SLP-76 and Vav Function in Separate, but Overlapping Pathways to Augment Interleukin-2 Promoter Activity*

SLP-76 and Vav, two hematopoietic cell specific molecules, are critical for T cell development and activation. Following T cell antigen receptor stimulation, SLP-76 and Vav both undergo tyrosine phosphorylation and associate with each other via the SH2 domain of Vav and phosphorylated tyrosines of SLP-76. Furthermore, SLP-76 and Vav have a synergistic effect on interleukin (IL)-2 promoter activity in T cells. In this report, we show that two tyrosines, Tyr-113 and Tyr-128, of SLP-76 are required for its binding to Vav, both in vitro and in intact cells. Surprisingly, we find also that the interaction between SLP-76 and Vav is not required for their cooperation in augmenting IL-2 promoter activity, as the two molecules appear to function in different signaling pathways upstream of IL-2 gene expression. Overexpression of SLP-76 in the Jurkat T cell line potentiates the activities of both nuclear factor of activated T cells and AP-1 transcription factors. In contrast, overexpression of Vav leads to enhanced nuclear factor of activated T cells activity without affecting AP-1. Additionally, overexpression of Vav, but not SLP-76, augments CD28-induced IL-2 promoter activity. These findings suggest that the synergy between SLP-76 and Vav in regulating IL-2 gene expression reflects the cooperation between different signaling pathways.

SLP-76 and Vav, two hematopoietic cell specific molecules, are critical for T cell development and activation. Following T cell antigen receptor stimulation, SLP-76 and Vav both undergo tyrosine phosphorylation and associate with each other via the SH2 domain of Vav and phosphorylated tyrosines of SLP-76. Furthermore, SLP-76 and Vav have a synergistic effect on interleukin (IL)-2 promoter activity in T cells. In this report, we show that two tyrosines, Tyr-113 and Tyr-128, of SLP-76 are required for its binding to Vav, both in vitro and in intact cells. Surprisingly, we find also that the interaction between SLP-76 and Vav is not required for their cooperation in augmenting IL-2 promoter activity, as the two molecules appear to function in different signaling pathways upstream of IL-2 gene expression. Overexpression of SLP-76 in the Jurkat T cell line potentiates the activities of both nuclear factor of activated T cells and AP-1 transcription factors. In contrast, overexpression of Vav leads to enhanced nuclear factor of activated T cells activity without affecting AP-1. Additionally, overexpression of Vav, but not SLP-76, augments CD28induced IL-2 promoter activity. These findings suggest that the synergy between SLP-76 and Vav in regulating IL-2 gene expression reflects the cooperation between different signaling pathways.
Recognition of specific antigen by the T cell antigen receptor (TCR) 1 initiates biochemical changes including protein tyrosine phosphorylation, calcium influx, metabolism of phospholipids, and activation of the Ras/MAPK cascade (1,2). Together with signaling events initiated by engagement of co-receptors on the T cell, these biological second messengers lead to cytokine production, proliferation, and differentiation, or conversely programmed cell death (3)(4)(5). Activation of protein tyrosine kinases (PTKs) of the Src and Syk families is the most membrane-proximal biochemical event known following TCR engagement (6). Phosphorylation of cellular proteins by these kinases is essential for all subsequent TCR-initiated signaling pathways. PTK activation may result in downstream signals through direct phosphorylation and modulation of the catalytic activities of effector molecules (such as phospholipase C-␥1) or the formation of signaling complexes by the interaction of proteins newly phosphorylated on tyrosine residues with other Src homology 2 (SH2) or phosphotyrosine-binding domain-containing proteins within the cell. A widely studied example of this in T cells is the recruitment of ZAP-70 to the newly phosphorylated tyrosines of immune receptor activation motifs (ITAMs) on the TCR and CD3 chains (7,8).
Given the central importance of PTKs in T cell activation, a number of laboratories have attempted to identify substrates of these PTKs in an effort to gain insight into how biochemical signals are integrated. Among the substrates identified are the hematopoietic cell-specific molecules SH2 domain containing leukocyte phosphoprotein of 76 kDa (SLP-76) and Vav, both of which play critical roles in the regulation of TCR signals (9,10). SLP-76 is an adapter protein which is comprised of three motifs allowing for protein-protein interactions: an amino-terminal acidic region containing tyrosine phosphorylation sites (11), a middle proline-rich motif that binds to the SH3 domain of Grb2 family members (9,12), and a carboxyl-terminal SH2 domain that associates with SLP-76-associated phosphoprotein of 130 kDa (SLAP-130 or FYB, for Fyn-binding protein) and another unidentified 62-kDa phosphoprotein (13)(14)(15). The importance of SLP-76 in T cell signaling has been demonstrated in experiments in the Jurkat human T cell leukemia line and in mice made deficient in SLP-76 expression through homologous recombination. When SLP-76 is overexpressed in Jurkat cells, there is a dramatic augmentation of TCR-mediated activation of the full-length IL-2 promoter or a reporter construct driven by the nuclear factor of activated T cells (NFAT) element from the IL-2 promoter (9,16). Each of the three structural domains of SLP-76 are essential for this enhanced promoter activity (17). Furthermore, TCR-mediated signals are abrogated in a mutant variant of Jurkat which has lost expression of SLP-76 (18). Studies of SLP-76-deficient mice indicate also that this adapter protein is critical for signaling via the pre-TCR, as these mice exhibit arrest of thymocyte development at the pro-T3, CD3 Ϫ CD4 Ϫ CD8 Ϫ stage (19,20).
Vav is a guanine nucleotide exchange factor which acts on members of the Rac/Rho family of small GTP-binding proteins (21,22). Vav has a pleckstrin homology, a Dbl homology, an SH2, and two Src homology 3 (SH3) domains (23,24). The Dbl homology domain of Vav is responsible for its nucleotide exchange activity while the pleckstrin homology domain regulates interactions with inositol phospholipids (25). The SH2 and SH3 domains mediate the interaction of Vav with other signaling molecules, including SLP-76 (26 -29). Like SLP-76, Vav is critical for effective TCR signaling. Its overexpression in Jurkat T cells also augments the transcriptional activity of the IL-2 promoter following TCR ligation (10). T cells from Vavdeficient mice do not respond effectively to TCR stimulation (30,31). Interestingly, overexpression of SLP-76 and Vav together has a synergistic effect on activation of the IL-2 promoter (29). However, previous studies have not addressed whether the interaction between SLP-76 and Vav is required for their synergy and if these two proteins function in the same signaling pathways.
In the experiments described in this report we investigated these questions first by identifying the tyrosines of SLP-76 responsible for the interaction with Vav both in vitro and in intact Jurkat cells. Using this information, we performed a series of co-transfection assays and determined that the intermolecular interaction between SLP-76 and Vav is not required for their functional synergy. This finding suggested the hypothesis that overexpression of SLP-76 versus Vav may impact distinct signaling pathways in T cells. Experiments are presented demonstrating that these two molecules modulate distinct signaling cascades to link T cell surface receptors with IL-2 gene expression.
cDNA Constructs and Fusion Proteins-Wild-type SLP-76 and each of the mutants were cloned into modified pEF-BOS vector containing a sequence encoding the FLAG epitope on the amino terminus of the cDNAs, as described previously (11,17). cDNAs encoding Vav and ERK2 with a Myc epitope on the amino terminus were also cloned into pEF-BOS vector (gifts of Dr. A. Weiss, University of California, San Francisco, CA). The NFAT luciferase reporter construct (NFAT-luc) was provided by Dr. G. Crabtree (Stanford University, Palo Alto, CA). The full-length IL-2 promoter driven luciferase construct (pIL-2luc2kb) was a gift of Dr. R. Abraham (Mayo Clinic, Rochester, MN). The AP-1 ␤-Gal construct (pAP-1-lacZ) was a gift of Dr. D. Mueller (University of Minnesota, Minneapolis, MN). The CD28RE luciferase construct was provided by Dr. K. L. McGuire (San Diego State University, San Diego, CA). The GST Vav SH2 was provided by Dr. S. Katzav (Lady Davis Institute, Jewish General Hospital, Montereal, Quebec, Canada). GST fusion protein was induced and affinity purified as described (33).
Reporter Assays-Luciferase or ␤-Gal assays were performed as described previously (17). Briefly, 24 h after transfection, triplicates of 5 ϫ 10 5 viable cells were stimulated as indicated at 37°C for 8 h, lysed, and subsequently assayed for luciferase or ␤-Gal activity with a monolight luminometer (Analytical Luminescence Laboratory, Ann Arbor, MI).
Immunoprecipitations, Protein Precipitations, and Immunoblots-24 h after transfection, cells were collected, washed in phosphate-buffered saline, and left unstimulated, or stimulated with C305 ascites (1:1,000) for the indicated time, or with pervanadate for 2 min, or with PMA for 5 min. Lysates were prepared in Nonidet P-40 lysis buffer containing protease and phosphatase inhibitors, as described previously (9), and precipitated with GST fusion proteins or indicated antibodies at 4°C for 2 h. Following precipitation, the protein complexes were washed extensively in Nonidet P-40 lysis buffer, subjected to SDS-PAGE, and transferred to nitrocellulose for immunoblot analysis with the indicated antibodies, followed by a horseradish peroxidase-conjugated secondary Ab (Bio-Rad) and developed by enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech).
ERK2 in Vitro Kinase Activity Assays-ERK2 kinase activity assays were performed as described previously (17). Briefly, Jurkat cells were transfected with pEF, pEF/SLP-76, pEF/Vav plus pEF/myc/ERK2. 24 h later, transfected cells were left unstimulated or stimulated with anti-TCR mAb C305 or phorbol myristate acetate (PMA), lysed in Triton X-100 lysis buffer, and precipitated with anti-Myc antibody for 2 h at 4°C after stimulation, The anti-Myc immune complexes were washed extensively and suspended in kinase buffer (20 mM Tris, pH 7.6, 13 mM MgCl 2 , and 1.5 mM EGTA) containing myelin basic protein (MBP, 1 mg/ml) and [␥-32 P]ATP (0.22 Ci/sample), incubated at room temperature for 15 min, and subjected to SDS-PAGE. Each gel was stained with Coomassie Blue to assure equal loading of the MBP substrate and phosphorylation of MBP was visualized by autoradiography and quantitated by densitometry using the NIH Image computer program.

RESULTS
Both Tyrosines 113 and 128 of SLP-76 Are Required for the SLP-76/Vav Interaction-The interaction between SLP-76 and Vav depends on the SH2 domain of Vav and tyrosine phosphorylation of SLP-76 (28,29). Using a phosphopeptide library, Songyang et al. (34) found that the SH2 domain of Vav preferentially binds phosphorylated tyrosines in a Y*XXP motif (where Y* represents a phosphorylated tyrosine, X is any amino acid, and P is a proline residue). There are three tyrosines within SLP-76 which fall into such a motif: tyrosines 113 (YESP), 128 (YESP), and 145 (YEPP). Several lines of evidence suggest that Tyr-113 and Tyr-128 are good candidates for mediating the association between SLP-76 and Vav. First, both Tyr-113 and Tyr-128 are substrates of the TCR-stimulated PTKs (11). Second, a similar YESP motif found in both ZAP-70 and Syk is known to be responsible for the interaction of these PTKs with Vav (35,36). Finally, phosphorylated peptides corresponding to the two YESP motifs of SLP-76 precipitate Vav from cell lysates and inhibit the SLP-76/Vav interaction in vitro (28,37).
To investigate further the importance of Tyr-113, Tyr-128, and Tyr-145 in mediating the interaction between full-length SLP-76 and Vav in intact T cells, FLAG-tagged SLP-76 constructs encoding wild-type SLP-76 (WT) or point mutants with tyrosines 113, 128, or 145 individually changed to phenylalanine (Y113F, Y128F, and Y145F) were transfected into Jurkat T cells. Transfected cells were stimulated via their TCR, lysed, and precipitated with anti-FLAG (Fig. 1A) or GST-Vav-SH2 fusion protein (Fig. 1B). Each construct was expressed at similar levels as revealed by immunoblot analysis for the FLAG epitope ( Fig. 1, A, top panel; B, left). As shown previously, wild-type SLP-76 and each single tyrosine mutant is phosphorylated upon TCR stimulation (Fig. 1A, bottom panel). Surprisingly, however, mutation of either Tyr-113 or Tyr-128 abrogates the binding of SLP-76 to the SH2 domain of Vav (Fig. 1B). In contrast, wild-type SLP-76 and the Y145F mutant still bind to the Vav-SH2 (Fig. 1B). To obtain optimal phosphorylation of SLP-76, we also stimulated cells with pervanadate, a tyrosine phosphatase inhibitor and found again that the Y113F and Y128F SLP-76 mutants fail to associate with the SH2 domain of Vav (data not shown).
These in vitro findings were followed by experiments in intact Jurkat cells. For these studies, cells were co-transfected with Myc-tagged Vav and FLAG-tagged SLP-76 constructs. Transfected Jurkat cells were treated with pervanadate, lysed, and immunoprecipitated with anti-Myc antibody. As shown, an equal amount of Vav is present in each immune complex (Fig.  1C, top panel). However, both SLP-76 mutants Y113F and Y128F fail to co-immunoprecipitate with Myc-Vav, while wildtype SLP-76 and the Y145F mutant associate with Vav (Fig.  1C, bottom panel). Thus, in agreement with our in vitro finding, both tyrosines 113 and 128 of SLP-76 are required for the association of SLP-76 with the Vav SH2 in intact cells.
Synergy between Vav and SLP-76 Does Not Require Their Interaction-Although it is clear that SLP-76 and Vav synergize to couple TCR ligation with cytokine gene expression (29), the mechanism for this cooperation is not clear. One possibility is that SLP-76, as an adaptor molecule, recruits Vav to the proper subcellular location where Vav executes its role as a guanine nucleotide exchange factor. If this were true, the synergy between SLP-76 and Vav should require their interaction. To test this possibility, we transfected cDNA encoding Vav along with SLP-76 mutants Y113F and Y128F and assessed the effect on IL-2-NFAT promoter activity. To optimize conditions for observing synergy between SLP-76 and Vav, limiting amounts of cDNA encoding SLP-76 and Vav were transfected into cells. As shown, overexpression of SLP-76 or Vav alone at this level fails to augment IL-2-NFAT promoter activity. However, co-transfection of Vav with wild-type SLP-76, or either SLP-76/Y113F or SLP-76/Y128F augments IL-2-NFAT promoter activity significantly (Fig. 2). In each experiment, Western blot analysis demonstrated that all of the SLP-76 constructs were expressed at similar levels and that Vav was expressed similarly in each transfection condition (not shown).
Overexpression of SLP-76, but Not Vav, Augments TCR-in-duced AP-1 Activity-Our observation that the association between SLP-76 and Vav is not required for their functional cooperation suggests the possibility that these two proteins modulate different TCR-induced signaling cascades. We and others have found that when overexpressed individually, both SLP-76 and Vav augment activity of the NFAT region of the IL-2 promoter (9, 10). Work from several laboratories has shown that this region of the IL-2 promoter includes two components; a sequence which binds NFAT family members and a contiguous region which binds AP-1 dimers (reviewed in Ref. 38). Thus, modulating signals leading to either NFAT translocation and activity or AP-1 function could increase responsiveness of an IL-2 promoter-derived NFAT reporter (39 -41).
We have shown that in the Jurkat T cell line, SLP-76 overexpression augments TCR-induced activation of a ␤-galactosidase reporter driven by 5 copies of an AP-1 response element (17). To test whether overexpression of Vav has a similar impact on AP-1 function, we transfected Jurkat cells with the AP-1 reporter construct along with either SLP-76 or Vav and measured ␤-galactosidase activity in resting and stimulated cells. As shown in Fig. 3, TCR or PMA stimulation results in only a modest increase in AP-1 reporter activity in cells transfected with vector control. TCR stimulation of cells transfected with SLP-76 cDNA results in marked augmentation of reporter activity. As expected, SLP-76 overexpression does not impact on PMA-stimulated AP-1 function as SLP-76 is not tyrosine phosphorylated following PMA stimulation ( Fig. 3 and data not shown). In contrast to results seen with SLP-76, overexpression of Vav does not increase TCR stimulated AP-1 activity above that seen with the vector transfected control cells. Thus it appears that although both SLP-76 and Vav can modulate some TCR signals, those leading to AP-1 activation are affected by SLP-76 but not Vav.
SLP-76, but Not Vav, Leads to ERK Activation-There is considerable evidence to suggest that extracellular signal-regulated kinase (ERK) family members stimulated by TCR ligation function upstream of AP-1 activation (40, 42, 43). We have found previously that overexpression of SLP-76 augments TCR induced activity of ERK2 (17). To investigate if Vav has a similar ability to modulate TCR-stimulated ERK function, a Myc-tagged ERK2 construct was co-transfected into Jurkat cells along with cDNA encoding SLP-76 or Vav. Cells were left unstimulated or stimulated via the TCR or PMA. Cellular lysates were then subjected to immunoprecipitation with anti-Myc and in vitro kinase assays were performed on each immune complex using MBP as an artificial substrate. Phosphorylation of MBP was quantitated by densitometry and normalized to PMA-induced MBP phosphorylation. Whereas overexpression of SLP-76 consistently enhances TCR-induced kinase activity of ERK2 to a modest degree, overexpression of Vav does not affect ERK2 enzymatic activity (data not shown).
We reasoned that if SLP-76 affects AP-1 indirectly via augmentation of ERK activity, overexpression of SLP-76 and ERK together should lead to additive enhancement of AP-1 activity in TCR-, but not PMA-stimulated cells. Furthermore, since Vav does not appear to modulate TCR-stimulated ERK2, we reasoned that overexpression of Vav plus ERK2 would not result in AP-1 function above that seen with overexpression of ERK2 alone. We tested these hypotheses in the experiments shown in Fig. 4. For this study, the AP-1 reporter construct was transfected into Jurkat cells along with cDNAs for ERK2 alone or with either SLP-76 or Vav. As predicted, overexpression of ERK2, but not SLP-76, impacts PMA-stimulated AP-1 activity (Fig. 4A). In contrast, overexpression of either SLP-76 or ERK2 augments TCR-induced AP-1 activity (Fig. 4A). Consistent with our hypothesis, overexpression of the two molecules together leads to further augmentation of AP-1 activity in TCRstimulated Jurkat cells. However, when we perform the same AP-1 reporter analysis in lysates from cells transfected with Vav, we find no augmentation of promoter activity above that seen with vector control transfectants. Additionally, transfection with Vav plus ERK2 provides no greater AP-1 response than transfection with ERK2 alone (Fig. 4B). For each experiment, Western blot analysis was performed and showed expression of Myc-tagged ERK2 and Vav, or FLAG-tagged SLP-76 constructs (data not shown).
Vav, but Not SLP-76, Augments CD28-induced IL-2 Promoter Activity-Our experiments assessing the effect of SLP-76 or Vav on ERK and AP-1 indicate that SLP-76 modulates TCR-induced signaling pathways which are not affected by Vav. We wondered, therefore, if the converse is true as well. In T cells, signals initiated from the TCR cooperate with those arising from co-receptors, such as CD28, to optimize IL-2 gene transcription (44,45). Work from a number of investigators has shown that the TCR and CD28 stimulate transactivation of different regions within the IL-2 promoter (46 -48). A CD28response element within the IL-2 promoter, CD28RE, has been defined which contains binding sites for Rel family members in conjunction with other transcription factors (48 -50). In combination with TCR ligation or mitogenic stimulators such as PMA or phytohemagglutinin, engagement of CD28 induces these transcription factors to bind to and transactivate the CD28RE (48,49).
To investigate the potential roles of SLP-76 and Vav in CD28-stimulated IL-2 transcription, we performed reporter assays using either a full-length IL-2 promoter construct (which includes the CD28RE) or a reporter construct driven by four copies of the CD28RE. The full-length IL-2 promoter construct was co-transfected into Jurkat T cells with cDNAs encoding either SLP-76 or Vav (Fig. 5A). Transfected cells were then left untreated or treated with TCR plus PMA or CD28 plus PMA. As shown, although overexpression of either SLP-76 or Vav augments IL-2 promoter activity induced by stimulation via the TCR plus PMA, overexpression of Vav but not SLP-76 augments the CD28 plus PMA signal. PMA by itself is not able to induce IL-2 promoter activity and overexpression of either Vav or SLP-76 does not affect its function (data not shown).
Thus, it appears that Vav, but not SLP-76, can mediate the signaling pathways linking CD28 to IL-2 gene expression. Expression of transfected Vav or SLP-76 constructs were verified by Western blot (data not shown).
To test more directly whether Vav can regulate CD28RE activation following CD28 ligation, we performed experiments with the CD28RE reporter (Fig. 5B). Jurkat cells were transfected with this reporter along with a vector control, SLP-76, or Vav. Cells were left untreated or were stimulated via their TCR or with anti-CD28 plus PMA. Consistent with previous findings of others (48,50), TCR stimulation or PMA by itself fails to induce CD28RE activity in vector control transfectants (Fig.  5B). Additionally, we find that overexpression of neither SLP-76 nor Vav modifies the ability of the TCR or PMA to induce this reporter. Interestingly, however, transfection of Vav, but not SLP-76, augments CD28/PMA-induced CD28RE activation, again supporting the notion that Vav and SLP-76 differ in their ability to impact on CD28-stimulated signaling pathways.

DISCUSSION
Numerous signaling cascades function together to link antigen recognition to gene expression in T cells. Recently, a number of studies have demonstrated the importance of adaptor molecules in regulating this network (reviewed in Refs. [51][52][53][54]. Having no intrinsic enzymatic activity, adaptor molecules recruit other signaling proteins to form multimeric complexes through intermolecular associations. SLP-76 is one such adaptor protein which has been shown to bind Gads (12), Vav (28,29,37), SLAP-130 (FYB) (14,15), and other as yet unidentified molecules. Although these protein-protein interactions have been documented, their physiological importance remains unclear. Because SLP-76 and Vav function synergistically in their ability to augment TCR signaling leading to activation of the IL-2 gene (29), we initiated a series of experiments to map the tyrosines of SLP-76 important for the SLP-76/Vav interaction and to ask about the functional consequences of their binding. These experiments have led to a number of surprising findings.
We and others have shown previously that the association between SLP-76 and Vav is mediated by the Vav SH2 domain binding to tyrosine-phosphorylated residues within the SLP-76 amino terminus (28,29,37). In experiments presented in this study we found that although the binding is mediated by a single Vav SH2 domain, two SLP-76 tyrosines, Tyr-113 and Tyr-128, both appear essential for this association. This is particularly surprising since both Tyr-113 and Tyr-128 are found in identical sequence motifs (DYESP), either of which would be predicted to bind the Vav SH2. Although we are not yet sure why both SLP-76 tyrosines are required for Vav binding, several models come to mind. One possibility is that tyrosine phosphorylation of SLP-76 is sequential, and that phosphorylation of either tyrosine 113 or 128 is required for induced or maintained phosphorylation of the second residue. Thus, although only one tyrosine may be involved in direct binding to Vav, the second tyrosine is required for the phosphorylation of the Vav-binding tyrosine. However, while mutation of both tyrosines 113 and 128 abolishes TCR-induced tyrosine phosphorylation of SLP-76, mutants with either Tyr-113 or Tyr-128 mutated individually are still phosphorylated following TCR or pervanadate stimulation, making this explanation unlikely. A second possibility is that one phosphorylated tyrosine is responsible for directing the interaction with the Vav SH2 domain, while the second tyrosine is required for stabilizing this interaction, perhaps through maintaining a structural microenvironment or via an interaction with a third molecule. In this regard, SLP-76 was recently shown to simultaneously bind to the SH2 domains of Vav and the adapter protein, Nck, to form a trimolecular complex (55). Thus, it is possible that the SLP-76/Nck interaction is important for stabilizing the association between SLP-76 and Vav. Crystal or solution nuclear magnetic resonance determination of the structures of SLP-76, Vav, and Nck, when bound together, will be necessary to test this hypothesis.
On the surface, a second finding from our studies, that the synergy between SLP-76 and Vav does not appear to require their association, seems surprising. One potential explanation could be that the SLP-76 tyrosine mutants dimerize with endogenous, wild-type SLP-76 and thus interact with Vav indirectly. However, multiple experimental approaches in our laboratory have failed to demonstrate evidence for a SLP-76/ SLP-76 interaction, 2 making this explanation unlikely. Moreover, our result is consistent with several other observations. First, when SLP-76 mutants Y113F and Y128F are overexpressed at high levels, they augment IL-2-NFAT promoter activity to a level similar to that of wild-type SLP-76 (11), suggesting that in this assay, SLP-76 can function independently of its ability to bind Vav. Second, SLP-76 and Vav synergize in augmenting basal IL-2-NFAT promoter activity in Jurkat cells, although under these conditions, SLP-76 is not phosphorylated and does not bind Vav (29). Third, it was reported that in the T cell hybridoma DC27.10, where there is no detectable association between SLP-76 and Vav (even following TCR stimulation), TCR ligation still induces IL-2 production (37). Finally, we have found recently that mice made deficient in SLP-76 expression by homologous recombination have a much more profound defect in T cell development than do mice which lack expression of Vav, suggesting that SLP-76 and Vav do not function entirely in the same pathway (19,20,30,31,56). Collectively, these observations, together with the experiments presented in our current study, suggest that the Vav/ SLP-76 interaction is not essential for their functional synergy, at least for inducing IL-2 gene transcription. This conclusion led us to ask if SLP-76 and Vav may work together by modulating different signaling pathways in T cells.
There are several examples of how different pathways may enhance signals leading to IL-2 gene expression in T cells. One involves the cooperation between signals initiated from the TCR and those stimulated by engagement of CD28 (47). Another is the interdependence of signals leading to translocation 2 E. J. Peterson  of NFAT from the cytoplasm to the nucleus with other signals leading to AP-1 activation resulting in stimulating promoter elements within many cytokine genes (38). Our studies suggest that the synergy between SLP-76 and Vav may reflect the cooperation of different, but overlapping signals initiated from the TCR and CD28 as Vav, but not SLP-76 impacts on the ability of CD28 to augment IL-2 gene promoter activity. This finding is consistent with the observation that while Vav is one of the major substrates for CD28-induced PTKs (57), we find that SLP-76 is phosphorylated to only a very limited extent, if at all following CD28 ligation (data not shown). Our data suggest also that SLP-76 and Vav function in different TCRstimulated pathways as SLP-76, but not Vav couples the TCR with AP-1. This is consistent with the recent observation that activation of ERK following TCR ligation is diminished markedly in SLP-76-deficient Jurkat cells (18) while stimulation of this second messenger is retained in Vav-deficient T cells (56,58).
Further insight into the potential mechanisms by which SLP-76 exerts its effects on TCR-mediated signaling have come from studies of the SLP-76 defective Jurkat mutant. In this cell line, TCR engagement results in activation of PTKs, but fails to couple effectively with the phosphatidylinositol as well as Ras signaling pathways (18). Thus, it appears that SLP-76, via its adaptor function, is critical for allowing TCR-stimulated PTK activation to result in optimal phosphorylation of phospholipase C ␥1 in addition to signals leading to ERK activation. It remains to be determined, however, exactly which proteinprotein interactions mediated by SLP-76 are critical for these more downstream signals to occur.
Although the experiments in this study indicate that for TCR-stimulated IL-2 gene regulation, the interaction between SLP-76 and Vav does not appear to play a critical role, it is possible, and perhaps likely, that other T cell activation events are influenced by SLP-76 and Vav and require their interaction. For example, it has been reported that the interaction between SLP-76 and Vav may be required for TCR-induced PAK activation as well as the capping process (55). Since several studies have shown that Vav plays a critical role in formation of the cap following TCR stimulation (56,58,59), it will be important to study capping function in SLP-76-deficient T cells. We are currently working to reconstitute our SLP-76deficient mice with wild-type and mutant variants of SLP-76 to begin to address this and other related questions.