA. Cells were left unstimulated or stimulated via their TCR, lysed, and then subjected to an anti-Flag immunoprecipitation. As shown, immunoprecipitation of SLAP-130/460 -595 or SLAP-130/460 -750 reveals an inducible association of these two mutants with SLP-76 (left panel). In contrast, little SLP-76 is present in immunoprecipitates of SLAP-130/460 -524 from lystates of unstimulated or stimulated Jurkat. Since SLAP-130/ 460 -524 associates poorly with SLP-76, we conclude that tyrosine 475 is not critical for the SLAP-130/SLP-76 association, and the sequence between amino acids 524 and 595, which contains 3 tyrosine residues, can mediate an association between SLAP-130 and SLP-76 both in vitro as well as in intact cells.
We next determined the ability of the carboxyl-terminal truncation mutants to affect SLP-76 mediated augmentation of NFAT/AP1 activity. Fig. 7A demonstrates that, in addition to encoding the SLP-76-binding domain, SLAP-130/460 -595 and SLAP-130/460 -750 also inhibit TCR-induced amplification of NFAT/AP1 activation by SLP-76. In contrast, co-expression of SLP-76 with SLAP-130/460 -524, the truncation mutant which appears to show little binding to SLP-76, has no effect on the ability of SLP-76 to augment NFAT/AP1 activity. Expression levels of the SLAP-130 truncations mutants in each transfection were determined by anti-HA immunoblot analysis (Fig.  7B) while the full-length SLAP-130 and the SLP-76 expression were detected using an anti-Flag antibody for blotting (Fig.  7C). None of the SLAP-130 mutants have an effect on reporter activity when expressed alone (data not shown). Together these data demonstrate that amino acids 524 -595 contain the key residues responsible for both binding and functional interactions with SLP-76.
Tyrosine 559 Is Essential for Optimal Binding of SLAP-130 to SLP-76 -We next sought the identity of the tyrosine residue(s) that mediate the inducible association of SLAP-130 with the SLP-76 SH2 domain. First, we used a series of peptides corresponding to the sequence demonstrated in previous experiments to be important for the association of SLAP-130 with SLP-76 (Figs. 6 and 7) to compete with SLAP-130 for binding to the SLP-76 SH2 domain in vitro. Peptides were generated, including tyrosines 559, 561, and 571, in either their phosphorylated or unphosphorylated forms (Fig. 8A). As shown in Fig.  8B, the peptide which is phosphorylated on Tyr-559 interferes dramatically with the association between SLAP-130 and the SLP-76 SH2 domain suggesting that Tyr-559 is important for this intermolecular interaction while the other phosphopeptides affected the binding of SLAP-130 with the SLP-76 SH2 domain minimally.
To confirm the importance of Tyr-559 in regulating the association between SLAP-130 and SLP-76, we introduced point mutations into the coding region of full-length SLAP-130 cDNA, substituting each of the tyrosines between amino acids 524 and 595 with phenylalanine (Fig. 9A). While mutation of Lysates were subjected to precipitation with the GST/SLP-76 SH2 domain glutathione-conjugated agarose beads for 2 h at 4°C. Precipitated complexes were washed 3 times in high salt lysis buffer, resolved by SDS-PAGE (15%) followed by immunoblot analysis using anti-HA mAb (left panel). Expression of the epitope-tagged constructs was confirmed by immunoblot analysis with anti-HA mAb (right panel). This experiment is representative of five independent transfections. C, Jurkat T cells were transfected with the indicated HA-tagged SLAP-130 constructs along with pEF/Flag/SLP-76. 24 h following transfection, cells were left unstimulated or stimulated with anti-TCR mAb for 5 min. Lysates were subjected to immunoprecipitation with anti-Flag mAb for 2 h at 4°C. Precipitated complexes were washed 3 times in high salt lysis buffer, then resolved by SDS-PAGE (15%) followed by immunoblot analysis using anti-Flag mAb to detect the SLAP-130 associated SLP-76 (left panel). Confirmation of the expression of the epitope-tagged constructs was determined by examining whole cell lysates by immunoblot analysis with anti-HA mAb (right panel). This experiment is representative of four independent transfections.

SLAP-130 Tyr-559 Is Important for Association with SLP-76
tyrosine 559 in SLAP-130 abolishes the binding interaction between SLAP-130 and the SH2 domain of SLP-76 in activated T cell lysates (Fig. 9B, left panel), mutation of either tyrosine 561 or 571 has no effect. In addition, the SLAP-130/Y559F mutant shows a striking loss in the ability to bind to SLP-76 in intact cells when overexpressed in Jurkat (Fig. 9C, left panel) while the other point mutations (SLAP-130/Y561F and SLAP-130/Y571F) do not affect the association between SLAP-130 and SLP-76.
Concomitant with the loss in binding, mutation of tyrosine 559 also leads to a decrease in the ability of SLAP-130 to inhibit SLP-76 function when co-expressed in wild type Jurkat (Fig.  10A, left panel). In contrast, the SLAP-130/Y561F and Y571F mutants remain potent inhibitors (comparable to wild type SLAP-130) of the augmentation of TCR signaling seen by overexpression of SLP-76. Similarly, we show in Fig. 10B that mutation of tyrosine 571 of SLAP-130 does not interfere with the ability of SLAP-130 to block the rescue of TCR-induced signals in J14-v-29 cells transfected with SLP-76. However, TCR signaling is intact in J14-v-29 cells co-transfected with SLP-76 and SLAP-130/Y559F. Together, these data suggest that the association of SLAP-130 and SLP-76 is important for the regulation of signaling events following TCR engagement. Additionally, phosphorylation of SLAP-130 on tyrosine 559 likely plays a key role in SLAP-130 function. DISCUSSION Much has been learned in recent years about the signal transduction events which occur following TCR engagement leading to the regulation of cytokine production and cellular proliferation or apoptosis. Early studies focused on cell surface receptors which initiate signaling upon ligand binding and on the most proximal signaling events which follow receptor engagement. Recently, it has become clear that adapter molecules play a key regulatory role in TCR signaling by recruiting effector molecules and by integrating different signaling pathways. One example of such an adapter protein is SLP-76, which appears to serve as a positive regulator of T cell activation (29,37,(43)(44)(45).
In an attempt to understand how SLP-76 may function to promote T cell signaling events, our laboratory has been interested in identifying SLP-76-associated proteins. We and others have shown that SLP-76 inducibly binds to Vav via the Vav

SLAP-130 Tyr-559 Is Important for Association with SLP-76
SH2 domain and the amino-terminal acidic region of SLP-76 following TCR ligation (45)(46)(47). Overexpression of either SLP-76 or Vav augments activation of the IL-2 gene following TCR ligation (45,48), and co-overexpression of SLP-76 with Vav has a synergistic effect on IL-2 gene regulation (49). Interestingly, however, the association of SLP-76 with Vav is not necessary for the effect that overexpression of each has on IL-2 gene regulation (50). In addition to inducible binding to Vav with its amino-terminal phosphorylation sites, SLP-76 associates constitutively with the Grb2 family member, Gads, via the SLP-76 central proline-rich region and the Gads SH3 domains (51). The functional significance of this association is suggested by experiments showing that the overexpression of Gads along with SLP-76 resulted in a synergistic augmentation of NFAT/ AP1 promoter activity.
SLP-76 also contains a single SH2 domain which mediates an interaction with two phosphoproteins of apparent molecular masses of 130 and 62 kDa. Recently, we and others have cloned the cDNA for the 130-kDa protein (SLAP-130) (30) (also known as FYB for Fyn-binding protein (31)). Sequence analysis reveals no obvious enzymatic activity but shows that SLAP-130 contains several domains predicted to mediate its association with other proteins. For example, Schraven and colleagues (32,52,53) recently showed that the SLAP-130 central proline-rich region binds SKAP-55 or a SKAP-55 related protein (SKAP-HOM), recently identified adapter proteins, through the SKAP-55 or SKAP-HOM SH3 domains. In addition, others have described an interaction between FYB/SLAP-130 with the Lysates were subjected to precipitation with the GST/SLP-76 SH2 domain glutathione-conjugated agarose beads for 2 h at 4°C. Precipitated complexes were washed 3 times in high salt lysis buffer, resolved by SDS-PAGE (10%) followed by immunoblot analysis using anti-Flag mAb (left panel). Expression of the epitope-tagged constructs was confirmed by immunoblot analysis with anti-Flag mAb (right panel). This experiment is representative of three independent transfections. C, Jurkat T cells were transfected with the indicated Flag-tagged SLAP-130 constructs along with pEF/HA/SLP-76. 24 h following transfection, cells were left unstimulated or stimulated with anti-TCR mAb for 5 min. Lysates were subjected to immunoprecipitation with anti-Flag mAb for 2 h at 4°C. Precipitated complexes were washed 3 times in high salt lysis buffer, then resolved by SDS-PAGE (10%) followed by immunoblot analysis using anti-HA mAb to detect the SLAP-130 associated SLP-76 (left panel). Confirmation of the expression of the epitopetagged constructs was determined by examining whole cell lysates by immunoblot analysis with anti-Flag mAb (right panel). This experiment is representative of three independent transfections. Jurkat T cells were transfected with pIL2-NFAT/AP1/luciferase, pCMV/␤-gal, and the indicated constructs. The amount of DNA was equalized with control DNA. 24 h following transfection, cells were left unstimulated, stimulated with anti-TCR mAb, or stimulated with PMA plus ionomycin (maximal response) for 16 h. The samples were assayed for luciferase activity as described for Fig. 1A (left panel). This experiment is representative of four independent transfections. Expression of the epitope-tagged proteins in the experiment represented by the solid bars was determined by examining whole cell lysates by immunoblot analysis with anti-Flag mAb (right panel). B, J14-v-29 cells were transfected with pIL2-NFAT/ AP1/luciferase, pCMV/␤-gal, and the indicated constructs. The amount of DNA was equalized with control DNA. 24 h following transfection, cells were left unstimulated, stimulated with anti-TCR mAb, or stimulated with PMA plus ionomycin (maximal response) for 16 h. The samples were assayed for luciferase activity as described for Fig. 1A (left  panel). This experiment is representative of three independent transfections. Expression of the epitope-tagged proteins in the experiment represented by the solid bars was determined by examining whole cell lysates by immunoblot analysis with anti-Flag mAb (right panel).
CD3 components of the TCR⅐CD3 complex (54). The functional significance of each of these interactions remains unclear.
When the association between SLAP-130 and SLP-76 was initially described, we speculated that SLAP-130 would function similarly to Vav and augment the ability of SLP-76 to promote TCR-mediated signaling. We were surprised to find, however, that in our model system overexpression of SLAP-130 along with SLP-76 abrogates the augmentation of NFAT/AP1 promoter activity by SLP-76 (30) (Fig. 1A, this study). In this report, we extend this finding by showing that overexpression of SLAP-130 along with SLP-76 abrogates the rescue of SLP-76-deficient Jurkat cells, J14-v-29.
We next choose to examine the structural components of SLAP-130 responsible for its interaction with SLP-76 and to ask if the association correlates with regulation of signaling events important for IL-2 gene expression. Our approach was to generate SLAP-130 mutants that fail to bind SLP-76 in T cells, then to determine whether the association of SLAP-130 with SLP-76 is important for the functional consequences seen when these two proteins are co-expressed. We found, in our model system, that tyrosine 559 is important for the TCRinduced association of SLP-76 with SLAP-130; and that a region of SLAP-130 containing this region is sufficient for the negative regulation of SLP-76 activity by SLAP-130. In support of this finding, mutation of that single tyrosine residue in SLAP-130 interferes with both the binding interaction between SLAP-130 and SLP-76 as well as the ability of SLAP-130 to affect NFAT/AP1 activity. In contrast, a recent report by Raab et al. (34) indicated that the region of SLAP-130 (FYB) important for its association with SLP-76 lies carboxyl-terminal to the site we describe. This report went on to identify tyrosine 651 as the residue critical for association with SLP-76. These discrepant results suggest that under different experimental conditions, different tyrosines of SLAP-130 may be phosphorylated and mediate binding to the SLP-76 SH2 domain. In this regard, Raab et al. (34) also found that instead of interfering with SLP-76 function, co-expression of SLAP-130 augments the effect of SLP-76 on IL-2 gene promoter activity. This is seen most clearly when the Fyn protein-tyrosine kinase is co-transfected into the responding cells or in the DC27.10 hybridoma which expresses high levels of Fyn (31). In our Jurkat cells, Fyn is present at very low levels. Thus, it is possible that at high levels, Fyn phosphorylates tyrosine residues other than 559 of SLAP-130, resulting also in SLP-76 binding, but a different functional outcome. Additionally, because SLAP-130 is an adapter molecule, it is possible that its function may differ based on what other proteins are present in the cell being studied. Further experiments in cell lines using transfected mutant constructs of SLAP-130 and its associated molecules, and studies utilizing cell lines and eventually animal models deficient in SLAP-130 expression will be necessary to define more precisely the role of SLAP-130 in T cell activation.
In addition to its tyrosine residues, SLAP-130 contains several motifs that may be important for its function in T cell signaling. There appears to be an SH3-like domain and two putative nuclear localization motifs in the SLAP-130 sequence that are conserved among species (54). Another recent report by the Rudd group suggests that these regions may be functional due to the subcellular localization SLAP-130 to perinuclear region of transfected cells (53). Further characterization of these sites may provide information into the role that SLAP-130 plays in lymphocyte signaling.
Note Added in Proof-While this manuscript was in the review process, a study by Geng et al. ((1999) J. Immunol. 163, 5753-5757) described the identification of additional tyrosine residues in SLAP-130/FYB, which are substrates of Fyn and can mediate the association of SLAP-130 with SLP-76. The Geng et al. study reported that although tyrosines 595 and 651 can mediate the association of SLP-76 with SLAP-130, SLAP-130 is still phosphorylated in Fyn-deficient cells, indicating that additional site(s) of phosphorylation in SLAP-130 exist. Together, our study and that of Geng et al. suggest that the association between SLAP-130 and SLP-76 is more complex than the recruitment of SLAP-130 to SLP-76 via a single phosphorylated tyrosine. Additionally, it appears likely that the regulation of the interaction between SLP-76 and SLAP-130 and whether SLAP-130 acts as a positive or negative regulator of TCR function depend on the experimental system employed.