Identification of the Minimal Tyrosine Residues Required for Linker for Activation of T Cell Function
2001; Elsevier BV; Volume: 276; Issue: 31 Linguagem: Inglês
10.1074/jbc.m102221200
ISSN1083-351X
Autores Tópico(s)Mast cells and histamine
ResumoThe linker for activation of T cells (LAT) is essential for signaling through the T cell receptor (TCR). Following TCR stimulation, LAT becomes tyrosine-phosphorylated, creating docking sites for other signaling proteins such as phospholipase C-γ1 (PLC-γ1), Grb2, and Gads. In this study, we have attempted to identify the critical tyrosine residues in LAT that mediate TCR activation-induced mobilization of intracellular Ca2+ and activation of the MAP kinase Erk2. By using the LAT-deficient Jurkat derivative, J.CaM2, stable cell lines were established expressing various tyrosine mutants of LAT. We show that three specific tyrosine residues (Tyr132, Tyr171, and Tyr191) are necessary and sufficient to achieve a Ca2+ flux following TCR stimulation. These tyrosine residues function by reconstituting PLC-γ1phosphorylation and recruitment to LAT. However, these same tyrosines can only partially reconstitute Erk activation. Full reconstitution of Erk requires two additional tyrosine residues (Tyr110 and Tyr226), both of which have the Grb2-binding motif YXN. This reconstitution of Erk activation requires that the critical tyrosine residues be on the same molecule of LAT, suggesting that a single LAT molecule nucleates multiple protein-protein interactions required for optimal signal transduction. The linker for activation of T cells (LAT) is essential for signaling through the T cell receptor (TCR). Following TCR stimulation, LAT becomes tyrosine-phosphorylated, creating docking sites for other signaling proteins such as phospholipase C-γ1 (PLC-γ1), Grb2, and Gads. In this study, we have attempted to identify the critical tyrosine residues in LAT that mediate TCR activation-induced mobilization of intracellular Ca2+ and activation of the MAP kinase Erk2. By using the LAT-deficient Jurkat derivative, J.CaM2, stable cell lines were established expressing various tyrosine mutants of LAT. We show that three specific tyrosine residues (Tyr132, Tyr171, and Tyr191) are necessary and sufficient to achieve a Ca2+ flux following TCR stimulation. These tyrosine residues function by reconstituting PLC-γ1phosphorylation and recruitment to LAT. However, these same tyrosines can only partially reconstitute Erk activation. Full reconstitution of Erk requires two additional tyrosine residues (Tyr110 and Tyr226), both of which have the Grb2-binding motif YXN. This reconstitution of Erk activation requires that the critical tyrosine residues be on the same molecule of LAT, suggesting that a single LAT molecule nucleates multiple protein-protein interactions required for optimal signal transduction. T cell receptor linker for activation of T cells phospholipase C-γ1 monoclonal antibody polyacrylamide gel electrophoresis wild type inositol 1,4,5-trisphosphate diacylglycerol glycolipid-enriched microdomains mitogen-activated protein B cell linker nuclear factor of activated T cells Engagement of the T cell receptor (TCR)1 triggers a complex cascade of events culminating in T cell proliferation, differentiation, and increased gene transcription (1Weiss A. Littman D.R. Cell. 1994; 76: 263-274Abstract Full Text PDF PubMed Scopus (1957) Google Scholar, 2Wange R.L. Samelson L.E. Immunity. 1996; 5: 197-205Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar). The initial steps of this process are carried out by the Src and Syk families of tyrosine kinases (3Kane L.P. Lin J. Weiss A. Curr. Opin. Immunol. 2000; 12: 242-249Crossref PubMed Scopus (428) Google Scholar). Targets of the Syk family of tyrosine kinases include an emerging class of proteins known as adaptors. Although these proteins lack intrinsic enzymatic activity, they function to promote intermolecular interactions utilizing multiple protein-protein interaction domains or motifs (4Tomlinson M.G. Lin J. Weiss A. Immunol. Today. 2000; 21: 584-591Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Examples of interaction domains include Src homology (SH) 2 domains, which bind phosphotyrosine residues, and SH3 domains, which bind proline-rich regions. One adaptor protein essential for T cell activation is the transmembrane adaptor protein, linker for activation of T cells (LAT) (5Zhang W. Sloan-Lancaster J. Kitchen J. Trible R.P. Samelson L.E. Cell. 1998; 92: 83-92Abstract Full Text Full Text PDF PubMed Scopus (1071) Google Scholar, 6Zhang W. Sommers C.L. Burshtyn D.N. Stebbins C.C. DeJarnette J.B. Trible R.P. Grinberg A. Tsay H.C. Jacobs H.M. Kessler C.M. Long E.O. Love P.E. Samelson L.E. Immunity. 1999; 10: 323-332Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar, 7Finco T.S. Kadlecek T. Zhang W. Samelson L.E. Weiss A. Immunity. 1998; 9: 617-626Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar). Following TCR engagement, LAT becomes phosphorylated on multiple tyrosine residues thereby allowing other proteins important for T cell activation to be recruited by SH2-phosphotyrosine interactions. Multiple proteins have been demonstrated by coimmunoprecipitation experiments to be recruited to LAT, either directly or indirectly, such as phospholipase C-γ1(PLC-γ1), Grb2, Sos, Gads, SLP-76, Vav, Cbl, Itk, and the p85 subunit of phosphatidylinositol 3-kinase (5Zhang W. Sloan-Lancaster J. Kitchen J. Trible R.P. Samelson L.E. Cell. 1998; 92: 83-92Abstract Full Text Full Text PDF PubMed Scopus (1071) Google Scholar, 8Liu S.K. Fang N. Koretzky G.A. McGlade C.J. Curr. Biol. 1999; 9: 67-75Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 9Shan X. Wange R.L. J. Biol. Chem. 1999; 274: 29323-29330Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 10Bunnell S.C. Diehn M. Yaffe M.B. Findell P.R. Cantley L.C. Berg L.J. J. Biol. Chem. 2000; 275: 2219-2230Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 11Ching K.A. Grasis J.A. Tailor P. Kawakami Y. Kawakami T. Tsoukas C.D. J. Immunol. 2000; 165: 256-262Crossref PubMed Scopus (62) Google Scholar). In addition to the many tyrosine residues, LAT also contains two cysteine residues proximal to the transmembrane domain. These two cysteine residues are palmitoylated resulting in localization of LAT into glycolipid-enriched microdomains (GEMs) within the plasma membrane (12Zhang W. Trible R.P. Samelson L.E. Immunity. 1998; 9: 239-246Abstract Full Text Full Text PDF PubMed Scopus (754) Google Scholar). Without correct localization into GEMs, LAT cannot mediate downstream signaling events induced by TCR stimulation (13Zhang W. Irvin B.J. Trible R.P. Abraham R.T. Samelson L.E. Int. Immunol. 1999; 11: 943-950Crossref PubMed Scopus (227) Google Scholar, 14Lin J. Weiss A. Finco T.S. J. Biol. Chem. 1999; 274: 28861-28864Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Since LAT recruits so many proteins to one location, it appears likely that LAT, with the help of other adaptor proteins, functions as a platform where many signal transduction proteins colocalize to propagate and integrate the signals initiated by TCR stimulation. The essential role of LAT in T cell development has been demonstrated using targeted gene disruption in mice. LAT-deficient mice have a block in thymocyte development at the immature CD25+CD44−, CD4− CD8− stage and a complete lack of mature peripheral T cells (6Zhang W. Sommers C.L. Burshtyn D.N. Stebbins C.C. DeJarnette J.B. Trible R.P. Grinberg A. Tsay H.C. Jacobs H.M. Kessler C.M. Long E.O. Love P.E. Samelson L.E. Immunity. 1999; 10: 323-332Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar). Blocks at this early stage of thymocyte development are similar to those observed in other mice lacking proteins, such as the Src kinases Lck and Fyn and the Syk kinases ZAP-70 and Syk, that mediate pre-TCR signaling events (15van Oers N.S. Semin. Immunol. 1999; 11: 227-237Crossref PubMed Scopus (43) Google Scholar). The LAT-deficient Jurkat derivative, J.CaM2, has provided considerable insight into the mechanism by which LAT mediates TCR-signaling events. This cell line is deficient in many pathways activated by TCR stimulation such as Ca2+ mobilization and Ras/MAP kinase activation (7Finco T.S. Kadlecek T. Zhang W. Samelson L.E. Weiss A. Immunity. 1998; 9: 617-626Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar, 16Goldsmith M.A. Dazin P.F. Weiss A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8613-8617Crossref PubMed Scopus (34) Google Scholar). These defects are, in part, due to an inability to recruit PLC-γ1 to the membrane where it is phosphorylated and becomes activated. PLC-γ1 activation is essential for the generation of the second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) from phosphatidylinositol 4,5-bisphosphate. IP3 binds receptors that regulate the release of stored Ca2+, whereas DAG is required to activate proteins such as protein kinase C and Ras guanyl nucleotide releasing protein (RasGRP) (17Downward J. Graves J.D. Warne P.H. Rayter S. Cantrell D.A. Nature. 1990; 346: 719-723Crossref PubMed Scopus (687) Google Scholar, 18Ebinu J.O. Stang S.L. Teixeira C. Bottorff D.A. Hooton J. Blumberg P.M. Barry M. Bleakley R.C. Ostergaard H.L. Stone J.C. Blood. 2000; 95: 3199-3203Crossref PubMed Google Scholar, 19Dower N.A. Stang S.L. Bottorff D.A. Ebinu J.O. Dickie P. Ostergaard H.L. Stone J.C. Nat. Immunol. 2000; 1: 317-321Crossref PubMed Scopus (12) Google Scholar). The absence of LAT may also result in the loss of Ras activation through the inability to recruit the Ras exchange factor Sos to the membrane via the adaptor Grb2. Analysis of the various coimmunoprecipitation studies involving LAT has led to a model where a complex of adaptors involving LAT, Grb2, Gads, and SLP-76 are localized to GEMs within the plasma membrane in order to recruit other signaling proteins (5Zhang W. Sloan-Lancaster J. Kitchen J. Trible R.P. Samelson L.E. Cell. 1998; 92: 83-92Abstract Full Text Full Text PDF PubMed Scopus (1071) Google Scholar, 8Liu S.K. Fang N. Koretzky G.A. McGlade C.J. Curr. Biol. 1999; 9: 67-75Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 20Zhang W. Trible R.P. Zhu M. Liu S.K. McGlade C.J. Samelson L.E. J. Biol. Chem. 2000; 275: 23355-23361Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar). Grb2 is an adaptor protein with a central SH2 domain flanked on both sides with SH3 domains, which bind Sos. Human LAT contains four tyrosine residues that fit the Grb2-SH2 binding motif of YXN that are conserved between human, mouse, and rat (Tyr110, Tyr171, Tyr191, and Tyr226). Three of these tyrosines (Tyr171, Tyr191, and Tyr226) when mutated have been shown to disrupt Grb2 recruitment to LAT. A Grb2-like molecule, known as Gads, also binds directly to LAT at only two of these sites (Tyr171 and Tyr191). Like Grb2, Gads contains two SH3 domains flanking a single SH2 domain. However, Gads also contains a proline-rich region between the SH2 domain and the C-terminal SH3 domain. Studies by Liu et al. (8Liu S.K. Fang N. Koretzky G.A. McGlade C.J. Curr. Biol. 1999; 9: 67-75Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar) have demonstrated that Gads is responsible for mediating the recruitment of SLP-76 to LAT. The SLP-76 adaptor protein contains proline-rich regions, a C-terminal SH2 domain, and three N-terminal phosphotyrosine residues. All have been demonstrated to recruit other signaling proteins. The C-terminal SH3 domain of Gads binds to proline-rich sequences within SLP-76 for its recruitment to LAT (8Liu S.K. Fang N. Koretzky G.A. McGlade C.J. Curr. Biol. 1999; 9: 67-75Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). SLAP-130/Fyb and HPK1 both contain phosphotyrosine residues that can bind the SH2 domain of SLP-76 (21Musci 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). 2K. Sauer, J. Liou, S. B. Singh, D. Yablonski, A. Weiss, and R. M. Perlmutter, submitted for publication. Proteins such as Itk, Rlk, and Vav are thought to bind directly to phosphotyrosine residues at the N terminus of SLP-76, explaining why they are detected in LAT immunoprecipitations (22Tuosto L. Michel F. Acuto O. J. Exp. Med. 1996; 184: 1161-1166Crossref PubMed Scopus (173) Google Scholar, 23Wu J. Motto D.G. Koretzky G.A. Weiss A. Immunity. 1996; 4: 593-602Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 24Su Y.W. Zhang Y. Schweikert J. Koretzky G.A. Reth M. Wienands J. Eur. J. Immunol. 1999; 29: 3702-3711Crossref PubMed Scopus (200) Google Scholar). Mice with targeted disruption of SLP-76 have a phenotype very similar to that of LAT-deficient mice, a profound block in thymic development with the absence of peripheral T cells (25Pivniouk 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 (324) Google Scholar, 26Clements J.L. Yang B. Ross-Barta S.E. Eliason S.L. Hrstka R.F. Williamson R.A. Koretzky G.A. Science. 1998; 281: 416-419Crossref PubMed Scopus (363) Google Scholar). Not surprisingly, the recently described Gads-deficient mice also have a similar, although less severe, phenotype (27Yoder J. Pham C. Iizuka Y. Kanagawa O. Liu S.K. McGlade J. Cheng A.M. Science. 2001; 291: 1987-1991Crossref PubMed Scopus (125) Google Scholar). This is most likely due to a partial compensation by Grb2 in the absence of Gads. A Jurkat mutant cell line lacking SLP-76 displays a phenotype similar to J.CaM2 cells, yet somewhat less drastic. These cells, named J14, have decreases in Ca2+ flux and Ras activation in response to TCR stimulation (28Yablonski D. Kuhne M.R. Kadlecek T. Weiss A. Science. 1998; 281: 413-416Crossref PubMed Scopus (357) Google Scholar). These findings suggest that SLP-76 plays a critical role in LAT-dependent signal transduction. Indeed, overexpression of a LAT-SLP-76 chimera was recently shown to substitute for LAT function (29Boerth N.J. Sadler J.J. Bauer D.E. Clements J.L. Gheith S.M. Koretzky G.A. J. Exp. Med. 2000; 192: 1047-1058Crossref PubMed Scopus (103) Google Scholar). The multiple protein interactions that have been demonstrated with LAT led us to examine further the role of different tyrosine residues and the activities that they mediate. Human LAT has 10 total tyrosines, and 9 are conserved between human, mouse, and rat. Previous studies have shown the importance of some of these residues for recruitment of PLC-γ1, Grb2, and Gads (8Liu S.K. Fang N. Koretzky G.A. McGlade C.J. Curr. Biol. 1999; 9: 67-75Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 20Zhang W. Trible R.P. Zhu M. Liu S.K. McGlade C.J. Samelson L.E. J. Biol. Chem. 2000; 275: 23355-23361Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar). In this study, we expand on the analysis of these and other tyrosine residues by identifying the minimal tyrosine residues required for LAT function. By utilizing the LAT-deficient J.CaM2 cell line, multiple stable lines expressing various tyrosine mutants of LAT were generated, and their abilities to mediate activation of downstream pathways were studied. Ca2+ mobilization is dependent on three of the tyrosines (Tyr132, Tyr171, and Tyr191), whereas full Ras pathway activation requires two additional tyrosine residues (Tyr110 and Tyr226). These two additional tyrosines contribute to the recruitment of Grb2 to the LAT complex. In addition, the critical tyrosine residues must also be on the same molecule of LAT for reconstitution of TCR-mediated signaling events. Therefore, these data provide evidence for the important function of an assembly of proteins on a single LAT molecule. Moreover, these studies show that the tyrosine residues have different roles in mediating events downstream of LAT. The LAT-deficient Jurkat mutant J.CaM2 (7Finco T.S. Kadlecek T. Zhang W. Samelson L.E. Weiss A. Immunity. 1998; 9: 617-626Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar, 16Goldsmith M.A. Dazin P.F. Weiss A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8613-8617Crossref PubMed Scopus (34) Google Scholar) and subsequent stable lines were maintained in RPMI 1640 supplemented with 10% fetal calf serum, 2 mm glutamine, penicillin, and streptomycin. For stable and transient transfections, 2 × 107 J.CaM2 cells were resuspended in 400 µl of RPMI 1640 and electroporated at 250 V, 960 microfarads using a Gene Pulser electroporator (Bio-Rad). For generation of stable lines, transfected cells were plated 48 h after electroporation in media containing 2 µg/ml G418. For transient assays, cells were utilized 24 h after transfection. Myc-tagged Erk2 was expressed using the pEF-BOS expression plasmid. Myc-tagged LAT was expressed using the pCDEF3 expression plasmid, which also encodes a neomycin resistance gene for production of stable lines. Point mutants of LAT were generated with the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Clones presented in this report are as follows: wt LAT (clone 70), Y-F6 (clone 107), Y-F7,8 (clone 73), Y-F6–8 (clone 55), F-Y6 (clone 35), F-Y7,8 (clone 15), F-Y6–8 (clone 40.8). TCR stimulation was performed with the anti-Jurkat TCR β-chain monoclonal antibody (mAb) C305 (30Weiss A. Stobo J.D. J. Exp. Med. 1984; 160: 1284-1299Crossref PubMed Scopus (388) Google Scholar). The polyclonal anti-LAT, anti-PLC-γ1 mixed mAb, and anti-phosphotyrosine 4G10 mAb were from Upstate Biotechnology, Inc. (Lake Placid, NY), whereas the polyclonal anti-Grb2 was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). The anti-phospho-Erk was from Cell Signaling Technology, Inc. (Beverly, MA), and the anti-Myc mAb was derived from the 9E10 hybridoma. To analyze intracellular Ca2+mobilization, 3 × 106 cells were resuspended in 1 ml of RPMI 1640 with 10% fetal bovine serum and labeled in 3 µm of the fluorescent Ca2+ indicator dye Indo-1AM (Molecular Probes, Eugene, OR) for 1 h at 37 °C. Cells were washed 3 times in Ca2+ buffer (0.1% glucose w/v, 25 mm HEPES, 125 mm NaCl, 5 mm KCl, 1 mm Na2HPO4, 0.1% bovine serum albumin, w/v, 50 mm MgCl2, 100 mmCaCl2, pH 7.4), and the cell pellet was kept on ice. Prior to the assay, cells were resuspended in Ca2+ buffer and warmed to 37 °C. 106 cells were stimulated with C305 followed by ionomycin (1 µm). The fluorescence emission at 400- and 500-nm wavelengths was measured with an excitation at 355 nm using a Hitachi F-4500 fluorescence spectrophotometer, and the intracellular Ca2+ concentration was calculated based on the ratio of the fluorescence at 400 and 500 nm. Cells, 2.5 × 107cells/ml, were first rested at 37 °C for 20 min and then stimulated with 1:250 dilution of purified anti-TCR (1 mg/ml stock solution, C305) in phosphate-buffered saline. Cells were lysed at 108 cells/ml in lysis buffer (1% Nonidet P-40, 150 mm NaCl, 10 mm Tris-HCl, pH 7.6). For coimmunoprecipitation assays, 1% Brij in 150 mm NaCl and 10 mm Tris, pH 7.6, was used instead. Both lysis buffers contained 2 mm EDTA and a combination of protease and phosphatase inhibitors as described previously (31Straus D.B. Weiss A. Cell. 1992; 70: 585-593Abstract Full Text PDF PubMed Scopus (935) Google Scholar). For immunoprecipitations, lysates were incubated with primary antibody for 45 min, followed by protein G-Sepharose beads for 45 min, and were then washed 3 times with lysis buffer. Samples were separated by SDS-PAGE, and proteins were analyzed by Western blotting. Membranes were incubated with the indicated primary antibodies followed by the appropriate secondary antibody conjugated to horseradish peroxidase. Reactive proteins were visualized by Renaissance, a chemiluminescence reagent (PerkinElmer Life Sciences). For quantitation of bands, chemiluminescence was assessed on a Kodak Imaging Station using Kodak one-dimensional image analysis software version 3.5 (Rochester, NY). J.CaM2 cells were transfected as before with 20 µg of a 3× NF-AT-luciferase reporter construct and 1 µg of the indicated LAT construct in pCDEF3. The TCR was stimulated with immobilized C305 for 8 h. Cells were harvested, lysed, and assayed for luciferase activity (32Shapiro V.S. Mollenauer M.N. Greene W.C. Weiss A. J. Exp. Med. 1996; 184: 1663-1669Crossref PubMed Scopus (69) Google Scholar). Lysates were also blotted for LAT expression. The LAT-deficient Jurkat-derived cell line J.CaM2 was used to produce cell lines stably expressing various tyrosine mutants of LAT. To aid in the description of the various tyrosine mutants of LAT, each of the 10 tyrosines was given a number. Fig.1 A depicts the location of the tyrosine residues in human LAT by amino acid numbers. The fourth tyrosine in human LAT was designated site 3.5 since it is not conserved between species. Throughout this paper, Y-F denotes mutant forms of LAT where tyrosine residues were changed to phenylalanine, whereas F-Y denotes mutants in which all tyrosines were initially changed to phenylalanine and then the specified residue mutated back to tyrosine. For example, F-Y6 describes a mutant of LAT that has only one tyrosine at position 132. Previous studies have identified multiple tyrosines within LAT that are phosphorylated and function as SH2-binding sites. Site 6 (YLVV) was shown to be important in the recruitment of PLC-γ1 to LAT (20Zhang W. Trible R.P. Zhu M. Liu S.K. McGlade C.J. Samelson L.E. J. Biol. Chem. 2000; 275: 23355-23361Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar), whereas site 7 and site 8 are identical motifs (YVNV) to which either of the adaptor proteins Gads or Grb2 can bind and thereby recruit SLP-76 or Sos, respectively (5Zhang W. Sloan-Lancaster J. Kitchen J. Trible R.P. Samelson L.E. Cell. 1998; 92: 83-92Abstract Full Text Full Text PDF PubMed Scopus (1071) Google Scholar, 8Liu S.K. Fang N. Koretzky G.A. McGlade C.J. Curr. Biol. 1999; 9: 67-75Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). Also conserved between human, mouse, and rat are two more YXN motifs found at site 4 and site 9 that could play roles in Grb2 recruitment. All of the tyrosine mutants of LAT we prepared contain a C-terminal Myc tag and were expressed using a vector containing the hEF-1a promoter. The cDNA constructs were electroporated into J.CaM2 cells, and clones with stable integration were selected for in G418-containing media. For each mutant, multiple clones were tested for LAT expression levels by Western blotting. Representative clones are presented here. Fig. 1 B shows that the clones presented in these studies had equivalent LAT expression. The levels of LAT in the transfectants were slightly higher than that of endogenous LAT in Jurkat. Uniform expression of the TCR was documented by staining for CD3 followed by analysis using flow cytometry (data not shown). Previous studies have demonstrated that the inability of J.CaM2 cells to flux Ca2+ is due to its deficiency in LAT expression (7Finco T.S. Kadlecek T. Zhang W. Samelson L.E. Weiss A. Immunity. 1998; 9: 617-626Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar). To determine which tyrosine residues are required for a Ca2+ flux, cell lines expressing the tyrosine mutants of LAT were stimulated via the TCR, and the subsequent Ca2+flux was measured on a fluorescence spectrophotometer. The Y-F6 cell line had a markedly decreased Ca2+ flux, whereas Y-F7,8 had a normal response when compared with wt LAT (Fig.2 A). However, Y-F6–8 had a complete loss in its ability to flux Ca2+ in response to TCR stimulation. These results implicate sites 6–8 in mediating the Ca2+ response. To extend further this analysis, cells expressing forms of LAT containing only certain tyrosine residues on an all phenylalanine background were tested in the same manner. The F-Y6 version of LAT did not reconstitute Ca2+ signaling, and the F-Y7,8 version of LAT only restored a slight, almost undetectable flux. However, the F-Y6–8 form of LAT reconstituted the Ca2+flux following TCR stimulation to the same extent as wt LAT (Fig.2 A). For these experiments, multiple clones of all mutants gave similar results. These data demonstrate that sites 6–8 are necessary and sufficient for Ca2+ mobilization in the LAT-deficient J.CaM2 cell line. A potential explanation could come from previous studies that have shown that site 6 binds to PLC-γ1 and sites 7 and 8 bind Gads (5Zhang W. Sloan-Lancaster J. Kitchen J. Trible R.P. Samelson L.E. Cell. 1998; 92: 83-92Abstract Full Text Full Text PDF PubMed Scopus (1071) Google Scholar, 8Liu S.K. Fang N. Koretzky G.A. McGlade C.J. Curr. Biol. 1999; 9: 67-75Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 20Zhang W. Trible R.P. Zhu M. Liu S.K. McGlade C.J. Samelson L.E. J. Biol. Chem. 2000; 275: 23355-23361Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar), thereby indirectly recruiting SLP-76 to LAT. This suggests that a LAT·PLC-γ1·Gads·SLP-76 complex may be necessary and sufficient for Ca2+mobilization. Since defective Ca2+ mobilization was observed with Y-F6, F-Y6, and F-Y7,8, we decided to investigate PLC-γ1 phosphorylation in these cell lines, given that PLC-γ1 is responsible for generating the IP3required for Ca2+ mobilization. Cells were stimulated with anti-TCR, PLC-γ1 immunoprecipitations were isolated from cellular lysates, and the samples were separated by SDS-PAGE. Western blotting with an anti-phosphotyrosine antibody indicated that PLC-γ1 was phosphorylated in J.CaM2 cells reconstituted with wt LAT, Y-F6, Y-F7,8, F-Y7,8, and F-Y6–8 (Fig.3 A, middle panel). Thus, the phosphorylation state of PLC-γ1 does not exactly correlate with the mobilization of intracellular Ca2+. In support of this observation, phosphorylation-independent mechanisms of PLC-γ1 activation have been noted previously in the literature (33Sekiya F. Bae Y.S. Rhee S.G. Chem. Phys. Lipids. 1999; 98: 3-11Crossref PubMed Scopus (61) Google Scholar). Moreover, our analysis fails to distinguish among the various reported sites of PLC-γ1 tyrosine phosphorylation (34Liao F. Shin H.S. Rhee S.G. Biochem. Biophys. Res. Commun. 1993; 191: 1028-1033Crossref PubMed Scopus (86) Google Scholar, 35Kim H.K. Kim J.W. Zilberstein A. Margolis B. Kim J.G. Schlessinger J. Rhee S.G. Cell. 1991; 65: 435-441Abstract Full Text PDF PubMed Scopus (448) Google Scholar). When the association of PLC-γ1 with phospho-LAT was examined, a direct correlation was observed between association and Ca2+ flux in response to TCR stimulation (Fig. 3 A, lower panel). Due to the poor ability of the LAT antibody to recognize phosphorylated LAT, we were not able to show inducible association of LAT with PLC-γ1. However, a phosphorylated band that comigrates with LAT can be detected by phosphotyrosine blotting. Samples were also blotted with an anti-PLC-γ1antibody to show equal protein levels in the immunoprecipitations (Fig.3 A, upper panel). To ensure that the lack of a phospho-LAT band in the PLC-γ1 immunoprecipitates is not due to an inability of these mutants of LAT to become phosphorylated, an additional study was done. Anti-Myc immunoprecipitates were performed on the LAT mutant lines in which an interaction of PLC-γ1with phospho-LAT could not be detected but still exhibited PLC-γ1 phosphorylation upon stimulation. Immunoprecipitates from unstimulated or TCR-stimulated cells reconstituted with wt LAT, Y-F6, and F-Y7,8 were separated by SDS-PAGE followed by blotting with an anti-phosphotyrosine antibody. Fig.3 B shows that the phosphorylation state of the Y-F6 mutant is very similar to that of wt LAT and that the F-Y7,8 mutant is still phosphorylated, although at a reduced level. These data suggest that phosphorylation of PLC-γ1 alone is not sufficient to elicit a Ca2+ flux. Mobilization of intracellular Ca2+ following TCR stimulation also requires PLC-γ1 recruitment to LAT. Since the F-Y6–8 form of LAT was sufficient to reconstitute the mobilization of intracellular Ca2+following stimulation through the TCR, we were interested to determine if any or all of these three sites could reconstitute other signaling pathways in J.CaM2. Previous studies performed with J.CaM2 have demonstrated a deficiency in the activation of the Ras pathway. One downstream indicator of this pathway is activation of the Erk MAP kinase. To investigate which of the tyrosines in LAT are sufficient for the activation of Erk, J.CaM2 cells were cotransfected with a Myc-tagged Erk2 construct and various LAT constructs that had sites 6–8 and 6–8 restored to tyrosines from an all-phenylalanine background. As controls, wt LAT and LAT with no tyrosines ("all-F") were used. Cells were then stimulated through the TCR followed by immunoprecipitation with an anti-Myc antibody to isolate the transfected proteins. An indicator of Erk kinase activity is phosphorylation on Thr202 and Tyr204, which can be followed with phospho-specific Erk Western blots. When J.CaM2 cells were transfected with Erk and a vector control or the all-F LAT, Erk did not become activated following TCR stimulation (Fig.4 A). If the cells were cotransfected with a wt LAT construct, Erk was phosphorylated following TCR stimulation. Neither the F-Y6 mutant nor the F-Y7,8 mutant form of LAT could reconstitute Erk phosphorylation in J.CaM2 cells. However, an F-Y6–8 form of LAT could partially reconstitute the defective signaling pathway, demonstrating the importance of all three of these sites (Fig. 4 A). For reasons unknown, mutants of LAT that have most or all of the tyrosine residues changed to phenylalanine reproducibly have a slightly faster mobility. One possible explanation is that the loss of hydroxyl groups on the tyrosine residues creates a protein that is now more hydrophobic resulting in a change in mobility. A time course of Erk activation was then performed to determine if the partial response at 3 min is due to a shift in kinetics or a decrease in th
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