Sequential phosphorylation of SLP-76 at tyrosine 173 is required for activation of T and mast cells
2011; Springer Nature; Volume: 30; Issue: 15 Linguagem: Inglês
10.1038/emboj.2011.213
ISSN1460-2075
AutoresMeirav Sela, Yaron Bogin, Dvora Beach, Thomas Oellerich, Johanna Lehne, Jennifer E. Smith‐Garvin, Mariko Okumura, Elina Starosvetsky, Rachelle Kosoff, Evgeny Libman, Gary A. Koretzky, Taku Kambayashi, Henning Urlaub, Jürgen Wienands, Jonathan Chernoff, Deborah Yablonski,
Tópico(s)PI3K/AKT/mTOR signaling in cancer
ResumoArticle1 July 2011free access Sequential phosphorylation of SLP-76 at tyrosine 173 is required for activation of T and mast cells Meirav Sela Meirav Sela Rappaport Family Institute for Research in the Medical Sciences, The Bruce Rappaport Faculty of Medicine, Technion—Israel Institute of Technology, Haifa, Israel Search for more papers by this author Yaron Bogin Yaron Bogin Rappaport Family Institute for Research in the Medical Sciences, The Bruce Rappaport Faculty of Medicine, Technion—Israel Institute of Technology, Haifa, Israel Search for more papers by this author Dvora Beach Dvora Beach Rappaport Family Institute for Research in the Medical Sciences, The Bruce Rappaport Faculty of Medicine, Technion—Israel Institute of Technology, Haifa, Israel Search for more papers by this author Thomas Oellerich Thomas Oellerich Institute of Cellular and Molecular Immunology, Georg August University of Göttingen, Göttingen, GermanyPresent address: Department of Medicine II, Hematology and Oncology, Goethe Universität Frankfurt, Frankfurt, Germany Search for more papers by this author Johanna Lehne Johanna Lehne Max Planck Institute of Biophysical Chemistry, Bioanalytical Mass Spectrometry Group, Göttingen, Germany Search for more papers by this author Jennifer E Smith-Garvin Jennifer E Smith-Garvin Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Search for more papers by this author Mariko Okumura Mariko Okumura Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Search for more papers by this author Elina Starosvetsky Elina Starosvetsky Rappaport Family Institute for Research in the Medical Sciences, The Bruce Rappaport Faculty of Medicine, Technion—Israel Institute of Technology, Haifa, Israel Search for more papers by this author Rachelle Kosoff Rachelle Kosoff Fox Chase Cancer Center, Philadelphia, PA, USA Cancer Biology Program, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Evgeny Libman Evgeny Libman Rappaport Family Institute for Research in the Medical Sciences, The Bruce Rappaport Faculty of Medicine, Technion—Israel Institute of Technology, Haifa, Israel Search for more papers by this author Gary Koretzky Gary Koretzky Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Search for more papers by this author Taku Kambayashi Taku Kambayashi Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Search for more papers by this author Henning Urlaub Henning Urlaub Max Planck Institute of Biophysical Chemistry, Bioanalytical Mass Spectrometry Group, Göttingen, Germany Search for more papers by this author Jürgen Wienands Jürgen Wienands Institute of Cellular and Molecular Immunology, Georg August University of Göttingen, Göttingen, Germany Search for more papers by this author Jonathan Chernoff Jonathan Chernoff Fox Chase Cancer Center, Philadelphia, PA, USA Search for more papers by this author Deborah Yablonski Corresponding Author Deborah Yablonski [email protected] Rappaport Family Institute for Research in the Medical Sciences, The Bruce Rappaport Faculty of Medicine, Technion—Israel Institute of Technology, Haifa, Israel Search for more papers by this author Meirav Sela Meirav Sela Rappaport Family Institute for Research in the Medical Sciences, The Bruce Rappaport Faculty of Medicine, Technion—Israel Institute of Technology, Haifa, Israel Search for more papers by this author Yaron Bogin Yaron Bogin Rappaport Family Institute for Research in the Medical Sciences, The Bruce Rappaport Faculty of Medicine, Technion—Israel Institute of Technology, Haifa, Israel Search for more papers by this author Dvora Beach Dvora Beach Rappaport Family Institute for Research in the Medical Sciences, The Bruce Rappaport Faculty of Medicine, Technion—Israel Institute of Technology, Haifa, Israel Search for more papers by this author Thomas Oellerich Thomas Oellerich Institute of Cellular and Molecular Immunology, Georg August University of Göttingen, Göttingen, GermanyPresent address: Department of Medicine II, Hematology and Oncology, Goethe Universität Frankfurt, Frankfurt, Germany Search for more papers by this author Johanna Lehne Johanna Lehne Max Planck Institute of Biophysical Chemistry, Bioanalytical Mass Spectrometry Group, Göttingen, Germany Search for more papers by this author Jennifer E Smith-Garvin Jennifer E Smith-Garvin Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Search for more papers by this author Mariko Okumura Mariko Okumura Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Search for more papers by this author Elina Starosvetsky Elina Starosvetsky Rappaport Family Institute for Research in the Medical Sciences, The Bruce Rappaport Faculty of Medicine, Technion—Israel Institute of Technology, Haifa, Israel Search for more papers by this author Rachelle Kosoff Rachelle Kosoff Fox Chase Cancer Center, Philadelphia, PA, USA Cancer Biology Program, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Evgeny Libman Evgeny Libman Rappaport Family Institute for Research in the Medical Sciences, The Bruce Rappaport Faculty of Medicine, Technion—Israel Institute of Technology, Haifa, Israel Search for more papers by this author Gary Koretzky Gary Koretzky Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Search for more papers by this author Taku Kambayashi Taku Kambayashi Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Search for more papers by this author Henning Urlaub Henning Urlaub Max Planck Institute of Biophysical Chemistry, Bioanalytical Mass Spectrometry Group, Göttingen, Germany Search for more papers by this author Jürgen Wienands Jürgen Wienands Institute of Cellular and Molecular Immunology, Georg August University of Göttingen, Göttingen, Germany Search for more papers by this author Jonathan Chernoff Jonathan Chernoff Fox Chase Cancer Center, Philadelphia, PA, USA Search for more papers by this author Deborah Yablonski Corresponding Author Deborah Yablonski [email protected] Rappaport Family Institute for Research in the Medical Sciences, The Bruce Rappaport Faculty of Medicine, Technion—Israel Institute of Technology, Haifa, Israel Search for more papers by this author Author Information Meirav Sela1, Yaron Bogin1, Dvora Beach1, Thomas Oellerich2, Johanna Lehne3, Jennifer E Smith-Garvin4, Mariko Okumura5, Elina Starosvetsky1, Rachelle Kosoff6,7, Evgeny Libman1, Gary Koretzky4,5, Taku Kambayashi5, Henning Urlaub3, Jürgen Wienands2, Jonathan Chernoff6 and Deborah Yablonski *,1 1Rappaport Family Institute for Research in the Medical Sciences, The Bruce Rappaport Faculty of Medicine, Technion—Israel Institute of Technology, Haifa, Israel 2Institute of Cellular and Molecular Immunology, Georg August University of Göttingen, Göttingen, Germany 3Max Planck Institute of Biophysical Chemistry, Bioanalytical Mass Spectrometry Group, Göttingen, Germany 4Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA, USA 5Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA 6Fox Chase Cancer Center, Philadelphia, PA, USA 7Cancer Biology Program, University of Pennsylvania, Philadelphia, PA, USA *Rappaport Family Institute for Research in the Medical Sciences, The Bruce Rappaport Faculty of Medicine, Technion—Israel Institute of Technology, POB 9649 Bat Galim, Haifa 31096, Israel. Tel.: +972 4 829 5393; Fax: +972 4 829 5255; E-mail: [email protected] The EMBO Journal (2011)30:3160-3172https://doi.org/10.1038/emboj.2011.213 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Cooperatively assembled signalling complexes, nucleated by adaptor proteins, integrate information from surface receptors to determine cellular outcomes. In T and mast cells, antigen receptor signalling is nucleated by three adaptors: SLP-76, Gads and LAT. Three well-characterized SLP-76 tyrosine phosphorylation sites recruit key components, including a Tec-family tyrosine kinase, Itk. We identified a fourth, evolutionarily conserved SLP-76 phosphorylation site, Y173, which was phosphorylated upon T-cell receptor stimulation in primary murine and Jurkat T cells. Y173 was required for antigen receptor-induced phosphorylation of phospholipase C-γ1 (PLC-γ1) in both T and mast cells, and for consequent downstream events, including activation of the IL-2 promoter in T cells, and degranulation and IL-6 production in mast cells. In intact cells, Y173 phosphorylation depended on three, ZAP-70-targeted tyrosines at the N-terminus of SLP-76 that recruit and activate Itk, a kinase that selectively phosphorylated Y173 in vitro. These data suggest a sequential mechanism whereby ZAP-70-dependent priming of SLP-76 at three N-terminal sites triggers reciprocal regulatory interactions between Itk and SLP-76, which are ultimately required to couple active Itk to its substrate, PLC-γ1. Introduction The adaptive immune system responds to antigens through a variety of receptor types, including the T-cell receptor (TCR), B-cell receptor (BCR) and Fc receptors. The latter are indirect antigen receptors whose specificity is determined by the bound antibody. An important example is the FcεRI of mast cells, which mediates immediate type hypersensitivity responses upon exposure to the cognate antigen of the bound IgE molecule. Antigen receptors signal through broadly similar pathways, in which Src-, Syk- and Tec-family tyrosine kinases form a cascade that results in tyrosine phosphorylation and activation of phospholipase C-γ isoforms (PLC-γ1 or PLC-γ2) (Carpenter and Ji, 1999). In addition to the kinases, cell type specific adaptor proteins are absolutely required for PLC-γ phosphorylation. In T cells and mast cells, this function is carried out by a heterotrimeric complex of adaptor proteins consisting of LAT, Gads and SLP-76 (reviewed in Koretzky et al, 2006; Alvarez-Errico et al, 2009; Kambayashi et al, 2009). LAT is a transmembrane adaptor that is heavily phosphorylated on tyrosine residues upon TCR or FcεRI stimulation. PLC-γ1 binds directly to LAT, whereas SLP-76 is indirectly recruited to LAT by Gads (Liu et al, 1999; Ishiai et al, 2000). Within this complex, SLP-76 binds and activates Itk, a Tec-family kinase that can phosphorylate PLC-γ1 at the sites required for its activation (Liu et al, 1998; Houtman et al, 2005; Bogin et al, 2007). In addition, SLP-76 binds to other proteins that regulate PLC-γ1 activation by incompletely understood mechanisms; the most prominent among these is Vav (Reynolds et al, 2002). Once activated, PLC-γ1 produces second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), that trigger calcium flux and Ras activation, respectively. In mast cells, increased intracellular calcium triggers rapid release of preformed mediators, through a process of vesicle exocytosis, known as degranulation. These mediators produce most of the symptoms of immediate type hypersensitivity. Over a longer time course, both T and mast cells transcribe and secrete cytokines, through processes that involve calcium–calcineurin-mediated dephosphorylation and activation of the NFAT transcription factor, and Ras-dependent activation of AP-1. As an essential regulator of PLC-γ1 activation, the SLP-76 adaptor protein is required for all of the above signalling events (Yablonski et al, 1998; Pivniouk et al, 1999). SLP-76 is expressed in all haematopoietic cells except B cells, where an analogous protein, SLP-65/BLNK is expressed (Fu et al, 1998; Wienands et al, 1998). SLP-76-deficient mice fail to develop mature T cells due to a block in pre-TCR signalling (Clements et al, 1998; Pivniouk et al, 1998). SLP-76-deficient mast cells develop normally, but exhibit defective responses to FcεRI activation (Pivniouk et al, 1999). In particular, FcεRI-induced PLC-γ1 activation is defective, as are the ensuing steps of degranulation and cytokine production. In addition, a SLP-76-deficient derivative of the Jurkat T cell line, known as J14, is useful for mechanistic studies of SLP-76. J14 cells fail to activate PLC-γ1 or to transcribe IL-2 in response to TCR stimulation, but signalling is restored upon reconstitution with wild-type SLP-76 (Yablonski et al, 1998). Based on these genetic models, SLP-76 has become an important paradigm for understanding adaptor protein function. SLP-76 contains three regions that mediate interactions with other signalling proteins: an N-terminal acidic domain that includes three well-characterized tyrosine phosphorylation sites, a central proline-rich domain and a C-terminal SH2 domain (Koretzky et al, 2006). An N-terminal SAM domain is also required for full functionality (Shen et al, 2009). Upon TCR stimulation, the three N-terminal tyrosines are phosphorylated by ZAP-70 (Wardenburg et al, 1996; Raab et al, 1997), and bind to three proteins, Nck, Vav and Itk (Tuosto et al, 1996; Wu et al, 1996; Raab et al, 1997; Bubeck Wardenburg et al, 1998; Su et al, 1999; Wunderlich et al, 1999; Bunnell et al, 2000). Mutation of all three tyrosines eliminates SLP-76 tyrosine phosphorylation (Fang et al, 1996; Wardenburg et al, 1996) and nearly abrogates its function (Myung et al, 2001; Yablonski et al, 2001; Kettner et al, 2003). The central proline-rich domain contains two additional regions that are required for SLP-76 function: a short Gads-binding motif (Musci et al, 1997; Berry et al, 2002), and the P-I region, which is found between the N-terminal tyrosine phosphorylation sites and the Gads-binding motif (Yablonski et al, 2001). The P-I region can bind to the SH3 domains of PLC-γ1, Itk and Lck (Sanzenbacher et al, 1999; Bunnell et al, 2000; Yablonski et al, 2001; Grasis et al, 2010); but its role in T-cell activation has been subject to multiple, often conflicting interpretations (Singer et al, 2004; Gonen et al, 2005; Kumar et al, 2005; Grasis et al, 2010). Of the proteins that bind to SLP-76, Itk is the most directly connected to PLC-γ1 activation, since it can phosphorylate PLC-γ1 at the sites that are required for its activation (Bogin et al, 2007). Catalytic activation of Itk depends on the inducible interaction of its SH2 domain with the N-terminal tyrosines of SLP-76 (Bogin et al, 2007). The additional interaction of its SH3 domain with the P-I region of SLP-76 appears to facilitate recruitment of Itk to the immune synapse (Bunnell et al, 2000; Grasis et al, 2010). An ongoing interaction of SLP-76 with Itk is required to maintain its catalytic activity (Bogin et al, 2007). This close interaction raises the possibility of reciprocal regulation; whereby SLP-76-activated Itk could feed back onto SLP-76 by phosphorylating it at other sites. Although SLP-76 is considered to have only three tyrosine phosphorylation sites, we suspected that these sites, by recruiting and activating Itk, might prime SLP-76 for phosphorylation at other sites. In this study, we identified a new tyrosine phosphorylation site on SLP-76 and characterized its importance for antigen receptor signalling in both T cells and mast cells. This new site, Y173, is located in the P-I region of SLP-76, a region that is critical for SLP-76-mediated signalling (Yablonski et al, 2001; Singer et al, 2004), but whose mechanistic role has been difficult to dissect (Gonen et al, 2005). By revealing an additional layer of regulation in the antigen receptor signalling pathways, this observation brings us closer to understanding the reciprocal interactions between enzymes and adaptor proteins that mediate the rapid and exquisite responsiveness of the immune system. Results SLP-76 tyrosine Y173 is selectively phosphorylated by Itk TCR stimulation triggers a cascade of kinases, each with a distinct role and substrate specificity. The N-terminus of SLP-76 is efficiently phosphorylated by ZAP-70, but not by Src-family kinases (Wardenburg et al, 1996; Raab et al, 1997). Phosphorylated SLP-76 recruits and activates Itk (Bunnell et al, 2000; Bogin et al, 2007); in turn, the PLC-γ1 sites required for its activation are efficiently phosphorylated by Itk, but not by ZAP-70 (Bogin et al, 2007). Despite their different substrate specificity, Itk and ZAP-70 exhibited comparable ability to phosphorylate a recombinant SLP-76 substrate in vitro (Lin et al, 2004; Bogin et al, 2007). This observation prompted us to search for tyrosine phosphorylation sites on SLP-76 that may be selectively phosphorylated by Itk. To map the sites targeted by each kinase, we immunopurified ZAP-70 and Itk from TCR-stimulated Jurkat cells and tested their ability to phosphorylate recombinant fragments of SLP-76 in vitro. An N-terminal fragment of SLP-76 encompassing residues 2–163 was efficiently phosphorylated by ZAP-70 but not by Itk (Figure 1A). Using phosphospecific antisera to SLP-76 Y113, 128 and 145, we detected ZAP-70-mediated phosphorylation of each of these sites (data not shown). This fragment was not phosphorylated by Itk; however, we noted that SLP-76 contains a fourth, evolutionarily conserved tyrosine, located at position 173 (Supplementary Figure S1). A substrate encompassing this residue was efficiently phosphorylated by Itk, but not by ZAP-70, and phosphorylation was abolished by mutation of Y173 to phenylalanine (Figure 1B). These experiments identify Y173 as a potential Itk-targeted phosphorylation site on SLP-76. Figure 1.Selective phosphorylation of SLP-76 sites by Itk and ZAP-70. (A, B) Itk (left panels) or ZAP-70 (right panels) immune complexes were prepared from the lysates of 8 × 106 TCR-stimulated cells, and their catalytic activity was assayed, using the indicated GST-SLP-76 recombinant fusion proteins as in vitro substrates, in the presence or absence of 100 μM ATP. Kinase reaction products were separated by SDS/PAGE, and substrate phosphorylation was detected by probing with anti-phosphotyrosine (4G10) antibody (top panels). The immune complex beads were probed by western blotting with anti-Itk and anti-ZAP-70 antibodies, as indicated (bottom panels). Recombinant substrates were GST alone (−), GST fused to the N-terminal region of SLP-76 (NT; residues 2–163), or GST fused to a fragment of the proline-rich region of SLP-76 (PR; residues 150–196, either wild-type or mutated at Y173). Migration of the NT and PR substrates is indicated with arrows to the right of (B). Results are representative of three independent experiments. See also Supplementary Figure S1. Download figure Download PowerPoint TCR-inducible phosphorylation of SLP-76 at Y173 proceeds by a sequential mechanism We postulated that the efficient phosphorylation of SLP-76 by Itk in vitro may recapitulate what happens upon TCR stimulation of intact cells. To test this idea, we performed mass spectrometric analysis of SLP-76, purified from TCR-stimulated cells. SLP-76 protein was digested with the endoprotease, Asp-N, chosen for its ability to cleave the acidic region of SLP-76, where the known tyrosine phosphorylation sites are found. Phosphorylated peptides were enriched by titanium oxide chromatography followed by MS and MSMS analysis. The expected Asp-N cleavage product, encompassing phosphorylated Y173, was unambiguously detected in this analysis (Figure 2A). In addition, we detected one of the previously known phosphorylation sites, Y145 (Supplementary Figure S2). Figure 2.TCR-inducible phosphorylation of Y173 in intact T cells. (A) Mass spectrometry analysis of a peptide derived from SLP-76 with Y173 being phosphorylated. FLAG-tagged SLP-76 was immunopurified from TCR-stimulated J14-76-11 cells and digested with Asp-N endoprotease, followed by enrichment of phosphopeptides with titanium oxide chromatography and LC-coupled MSMS analysis. Shown is the MSMS analysis of the peptide DEEALQNSILPAKPFPNSNSMpYI derived from SLP-76. The insert shows the intact mass-to-charge ratios of the particular phosphopeptides selected for analysis by MSMS, the sequence of the peptide, and the corresponding b-type ions that unambiguously identified Tyr173 to be phosphorylated. The mass of the intact peptide (MS) and the mass deviation between calculated and experimental mass are shown. See also Supplementary Figure S2. (B) TCR-inducible phosphorylation of Y173 in primary T cells. Murine thymocytes (left panels) and negatively purified splenic T cells (right panels) were stimulated for the indicated time with avidin-crosslinked anti-CD3 and anti-CD4 (thymocytes) or avidin-crosslinked anti-CD3 (splenic T cells) and lysed. Lysates were probed with anti-SLP-76 phospho-Y173, then stripped and reprobed with anti-murine SLP-76. Results are representative of three independent experiments. (C) Y173-independent phosphorylation of the N-terminal tyrosines. J14 cells, retrovirally reconstituted wild-type or Y173F-mutated, FLAG-tagged SLP-76, were stimulated with anti-TCR for the indicated time and lysed. Anti-FLAG immunoprecipitates from 15 million cells (top two panels) or lysates from 0.5 million cells (third and fourth panels) were probed with the indicated anti-SLP-76 phosphospecific antibodies. Subsequent stripping and reprobing of membranes with anti-FLAG indicated equivalent loading of SLP-76 in all lanes (bottom panel and data not shown). Results are representative of two independent experiments. Download figure Download PowerPoint For routine detection of Y173 phosphorylation, we prepared an affinity-purified, polyclonal, phospho-Y173-specific antiserum. Using this reagent, we observed rapid and transient phosphorylation of Y173 in primary murine thymocytes upon co-crosslinking of CD3 and CD4, whereas CD3 crosslinking was sufficient to induce phosphorylation of Y173 in primary murine splenic T cells (Figure 2B). To confirm the specificity of this reagent, we probed lysates from TCR-stimulated J14 cells, stably reconstituted with FLAG-tagged wild-type or Y173-mutated SLP-76. Wild-type SLP-76 was inducibly phosphorylated at Y173, with a time course roughly parallel to that of the three previously known phosphorylation sites (Figure 2C, left four lanes). Mutation of Y173 to phenylalanine abolished the signal detected with the phosphoY173 reagent, but did not affect phosphorylation of the three previously known phosphorylation sites (Figure 2C). This result provides strong evidence for TCR-inducible phosphorylation of Y173. Incidentally, this result also shows that phosphorylation of the three N-terminal sites proceeds independently of Y173. Phosphorylation of many proteins proceeds according to a stepwise mechanism, whereby one site primes the protein for subsequent phosphorylation at additional sites. In the case of SLP-76, the three N-terminal sites are required for recruitment and activation of Itk (Bogin et al, 2007), suggesting that they may be required for phosphorylation of Y173. Consistent with this idea, Y173 was not phosphorylated in J14 cells that stably express the Y3F mutant of SLP-76, in which tyrosines 113, 128 and 145 are mutated to phenylalanine (Figure 3A). Figure 3.Phosphorylation of Y173 is primed by three N-terminal tyrosines. The indicated cell types were stimulated and lysed. Western blots of the lysates were probed with the indicated phosphospecific antibodies, then stripped and reprobed for the total protein, as indicated. All results shown are representative of at least three independent experiments. The cell types used in these experiments were as follows: (A) J14 cells, stably reconstituted with wild-type or Y3F-mutated (Y113,128,145F) human SLP-76. (B) One day before stimulation and lysis, J14 cells were transiently transfected with the indicated alleles of FLAG-tagged human SLP-76: wild-type; Y3F (Y113,128,145F) Y2F (Y113, 128F) or Y145F. (C) Thymocytes were isolated from gene-targeted ‘knockin’ mice bearing the indicated point mutations in SLP-76, and stimulated with avidin-crosslinked anti-CD3 and anti-CD4. (D) CD90.2+ purified splenic T cells were isolated from the strains of mice shown in (C), and stimulated with crosslinked anti-CD3. Download figure Download PowerPoint The N-terminal phosphorylation sites of SLP-76 have been divided into two groups according to the sequence immediately surrounding the phosphorylated tyrosine. Y113 and 128 are embedded in the sequence DYESP, whereas Y145 occurs in the sequence DYEPPP (Fang et al, 1996). To address their contribution to Y173 phosphorylation, J14 cells were transiently transfected with SLP-76 that was either wild-type or mutated at one (Y145F), two (Y2F; Y113,128F) or three (Y3F; Y113,128,145F) tyrosines. As previously reported (Jordan et al, 2006), TCR-induced phosphorylation of PLC-γ1 was markedly reduced by the single and double mutations of SLP-76 and abrogated by the triple mutation. Phosphorylation of Y173 followed a similar pattern (Figure 3B), suggesting that both the DYESP and the DYEPPP motifs contribute to Y173 phosphorylation. Broadly similar results were obtained upon stimulation of thymocytes (Figure 3C) or splenic T cells (Figure 3D) from gene-targeted mice that bear genomic Y145F or Y112,128F point mutations on SLP-76 (Jordan et al, 2008). Whereas the Y145F mutation produced a substantial reduction in Y173 phosphorylation, it was virtually eliminated in mice bearing the Y112,128F allele of SLP-76 (Figure 3C and D). Taken together, these results strongly support TCR-induced sequential phosphorylation of SLP-76 on at least four tyrosines. Activation of PLC-γ1 by the TCR depends on tyrosine 173 of SLP-76 To explore the role of Y173 in TCR signalling, we stably reconstituted J14 cells with wild-type or mutant FLAG-tagged SLP-76, by infection with an IRES-GFP-containing retroviral vector, followed by FACS-based cell sorting to remove noninfected cells. Y173 was disrupted by mutation to phenylalanine (Y173F), to alanine (Y173A) or by a short deletion (Δ158–180). GFP and TCR expression in each of the cell lines is presented in Supplementary Figure S3. We first tested whether Y173 is required for recruitment of proteins to the SLP-76- and LAT-nucleated signalling complex. Upon TCR stimulation, wild-type SLP-76 associated with a number of proteins, including PLC-γ1, Vav, Itk, Lck and Nck. None of these interactions was disrupted by the Y173F mutation; however, we reproducibly observed a somewhat extended association of the Y173F mutant with PLC-γ1 and Vav (Figure 4A). Taken together with Figure 2C, these experiments demonstrate that the Y173F mutation does not affect phosphorylation of SLP-76 at its N-terminal tyrosine phosphorylation sites, nor does it affect the recruitment of Nck, Vav and Itk to these sites. Even the indirect association of SLP-76 with PLC-γ1 was not reduced by the Y173F mutation, suggesting that the SLP-76- and LAT-nucleated signalling complex is largely intact. Figure 4.Y173 is required for TCR-induced phosphorylation of PLC-γ1. J14 cells were retrovirally reconstituted with wild-type or Y173F-mutated, FLAG-tagged SLP-76 (see Supplementary Figure S3). Cells were stimulated for the indicated time with anti-TCR and lysed. All results shown are representative of at least three independent experiments. (A) TCR-inducible recruitment of signalling proteins to the SLP-76-nucleated complex. Anti-FLAG immunoprecipitates prepared from the lysates of 20 million cells were separated by SDS–PAGE on a 9–12% gradient gel, and were analysed by probing the western blot with the indicated antibodies. (B) TCR-induced tyrosine phosphorylation. Lysates were probed with anti-phosphotyrosine (4G10). (C) TCR-induced phosphorylation of PLC-γ1 and Erk1/2. Anti-PLC-γ1 immunoprecipitates (top two panels) or lysates (bottom two panels) were probed with the indicated phosphospecific antibodies, then stripped and reprobed to detect total PLC-γ1or Erk1/2. See also Supplementary Figure S4. Download figure Download PowerPoint The overall pattern of TCR-induced tyrosine-phosphorylated proteins was not grossly affected by mutation of Y173; however, phosphorylation of the 150-kDa band corresponding to PLC-γ1 was abrogated (Figure 4B). The marked dependence of PLC-γ1 phosphorylation on tyrosine 173 was more convincingly shown by using a phosphospecific antibody for PLC-γ1 Y783, one of the sites required for its activation (Serrano et al, 2005) (Figure 4C, top two panels). A similar impairment of PLC-γ1 phosphorylation was observed upon mutation of Y173 to alanine, or upon deletion of the region of SLP-76 surrounding Y173 (Δ158–180) (Supplementary Figure S4). This impairment was quite profound; indeed, PLC-γ1 phosphorylation in the Y173F mutant cells was only slightly higher than in the absence of SLP-76 (Figure 4C). Unlike the profound decrease in PLC-γ1 phosphorylation, TCR-induced tyrosine phosphorylation of Itk did not depend on
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