T Cell Receptor Engagement Leads to the Recruitment of IBP, a Novel Guanine Nucleotide Exchange Factor, to the Immunological Synapse
2003; Elsevier BV; Volume: 278; Issue: 44 Linguagem: Inglês
10.1074/jbc.m308960200
ISSN1083-351X
AutoresSanjay Gupta, Jessica Fanzo, Chuanmin Hu, Dianne Cox, So Young Jang, Andrea E. Lee, Steven M. Greenberg, Alessandra B. Pernis,
Tópico(s)Cell Adhesion Molecules Research
ResumoReorganization of the actin cytoskeleton is crucial to the formation and function of the immunological synapse. Rho GTPases are critical mediators of cytoskeletal reorganization, and their activity at the synapse is likely to be stringently regulated. Guanine nucleotide exchange factors (GEFs) belonging to the Dbl family of proteins represent one major class of proteins that regulate the activity of Rho GTPases. Here we demonstrate that IBP, a homologue of SWAP-70, is a novel GEF for Rac1 and Cdc42 in T lymphocytes, which is recruited to the immunological synapse upon engagement of the antigen receptor. Mutational analysis supports a model whereby IBP is inactive in unstimulated cells. Upon engagement of the T cell receptor, its GEF activity is enhanced by tyrosine phosphorylation, as well as by binding newly generated phosphatidylinositol 3,4,5-trisphosphate. Although it is known that T cell receptor engagement leads to the recruitment of Vav to the immunological synapse, these findings indicate that other GEFs, such as IBP, also relocalize to this intercellular region. The recruitment and activation of distinct classes of GEFs may allow for precise control of Rho GTPase function at the crucial interface between T cells and antigen presenting cells. Reorganization of the actin cytoskeleton is crucial to the formation and function of the immunological synapse. Rho GTPases are critical mediators of cytoskeletal reorganization, and their activity at the synapse is likely to be stringently regulated. Guanine nucleotide exchange factors (GEFs) belonging to the Dbl family of proteins represent one major class of proteins that regulate the activity of Rho GTPases. Here we demonstrate that IBP, a homologue of SWAP-70, is a novel GEF for Rac1 and Cdc42 in T lymphocytes, which is recruited to the immunological synapse upon engagement of the antigen receptor. Mutational analysis supports a model whereby IBP is inactive in unstimulated cells. Upon engagement of the T cell receptor, its GEF activity is enhanced by tyrosine phosphorylation, as well as by binding newly generated phosphatidylinositol 3,4,5-trisphosphate. Although it is known that T cell receptor engagement leads to the recruitment of Vav to the immunological synapse, these findings indicate that other GEFs, such as IBP, also relocalize to this intercellular region. The recruitment and activation of distinct classes of GEFs may allow for precise control of Rho GTPase function at the crucial interface between T cells and antigen presenting cells. Engagement of the T cell receptor (TCR) 1The abbreviations used are: TCR, T cell receptor; PTK, protein tyrosine kinase; PI3K, phosphoinositide 3-kinase; PI(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PI(3,4)P2, phosphatidylinositol 3,4-biphosphate; PH, pleckstrin homology; APC, antigen presenting cells; IS, immunological synapse; GEF, guanine nucleotide exchange factor; DH, Dbl homology; mAb, monoclonal antibody; HA, hemagglutinin; GST, glutathione S-transferase; PKC, protein kinase C; FITC, fluorescein isothiocyanate; TRITC, tetramethylrhodamine isothiocyanate; Ab, antibody; PI(4,5)P2, phosphatidylinositol 4,5-biphosphate; SEE, Staphylococcus enterotoxin E; PAK1, p21-activated kinase 1.1The abbreviations used are: TCR, T cell receptor; PTK, protein tyrosine kinase; PI3K, phosphoinositide 3-kinase; PI(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PI(3,4)P2, phosphatidylinositol 3,4-biphosphate; PH, pleckstrin homology; APC, antigen presenting cells; IS, immunological synapse; GEF, guanine nucleotide exchange factor; DH, Dbl homology; mAb, monoclonal antibody; HA, hemagglutinin; GST, glutathione S-transferase; PKC, protein kinase C; FITC, fluorescein isothiocyanate; TRITC, tetramethylrhodamine isothiocyanate; Ab, antibody; PI(4,5)P2, phosphatidylinositol 4,5-biphosphate; SEE, Staphylococcus enterotoxin E; PAK1, p21-activated kinase 1. initiates a complex cascade of biochemical events that culminates in the expansion and differentiation of T cells (1van Leeuwen J. Samelson L.E. Curr. Opin. Immunol. 1999; 11: 242-248Crossref PubMed Scopus (217) Google Scholar, 2Kane L.P. Lin J. Weiss A. Curr. Opin. Immunol. 2000; 12: 242-249Crossref PubMed Scopus (426) Google Scholar). Activation of protein tyrosine kinases (PTKs) of the Src family, Lck and Fyn, constitutes one of the most proximal and crucial signaling events that couples receptor engagement to downstream biochemical pathways (3Clements J.L. Koretzky G.A. J. Clin. Invest. 1999; 103: 925-929Crossref PubMed Scopus (39) Google Scholar). These Src family kinases phosphorylate specific tyrosine residues within the immunoreceptor tyrosine-based activation motifs of the CD3 and TCR ζ chain leading to the recruitment and activation of additional kinases, adaptor proteins, and enzymes. Pharmacological and genetic studies have indicated that stimulation of the activity of one enzyme, phosphoinositide 3-kinase (PI3K), is particularly important in mediating the propagation and amplification of the TCR-mediated signaling cascade (4Cantley L.C. Science. 2002; 296: 1655-1657Crossref PubMed Scopus (4639) Google Scholar, 5Ward S.G. Cantrell D.A. Curr. Opin. Immunol. 2001; 13: 332-338Crossref PubMed Scopus (89) Google Scholar). The products of PI3K, PI(3,4,5)P3, and PI(3,4)P2 bind to pleckstrin homology (PH) domains contained in a variety of crucial signaling intermediates inducing the relocalization of these proteins to specific areas of the plasma membrane. T cell recognition of antigen presenting cells (APCs) results in the formation of a specialized interface, termed the immunological synapse (IS) (6Bromley S.K. Burack W.R. Johnson K.G. Somersalo K. Sims T.N. Sumen C. Davis M.M. Shaw A.S. Allen P.M. Dustin M.L. Annu. Rev. Immunol. 2001; 19: 375-396Crossref PubMed Scopus (746) Google Scholar, 7van der Merwe P.A. Curr. Opin. Immunol. 2002; 14: 293-298Crossref PubMed Scopus (97) Google Scholar, 8Delon J. Germain R.N. Curr. Biol. 2000; 10: R923-R933Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). The immunological synapse may function to integrate and/or stabilize TCR-generated signaling pathways and to promote the restricted delivery of secretory products, such as cytokines, to the target cell. Assembly of the immunological synapse is accompanied by the large scale redistribution of receptors and signaling proteins, processes that are critically dependent on TCR-mediated actin cytoskeletal remodeling and polarization. Actin cytoskeletal reorganization in the immunological synapse requires the activity of the Rho family of GTPases (9Dustin M.L. Cooper J.A. Nat. Immunol. 2000; 1: 23-29Crossref PubMed Scopus (556) Google Scholar, 10Krawczyk C. Penninger J.M. J. Leukocyte Biol. 2001; 69: 317-330PubMed Google Scholar). Rho GTPases function as molecular switches that cycle between an inactive GDP-bound form and an active GTP-bound form (11Symons M. Settleman J. Trends Cell Biol. 2000; 10: 415-419Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 12Ridley A.J. Trends Cell Biol. 2001; 11: 471-477Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar, 13Schmidt A. Hall A. Genes Dev. 2002; 16: 1587-1609Crossref PubMed Scopus (978) Google Scholar). Guanine nucleotide exchange factors (GEFs) belonging to the Dbl family of proteins represent major regulators of the activation state of Rho GTPases (14Zheng Y. Trends Biochem. Sci. 2001; 26: 724-732Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). One member of this family, Vav, is critically important in orchestrating TCR-mediated actin cytoskeletal reorganization (15Fischer K.-D. Tedford K. Penninger J.M. Semin. Immunol. 1998; 10: 317-327Crossref PubMed Scopus (73) Google Scholar, 16Bustelo X.R. Oncogene. 2001; 20: 6372-6381Crossref PubMed Scopus (178) Google Scholar, 17Turner M. Billadeau D.D. Nat. Rev. Immunol. 2002; 2: 476-486Crossref PubMed Scopus (263) Google Scholar). Like other members of the Dbl family, Vav contains a sequence of ∼200 amino acids termed the Dbl homology (DH) domain, responsible for catalyzing the GDP/GTP exchange reactions, followed by a C-terminal PH domain necessary for proper intracellular localization and function (18Hoffman G.R. Cerione R.A. FEBS Lett. 2002; 513: 85-91Crossref PubMed Scopus (117) Google Scholar). Binding of PI(3,4,5)P3 to the PH domain serves to activate the GEF activity of Vav, which is further enhanced by multiple tyrosine kinases. Interestingly, recent studies in Drosophila melanogaster indicate that cells employ multiple GEFs to elicit specific subsets of Rho GTPase-mediated responses (19Hakeda-Suzuki S. Ng J. Tzu L. Dietzl G. Sun Y. Harms M. Nardine T. Luo L. Dickson B.J. Nature. 2002; 416: 438-442Crossref PubMed Scopus (298) Google Scholar). Whether GEFs other than Vav are activated and recruited to the immunological synapse is, however, not known. Although the great majority of GEFs for Rho GTPases contain the canonical DH-PH module, recent studies (13Schmidt A. Hall A. Genes Dev. 2002; 16: 1587-1609Crossref PubMed Scopus (978) Google Scholar) have revealed the existence of novel classes of these regulators. In particular, SWAP-70 is a novel type of Rac-GEF, in which the catalytic domain is flanked at its N terminus, rather than at its C terminus, by a PH domain (20Shinohara M. Terada Y. Iwamatsu A. Shihora A. Mochizuki N. Higuchi M. Gotoh Y. Ihara S. Nagata S. Itoh H. Fukui Y. Jessberger R. Nature. 2002; 416: 759-763Crossref PubMed Scopus (185) Google Scholar). The DH domain of SWAP-70 exhibits only a very low degree of homology to that of other Rho-GEFs such as Vav, further suggesting that this class of GEFs may activate a unique subset of Rho GTPase-mediated functions. We recently identified a novel protein termed IBP (IRF-4-binding protein) that exhibits significant similarity to SWAP-70 (21Gupta S. Lee A. Hu C. Fanzo J. Goldberg I. Cattoretti G. Pernis A.B. Hum. Immunol. 2003; 64: 389-401Crossref PubMed Scopus (69) Google Scholar). IBP is highly expressed in lymphoid tissues, and, interestingly, the T cell compartment preferentially expresses IBP rather than SWAP-70 (21Gupta S. Lee A. Hu C. Fanzo J. Goldberg I. Cattoretti G. Pernis A.B. Hum. Immunol. 2003; 64: 389-401Crossref PubMed Scopus (69) Google Scholar, 22Borggrefe T. Masat L. Wabl M. Riwar B. Cattoretti G. Jessberger R. Eur. J. Immunol. 1999; 29: 1812-1822Crossref PubMed Scopus (48) Google Scholar). This finding prompted us to investigate the regulation and function of IBP in T cells. Here we report that IBP is rapidly tyrosine-phosphorylated by Lck in response to T cell activation. Tyrosine phosphorylation of IBP enables it to bind PI(3,4,5)P3. IBP is recruited to the T cell:APC contact region in a manner that is dependent on the activities of both Lck and PI3K, and these signals control the ability of IBP to function as a GEF toward Rac1 and Cdc42. The recruitment of IBP to the immunological synapse coupled with the finding that its GEF activity is controlled by TCR-mediated signals suggest that IBP is a novel type of GEF that participates in lymphocyte activation. Cell Cultures and Transfections—The various Jurkat (human T cell leukemia) cell lines, including JE6–1 (wild-type), J.CaM1.6 (Lck-deficient), and J-TAg (SV40 large T-antigen-transfected) cell lines, were obtained from American Type Culture Collection (ATCC, Manassas, VA). All the cell lines were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (Atlanta Biologicals, Inc.), 2 mm l-glutamine, 10 mm HEPES, and antibiotics. 293T (a human embryonic kidney cell line) cells were a kind gift of Dr. Chris Schindler, Columbia University, New York, NY and were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. COS-7 cells (a generous gift of Dr. Steven Greenberg, Columbia University, New York, NY) were grown in RPMI 1640 medium supplemented with 10% fetal calf serum. Raji cells were obtained from Dr. Raphael Clynes (Columbia University) and were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum. Jurkat T cells were washed with serum-free RPMI 1640 medium, serum-starved for 5 h, and then stimulated at 37 °C for the indicated time periods with a mouse anti-human CD3 mAb (clone OKT3) followed by cross-linking with a secondary goat anti-mouse Ig antibody, as described previously (23Liu Y. Witte S. Liu Y.C. Doyle M. Elly C. Altman A. J. Biol. Chem. 2000; 275: 3603-3609Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). For expression of recombinant proteins, 293T cells and COS-7 cells were transfected with expression plasmids by the calcium phosphate/DNA precipitation method or the SuperFect transfection reagent (Qiagen, Inc.), respectively. After 24 h of incubation, the transfected cells were harvested for cell extract preparation. DNA Constructs—The full-length wild-type human IBP expression plasmids (pCEP4-HA-IBP and pIRES2-EGFP-HA-IBP) were constructed by cloning the entire coding region of the human IBP cDNA, fused in-frame with a hemagglutinin (HA) epitope coding sequence at its 5′ terminus, into the pCEP4 expression vector (Invitrogen) or pIRES2-EGFP bicistronic expression vector (Clontech), respectively. The point mutations Y210F or R236C in the full-length IBP were introduced by the site-directed mutagenesis method (Stratagene) and confirmed by DNA sequencing. Various deletion mutants of human IBP were generated by PCR using appropriate primers and confirmed by DNA sequencing. For preparation of various glutathione S-transferase (GST)-IBP fusion proteins, the corresponding GST-IBP expression plasmids were generated by cloning either the entire coding sequence of the human IBP cDNA or its appropriate segments, in-frame, into the pGEX-KG Escherichia coli expression vector (Amersham Biosciences). The in-frame junction in the GST-IBP fusion constructs was confirmed by DNA sequencing. The constitutively active Lck(Y505F) expression construct in pcDNA3 mammalian expression vector (24Abraham K. Levin S. Marth J. Forbush K. Perlmutter R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3977-3981Crossref PubMed Scopus (165) Google Scholar) was a kind gift of Dr. Jerry Siu. Protein Purification, Antibodies, Cell Extracts, and Protein Assays— GST-IBP fusion proteins were expressed in E. coli DH5α and affinity-purified on glutathione-agarose beads (Sigma), as described previously (25Gupta S. Jiang M. Anthony A. Pernis A. J. Exp. Med. 1999; 190: 1837-1848Crossref PubMed Scopus (106) Google Scholar). HA epitope-tagged IBP (full-length wild-type) was expressed in 293T cells and affinity-purified on immobilized anti-HA monoclonal antibody (clone 3F10; anti-HA affinity matrix; Roche Applied Science) according to the procedures recommended by the manufacturer. The polyclonal anti-IBP antibody was generated by immunizing rabbits with purified GST-IBP (amino acids 410–631) fusion protein (Covance, Inc., Princeton, NJ). This GST fusion protein contains a portion of the human IBP protein, which is least homologous to SWAP-70 and thus minimizes cross-reactivity of the antibody with SWAP-70 (21Gupta S. Lee A. Hu C. Fanzo J. Goldberg I. Cattoretti G. Pernis A.B. Hum. Immunol. 2003; 64: 389-401Crossref PubMed Scopus (69) Google Scholar). The anti-IBP antibody was utilized at 1:1000 in Western blotting and at 1:200 in the immunofluorescence experiments. An anti-phosphotyrosine monoclonal antibody (clone 4G10) was obtained from Upstate Biotechnology. The rat monoclonal antibody against HA epitope (clone 3F10) was purchased from Roche Applied Science. The PKC-θ and β-actin antibodies were purchased from Santa Cruz Biotechnology, Inc. Whole cell extracts were prepared as described previously (26Gupta S. Xia D. Jiang M. Lee S. Pernis A. J. Immunol. 1998; 161: 5997-6004PubMed Google Scholar). Cell lysates were immunoprecipitated with an anti-IBP antibody or anti-HA mAb (clone 3F10) as described previously (26Gupta S. Xia D. Jiang M. Lee S. Pernis A. J. Immunol. 1998; 161: 5997-6004PubMed Google Scholar). The immunoprecipitates were resolved by 7% SDS-PAGE. The gel was transferred to a nitrocellulose membrane and then immunoblotted with an anti-phosphotyrosine antibody (4G10) or the anti-IBP antiserum. The bands were visualized by ECL (Amersham Biosciences). Pull-down assays with phosphoinositide analogue beads (Echelon Research Laboratories Inc.) were performed according to the manufacturer's instructions. Lck-mediated tyrosine phosphorylation of IBP was assessed by in vitro Lck kinase assay using purified Lck kinase (Upstate Biotechnology) and either purified HA epitope-tagged IBP (wild-type) or immunoprecipitated recombinant IBP proteins (wild-type or Y210F mutant) according to previously described protocols (27Qian D. Lev S. van Oers N.S. Dikic I. Schlessinger J. Weiss A. J. Exp. Med. 1997; 185: 1253-1259Crossref PubMed Scopus (151) Google Scholar). Briefly, purified HA-IBP (∼150 ng) or immunoprecipitates of recombinant IBP proteins were incubated with 5 units of purified Lck in 30 μl of 1× kinase buffer (20 mm HEPES, pH 7.4, 100 mm NaCl, 5 mm MnCl2, 10 mm MgCl2, 50 μm Na3VO4, 1 mm dithiothreitol) containing 1 μm cold ATP and 10 μCi of [γ-32P]ATP for 30 min at 30 °C. The reactions were terminated by adding SDS-PAGE sample buffer and boiling. The reaction samples were resolved on a 7% SDS-polyacrylamide gel. The gel was fixed, soaked in 1 n KOH at 55 °C for 2 h, refixed, dried, and then autoradiographed to visualize tyrosine-phosphorylated products. In Vivo Rac1/Cdc42 Activation Assay and in Vitro GDP Release Assay— For the in vivo activation assays for Rac1 and Cdc42, COS-7 cells were transiently transfected with an appropriate expression vector for either wild-type HA-IBP or various IBP deletion mutants. The cells were washed with ice-cold phosphate-buffered saline containing 5 mm MgCl2 and then lysed in 1× lysis buffer (25 mm HEPES, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, 10 mm MgCl2, 1 mm EDTA, 10% glycerol, 2 mm dithiothreitol, 1 mm Na3VO4, protease inhibitors). Activated GTP-bound Rac1 (or Cdc42) was affinity-precipitated from the cell lysates by using GST-PAK1 PBD (p21 Rac/Cdc42-binding domain) fusion protein immobilized onto glutathione-agarose beads (Upstate Biotechnology) according to the manufacturer's instructions. The precipitated Rac1-GTP or Cdc42-GTP was resolved by 12.5% SDS-PAGE and then visualized by Western blot analysis with either an anti-Rac1 antibody (Upstate Biotechnology) or an anti-Cdc42 antibody (Transduction Laboratories), respectively. The in vitro GDP release assays were carried out by filter-binding method as described previously (28Shou C. Farnsworth C.L. Neel B.G. Feig L.A. Nature. 1992; 358: 351-354Crossref PubMed Scopus (290) Google Scholar, 29Crespo P. Schuebel K.E. Ostrom A.A. Gutkind J.S. Bustelo X.R. Nature. 1997; 385: 169-172Crossref PubMed Scopus (680) Google Scholar, 30Han J. Luby-Phelps K. Das B. Shu X. Xia Y. Mosteller R.D. Krishna U.M. Falck J.R. White M.A. Broek D. Science. 1998; 279: 558-560Crossref PubMed Scopus (710) Google Scholar, 31Abe K. Rossman K.L. Liu B. Ritola K.D. Chiang D. Campbell S.L. Burridge K. Der C.J. J. Biol. Chem. 2000; 275: 10141-10149Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). To prepare [3H]GDP-loaded GTPases, 40 pmol of bacterially expressed and purified GST-Rac1, GST-Cdc42, or His-RhoA (Calbiochem-Novabiochem) was incubated with 1 μm [3H]GDP in 100 μl of a binding buffer (10 mm HEPES, pH 7.5, 100 mm NaCl, 5 mm MgCl2, 1 mm EGTA, 1 mm dithiothreitol, 50 μg/ml bovine serum albumin) for 60 min at 30 °C. The nucleotide exchange reaction was initiated by adding 100 μm nonradioactive GTP and 4 pmol of purified GST alone or GST-IBP fusion proteins (or when appropriate, 4 pmol of purified wild-type HA-IBP either non-phosphorylated or in vitro phosphorylated by purified Lck and cold ATP) to the [3H]GDP-loaded GTPase and then equally splitting and incubating the reaction mixture for the indicated time periods at room temperature. When indicated, control exchange reactions were also performed with purified Lck alone using the same amount of Lck as was used to phosphorylate purified HA-IBP. In some experiments, a water-soluble analogue of PI(3,4,5)P3 (Echelon Research Laboratories Inc.) was added to the exchange reaction mixtures to a final concentration of 1 μm. The exchange reactions were stopped by adding 1 ml of ice-cold dilution buffer (20 mm Tris-HCl, pH 7.5, 100 mm NaCl, 20 mm MgCl2) to the reaction mixtures. The amount of [3H]GDP remaining bound to the GTPases was determined by filtering the quenched reaction samples over nitrocellulose membranes followed by extensive washing of the filters and then quantification of the membrane-bound radioactivity by scintillation counting. Conjugate Formation and CD3 Capping—Raji cells were used as the APCs for conjugation with Jurkat T cells. Raji cells were labeled with 10 μm 7-amino-4-chloromethylcoumarin cell tracker blue dye (Molecular Probes, Eugene, OR) followed by pulsing with or without 5 μg/ml SEE (Toxin Technology, Sarasota, FL) for 30 min at 37 °C. To induce conjugate formation, 1 × 105 B cells were combined with 1 × 105 Jurkat E6–1 or 1 × 105 Lck-deficient Jurkat (J.CaM1.6) T cells at 37 °C for 5 min. Conjugates were pipetted onto poly-l-lysine-coated coverslips and then fixed in 3.7% formaldehyde, washed, and permeabilized with 0.5% Triton X-100/phosphate-buffered saline. Conjugates were then stained with antibodies to IBP and PKC-θ followed by a secondary staining with Alexa-Fluor 568-conjugated donkey anti-rabbit (Molecular Probes) and FITC-conjugated donkey anti-mouse (Jackson Immunoresearch Laboratories, IgAb), respectively. For treatments with wortmannin (Calbiochem), T cells were resuspended in serum-free medium containing 100 nm wortmannin and incubated for 30 min at 37 °C followed by incubation with APCs. Conjugates were examined by a Zeiss LSM 510 laser scanning confocal microscope (Thornwood, NY) with a ×100/1.3 Plan-Neofluor objective lens. FITC, TRITC, and 7-methyl-4-chloromethylcoumarin (in two-photon mode) were excited at 488, 543, and 800 nm, respectively, and emission was collected at 500–550, above 585, and 435–485 nm, respectively. Optical section thickness was ∼1 μm. Image enhancement and analysis were performed using the public domain program NIH Image 1.6 and Adobe Photoshop 6.0. Approximately 100 conjugates were scored visually for polarized IBP or PKC-θ at the synapse from two independent scores of three different experiments. For capping experiments, naïve CD4+ T cells were isolated from splenocytes of D0.11.10 TCR transgenic mice by negative selection using naïve CD4+-specific T cell enrichment columns (R & D Systems). The purity of naïve CD4+ cells was assessed by flow cytometry and was found to be >90%. T cells were stimulated with 5 μg/ml anti-CD3ϵ Ab (Pharmingen) for 1 h on ice, followed by cross-linking with FITC-labeled mouse anti-hamster Ig Ab (Molecular Probes, Eugene, OR) for 5 min at 4 or 37 °C. Cells were pipetted onto poly-l-lysine-coated coverslips and then fixed with 3.7% formaldehyde. Cells were then washed, permeabilized, and stained with antibodies against IBP followed by a secondary antibody stain of anti-rabbit Alexa-Fluor 568 (Molecular Probes). Capped T cells were examined by a Zeiss LSM 510 laser scanning confocal microscope (Thornwood, NY) with a ×100/1.3 Plan-Neofluor objective lens. FITC and TRITC were excited at 488 and 543 nm, respectively, and emission was collected at 500–550 and above 585 nm, respectively. Optical section thickness was ∼1 μm. Image enhancement and analysis were performed using the public domain program NIH Image 1.6 and Adobe Photoshop 6.0. The percentage of cells displaying caps, in which the Alexa-Fluor condenses to less than 25% of the cell surface was determined by counting 10 fields per treatment composed of ∼150–250 cells. D0.11.10 TCR transgenic mice were obtained from Jackson Immunoresearch Laboratories and were maintained under specific pathogen-free conditions. IBP Is Tyrosine-phosphorylated upon TCR Stimulation in an Lck-dependent Manner—The rapid activation of PTKs of the Src family is one of the earliest signaling events triggered by engagement of the TCR (1van Leeuwen J. Samelson L.E. Curr. Opin. Immunol. 1999; 11: 242-248Crossref PubMed Scopus (217) Google Scholar, 2Kane L.P. Lin J. Weiss A. Curr. Opin. Immunol. 2000; 12: 242-249Crossref PubMed Scopus (426) Google Scholar). Because a survey of the IBP sequence utilizing the Scansite algorithm (32Yaffe M.B. Leparc G.G. Lai J. Obata T. Volinia S. Cantley L.C. Nat. Biotechnol. 2001; 19: 348-353Crossref PubMed Scopus (464) Google Scholar) revealed the presence of a potential tyrosine phosphorylation site, which fits the consensus motif for Lck-mediated phosphorylation (Fig. 1A) (33Latour S. Veillette A. Curr. Opin. Immunol. 2001; 13: 299-306Crossref PubMed Scopus (171) Google Scholar), we first determined whether IBP underwent enhanced tyrosine phosphorylation in response to TCR stimulation. Jurkat T cells, which express endogenous IBP, were stimulated with anti-CD3 mAb for varying periods of time, and whole cell lysates were obtained and then immunoprecipitated with an Ab directed against IBP (Fig. 1B). Western blot analysis of the immunoprecipitates with an anti-phosphotyrosine Ab revealed that TCR engagement led to the rapid and transient tyrosine phosphorylation of IBP. To address more directly the possibility that IBP might be a substrate for the Src family of tyrosine kinases, we determined whether purified Lck could phosphorylate purified recombinant IBP in an in vitro kinase assay (Fig. 1C). Coincubation of IBP and Lck resulted in the phosphorylation of IBP in addition to the known autophosphorylation of Lck. Furthermore, transient cotransfection of an IBP expression construct in 293T cells, together with a constitutively active form of Lck (LckY505F) (24Abraham K. Levin S. Marth J. Forbush K. Perlmutter R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3977-3981Crossref PubMed Scopus (165) Google Scholar), led to the tyrosine phosphorylation of IBP (data not shown). Given that the N terminus of IBP contains a tyrosine (Tyr-210) that represents a potential consensus motif for Lck-mediated phosphorylation, we then proceeded to determine whether inactivating this residue would affect the ability of Lck to phosphorylate IBP. As shown in Fig. 1D, in vitro kinase assays indeed demonstrated that Lck can phosphorylate wild-type IBP but not the Y210F mutant. To assess whether Lck was required for TCR-induced phosphorylation of IBP in lymphocytes, we compared the ability of IBP to undergo tyrosine phosphorylation in cells from the Lck+ Jurkat cell line JE6.1 and in cells from J.CaM1.6, an Lck-deficient subline of JE6.1 (34Straus D.B. Weiss A. Cell. 1992; 70: 585-593Abstract Full Text PDF PubMed Scopus (933) Google Scholar). The anti-CD3-induced tyrosine phosphorylation of IBP was markedly diminished in the Lck-deficient as compared with the Lck+ Jurkat T cells (Fig. 1E). Taken together these data indicate that IBP is rapidly tyrosine-phosphorylated upon TCR stimulation and that IBP can serve as a substrate for Src kinases. IBP Binds PI(3,4,5)P3 upon Phosphorylation by Lck—Because IBP contains a PH domain we determined whether IBP binds specific phosphoinositides. We prepared whole cell lysates from 293T cells cotransfected with an HA-tagged IBP expression construct and either an empty vector or a vector expressing a constitutively active Lck (LckY505F). Lysates were then subjected to pull-down assays with a panel of different phosphoinositides conjugated to agarose beads (Fig. 2A). IBP bound efficiently to the PI3K product, PI(3,4,5)P3, only when coexpressed with constitutively active Lck. We did not detect association of IBP with PI(4,5)P2. To confirm the specificity of the interaction of PI(3,4,5)P3 with the PH domain of IBP, we generated a point mutation within the PH domain of IBP (R236C). This residue has been shown previously (35Isakoff S.J. Cardozo T. Andreev J. Li Z. Ferguson K.M. Abagyan R. Lemmon M.A. Aronheim A. Skolnik E.Y. EMBO J. 1998; 17: 5374-5387Crossref PubMed Scopus (282) Google Scholar) to be critical for the interaction of PH domains with PI(3,4,5)P3, and a similar mutation was previously demonstrated to prevent the association of SWAP-70 with phosphoinositides (20Shinohara M. Terada Y. Iwamatsu A. Shihora A. Mochizuki N. Higuchi M. Gotoh Y. Ihara S. Nagata S. Itoh H. Fukui Y. Jessberger R. Nature. 2002; 416: 759-763Crossref PubMed Scopus (185) Google Scholar). As shown in Fig. 2B, the R236C mutation completely abolished the ability of IBP to interact with PI(3,4,5)P3. To further investigate whether phosphorylation of Tyr-210 by Lck was critical for the ability of IBP to efficiently associate with PI(3,4,5)P3 we then assayed the ability of the IBPY210F mutant to bind PI(3,4,5)P3 (Fig. 2C). Consistent with the notion that Tyr-210 constitutes the major Lck phosphorylation site within the IBP molecule, cotransfection of Lck with IBPY210F did not lead to binding of this mutant to PI(3,4,5)P3. Interestingly, a deletion mutant of IBP lacking the entire N terminus (IBP ΔN) bound to PI(3,4,5)P3 even in the absence of constitutively active Lck (Fig. 2D). Surprisingly, this mutant also acquired the ability to bind PI(4,5)P2 and PI(3,4)P2. Further studies are in progress to determine whether the binding of IBP ΔN to PI(4,5)P2 and PI(3,4)P2 is mediated directly by the PH domain of IBP or by association of this mutant with additional PH domain-containing proteins of different specificities. Collectively, these data are thus consistent with a model whereby phosphorylation of Tyr-210 by Lck leads to a conformational change in IBP that allows its PH domain to bind to the appropriate phosphoinositide molecule. Recruitment of IBP to the IS Requires the Activities of L
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