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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

2006; Elsevier BV; Volume: 281; Issue: 50 Linguagem: Inglês

10.1074/jbc.m608913200

ISSN

1083-351X

Autores

Ana V. Miletic, Kumiko Sakata-Sogawa, Michio Hiroshima, Michael J. Hamann, Timothy S. Gomez, Naruhisa Ota, Tracie Kloeppel, Osami Kanagawa, Makio Tokunaga, Daniel D. Billadeau, Wojciech Swat,

Tópico(s)

Monoclonal and Polyclonal Antibodies Research

Resumo

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 M.K. Cell. 2000; 102: 625-633Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 22Llorca O. Arias-Palomo E. Zugaza J.L. Bustelo X.R. EMBO J. 2005; 24: 1330-1340Crossref PubMed Scopus (36) Google Scholar). One mechanism for relieving such an autoinhibitory constraint is via phosphorylation of three conserved AC tyrosines: Tyr142, Tyr160, and Tyr174, which results in unraveling of the autoinhibitory N-terminal extension and its release from the DH domain (21Aghazadeh B. Lowry W.E. Huang X.Y. Rosen M.K. Cell. 2000; 102: 625-633Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 23Amarasinghe G.K. Rosen M.K. Biochemistry. 2005; 44: 15257-15268Crossref PubMed Scopus (30) Google Scholar). Given that tyrosine phosphorylation distinguishes Vav from the plethora of other Rho GEFs (24Bustelo X.R. Ledbetter J.A. Barbacid M. Nature. 1992; 356: 68-71Crossref PubMed Scopus (242) Google Scholar, 25Margolis B. Hu P. Katzav S. Li W. Oliver J.M. Ullrich A. Weiss A. Schlessinger J. Nature. 1992; 356: 71-74Crossref PubMed Scopus (304) Google Scholar), it is possible that Vav has evolved a complex mechanism for regulation of GEF activity in which tyrosine phosphorylation represents a specific adaptation of Vav proteins to function downstream of ITAM-containing antigen receptors in lymphocytes. It has also been suggested that binding of the Vav PH domain to phosphatidylinositols may contribute to regulation of GEF activity. For example, phosphatidylinositol 3,4,5-trisphosphate (PIP3) binding to the PH domain of Vav may lead to activation of DH domain activity, whereas phosphatidylinositol 4,5-bisphosphate (PIP2) binding has been suggested to negatively regulate DH domain activity (26Han J. Luby-Phelps K. Das B. Shu X. Xia Y. Mosteller R.D. Krishna U.M. Falck J.R. White M.A. Broek D. Science. 1998; 279: 558-560Crossref PubMed Scopus (710) Google Scholar). A thermodynamic model has been proposed in which PIP3 binding to the PH domain may relax interactions between the Vav PH domain and the autoinhibited DH domain, thereby allowing access of protein-tyrosine kinases to the AC tyrosines, with the resulting phosphorylation of AC tyrosines and activation of GEF activity (21Aghazadeh B. Lowry W.E. Huang X.Y. Rosen M.K. Cell. 2000; 102: 625-633Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). In this regard, GEF activity is diminished in a mutant of Vav1 in which the PH domain is rendered incapable of interacting with phosphatidylinositols (27Prisco A. Vanes L. Ruf S. Trigueros C. Tybulewicz V.L.J. Immunity. 2005; 23: 263-274Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). In addition, binding of proteins to the N-terminal CH domain of Vav may contribute to activation of the DH domain. For example, interaction of the adaptor protein APS with the CH domain of Vav3 has been implicated in activation of Vav3 (28Yabana N. Shibuya M. Oncogene. 2002; 21: 7720-7729Crossref PubMed Scopus (22) Google Scholar). In this report, we show that the tyrosine residues in the Vav1 AC are evolutionarily conserved and conform to consensus SH2 domain binding motifs for several proteins implicated in TCR signaling (including Lck, PI3K p85α, and PLCγ1). Indeed, Tyr142, Tyr160, and Tyr174 can bind these proteins through direct interactions with their SH2 domains. Moreover, the AC tyrosines are essential for total tyrosine phosphorylation of Vav1. Our data also indicate that Tyr174 is critical for TCR-induced Vav1 microcluster stability and for normal T cell development and activation, because the Y174F mutation disrupts TCR signaling. Generation of Reconstituted J.Vav Cell Lines, Stimulation, and Immunoblotting—The Vav1-deficient J.Vav cell line was previously described (29Cao Y. Janssen E.M. Duncan A.W. Altman A. Billadeau D.D. Abraham R.T. EMBO J. 2002; 21: 4809-4819Crossref PubMed Scopus (88) Google Scholar). To generate J.Vav cell lines expressing Vav1WT, Vav1 AC Tyr → Phe mutants, and Vav1Y174F/GEF–, GFP-tagged Vav1 expression constructs were transduced into J.Vav cells via “spinfection” with retroviral particles at room temperature, 2000 rpm for 90 min. GFP+ cells were fluorescent-activated cell-sorted and subcloned. Vav1-GFP constructs were generated by ligation of an XbaI-BamHI Vav1-GFP cDNA fragment into IRES-GFP-RV digested with XhoI-BamHI replacing IRES-GFP. Mutagenesis was performed by PCR (Stratagene, La Jolla, CA) and confirmed by sequencing. Cells were stimulated with anti-CD3ϵ (clone HIT3a; 1 μg/ml, BD Biosciences, San Diego, CA) and anti-IgG2a cross-linker (1 μg/ml, Southern Biotechnology Assoc., Birmingham, AL), and lysed in radioimmune precipitation assay buffer supplemented with a protease inhibitor mixture (Boehringer, Ridgefield, CT), 10 mm NaF, and 1 mm Na3VO4. Western blotting was performed following standard procedures. Primary antibodies were developed with horseradish peroxidase-conjugated secondary antibodies (anti-mouse, Zymed Laboratories Inc., San Francisco, CA; anti-rabbit, Amersham Biosciences, Piscataway, NJ). Immune complexes were detected by enhanced chemiluminescence (Amersham Biosciences). Peptide Immunoprecipitation Assays—For GST fusion protein immunoprecipitations, peptide-bead complexes were generated by mixing 30 nmol of biotinylated peptide (QCB, Hopkinton, MA) and 20-μl SA-conjugated beads (Sigma) in 500 μl of phosphate-buffered saline at 4 °C for 1 h. Complexes were washed four times by adding 500 μl of phosphate-buffered saline followed by centrifugation at 4 °C and 13,000 rpm for 1 min. To peptide-bead complex, 500 μl of Triton X-100 lysis buffer (1% Triton X-100, 0.15 m NaCl, 25 mm HEPES pH 7.5) supplemented with a protease inhibitor mixture (Boehringer), 10 mm NaF, and 1 mm Na3VO4, and 1 μg of GST fusion proteins were added followed by rotation at 4 °C for 1 h. Immunoprecipitates were washed four times with cold Triton X-100 lysis buffer, resuspended in SDS sample buffer, and analyzed by Western blotting with anti-GST antibodies (Upstate Biotechnology, Lake Placid, NY) following standard procedures. For immunoprecipitation from cell lysates, 50 × 106 Jurkat T cells were lysed in Triton X-100 lysis buffer for 20 min on ice. Postnuclear lysates were obtained by centrifugation at 4 °C and 13,000 rpm for 10 min. Clarified lysates were precleared by adding 20 μl of SA-conjugated beads followed by rotation at 4 °C for 1 h. During preclearance, peptide-bead complexes were generated by mixing 30 nmol of biotinylated peptide and 200 μl of beads with 500 μl of PBS with rotation at 4 °C for 1 h. Complexes were washed as described above. Precleared lysates were centrifuged at 4 °C and 13,000 rpm for 1 min and incubated with washed peptide-bead complexes at 4 °C for 1–2 h. Immunoprecipitates were washed and Western blotting performed as above. TIRFM Imaging—Imaging of dynamic Vav1-GFP microcluster assembly and movement was performed using TIRF microscopy as described in Ref. 30Tokunaga M. Kitamura K. Saito K. Iwane A.H. Yanagida T. Biochem. Biophys. Res. Commun. 1997; 235: 47-53Crossref PubMed Scopus (277) Google Scholar. 1 × 106 cells were resuspended in non-fluorescent medium, dropped onto glass bottom dish coverslips (MatTek, Ashland, MA) coated overnight at 4 °C with 1 μg/ml anti-CD3ϵ (clone HIT3a; BD Biosciences) as previously described (31Bunnell S.C. Barr V.A. Fuller C.L. Samelson L.E. Sci. STKE. 2003; 177: PL8Google Scholar, 32Bunnell S.C. Kapoor V. Trible R.P. Zhang W. Samelson L.E. Immunity. 2001; 14: 315-329Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar). A beam from a solid state laser (488 nm, 20 milliwatt, SAPPHIRE 488-20-OPS, Coherent) was introduced into an inverted microscope (IX-81, Olympus) for illumination. Images were captured using an EB-CCD camera (C-7190-23, Hamamatsu Photonics) equipped with an image intensifier (C8600-05, Hamamatsu Photonics). Image recording and processing were performed using AQUACOSMOS software (Hamamatsu Photonics), and image analyses were performed using Metamorph Software (Molecular Devices, Sunnyvale, CA). Kymographic analysis was performed 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 Scholar. In brief, an arbitrary “slice” was drawn through a cell and was then applied to all frames of a movie using MetaMorph software. Subsequently, the fluorescence over time of individual Vav1-GFP microclusters contained within the “slice” was visualized as white streaks. For analyses of fluorescence of single microclusters over time, a gate was drawn around individual, randomly chosen microclusters and was applied to all frames of a movie beginning with the first frame in which the microcluster was visible to the last frame of the movie. Fluorescence of a selected microcluster within the gate was reflected in arbitrary units. Mice, Cell Suspensions, Antibodies, and Flow Cytometry—Germline VavNULL mice have been previously described (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) and were maintained in the SPF facility of Washington University School of Medicine according to institutional protocols. Cell suspensions were prepared, counted, and stained with antibodies following standard procedures. The following antibody conjugates were used (BD Biosciences): phycoerythrin (PE), allophyocyanin (APC)-H129.19 (anti-CD4), and cytochrome c (CyC)-53-6.7 (anti-CD8α). All samples were analyzed on a FACSCalibur flow cytometer (BD Biosciences) with FlowJo software. VavNULL Hematopoietic Stem Cell Complementation (VavNULL-HSCC)—A single dose of 150 mg/kg of 5-flurouracil (10 mg/ml in phosphate-buffered saline, Sigma) was injected into donor mice intraperitoneally. 4–5 days postinjection, donors were sacrificed, and bone marrow (BM) harvested. BM cells were expanded in medium containing 15% fetal calf serum and supplemented with stem cell factor (100 ng/ml, PeproTech, Rocky Hill, NJ), IL-3 (6 ng/ml, PeproTech), and IL-6 (10 ng/ml, PeproTech). After 2 days in culture, the cells were retrovirally transduced via spinfection. Infection efficiency and viability of BM cells were assessed by flow cytometry. RAG2–/–-recipient mice were lethally irradiated with 950 Rad (γ irradiation (Cs137), MDS Nordion, Ottawa, Ontario, Canada) and injected with a 250-μl cell suspension (∼.25 × 106 cells), intravenous. Chimera were sacrificed and analyzed 5–7 weeks following reconstitution. T Cell Stimulation and Proliferation Assays—Purified T cells were stimulated with soluble anti-CD3ϵ antibodies (clone 145-2C11, 1 μg/ml, BD Biosciences), as indicated, and [3H]thymidine incorporation was performed as described in Ref. 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. Rac Assay—Purified LN T cells were starved for 30 min in medium lacking serum. Cells were treated with 1 μg/ml anti-CD3 antibodies for 2 min, and the Rac assay was performed using the EZ-Detect Rac1 Activation kit (Pierce) according to the manufacturer's instructions. Vav1 AC Tyrosines Are Evolutionarily Conserved and Conform to SH2 Domain Binding Motifs—The Vav1 AC of several species was analyzed by sequence alignment (Fig. 1A). This alignment shows that amino acid sequences surrounding AC (Tyr142, Tyr160, and Tyr174; numbering relative to the human sequence) are conserved within several mammalian and non-mammalian species, including frog (Xenopus laevis), zebrafish (Danio rerio), fruit fly (Drosophila melanogaster), and nematode (Caenorhabditis elegans) (Fig. 1A). Moreover, AC tyrosines are found within consensus binding motifs for the SH2 domains of several proteins implicated in TCR signaling, including the Src family kinase Lck (YXXΦ), the p85α subunit of PI3K (YXXM/Φ), and PLCγ1 (YΦXΦ)(X is any residue, Φ is Val, Ile, or Leu) (Fig. 1A and Refs. 33Songyang Z. Cantley L.C. Cell. 2004; 116: S41-S43Abstract Full Text PDF PubMed Google Scholar, 35Obenauer J.C. Cantley L.C. Yaffe M.B. Nucleic Acids Res. 2003; 31: 3635-3641Crossref PubMed Scopus (1330) Google Scholar). These data suggest that the AC tyrosines may be involved in Vav interactions with these SH2 domain-containing proteins during TCR signaling. AC Tyrosines Are Critical for Inducible Tyrosine Phosphorylation of Vav1—AC tyrosines have been implicated in regulation of Vav tyrosine phosphorylation and function in T lymphocytes (36Lopez-Lago M. Lee H. Cruz C. Movilla N. Bustelo X.R. Mol. Cell Biol. 2000; 20: 1678-1691Crossref PubMed Scopus (142) Google Scholar, 40Billadeau D.D. Mackie S.M. Schoon R.A. Leibson P.J. J. Immunol. 2000; 164: 3971-3981Crossref PubMed Scopus (46) Google Scholar), and as potential phosphorylation sites for Src and Syk family protein-tyrosine kinases (23Amarasinghe G.K. Rosen M.K. Biochemistry. 2005; 44: 15257-15268Crossref PubMed Scopus (30) Google Scholar, 26Han J. Luby-Phelps K. Das B. Shu X. Xia Y. Mosteller R.D. Krishna U.M. Falck J.R. White M.A. Broek D. Science. 1998; 279: 558-560Crossref PubMed Scopus (710) Google Scholar, 41Deckert M. Tartare-Deckert S. Couture C. Mustelin T. Altman A. Immunity. 1996; 5: 591-604Abstract Full Text PDF PubMed Scopus (245) Google Scholar). To determine whether the AC tyrosines can function in T cells as potential SH2 domain docking sites, we first tested whether these residues are phosphorylated during T cell activation using antibodies that specifically recognize phosphorylated-AC Tyr142, Tyr160, or Tyr174, with minimal cross-reactivity. Using Vav1-deficient Jurkat T cells (29Cao Y. Janssen E.M. Duncan A.W. Altman A. Billadeau D.D. Abraham R.T. EMBO J. 2002; 21: 4809-4819Crossref PubMed Scopus (88) Google Scholar) in which endogenous Vav1 was replaced by wild-type Vav1-GFP (Vav1WT) or with Vav1 harboring tyrosine-to-phenylalanine (Tyr → Phe) substitutions of the AC tyrosines, we found that consistent with previously published studies (27Prisco A. Vanes L. Ruf S. Trigueros C. Tybulewicz V.L.J. Immunity. 2005; 23: 263-274Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 36Lopez-Lago M. Lee H. Cruz C. Movilla N. Bustelo X.R. Mol. Cell Biol. 2000; 20: 1678-1691Crossref PubMed Scopus (142) Google Scholar) Vav1 AC tyrosines were rapidly phosphorylated during T cell activation (Fig. 1B and supplemental Fig. S1A). To determine if the AC tyrosines are required for inducible tyrosine phosphorylation of Vav1 in activated T cells, J.Vav cell lines reconstituted with either Vav1WT or AC tyrosine-mutated Vav1 were stimulated with orthovanadate, and tyrosine phosphorylation of immunoprecipitated Vav1 was examined by immunoblotting with total anti-phosphotyrosine antibodies (Fig. 1C). These experiments reveal that while mutation of any single AC tyrosine has no discernible effect on Vav1 phosphorylation, mutation of Tyr174 in combination with Tyr142 and/or Tyr160, results in severely diminished phosphorylation of Vav1 (Fig. 1C). Strikingly, loss of all three AC tyrosines (Vav1Y3F) leads to a virtually complete disruption of Vav1 total tyrosine phosphorylation (Fig. 1C), even though in addition to the AC tyrosines, there are six tyrosine residues outside of the AC (Tyr267, Tyr482, Tyr603, Tyr604, Tyr826, Tyr841) that conform to consensus tyrosine phosphorylation motifs (35Obenauer J.C. Cantley L.C. Yaffe M.B. Nucleic Acids Res. 2003; 31: 3635-3641Crossref PubMed Scopus (1330) Google Scholar). Importantly, we observed similar results when cells were activated by TCR cross-linking with anti-CD3 antibodies (supplemental Fig. S1B), indicating that these results are not an artifact of orthovanadate treatment. Diminished Vav tyrosine phosphorylation is not caused by altered levels of Vav protein expression because the reconstituted J.Vav cell lines express recombinant Vav1-GFP at a level similar to endogenous Vav1 (Fig. 1 and data not shown). Together, these data suggest that the AC tyrosines are critical for TCR-induced phosphorylation of Vav1, suggesting that the AC tyrosines may function as docking sites and/or facilitate access for kinases such as Lck to phosphorylate additional Vav tyrosines. In this context, tyrosine residues outside of the AC may not be accessible to protein-tyrosine kinases as they may not be surface-exposed, or they may be masked by intramolecular interactions. Thus, the AC tyrosines appear to control Vav1 tyrosine phosphorylation and may be required for protein-tyrosine kinase recruitment. Vav1 AC Tyrosines Bind SH2 Domains of TCR-signaling Proteins—To examine AC tyrosine interactions with the SH2 domains of Lck, PI3K p85α, and PLCγ1, we first tested binding of biotinylated peptides derived from the AC of Vav1 containing either phosphorylated or unphosphorylated Tyr142, Tyr160, or Tyr174 (supplemental Table S1) to purified GST-SH2 domain(s) of Lck, PI3K p85α, PLCγ1, or Grb2. As a positive control, we used the previously characterized binding of CD22- or BTLA-derived peptides to the SH2 domain of Grb2 (supplemental Fig. S2A and Refs. 42Yohannan J. Wienands J. Coggeshall K.M. Justement L.B. J. Biol. Chem. 1999; 274: 18769-18776Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar and 43Gavrieli M. Murphy K.M. Biochem. Biophys. Res. Commun. 2006; 345: 1440-1445Crossref PubMed Scopus (57) Google Scholar). These experiments show that, as expected based on the predicted sequence motif binding (Fig. 1A), each of the phosphorylated but not unphosphorylated AC tyrosines can bind to the SH2 domain of Lck, the C-terminal SH2 domain of PLCγ1, or to either of the two SH2 domains of PI3K p85α (Fig. 2A). In contrast, the N-terminal SH2 domain of PLCγ1 did not associate with any of the AC tyrosines nor did the SH2 domain of Grb2 (Fig. 2A), consistent with the preference of the Grb2 SH2 domain for the YXNM motif (Fig. 1 and Ref. 44Schneider H. Cai Y.C. Prasad K.V. Shoelson S.E. Rudd C.E. Eur. J. Immunol. 1995; 25: 1044-1050Crossref PubMed Scopus (137) Google Scholar). Taken together, these data indicate that phosphorylated Vav1 AC tyrosines can directly bind SH2 domains of several proteins implicated in TCR signaling, consistent with a recent report showing association of the Lck SH2 domain with AC tyrosines (23Amarasinghe G.K. Rosen M.K. Biochemistry. 2005; 44: 15257-15268Crossref PubMed Scopus (30) Google Scholar). To determine whether the AC tyrosines could interact with SH2 domain-containing proteins in their native conformation, we performed immunoprecipitation experiments using the biotinylated AC tyrosine-containing phosphopeptides and cytoplasmic extracts generated from Jurkat cells. In these experiments, we used the previously characterized interaction between Grb2 and CD22 Tyr828 as a positive control (supplemental Fig. S2B and Ref. 42Yohannan J. Wienands J. Coggeshall K.M. Justement L.B. J. Biol. Chem. 1999; 274: 18769-18776Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). These experiments confirmed binding of phosphorylated Tyr142, Tyr160, and Tyr174 AC tyrosine-containing peptides to native Lck protein (Fig. 2B). Moreover, we find that PI3K p85α and PLCγ1 bind specifically to phosphorylated but not unphosphorylated AC tyrosines (Fig. 2B). As expected, Grb2 fails to bind to any of the Vav1 AC tyrosines in these assays (Fig. 2B). Thus, while associations of Vav with Lck, PI3K p85α, and PLCγ1 have been previously shown by co-immunoprecipitation experiments (45Reynolds L.F. de Bettignies C. Norton T. Beeser A. Chernoff J. Tybulewicz V.L. J. Biol. Chem. 2004; 279: 18239-18246Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 46Reynolds L.F. Smyth L.A. Norton T. Freshney N. Downward J. Kioussis D. Tybulewicz V.L. J. Exp. Med. 2002; 195: 1103-1114Crossref PubMed Scopus (178) Google Scholar), 3W. Swat, unpublished observations. these data indicate the direct binding of Vav with SH2 domain-containing proteins via phosphorylated AC tyrosines. Vav1 Tyrosine 174 Controls Dynamic Redistribution of Vav1 upon TCR Activation—Recent imaging studies examining activation of T cells on stimulatory planar surfaces have identified microclusters of signaling proteins at the sites of TCR contacts (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). It has been proposed that within these signaling complexes, TCR signals leading to tyrosine phosphorylation of signaling proteins, generation of second messengers, and rearrangements of the actin and microtubule cytoskeleton are initiated and sustained (5Campi G. Varma R. Dustin M.L. J. Exp. Med.

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