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• W2044749541 abstract "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. 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. T cell receptor protein-tyrosine kinase guanine nucleotide exchange factor Dbl homology Src homology 2 Src homology 3 glutathione S-transferase extracellular signal-regulated kinase calcineurin constitutively active Cn serum response element nuclear factor of activated T cells interleukin-2 phorbol 12-myristate 13-acetate monoclonal antibody polymerase chain reaction Antigen receptor engagement stimulates the activity of T cell receptor (TCR)1-coupled protein-tyrosine kinases (PTKs) of the Src and Syk families, which induce the assembly of large signaling complexes composed of cellular enzymes, adaptors, and other cytoplasmic transducers (1Weiss A. Littman D.R. Cell. 1994; 76: 263-274Abstract Full Text PDF PubMed Scopus (1955) Google Scholar, 2van Leeuwen J.E. Samelson L.E. Curr. Opin. Immunol. 1999; 11: 242-248Crossref PubMed Scopus (217) Google Scholar). These complexes initiate multiple signaling pathways coordinating the activation of immediate-early genes (3Treisman R. Curr. Opin. Genet. Dev. 1994; 4: 96-101Crossref PubMed Scopus (620) Google Scholar) and nuclear factors that control the transcription of several immunomodulatory genes, including the interleukin-2 (IL-2) gene (4Crabtree G.R. Cell. 1999; 96: 611-614Abstract Full Text Full Text PDF PubMed Scopus (669) Google Scholar). 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 (5Penninger J.M. Crabtree G.R. Cell. 1999; 96: 9-12Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). 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 (6Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3732) Google Scholar, 7Altman A. Deckert M. Adv. Immunol. 1999; 72: 1-101Crossref PubMed Google Scholar). 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 (8Collins T. Deckert M. Altman A. Immunol. Today. 1997; 18: 221-225Abstract Full Text PDF PubMed Scopus (96) Google Scholar). 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 (7Altman A. Deckert M. Adv. Immunol. 1999; 72: 1-101Crossref PubMed Google Scholar, 8Collins T. Deckert M. Altman A. Immunol. Today. 1997; 18: 221-225Abstract Full Text PDF PubMed Scopus (96) Google Scholar, 9Bustelo X.R. Mol. Cell. Biol. 2000; 20: 1461-1477Crossref PubMed Scopus (447) Google Scholar). 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 (10Fischer K.D. Zmuldzinas A. Gardner S. Barbacid M. Bernstein A. Guidos C. Nature. 1995; 374: 474-477Crossref PubMed Scopus (286) Google Scholar, 11Tarakhovsky A. Turner M. Schaal S. Mee P.J. Duddy L.P. Rajewsky K. Tybulewicz L.J. Nature. 1995; 374: 467-470Crossref PubMed Scopus (390) Google Scholar, 12Zhang R. Alt F.W. Davidson L. Orkin S.H. Swat W. Nature. 1995; 374: 470-473Crossref PubMed Scopus (374) Google Scholar). Vav1 activity is also required for TCR clustering and actin cytoskeleton reorganization (13Fischer K.D. Kong Y.Y. Nishina H. Tedford K. Marengere L.E. Kozieradzki I. Sasaki T. Starr M. Chan G. Gardener S. Nghiem M.P. Bouchard D. Barbacid M. Bernstein A. Penninger J.M. Curr. Biol. 1998; 8: 554-562Abstract Full Text Full Text PDF PubMed Google Scholar, 14Holsinger L.J. Graef I.A. Swat W. Chi T. Bautista D.M. Davidson L. Lewis R.S. Alt F.W. Crabtree G.R. Curr. Biol. 1998; 8: 563-572Abstract Full Text Full Text PDF PubMed Google Scholar), Ca2+ signaling, activation of mitogen-activated protein kinase ERKs and transcription factors NF-AT and NF-κB, up-regulation of CD69, and IL-2 or IL-4 production (13Fischer K.D. Kong Y.Y. Nishina H. Tedford K. Marengere L.E. Kozieradzki I. Sasaki T. Starr M. Chan G. Gardener S. Nghiem M.P. Bouchard D. Barbacid M. Bernstein A. Penninger J.M. Curr. Biol. 1998; 8: 554-562Abstract Full Text Full Text PDF PubMed Google Scholar, 14Holsinger L.J. Graef I.A. Swat W. Chi T. Bautista D.M. Davidson L. Lewis R.S. Alt F.W. Crabtree G.R. Curr. Biol. 1998; 8: 563-572Abstract Full Text Full Text PDF PubMed Google Scholar, 15Wu J. Katzav S. Weiss A. Mol. Cell. Biol. 1995; 15: 4337-4346Crossref PubMed Scopus (166) Google Scholar, 16Deckert M. Tartare-Deckert S. Couture C. Mustelin T. Altman A. Immunity. 1996; 5: 591-604Abstract Full Text PDF PubMed Scopus (245) Google Scholar, 17Costello P.S. Walters A.E. Mee P.J. Turner M. Reynolds L.F. Prisco A. Sarner N. Zamoyska R. Tybulewicz V.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3035-3040Crossref PubMed Scopus (215) Google Scholar, 18Krawczyk C. Bachmaier K. Sasaki T. Jones R.G. Snapper S.B. Bouchard D. Kozieradzki I. Ohashi P.S. Alt F.W. Penninger J.M. Immunity. 2000; 13: 463-470Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 19Villalba M. Hernandez J. Deckert M. Tanaka Y. Altman A. Eur. J. Immunol. 2000; 30: 1587-1596Crossref PubMed Scopus (52) Google Scholar, 20Villalba M. Coudronniere N. Deckert M. Teixeiro E. Mas P. Altman A. Immunity. 2000; 12: 151-160Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 21Hehner S.P. Li-Weber M. Giaisi M. Droge W. Krammer P.H. Schmitz M.L. J. Immunol. 2000; 164: 3829-3836Crossref PubMed Scopus (54) Google Scholar). Recent studies have identified other members of the Vav family, Vav2 and Vav3, which display a much broader tissue expression (9Bustelo X.R. Mol. Cell. Biol. 2000; 20: 1461-1477Crossref PubMed Scopus (447) Google Scholar). Vav2 transcripts are nearly ubiquitously expressed in mouse, from embryonic to adult stage (22Schuebel K.E. Bustelo X.R. Nielsen D.A. Song B.J. Barbacid M. Goldman D. Lee I.J. Oncogene. 1996; 13: 363-371PubMed Google Scholar). The enzymatic activities of all three isoforms are subjected to a phosphorylation-dependent regulation (9Bustelo X.R. Mol. Cell. Biol. 2000; 20: 1461-1477Crossref PubMed Scopus (447) Google Scholar). 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 (23Movilla N. Bustelo X.R. Mol. Cell. Biol. 1999; 19: 7870-7885Crossref PubMed Scopus (227) Google Scholar, 24Pandey A. Podtelejnikov A.V. Blagoev B. Bustelo X.R. Mann M. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 179-184Crossref PubMed Scopus (376) Google Scholar, 25Liu B.P. Burridge K. Mol. Cell. Biol. 2000; 20: 7160-7169Crossref PubMed Scopus (171) Google Scholar, 26Moores S.L. Selfors L.M. Fredericks J. Breit T. Fujikawa K. Alt F.W. Brugge J.S. Swat W. Mol. Cell. Biol. 2000; 20: 6364-6373Crossref PubMed Scopus (126) Google Scholar). 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 (27Schuebel K.E. Movilla N. Rosa J.L. Bustelo X.R. EMBO J. 1998; 17: 6608-6621Crossref PubMed Scopus (223) Google Scholar). In this regard, the morphological phenotypes induced by Vav1 and Vav2 expression in fibroblasts are distinct (27Schuebel K.E. Movilla N. Rosa J.L. Bustelo X.R. EMBO J. 1998; 17: 6608-6621Crossref PubMed Scopus (223) Google Scholar). However, these findings were not confirmed in another study (28Abe K. Rossman K.L. Liu B. Ritola K.D. Chiang D. Campbell S.L. Burridge K. Der C.J. J. Biol. Chem. 2000; 275: 10141-10149Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). 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 TCR-associated 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. 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 anti-phospho-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. Plasmid construction, cloning, and DNA sequencing were carried out according to standard protocols. The yeast two-hybrid constructs LexA-Syk, LexA-Fyn, LexA-lamin, GAD-Vav1, and GAD-raf were previously described (16Deckert M. Tartare-Deckert S. Couture C. Mustelin T. Altman A. Immunity. 1996; 5: 591-604Abstract Full Text PDF PubMed Scopus (245) Google Scholar, 29Deckert M. Tartare-Deckert S. Hernandez J. Rottapel R. Altman A. Immunity. 1998; 9: 595-605Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The cDNA encoding Myc-tagged Vav1 (16Deckert M. Tartare-Deckert S. Couture C. Mustelin T. Altman A. Immunity. 1996; 5: 591-604Abstract Full Text PDF PubMed Scopus (245) Google Scholar) 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 (19Villalba M. Hernandez J. Deckert M. Tanaka Y. Altman A. Eur. J. Immunol. 2000; 30: 1587-1596Crossref PubMed Scopus (52) Google Scholar). The NF-AT and IL-2 luciferase reporter plasmids have been described (29Deckert M. Tartare-Deckert S. Hernandez J. Rottapel R. Altman A. Immunity. 1998; 9: 595-605Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The c-fos SRE luciferase reporter plasmid was a kind gift from R. Janknecht (Rochester, MN) and has been described (30Janknecht R. Ernst W.H. Pingoud V. Nordheim A. EMBO J. 1993; 12: 5097-5104Crossref PubMed Scopus (508) Google Scholar). Mammalian expression vectors encoding Syk, Lck, Fyn, and ZAP-70 have been previously described (31Williams S. Couture C. Gilman J. Jascur T. Deckert M. Altman A. Mustelin T. Eur. J. Biochem. 1997; 245: 84-90Crossref PubMed Scopus (45) Google Scholar, 32Deckert M. Elly C. Altman A. Liu Y.-C. J. Biol. Chem. 1998; 273: 8867-8873Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). 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. 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)+ 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 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 mmsodium pyruvate, 10 mm HEPES, 1× 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 (16Deckert M. Tartare-Deckert S. Couture C. Mustelin T. Altman A. Immunity. 1996; 5: 591-604Abstract Full Text PDF PubMed Scopus (245) Google Scholar). Growth and transformation of the yeast strain L40 and the β-galactosidase filter assay were performed as previously described (16Deckert M. Tartare-Deckert S. Couture C. Mustelin T. Altman A. Immunity. 1996; 5: 591-604Abstract Full Text PDF PubMed Scopus (245) Google Scholar). Cells were stimulated for 5 min with 5 μg/ml of anti-CD3 mAb, washed twice, and lysed at 1 × 108 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 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride) for 15 min. Lysates were clarified by centrifugation at 15,000 × gfor 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 fusion proteins were expressed in BL21 bacterial cells and produced as described (33Sawka-Verhelle D. Baron V. Mothe I. Filloux C. White M.F. Van Obberghen E. J. Biol. Chem. 1997; 272: 16414-16420Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Jurkat cells (1 × 108 cells) were lysed in 1 ml of ice-cold lysis buffer for 20 min. After centrifugation, lysates were incubated with 5 μg 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. 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 μl of reporter lysis buffer (Promega). Luciferase activity was assayed by luminometry (Lumat, EG&G Berthold) using the Promegaluciferase assay system. Normalization of transfection efficiency was done using a co-transfected β-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. Jurkat-TAg cells were transfected with the indicated plasmids together with the pEGFP-N1 reporter plasmid (CLONTECH) 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 (19Villalba M. Hernandez J. Deckert M. Tanaka Y. Altman A. Eur. J. Immunol. 2000; 30: 1587-1596Crossref PubMed Scopus (52) Google Scholar). CD69 expression was analyzed by flow cytometry (FACScan, Becton Dickinson) after gating on GFP-positive cells. 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.1 A, 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 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.1 B). The distinct expression pattern of Vav1 and Vav2 might account for distinct functions of the two proteins in different cell types. 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.2 A, lanes 1–5). Tyrosine phosphorylation of Vav2 was also observed in pervanadate-treated Jurkat E6.1 T cells (Fig. 2 A, 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 determined by immunoblotting with an anti-Myc mAb (Fig. 2 B). Both Vav1 and Vav2 were found tyrosine-phosphorylated upon TCR stimulation (Fig. 2 B,upper panel, lanes 4 and 6). Of note, basal tyrosine phosphorylation of Vav1 and Vav2 could be detected in resting cells after prolonged exposure of the membrane (Fig.2 B, upper panel, lanes 3 and5, 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. 2 C, 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 β-galactosidase filter assay (16Deckert M. Tartare-Deckert S. Couture C. Mustelin T. Altman A. Immunity. 1996; 5: 591-604Abstract Full Text PDF PubMed Scopus (245) Google Scholar). GAD-Vav2 and GAD-Vav1 similarly interacted with LexA-Syk and LexA-Fyn (Fig. 2 D). 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 SH2-inactivating point mutation (R688Q) abolished the interaction between Vav2 and Syk (Fig. 2 D). As a negative control, no interactions between GAD-Vav2 constructs and a LexA DNA-binding 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. 3 A) 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.3 B, 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. 3 B, lane 2 versus lane 5). In contrast, none of these proteins bound the SH2-mutated GST-Vav2 protein or the GST alone (Fig. 3 B, 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. 3 B, 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 TCR-mediated cell activation. Vav1 plays an important role in the immune system as an inducer of gene transcription (8Collins T. Deckert M. Altman A. Immunol. Today. 1997; 18: 221-225Abstract Full Text PDF PubMed Scopus (96) Google Scholar, 9Bustelo X.R. Mol. Cell. Biol. 2000; 20: 1461-1477Crossref PubMed Scopus (447) Google Scholar), and a recent report indicated that Vav2 might play a similar role (26Moores S.L. Selfors L.M. Fredericks J. Breit T. Fujikawa K. Alt F.W. Brugge J.S. Swat W. Mol. Cell. Biol. 2000; 20: 6364-6373Crossref PubMed Scopus (126) Google Scholar). 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. 4 A, expression of Vav2 resulted in a significant increase of the activation of ERK1/2 following TCR stimulation (compare lanes 2–4 withlanes 6–8). Next, we compared the ability of Vav1 and Vav2 to promote ERK2 activation. 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. 4 B, 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.5 A). As a control, PMA plus ionomycin stimulation caused maximal SRE activation, which was not affected by Vav1 or Vav2 expression (Fig. 5 A). Proper expression of the transfected Vav proteins was confirmed by immunoblot analysis (Fig. 5 A, inset). Recently, we reported that Vav1 plays a role in the induction of the early activation marker CD69 (19Villalba M. Hernandez J. Deckert M. Tanaka Y. Altman A. Eur. J. Immunol. 2000; 30: 1587-1596Crossref PubMed Scopus (52) Google Scholar, 20Villalba M. Coudronniere N. Deckert M. Teixeiro E. Mas P. Altman A. Immunity. 2000; 12: 151-160Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). 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. 5 B). 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. 5 B). Taken together, our results indicate that Vav2 shares with Vav1 the ability to activate pathways that stimulate ERK" @default.
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• W2044749541 date "2001-06-01" @default.
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