Immunophilins Control T Lymphocyte Adhesion and Migration by Regulating CrkII Binding to C3G

This information is current as Pulak Ranjan Nath, Guangyu Dong, Alex Braiman and Noah of September 23, 2021. Isakov J Immunol published online 15 September 2014 http://www.jimmunol.org/content/early/2014/09/13/jimmun ol.1303485 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published September 15, 2014, doi:10.4049/jimmunol.1303485 The Journal of Immunology

Immunophilins Control T Lymphocyte Adhesion and Migration by Regulating CrkII Binding to C3G

Pulak Ranjan Nath, Guangyu Dong,1 Alex Braiman, and Noah Isakov

Crk adaptor proteins are key players in signal transduction from a variety of cell surface receptors. CrkI and CrkII, the two al- ternative spliced forms of CRK, possess an N-terminal Src homology 2 domain, followed by a Src homology 3 (SH3) domain, whereas CrkII possesses in addition a C-terminal linker region plus a SH3 domain, which operate as regulatory moieties. In this study, we investigated the ability of immunophilins, which function as peptidyl-prolyl isomerases, to regulate Crk proteins in human T lymphocytes. We found that endogenous CrkII, but not CrkI, associates with the immunophilins, cyclophilin A, and 12- kDa FK506-binding protein, in resting human Jurkat T cells. In addition, cyclophilin A increased Crk SH3 domain–binding guanine-nucleotide releasing factor (C3G) binding to CrkII, whereas inhibitors of immunophilins, such as cyclosporine A (CsA)

and FK506, inhibited CrkII, but not CrkI association with C3G. Expression in Jurkat T cells of phosphorylation indicator of Crk Downloaded from

chimeric unit plasmid, a plasmid encoding the human CrkII1–236 sandwiched between cyan fluorescent protein and yellow fluorescent protein, demonstrated a basal level of fluorescence resonance energy transfer, which increased in response to cell treatment with CsA and FK506, reflecting increased trans-to-cis conversion of CrkII. Crk-C3G complexes are known to play an important role in integrin-mediated cell adhesion and migration. We found that overexpression of CrkI or CrkII increased adhesion and migration of Jurkat T cells. However, immunophilin inhibitors suppressed the ability of CrkII- but not CrkI-

overexpressing cells to adhere to fibronectin-coated surfaces and migrate toward the stromal cell-derived factor 1a chemokine. http://www.jimmunol.org/ The present data demonstrate that immunophilins regulate CrkII, but not CrkI activity in T cells and suggest that CsA and FK506 inhibit selected effector T cell functions via a CrkII-dependent mechanism. The Journal of Immunology, 2014, 193: 000–000.

embers of the Crk family of adaptor proteins play WIP–WASP complex, thereby releasing it from WIP inhibition important roles in signal transduction from numerous and enabling the activation of cellular events leading to actin M cell surface receptors (1). Adaptor proteins can or- polymerization and cytoskeletal rearrangement (5). chestrate the assembly of multimolecular complexes at receptor All Crk adaptor proteins possess a single N-terminal Src ho- sites, thereby linking receptors to their downstream signaling mology 2 (SH2) domain, which binds phosphotyrosine-containing cascades (2). In T lymphocytes, Crk proteins were found to as- sequences, followed by one (CrkI) or two (CrkII and CrkL) Src by guest on September 23, 2021 sociate with the ZAP70 protein tyrosine kinase in a cell activation- homology 3 (SH3) domains, which bind proline-rich motifs (8, 9). dependent manner (3, 4). A ZAP70-associated CrkL was found to The two SH3 domains are connected via a linker region containing interact with the Wiskott–Aldrich syndrome protein (WASP)/ a single, highly conserved, protein conformation-regulating tyro- WASP-interacting protein (WIP) complex and induce its recruit- sine residue, plus several proline residues (10, 11). Phosphorylation ment to the immunological synapse (5). Concomitant recruitment of the tyrosine residue (Tyr211 and Tyr207 in human CrkII and of protein kinase Cu to the immunological synapse (6, 7) results in CrkL, respectively) by Abl, or by other protein tyrosine kinases, phosphorylation of WIP and disengagement of WASP from the promotes intramolecular binding of the linker region to the self SH2 domain, thereby sequestering the SH2 and N-terminal SH3 (SH3N) domains and preventing them from interaction with phys- Shraga Segal Department of Microbiology, Immunology, and Genetics, Faculty of iological binding partners (11, 12). Health Sciences, Ben Gurion University of the Negev, Beer Sheva 84105, Israel Recent in vitro studies suggested an additional regulatory role 1Current address: Department of Periodontics, School of Dental Medicine, University for the proline-rich motif within the CrkII linker region, which of Pennsylvania, Philadelphia, PA. is under the control of peptidyl-prolyl cis-trans isomerases Received for publication January 6, 2014. Accepted for publication August 12, 2014. (PPIases) (13–16). In these studies, a truncated recombinant This work was supported in part by the USA–Israel Binational Science Foundation, protein consisting of the SH3N-linker-SH3C of the chicken CrkII the Israel Science Foundation administered by the Israel Academy of Science and SLS Humanities, and donations by Martin Kolinsky and Linda Osofsky. N.I. holds the (CrkII ) was found to be sensitive to PPIases, and undergo 238 Joseph H. Krupp Chair in Cancer Immunobiology. cis-trans isomerization at Pro located within the linker region. Address correspondence and reprint requests to Prof. Noah Isakov, Shraga Segal Combined chemical shift and relaxation rate analyses revealed Department of Microbiology, Immunology, and Genetics, Faculty of Health Sciences, that CrkIISLS exists in solution in two different geometric con- Ben-Gurion University, P.O.B. 653, Beer Sheva 84105, Israel. E-mail address: 238 [email protected] formations, as follows: a major one (90%) in which Pro adopts a cis conformation and CrkIISLS is folded in a closed and Abbreviations used in this article: CFP, cyan fluorescent protein; C3G, Crk SH3 domain–binding guanine-nucleotide releasing factor; CsA, cyclosporine A; CypA, inactive conformation, and a second minor conformation (10%) in cyclophilin A; FKBP12, 12-kDa FK506-binding protein; FRET, fluorescence reso- which Pro238 acquires a trans conformation and CrkIISLS adopts nance energy transfer; h, human; PICCHUx, phosphorylation indicator of Crk chi- meric unit plasmid; PPIase, peptidyl-prolyl cis-trans isomerase; SDF1a, stromal cell- an open, active conformation, allowing the protein to interact derived factor 1a; SH2, Src homology 2; SH3, Src homology 3; SH3C, C-terminal with its binding partners. SH3 domain; SH3N, N-terminal SH3; WASP, Wiskott–Aldrich syndrome protein; Despite the in vitro responsiveness of CrkIISLS to PPIase- WIP, WASP-interacting protein; WT, wild-type; YFP, yellow fluorescent protein. induced conformational changes, the direct effect of PPIases on Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 full-length CrkII has not yet been resolved, nor has the ability of

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1303485 2 IMMUNOPHILINS REGULATE CrkII ACTIVITY IN HUMAN T LYMPHOCYTES

CrkII to serve as a substrate for PPIases in vivo. In addition, the (hCypA), and J. Buchner (University of Technology, Munich, Germany) sequence around Pro238 in the chicken CrkII is only partially (hCyp40). conserved in mammalians, raising questions about the ability of Bacterial expression vectors were transformed into Escherichia coli DH5a-competent cells, and GST-fusion proteins were prepared as de- PPIases to isomerase CrkII and regulate its function in human cells. scribed (4). Pull-down assays were performed by incubation of bead- The aim of the current study is to analyze the ability of adsorbed GST or GST fusion proteins (2–10 mg, as indicated) with cell immunophilins, predominantly cyclophilin A (CypA) and 12-kDa lysates at 4˚C on a rotator for 4 h. The beads were washed (three times) in 3 FK506-binding protein (FKBP12), to affect CrkII activity in human lysis buffer, resuspended in 2 Laemmli sample buffer, and boiled for 5 min. The eluents were subjected to SDS-PAGE under reducing condi- T cells, using the Jurkat leukemia T cell line as a model system. tions, followed by immunoblotting. Our findings demonstrate that the in vivo activity of human CrkII, but not of CrkI, is regulated by the immunophilins, CypA Matrix-assisted laser desorption/ionization mass spectrometry and FK506-binding protein. The results are further substantiated Lysates of Jurkat T cells (∼108 cells) were incubated with bead- by experiments that use cyclosporine A (CsA) and FK506, two immobilized GST or GST-CrkII (10 mg/sample) for 3 h at 4˚C. The distinct immunophilin inhibitors that are known to inhibit the beads were extensively washed with lysis buffer, and bound proteins were calcineurin–NFAT signaling pathway. The overall findings suggest eluted by boiling for 5 min in sample buffer and subjected to SDS-PAGE (on a 10% polyacrylamide gel) under reducing conditions. The gel was that a mixture of immunophilin inhibitors, containing the CsA stained with GelCode Blue reagent, followed by extensive washes in and FK506, can inhibit selected effector T cell functions via double-distilled water. A gel slice including the 14 kDa up to 22-kDa a CrkII-dependent, and NFAT-independent mechanism. protein bands was excised, reduced, alkylated, and trypsin digested. Mass spectrometry analysis of the peptides was performed on MALDI mass spectrometer (Biological Mass Spectrometry Unit, Weizmann Institute of Materials and Methods Science, Rehovot, Israel), followed by peptide sequence comparison with Downloaded from Reagents and Abs protein database using the Sonar MS/MS search engine (ProteoMetrics; Genomic Solutions). Sandimmune (CsA; 50 mg/ml) was from Novartis Pharma AG (Basel, Switzerland), and FK506 (Prograf or tacrolimus, 5 mg/ml) was from Expression vectors and transient transfection of Jurkat T cells Astellas Pharma. Recombinant human CypA, 2-ME, and Triton X-100 were from Sigma-Aldrich. AEBSF, aprotinin, and leupeptin were from ICN The phosphorylation indicator of Crk chimeric unit plasmid (PICCHUx, Biomedicals (Aurora, OH). ECL, glutathione-coupled Sepharose beads, a gift of M. Matsuda, National Institutes of Health, Tokyo, Japan) consists of http://www.jimmunol.org/ and protein A–Sepharose were from Amersham Pharmacia Biotech human CrkII (aa 1–236) sandwiched between cyan fluorescent protein (Uppsala, Sweden). Recombinant stromal cell-derived factor 1a (SDF1a) (CFP)– and yellow fluorescent protein (YFP)–emitting variants of GFP, in protein and human fibronectin were from R&D Systems (Minneapolis, addition to the ki-Ras–derived CAAX box, which anchors the protein MN) and Sigma-Aldrich, respectively. Trypsin (0.25%-EDTA [1:200]) was predominantly to the plasma membrane (18). A cDNA plasmid encoding from Biological Industries (Beit Haemek, Israel). Mouse anti-Crk (I/II) a constitutively active Lck was a gift of A. Altman (La Jolla Institute for mAb was from BD Transduction Laboratories (Lexington, KY), and Allergy and Immunology). The eukaryotic vectors pCEFL-GST-CrkII- mouse anti-phosphotyrosine mAb (4G10) was from Upstate Biotechnology wild-type (WT), pCEFL-GST-CrkII-R38L, pCEFL-GST-CrkII-W169L, (Lake Placid, NY). Mouse anti-CD3ε mAb (OKT3) was prepared in nude and pCEFL-GST-CrkII-Y221F were a gift of K. Yamada (National Insti- mice by i.p. injection of the OKT3 hybridoma and collection of the ascites tute of Dental and Craniofacial Research, National Institutes of Health, fluid. Rabbit polyclonal anti-C3G Ab and mouse mAb specific to CypA Bethesda, MD) (19). For DNA transfer into cells, Jurkat TAg cells were were from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse anti–b- maintained at a logarithmic growth phase, washed in serum-free RPMI by guest on September 23, 2021 6 actin mAb (AC-15) was from Sigma-Aldrich. Mouse anti-human Myc 1640, resuspended at 7 3 10 cells/ml in transfection buffer (1.8 g/ml mAb (clone 9E102)–producing hybridoma was obtained from the Amer- sucrose, 2 nM DTT in RPMI 1640), and transferred into 0.4-cm–gap 6 ican Type Culture Collection (CLR 1729), and ascites was prepared in Gene Pulser cuvettes (Bio-Rad) (5 3 10 cells/700 ml/cuvette). Plasmid BALB/c mice. Cy3-conjugated goat Abs directed against mouse or rabbit DNA (10 mg/group, unless otherwise indicated) was added to each cuvette, IgG were from Jackson ImmunoResearch Laboratories, and HRP-conjugated and electroporation was performed using a Bio-Rad Gene Pulser (250 V, goat Abs directed against mouse or rabbit IgG were from Amersham 950 mF). The cells were then cultured in 50 ml complete RPMI 1640 in 2 Pharmacia Biotech. 145-mm tissue culture plates for 36 h. Cell lines and culture conditions Preparation of cell lysates and immunoprecipitation Human leukemia cell lines used in this study include the Jurkat T cell, clone Cell lysates were prepared by resuspension of cells in lysis buffer (25 mM 6.1, and Jurkat TAg T cell, which stably expresses the SV40-derived large T Tris/HCl [pH 7.5], 150 mM NaCl, 5 mM EDTA, 1 mM Na3VO4,50mM Ag. In addition, Jurkat T cell subclones, JKCrkI and JKCrkII, were prepared NaF, 10 mg/ml each of leupeptin and aprotinin, 2 mM AEBSF, and 1% by S. Gelkop from our laboratory by transfecting Jurkat TAg T cells with Triton X-100), followed by a 20-min incubation on ice. Lysates were Myc-tag–containing expression vectors for CrkI and CrkII, respectively, centrifuged at 13,000 3 g for 30 min at 4˚C, and the nuclear-free super- and selection of clones that express ∼2-fold CrkI or CrkII, compared with natants were used for immunoprecipitation. the cell of origin. All cell lines were maintained at a logarithmic growth For immunoprecipitation, primary Abs were preadsorbed to protein A– phase in complete RPMI (RPMI 1640 supplemented with 5% heat- Sepharose beads for 1 h at 4˚C. Excess Abs were removed by three washes inactivated FCS, 2 mM L-glutamine, 50 U/ml penicillin, 50 mg/ml strepto- in cold PBS, and Ab-coated beads were incubated with cell lysates for 3 h mycin [all from Biological Industries], and 0.5 mM 2-ME (Sigma-Aldrich). at 4˚C. Immune complexes were precipitated by centrifugation, followed Cells were grown in 75-cm2 growth-area tissue culture flasks (Cellstar, by extensive washing in a lysis buffer. Equal volumes of 23 SDS sample Greiner, Germany) in an atmosphere of 5% CO2, at 37˚C. Stimulation by buffer were added to immunoprecipitates or whole-cell lysates, which were TCR triggering was carried out using anti-CD3ε mAbs (OKT3; 1:1000 vortexed, boiled for 5 min, and fractionated by SDS-PAGE. dilution of ascites, 30-min incubation on ice) plus cross-linking with a secondary Ab (goat anti-mouse IgG, 1:200) for 1 min at 37˚C. Electrophoresis and immunoblotting GST fusion proteins and pull-down assay Samples of whole-cell lysates, immunoprecipitates, or pulled-down proteins were resolved by electrophoresis on 10% polyacrylamide gels using Bio- pGEX-5X vectors were from Amersham Pharmacia Biotech, and pGEX Rad Mini-PROTEAN II cells. Proteins from the gel were electroblotted plasmids encoding different GST-Crk fusion proteins were gifts of onto a nitrocellulose membrane (Schleicher & Schuell) at 100 V for 1 h in M. Matsuda (National Institutes of Health, Tokyo, Japan). The pGST-C3G and BioRad Mini Trans-Blot transfer cells. After 1-h blocking with 3% BSA in pGST-SH3b were gifts of C. Guerrero (Universidad de Salamanca-Consejo PBS at 37˚C, the nitrocellulose membranes were incubated with the in- Superior de Investigaciones Cientificas, Salamanca, Spain) (17). GST- dicated primary Abs, followed by incubation with HRP-conjugated sec- isomerase vectors were gifts of J. Bolstad and W. Chen (University of ondary Abs or protein A. Immunoreactive protein bands were visualized Calgary, Calgary, AB, Canada) (human [h]FKBP12 and hFKBP12.6), using an ECL reagent and autoradiography. Whenever required, nitrocel- T. Ratajczak (University of Western Australia, Crawley, WA, Australia) lulose membranes were stripped by incubation in stripping buffer (100 mM (hFKBP51 and hCyp40), B. Chambraud (INSERM U488, Paris, France) 2-ME, 2% SDS, and 62.5 mM Tris/HCl [pH 6.8]) for 30 min at 50˚C, (hFKBP52), M. Emerman (University of Washington, Seattle, WA) followed by 1-h incubation with blocking buffer (3% BSA in PBS). The Journal of Immunology 3

Binding studies using CypA-treated recombinant CrkII or was acquired from single-labeled CFP- or YFP-expressing cells for each whole-cell lysate-derived CrkII set of acquisition parameters, and a calibration curve was derived to allow elimination of the non-FRET components from the FRET channel. The Treatment with CypA was performed according to Kofron et al. (20), with FRET efficiency was calculated on a pixel-by-pixel basis using the fol- some modifications. For a pull-down assay, bead-immobilized GST-CrkII lowing equation: fusion proteins (10 mg) were incubated with recombinant CypA (6 mM, final concentration) in assay buffer (50 mM HEPES, 100 mM NaCl [pH 8]) FRETeff ¼ FRETcorr=ðFRETcorr þ CFPÞ 3 100%, for 1 h at room temperature. The beads were then washed and incubated 7 with 280 ml Jurkat T cell lysate (equivalent to 2 3 10 cells) for an ad- where FRETcorr is the pixel intensity in the corrected FRET image, and ditional 3 h at 4˚C. After washings, bound proteins were eluted from the CFP is the intensity of the corresponding pixel in the CFP channel image. beads by 5-min boiling in sample buffer, followed by SDS-PAGE. For coimmunoprecipitation studies, Jurkat T cells (8 3 107) were lysed in 100 Adhesion assay ml lysis buffer diluted in assay buffer in a final volume of 250 ml. CypA Flat-bottom 96-well microtiter plates (Costar, Cambridge, MA) were coated (6 mM, final concentration) was added to one of the two test tubes for 1-h overnight at 4˚C with 100 mg/ml soluble human fibronectin in PBS, fol- incubation at room temperature. Protein A beads (10 ml/group) that were lowed by blocking with 2% BSA/PBS for 1 h at 37˚C, and then washed preincubated with anti-Crk mAbs (1 mg/group) for 1 h at room temperature with medium (RPMI 1640 without additives). Cells (2 3 105/100 ml) were were added to each test tube for 3 h of incubation at 4˚C. The beads were allowed to attach for 2 h at 37˚C, and nonadherent cells were then removed extensively washed, and bound proteins were eluted by 5-min boiling in by gentle aspiration and rinsing with prewarmed RPMI 1640. Adherent sample buffer and subjected to SDS-PAGE. Proteins that were pulled down cells were quantified, and data are expressed as the mean of the percentage by CypA-treated GST-CrkII, or coimmunoprecipitated with Crk from of adherent cells relative to total cell input, in three replicate wells. lysates of CypA-treated Jurkat T cells, were electroblotted onto nitrocel- lulose membranes that were immunoblotted with anti-Crk and anti-C3G Migration assay

Abs. Downloaded from Cell migration was assessed in disposable ThinCert cell culture inserts Cell treatment with CsA and FK506 (Greiner) placed in a 24-well multiwell cell culture plate in which the two compartments in each migration chamber are separated by a porous CsA (Sandimmun, 50 mg/ml in oil solution) and FK506 (Prograf, 5 mg/ml positron-emission tomography membrane with 5.0 mm pore size. One in ethanol) were diluted in RPMI 1640 culture medium before each ex- hundred microliters of cell suspension (5 3 106 cells/ml) in migration periment. Jurkat TAg, JKCrkI, and JKCrkII cells were cultured in 75-cm2 buffer (RPMI 1640 containing 50 U/ml penicillin, 50 mg/ml streptomycin, growth-area tissue culture flasks at a concentration of 50 3 106 cells/group 10 mM HEPES, and 5% FCS) was seeded into the upper compartment of in the presence or absence of CsA (5 mg/ml) and/or FK506 (5 ng/ml), or as each chamber, and 600 ml migration buffer was placed in the lower http://www.jimmunol.org/ indicated in the figure legend, and tested on the next day. In longer chambers. Active cell migration into the lower compartment was induced treatments, the cells were incubated with the same drug concentrations, by the addition of recombinant SDF1a (100 ng/ml) chemoattractant into and half of the volume of the culture medium was replaced once every 2 d the lower chamber. After 3 h of incubation at 37˚C and 5% CO2, cells that with fresh culture medium containing the same drug concentration. migrated through the membrane to the lower chamber were counted using a hemocytometer. Fluorescent staining and confocal microscopy Statistical analysis PICCHUx-transfected Jurkat T cells were grown on coverslips preincubated with poly-L-lysine in a 24-well culture dish. Cells were fixed with 4% Statistical analyses were performed by one-way ANOVA using SIGMA paraformaldehyde (Sigma-Aldrich)/PBS for 15 min at room temperature STAT 3.11 (Systat Software 2004, San Jose, CA) or t test. Values are and permeabilized with 0.2% Triton X-100/PBS for 5 min. Cells were then presented as average 6 SD. Data were considered significantly different at by guest on September 23, 2021 blocked in 1% BSA/PBS for 1 h, followed by staining with rabbit anti-C3G p , 0.05. (Santa Cruz Biotechnology) for 1 h. Subsequently, the cells were washed three times in PBS and incubated with Cy3-conjugated goat anti-rabbit Results Abs. The coverslips were mounted on slides using DAKO mounting me- dium and imaged by Olympus FluoView FV1000 laser-scanning confocal CrkII associates with PPIases in Jurkat T cells microscope. Previous studies demonstrated the ability of CypA to catalyze Fluorescence resonance energy transfer analysis by FACS and in vitro the conformational change of a recombinant molecule confocal microscopy possessing the SH3N-linker-SH3C region of the chicken CrkII adaptor protein (13). In addition, pull-down and coimmunopreci- Fluorescence resonance energy transfer (FRET) analysis of living cells by pitation studies demonstrated the physical interaction of CypA flow cytometry and confocal microscopy was performed according to He et al. (21). Briefly, cells were transfected with the PICCHUx plasmid with some of its substrate protein (24–26). constructed to include the human CrkII protein (aa 1–236) and YFP and To test whether CrkII can serve as an in vivo substrate for CypA, CFP on both ends of CrkII, plus an N-terminal CAAX motif (18). Cells we first analyzed the ability of CrkII to associate with CypA in were cultured for 48 h and then split into two identical groups that were a pull-down assay. Bead-immobilized GST-CrkII fusion proteins, cultured for an additional 24 h in the presence or absence of CsA (5 mM) or GST as a negative control, were used to pull down binding plus FK506 (5 nM). Cells were then resuspended in FACS buffer (PBS proteins from a human Jurkat T cell lysate. Proteins were then plus 0.5% BSA and 0.1% NaN3) for analysis on a FACSCanto II device (BD Biosciences). Excitation of CFP occurred at 405 nm, whereupon fractionated by SDS-PAGE, and the gel was stained with GelCode emission was detected in the CFP emission window and simultaneously in Blue reagent. Because human CypA possesses 165 aa and a cal- the YFP emission window. If FRET occurs, CFP emission decreases, while culated molecular mass of 18,012 Da, a gel slice that includes simultaneously YFP emission increases. This can be visualized by a shift in ratio of YFP/CFP emission intensities. The data were analyzed using proteins with a molecular mass of 14–22 kDa was excised, re- Flowjo7.6.3 software. duced, alkylated, and trypsin digested. Proteins were then ana- Alternatively, another group of transfected cells was similarly grown on lyzed on a MALDI mass spectrometer, followed by a peptide Fluorodish for imaging by a confocal microscope (Olympus FluoView sequence comparison with a protein database using the Sonar MS/ FV1000), and FRET was measured by the donor-sensitized acceptor MS search engine. Results revealed that CrkII associates with fluorescence technique, as previously described (22, 23). Briefly, three images were acquired for each set of measurements, as follows: YFP a number of proteins, including CypA, and a closely related excitation/YFP emission image (YFP channel); CFP excitation/CFP protein termed pepityl-prolyl cis-trans isomerase-like-1 (Fig. 1). emission image (CFP channel); and CFP excitation/YFP emission image MALDI mass spectrometry is an extremely sensitive method that (FRET channel). Single-labeled (CFP or YFP) cells were used to calculate can hardly distinguish between low amounts of high-affinity CrkII- the non-FRET fluorescence bleed-through produced by the fluorophores into the FRET channel, and the non-FRET fluorescent intensity values binding proteins versus large amounts of low-affinity CrkII-binding were subtracted from the apparent FRET intensities obtained from the proteins in a tested sample. To demonstrate CrkII–CypA interac- double-labeled cells under the same conditions. A set of reference images tion using a less sensitive method, we performed a pull-down 4 IMMUNOPHILINS REGULATE CrkII ACTIVITY IN HUMAN T LYMPHOCYTES

FIGURE 1. CrkII associates with distinct human T cell–derived immunophilins. (A) Jurkat T cell lysates were incubated with bead-immobilized GST or Downloaded from GST-CrkII fusion proteins, and bound proteins were subjected to SDS-PAGE under reducing conditions. After gel staining with GelCode Blue, a gel slice that includes the 14-kDa up to 22-kDa protein bands was excised, reduced, alkylated, and trypsin digested. The peptides obtained were analyzed on a MALDI mass spectrometer, followed by peptide sequence comparison with protein database using the Sonar MS/MS search engine. The table includes names, accession numbers, and calculated molecular mass (size) of proteins predicted to associate with CrkII. The number of trypsin-cleaved MALDI-mass spectrometer–identified nonredundant peptides corresponding to each of the proteins and the ratio of the protein score are also included. (B) Bead- immobilized GST proteins fused to distinct immunophilins (2 mg/sample) were incubated with a lysate of Jurkat T cell subclone, JKCrkII, which con- stitutively expresses Myc-CrkII (equal to 4 3 107 cells/group). After 3 h of incubation on a rotator at 4˚C, the beads were extensively washed with cold lysis http://www.jimmunol.org/ buffer and bound proteins were eluted and subjected to SDS-PAGE under reducing conditions. Whole-cell lysate (WCL) was boiled and electrophoresed in parallel on the same gel. Proteins were then electroblotted onto a nitrocellulose membrane that was immunoblotted with anti-Crk mAb. (C) The experiment was repeated as in (B), with 4-fold excess of cell lysate and longer exposure of the x-ray film. (D) Ponceau S staining of the gel used in (B) for the immunoblot. Molecular mass markers (in kDa) are indicated on the left, and arrows mark positions of the indicated protein bands. Results are representative of two independent experiments. assay in which bead-adsorbed CypA was used to pull down CrkII we selected the Crk SH3 domain–binding guanine-nucleotide re- from a lysate of Jurkat T cells. Gel filtration and Western blot leasing factor (C3G), a guanine nucleotide exchange factor for by guest on September 23, 2021 analysis revealed that CrkII, but not CrkI, associates with CypA Rap1 (32), which is activated following binding to Crk adaptor (Fig. 1B, 1C). Because a single substrate might be subjected to proteins (33–35), and tested its ability to bind CrkII-SH3N under isomerization by different isomerases, we tested whether CypA different experimental conditions. As a first step, we verified that interaction with CrkII is novel, or whether other immunophilins C3G binds the CrkII–SH3N domain and not other regions within exhibit similar capabilities. A pull-down assay using different bead- the molecule. Jurkat T cells were transfected with eukaryotic immobilized GST immunophilins revealed that, in addition to vectors encoding GST-tagged WT CrkII, CrkII mutants that CypA, FKBP12 and Cyp40, but not FKBP12.6, FKBP51, or abolish SH2 (R38L) or SH3N (W169L) binding to their ligands, or FKBP52, pulled down CrkII from a Jurkat T cell lysate (Fig. 1B, a CrkII mutant (Y221F) that is unable to undergo activation- 1C). CrkI, which lacks the C-terminal SH3 domain, was observed in dependent tyrosine phosphorylation in vivo (19). The cells were whole-cell lysates of Jurkat T cells, although in a significantly lower then lysed and subjected to immunoprecipitation with anti-GST amount compared with CrkII. However, CrkI was undetectable in all Abs. Protein fractionation by SDS-PAGE and immunoblot anal- pull-down groups, suggesting no binding to any of the immuno- ysis demonstrated binding of C3G to WT and mutant CrkII, but philins tested (compare lane 1 with all other lanes in Fig. 1B, 1C). not to CrkII (W169L) that possesses an inactive mutant SH3N domain (Fig. 2). CrkII binding to C3G is mediated via the N-terminal SH3 domain CypA increases CrkII binding to C3G Structure–function studies demonstrated that the CrkII C-terminal The ability of CrkII to associate with both C3G and CypA, and SH3 domain (SH3C) possesses an amino acid sequence and an serve as a putative substrate for CypA, raises the possibility that overall conformation of a classical SH3 domain (27). However, the CypA-mediated isomerization of CrkII may affect CrkII binding to canonical-binding surface of the CrkII-SH3C domain is unusually C3G. To test this hypothesis, we incubated bead-immobilized GST- polar, suggesting that its interaction with typical proline-rich ligands CrkII in the presence or absence of CypA for 1 h at room tem- is not possible or occurs at a very low affinity (27). Other studies perature, and then added a Jurkat T cell lysate for additional 3 h of showed a negative regulatory role for the CrkII-SH3C domain, incubation. GST–CrkII–binding proteins were pulled down and which affects ligand binding to the CrkII-SH2 or -SH3N domains analyzed by Western blot for the presence of C3G. The results (13, 28–31). The negative regulation by the CrkII-SH3C domain (Fig. 3A) showed that GST–CrkII preincubation with CypA led to may reflect an isomerase-mediated trans-to-cis conversion of CrkII a ∼5-fold increase of the amount of C3G that interacted with at the linker region, resulting in steric hindrance that prevents ligand CrkII. binding by the CrkII-SH2 and/or -SH3N domains. We further tested whether Jurkat T cell lysate preincubation with To test whether binding to immunophilin alters the ability of CypA would affect the ability of the endogenous CrkII to associate CrkII to interact with known CrkII-SH3N binding partners in vivo, with C3G. Thus, we incubated Jurkat T cell lysates with or without The Journal of Immunology 5 Downloaded from

FIGURE 3. CypA increases CrkII binding to C3G. (A–C) Bead-immo- bilized GST-CrkII proteins (10 mg) in 250 ml assay buffer (50 mM HEPES

and 100 mM NaCl [pH 8]) were incubated with 6 mM recombinant CypA http://www.jimmunol.org/ for 1 h at room temperature. After centrifugation, the supernatant was removed and 250 ml Jurkat T cell lysate (equivalent to 2 3 107 cells) was added to the beads for an additional 3 h of incubation at 4˚C. The beads were washed extensively, and bound proteins were eluted by boiling for 5 min in sample buffer and subjected to SDS-PAGE under reducing con- ditions. (D–G) Jurkat T cells (8 3 107) were lysed in 100 ml lysis buffer that was diluted in an assay buffer to a final volume of 250 ml, containing 50 mM HEPES and 100 mM NaCl (pH 8.0). CypA (6 mM, final con- centration) was added to one of the two test tubes that were incubated for

FIGURE 2. Crk binding to C3G is mediated by the Crk-SH3N domain. by guest on September 23, 2021 1 h at room temperature. Protein A beads (10 ml/group) that were pre- Jurkat TAg cells (4 3 107/group) were transiently transfected with incubated with anti-Crk mAbs (1 mg/group) for 1 h at room temperature eukaryotic expression vectors (pCEFL-GST-CrkII) encoding WT or mu- were added to each of the test tubes for an additional 3 h of incubation at tated CrkII, as indicated above (A). After 36 h in culture, cells were lysed 4˚C. The beads were extensively washed, and bound proteins were eluted and GST-Crk fusion proteins were immunoprecipitated using anti-GST by boiling for 5 min in sample buffer and subjected to SDS-PAGE under Abs. Samples of whole-cell lysates (WCL; representing 0.5 3 106 cells/ reducing conditions. Proteins from both experiments were then electro- group) and immunoprecipitates were resolved by SDS-PAGE under re- blotted onto nitrocellulose membranes that were sequentially immuno- ducing conditions, followed by electrotransfer to nitrocellulose mem- blotted with Abs directed against C3G (A and D), CypA (B and F), and Crk branes. Membranes were then stained with Ponceau S to validate the (E). Ponceau S staining (bottom panels) was used to validate equal loading loading of equal amount of proteins (C). After washings, membranes were of proteins. Molecular mass markers (kDa) are indicated on the left, and sequentially immunoblotted with anti-C3G (A) and anti-GST (B) Abs. arrows on right show positions of indicated protein bands. Results in both Molecular mass markers (kDa) are indicated on the left, and arrows mark panels are representative of three independent experiments. positions of the indicated proteins. IB, Immunoblot. Results are repre- sentative of two independent experiments. *Indicates the position of GST- CrkII WT and mutants. motes a conformational change in CrkII, thereby increasing ac- cessibility of the CrkII-SH3N to the C3G poly-proline region. recombinant CypA for 1 h at room temperature, followed by in- To distinguish between these two potential mechanisms, we cubation with anti-Crk mAb-coated protein A beads. Immuno- precultured Jurkat T cell lysates for 24 h in the presence or absence precipitation and Western blot analysis demonstrated that the of immunophilin inhibitors, CsA and/or FK506, and pulled down addition of CypA to the Jurkat T cell lysate resulted in ∼3-fold binding proteins using bead-immobilized GST-C3G or GST-SH3b increase in the amount of C3G that coimmunoprecipitates with [a truncated C3G corresponding to aa 208–662, which possesses CrkII (Fig. 3D). multiple copies of proline-rich regions (17, 33)] fusion proteins. Immunoblot analysis revealed that each of the two fusion proteins, PPIase-mediated increase in CrkII binding to C3G is GST-C3G and -SH3b, pulled down both CrkI and CrkII (Fig. 4A). dependent on the PPIase catalytic activity Cell pretreatment with either CsA or FK506 had a negligible ef- The results in Fig. 3 demonstrated that the presence of CypA in the fect on the expression levels of Crk I and CrkII (see Fig. 5C). assay system increases C3G association with CrkII. This may However, the amount of CrkII pulled down by each of the fusion reflect an increase in the affinity of CrkII to C3G due to physical proteins was significantly reduced following drug pretreatment interaction of immunophilins with CrkII. Alternatively, increased (Fig. 4A). In contrast, the drugs had no effect on the amount of binding of C3G to CrkII may occur by immunophilin-dependent CrkI pulled down by GST-C3G and -SH3b. The results indicate cis-to-trans isomerization of CrkII. The latter possibility is likely that the effect of CsA and FK506 on CrkII binding to C3G is not to be dependent on the immunophilin catalytic activity that pro- due to some unknown effects of the drugs on protein synthesis, 6 IMMUNOPHILINS REGULATE CrkII ACTIVITY IN HUMAN T LYMPHOCYTES Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021 FIGURE 4. Immunophilin-mediated increase in CrkII binding to C3G is dependent on the immunophilin enzymatic activity. (A)JurkatTAg T cells (107/group) were cultured in the presence or absence of CsA (5 mg/ml) plus FK506 (5 ng/ml) for 24 h. After 30 min of incubation in lysis buffer on ice, equal volumes of cell lysates were incubated with bead-immobilized GST-C3G or GST-SH3b fusion proteins (10 mg/ group). After 4 h of incubation on a rotator at 4˚C, the beads were washed extensively and proteins were eluted by boiling for 5 min in sample buffer and subjected to SDS-PAGE under reducing conditions. Proteins were then electroblotted onto a nitrocellulose membrane that FIGURE 5. Treatment of Jurkat T cell subclones with immunophilin was immunoblotted with mouse anti-Crk mAbs. (B) Reblotting of whole- inhibitors downregulates C3G binding to CrkII, but not to CrkI. Cells of cell lysates (WCL) with anti–b-actin served as a control for equal protein Jurkat-derived subclones, which stably express Myc-CrkI or Myc-CrkII loading. (C) The relative intensity of the protein band signals was de- (5 3 107/group), were cultured in the presence or absence of CsA (5 mg/ml) termined by ImageJ (v1.45) and presented in a bar graph. (D) Ponceau plus FK506 (5 ng/ml) for 16 h. After additional 20 min of incubation in S staining of the nitrocellulose used in (A)–(C). SH3b corresponds to aa lysis buffer on ice, cell lysates were incubated with protein A–agarose 208–662 of human C3G and possesses poly proline-rich regions that bead-bound mouse anti-Myc mAbs for 16 h on a rotator at 4˚C. The beads mediate the binding to the Crk-SH3N (17). Results are representative of were washed extensively, and bound proteins were eluted by boiling for two independent experiments. 5 min in sample buffer and subjected to SDS-PAGE under reducing con- ditions. Proteins were then electroblotted onto a nitrocellulose membrane, and C3G-coimmunoprecipitating proteins were detected using rabbit anti- C3G Abs (A). Anti-C3G immunoblot of whole-cell lysates was performed content, or activity, because the drugs had no effect on the extent to show that both cell lines express similar amounts of C3G, and that drug of CrkI binding to C3G. treatment did not alter C3G expression levels (B). Presence of Myc-CrkI or To substantiate these findings in a more physiological system, we -CrkII in the immunoprecipitates was observed by reblotting of striped analyzed the effect of immunophilin inhibitors on Crk-C3G in- membrane with anti-Crk mAbs (C). Expression levels of endogenous and teraction using coimmunoprecipitation studies. In this study, Jurkat overexpressed Crk proteins in whole cell lysates were observed using anti- T cells were pretreated as previously with a mixture of CsA and Crk mAbs (D). Ponceau S staining validated the equal amounts of Abs FK506 for 16 h, and C3G binding to Crk adaptor proteins was used in all groups (E). IP, Immunoprecipitation. Results are representative tested by immunoprecipitation of Crk and immunoblot with anti- of three independent experiments. C3G Abs. Because Abs that distinguish between CrkI and CrkII do not exist, we used Jurkat T cell lines that constitutively express We found that anti-Myc mAbs coimmunoprecipitated C3G from either Myc-CrkI (JKCrkI) or Myc-CrkII (JKCrkII) at levels that JKCrkII cells and that cell pretreatment with CsA and FK506 resemble those of the endogenous Crk proteins (see Fig. 5D). reduced the amount of coimmunoprecipitating C3G by ∼2-fold. In The Journal of Immunology 7 contrast, no effect of the drugs was observed on the amount of (Fig. 6). A fraction of the C3G proteins in untreated cells was C3G coimmunoprecipitated with Myc-CrkI from JKCrkI cells associated with the cell membrane in addition to a smaller amount (Fig. 5A). This is despite the fact that similar levels of C3G were of C3G that distributed unevenly in the cytoplasm. Cell treatment observed in both cell lines, and that C3G levels were not affected with CsA/FK506 did not affect CrkII distribution within the cell’s by the drug treatment (Fig. 5B). outer membrane, but resulted in a significant redistribution of the Anti-Crk immunoblot demonstrated that anti-Myc mAbs im- membrane-bound C3G back into the cell’s cytoplasm. Quantifi- munoprecipitated only Myc-CrkI from JKCrkI cells, and only cation of CFP and YFP colocalization using the ImageJ colocal- Myc-CrkII from JKCrkII cells (Fig. 5C). Furthermore, drug ization plugin JACoP (39) demonstrated that the fraction of red treatment of both cell lines did not alter the amount of Myc-CrkI overlapping with green in untreated cells (Mander’s M2 coeffi- or Myc-CrkII expressed in the two cell lines, respectively cient of 0.394) was reduced upon cell treatment with CsA/FK506 (Fig. 5C). (M2 = 0.095). The colocalization of C3G and CrkII in Jurkat T cells is Treatment of PICCHUx-transfected Jurkat T cells with CsA inhibited by cell pretreatment with CsA and FK506 plus FK506 increases FRET efficiency To analyze whether immunophilin inhibitors affect the association The PICCHUx plasmid was designed to serve as a phosphorylation of CrkII with C3G in vivo, we used drug-treated or untreated Jurkat indicator for active Abl, because Abl-mediated phosphorylation T cells and performed a fluorescence colocalization assay. Staining of PICCHUx (on Tyr221) leads to intramolecular interaction of of Jurkat T cells confirmed that both CrkII (19, 36) and C3G (37, phosphotyrosine221 with the internal SH2 domain, which results in

38) localize predominantly at the cytoplasm of resting cells (data folding of the protein and increased FRET emission (18). Downloaded from not shown). However, due to the wide distribution of CrklI and We tested whether FRET activity of PICCHUx is also regulated C3G all over the cytoplasm, we were unable to distinguish be- by immunophilins, and therefore sensitive to inhibition by im- tween CrkII-associated C3G or free C3G, as both would have munophilin inhibitors. In this experiment, PICCHUx-transfected shown a very similar staining pattern. Jurkat T cells were treated or untreated with CsA/FK506, and Trying a different approach, we transiently transfected Jurkat their FRET activity, in which CFP and YFP fluorophores served as

T cells with a CrkII-encoding plasmid, termed PICCHUx (18), a donor and acceptor for FRET, was monitored. The results http://www.jimmunol.org/ which possesses a C-terminal CAAX box (in which C is cysteine, demonstrated a significantly higher FRETefficiency in drug-treated A is an aliphatic residue, and X is a variety of residues) of ki-Ras cells compared with the control cells (Fig. 7A, 7B, control = that allows posttranslational addition of a lipid group and thereby 29.2 6 1.8, drug treated = 45.9 6 1.9, n =10). increases the protein’s affinity to the cell membrane. In addition, We also established a FACS-based FRET analysis method of the PICCHUx chimeric construct encodes the human CrkII1–236 PICCHUx-expressing Jurkat T cells, in which the transfected and sandwiched between cyan- and yellow-emitting variants of GFP drug-treated cells were analyzed using FACSCanto II. Live cells (CFP and YFP, respectively). Expression of this fluorescently la- were gated according to forward and sideward scatter and adjusted beled CrkII at the plasma membrane allows the distinction be- photomultiplier tube voltages and compensation for CFP and YFP tween CrkII-bound membrane-associated C3G versus free C3G to specifically assess FRET in double-positive cells. Five-minute by guest on September 23, 2021 that is distributed in the cell’s cytoplasm. kinetics of FRET emission ratios were analyzed using FlowJo Two days after transfection of Jurkat T cells with PICCHUx, the Ver. 7.6.3 software. An increase in FRET emission ratio was found cells were incubated for an additional 24 h in the presence or in all time points of the measured kinetics in drug-treated group absence of CsA plus FK506 (CsA/FK506), and then stained with compared with the control (Fig. 7C, 7D). The results correlate with C3G-specific Cy3-labeled Abs. The results demonstrated that the the confocal microscopy FRET data, supporting the assumption majority of the overexpressed CrkII protein was membrane bound that CrkII is a target for immunophilins, and that inhibitors of

FIGURE 6. Treatment of Jurkat T cells with CsA plus FK506 inhibits C3G colocalization with a membrane-bound heterologous Crk protein. Jurkat 6 T cells (5 3 10 cells/700 ml/cuvette) were transfected with 10 mg PICCHUx plasmid that encodes the human CrkII1–236 sandwiched between CFP and YFP, and a CAAX box that anchors the protein to the plasma membrane. After 48 h in culture, cells were incubated for an additional 24 h in the presence or absence of CsA (5 mM) and FK506 (5 nM), fixed, permeabilized, and stained with rabbit anti-C3G and Cy3-conjugated anti-rabbit Ig Abs. Localization of PICCHUx-expressed Crk proteins (Channel 1, GFP) and C3G (Channel 2, Cy3) was determined by confocal microscopy. Channel 3 demonstrates a dif- ferential interference contrast (DIC), and the rightmost panels include an overlay photo of Channels 1 + 2. Quantification of red and green colocalization was performed using the ImageJ colocalization plugin JACoP, and values are shown on the right. Mander’s M2 coefficient, which indicates the fraction of red overlapping with green, has a significantly lower value in CsA/FK506-treated cells. Scale bars, 10 mm. 8 IMMUNOPHILINS REGULATE CrkII ACTIVITY IN HUMAN T LYMPHOCYTES Downloaded from http://www.jimmunol.org/

FIGURE 7. Treatment of Jurkat T cells with CsA plus FK506 increases FRET activity of a heterologously expressed PICCHUx cDNA. (A) Jurkat TAg T cells (2 3 107) were transiently transfected with the PICCHUx vector by electroporation. (18). After 48 h in culture, the cells were washed and resuspended in complete RPMI 1640. Cell suspension was split into two identical volumes, and cells were recultured for 24 h on coverslips in the absence or presence of CsA (5 mM) plus FK506 (5 nM). Cells were then fixed and scanned under FV1000 confocal microscope, and protein fluorescence in CFP and FRET channels was measured. Three cells that express the PICCHUx-encoded protein were randomly selected, and 10 regions of interest were randomly taken from the membrane of each cell to measure the emission ratio. Original magnification 3400. (B) The values obtained are presented in a bar graph. (C) Jurkat TAg T cells were transfected with PICCHUx, cultured, and treated, as in (A). Cells were analyzed on a FACSCanto II in which CFP was

exited at 405 nm and its emission was detected using a BP450/50 filter (for CFP only) and BP585/42 filter (for CFP and YFP). (D) Emission values were by guest on September 23, 2021 analyzed by Flowjo7.6.3 software and presented in a bar graph. The relative amount of PICCHUx protein expressed by untreated and drug-treated cells was evaluated by immunoblot (E and F). The ratios between CFP and YFP in untreated and drug-treated cells were 1.37 and 1.31, respectively. Results of the confocal and FACS studies are each representative of two independent experiments. *p , 0.05, using t test.

immunophilin catalytic activity increase the proportions of cis express either CrkII or CrkI and analyzed their ability to adhere to conformers of CrkII. fibronectin-coated microtiter wells before or after treatment with immunophilin inhibitors. CrkI or CrkII overexpression in Jurkat Adhesion and migration of Jurkat T cells that overexpress T cells increased their adherence by ∼2-fold (Fig. 8A). Pretreat- CrkII, but not CrkI, are inhibited by cell pretreatment with CsA ment with CsA/FK506 inhibited the adhesion of Jurkat, JKCrkI, plus FK506 and JKCrkII T cells by 44.9, 20.1, and 55.4%, respectively Integrin-mediated cell adhesion is a critical process required for the (Fig. 8A). Because all three cell lines express similar levels of migration of normal cells during biological processes, including endogenous CrkI and CrkII, we used the adhesion values of Jurkat embryonic development, wound healing, and immune responses T cells as basal levels and subtracted them from the values ob- (40, 41). In addition, cell migration plays an important role in tained by JKCrkI and JKCrkII cells to calculate the increment in various pathological conditions, such as tumor metastasis to re- adhesion reflecting the effect of the overexpressed genes (Fig. 8B). mote tissues and organs (42, 43). We found that CsA/FK506 had almost no effect on Myc-CrkI– Previous studies demonstrated that independent overexpression dependent adhesion of JKCrkI cells, but downregulated the Myc- of Crk or C3G, which form constitutive functional complexes CrkII–dependent adhesion of JKCrkII cells by ∼58% (Fig. 8B). in vivo, augments integrin-mediated adhesion of hematopoietic Adhesion of cells in different biological systems precedes their cells to fibronectin (44, 45). The Crk-SH3N, which mediates C3G migration, as observed during embryogenesis, wound healing, and binding, plays an important role in this process, because expres- immune responses. Both Crk and C3G are important components sion of a SH3N-defective mutant inhibited cell adhesion to fi- of both processes, and overexpression of genetically altered Crk bronectin (44–46). In addition, both Crk and C3G function as or C3G impairs both cell adhesion and migration (46, 50–52). focal adhesion proteins and Crk-C3G complexes recruit to the To test the effect of immunophilin inhibitors on CrkI- versus leading edge of polarized cells, where they activate additional CrkII-dependent cell migration, we used the same Jurkat sublines effector molecules and provide spatially instructive cues required and tested their ability to migrate in vitro in response to the for cell adhesion and migration (47–50). chemoattractant, SDF1a. The cells were introduced into chambers To test the effect of CsA/FK506 on C3G-CrkII– versus C3G- that are divided into two compartments by a microporous mem- CrkI–dependent cell adhesion, we used Jurkat T cells that over- brane. The assay ascertains efficiency of cell migration by deter- The Journal of Immunology 9 Downloaded from

FIGURE 8. Overexpression of either CrkI or CrkII increases both fibronectin-induced adhesion and SDF1a-induced migration of Jurkat T cells, which is selectively inhibited by CsA plus FK506 in CrkII-overexpressing cells. (A) Adhesion assay was performed in wells of 96-well microtiter plates precoated with human fibronectin. The wells were washed and blocked for 2 h by incubation with 100 ml PBS/2% BSA at room temperature. Jurkat T cells and Jurkat- derived cell lines that constitutively express Myc-CrkI and Myc-CrkII were untreated (Control) or cultured in the presence of CsA (5 mM) plus FK506 (5 nM) (drug treated) for 24 h. The cells (105/50 ml prewarmed additive-free RPMI 1640/well) were placed in BSA-blocked/RPMI 1640–washed wells and allowed to adhere for 2 h at 37˚C. Nonadherent cells were then removed by gentle aspiration and rinsing with prewarmed RPMI 1640, and adherent cells http://www.jimmunol.org/ were counted using a hemocytometer. Experiments were conducted using triplicate wells per group, and results are presented as a bar graph showing average 6 SD of the percentage of adherent cells obtained in two independent experiments. (B) The net change in the percentage of adherent Myc-CrkI– and Myc-CrkII–overexpressing cells after subtraction of the values obtained in normal Jurkat T cells. The net value of the percentage of adherent cells in each group is indicated above the bar. (C) Transwell migration assay was performed using ThinCert cell culture inserts (with a 5.0-mm pore-size positron- emission tomography membrane) placed in wells of 24-well cell culture plates. A half million cells in 100 ml migration buffer were seeded into the upper compartment of individual chambers, and 600 ml SDF1a (100 ng/ml) in migration buffer was placed into the lower chambers. After 3 h of incubation at 37˚C, the migrating cells were counted by hemocytometer. Experiments were conducted using triplicate wells per group, and results are presented as a bar graph showing average 6 SD of the percentage of migrating cells obtained in two independent experiments. (D) The net change in the percentage of

migration of Myc-CrkI– and Myc-CrkII–overexpressing cells after subtraction of the values obtained in normal Jurkat T cells. The net value of by guest on September 23, 2021 the percentage of migrating cells in each group is indicated above the bar. Asterisks indicate statistically significant differences (*p , 0.05 by one-way ANOVA). mining the percentage of cells that migrate from one chamber, mechanisms that control their expression levels and relative pro- across a microporous membrane, into the second chamber in re- portions. Structure–function studies revealed that CrkII and CrkL, sponse to SDF1a, which binds to CXCR4 that signals the cells to but not CrkI, can undergo autoinhibition following phosphoryla- respond by chemotaxis (53) migration (54, 55). tion of a critical tyrosine residue in the linker region connecting Overexpression of either CrkI or CrkII significantly increased the the two SH3 domains (Tyr221 and Tyr207 in CrkII and CrkL, re- migration rate of the Jurkat T cells (Fig. 8C), and cell treatment with spectively) that can interact intramolecularly with the N-terminal CsA/FK506 inhibited the migration of all three cell types (Fig. 8C). SH2 domain. We used the migration values of Jurkat T cells as basal levels and Recent studies suggested that activity of the CrkII adaptor subtracted them from the values obtained by JKCrkI and JKCrkII protein can be regulated by an additional mechanism, mediated by cells to calculate the increment in migration reflecting the effect of immunophilins that reversibly catalyze CrkII and determine the the overexpressed genes (Fig. 8D). We found that CsA/FK506 did interconversion between cis (closed and inactive) and trans (open not inhibit Myc-CrkI–dependent migration of JKCrkI cells, while and active) conformers (13, 14). These in vitro studies were per- inhibiting Myc-CrkII–dependent migration of JKCrkII cells by formed on a truncated recombinant protein consisting of the ∼46% (Fig. 8D). SH3N-linker-SH3C of the chicken CrkII (CrkIISLS) and raise The results indicate that CsA/FK506 significantly inhibited questions about the physiological relevance of these findings in CrkII- but not CrkI-dependent adhesion and migration of the Jurkat mammals. For example, it is not clear whether immunophilins can T cells. catalyze the in vitro isomerization of full-length chicken CrkII, whether this isomerization can occur in vivo, and what mecha- Discussion nisms regulate the cis into trans isomerization of CrkII and vice T lymphocytes express the three major types of Crk adaptor versa. In addition, it is not clear whether immunophilins can proteins, and all three are involved in constitutive and induced catalyze the in vivo isomerization of human CrkII, which proline protein–protein interactions. The fact that Crk proteins interact residue(s) may serve as target(s) for PPIases, and what is the with signaling molecules in T cells in a TCR activation-dependent physiological impact of this conformational change. manner (3–5, 56–60) supports a role for these proteins in signaling In this work, we used the human leukemia T cell line, Jurkat, cascades operating downstream of the activated TCR and in reg- to analyze the potential in vivo regulation of CrkII and CrkII- ulation of T cell responsiveness. However, little is known about dependent functions by immunophilins and immunophilin-specific the biological function of the distinct Crk proteins in T cells or the inhibitors. 10 IMMUNOPHILINS REGULATE CrkII ACTIVITY IN HUMAN T LYMPHOCYTES

Pull-down assays and Western blot analyses demonstrated the degree of colocalization of Crk and C3G at one pole of the cell in vivo physical association between CrkII and immunophilins, membrane. In contrast, this colocalization was partially disrupted including CypA, Cyp40, and FKBP12 in resting Jurkat T cells by cell treatment with CsA/FK506 (Fig. 6). These findings are in (Fig. 1). Although ∼90% of the CrkII proteins acquire a cis agreement with the assumption that CsA/FK506 shifts the balance conformation in solution, in which the protein possesses a closed of CrkII proteins toward the cis conformation, which does not sup- conformation that is not accessible to interaction with its SH3N- port CrkII-SH3N association with binding partners, such as C3G. binding partners (14), it is yet unclear whether excess of immu- The assumption that CrkII is a substrate for catalytically active nophilins will change the balance between the cis and trans PPIases was further analyzed on PICCHUx-expressing Jurkat conformers of CrkII, and toward which direction. T cells. PICCHUx encodes a chimeric CrkII1–236 that is targeted to One way to approach this question is to test the effect of excess the cell membrane via a CAAX domain, and is sandwiched be- of immunophilins on the ability of CrkII to associate with a bind- tween CFP and YFP (18). Confocal analysis demonstrated a basal ing partner that interacts with the CrkII-SH3N domain. level of FRET emission from these cells, which was significantly One such candidate is C3G, a guanine nucleotide exchange increased by cell pretreatment with CsA/FK506 (Fig. 7A, 7B). factor essential for Rap-GTPase activation (32), which interacts Similar results were obtained when cell analysis was performed on with the Crk-SH3N domain (35), and is critical for integrin- a FACS (Fig. 7C, 7D). mediated cell adhesion and migration (50, 61). Our findings, These results support the assumption that CrkII is a target for showing that CypA increases the in vitro association of CrkII with catalytically active immunophilins, and that inhibition of their isom- C3G (Fig. 3), suggest that excess of immunophilins shifts the erase activity (by CsA/FK506) promotes the accumulation of cis cis-trans balance of CrkII proteins toward the trans conformation. conformer of CrkII, as reflected by the increased FRET emission. Downloaded from Increase in the proportions of trans CrkII conformers increases the The accumulation of cis conformers of CrkII normally occurs number of open CrkII proteins that are available for interaction following Abl kinase activation that phosphorylates CrkII and with the C3G molecule via their SH3N domain. promotes the intramolecular phospho-Tyr221 binding to the CrkII- To find out whether the effect of immunophilins on CrkII binding SH2 domain. One possible explanation for the FRET data assumes to C3G is dependent on the isomerase catalytic activity, we used that CsA/FK506 affects some unknown tyrosine kinase(s) that

GST-C3G to pull down CrkII from lysates of Jurkat T cells that phosphorylates CrkII on a tyrosine residue and promotes the http://www.jimmunol.org/ were pretreated with the most commonly used immunophilin intramolecular folding, rather than an immunophilin-mediated inhibitors, CsA and FK506. We found that neither CsA nor FK506 trans-to-cis conformational change of CrkII. This assumption is had a dramatic effect on CrkII binding to GST-C3G, apparently likely to be incorrect, because tyrosine phosphorylation levels of because each drug by itself suppressed just one group of immu- CrkII in Jurkat T cell lysates were unaltered by cell treatment with nophilins, leaving the other group uninterrupted and free to induce CsA/FK506 (results not shown). a conformational change in C3G. However, the combined effect of The CsA/FK506-induced increase in FRET emission is not as CsA/FK506 significantly inhibited CrkII binding to GST-C3G dramatic as in other systems, apparently reflecting the previously (Fig. 4). The drug effect was CrkII specific because binding published observation that ∼90% of the CrkIISLS in solution of CrkI, a SH3C-deficient alternatively spliced form, to GST-C3G possesses the cis conformation (14). Furthermore, the ability of by guest on September 23, 2021 was not altered under similar experimental conditions. Further- CsA/FK506 to modulate the conformation of the PICCHUx pro- more, a shorter GST fusion protein (GST-SH3b) possessing the tein supports the hypothesis that PPIases act on a Pro residue, C3G proline-rich regions (aa 208–662) (17, 33), which is suffi- which is distinct from Pro238 that is predicted to be buried in the cient for interaction with Crk, pulled down less CrkII proteins SH3N-SH3C binding interface in the CrkIISLS cis isomer (16). from a lysate of CsA/FK506-treated Jurkat T cells. In contrast, C3G is a guanine nucleotide exchange factor, which regulates the CrkI interaction with GST-SH3b was unaltered by cell pretreat- activity of the Rap1-GTPase (32), and is the first protein identified ment with CsA/FK506. to physically associate with Crk, specifically with the Crk-SH3N A support for the assumption that a relatively large fraction of the domain (33, 35). Because Crk-C3G interaction plays a critical role cellular CrkII acquires a cis (closed) conformation that is inacces- in integrin-mediated cell adhesion and migration (49, 50, 61), we sible to C3G (or other CrkII-SH3N– binding partners) comes from tested whether immunophilin-mediated regulation of Crk adaptor the observation in Jurkat T cells that, despite having excess of CrkII proteins may alter the Crk-C3G–dependent adhesion and migra- over CrkI (see Fig. 4A, whole-cell lysates), more CrkI molecules tion of human T lymphocytes. Studies in this paper demonstrated associate with GST-SH3b compared with CrkII in the pull-down that cell treatment with immunophilin inhibitors increased the assay (see Fig. 4A, two right lanes). These findings suggest that proportions of cis conformers of CrkII, representing a closed con- a fraction of the CrkII molecules that is detectable by Western blot formation, which is not accessible for C3G binding. In agreement, in the whole-cell lysate is incapable of associating with GST-SH3b, immunophilin inhibitors suppressed the ability of Myc-CrkII– apparently because of the cis conformation of the proteins. overexpressing JKCrkII cells to adhere to a fibronectin-coated sur- To further verify that immunophilin inhibitors can alter the face (Fig. 8A, 8B) and migrate via a porous membrane toward conformation of CrkII, but not CrkI, we first established two Jurkat a chamber rich in SDF1a chemokines (Fig. 8C, 8D). However, the sublines that constitutively express either Myc-CrkI or Myc-CrkII. same immunophilin inhibitors had a minimal effect on adhesion Immunoprecipitation of Myc-tagged proteins from these cells and migration of Myc-CrkI–overexpressing JKCrkI cells. This min- coimmunoprecipitated C3G (Fig. 5A), and pretreatment of the imal inhibition may reflect the drug effects on the endogenous CrkII. cells with CsA/FK506 did not alter the amount of C3G that was Although the cell adhesion data support a role for immunophilin- expressed by the cells (Fig. 5B). However, cell pretreatment with mediated regulation of CrkII in integrin binding to fibronectin, results immunophilin inhibitors decreased the amount of C3G that co- of the migration assay may also indicate the involvement of immu- immunoprecipitated with Myc-CrkII by ∼3-fold, suggesting that nophilins and CrkII in the regulation of SDF1a-mediated signaling the immunophilin inhibitors increased the proportions of cis pathway. conformers of CrkII without altering the conformation of CrkI. Both CsA and FK506 are known to inhibit NFAT in activated Fluorescent analysis of Jurkat T cells, ectopically expressing T cells and induce immunosuppression by preventing NFAT- membrane-bound Crk (PICCHUx) proteins, demonstrated a high mediated activation of essential genes, including the IL2 (62). The Journal of Immunology 11

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