Vav1 Acidic Region Tyrosine 174 Is Required for the Formation of T Cell Receptor-induced Microclusters and Is Essential in T Cell Development and Activation

Vav proteins are multidomain signaling molecules critical for mediating signals downstream of several surface receptors, including the antigen receptors of T and B lymphocytes. The catalytic guanine nucleotide exchange factor (GEF) activity of the Vav Dbl homology (DH) domain is thought to be controlled by an intramolecular autoinhibitory mechanism involving an N-terminal extension and phosphorylation of tyrosine residues in the acidic region (AC). Here, we report that the sequences surrounding the Vav1 AC: Tyr142, Tyr160, and Tyr174 are evolutionarily conserved, conform to consensus SH2 domain binding motifs, and bind several proteins implicated in TCR signaling, including Lck, PI3K p85α, and PLCγ1, through direct interactions with their SH2 domains. In addition, the AC tyrosines regulate tyrosine phosphorylation of Vav1. We also show that Tyr174 is required for the maintenance of TCR-signaling microclusters and for normal T cell development and activation. In this regard, our data demonstrate that while Vav1 Tyr174 is essential for maintaining the inhibitory constraint of the DH domain in both developing and mature T cells, constitutively activated Vav GEF disrupts TCR-signaling microclusters and leads to defective T cell development and proliferation. Vav proteins are multidomain signaling molecules critical for mediating signals downstream of several surface receptors, including the antigen receptors of T and B lymphocytes. The catalytic guanine nucleotide exchange factor (GEF) activity of the Vav Dbl homology (DH) domain is thought to be controlled by an intramolecular autoinhibitory mechanism involving an N-terminal extension and phosphorylation of tyrosine residues in the acidic region (AC). Here, we report that the sequences surrounding the Vav1 AC: Tyr142, Tyr160, and Tyr174 are evolutionarily conserved, conform to consensus SH2 domain binding motifs, and bind several proteins implicated in TCR signaling, including Lck, PI3K p85α, and PLCγ1, through direct interactions with their SH2 domains. In addition, the AC tyrosines regulate tyrosine phosphorylation of Vav1. We also show that Tyr174 is required for the maintenance of TCR-signaling microclusters and for normal T cell development and activation. In this regard, our data demonstrate that while Vav1 Tyr174 is essential for maintaining the inhibitory constraint of the DH domain in both developing and mature T cells, constitutively activated Vav GEF disrupts TCR-signaling microclusters and leads to defective T cell development and proliferation. The cells of the αβ T cell lineage progress through a developmental program that links ordered V(D)J gene rearrangement and antigen receptor protein expression to further developmental progression in which CD4–8– cells give rise to CD4+8+ cells that are precursors to CD4+8– or CD4–8+ single positive thymocytes (reviewed in Ref. 1Borowski C. Martin C. Gounari F. Haughn L. Aifantis I. von Grassi F. Boehmer H. Curr. Opin. Immunol. 2002; 14: 200-206Crossref PubMed Scopus (66) Google Scholar). In developing and mature T cells, the T cell receptor (TCR) 2The abbreviations used are: TCR, T cell receptor; ITAM, immunoreceptor tyrosine-based activation motif; AC, acidic region; CH, calponin homology; DH, Dbl homology; GEF, guanine nucleotide exchange factor; GFP, green fluorescent protein; PH, pleckstrin homology; PIP3, phosphatidylinositol 3,4,5-trisphosphate; SH2, Src homology 2; SH3, Src homology 3; TIRFM, total internal reflection fluorescence microscopy; GST, glutathione S-transferase; PI3K, phosphatidylinositol 3-kinase; PLC, phospholipase C; IL, interleukin; WT, wild type. 2The abbreviations used are: TCR, T cell receptor; ITAM, immunoreceptor tyrosine-based activation motif; AC, acidic region; CH, calponin homology; DH, Dbl homology; GEF, guanine nucleotide exchange factor; GFP, green fluorescent protein; PH, pleckstrin homology; PIP3, phosphatidylinositol 3,4,5-trisphosphate; SH2, Src homology 2; SH3, Src homology 3; TIRFM, total internal reflection fluorescence microscopy; GST, glutathione S-transferase; PI3K, phosphatidylinositol 3-kinase; PLC, phospholipase C; IL, interleukin; WT, wild type. activates Src family kinases that phosphorylate immunoreceptor tyrosine-based activation motifs (ITAMs) in CD3/TCRζ proteins, providing docking sites for Syk/ZAP-70 family protein-tyrosine kinases. Subsequently, the recruitment of the adaptors LAT, GADS, and SLP-76, and enzymes such as Tec family kinases, phosphoinositol 3-kinase (PI3K), phospholipase Cγ1 (PLCγ1), and Vav family guanine nucleotide exchange factors (GEF) leads to generation of the secondary signaling intermediates, 1,4,5-inositol trisphosphate (IP3) and diacylglycerol (DAG), and activation of intracellular Ca2+, Rho GTPases, and mitogen-activated protein kinases (MAPK) (reviewed in Ref. 2Kane L.P. Lin J. Weiss A. Curr. Opin. Immunol. 2000; 12: 242-249Crossref PubMed Scopus (425) Google Scholar). Together, these events promote the transcription of genes involved in T cell proliferation, differentiation, and cytoskeletal reorganization. Live cell imaging studies using T cells stimulated on peptide:MHC-containing planar bilayers or on anti-CD3 antibodies immobilized on a planar surface revealed formation of microclusters of signaling proteins, including the TCR, ZAP-70, LAT, and SLP-76, within seconds of contact (3Barda-Saad M. Braiman A. Titerence R. Bunnell S.C. Barr V.A. Samelson L.E. Nat. Immunol. 2005; 6: 80-89Crossref PubMed Scopus (260) Google Scholar, 4Bunnell S.C. Hong D.I. Kardon J.R. Yamazaki T. McGlade C.J. Barr V.A. Samelson L.E. J. Cell Biol. 2002; 158: 1263-1275Crossref PubMed Scopus (502) Google Scholar), and recent reports suggested that TCR signal transduction may be initiated and sustained within these dynamically regulated microclusters (5Campi G. Varma R. Dustin M.L. J. Exp. Med. 2005; 202: 1031-1036Crossref PubMed Scopus (453) Google Scholar, 6Yokosuka T. Sakata-Sogawa K. Kobayashi W. Hiroshima M. Hashimoto-Tane A. Tokunaga M. Dustin M.L. Saito T. Nat. Immunol. 2005; 6: 1253-1262Crossref PubMed Scopus (557) Google Scholar). The Vav family of Rho GEFs consists of three members: Vav1, Vav2, and Vav3, which are expressed in both T and B lymphocytes. Vav proteins contain multiple domains, including a calponin homology (CH) domain, an acidic region (AC), a catalytic Dbl homology domain (DH), a pleckstrin homology domain (PH), a cysteine-rich region (CR), and a Src homology 2 (SH2) domain flanked by two SH3 domains. The importance of Vav1 in lymphocytes was first demonstrated in mice lacking Vav1 (Vav1–/–), which show activation defects in T and B lymphocytes (7Fischer K.D. Zmuldzinas A. Gardner S. Barbacid M. Bernstein A. Guidos C. Nature. 1995; 374: 474-477Crossref PubMed Scopus (286) Google Scholar, 10Turner M. Mee P.J. Walters A.E. Quinn M.E. Mellor A.L. Zamoyska R. Tybulewicz V.L. Immunity. 1997; 7: 451-460Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar), γδ T cells (11Swat W. Xavier R. Mizoguchi A. Mizoguchi E. Fredericks J. Fujikawa K. Bhan A.K. Alt F.W. Int. Immunol. 2003; 15: 215-221Crossref PubMed Scopus (22) Google Scholar), and NK cells (12Colucci F. Rosmaraki E. Bregenholt S. Samson S.I. Di Bartolo V. Turner M. Vanes L. Tybulewicz V. Di Santo J.P. J. Exp. Med. 2001; 193: 1413-1424Crossref PubMed Scopus (72) Google Scholar, 16Graham D.B. Cella M. Giurisato E.K.F. Miletic A.V. Kloeppel T. Brim K. Takai T. Shaw A.S. Colonna M. Swat W. J. Immunol. 2006; 177: 2349-2355Crossref PubMed Scopus (73) Google Scholar). However, Vav1–/– lymphocytes retain significant functional ability, presumably because of redundancy between Vav family members. Indeed, mice with combined Vav deficiencies show more severe lymphocyte defects (17Fujikawa K. Miletic A.V. Alt F.W. Faccio R. Brown T. Hoog J. Fredericks J. Nishi S. Mildiner S. Moores S.L. Brugge J. Rosen F.S. Swat W. J. Exp. Med. 2003; 198: 1595-1608Crossref PubMed Scopus (195) Google Scholar, 19Doody G.M. Bell S.E. Vigorito E. Clayton E. McAdam S. Tooze R. Fernandez C. Lee I.J. Turner M. Nat. Immunol. 2001; 2: 542-547Crossref PubMed Scopus (156) Google Scholar), whereas mice lacking all three Vav proteins (VavNULL) lack functional T and B lymphocytes (17Fujikawa K. Miletic A.V. Alt F.W. Faccio R. Brown T. Hoog J. Fredericks J. Nishi S. Mildiner S. Moores S.L. Brugge J. Rosen F.S. Swat W. J. Exp. Med. 2003; 198: 1595-1608Crossref PubMed Scopus (195) Google Scholar). Control of Vav GEF activity is complex and involves multiple modes of regulation (reviewed in Ref. 20Schmidt A. Hall A. Genes Dev. 2002; 16: 1587-1609Crossref PubMed Scopus (977) Google Scholar). Structural studies suggested that autoinhibitory intramolecular interactions between the CH domain and the CR region and the AC and the DH domain are responsible for maintaining the Vav DH domain in an inactive state (21Aghazadeh B. Lowry W.E. Huang X.Y. Rosen 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, E. J. 2005; PubMed Scopus Google Scholar). mechanism for such an autoinhibitory constraint is phosphorylation of three AC Tyr142, Tyr160, and which in of the autoinhibitory N-terminal extension and the DH domain (21Aghazadeh B. Lowry W.E. Huang X.Y. Rosen 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, Rosen 2005; PubMed Scopus Google Scholar). that tyrosine phosphorylation Vav the of Rho GEFs Barbacid M. Nature. PubMed Scopus Google Scholar, B. S. W. A. Weiss A. J. Nature. PubMed Scopus Google Scholar), is that Vav a complex mechanism for regulation of GEF activity in which tyrosine phosphorylation a of Vav proteins to downstream of antigen receptors in lymphocytes. also suggested that binding of the Vav domain to may to regulation of GEF phosphatidylinositol binding to the domain of Vav may to activation of DH domain whereas phosphatidylinositol binding suggested to regulate DH domain activity J. K. B. J.R. PubMed Scopus Google Scholar). in which binding to the domain may interactions between the Vav domain and the DH domain, of protein-tyrosine kinases to the AC with the phosphorylation of AC tyrosines and activation of GEF activity (21Aghazadeh B. Lowry W.E. Huang X.Y. Rosen 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). In this regard, GEF activity is in a of Vav1 in which the domain is of with A. Vanes L. S. C. Tybulewicz Immunity. 2005; Full Text Full Text PDF PubMed Scopus (22) Google Scholar). In addition, binding of proteins to the N-terminal CH domain of Vav may to activation of the DH of the protein with the CH domain of implicated in activation of M. 2002; PubMed Scopus (22) Google Scholar). In this we show that the tyrosine residues in the Vav1 AC are evolutionarily and conform to consensus SH2 domain binding motifs for several proteins implicated in TCR signaling Lck, PI3K p85α, and Indeed, Tyr142, Tyr160, and Tyr174 bind these proteins through direct interactions with their SH2 domains. the AC tyrosines are essential for total tyrosine phosphorylation of Vav1. data also that Tyr174 is critical for Vav1 and for normal T cell development and because the disrupts TCR of Cell and cell was A. J. 2002; PubMed Scopus Google Scholar). cell Vav1 AC and Vav1 expression cells with for cells and by of an with was by and by stimulated with and and in with a and was antibodies with secondary antibodies by protein by of and in of for by of by and for of with a and and of proteins by for with in and by with antibodies cell T cells in for on by and for by of by for by of and of with of with for as and for and with for and as of and was using as in Ref. M. K. Saito K. T. 1997; PubMed Scopus Google cells in with as S.C. Barr V.A. Samelson L.E. 2003; 177: Scholar, S.C. V. W. Samelson L.E. Immunity. 2001; 14: Full Text Full Text PDF PubMed Scopus Google Scholar). a state was an for using an with an and using and using was as in Ref. 6Yokosuka T. Sakata-Sogawa K. Kobayashi W. Hiroshima M. Hashimoto-Tane A. Tokunaga M. Dustin M.L. Saito T. Nat. Immunol. 2005; 6: 1253-1262Crossref PubMed Scopus (557) Google In an was through a cell and was to all of a using Subsequently, the fluorescence of microclusters within the was as of fluorescence of single microclusters a was microclusters and was to all of a with the first in which the was to the of the of a within the was in Cell and mice (17Fujikawa K. Miletic A.V. Alt F.W. Faccio R. Brown T. Hoog J. Fredericks J. Nishi S. Mildiner S. Moores S.L. Brugge J. Rosen F.S. Swat W. J. Exp. Med. 2003; 198: 1595-1608Crossref PubMed Scopus (195) Google and in the of of to Cell and with antibodies The used and on a with Cell single of of in was mice and cells in and with cell factor and 2 in the cells and of cells by mice with and with a cell and T Cell and T cells stimulated with antibodies as and was as in Ref. K. Miletic A.V. Alt F.W. Faccio R. Brown T. Hoog J. Fredericks J. Nishi S. Mildiner S. Moores S.L. Brugge J. Rosen F.S. Swat W. J. Exp. Med. 2003; 198: 1595-1608Crossref PubMed Scopus (195) Google T cells for in lacking with anti-CD3 antibodies for 2 and the was using the to the Vav1 AC and to SH2 Vav1 AC of several was by that sequences surrounding AC Tyr160, and to the are within several and including and AC tyrosines are within consensus binding motifs for the SH2 of several proteins implicated in TCR signaling, including the Src family the of PI3K and is is or and Full Text PDF PubMed Google Scholar, 2003; PubMed Scopus Google Scholar). data that the AC tyrosines may be involved in Vav interactions with these SH2 proteins TCR AC for of tyrosines implicated in regulation of Vav tyrosine phosphorylation and in T lymphocytes M. Lee H. C. Cell Biol. 2000; PubMed Scopus Google Scholar, P.J. J. Immunol. 2000; PubMed Scopus Google Scholar), and as phosphorylation sites for Src and family protein-tyrosine kinases Rosen 2005; PubMed Scopus Google Scholar, J. K. B. J.R. PubMed Scopus Google Scholar, M. S. C. T. A. Immunity. Full Text PDF PubMed Scopus Google Scholar). the AC tyrosines in T cells as SH2 domain docking we first these residues are T cell activation using antibodies that Tyr142, Tyr160, or with T cells A. J. 2002; PubMed Scopus Google in which Vav1 was by or with Vav1 of the AC we that with studies A. Vanes L. S. C. Tybulewicz Immunity. 2005; Full Text Full Text PDF PubMed Scopus (22) Google Scholar, M. Lee H. C. Cell Biol. 2000; PubMed Scopus Google Vav1 AC tyrosines T cell activation and the AC tyrosines are required for tyrosine phosphorylation of Vav1 in activated T cells, cell with or AC Vav1 stimulated with and tyrosine phosphorylation of Vav1 was by with total antibodies that while of single AC tyrosine on Vav1 of Tyr174 in with Tyr160, in phosphorylation of Vav1 of all three AC tyrosines leads to a of Vav1 total tyrosine phosphorylation in to the AC are tyrosine residues of the AC that conform to consensus tyrosine phosphorylation motifs 2003; PubMed Scopus Google Scholar). we cells activated by TCR with anti-CD3 antibodies that these are an of Vav tyrosine phosphorylation is by of Vav protein expression because the cell a to Vav1 and data Together, these data that the AC tyrosines are critical for phosphorylation of Vav1, that the AC tyrosines may as docking sites for kinases such as to phosphorylate Vav In this tyrosine residues of the AC may be to protein-tyrosine kinases as may be or may be by intramolecular the AC tyrosines to Vav1 tyrosine phosphorylation and may be required for protein-tyrosine Vav1 AC SH2 of TCR-signaling AC tyrosine interactions with the SH2 of Lck, PI3K p85α, and PLCγ1, we first binding of the AC of Vav1 or Tyr142, Tyr160, or Tyr174 to of Lck, PI3K p85α, PLCγ1, or a positive we used the binding of or to the SH2 domain of and J. J. J. Biol. Full Text Full Text PDF PubMed Scopus Google and M. 2006; PubMed Scopus Google Scholar). show as on the binding of the AC tyrosines bind to the SH2 domain of Lck, the SH2 domain of PLCγ1, or to of the two SH2 of PI3K In the N-terminal SH2 domain of with of the AC tyrosines the SH2 domain of with the of the SH2 domain for the and Ref. H. S.E. J. Immunol. 1995; PubMed Scopus Google Scholar). these data that Vav1 AC tyrosines bind SH2 of several proteins implicated in TCR signaling, with a recent report of the SH2 domain with AC tyrosines Rosen 2005; PubMed Scopus Google Scholar). the AC tyrosines with SH2 proteins in their we using the AC and In these we used the between and as a positive and Ref. J. J. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). binding of Tyr142, Tyr160, and Tyr174 AC to protein we that PI3K and bind to AC tyrosines to bind to of the Vav1 AC tyrosines in these while of Vav with Lck, PI3K p85α, and by C. T. A. J. Tybulewicz V.L. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, T. J. Tybulewicz V.L. J. Exp. Med. 2002; PubMed Scopus Google Scholar), these data the direct binding of Vav with SH2 proteins AC Vav1 of Vav1 TCR imaging studies activation of T cells on planar microclusters of signaling proteins the sites of TCR (3Barda-Saad M. Braiman A. Titerence R. Bunnell S.C. Barr V.A. Samelson L.E. Nat. Immunol. 2005; 6: 80-89Crossref PubMed Scopus (260) Google Scholar, 4Bunnell S.C. Hong D.I. Kardon J.R. Yamazaki T. McGlade C.J. Barr V.A. Samelson L.E. J. Cell Biol. 2002; 158: 1263-1275Crossref PubMed Scopus (502) Google Scholar). that within these signaling TCR signals to tyrosine phosphorylation of signaling proteins, generation of and of the and are initiated and sustained (5Campi G. Varma R. Dustin M.L. J. Exp. Med. 2005; 202: 1031-1036Crossref PubMed Scopus (453) Google Scholar, 6Yokosuka T. Sakata-Sogawa K. Kobayashi W. Hiroshima M. Hashimoto-Tane A. Tokunaga M. Dustin M.L. Saito T. Nat. 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Vav is involved in the generation of signaling we the of Vav1 in T cells using cells or with of Tyr142, Tyr160, or Tyr174 cells by TIRFM, which imaging of in single cells in the direct of on we that Vav1 microclusters the within of contact and that such microclusters are as a cell and the importance of Vav1 AC Tyr174 for total Vav tyrosine we to the for Tyr174 for In this regard, we that in to microclusters are and TCR the within of contact by which show that in to the of of is as these microclusters B and The of microclusters by the Vav tyrosine phosphorylation because be to Vav1 and In this Tyr174 may be required for the maintenance of Vav1 for with signaling and microclusters TCR and of all three AC tyrosines to further of defects with of Tyr174 a for Tyr174 for normal formation of Vav1 Vav1 for T Cell and TCR that Tyr174 is required for formation of Vav1 microclusters we to this is required for T cell development and T cell proliferation. this we T cells that in the of Vav proteins to the of of Vav We a cell in which we expressed or Vav1 the and that expression T cell as a of thymocytes and T lymphocytes are and data In thymocytes show total and of cells Tyr174 is required in of T cells, we of such T cells and that in to T cells or T cells with these cells to in to anti-CD3 protein is expressed the as Vav1 in normal T cells that these developmental and functional defects are by protein Together, these that Tyr174 is essential for T cell development and for in T our with because of expression of the protein in this Vav1 Vav GEF Tyr174 is and as a docking for signaling proteins of this the of Vav1 to bind to TCR-signaling proteins, in microclusters and TCR defects in T cells may be by activation of Vav1 DH domain GEF activity because Tyr174 implicated in inhibitory constraint of GEF activity M. Lee H. C. Cell Biol. 2000; PubMed Scopus Google Scholar, M. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). this we to activation in T cells in the or of anti-CD3 Indeed, we that expression of to which is in T cells, in to T cells data that the of the may be by activation of Vav GEF we Vav1 formation using a which also a and M. Barbacid M. Google and Nature. 1997; PubMed Scopus Google Scholar). that the of the the defects in formation of to Vav1, microclusters and in to Together, these data show the for Tyr174 in the of Vav1 DH domain activity (21Aghazadeh B. Lowry W.E. Huang X.Y. Rosen 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, Rosen 2005; PubMed Scopus Google Scholar), a for Tyr174 in TCR Vav1 was the first proteins in signaling downstream of the TCR Barbacid M. Nature. PubMed Scopus Google Scholar, B. S. W. A. Weiss A. J. Nature. PubMed Scopus Google Scholar). In all three Vav proteins be and are to the TCR antigen receptor (17Fujikawa K. Miletic A.V. Alt F.W. Faccio R. Brown T. Hoog J. Fredericks J. Nishi S. Mildiner S. Moores S.L. Brugge J. Rosen F.S. Swat W. J. Exp. Med. 2003; 198: 1595-1608Crossref PubMed Scopus (195) Google Scholar). tyrosine phosphorylation Vav family proteins the of DH Rho the mechanism of Vav regulation by tyrosine phosphorylation is was that AC Tyr142, Tyr160, and Tyr174 bind to and tyrosine kinases such as and Rosen 2005; PubMed Scopus Google Scholar, A. Vanes L. S. C. Tybulewicz Immunity. 2005; Full Text Full Text PDF PubMed Scopus (22) Google Scholar, M. Lee H. C. Cell Biol. 2000; PubMed Scopus Google Scholar, M. S. C. T. A. Immunity. Full Text PDF PubMed Scopus Google is the AC tyrosines are required for tyrosine phosphorylation of Vav as reports J. K. B. J.R. PubMed Scopus Google Scholar, M. Lee H. C. Cell Biol. 2000; PubMed Scopus Google Scholar, G. Weiss A. J. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). that an Vav and the be by in that Tyr174 is required for Vav phosphorylation J. K. B. J.R. PubMed Scopus Google Scholar), whereas studies that Tyr174 is required for phosphorylation of Vav M. Lee H. C. Cell Biol. 2000; PubMed Scopus Google Scholar, G. Weiss A. J. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, P.J. J. Immunol. 2000; PubMed Scopus Google Scholar). In this using a in which Vav1 is expressed we show that Tyr142, Tyr160, and Tyr174 are critical for regulation of tyrosine phosphorylation of Vav1, because combined of all three AC tyrosines to a of Vav1 a total of tyrosine in to the AC tyrosines are within consensus motifs for tyrosine phosphorylation 2003; PubMed Scopus Google Scholar). However, of these residues may be or may be involved in intramolecular tyrosine an between and is for the of the N-terminal SH3 domain of Vav1 M. K. M. K. V. J. F. J. 2001; PubMed Scopus Google Scholar, K. K. M. E. T. S. M. H. F. J. 2002; PubMed Scopus Google Scholar). phosphorylation of the acidic region tyrosines may Tyr142, Tyr160, and Tyr174 to docking sites for kinases, such as Lck, which phosphorylate tyrosine residues in Vav Rosen 2005; PubMed Scopus Google Scholar). is also that phosphorylation of Tyr142, Tyr160, Tyr174 in of Vav1 that Vav1 tyrosine residues to to protein-tyrosine this tyrosine phosphorylation of the AC tyrosines in the of the regulation of the GEF studies in and cell suggested that phosphorylation of AC tyrosine residues may be required for regulation of activity of the GEF activity (21Aghazadeh B. Lowry W.E. Huang X.Y. Rosen 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, Rosen 2005; PubMed Scopus Google Scholar, M. Lee H. C. Cell Biol. 2000; PubMed Scopus Google Scholar, M. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). whereas expression of leads to of antigen expression of a constitutively Vav1 the CH M. Lee H. C. Cell Biol. 2000; PubMed Scopus Google Scholar, G. Weiss A. J. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). is that the Vav CH domain binding to proteins may Vav to signals downstream of the TCR to activation of the GEF in this report that Tyr174 to a of Vav downstream of the TCR by the catalytic activity of the DH domain in through phosphorylation with SH2 In to required for phosphorylation of we that the AC tyrosines as docking sites for SH2 TCR-signaling proteins including Lck, PI3K p85α, and PLCγ1, with a of Vav as a a NK cell the receptor signaling D.B. Cella M. Giurisato E.K.F. Miletic A.V. Kloeppel T. Brim K. Takai T. Shaw A.S. Colonna M. Swat W. J. Immunol. 2006; 177: 2349-2355Crossref PubMed Scopus (73) Google Scholar, C.J. P.J. Nat. Immunol. 2006; 7: PubMed Scopus Google Scholar), may AC tyrosine interactions to the of this to be In this in T cells for the AC tyrosines for binding to PI3K p85α, Lck, or whereas studies that Vav1 to is the SH2 domain R. Brown J. J. J. Exp. Med. 2000; PubMed Scopus Google Scholar). is that the AC tyrosines are for formation of a TCR-signaling because the TCR is of proteins that several proteins in to Vav1, in binding in the of Tyr142, Tyr160, Tyr174 may be in studies using cell imaging of T cells activated on planar revealed the formation of microclusters of signaling proteins including the TCR, ZAP-70, LAT, SLP-76, and within seconds of contact (3Barda-Saad M. Braiman A. Titerence R. Bunnell S.C. Barr V.A. Samelson L.E. Nat. Immunol. 2005; 6: 80-89Crossref PubMed Scopus (260) Google Scholar, 6Yokosuka T. Sakata-Sogawa K. Kobayashi W. Hiroshima M. Hashimoto-Tane A. Tokunaga M. Dustin M.L. Saito T. Nat. Immunol. 2005; 6: 1253-1262Crossref PubMed Scopus (557) Google Scholar, S.C. V. W. Samelson L.E. Immunity. 2001; 14: Full Text Full Text PDF PubMed Scopus Google Scholar). In this regard, is thought that signal transduction may be initiated and sustained within such microclusters (5Campi G. Varma R. Dustin M.L. J. Exp. Med. 2005; 202: 1031-1036Crossref PubMed Scopus (453) Google Scholar, 6Yokosuka T. Sakata-Sogawa K. Kobayashi W. Hiroshima M. Hashimoto-Tane A. Tokunaga M. Dustin M.L. Saito T. Nat. Immunol. 2005; 6: 1253-1262Crossref PubMed Scopus (557) Google Scholar). in this report show that while Vav1 microclusters to TCR-signaling proteins, the in defects in for these is that Tyr174 is required for Vav1 maintenance by mediating of Vav with SH2 signaling molecules for TCR However, a of the is that the of Tyr174 leads to GEF activity in TCR with the and with reports M. Lee H. C. Cell Biol. 2000; PubMed Scopus Google Scholar, M. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), we that the leads to of Vav GEF activity and of activated these defects be by the of a which in a while to be Vav GEF activity is required for T cell our data that Tyr174 Vav1 by maintaining the inhibitory constraint of DH domain We and for critical of the and antibodies a with