T Cell Receptor-dependent Tyrosine Phosphorylation of β2-Chimaerin Modulates Its Rac-GAP Function in T Cells
2009; Elsevier BV; Volume: 284; Issue: 17 Linguagem: Inglês
10.1074/jbc.m806098200
ISSN1083-351X
Autores Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoThe actin cytoskeleton has an important role in the organization and function of the immune synapse during antigen recognition. Dynamic rearrangement of the actin cytoskeleton in response to T cell receptor (TCR) triggering requires the coordinated activation of Rho family GTPases that cycle between active and inactive conformations. This is controlled by GTPase-activating proteins (GAP), which regulate inactivation of Rho GTPases, and guanine exchange factors, which mediate their activation. Whereas much attention has centered on guanine exchange factors for Rho GTPases in T cell activation, the identity and functional roles of the GAP in this process are largely unknown. We previously reported β2-chimaerin as a diacylglycerol-regulated Rac-GAP that is expressed in T cells. We now demonstrate Lck-dependent phosphorylation of β2-chimaerin in response to TCR triggering. We identify Tyr-153 as the Lck-dependent phosphorylation residue and show that its phosphorylation negatively regulates membrane stabilization of β2-chimaerin, decreasing its GAP activity to Rac. This study establishes the existence of TCR-dependent regulation of β2-chimaerin and identifies a novel mechanism for its inactivation. The actin cytoskeleton has an important role in the organization and function of the immune synapse during antigen recognition. Dynamic rearrangement of the actin cytoskeleton in response to T cell receptor (TCR) triggering requires the coordinated activation of Rho family GTPases that cycle between active and inactive conformations. This is controlled by GTPase-activating proteins (GAP), which regulate inactivation of Rho GTPases, and guanine exchange factors, which mediate their activation. Whereas much attention has centered on guanine exchange factors for Rho GTPases in T cell activation, the identity and functional roles of the GAP in this process are largely unknown. We previously reported β2-chimaerin as a diacylglycerol-regulated Rac-GAP that is expressed in T cells. We now demonstrate Lck-dependent phosphorylation of β2-chimaerin in response to TCR triggering. We identify Tyr-153 as the Lck-dependent phosphorylation residue and show that its phosphorylation negatively regulates membrane stabilization of β2-chimaerin, decreasing its GAP activity to Rac. This study establishes the existence of TCR-dependent regulation of β2-chimaerin and identifies a novel mechanism for its inactivation. T cell activation requires presentation of an antigen by antigen-presenting cells (APC) 2The abbreviations used are: APC, antigen-presenting cell; Ab, antibody; CFP, cyan fluorescent protein; EGF, epidermal growth factor; GAP, GTPase-activating protein; GEF, guanosine exchange factor; IL-2, interleukin 2; IS, immunological synapse; TCR, T cell receptor; RFP, red fluorescent protein; PMA, phorbol myristate acetate; DAG, diacylglycerol; GFP, green fluorescent protein; PBS, phosphate-buffered saline; SEE, Staphylococcus enterotoxin E; PLC, phospholipase C; SH, Src homology; GTP, guanosine triphosphate; GDP, guanosine diphosphate; Tyr(P), phosphotyrosine. 2The abbreviations used are: APC, antigen-presenting cell; Ab, antibody; CFP, cyan fluorescent protein; EGF, epidermal growth factor; GAP, GTPase-activating protein; GEF, guanosine exchange factor; IL-2, interleukin 2; IS, immunological synapse; TCR, T cell receptor; RFP, red fluorescent protein; PMA, phorbol myristate acetate; DAG, diacylglycerol; GFP, green fluorescent protein; PBS, phosphate-buffered saline; SEE, Staphylococcus enterotoxin E; PLC, phospholipase C; SH, Src homology; GTP, guanosine triphosphate; GDP, guanosine diphosphate; Tyr(P), phosphotyrosine. to the T cell receptor (TCR); this event involves the reorganization of several scaffolds and signaling proteins, leading to formation of the immunological synapse (IS) (1.Cemerski S. Shaw A. Curr. Opin. Immunol. 2006; 18: 298-304Crossref PubMed Scopus (102) Google Scholar). Correct protein redistribution during synapse formation is critical for an efficient T cell response, and it is largely regulated by actin polymerization at the T cell/APC contact site as a result of TCR-regulated Rac-dependent signals (2.Dustin M.L. Cooper J.A. Nat. Immunol. 2000; 1: 23-29Crossref PubMed Scopus (552) Google Scholar, 3.Billadeau D.D. Nolz J.C. Gomez T.S. Nat. Rev. Immunol. 2007; 7: 131-143Crossref PubMed Scopus (271) Google Scholar). Like other Rho GTPases, Rac cycles between a GTP-bound active state and a GDP-bound inactive state. This continuous recycling is regulated by the concerted action of two proteins as follows: GEF, which activates Rac by mediating GDP/GTP exchange (4.Rossman K.L. Der C.J. Sondek J. Nat. Rev. Mol. Cell Biol. 2005; 6: 167-180Crossref PubMed Scopus (1290) Google Scholar), and GAP, which induces Rac inactivation by accelerating intrinsic Rac GTPase activity, converting GTP to GDP (5.Moon S.Y. Zheng Y. Trends Cell Biol. 2003; 13: 13-22Abstract Full Text Full Text PDF PubMed Scopus (480) Google Scholar). Vav-1 is the best studied GEF for Rac, and it has critical roles in T cell-dependent functions (6.Tybulewicz V.L. Curr. Opin. Immunol. 2005; 17: 267-274Crossref PubMed Scopus (272) Google Scholar). In naive, unstimulated T cells, Vav-1 is in an inactive state through autoinhibition, as the GEF domain is blocked by the N-terminal region (7.Aghazadeh B. Lowry W.E. Huang X.Y. Rosen M.K. Cell. 2000; 102: 625-633Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). This autoinhibition is relieved by TCR-mediated tyrosine phosphorylation (8.Han J. Das B. Wei W. Van Aelst L. Mosteller R.D. Khosravi-Far R. Westwick J.K. Der C.J. Broek D. Mol. Cell. Biol. 1997; 17: 1346-1353Crossref PubMed Scopus (276) Google Scholar, 9.Salojin K.V. Zhang J. Delovitch T.L. J. Immunol. 1999; 163: 844-853PubMed Google Scholar). Thymocytes from Vav-1-deficient mice have a developmental block, and their mature T cells show severe defects in IS formation, as well as reduced Ca2+ influx, IL-2 production, T cell proliferation, and cytotoxic activity (10.Holsinger 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, 11.Fischer 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, 12.Costello P.S. Walters A.E. Mee P.J. Turner M. Reynolds L.F. Prisco A. Sarner N. Zamoyska R. Tybulewicz V.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3035-3040Crossref PubMed Scopus (215) Google Scholar, 13.Penninger J.M. Fischer K.D. Sasaki T. Kozieradzki I. Le J. Tedford K. Bachmaier K. Ohashi P.S. Bachmann M.F. Eur. J. Immunol. 1999; 29: 1709-1718Crossref PubMed Scopus (33) Google Scholar). Although several studies have shown a key role for Vav-1, the mechanisms that govern Rac inactivation downstream of the TCR remain elusive. The chimaerins are a family of Rho-GAP, with specific activity for Rac. In addition to their catalytic domain, they have an N-terminal SH2 domain and a C1 domain required for interaction with the lipid messenger diacylglycerol (DAG) and with phorbol esters (14.Yang C. Kazanietz M.G. Biochem. J. 2007; 403: 1-12Crossref PubMed Scopus (12) Google Scholar). There are two mammalian chimaerin genes (CHN1 and CHN2), which encode the full-lengthα2-(ARHGAP2) and β2-chimaerins (ARHGAP3), and at least one splice variant each (α1 and β1) that lack the SH2 domain. The α-chimaerins are expressed abundantly in brain and are linked to neuritogenesis and axon guidance (15.Hall C. Michael G.J. Cann N. Ferrari G. Teo M. Jacobs T. Monfries C. Lim L. J. Neurosci. 2001; 21: 5191-5202Crossref PubMed Google Scholar, 16.Brown M. Jacobs T. Eickholt B. Ferrari G. Teo M. Monfries C. Qi R.Z. Leung T. Lim L. Hall C. J. Neurosci. 2004; 24: 8994-9004Crossref PubMed Scopus (178) Google Scholar, 17.Beg A.A. Sommer J.E. Martin J.H. Scheiffele P. Neuron. 2007; 55: 768-778Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 18.Wegmeyer H. Egea J. Rabe N. Gezelius H. Filosa A. Enjin A. Varoqueaux F. Deininger K. Schnutgen F. Brose N. Klein R. Kullander K. Betz A. Neuron. 2007; 55: 756-767Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 19.Iwasato T. Katoh H. Nishimaru H. Ishikawa Y. Inoue H. Saito Y.M. Ando R. Iwama M. Takahashi R. Negishi M. Itohara S. Cell. 2007; 130: 742-753Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 20.Shi L. Fu W.Y. Hung K.W. Porchetta C. Hall C. Fu A.K. Ip N.Y. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 16347-16352Crossref PubMed Scopus (81) Google Scholar). β2-Chimaerin expression is ubiquitous (21.Yuan S. Miller D.W. Barnett G.H. Hahn J.F. Williams B.R. Cancer Res. 1995; 55: 3456-3461PubMed Google Scholar) and is involved in EGF-dependent Rac regulation (22.Wang H. Yang C. Leskow F.C. Sun J. Canagarajah B. Hurley J.H. Kazanietz M.G. EMBO J. 2006; 25: 2062-2074Crossref PubMed Scopus (43) Google Scholar, 23.Yang C. Liu Y. Lemmon M.A. Kazanietz M.G. Mol. Cell. Biol. 2006; 26: 831-842Crossref PubMed Scopus (65) Google Scholar). Experiments in T cells showed that β2-chimaerin participates in chemokine-dependent regulation of T cell migration and adhesion (24.Siliceo M. Garcia-Bernal D. Carrasco S. Diaz-Flores E. Coluccio Leskow F. Teixido J. Kazanietz M.G. Merida I. J. Cell Sci. 2006; 119: 141-152Crossref PubMed Scopus (24) Google Scholar). A very recent study implicates chimaerins in the modulation of Rac activity during T cell synapse formation, suggesting that this protein family contributes to DAG-mediated regulation of cytoskeletal remodeling during T cell activation (25.Caloca M.J. Delgado P. Alarcon B. Bustelo X.R. Cell. Signal. 2008; 20: 758-770Crossref PubMed Scopus (22) Google Scholar). Determination of the β2-chimaerin crystal structure provided important clues regarding its mechanism of action. In the absence of stimulation, the protein is in an inactive state in which the N-terminal domain maintains a “closed” conformation, blocking Rac binding and concealing the C1 domain (26.Canagarajah B. Leskow F.C. Ho J.Y. Mischak H. Saidi L.F. Kazanietz M.G. Hurley J.H. Cell. 2004; 119: 407-418Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). These structural data were fully supported by experiments in live T lymphocytes showing that phorbol myristate acetate (PMA)-dependent translocation of β2-chimaerin was less effective than that of its isolated C1 domain (24.Siliceo M. Garcia-Bernal D. Carrasco S. Diaz-Flores E. Coluccio Leskow F. Teixido J. Kazanietz M.G. Merida I. J. Cell Sci. 2006; 119: 141-152Crossref PubMed Scopus (24) Google Scholar). These data not only confirmed the lack of accessibility of the β2-chimaerin C1 domain but also suggested that there are negative regulatory mechanisms that promote β2-chimaerin release from the membrane. DAG-dependent signaling is critical for the modulation of T cell functions, by virtue of its ability to bind and regulate C1 domain-containing proteins such as protein kinase Cθ, protein kinase D, and RasGRP1 (27.Hall C. Lim L. Leung T. Trends Biochem. Sci. 2005; 30: 169-171Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). An important issue is to determine how the different DAG-binding proteins discriminate between distinct DAG pools, and how DAG activates certain C1-containing proteins and not others. Some mechanisms that allow discrimination between DAG receptors include the distinct affinity of C1 domains for different DAG pools, association of C1 domain-containing proteins to specific scaffolds, and/or structural determinants in these proteins that limit C1 domain accessibility to membrane DAG (28.Kazanietz M.G. Caloca M.J. Eroles P. Fujii T. Garcia-Bermejo M.L. Reilly M. Wang H. Biochem. Pharmacol. 2000; 60: 1417-1424Crossref PubMed Scopus (69) Google Scholar, 29.Carrasco S. Merida I. Mol. Biol. Cell. 2004; 15: 2932-2942Crossref PubMed Scopus (106) Google Scholar, 30.Colon-Gonzalez F. Kazanietz M.G. Biochim. Biophys. Acta. 2006; 1761: 827-837Crossref PubMed Scopus (224) Google Scholar). To explore the events that contribute to the specific regulation of β2-chimaerin, we studied β2-chimaerin phosphorylation in the context of TCR stimulation. We show that β2-chimaerin is phosphorylated in tyrosine residues after TCR stimulation, and we identify Lck as the Tyr kinase responsible for this phosphorylation. Generation of point mutants identified Tyr-153, at the hinge of the SH2 and C1 domains, as the main tyrosine residue phosphorylated in response to TCR stimulation. Cells expressing a β2-chimaerin mutant defective for Tyr-153 phosphorylation show anomalies in TCR clustering, conjugate formation, NF-AT activation, and IL-2 production that correlate with elevated Rac-GAP activity in this mutant. Subcellular localization analysis of the β2-chimaerin mutants reveals that impairment of β2-chimaerin phosphorylation at Tyr-153 promotes C1-mediated β2-chimaerin stabilization at the plasma membrane, providing a mechanistic explanation for its higher Rac-GAP activity. In summary, our results demonstrate for the first time that tyrosine kinase-mediated negative regulation of β2-chimaerin is elicited by physiological stimulation in T lymphocytes, and suggest that TCR stimulation provides both positive and negative signals for β2-chimaerin activation. Reagents and Antibodies—Poly-dl-lysine and PMA were from Sigma; Gamma-bind-G-Sepharose beads were from GE Healthcare, and Rac1 activation assay kit was from Upstate Biotechnology, Inc. U73122, PP2, R59949, and Triton X-100 were from Calbiochem, and chamber slides were from Nunc (Lab-Tek). BODIPY-TR-ceramide, rhodamine-phalloidin, and anti-GFP polyclonal antibody were from Invitrogen. Polyclonal anti-MAPK was from Zymed Laboratories Inc. and phospho-p44/42 MAPK was from Cell Signaling. Monoclonal antibodies were anti-p56Lck, -CD3, and -CD28 from Pharmingen; phosphotyrosine 4G10 was from Upstate Biotechnology; GFP was from Roche Applied Science, and Rac1 was from BD Transduction Laboratories. Horseradish peroxidase-conjugated goat anti-mouse and -rabbit IgG were from Dako. Plasmid Constructs—Generation of plasmids encoding pECFP-β2-chimaerin WT and the point mutants ECFP-β2-chimaerin Y21F, Y31F, and Y153F have been described (31.Kai M. Yasuda S. Imai S. Kanoh H. Sakane F. Biochim. Biophys. Acta. 2007; 1773: 1407-1415Crossref PubMed Scopus (15) Google Scholar) and were a generous gift of Dr. F. Sakane (Sapporo Medical University, Japan). β2-Chimaerin WT and β2-chimaerinY153F full-length cDNAs were subcloned at the EcoRI/BamHI site of the pEF-EGFP and pEF-RFP expression vectors modified from pEGFP (Clontech) and pmRFP (kindly provided by Dr. S. Mañes, Centro Nacional de Biotecnología, Madrid, Spain), respectively. The GFP-fused construct of the β2-chimaerin C1 domain was described (29.Carrasco S. Merida I. Mol. Biol. Cell. 2004; 15: 2932-2942Crossref PubMed Scopus (106) Google Scholar). The pEF-GFP-β2-chimaerin F215G mutant was generated as described (24.Siliceo M. Garcia-Bernal D. Carrasco S. Diaz-Flores E. Coluccio Leskow F. Teixido J. Kazanietz M.G. Merida I. J. Cell Sci. 2006; 119: 141-152Crossref PubMed Scopus (24) Google Scholar). The pEGFP-β2-chimaerin Q32A and I130A mutants were kindly provided by Dr. M. G. Kazanietz (University of Pennsylvania Medical School, Philadelphia). pXJ40-GFP-α2-chimaerin was a gift of Dr. C. Hall (Institute of Neurology, London, UK). The plasmid encoding GFP-fused Vav1 was from Dr. X. R. Bustelo (Centro de Investigación del Cancer, Salamanca, Spain) and that encoding p56Lck was from Dr. A. C. Carrera (Centro Nacional de Biotecnología). Cell Culture and Transfection—Human leukemic Jurkat T cells, the Jurkat-derived cell line JCaM-1.6 (defective for Lck expression), and Raji B cells were maintained in RPMI 1640 medium (BioWhittaker) supplemented with 10% fetal bovine serum (Sigma) and 2 mm glutamine (BioWhittaker). Jurkat and JCaM-1.6 cells in logarithmic growth were transiently transfected (1.2 × 107 in 400 μl of complete medium) with 20 μg of plasmid DNA by electroporation using a Gene Pulser (Bio-Rad; 270 V, 975 microfarads). Cells were immediately transferred to 10 ml of complete medium and assayed after 24 h. The human embryonic kidney cell line HEK293 was cultured in Dulbecco's modified Eagle's medium (BioWhittaker) supplemented as above. Cells were transfected using jet-PEI reagent (Polyplus Transfection SAS, Illkirch, France) and assayed 24 h later. Immunoprecipitation and Western Blot—Transfected cells (4 × 106 cells/ml) were stimulated with PMA (800 ng/ml) or anti-CD3/CD28 antibodies (1 μg/ml each) in complete medium (37 °C, 5 min). For inhibitor treatment, before stimulation cells were incubated with U73122 (1 μm; 37 °C, 30 min) or with PP2 or R59949 (30 μm; 37 °C, 1 h). Stimulated cells were pelleted and lysed in lysis buffer (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 10 mm NaF, 5 mm EDTA, 5 mm Na4P2O7, 1 mm Na3VO4, 1% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, and 10 μg/ml each aprotinin and leupeptin) for 15 min on ice. After centrifugation (15,000 × g, 4 °C, 15 min), supernatants were assayed for total protein (DC protein assay, Bio-Rad). For immunoprecipitation of CFP or GFP fusion proteins, equal amounts of total cell lysates (300–600 μg) were precleared with Gamma-bind-Sepharose beads (4 °C, 30 min) and incubated with polyclonal anti-GFP antibody that recognizes both fluorescent tags (2 μg, 4 °C, 2 h), and then with Gamma-bind-Sepharose beads (30 μl, 4 °C, 1 h). Beads were washed twice in lysis buffer, once in 0.5 m LiCl, in 150 mm Tris-HCl, and twice in 150 mm Tris-HCl, and immunoprecipitated proteins were eluted in sample buffer. Bound proteins and total lysates were separated by SDS-PAGE and transferred to nitrocellulose membranes (Amersham Biosciences). Membranes were blocked, and blots were developed with the indicated antibodies and horseradish peroxidase-conjugated anti-rabbit or -mouse antibodies using the ECL detection kit (GE Healthcare). Rac1 Activation Assay—The Rac1 activation assay kit (Upstate Biotechnology, Inc.) was used to precipitate Rac-GTP from cell lysates by binding to the RAC binding domain of PAK-1 fused to glutathione S-transferase (32.Benard V. Bohl B.P. Bokoch G.M. J. Biol. Chem. 1999; 274: 13198-13204Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar). Jurkat cells transfected with the indicated plasmids were plated (5 × 106 cells/ml) onto anti-CD3/CD28 antibody-coated plates (5 μg/ml each) and incubated (37 °C, 15 min). After incubation, medium was removed, and adhered cells were lysed, and equal amounts of cell lysates were processed according to the manufacturer's protocol. Bound proteins were eluted in loading buffer. The p21-binding domain of PAK-1-PAK-GST-associated Rac was detected with an anti-Rac1 monoclonal antibody; 30 μg of each lysate was probed with the same antibody to detect total Rac. Time-lapse Confocal Microscopy—For imaging of live Jurkat cells, cells were pelleted at 24 h post-transfection and suspended in HEPES-balanced salt solution (HBSS: 25 mm HEPES-KOH, pH 7.4, 1 mm MgCl2, 1 mm CaCl2, 132 mm NaCl, 0.1% bovine serum albumin) and plated on poly-dl-lysine-coated chamber slides. Slides were mounted on a 37 °C plate on an Olympus Fluoview 1000 confocal microscope, and PMA (200 ng/ml) was added after the first frame; images were captured every 10 s and processed using Adobe Photoshop and ImageJ software. To monitor Jurkat:APC conjugate formation, Raji cells stably expressing CFP were suspended in HBSS (10 × 106 cells/ml) and incubated alone or with Staphylococcus enterotoxin E (SEE, 1 μg/ml; 37 °C, 1 h). At 24 h post-transfection with the indicated GFP- and RFP-fused constructs, Jurkat cells were suspended (0.3 × 106 cells/ml) in HBSS containing 2% fetal bovine serum, transferred to poly-dl-lysine-coated chamber slides, and allowed to attach at 37 °C. CFP-expressing Raji cells (2.5 × 106 cells/ml) were added to the attached Jurkat cells, and images were recorded every 20 s to monitor conjugate formation. Images were processed as above. Confocal Microscopy of Fixed Jurkat Cells—For F-actin staining of transfected Jurkat cells conjugated with APC, we incubated Raji cells (10 × 106 cells/ml in HBSS) alone or with SEE (1 μg/ml) for 1 h at 37 °C. Cells were washed and suspended in HBSS (106 cells/ml). Transfected Jurkat cells were suspended in HBSS (106 cells/ml) and incubated with pulsed or unpulsed APC (1:1 Jurkat:APC; 37 °C, 10 min). Cells were then transferred to poly-dl-lysine-coated coverslips (50 μg/ml) and allowed to attach for 10 min. Cells were fixed with 2% paraformaldehyde (10 min), washed three times with 150 mm Tris-HCl, pH 7.4, permeabilized with PBS, 0.2% Triton X-100 (5 min), washed twice with PBS, blocked in PBS, 1% bovine serum albumin, and incubated with rhodamine-phalloidin (15 min). Coverslips were mounted on an Olympus Fluoview 1000 confocal microscope. Images were processed using Adobe Photoshop software. Conjugation Assay—Raji cells were incubated alone or with SEE (1 μg/ml, 37 °C, 1–2 h) in HBSS, stained with BODIPY-TR ceramide (5 μm in HBSS, 4 °C, 30 min), washed, and suspended in HBSS (106 cells/ml). Jurkat cells transfected with GFP-fused constructs were suspended in HBSS (106 cells/ml); 0.25 ml Raji cells were combined with 0.25 ml of Jurkat cells in 6-ml round-bottom tubes and incubated (37 °C, 90 min). Tubes were then vortexed (10 s) to resuspend cells, which were fixed with 0.5 ml of 4% paraformaldehyde. The relative proportion of green/red events in each tube was determined by flow cytometry (Beckman-Coulter). The percentage of conjugation was calculated as the number of green/red events (conjugated transfected Jurkat cells) divided by total green events (total transfected Jurkat cells). IL-2 Detection Assay—Jurkat T cells were transfected with GFP-fused constructs and sorted 24 h later to recover GFP-positive cells (Altra Hypersort, Beckman-Coulter). Cells were then added (3 × 105 in 200 μl of culture medium) in duplicate to 48-well plates (Costar) and stimulated with soluble anti-CD3/CD28 Ab (1 μg/ml each) or with PMA (100 ng/ml) alone, PMA with anti-CD3, or PMA with anti-CD3/CD28 Ab. At 24 h post-stimulation, cells were pelleted and supernatants used to determine IL-2 levels with the Bead Immunoassay IL-2 kit (BIOSOURCE) and the xMAP system (Luminex). The cytokine concentration in each sample was calculated from a standard curve prepared using known concentrations of recombinant cytokines. NF-AT Reporter Assay—Jurkat cells were transfected with 15 μg of the NF-AT promoter construct (Adgene), 10 ng of Renilla luciferase vector (Promega) as internal control, and 20 μg of the indicated GFP-fused constructs. After 24 h, cells were washed, allowed to recover (20 h), and stimulated with anti-CD3 or anti-CD3/CD28 (1 μg/ml each) for 8 h (37 °C). Cells were harvested and assayed for luciferase activity using the Dual-Luciferase Reporter Assay (Promega). Luciferase activity was reported relative to Renilla luciferase activity and, where indicated, normalized to the luciferase activity of unstimulated cells. TCR Triggering Induces Tyr Phosphorylation of β2-Chimaerin—β2-chimaerin is expressed in T cells, where it is proposed to regulate Rac activation during T cell synapse formation (25.Caloca M.J. Delgado P. Alarcon B. Bustelo X.R. Cell. Signal. 2008; 20: 758-770Crossref PubMed Scopus (22) Google Scholar). Moreover, a recent study using COS-1 cells showed that Src-dependent tyrosine phosphorylation negatively regulates β2-chimaerin Rac-GAP functions (31.Kai M. Yasuda S. Imai S. Kanoh H. Sakane F. Biochim. Biophys. Acta. 2007; 1773: 1407-1415Crossref PubMed Scopus (15) Google Scholar). This led us to determine whether this type of regulation occurs after physiological stimulation of T cells. We transfected Jurkat T cells with a plasmid encoding CFP (cyan fluorescent protein)-β2-chimaerin, stimulated them with anti-CD3/CD28 antibodies, and immunoprecipitated the CFP-fused proteins with an anti-GFP Ab. Phosphorylation levels in the immunoprecipitated proteins were determined with anti-Tyr(P) Ab. We observed robust phosphorylation of β2-chimaerin following activation of the TCR and its co-stimulatory molecule CD28 (Fig. 1A). Addition of PP2, Src family kinase inhibitor (33.Hanke J.H. Gardner J.P. Dow R.L. Changelian P.S. Brissette W.H. Weringer E.J. Pollok B.A. Connelly P.A. J. Biol. Chem. 1996; 271: 695-701Abstract Full Text Full Text PDF PubMed Scopus (1775) Google Scholar), prevented this phosphorylation, suggesting that Src family Tyr kinase is responsible for β2-chimaerin phosphorylation. Lck is Src kinase that is pivotal to TCR function, because it phosphorylates several signaling molecules in the TCR pathway and allows assembly of the scaffolds required for correct T lymphocyte activation (34.Palacios E.H. Weiss A. Oncogene. 2004; 23: 7990-8000Crossref PubMed Scopus (507) Google Scholar). To determine the relevance of this Tyr kinase for phosphorylation of β2-chimaerin, we performed immunoprecipitation experiments in JCaM 1.6 cells, a Jurkat T cell variant that lacks functional Lck but expresses Fyn and other Src family kinases (35.Straus D.B. Weiss A. Cell. 1992; 70: 585-593Abstract Full Text PDF PubMed Scopus (920) Google Scholar). We observed no phosphorylation of immunoprecipitated β2-chimaerin after CD3/CD28 stimulation of the cells (Fig. 1B). Reintroduction of Lck induced intense phosphorylation of the protein after anti-CD3/CD28 Ab treatment (Fig. 1B). These experiments did not discard β2-chimaerin phosphorylation by an Lck-regulated Tyr kinase. We therefore examined tyrosine phosphorylation of β2-chimaerin in HEK293 cells that express neither Lck nor any other T cell lineage-specific Tyr kinase. Phosphorylation of immunoprecipitated CFP-β2-chimaerin was strictly dependent on p56Lck expression (Fig. 1C), suggesting that this is a direct event independent of other T cell Tyr kinases. These results suggest that Lck is largely responsible for TCR-dependent tyrosine phosphorylation of β2-chimaerin in T cells. The other chimaerin isoform, α2-chimaerin, has recently received considerable attention, as it is an essential downstream effector of EphA4-dependent axon navigation (17.Beg A.A. Sommer J.E. Martin J.H. Scheiffele P. Neuron. 2007; 55: 768-778Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 18.Wegmeyer H. Egea J. Rabe N. Gezelius H. Filosa A. Enjin A. Varoqueaux F. Deininger K. Schnutgen F. Brose N. Klein R. Kullander K. Betz A. Neuron. 2007; 55: 756-767Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 19.Iwasato T. Katoh H. Nishimaru H. Ishikawa Y. Inoue H. Saito Y.M. Ando R. Iwama M. Takahashi R. Negishi M. Itohara S. Cell. 2007; 130: 742-753Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 20.Shi L. Fu W.Y. Hung K.W. Porchetta C. Hall C. Fu A.K. Ip N.Y. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 16347-16352Crossref PubMed Scopus (81) Google Scholar). As this isoform is also reported to act as a negative modulator of T cell responses (25.Caloca M.J. Delgado P. Alarcon B. Bustelo X.R. Cell. Signal. 2008; 20: 758-770Crossref PubMed Scopus (22) Google Scholar), we analyzed whether α2-chimaerin is also subject to tyrosine phosphorylation following T cell activation. Analysis of Jurkat cells expressing green fluorescent protein (GFP)-α2-chimaerin showed TCR/CD28-induced tyrosine phosphorylation, which was not observed in JCaM cells (Fig. 1D). These data suggest that this isoform is also a substrate of Lck-dependent phosphorylation. Identification of Tyr-153 in β2-Chimaerin as the Lck-phosphorylated Residue—Analysis of the β2-chimaerin amino acid sequence (NCBI NP_004058) with the Motifscanner program reveals Tyr-21, Tyr-31, and Tyr-153 as three potential sites of phosphorylation by Src family kinases. To identify the Tyr residue phosphorylated by Lck in response to TCR triggering, we transfected cells with distinct CFP-β2-chimaerin constructs in which each Tyr residue was mutated independently to Phe. Y21F and Y31F replacements did not impair tyrosine phosphorylation in response to TCR, whereas the Y153F mutation resulted in severe impairment of TCR-mediated tyrosine phosphorylation (Fig. 2). This suggests Tyr-153 as the main β2-chimaerin phosphorylation site in Jurkat T cells after TCR triggering. Tyr-153 Phosphorylation of β2-Chimaerin Negatively Regulates Its Rac-GAP Activity—As tyrosine phosphorylation is proposed to impair β2-chimaerin Rac-GAP function (31.Kai M. Yasuda S. Imai S. Kanoh H. Sakane F. Biochim. Biophys. Acta. 2007; 1773: 1407-1415Crossref PubMed Scopus (15) Google Scholar), we next compared the Rac-GAP activity of the β2-chimaerin Y153F mutant with that of its wild type (WT) counterpart. We compared Rac-GTP levels induced by CD3/CD28 stimulation in GFP-expressing control Jurkat cells with those of cells transfected with WT β2-chimaerin or the Y153F mutant fused to GFP. In a pulldown assay, WT β2-chimaerin expression reduced TCR-induced Rac-GTP levels, confirming the Rac-GAP function of β2-chimaerin (Fig. 3A). Cells expressing the Y153F mutant showed a marked decrease in Rac-GTP levels, suggesting that Lck-dependent phosphorylation of β2-chimaerin at Tyr-153 negatively regulates its Rac-GAP activity. To confirm that Tyr-153 phosphorylation indeed represents a mechanism that limits β2-chimaerin Rac-GAP function, we compared the effect of WT and mutant proteins in TCR-triggered Rac-regulated responses. After TCR stimulation, Rac activation is critical for NF-AT transcription, which in turn is necessary for IL-2 production and, finally, cell proliferation (10.Holsinger L.J. Graef I.A. Swat W. Chi T. Bautista D.M. Davidson L. Lewis R.S. Alt F.W.
Referência(s)