T Cell Receptor-mediated Signal Transduction Controlled by the β Chain Transmembrane Domain
2002; Elsevier BV; Volume: 277; Issue: 6 Linguagem: Inglês
10.1074/jbc.m107797200
ISSN1083-351X
AutoresEmma Teixeiro, Patricia Fuentes, Begoña Galocha, Balbino Alarcón, Rafael Bragado,
Tópico(s)CAR-T cell therapy research
ResumoThe bases that support the versatility of the T cell receptor (TCR) to generate distinct T cell responses remain unclear. We have previously shown that mutant cells in the transmembrane domain of TCRβ chain are impaired in TCR-induced apoptosis but are not affected in other functions. Here we describe the biochemical mechanisms by which this mutant receptor supports some T cell responses but fails to induce apoptosis. Extracellular signal-regulated protein kinase (ERK) is activated at higher and more sustained levels in TCRβ-mutated than in wild type cells. Conversely, activation of both c-Jun N-terminal kinase and p38 mitogen-activated protein kinase is severely reduced in mutant cells. By attempting to link this unbalanced induction to altered upstream events, we found that ZAP-70 is normally activated. However, although SLP-76 phosphorylation is normally induced, TCR engagement of mutant cells results in lower tyrosine phosphorylation of LAT but in higher tyrosine phosphorylation of Vav than in wild type cells. The results suggest that an altered signaling cascade leading to an imbalance in mitogen-activated protein kinase activities is involved in the selective impairment of apoptosis in these mutant cells. Furthermore, they also provide new insights in the contribution of TCR to decipher the signals that mediate apoptosis distinctly from proliferation. The bases that support the versatility of the T cell receptor (TCR) to generate distinct T cell responses remain unclear. We have previously shown that mutant cells in the transmembrane domain of TCRβ chain are impaired in TCR-induced apoptosis but are not affected in other functions. Here we describe the biochemical mechanisms by which this mutant receptor supports some T cell responses but fails to induce apoptosis. Extracellular signal-regulated protein kinase (ERK) is activated at higher and more sustained levels in TCRβ-mutated than in wild type cells. Conversely, activation of both c-Jun N-terminal kinase and p38 mitogen-activated protein kinase is severely reduced in mutant cells. By attempting to link this unbalanced induction to altered upstream events, we found that ZAP-70 is normally activated. However, although SLP-76 phosphorylation is normally induced, TCR engagement of mutant cells results in lower tyrosine phosphorylation of LAT but in higher tyrosine phosphorylation of Vav than in wild type cells. The results suggest that an altered signaling cascade leading to an imbalance in mitogen-activated protein kinase activities is involved in the selective impairment of apoptosis in these mutant cells. Furthermore, they also provide new insights in the contribution of TCR to decipher the signals that mediate apoptosis distinctly from proliferation. T cell receptor interleukin extracellular signal-regulated kinase cytotoxic T lymphocyte mitogen-activated protein kinase c-Jun N-terminal kinase glutathioneS-transferase Ras-binding domain protein kinase C phospholipase C immunoreceptor tyrosine-based activation motif The TCR1 expressed in the majority of peripheral T lymphocytes is a complex composed of the clonotypic TCR αβ heterodimer, responsible for antigen and superantigen recognition, linked to the monomorphic CD3-γ, -δ, -ε and -ζ chains, which are involved in signal transduction. This structural complexity, in comparison with other families of cytokines and growth receptors, might be responsible for the different outcomes of TCR engagement. The fact that TCR engagement triggers either cytokine-driven proliferation or cell death is particularly intriguing and raises the question of how these outcomes are differentially regulated. It has promoted the concept that the TCR does not function as a simple on-off switch upon activation. In fact, several approaches have provided evidence that TCR could signal to some activation while keeping other pathways functionally inactive (reviewed in Ref. 1Germain R.N. J. Biol. Chem. 2001; 276: 35223-35226Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Because of the short length of the cytoplasmic tails and the apparent lack of inherent activity of both clonotypic chains, it is assumed that the heterodimer transmits the signal through the CD3/ζ chains. In this context, questions that remain to be answered are the following. 1) How do engaged TCR transfer signals to initiate signal transduction? 2) Which domains of the TCR conserved regions are involved in such transmission? 3) What effector functions are modulated through the integrity of such domains? Only a few reports have stressed the contribution of individual structural domains of the αβ clonotypic module to specific signal transduction. TCRα chain tail seems to be required for down-regulation of the TCR-CD3 complex (2Backstrom B.T. Rubin B. Peter A. Tiefenthaler G. Palmer E. Eur. J. Immunol. 1997; 27: 1433-1441Crossref PubMed Scopus (21) Google Scholar), and chimeric TCRs containing clonotypic δ instead of α residues in the connecting peptide of the TCRα chain fail to trigger IL-2 production upon cell activation (3Backstrom B.T. Milia E. Peter A. Jaureguiberry B. Baldari C.T. Palmer E. Immunity. 1996; 5: 437-447Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). In addition, double positive thymocytes bearing this chimeric TCR undergo negative, but not positive, selection because of a failure in the activation of the extracellular signal-regulated kinase (ERK) (4Backstrom B.T. Muller U. Hausmann B. Palmer E. Science. 1998; 281: 835-838Crossref PubMed Scopus (102) Google Scholar, 5Werlen G. Hausmann B. Palmer E. Nature. 2000; 406: 422-426Crossref PubMed Scopus (179) Google Scholar). A single mutation in the β chain connecting peptide results in a TCR that is specifically deficient in activating both the calcium pathway and IL-2 secretion (6Backstrom B.T. Hausmann B.T. Palmer E. J. Exp. Med. 1997; 186: 1933-1938Crossref PubMed Scopus (25) Google Scholar). Furthermore, individual mutations at either of the two conserved Tyr residues in β chain transmembrane domain allow TCR-CD3 expression and are compatible with normal signaling. On the contrary, dual mutations affected signaling efficiency to a greater extent than predicted by surface expression alone and resulted in severe reductions in IL-2 production and apoptosis (7Fuller-Espie S. Hoffman T.P. Wiest D.L. Tietjen I. Spain L.M. Int. Immunol. 1998; 10: 923-933Crossref PubMed Scopus (20) Google Scholar, 8Kunjibettu S. Fuller-Espie S. Carey G.B. Spain L.M. Int. Immunol. 2001; 13: 211-222Crossref PubMed Scopus (6) Google Scholar). Notwithstanding, although different mouse T cell hybridomas dramatically differed in their functional response to the same mutations, these results suggest that membrane-spanning domains in TCRβ are relevant for signal transduction (8Kunjibettu S. Fuller-Espie S. Carey G.B. Spain L.M. Int. Immunol. 2001; 13: 211-222Crossref PubMed Scopus (6) Google Scholar). In all works, however, information on how mutations might affect biochemical events involved in the activation of upstream intermediates and signaling pathways is basically lacking. The ability of the TCR to transduce quantitatively and qualitatively different signals is strongly supported by the existence of partial agonists, peptide analogues that can selectively stimulate only some T cell effector functions (9Sloan-Lancaster J. Allen P.M. Annu. Rev. Immunol. 1996; 14: 1-27Crossref PubMed Scopus (614) Google Scholar). Partial agonists, usually related to low affinity ligands, have been proved to be a useful tool to understand mechanisms regulating TCR differential signaling. In fact, a broad variety of such peptides has been described that trigger TCR to usually result in anergy, cytokine production, cytolytic activity, or Fas-mediated cytotoxicity as opposed to perforin-dependent cytotoxicity or proliferation (10Brossart P. Bevan M.J. J. Exp. Med. 1996; 183: 2449-2458Crossref PubMed Scopus (83) Google Scholar, 11Cao W. Braciale T.J. J. Mol. Med. 1996; 74: 573-582Crossref PubMed Scopus (3) Google Scholar, 12Sloan-Lancaster J. Evavold B.D. Allen P.M. J. Exp. Med. 1994; 180: 1195-1205Crossref PubMed Scopus (174) Google Scholar). Analogues that selectively impair triggering to apoptosis have not been reported, but, singularly, TCR ligands that uniquely trigger apoptosis in CD4+lymphocytes have been described (13Combadiere B. Sousa C.R. Germain R.N. Lenardo M.J. J. Exp. Med. 1998; 187: 349-355Crossref PubMed Scopus (61) Google Scholar). No similar selective peptides have been reported for CD8+ cells. However, some partial agonists, being inducers of T cell cytotoxicity, can dissociate the induction of Fas-L-dependent CTL death from CTL activation and perforin-dependent (14Wei C.H. Beeson C. Masucci M.G. Levitsky V. J. Immunol. 1999; 163: 2601-2609PubMed Google Scholar) or Fas-L-dependent target cell death (10Brossart P. Bevan M.J. J. Exp. Med. 1996; 183: 2449-2458Crossref PubMed Scopus (83) Google Scholar, 11Cao W. Braciale T.J. J. Mol. Med. 1996; 74: 573-582Crossref PubMed Scopus (3) Google Scholar). In some experimental systems, the ability to divorce killing of the targets and Fas-L-mediated CTL apoptosis has been demonstrated to occur by blocking CD8/MHC class I interaction (15Xu X.N. Purbhoo M.A. Chen N. Mongkolsapaya J. Cox J.H. Meier U.C. Tafuro S. Dunbar P.R. Sewell A.K. Hourigan C.S. Appay V. Cerundolo V. Burrows S.R. McMichael A.J. Screaton G.R. Immunity. 2001; 14: 591-602Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Two major biochemical events are characteristics of triggering with partial agonist: 1) the phosphorylation of CD3-ζ chains, which results in the predominant phosphorylation of the higher mobility phospho-ζ isoform with the almost absent phosphorylation of the lower mobility isoform, was incomplete, and 2) although ZAP-70 was seen to bind to CD3-ζ following signaling, the ZAP-70 molecule itself was not phosphorylated, and its kinase activity was not induced (16Chau L.A. Bluestone J.A. Madrenas J. J. Exp. Med. 1998; 187: 1699-1709Crossref PubMed Scopus (82) Google Scholar, 17Madrenas J. Wange R.L. Wang J.L. Isakov N. Samelson L.E. Germain R.N. Science. 1995; 267: 515-518Crossref PubMed Scopus (501) Google Scholar). In this regard, the fact that a consistent pattern of early signaling is not elicited by ligands capable of inducing similar functional responses (9Sloan-Lancaster J. Allen P.M. Annu. Rev. Immunol. 1996; 14: 1-27Crossref PubMed Scopus (614) Google Scholar, 13Combadiere B. Sousa C.R. Germain R.N. Lenardo M.J. J. Exp. Med. 1998; 187: 349-355Crossref PubMed Scopus (61) Google Scholar) suggests that differential signaling would take place after CD3-ζ and ZAP-70 phosphorylation. Signal transduction through the TCR is initiated by Src kinase-mediated phosphorylation of the ITAMs of the CD3 and ζ chains, followed by the recruitment, phosphorylation, and activation of ZAP-70. The coordinated action of Src kinases and ZAP-70 results in the phosphorylation of multiple substrates. Among these substrates, LAT becomes heavily tyrosine-phosphorylated upon TCR activation, a fact that endows LAT with the capacity for recruiting multiple signaling molecules. It has been hypothesized that signaling through Ras is exquisitely dependent on tyrosine-phosphorylated LAT (18Finco T.S. Kadlecek T. Zhang W. Samelson L.E. Weiss A. Immunity. 1998; 9: 617-626Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). However, several lines of evidence suggest that Ras activation involves additional interactions and that the Ras-MAPK activation can occur through alternative pathways independent of LAT phosphorylation (19Chau L.A. Madrenas J. J. Immunol. 1999; 163: 1853-1858PubMed Google Scholar). These alternative interactions might lead to distinct patterns of ERK activation, which could be associated with the regulation of T cell death or survival (20Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1331Crossref PubMed Scopus (5074) Google Scholar, 21Penninger J.M. Crabtree G.R. Cell. 1999; 96: 9-12Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). In addition to Ras, other small GTPases belonging to the Rho family (Rac-1, Cdc42, and RhoA) are activated upon stimulation of the TCR. These GTPases can activate other MAPK cascades, such as the c-Jun N-terminal kinase (JNK) and p38MAPKactivation pathways that have also been related to apoptosis (22Zhang J. Gao J.X. Salojin K. Shao Q. Grattan M. Meagher C. Laird D.W. Delovitch T.L. J. Exp. Med. 2000; 191: 1017-1030Crossref PubMed Scopus (83) Google Scholar). Vav is a guanosine nucleotide exchange factor for Rho family GTPases whose exchange activity can be modulated by TCR signaling. Such a mechanism, on operating over Rac-1, has been reported to link the TCR to the activation of JNK and p38MAPK (23Salojin K.V. Zhang J. Delovitch T.L. J. Immunol. 1999; 163: 844-853PubMed Google Scholar, 24Kaminuma O. Deckert M. Elly C. Liu Y.C. Altman A. Mol. Cell. Biol. 2001; 21: 3126-3136Crossref PubMed Scopus (74) Google Scholar). LAT phosphorylation appears to play a critical role on the assembly of ZAP-70-LAT-Vav complexes in lipids rafts and on its translocation in the vicinity of Vav downstream effectors (25Salojin K.V. Zhang J. Meagher C. Delovitch T.L. J. Biol. Chem. 2000; 275: 5966-5975Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Additionally, it has been reported that Vav plays TCR-mediated roles other than acting as the GDP/GTP exchanger (26Villalba M. Hernandez J. Deckert M. Tanaka Y. Altman A. Eur. J. Immunol. 2000; 30: 1587-1596Crossref PubMed Scopus (53) Google Scholar, 27Tartare-Deckert S. Monthouel M.N. Charvet C. Foucault I. Van Obberghen E. Bernard A. Altman A. Deckert M. J. Biol. Chem. 2001; 276: 20849-20857Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 28Fischer K.D. Kong Y.Y. Nishina H. Tedford K. Marengere L.E. 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), but the mechanisms involved are poorly understood. We have previously reported that Jurkat-derived mutant T cell clones, bearing a mutation (Tyr to Leu) in the C-terminal tyrosine of the conserved ITAM-like motif of the transmembrane domain of ΤCRβ chain, show normal ability: to secrete cytokines (IL-2 and interferon-γ); to express activation markers; to down-modulate their TCR-CD3 complex; and to mobilize intracellular Ca2+ upon TCR activation. However, these mutant cells show a resistance to TCR-induced apoptosis, a defect that is not observed upon direct Fas stimulation or after stimulating with reagents that bypass TCR engagement (i.e. phorbol esters plus calcium ionophore) (29Rodriguez-Tarduchy G. Sahuquillo A.G. Alarcon B. Bragado R. J. Biol. Chem. 1996; 271: 30417-30425Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 30Sahuquillo A.G. Roumier A. Teixeiro E. Bragado R. Alarcon B. J. Exp. Med. 1998; 187: 1179-1192Crossref PubMed Scopus (37) Google Scholar, 31Teixeiro E. Garcia-Sahuquillo A. Alarcon B. Bragado R. Eur. J. Immunol. 1999; 29: 745-754Crossref PubMed Scopus (28) Google Scholar). Altogether, these findings prompted us to further investigate the effects of the mutation on TCR signaling, in an attempt to clarify the mechanisms by which the integrity of the transmembrane domain of the TCRβ chain contributes to the early biochemical events and MAPK activation pathways associated with TCR-mediated signaling efficiency. Wild type and mutant clones have been previously described (29Rodriguez-Tarduchy G. Sahuquillo A.G. Alarcon B. Bragado R. J. Biol. Chem. 1996; 271: 30417-30425Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar,30Sahuquillo A.G. Roumier A. Teixeiro E. Bragado R. Alarcon B. J. Exp. Med. 1998; 187: 1179-1192Crossref PubMed Scopus (37) Google Scholar). Briefly, they were obtained by reconstitution of the TCRβ-negative Jurkat variant 31.13 with either a wild type Vβ3 TCR cDNA or with a mutant cDNA that contains a tyrosine to leucine mutation in the C-terminal tyrosine of the transmembrane domain. No clonal variation was observed between different clones derived from independent wild type or mutant cDNA transfections (29Rodriguez-Tarduchy G. Sahuquillo A.G. Alarcon B. Bragado R. J. Biol. Chem. 1996; 271: 30417-30425Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 30Sahuquillo A.G. Roumier A. Teixeiro E. Bragado R. Alarcon B. J. Exp. Med. 1998; 187: 1179-1192Crossref PubMed Scopus (37) Google Scholar). The cells were maintained in RPMI supplemented with 10% fetal calf serum and antibiotics. Expression of TCR-CD3 complex was routinely tested by flow cytometry before each assay. The following antibodies were used: 1) for cytometry, anti-CD3ε (UCHT-1-fluorescein isothiocyanate (Caltag, Burlingame, CA); anti-TCRVβ3 (JOVI-3, a gift from Dr. M. Owen); and anti-CD69-fluorescein isothiocyanate (Becton Dickinson, Madrid, Spain); 2) for stimulation, anti-CD3ε (UCHT-1, Immunokontact, Switzerland; used for stimulation at 10 μg/ml); anti-CD28 (Pharmingen, San Diego, CA; used for stimulation at 1 μg/ml); and the cross-linking antibody goat anti-mouse (Pierce; used for stimulation at 10 μg/ml); 3) for immunoprecipitation assays, anti-ZAP-70 (ZAP-4, a gift from Dr. S. Ley, NIMR, London); anti-LAT and anti-Vav (Upstate Biotechnology, Inc., Lake Placid, NY); and anti-SLP-76 (kindly provided by Dr. G. Koretzky, University of Pennsylvania School of Medicine); 4) for Western blot, anti-PLCγ1 and anti-Vav (BD Transduction Laboratories, Lexington, KY); anti-c-Cbl and anti-Raf-1 kinase (Santa Cruz, Santa Cruz, CA); anti-SLP-76 (kindly provided by Dr. G. Koretzky); anti-ERK (Zymed Laboratories Inc., San Francisco, CA); anti-phospho-p44/42MAPK, anti-phospho-p38MAPK, anti-p38MAPK, and anti-JNK (New England Biolabs, Beverly, CA); anti-α-tubulin (Sigma-Aldrich); and anti-LAT, anti-Ras, and anti-phosphotyrosine 4G10 (Upstate Biotechnology, Inc.). Staphylococcal enterotoxin B was purchased from Toxin Technology (Sarasota, FL; used for stimulation at 10 μg/ml). Biotinylated annexin-V was from Roche Molecular Biochemicals, and phycoerythrin-labeled streptavidin was from Caltag (Burlingame, CA). The construct for the glutathione S-transferase (GST) fusion protein containing the Ras-binding domain (RBD) of Raf was from Upstate Biotechnology, Inc. 0.5–2.5 × 107 cells/ml were resuspended in RPMI containing 15 mm HEPES. Unless otherwise detailed, stimulation was performed by incubation with anti-CD3 (10 μg/ml) together with anti-CD28 (1 μg/ml) antibodies for 10 min on ice followed by cross-linking with goat anti-mouse antibodies at 37 °C for the indicated times. 106 cells were incubated on ice for 1 h with a fluorescein isothiocyanate-conjugated specific antibody, washed, and analyzed in a flow cytometer (EPICS-XL MCL, Coulter). 2.5 × 107 cells were unstimulated or stimulated as described and lysed for 30 min at 4 °C in a buffer containing 1% Brij-96 (or 1% Nonidet P-40), 20 mmTris-HCl, pH 7.6, 150 mm NaCl, leupeptin, pepstatin, and aprotinin (1 μg/ml each), 1 mm phenylmethylsulfonyl fluoride, 20 mm NaF, and 1 mmNaVO4. After centrifugation, the lysates were precleared sequentially with Sepharose beads and with protein A- or G-Sepharose beads coated with the appropriate control antibody and afterward subjected to immunoprecipitation for 4 h at 4 °C with protein A- or G-Sepharose beads coated with specific antibodies. Immunoprecipitates were then washed four times with lysis buffer and subjected to SDS-PAGE followed by standard immunoblot analysis with the indicated antibodies. Upon stimulation, the cells were resuspended in ice-cold hypothonic buffer (42 mm KCl, 10 mm HEPES, pH 7.4, 5 mm MgCl2, 10 μg/ml each aprotinin and leupeptin) and incubated on ice for 15 min. The cells were transferred to a 1-ml syringe and sheared by being passed five times through a 30-gauge needle. Whole cell lysates were centrifuged at 200 ×g for 10 min at 4 °C to remove nuclei and cell debris, and the supernatant was collected and centrifuged at 13,000 ×g for 60 min at 4 °C. The supernatant (cytosol) was collected, and the pellet was resuspended in lysis buffer (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, and 10 μg/ml each aprotinin and leupeptin), vortexed for 5 min at 4 °C, and centrifuged again at 13,000 × g for 60 min at 4 °C. The supernatant representing the particulate (membrane) fraction was then saved. Each fraction was then diluted to a final concentration of 1× Laemmli buffer and separated by SDS-PAGE, and the Western blots were analyzed with anti-PKCθ antibodies. 106 cells/time point were unstimulated or stimulated for the indicated times and lysed for 30 min at 4 °C in 50 μl of a buffer containing 1% Nonidet P-40, 20 mmHEPES, 10 mm EGTA, 2.5 mm MgCl2, 40 mm β-glycerophosphate, 1 mm dithiothreitol, and the following protease and phosphatase inhibitors (1 μg/ml each): leupeptin, pepstatin, and aprotinin, 1 mmphenylmethylsulfonyl fluoride, 20 mm NaF, and 1 mm NaVO4. Lysates were then centrifuged, and the supernatants were subjected to standard 10% SDS-PAGE for ERK and p38MAPK or 8% (acrylamide/bisacrylamide 24/0.6) for Raf kinase to improve resolution of phosphorylated band shifts. Electrophoresed proteins were transferred to nitrocellulose membrane for immunoblot analysis with specific antibodies. In the cases of ERK and p38MAPK, their phosphorylation status, as determined by immunoblotting with antibodies that specifically recognize the phosphorylated forms of these kinases, is assumed to be an indication of their activation state. Raf kinase activation was evaluated by the band shifts corresponding to the protein phosphorylated forms, which can be detected by immunoblotting with specific anti-Raf-1 kinase antibodies. 5 × 106 cells/time point were not stimulated or stimulated for the indicated times and lysed for 30 min at 4 °C in 300 μl of a buffer containing 1% Nonidet P-40, 0.25% sodium deoxycholate, 20 mm HEPES, pH 7.5, 150 mm NaCl, 1 mm EDTA, 25 mm NaF, 10 mm MgCl2, 10% glycerol, leupeptin, pepstatin, and aprotinin (1 μg/ml each), 1 mm phenylmethylsulfonyl fluoride, and 1 mm NaVO4. The lysates were then centrifuged, and the supernatants were mixed with 20 μl of a freshly prepared GST fusion protein containing the Ras-binding domain of Raf-1 (GST-RBD), immobilized in glutathione-Sepharose beads (Amersham Biosciences, Inc.). The samples were incubated by rotating for 90 min at 4 °C, and the beads were then washed three times with lysis buffer. The bound Ras-GTP protein was eluted in Laemmli sample buffer and subjected to 12.5% SDS-PAGE. The levels of active (GTP-bound) Ras were assessed by immunoblotting with specific anti-Ras antibodies. 5 × 106 cells were not stimulated or stimulated for the indicated times and lysed for 15 min at 4 °C in 200 μl of lysis buffer ( 0.1% Triton X-100, 25 mm HEPES, pH 7.5, 300 mm NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 0.5 mm dithiothreitol, 20 mm β-glycerolphosphate, 0.1 mm NaVO4, 2 μg/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride). After centrifuging, solid phase kinase assays were performed on supernatants by pull-down precipitation as previously described (32Hibi M. Lin A. Smeal T. Minden A. Karin M. Genes Dev. 1993; 7: 2135-2148Crossref PubMed Scopus (1723) Google Scholar). Briefly, cell extracts were mixed with 10 μl of immobilized GST-c-Jun 1–79, and the samples were rotated at 4 °C for 3 h. After centrifugation, GST-c-Jun-Sepharose beads were washed four times and resuspended in 30 μl of kinase buffer (20 mm HEPES, pH 7.6, 20 mm MgCl2, 20 mmβ-glycerophosphate, 0.1 mm NaVO4, 2 mm dithiothreitol) supplemented with 20 μmATP and 5 μCi of [γ-32P]ATP. After incubation for 20 min at 30 °C, the reaction was stopped by washing with kinase buffer, and the proteins were eluted in Laemmli buffer and subjected to SDS-PAGE followed by autoradiography. The phosphorylation of GST-c-Jun was quantified using a laser densitometer (Molecular Dynamics, Kent, UK) and normalized to JNK as a loading control. We have previously shown that T cell clones bearing a single replacement in the ITAM-like motif of the TCRβ chain transmembrane domain express high amounts of TCR-CD3 complexes, similar to those of wild type cells. In these mutant clones, ZAP-70 becomes normally tyrosine-phosphorylated upon TCR activation. Furthermore, ZAP-70 activity was similar in wild type and mutant cells, as shown in an in vitro kinase assay using an exogenous specific substrate (29Rodriguez-Tarduchy G. Sahuquillo A.G. Alarcon B. Bragado R. J. Biol. Chem. 1996; 271: 30417-30425Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 30Sahuquillo A.G. Roumier A. Teixeiro E. Bragado R. Alarcon B. J. Exp. Med. 1998; 187: 1179-1192Crossref PubMed Scopus (37) Google Scholar). To further characterize the effects of the mutation in TCR-mediated signal transduction, we tested whether this alteration might affect the in vivo tyrosine phosphorylation of ZAP-70 substrates in mutant cells. Among these substrates, LAT is an adapter protein that constitutes the foremost scaffold that links the proximal and distal events of the TCR signaling pathway. Immunoprecipitation with specific anti-LAT antibodies followed by immunoblot with anti-phosphotyrosine showed that in comparison with wild type cells, TCR-inducible tyrosine phosphorylation of LAT was clearly diminished in mutant cells (Fig. 1A). A longer exposure of the LAT immunoprecipitation membrane revealed the presence of previously described tyrosine-phosphorylated proteins that coprecipitate with LAT upon TCR triggering (33Zhang W. Sloan-Lancaster J. Kitchen J. Trible R.P. Samelson L.E. Cell. 1998; 92: 83-92Abstract Full Text Full Text PDF PubMed Scopus (1082) Google Scholar) identified as PLCγ1, c-Cbl, Vav, and SLP76. Interestingly, although the levels of LAT-associated tyrosine-phosphorylated SLP76, Vav, and PLCγ1 were reduced in mutant cells, the levels of LAT-associated phospho-c-Cbl were higher. Furthermore, blotting with specific antibodies indicated that mutant cells are defective in the recruitment of PLCγ1, Vav, and SLP-76 to LAT and in a lesser extent in the recruitment of c-Cbl (Fig.1B). Because ZAP70 was not detectable in LAT immunoprecipitates (33Zhang W. Sloan-Lancaster J. Kitchen J. Trible R.P. Samelson L.E. Cell. 1998; 92: 83-92Abstract Full Text Full Text PDF PubMed Scopus (1082) Google Scholar) and we had reported that ZAP-70 was scarcely recruited to the plasma membrane in TCR-activated mutant cells (30Sahuquillo A.G. Roumier A. Teixeiro E. Bragado R. Alarcon B. J. Exp. Med. 1998; 187: 1179-1192Crossref PubMed Scopus (37) Google Scholar), we performed reverse immunoprecipitation with specific anti-ZAP-70 antibodies to determine whether the low level of induced phospho-LAT in mutant cells was the result of poor activity of ZAP-70 on its membrane substrate. The immunoprecipitation revealed lower levels of coprecipitated phospho-LAT in mutant cells than in wild type cells (Fig. 1C). Furthermore and as expected, the immunoprecipitation revealed similar levels of tyrosine-phosphorylated ZAP-70 in both stimulated cells (Fig.1C). Thus, despite the fact that both the tyrosine phosphorylation level of ZAP-70 and in vitro ZAP-70 activity were similar in both cell types, our results show that the in vivo phosphorylation of LAT and the recruitment to LAT of other signaling molecules are impaired in TCR-stimulated mutant cells. Most of the current evidence suggests that the activation of the Ras-Raf-ERK pathway, after TCR signaling, requires tyrosine-phosphorylated LAT (18Finco T.S. Kadlecek T. Zhang W. Samelson L.E. Weiss A. Immunity. 1998; 9: 617-626Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). If this dependence were absolute, in light of the aforementioned results, we would expect that the impaired LAT phosphorylation would lead downstream to a deficient activation of the ERK pathway in mutant cells. We therefore studied whether the mutation could influence the activation of the Ras-ERK signaling pathway. ERK is the last member in the MAPK cascade that is initiated when Raf is activated after its recruitment by the activated form of Ras. The analysis of the phosphorylation status of ERK, an indication of their activation state (34Schaeffer H.J. Weber M.J. Mol. Cell. Biol. 1999; 19: 2435-2444Crossref PubMed Scopus (1420) Google Scholar), upon TCR engagement revealed not only that the induced ERK activity was higher in mutant cells than in wild type cells but also that this activity lasted longer. Thus, although both activities peaked at 5 min, a high phosphorylation in both p42 and p44 bands was still observed at 60 min in mutant cells, whereas in wild type cells, ERK phosphorylation was almost undetectable after 20 min of stimulation (Fig. 2A). Expression of ERK, assessed by immunoblotting with anti-ERK antibodies, was unaffected by TCR stimulation. As controls, phorbol 12-myristate 13-acetate alone or in combination with ionomycin induced similar kinetics and ERK activities in both wild type and mutant cells (not shown). It was then reasonable to test whether this altered activity of ERK in mutant cells could be the consequence of a similar activation pattern of the upstream intermediates Raf and Ras. Interestingly, the activation of Raf in mutant cells was higher and was sustained longer compared with that in wild type cells, as evidenced by the band shift corresponding to the phosphorylated forms of Raf (Fig. 2B). Finally, we evaluated the levels of active (GTP-bound) Ras by pull-down experiments using an immobilized GST fusion protein containing the RBD of Raf. As shown in Fig. 2C (upper panels), the levels of Ras-GTP significantly increased as early as 1 min in mutant cells, were similarly maintaine
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