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• W2017068352 abstract "Protein-tyrosine kinases p56Lck, SYK, and ZAP-70 and downstream adaptors LAT and SLP-76 have been implicated as essential components in T-cell activation. Another lymphoid-specific adaptor FYB/SLAP has also been identified as a predominant binding partner of SLP-76 and the Src kinase FYN-T, although its role in the activation process has been unclear. In this study, we demonstrate that FYN-T selectively phosphorylates FYB providing a template for the recruitment of FYN-T and SLP-76 SH2 domain binding. This interaction is unusual in its distinct cytoplasmic localization and its long term stable kinetics of phosphorylation. Furthermore, we demonstrate for the first time that the co-expression of all three components of the FYN-T-FYB-SLP-76 matrix can synergistically up-regulate T-cell receptor-driven interleukin 2 transcription activity. These findings document the existence of a T-cell receptor-regulated FYN-T-FYB pathway that interfaces with the adaptor SLP-76 and up-regulates lymphokine production in T-cells. Protein-tyrosine kinases p56Lck, SYK, and ZAP-70 and downstream adaptors LAT and SLP-76 have been implicated as essential components in T-cell activation. Another lymphoid-specific adaptor FYB/SLAP has also been identified as a predominant binding partner of SLP-76 and the Src kinase FYN-T, although its role in the activation process has been unclear. In this study, we demonstrate that FYN-T selectively phosphorylates FYB providing a template for the recruitment of FYN-T and SLP-76 SH2 domain binding. This interaction is unusual in its distinct cytoplasmic localization and its long term stable kinetics of phosphorylation. Furthermore, we demonstrate for the first time that the co-expression of all three components of the FYN-T-FYB-SLP-76 matrix can synergistically up-regulate T-cell receptor-driven interleukin 2 transcription activity. These findings document the existence of a T-cell receptor-regulated FYN-T-FYB pathway that interfaces with the adaptor SLP-76 and up-regulates lymphokine production in T-cells. Ligation of CD4/CD8-p56Lck and the T-cell receptor complex (TcRζ 1The abbreviations used are: TcR, T-cell receptor; ITAMs, immunoreceptor tyrosine-based activation motifs; IL-2, interleukin 2; SH2, Src homology 2; SH3, Src homology 3; FCS, fetal calf serum; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; PMA, phorbol 12-myristate 13-acetate; HA, hemagglutinin/CD3) activates Src protein-tyrosine kinases p56Lck and p59Fyn-T (1Rudd C.E. Janssen O. Cai Y.-C. da Silva A.J. Raab M. Prasad K.V.S. Immunol. Today. 1994; 15: 225-234Abstract Full Text PDF PubMed Scopus (183) Google Scholar) leading to the phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) of the TcRζ and CD3 chains (2Weiss A. Littman D.R. Cell. 1994; 76: 263-274Abstract Full Text PDF PubMed Scopus (1955) Google Scholar, 3Mustelin T. Immunity. 1994; 1: 351-356Abstract Full Text PDF PubMed Scopus (82) Google Scholar, 4Wange R.L. Samelson L.E. Immunity. 1996; 5: 197-205Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar). Phosphorylated ITAMs allow for ZAP-70 recruitment by means of tandem SH2 domain binding (5Chan A.C. Iwashima M. Turck C.W. Weiss A. Cell. 1992; 71: 649-662Abstract Full Text PDF PubMed Scopus (884) Google Scholar, 6Iwashima M. Irving B.A. Van Oers N.S.C. Chan A.C. Weiss A. Science. 1994; 263: 1136-1139Crossref PubMed Scopus (2) Google Scholar). Activation of ZAP-70 catalytic activity then requires phosphorylation of the kinase at residue Tyr-493 by p56Lck (7Chan A.C. Dalton M. Johnson R. Kong G.H. Wang T. Thoma R. Kurosaki T. EMBO J. 1995; 14: 2499-2508Crossref PubMed Scopus (325) Google Scholar, 8Wange R.L. Guitian R. Isakov N. Watts J.D. Aebersold R. Samelson L.E. J. Biol. Chem. 1995; 270: 18730-18733Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). The p56LckSH2 domain can also bind to ZAP-70, thereby consolidating CD4-p56Lck within the TcR aggregate (9Duplay P. Thome M. Herve F. Acuto O. J. Exp. Med. 1994; 179: 1163-1172Crossref PubMed Scopus (156) Google Scholar). The importance of p56Lck and ZAP-70 has been shown in kinase negative Jurkat cells that show defects in Ca2+ mobilization, tyrosine phosphorylation, and IL-2 transcription (10Straus D. Weiss A. Cell. 1992; 70: 585-593Abstract Full Text PDF PubMed Scopus (935) Google Scholar, 11Williams B.L. Schreiber K.L. Zhang W. Wange R. Samelson L. Leibson P.J. Abraham R.T. Mol. Cell. Biol. 1998; 18: 1388-1399Crossref PubMed Scopus (224) Google Scholar). Recent studies have identified an array of immune cell-specific adaptor proteins in T-cells that act as substrates for upstream kinases and play important roles in T-cell function (12Rudd C.E. Cell. 1999; 96: 1-20Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 13Peterson E.J. Clements J.L. Fang N. Koretzky G.A. Curr. Opin. Immunol. 1998; 10: 337-344Crossref PubMed Scopus (79) Google Scholar). These include the LAT (linker for activation of T-cells), VAV, SLP-76 (SH2 domain-containing leukocyteprotein of 76 kDa), and FYB/SLAP (Fyn T-bindingprotein/SLP-76-associatedprotein)). LAT (pp36) is a transmembrane type III surface protein with a small extracellular region attached to a long cytoplasmic tail (14Weber J.R. Orstavik S. Torgersen K.M. Danbolt N.C. Berg S.F. Ryan J.C. Tasken K. Imboden J.B. Vaage J.T. J. Exp. Med. 1998; 187: 1157-1161Crossref PubMed Scopus (128) Google Scholar, 15Zhang W.J. Sloan-Lancaster J. Kitchen J. Trible R.P. Samelson L.E. Cell. 1998; 92: 83-92Abstract Full Text Full Text PDF PubMed Scopus (1068) Google Scholar). It is expressed exclusively in hematopoietic cells, primarily in T-cells, natural killer cells, mast cells but not B-cells. Rather than binding extracellular ligands, LAT has the hallmarks of a specialized surface protein that acts as an anchor for multiple intracellular proteins. LAT-binding proteins include the established transducing molecules such as growth factor binding protein-2 (GRB-2), Son of sevenless (SOS), VAV, phosphatidylinositol 3-kinase, phospholipase Cγ, the cellular homologue of Casitas B-lineage lymphoma protein (c-Cbl), and SLP-76. Consensus motifs for direct SH2 domain binding exist for GRB-2 and phospholipase Cγ, whereas other components may be indirectly recruited. Although the exact mechanism by which LAT is coupled to the TcRζ·CD3 complex remains to be defined, it is involved in integrating signals from the receptor as demonstrated by the ability of dominant negative form of LAT to inhibit NF-AT transcriptional activity (15Zhang W.J. Sloan-Lancaster J. Kitchen J. Trible R.P. Samelson L.E. Cell. 1998; 92: 83-92Abstract Full Text Full Text PDF PubMed Scopus (1068) Google Scholar). LAT is also needed for the activation of phospholipase Cγ and the Ras pathway (16Finco T.S. Kadlecek T. Zhang W. Samelson L.E. Weiss A. Immunity. 1998; 9: 617-626Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar). One potentially important link to LAT is SLP-76, another immune cell-specific cytoplasmic adaptor (17Jackman J.K. Motto D.G. Sun Q. Tanemoto M. Turck C.W. Peltz G.A. Koretzky G.A. Findell P.R. J. Biol. Chem. 1995; 270: 7029-7032Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). It is a relatively hydrophilic protein with an acidic amino-terminal region, several tyrosine consensus motifs, a central proline-rich region, and a carboxyl-terminal SH2 domain. As expected of an adaptor, SLP-76 also binds multiple proteins including Nck, c-Cbl, phospholipase Cγ, and VAV. SLP-76−/− knock-out mice show that SLP-76 is required for pre-TcR signaling with a block at the double negative stage of differentiation (18Clements J.L. Yang B. Ross-Barta S.E. Eliason S.L. Hirstka R.F. Williamson R.A. Koretzky G.A. Science. 1998; 281: 416-419Crossref PubMed Scopus (362) Google Scholar, 19Pivniouk V. Tsitsikov E. Swinton P. Rathbun G. Alt F.W. Geha R.S. Cell. 1998; 94: 229-238Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). Jurkat T-cells lacking SLP-76 also show defects in Ras activation, CD69 expression, and the activation of NFAT transcriptional activity of the IL-2 promoter (20Yablonski D. Kuhne M.R. Kadlecek T. Weiss A. Science. 1998; 218: 413-416Crossref Scopus (355) Google Scholar). SLP-76 serves as a substrate for ZAP-70 (21Wardenburg J.B. Fu C. Jackman J.K. Flotow H. Wilkinson S.E. Williams D.H. Johnson R. Kong G. Chan A.C. Findell P.R. J. Biol. Chem. 1996; 271: 19641-19644Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 22Raab M. da Silva A.J. Findell P.R. Rudd C.E. Immunity. 1997; 6: 1-11Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar), an event that facilitates binding to the SH2 domain of VAV (22Raab M. da Silva A.J. Findell P.R. Rudd C.E. Immunity. 1997; 6: 1-11Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 23Wu J. Motto D.G. Koretsky G.A. Weiss A. Immunity. 1996; 4: 593-602Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 24Onodera H. Motto D.G. Koretzky G.A. Rothstein D.M. J. Biol. Chem. 1996; 271: 22225-22230Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 25Motto D.G. Ross S.E. Wu J. Hendriks-Taylor L.R. Koretzky G.A. J. Exp. Med. 1996; 183: 1937-1943Crossref PubMed Scopus (180) Google Scholar). SYK also promotes the binding of the adaptor NCK to the same region, leading to the formation of a SLP-76·VAV·NCK trimolecular complex that may regulate cytoskeletal organization (26Wardenburg J.B. Pappu R. Bu J.-Y. Mayer B. Chernoff J. Straus D. Chan A.C. Immunity. 1998; 9: 607-616Abstract Full Text Full Text PDF PubMed Google Scholar). Consistent with this, overexpression of SLP-76, NCK, and VAV enhanced TcR-induced actin polymerization, whereas dominant negative forms of these components inhibited polymerization (26Wardenburg J.B. Pappu R. Bu J.-Y. Mayer B. Chernoff J. Straus D. Chan A.C. Immunity. 1998; 9: 607-616Abstract Full Text Full Text PDF PubMed Google Scholar). SLP-76 is also connected with two other lymphoid-specific proteins, Vav and FYB/SLAP. VAV has a guanine nucleotide exchange factor domain for the Rho and Rac GTPases (27Crespo P. Schuebel K.E. Ostrom A.A. Gutkind J.S. Bustelo X.R. Nature. 1997; 385: 169-172Crossref PubMed Scopus (680) Google Scholar), a pleckstrin homology domain, a cysteine-rich (Cys-rich) domain, as well as two Src homology 3 (SH3) domains and a Src homology 2 (SH2) domain at its carboxyl terminus (28Koch C.A. Anderson D. Moran M.F. Science. 1991; 252: 668-674Crossref PubMed Scopus (1438) Google Scholar). The regulation of Rac is in turn correlated with defects in actin-dependent TcRζ/CD3 cap formation in Vav−/− T-cells (29Holsinger 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, 30Fischer K.-D. Kong Y.-Y. Nichina H. Tedford K. Marenger L.E.M. 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). VAV becomes tyrosine-phosphorylated in response to antigen-receptor ligation (31Bustelo X.R. Ledbetter J.A. Barbacid M. Nature. 1992; 356: 68-74Crossref PubMed Scopus (244) Google Scholar) and binds via its SH2 domain to SLP-76 (23Wu J. Motto D.G. Koretsky G.A. Weiss A. Immunity. 1996; 4: 593-602Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 24Onodera H. Motto D.G. Koretzky G.A. Rothstein D.M. J. Biol. Chem. 1996; 271: 22225-22230Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 25Motto D.G. Ross S.E. Wu J. Hendriks-Taylor L.R. Koretzky G.A. J. Exp. Med. 1996; 183: 1937-1943Crossref PubMed Scopus (180) Google Scholar). Overexpression of VAV and SLP-76 increases TcR-mediated IL-2 transcription in a cooperative manner (23Wu J. Motto D.G. Koretsky G.A. Weiss A. Immunity. 1996; 4: 593-602Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 25Motto D.G. Ross S.E. Wu J. Hendriks-Taylor L.R. Koretzky G.A. J. Exp. Med. 1996; 183: 1937-1943Crossref PubMed Scopus (180) Google Scholar), although VAV·SLP-76 complex formation itself is not needed for TcR-induced IL-2 production in all T-cells (22Raab M. da Silva A.J. Findell P.R. Rudd C.E. Immunity. 1997; 6: 1-11Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Another connection to SLP-76 signaling involves its SH2 domain binding to FYB/SLAP. FYB/SLAP was independently cloned on the basis of its ability to bind to the Src-related kinase FYN-T and SLP-76 (32Musci M.A. Hendricks-Taylor L.R. Motto D.G. Paskind M. Kamens J. Turck C.W. Koretzky G.A. J. Biol. Chem. 1997; 272: 11674-11677Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 33da Silva A.J. Li Z. de Vera C. Canto E. Findell P. Rudd C.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7493-7498Crossref PubMed Scopus (234) Google Scholar). It also has the hallmarks of an adaptor protein with several proline-rich regions, multiple tyrosine-containing motifs, and two putative nuclear localization sequences linked to a carboxyl-terminal SH3 domain. As with LAT and SLP-76, FYB is restricted in expression to lymphoid cells, in particular T-cells and macrophages, but not in B-cells. Two FYB isoforms at 120 and 130 kDa are known, which differ due to a 46-amino acid insert in the 130-kDa form between the two nuclear localization sequences. 2M. Veale, M. Raab, Z. Li, A. da Silva, S.-K. Kraeft, S. Weremowicz, C. C. Morton, and C. E. Rudd, submitted for publication. FYB also undergoes tyrosine phosphorylation in response to TcR ligation, an event diminished in FYN-T-deficient T-cells (35da Silva A.J. Rosenfield J.M. Mueller I. Bouton A. Hirai H. Rudd C.E. J. Immunol. 1997; 158: 2007-2016PubMed Google Scholar). However, unlike with SLP-76, transfection studies with FYB have yielded conflicting results on the role of FYB/SLAP in the regulation of IL-2 production. One report found that FYB/SLAP potentiated IL-2 production (33da Silva A.J. Li Z. de Vera C. Canto E. Findell P. Rudd C.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7493-7498Crossref PubMed Scopus (234) Google Scholar), and another report has postulated a negative function for the protein (32Musci M.A. Hendricks-Taylor L.R. Motto D.G. Paskind M. Kamens J. Turck C.W. Koretzky G.A. J. Biol. Chem. 1997; 272: 11674-11677Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). Part of the uncertainty related to the effects of FYB/SLAP on lymphokine production may be related to its binding to the Src kinase FYN-T, an immune cell-specific variant of the Src kinase FYN. FYN-T associates with the antigen-receptor in T-cells (36Samelson L.E. Phillips A.F. Luong E.T. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4358-4362Crossref PubMed Scopus (533) Google Scholar, 37Sarosi G.A. Thomas P.M. Egerton M. Phillips A.H. Kim K.W. Bonvini E. Samelson L.E. Int. Immunol. 1992; 4: 1211-1217Crossref PubMed Scopus (41) Google Scholar, 38Gauen L.K.T. Kong A.-N.T. Samelson L.E. Shaw A.S. Mol. Cell. Biol. 1992; 12: 5438-5446Crossref PubMed Scopus (163) Google Scholar, 39Gauen L.K.T. Linder M.E. Shaw A.S. J. Cell Biol. 1996; 133: 1007-1015Crossref PubMed Scopus (38) Google Scholar) and acts as a weak substitute for LCK in the phosphorylation of TcRζ ITAMs (6Iwashima M. Irving B.A. Van Oers N.S.C. Chan A.C. Weiss A. Science. 1994; 263: 1136-1139Crossref PubMed Scopus (2) Google Scholar, 22Raab M. da Silva A.J. Findell P.R. Rudd C.E. Immunity. 1997; 6: 1-11Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Although FYN−/− mice exhibit normal thymic development (40Stein P. Lee H. Rich S. Soriano P. Cell. 1992; 70: 741-750Abstract Full Text PDF PubMed Scopus (500) Google Scholar, 41Appleby M. Gross J. Cooke M. Levin S. Qian X. Perlmutter R. Cell. 1992; 70: 751-763Abstract Full Text PDF PubMed Scopus (444) Google Scholar), the loss of FYN in LCK−/− mice contributes to the block at the double negative stage of thymic development (42Groves T. Smiley P. Cooke M.P. Forbush K. Perlmutter R.M. Guidos C.J. Immunity. 1996; 5: 417-428Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 43Van Oers N.S.C. Lowen-Kropf B. Finlay D. Connolly K. Weiss A. Immunity. 1996; 5: 429-436Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). In addition, expression of constitutively active FYN in LCK−/− mice can restore the differentiation of single positive thymocytes (42Groves T. Smiley P. Cooke M.P. Forbush K. Perlmutter R.M. Guidos C.J. Immunity. 1996; 5: 417-428Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). However, the selective binding of the SH2 domain of FYN-T (and not the other Src kinase LCK) to FYB provides a novel avenue by which the FYN-T kinase could intervene in TcR signaling (33da Silva A.J. Li Z. de Vera C. Canto E. Findell P. Rudd C.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7493-7498Crossref PubMed Scopus (234) Google Scholar, 44da Silva A.J. Janssen O.J. Rudd C.E. J. Exp. Med. 1993; 178: 2107-2113Crossref PubMed Scopus (53) Google Scholar). In this paper, we demonstrate that the Src kinase FYN-T (but not LCK or ZAP-70) regulates the binding of the FYN-T and SLP-76 SH2 domains to distinct sites on FYB. Furthermore, we show that members of the matrix co-localize in the cytoplasm of cells and cooperatively up-regulate TcR-initiated IL-2 promoter activity. Spodoptera frugiperda (Sf) cell line IPLB-SF21 was obtained from Invitrogen and was propagated as a monolayer culture in Sf900 insect medium (Life Technologies, Inc.) supplemented with 10% (v/v) fetal bovine serum and 50 μg/ml gentamycin according to the procedure of Brown and Faulkner (60Brown M. Faulkner P. J. Gen. Virol. 1977; 36: 361-364Crossref Scopus (147) Google Scholar). Viral infections were performed at a multiplicity of infection of 5 for protein production and of 0.1 for virus production. COS cells were maintained in RPMI 1640 media supplemented with 5% (v/v) FCS, 1% (w/v) penicillin and streptomycin, and 1 (v/v) l-glutamine. COS cells were transfected with cDNAs inserted into the SRα2 expression vector (gift of Dr. M. Streuli, Dana Farber Cancer Institute, Boston). COS cell transfections were conducted according to standard protocols. The murine T-cell hybridoma, DC27.10 (gift of Dr. R. Zamoyska, Medical Research Council, London, UK), Jurkat cells were cultured in RPMI 1640 medium supplemented with 10% (v/v) FCS and 1% (w/v) penicillin/streptomycin at 37 °C in an atmosphere containing 5% CO2. mAb to SLP-76 was kindly provided by Dr. Paul R. Findell (Syntex, Palo Alto, CA). Anti-phosphotyrosine mAb 4G10 was kindly provided by Dr. Tom Roberts (Dana Farber Cancer Institute, Boston). Anti-FYN mAb was purchased from Transduction Laboratories (Lexington, KY). Rabbit antisera against p59Fyn-T and p56Lck were generated against synthetic peptides corresponding to residues 35–51 and 39–64, respectively. Anti-murine CD3 (2C11) was obtained from American Type Culture Collection. Immunoprecipitations were conducted as described previously (22Raab M. da Silva A.J. Findell P.R. Rudd C.E. Immunity. 1997; 6: 1-11Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 61Raab M. Rudd C.E. Biochem. Biophys. Res. Commun. 1996; 222: 50-57Crossref PubMed Scopus (38) Google Scholar). Briefly, either 1.5 × 106 Sf21 cells were infected with the baculovirus encoding the different proteins, 2 × 105COS cells were transfected with the different DNAs using DEAE-dextran, or 25 × 106 DC27.10 were electroporated with the different DNAs. After 2 days cells were harvested and lysed with 200 μl of lysis buffer (20 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1% (v/v) Triton X-100, 1 mm sodium vanadate, 1 mm phenylmethylsulfonyl fluoride, 1 mm leupeptin). Immunoprecipitation was carried out by incubation of the lysate with the antibody for 1 h at 4 °C, followed by incubation with 50 μl of protein A-Sepharose beads (10% w/v) for 1 h at 4 °C. Immunoprecipitates were washed 3 times with ice-cold lysis buffer and subjected to SDS-PAGE. For immunoblotting the immunoprecipitates were separated by SDS-PAGE and transferred onto nitrocellulose filters (Schleicher & Schuell). Filters were blocked with 5% (w/v) skim milk for 1 h in Tris-buffered saline, pH 8.0, and then probed with the indicated antibody. Bound antibody was revealed with horseradish peroxidase-conjugated rabbit anti-mouse or donkey anti-rabbit antibodies using enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech). Peptides were synthesized and high pressure liquid chromatography purified by the Molecular Biology Core Facility (Dana Farber Cancer Institute, Boston). The sequences of the peptides used were as follows (with pY indicating the phosphorylated residue): phosphorylated peptide III, DDIpYDGIEEED; unphosphorylated peptide III, DDIYDGIEEED; phosphorylated peptide II, DEVpYDDVDTSD; unphosphorylated peptide II, DEVYDDVDTSD; phosphorylated peptide I, RGSpYGYIKTTA; unphosphorylated peptide I, RGSYGYIKTTA. For the binding analysis, peptides were coupled to AminoLink Plus gel beads (Pierce). The beads were added to the lysate and incubated for 2 h at 4 °C. The precipitates were then washed 3 times with ice-cold lysis buffer and subjected to SDS-PAGE and immunoblotting. To activate Jurkat and the DC27.10 murine T-cell hybridoma, 50 × 106 cells were incubated with pre-warmed RPMI media supplemented with 2% FCS, containing either 1 μg/ml OKT3 and 2 μg/ml rabbit anti-mouse for Jurkat or 5 μg/ml 145-2C11 (anti-CD3ε) and 10 μg/ml of the rabbit anti-hamster antibody for DC27.10 at 37 °C for varying lengths of time. Following activation, cells were then rapidly pelleted and solubilized in lysis buffer, as described above. Cells were plated on glass coverslips and grown for 1 day prior to staining. Cells were rinsed with PBS, fixed with 2% paraformaldehyde in PBS for 10 min, and permeabilized for 10 min in 0.5% (v/v) Triton X-100 in PBS. To detect FYN-T, FYB, and SLP-76, permeabilized cells were exposed to the antigen-specific antibodies in blocking buffer (2% (v/v) normal goat and 10% (v/v) rabbit serum in PBS) for 1 h, followed by a brief wash and incubation with isotype-specific secondary antibody (Texas Red or FITC-labeled, Southern Biotechnology Associates, Birmingham, AL) and 0.5 mg/ml Hoechst dye 33238 (Sigma, to visualize DNA (blue)). Slides were mounted in a polyvinyl alcohol medium and viewed on a Nikon FXA microscope equipped for epifluorescence. Photographs were taken using Fujichrome ASA 400 film. Jurkat cells (2 × 107) were co-transfected with 20 μg of FYN-T, FYB, and SLP-76 cDNAs alone or in combinations plus 5 μg of IL-2 full-length LUC reporter plasmid or 3× NFAT/AP-1-LUC reporter plasmid (kindly provided by Dr. Burakoff, Dana Farber Cancer Institute, Boston) and 0.2 μg of a control reporter plasmid (pRL-TK from Promega). Cells were pulsed using BTX Gene Pulser at 250 V, 800 microfarads in 10% FCS. Cells (1 × 106) were aliquoted into a 12-well plate 16 h after transfection and cultured in a final volume of 1 ml of RPMI growth medium. Cells were unstimulated or stimulated at 37 °C with OKT3 or 10 ng/ml PMA. After 6 h stimulation cells were lysed in 100 μl of lysis buffer (Promega kit). Luciferase activity was determined using the luminometer (MicroLumat, EG7G Berthold) immediately after the addition of 100 μl of luciferase substrate (Promega kit) followed by a Stop and Go reaction to measure the control reporter plasmid (dual luciferase system kit from Promega). Luciferase units of the experimental vector were normalized to the level of the control vector in each sample. Previous studies have shown that FYB preferentially associates with the Src kinase FYN-T and the intracellular signaling protein SLP-76 (32Musci M.A. Hendricks-Taylor L.R. Motto D.G. Paskind M. Kamens J. Turck C.W. Koretzky G.A. J. Biol. Chem. 1997; 272: 11674-11677Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 33da Silva A.J. Li Z. de Vera C. Canto E. Findell P. Rudd C.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7493-7498Crossref PubMed Scopus (234) Google Scholar, 35da Silva A.J. Rosenfield J.M. Mueller I. Bouton A. Hirai H. Rudd C.E. J. Immunol. 1997; 158: 2007-2016PubMed Google Scholar). Preliminary data also implicated FYN-T in the phosphorylation of FYB (33da Silva A.J. Li Z. de Vera C. Canto E. Findell P. Rudd C.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7493-7498Crossref PubMed Scopus (234) Google Scholar). To determine the specificity of FYN-T phosphorylation of FYB and to assess whether it regulates FYB·SLP-76 complex formation, FYB was co-expressed with either the LCK, FYN-T, or ZAP-70 kinase in COS cells and was assessed for phosphorylation by anti-phosphotyrosine blotting (Fig. 1 A). Only FYN-T was found to consistently phosphorylate FYB (lane 3). ZAP-70 failed to phosphorylate the protein in each of five experiments (lane 4). Similar negative results were obtained using the combination of LCK and ZAP-70 (data not shown). In addition, LCK showed little phosphorylation of FYB (lane 2). Specificity was also seen in a comparison with SLP-76 as substrate (Fig. 1 B). In this case, as previously reported (21Wardenburg J.B. Fu C. Jackman J.K. Flotow H. Wilkinson S.E. Williams D.H. Johnson R. Kong G. Chan A.C. Findell P.R. J. Biol. Chem. 1996; 271: 19641-19644Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 22Raab M. da Silva A.J. Findell P.R. Rudd C.E. Immunity. 1997; 6: 1-11Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar), ZAP-70 preferentially phosphorylated SLP-76 (lane 4). Some FYN-T phosphorylation of SLP-76 was observed but at levels some 2–5-fold less than ZAP-70 (lane 3). No phosphorylation by LCK was evident (lane 2), although using the same system, LCK has previously been found to phosphorylate selectively the TcR ζ chain (6Iwashima M. Irving B.A. Van Oers N.S.C. Chan A.C. Weiss A. Science. 1994; 263: 1136-1139Crossref PubMed Scopus (2) Google Scholar, 22Raab M. da Silva A.J. Findell P.R. Rudd C.E. Immunity. 1997; 6: 1-11Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). As an internal control for expression, FYB and SLP-76 (Fig. 1, A andB, lower panels), and the kinases (Lck, FYN-T, and ZAP-70) in each co-transfectant was expressed at significant levels (data not shown). These observations demonstrate that among the three TcR associated kinases, FYN-T preferentially phosphorylates FYB. In order for FYN-T phosphorylation of FYB to be of functional importance, one would expect the kinase to phosphorylate sites that are needed for FYN-T and/or SLP-76 SH2 domain binding. To examine this, it was first necessary to identify the site(s) of FYN-T phosphorylation in FYB. To accomplish this, several deletion mutants of FYB were generated from residues 450, 585, 670, and 707 to the carboxyl terminus and were assessed for phosphorylation. The structure of each mutant is illustrated in Fig. 1 C. Deletion mutant 707 selectively deletes the SH3-like domain (Δ707); 670 deletes the putative second nuclear localization motif and the SH3-like domain (Δ670); 585 additionally deletes the above plus two YDDV motifs and a YDGI motif (Δ585); and 450 deletes the above plus a YEDI motif and another putative nuclear localization motif (Δ450). Each mutant was HA epitope-tagged, co-expressed with FYN-T in COS cells, and examined for phosphorylation (Fig.1 C, lower left panels). Although the Δ707 and Δ670 mutants underwent phosphorylation at levels comparable to wild-type (lanes 2–4), deletion from residue 585 resulted in an abrupt loss of FYB phosphorylation by FYN-T (lanes 4–6). FYB expression was found to be equivalent for the different mutants as detected by anti-HA blotting (lower left panel). Importantly, these results could be duplicated in T-cells. Using the T-cell hybridoma DC27.10, wild-type FYB, Δ707, and Δ670 mutants showed similar levels of phosphorylation (right lower panel, lanes 7, 3, and 4, respectively). Again, the majority of phosphorylation was lost with the Δ585 mutant (lane 5). The Δ450 mutant also showed no phosphorylation (lane 6). Each of the proteins was expressed at high levels as observed by anti-HA blotting (lower right panel, lanes 1–7). Background phosphorylation by endogenous FYN-T was also observed but only with overexposure of the film (data not shown). These data confirm that in both COS and T-cells, the primary FYN-T phosphorylation site(s) is located between residues 585 and 670. The above result was initially found to be somewhat surprising given the ideal nature of the YEDI motif (at residues 472–475) for Src-SH2 domain binding. Therefore, to exclude more rigorously whether the YEDI site served as a target, we analyzed another clone of FYB (termed J12) that encodes a limited stretch between residues 395 and 545 (Fig.1 D). In this construct, the YEDI motif is the only possible tyrosine phosphorylation site. However, J12 failed to become phosphorylated when co-expressed with FYN-T (lane 2). As controls, a mutant form of J12 lacking the tyrosine failed to undergo phosphorylation (lane 3), whereas full-length FYB became phosphorylated (lane 4). This observation indicates that the YEDI motif is unlikely to serve as a phosphorylation site for FYN-T. Taken together, these data identified the region between residues 585 and 670 as the region of FYN-T phosphorylation. In order to assess whether phosphorylation by FYN-T leads to SH2 domain binding of FYN-T and/or SLP-76, lysates from cells transfected with FYN-T and the deletion mutants were subjected to precipitation using GST-SH2 fusion proteins. Under these conditions, FYN-T phosphorylation of FYB was found to induce the binding of the SH2 domains of both FYN-T and SLP-76 (Fig. 2 A, lan" @default.
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• W2017068352 title "FYN-T-FYB-SLP-76 Interactions Define a T-cell Receptor ζ/CD3-mediated Tyrosine Phosphorylation Pathway That Up-regulates Interleukin 2 Transcription in T-cells" @default.
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• W2017068352 doi "https://doi.org/10.1074/jbc.274.30.21170" @default.
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