Vav2 activates c-fos serum response element and CD69 expression but negatively regulates nuclear factor of activated T cells and interleukin-2 gene activation in T lymphocyte.

Vav1 and Vav2 are members of the Dbl family of guanine nucleotide exchange factors for the Rho family of small GTPases. Although the role of Vav1 during lymphocyte development and activation is well characterized, the function of Vav2 is still unclear. In this study, we compared the signaling pathways regulated by Vav1 and Vav2 following engagement of the T cell receptor (TCR). We show that Vav2 is tyrosine-phosphorylated upon TCR stimulation and by co-expressed Src and Syk family kinases. Using glutathione S-transferase fusion proteins, we observed that the Src homology 2 domain of Vav2 binds tyrosine-phosphorylated proteins from TCR-stimulated Jurkat T cell lysates, including c-Cbl and SLP-76. Like Vav1, Vav2 cooperated with TCR stimulation to increase extracellular signal-regulated kinase activation and to promote c-fos serum response element transcriptional activity. Moreover, both proteins displayed a similar action in increasing the expression of the early activation marker CD69 in Jurkat T cells. However, in contrast to Vav1, Vav2 dramatically suppressed TCR signals leading to nuclear factor of activated T cells (NF-AT)-dependent transcription and induction of the interleukin-2 promoter. Vav2 appears to act upstream of the phosphatase calcineurin because a constitutively active form of calcineurin rescued the effect of Vav2 by restoring TCR-induced NF-AT activation. Interestingly, the Dbl homology and Src homology 2 domains of Vav2 were necessary for its inhibitory effect on NF-AT activation and for induction of serum response element transcriptional activity. Taken together, our results indicate that Vav1 and Vav2 exert overlapping but nonidentical functions in T cells. The negative regulatory pathway elicited by Vav2 might play an important role in regulating lymphocyte activation processes.

Src and Syk families, which induce the assembly of large signaling complexes composed of cellular enzymes, adaptors, and other cytoplasmic transducers (1,2). These complexes initiate multiple signaling pathways coordinating the activation of immediate-early genes (3) and nuclear factors that control the transcription of several immunomodulatory genes, including the interleukin-2 (IL-2) gene (4).
Optimal T cell activation requires the formation of a T cell synapse. This process depends on reorganization of the actin cytoskeleton, which is controlled by small GTPases of the Rho/ Rac family (5). Upon stimulation by extracellular signals, these proteins cycle from an inactive, GDP-bound form to an active, GTP-bound enzyme that translocates to the membrane and interacts with downstream effector proteins. These proteins, in turn, regulate adhesion, motility, and gene transcription in lymphocytes and other cell types (6,7).
In lymphocytes, the activation-dependent exchange of GDP for GTP is regulated by the guanosine nucleotide exchange factor (GEF) Vav1. Vav1 contains several functional domains, including a Dbl homology (DH) domain, a Pleckstrin homology domain, a cysteine-rich domain, one Src homology 2 (SH2) domain, and two Src homology 3 (SH3) domains (8). Vav1 expression is mostly restricted to hematopoietic cells, and different studies have shown that it is a critical link between TCR-coupled PTKs of the Src and Syk families and the signaling pathways regulated by Rho/Rac proteins (7)(8)(9). Analysis of Vav1-deficient mice indicated that Vav1 is required for T cell development and antigen receptor-mediated T or B lymphocytes activation or apoptosis (10 -12). Vav1 activity is also required for TCR clustering and actin cytoskeleton reorganization (13,14), Ca 2ϩ signaling, activation of mitogen-activated protein kinase ERKs and transcription factors NF-AT and NF-B, up-regulation of CD69, and IL-2 or IL-4 production (13)(14)(15)(16)(17)(18)(19)(20)(21).
Recent studies have identified other members of the Vav family, Vav2 and Vav3, which display a much broader tissue expression (9). Vav2 transcripts are nearly ubiquitously expressed in mouse, from embryonic to adult stage (22). The enzymatic activities of all three isoforms are subjected to a phosphorylation-dependent regulation (9). Tyrosine phosphorylation of Vav proteins can be induced through the stimulation of different receptors, including immune recognition receptors, cytokine receptors, integrins, and PTK receptors such as the epidermal growth factor and the platelet-derived growth factor receptors (23)(24)(25)(26). Despite the fact that each member of the Vav family can induce cytoskeletal reorganization and transform rodent fibroblasts, their catalytic specificity toward Rho/Rac proteins appears to differ. Whereas Vav1 displays GEF activity for Rac1, Cdc42, RhoA, and RhoG, Vav2 was shown to exhibit GEF activity for RhoA, RhoB, and RhoG but not for Rac1 or Cdc42 (27). In this regard, the morphological phenotypes induced by Vav1 and Vav2 expression in fibroblasts are distinct (27). However, these findings were not confirmed in another study (28). Together these studies suggest that the Vav family might use overlapping but nonidentical signal transduction pathways.
Although the physiological role of Vav1 during lymphocyte development and activation is well established, the function of Vav2 is poorly documented. In particular, it is not known whether Vav1 and Vav2 elicit similar responses in lymphocytes. Here, we compared the involvement of Vav1 and Vav2 in TCR signaling. We show that Vav2 shares with Vav1 several biological features, including tyrosine phosphorylation by TCRassociated PTKs of the Src and Syk families, binding to tyrosine-phosphorylated c-Cbl and SLP-76, and a positive effect on activation of ERKs, c-fos serum response element (SRE), and CD69 expression. However, in contrast to Vav1, Vav2 negatively regulates TCR-induced NF-AT and IL-2 gene activation. We also demonstrate that Vav2 functions upstream of calcineurin (Cn) and that the intact DH and SH2 domains of Vav2 are required for activation of c-fos SRE and inhibition of NF-AT induction. Therefore, Vav1 and Vav2, two closely related members of the Vav family, are functionally distinct in promoting gene activation in T cells.

MATERIALS AND METHODS
Antibodies and Reagents-The anti-CD3 monoclonal antibody (mAb) OKT3 was purified from the corresponding hybridoma supernatant by protein A-Sepharose chromatography. The anti-phosphotyrosine (Tyr(P)) and the anti-Myc mAbs were derived from the 4G10 and 9E10 hybridomas, respectively. The anti-hemagglutinin mAb (12CA5) was from Roche Molecular Biochemicals. The anti-SLP-76 mAb was provided by P. Findell (Palo Alto, CA). The anti-human Vav2 mAb was a kind gift from J. Downward (London, UK). The anti-ERK1/2 polyclonal and anti-Cbl mAb were from Santa Cruz Biotechnology, Inc. The antiphospho-ERK polyclonal antibody and anti-human Vav1 mAb were obtained from Upstate Biotechnology, Inc. The phycoerythrin-conjugated anti-human CD69 mAb was from PharMingen. Culture media and oligonucleotides were from Life Technologies, Inc. Chemicals were obtained from Sigma, and enzymes were from New England Biolabs, Inc.
DNA Constructs-Plasmid construction, cloning, and DNA sequencing were carried out according to standard protocols. The yeast twohybrid constructs LexA-Syk, LexA-Fyn, LexA-lamin, GAD-Vav1, and GAD-raf were previously described (16,29). The cDNA encoding Myctagged Vav1 (16) was cloned into the pCDNA3 vector (Invitrogen). The cDNA encoding Vav2 (amino acids 1-868) was amplified by polymerase chain reaction (PCR) from a cDNA library of mouse liver and cloned into pCDNA3-Myc vector (Invitrogen) or pACT2 vector (CLONTECH) yielding Vav2-Myc and GAD-Vav2, respectively. PCRs were performed using the thermostable Pwo DNA polymerase (Roche Molecular Biochemicals). Myc-ERK2 has been described elsewhere (19). The NF-AT and IL-2 luciferase reporter plasmids have been described (29). The c-fos SRE luciferase reporter plasmid was a kind gift from R. Janknecht (Rochester, MN) and has been described (30). Mammalian expression vectors encoding Syk, Lck, Fyn, and ZAP-70 have been previously described (31,32). A bacterial expression plasmid of a GST fusion protein containing the SH3-SH2-SH3 domains of Vav2 was generated by PCR amplification of nucleotides 1797-2604 (encoding amino acid residues 599 -868) from the pCDNA3-Vav2-Myc vector, followed by in-frame insertion into the pGEX-3X plasmid (Amersham Pharmacia Biotech). Mutants were created with the Quick Change site-directed mutagenesis kit (Stratagene), and mutations were verified by DNA sequence analysis.
Reverse Transcriptase-PCR Analysis-Sets of primers were designed for specific PCR amplification of human Vav1 or Vav2 fragments. These primers were used to screen a panel of cDNAs generated using poly(A) ϩ RNA from different human tissues (CLONTECH). PCRs were performed with Superscript DNA polymerase (Life Technologies, Inc.), and GAPDH amplification was used as an internal control. Products were analyzed on a 1.5% agarose gel, stained with ethidium bromide, and photographed using UV light.
Cell Culture, Transfection, and Yeast Manipulation-Cell lines were obtained from American Type Culture Collection. Cells were grown in RPMI 1640 medium (Life Technologies, Inc.), supplemented with 10% fetal bovine serum, 2 mM glutamine, 1 mM sodium pyruvate, 10 mM HEPES, 1ϫ minimum Eagle's medium nonessential amino acids solution (Life Technologies, Inc.), and 100 units/ml each of penicillin G and streptomycin. Simian virus 40 T antigen-transfected human leukemic Jurkat T cells (Jurkat-TAg) were kindly provided by G. Crabtree (Stanford, CA). Jurkat (clone JE6.1) and Jurkat-TAg cells were transfected with the indicated plasmids by electroporation as described previously (16). Growth and transformation of the yeast strain L40 and the ␤-galactosidase filter assay were performed as previously described (16).
Immunoprecipitation and Immunoblotting-Cells were stimulated for 5 min with 5 g/ml of anti-CD3 mAb, washed twice, and lysed at 1 ϫ 10 8 cells/ml in ice-cold lysis buffer (1% Nonidet P-40 in 150 mM NaCl, 50 mM HEPES, pH 7.4, 5 mM NaF, 5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 10 g/ml aprotinin, 10 g/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride) for 15 min. Lysates were clarified by centrifugation at 15,000 ϫ g for 10 min at 4°C, and protein concentration was determined using the bicinchoninic acid protein assay (Pierce). Cleared lysates were incubated for 3 h at 4°C with the indicated antibodies and protein G-Sepharose beads (Sigma). Pellets were then washed three times with ice-cold lysis buffer containing 0.2% Nonidet P-40 and resuspended in SDS sample buffer. Eluted immunoprecipitates or whole cell lysates were separated by SDS-polyacrylamide gel electrophoresis and analyzed by immunoblotting. Reactive proteins were visualized by ECL.
GST Pulldown Assays-GST fusion proteins were expressed in BL21 bacterial cells and produced as described (33). Jurkat cells (1 ϫ 10 8 cells) were lysed in 1 ml of ice-cold lysis buffer for 20 min. After centrifugation, lysates were incubated with 5 g of the indicated fusion proteins for 3 h at 4°C, followed by incubation with glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) for 1 h. Bound proteins were washed four times with 1 ml of lysis buffer and resuspended in SDS sample buffer. Samples were resolved by SDS-polyacrylamide gel electrophoresis and analyzed by immunoblotting.
Reporter Assays-For luciferase assays, transfected Jurkat cells were left unstimulated or stimulated with anti-CD3 mAb for the indicated times, as described in the legend to each figure. Cells were washed twice in phosphate-buffered saline, pH 7.2, and lysed in 100 l of reporter lysis buffer (Promega). Luciferase activity was assayed by luminometry (Lumat, EG&G Berthold) using the Promega luciferase assay system. Normalization of transfection efficiency was done using a co-transfected ␤-galactosidase expression vector. Luciferase activity was determined in triplicate and expressed as fold increase relative to the basal activity seen in unstimulated mock-transfected cells.
For ERK2 phosphorylation assays, cells were lysed in lysis buffer containing 1% Triton X-100 in place of Nonidet P-40. After centrifugation, the supernatants were incubated overnight at 4°C with antibodies to Myc and protein G-Sepharose beads. Immunoprecipitates were resolved on SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose, and the membranes were probed with antibodies to phospho-ERK according to the manufacturer's instructions. After stripping, the membranes were reprobed with antibodies to Myc to confirm equal levels of ERK2 in the immunoprecipitates.
CD69 Expression-Jurkat-TAg cells were transfected with the indicated plasmids together with the pEGFP-N1 reporter plasmid (CLON-TECH) and cultured for 24 h at 37°C. Cells were incubated with medium alone or with PMA (50 ng/ml) for the final 18 h of culture and stained with a phycoerythrin-conjugated anti-human CD69 mAb as described (19). CD69 expression was analyzed by flow cytometry (FACScan, Becton Dickinson) after gating on GFP-positive cells.

Expression Pattern of Vav2 in Human Cells and Tissues from
Immune Origin-To address the role of Vav2 in the immune system, we first examined its expression pattern by reverse transcriptase-PCR analysis on various immune tissues. As shown in Fig. 1A, the Vav2 message was predominantly expressed in spleen, fetal liver, tonsil, and peripheral blood leukocytes and to a lesser extend in thymus and bone marrow. As

Vav2 Functions in Lymphocyte
previously reported, Vav1 message was detected in all the immune tissues tested. We also examined the expression of Vav2 at the protein level in different hematopoietic cell lines. Vav2 was well expressed in several human T (Jurkat, CEM, HPBall) or B (88.66, Raji, SKW6.4) cell lines but poorly expressed in the monocytic cell line U937 (Fig. 1B). The distinct expression pattern of Vav1 and Vav2 might account for distinct functions of the two proteins in different cell types.
Interaction of Vav2 with T Cell Signaling Molecules-Jurkat T cells are a widely used model for studying TCR signaling. Therefore, we examined whether Vav2 undergoes tyrosine phosphorylation following TCR stimulation. Jurkat E6.1 T cells were stimulated with an anti-CD3 mAb, and after immunoprecipitation with Vav2-specific antibodies, the level of tyrosine phosphorylation was assayed by immunoblotting with antibodies to Tyr(P). Increased Vav2 tyrosine phosphorylation was detectable after 30 s of stimulation, with peak levels detected 2-5 min following TCR ligation ( Fig. 2A, lanes 1-5). Tyrosine phosphorylation of Vav2 was also observed in pervanadatetreated Jurkat E6.1 T cells ( Fig. 2A, lane 6). We then compared the abilities of Vav1 and Vav2 to become tyrosine-phosphorylated upon TCR stimulation. To do this, Jurkat-TAg cells were transfected with the Myc-tagged forms of Vav1 or Vav2 and stimulated with an anti-CD3 mAb. The level of tyrosine phosphorylation was assayed by immunoblotting cell lysates and anti-Myc immunoprecipitates with an anti-Tyr(P) mAb. Similar expression levels of the transfected proteins were deter-mined by immunoblotting with an anti-Myc mAb (Fig. 2B). Both Vav1 and Vav2 were found tyrosine-phosphorylated upon TCR stimulation (Fig. 2B, upper panel, lanes 4 and 6). Of note,  6). Cells were lysed, and proteins were immunoprecipitated using an anti-Myc mAb. Immunoprecipitated proteins were immunoblotted with antibodies to Tyr(P) (top panel) or to Myc (bottom panel). The mobility of Vav2 protein is indicated by arrows. D, interaction of Vav2 with Syk and Fyn in the yeast two-hybrid system. Syk or Fyn cDNAs were fused to the DNA-binding domain of LexA, whereas the indicated Vav2 constructs were fused to the Gal4 activation domain (GAD). Yeast (L40) was co-transformed with the indicated plasmids combinations, and interactions were assayed using a ␤-galactosidase filter test. The panel summarizes three independent experiments. Ϫ, no interaction; ϩϩϩ, strong interaction. Co-expression of GAD-Vav1 with LexA-Syk or LexA-Fyn is shown as a positive control. Co-expression of GAD constructs with LexA-lamin is shown as a negative control. Vav2 599 -868 consists of the two SH3 domains and the SH2 domain of Vav2. R688Q variant has a mutation in a conserved arginine residue within the SH2 domain.

Vav2 Functions in Lymphocyte
basal tyrosine phosphorylation of Vav1 and Vav2 could be detected in resting cells after prolonged exposure of the membrane (Fig. 2B, upper panel, lanes 3 and 5, and data not shown). To identify candidate PTKs involved in the TCR-mediated phosphorylation of Vav2, we co-transfected Jurkat-TAg cells with Vav2 and Lck, Fyn, Syk, or Zap-70. As shown in Fig. 2C, Vav2 was prominently phosphorylated in intact T cells by Lck, Fyn, and Syk and to a lesser extent by Zap-70. These results indicate that, like Vav1, Vav2 is tyrosine-phosphorylated by T cell nonreceptor PTKs.
Next, we used the yeast two-hybrid system to further examine the physical interaction of Vav2 with Syk and Fyn. The interactions in co-transformed L40 yeast cells were monitored by a ␤-galactosidase filter assay (16). GAD-Vav2 and GAD-Vav1 similarly interacted with LexA-Syk and LexA-Fyn (Fig.  2D). As expected, a construct containing the C-terminal SH3-SH2-SH3 domain of Vav2 (Vav2 599 -868) still interacted with Syk. The SH2 domain of Vav2 was required because an SH2inactivating point mutation (R688Q) abolished the interaction between Vav2 and Syk (Fig. 2D). As a negative control, no interactions between GAD-Vav2 constructs and a LexA DNAbinding domain fused to lamin were detected.
Finally, we assessed the nature of proteins interacting with the SH2 domain of Vav2 in lymphocytes. Two GST-Vav2 fusion proteins were generated (Fig. 3A) and used in GST pull-down assays. When GST-Vav2 599 -868 was incubated with lysates of resting Jurkat cells, it associated with two major tyrosine-phosphorylated proteins of 120 and 62 kDa, respectively (Fig.  3B, lane 2). TCR stimulation did not significantly increase the association of these two proteins with Vav2, but it induced an additional interaction with a 75-kDa Tyr(P)-containing protein (Fig. 3B, lane 2 versus lane 5). In contrast, none of these proteins bound the SH2-mutated GST-Vav2 protein or the GST alone (Fig. 3B, lanes 1, 3, 4, and 6). Probing the membranes with a panel of antibodies to different candidate proteins allowed us to identified pp120 and pp75 as c-Cbl and SLP-76, respectively (Fig. 3B, two lower panels, lane 5). Together, these results suggest that Vav2 may play an important role as a component of the signaling complex assembled during TCRmediated cell activation.
Vav2 Promotes Activation of ERKs and SRE and Expression of CD69 in T Cells-Vav1 plays an important role in the immune system as an inducer of gene transcription (8,9), and a recent report indicated that Vav2 might play a similar role (26). Therefore, we wished to compare the impact of Vav1 and Vav2 overexpression on several TCR-mediated downstream activation events, beginning with ERK activation. First, we examined the activation of endogenous ERK proteins by Vav2 in Jurkat E6.1 cells. Cells were transfected with the Myc-tagged form of Vav2 or empty vector and stimulated with an anti-CD3 mAb for different times. Activation of endogenous ERK1 and 2 was monitored by immunoblotting with a phospho-ERK-specific antibody. As shown in Fig. 4A, expression of Vav2 resulted in a significant increase of the activation of ERK1/2 following TCR stimulation (compare lanes 2-4 with lanes 6 -8). Next, we compared the ability of Vav1 and Vav2 to promote ERK2 acti-

Vav2 Functions in Lymphocyte
vation. Jurkat-TAg cells were co-transfected with a Myc-tagged ERK2 reporter, plus Vav1-Myc or Vav2-Myc. The transfected cells were left unstimulated or stimulated with an anti-CD3 antibody. Expression of MEK1 was used as a positive control for ERK2 activation. Activation of ERK2 was monitored by immunoblotting with a phospho-ERK-specific antibody. As shown in Fig. 4B, expression of Vav1 and Vav2 induced no significant activation of ERK2 in unstimulated T cells (lanes 3 and 5). However, either Vav1 or Vav2 further increased ERK2 activation following TCR stimulation (lanes 4 and 6).
Next, we examined whether Vav2 overexpression results in stimulation of c-fos SRE transcriptional activity. We transfected Jurkat-TAg cells with Myc-tagged Vav1 or Vav2 along with a luciferase reporter driven by SRE-binding sequences. Similarly both Vav1 and Vav2 induced a marked increase of either the basal or TCR-stimulated activities of SRE reporter plasmid (4-and 8-fold increase over basal activity, respectively) (Fig. 5A). As a control, PMA plus ionomycin stimulation caused maximal SRE activation, which was not affected by Vav1 or Vav2 expression (Fig. 5A). Proper expression of the transfected Vav proteins was confirmed by immunoblot analysis (Fig. 5A,  inset).
Recently, we reported that Vav1 plays a role in the induction of the early activation marker CD69 (19,20). Therefore, we examined the possibility that Vav2 could mediate a similar function in T cells. Jurkat-TAg cells were transfected with Myc-tagged Vav1 or Vav2 together with a pEF-GFP reporter plasmid and stimulated or not with PMA. CD69 expression of GFP-gated cells was then determined by FACScan analysis. Interestingly, Vav2 overexpression led to a 4-fold increase of CD69 expression in the absence of stimulation (Fig. 5B). This effect was similar to the one induced by Vav1. Moreover, Vav2, like Vav1, further increased PMA stimulation to induce the surface expression of CD69 (Fig. 5B). Taken together, our results indicate that Vav2 shares with Vav1 the ability to activate pathways that stimulate ERKs, SRE, and up-regulation of CD69 expression in T cells.
Vav2 Inhibits TCR-induced NF-AT and IL-2 Promoter Activation-Vav1 plays a crucial role in the signaling pathways leading to NF-AT activation in T cells (13,15,16). To further explore the function of Vav2 in T cell signaling, we transfected Jurkat-TAg cells with Vav1 or Vav2 along with luciferase reporters driven by the complete IL-2 promoter or its NF-ATbinding sequences. As reported earlier (15,16), overexpression of Vav1 increased the basal and TCR-stimulated activity of both NF-AT and IL-2 promoter (Fig. 6, A and B). However, in contrast to Vav1, Vav2 expression suppressed the TCR-induced NF-AT and IL-2 promoter activation. Vav2 reduced the TCRinduced activation of NF-AT and IL-2 promoter by 80 and 60%, respectively. However, Vav2 had no significant effect on the basal activities of these two reporters (Fig. 6, A and B). Immunoblotting with anti-Myc mAb revealed similar expression levels of the two Vav proteins (Fig. 6B, inset). We also examined the effect of Vav2 on NF-AT activation following TCR stimulation in the parental Jurkat clone E6.1. As shown in Fig. 6C, Vav2 reduced TCR-induced activation of NF-AT by 80%, ruling out the possibility that the effect of Vav2 on NF-AT was due to a cell type effect. As a control, PMA plus ionomycin stimulation caused maximal NF-AT activation, which was not affected by Vav2 overexpression, either in Jurkat-TAg (Fig. 6A) or in Jurkat E6.1 cells (Fig. 6C, inset). Of note, Vav2 was also capable of blocking the stimulatory effect of Vav1 on NF-AT activation (data not shown). These data provide evidence that Vav2 exerts a negative regulatory effect on TCR-induced NF-AT and IL-2 promoter activation.
Vav2 Functions Upstream of Cn to Inhibit TCR-induced NF-AT Activation-Cn is a calcium-activated serine/threonine phosphatase that promotes the dephosphorylation of NF-AT and, thereby, its nuclear translocation and activation (4). Because NF-AT activation by Vav1 is sensitive to cyclosporin A (15) and Vav1 modulates Ca 2ϩ signaling pathways (17), Cn function has been implicated in the Vav1 signaling pathway leading to activation of the IL-2 gene. These findings prompted us to examine whether the suppression of TCR-mediated NF-AT activation by Vav2 can be rescued by co-expression of a constitutively active Cn mutant (CA-Cn). As shown in Fig. 7A, expression of CA-Cn increased the basal activity of NF-AT in Jurkat-TAg cells and further enhanced anti-CD3 stimulation. Interestingly, the blocking effect of Vav2 on TCR-induced NF-AT activation was not observed when CA-Cn was co-expressed with Vav2. Of note, under these conditions, a 2-fold increase in the basal activity of NF-AT was observed, suggesting the existence of a functional interaction between the two proteins in T cells. The absence of the Vav2 inhibitory effect was not due to decreased protein expression as revealed by immunoblotting with the relevant antibodies (Fig. 7B). These results indicate that Vav2 functions upstream of Cn to inhibit TCR-induced NF-AT activation.
Involvement of DH and SH2 Domains of Vav2 in Regulation of NF-AT and SRE Activities-The activation of distinct signaling pathways by Vav1 requires different structural domains

Vav2 Functions in Lymphocyte
of the protein, including the catalytic DH domain and the SH2 domain (8). Because these two domains are highly conserved in Vav2, we were interested in determining their potential involvement in Vav2-mediated transcriptional activities. To inactivate the DH and the SH2 domains, we introduced point mutations in the conserved leucine 212 and arginine 688 residues, respectively. The Myc-tagged mutated forms of Vav2 were transfected into Jurkat-TAg cells along with NF-AT-or SRE-luciferase reporters. Wild-type Vav2 reduced the TCRinduced activation of NF-AT by 60% (Fig. 8A). In contrast, overexpression of the SH2-mutated Vav2 (R688Q) had a minimal effect on the TCR-induced NF-AT activation (15% reduction), whereas overexpression of DH-mutated Vav2 (L212A) slightly increased NF-AT activation (Fig. 8A). We also examined the effect of these two mutations on the ability of Vav2 to increase SRE activity. As shown before (Fig. 5A), Vav2 increased the basal and TCR-stimulated activities of SRE. However, the ability of the L212A or R688Q Vav2 mutants to stimulate the SRE reporter was severely impaired (Fig. 8B). Immunoblotting with antibodies to Myc confirmed the identical expression of the different proteins (Fig. 8C). These results indicate that activation of gene transcription and suppression of TCR-induced NF-AT activation by Vav2 requires its intact DH and SH2 domains. DISCUSSION Recently, new members of the Vav family of Rho/Rac GEFs have been identified, but little is known about their functions during T cell activation. In this study, we investigated the role of Vav2 in TCR-stimulated T cells. Tyrosine phosphorylation of Vav proteins is thought to play a critical role in stimulating their catalytic activation (7-9). Vav2 and other Vav proteins are tyrosine-phosphorylated following stimulation of diverse membrane receptors, including the epidermal and plateletderived growth factor receptors (24 -26) and lymphocyte antigen receptors (23,26). Consistent with the latter studies, we observed that Vav2 was tyrosine-phosphorylated following TCR stimulation and that this phosphorylation can be mediated by co-expressed PTKs of the Src (Lck and Fyn) and Syk (Zap-70 and Syk) families in Jurkat T cells. Moreover, we showed that Vav2 interacts with Syk and Fyn via its SH2 domain. Thus, similar mechanisms appear to be involved in the recruitment and activation of Vav1 and Vav2 induced by TCR engagement. Although additional studies are required to determine whether the interaction between Vav2 and members of the Src and Syk families is direct, it is likely that TCR engagement stimulates the catalytic activity of Vav2 through direct phosphorylation by Src and/or the Syk PTKs. Therefore, our findings connect Vav2 (in addition to Vav1) to TCR signaling pathways.
TCR engagement generates multiple signaling pathways that promote the activation of immediate early genes (i.e. c-fos and egr-1) and nuclear factors contributing to cytokine gene expression (3,4). The best characterized Vav family member, Vav1, plays a critical role in TCR signal transduction during lymphocyte development and activation (10 -12). Analysis of vav1 Ϫ/Ϫ mice indicated that Vav1 activity is required for TCR capping and cytoskeleton reorganization, maximal Ca 2ϩ signaling, ERK activation, and IL-2 gene induction (13,14,17). However, it is unclear whether other Vav family members can compensate for the absence of Vav1 and stimulate at least a subset of the Vav1-dependent pathways (13, 14, 17). A very recent study suggested that other Vav proteins may substitute for Vav1 function in pathways involved in TCR capping, actin cytoskeleton reorganization, and lipid raft clustering (18). However, the identity of the relevant Vav protein was not determined in this study.
Our studies provide evidence that Vav1 and Vav2 exert overlapping but nonidentical functions in T cells. Indeed, we showed that, similar to Vav1, Vav2 cooperates with TCR stimulation to activate ERKs and c-fos SRE and increases the expression of CD69. There was no significant difference in the extent of c-fos SRE activation in response to either Vav1 or Vav2. Transcriptional activation of c-fos involves several control elements, one of which, the SRE, is regulated by the activation of a Ras/MEK/ERK pathway (34). Because the transcription factors that bind SRE or AP-1-binding sites are major targets for ERK pathways, our studies suggest that Vav2 may trigger in T cells an ERK pathway involved in the activation of c-fos SRE and CD69 expression. Considering the broad tissue expression of Vav2, it would be interesting to investigate whether Vav2 is also involved in the activation of ERK-dependent pathways in cells of nonhematopoietic origins.
Our work also illustrates a major difference between Vav1 and Vav2 in the way they regulate the transcription factor NF-AT and the IL-2 gene. NF-AT proteins represent a large family of Ca 2ϩ /Cn-dependent nuclear factors, which are involved in the regulation of several cytokine genes, including IL-2 (4). During the preparation of this manuscript, two studies reported that Vav2 does not activate NF-AT in Jurkat T cells (26,35). In agreement with these reports, we found that Vav2 did not affect the basal activity of NF-AT. However, our study revealed that Vav2 blocked the activation of NF-AT induced by TCR stimulation. As a direct consequence of this inhibition, the TCR-stimulated activity of the IL-2 promoter was also severely impaired in T cells overexpressing Vav2. Moreover, our results imply that Vav2 might suppress NF-AT activation by interfering with early TCR signals proximal to Cn activation. However, this role of Vav2 was observed when the protein was transiently overexpressed in Jurkat cells. When we compared the expression level of Vav1 and Vav2, we found that Vav1 and Vav2 proteins could be simultaneously expressed in native leukocytes and in various immune cell lines, including Jurkat cells. Although the determination of the exact stoichiometry of the two molecules might require specific experimentations such as Real-Time PCR analyses, our results indicate that Vav2 was expressed in Jurkat cells at a lower level relative to Vav1 expression. Thus, ascertaining Vav2 functions in T cells will require further experimentations in native lymphocytes and/or animal models. Nevertheless, our observations may partially account for the loss of TCR-induced NF-AT and IL-2 gene activation in T cells from vav1 Ϫ/Ϫ mice (13) and the inability of other Vav proteins to rescue NF-AT/IL-2 gene activation in the Vav1ϫCbl-b double knockout mice (18). Taken together, our results further support the notion that Vav1 and Vav2 are functionally distinct in promoting gene activation.
A major question is how two highly homologous proteins such as Vav1 and Vav2 can simultaneously exert antagonistic effects on Cn/NF-AT-dependent pathways and similar effects on ERK-dependent pathways? Cantrell and co-workers (36) have shown that TCR-stimulated pathways leading to NF-AT and ERK2 activation diverge early after Ras activation in T cells, at a level implicating the GTPase Rac. Another recent study has shown that Vav1 and Vav2 have distinct specificities toward Rac and Rho GTPases (27). Although these findings have not been confirmed in another study (28), such a differential recruitment of Rho-regulated and Rac-regulated pathways by individual Vav family members may provide one explanation for the observed different biological responses in T cells. Our finding that the GEF activity of Vav2 is required for NF-AT inhibition strongly suggests that this is an active process and further supports the above notion. The use of dominant-negative mutants of different small GTPases of the Rho/ Rac family should help to determine whether preferential activation of Rho versus Rac GTPases by Vav2 underlies the inhibition of NF-AT by Vav2.
Our results also indicate that the SH2 domain of Vav2 is required for its function in T cells. Thus, an alternative mechanism for the inhibition of NF-AT activity is that an SH2-dependent interaction of Vav2 with either stimulatory or inhibitory proteins modulates TCR signals to uncouple NF-AT activation from ERK activation, thereby blocking IL-2 gene activation but enhancing c-fos SRE transcription and CD69 expression. This second mechanism is supported by our observations that TCR stimulation increased the association of Vav2 with proteins reminiscent of those interacting with Vav1, including Syk, Fyn, c-Cbl, and SLP-76 (2,8,37). The association of Vav2 with SLP-76 might play a critical role in Vav2-mediated signaling pathways in T cells. Indeed, SLP-76 is a hematopoietic-specific adapter protein that is tyrosine-phosphorylated by ZAP-70 following TCR engagement. Phosphorylated SLP-76 associates with a large number of signal transducers, including Vav1, LAT, Nck, Grb2, and the Grb2-related protein Gads (38). SLP-76 cooperates with Vav1 to induce NF-AT activity (39), and it is also involved in the activation of ERK and AP-1 in T cells (40 -42). These observations raise the possibility that association of Vav2 with SLP-76 may account for the Vav2 Functions in Lymphocyte positive effect of Vav2 on a Ras-regulated pathway leading to ERK and SRE activation. We consistently found that an SH2inactive Vav2 mutant, which does not bind SLP-76, failed to activate SRE (and also NF-B; data not shown), suggesting that binding to SLP-76 may be critical for Vav2 function in T cells. Moreover, the expression of CD69, a known target of the Ras pathway in T cells (43)(44)(45), was also up-regulated by Vav2. Because a Ras-dependent pathway is required for CD69 upregulation by Vav1 in T cells (19), Vav2 may also promote an increase of CD69 expression by recruiting the Ras pathway through a direct association with SLP-76.
Another important signal transducer in T cells, which we found to be associated with the SH2 domain of Vav2 is c-Cbl. This finding might provide a possible explanation for the negative regulation of NF-AT and IL-2 transcription by Vav2. c-Cbl is a negative regulator of PTK-mediated signaling pathways, and it inhibits Syk family PTK-dependent signals in hematopoietic cells (46 -50). However, recent studies have shown that c-Cbl plays an important role during T cell development but not during mature T cell activation in the periphery (51,52). Conversely, the Cbl-related protein Cbl-b negatively regulates mature T cell activation (53,54). In addition, Cbl-b mutation in mouse T cells has revealed compensation of Vav1 function by other Vav proteins (18). Therefore, it would be interesting to determine whether Vav2 also associates Cbl-b in mature T cells. Finally, we cannot rule out the possibility that Vav2 interacts with yet unidentified regulatory proteins that uncouple NF-AT activation from ERK activation, thereby blocking IL-2 gene activation. In this regard, it would be of interest to identify the phosphoprotein at 62 kDa that we found interacting with Vav2.
Taken together, our results show that, whereas Vav1 displays a positive action on TCR-induced signaling pathways, Vav2 uncouples the activation of a ERK/SRE/CD69 pathway from a NF-AT/IL-2 gene activation pathway. In support of this notion, a recent study has shown that different Vav1 functions can be uncoupled in the Vav1ϫCbl-b double knockout mice, reflecting potential compensation by other Vav proteins (18). Although the physiological relevance of the Vav2-regulated signaling pathways are presently unknown, our results raise the possibility that Vav2 negatively regulates major aspects of T cell activation associated with IL-2 production. Alternatively, the balance between expression levels of different Vav proteins could determine T cell fates by fine tuning TCR-mediated gene activation. Further studies are required to determine the relative expression and role of each Vav isoform during T cell development and activation. Finally, the identification of proteins that specifically interact with different Vav proteins should help to understand the functions of the Vav family in immune cell signaling pathways.