Bcl10 Controls TCR- and FcγR-Induced Actin Polymerization Daniel Rueda, Olivier Gaide, Liza Ho, Elodie Lewkowicz, Florence Niedergang, Stephan Hailfinger, Fabien Rebeaud, This information is current as Montserrat Guzzardi, Béatrice Conne, Marcus Thelen, of September 29, 2021. Jérôme Delon, Uta Ferch, Tak W. Mak, Jürgen Ruland, Jürg Schwaller and Margot Thome J Immunol 2007; 178:4373-4384; ;
doi: 10.4049/jimmunol.178.7.4373 Downloaded from http://www.jimmunol.org/content/178/7/4373
References This article cites 56 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/178/7/4373.full#ref-list-1 http://www.jimmunol.org/
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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 © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology
Bcl10 Controls TCR- and Fc␥R-Induced Actin Polymerization1
Daniel Rueda,* Olivier Gaide,2,3* Liza Ho,2† Elodie Lewkowicz,‡§¶ʈ Florence Niedergang,‡§¶ʈ Stephan Hailfinger,* Fabien Rebeaud,* Montserrat Guzzardi,* Be´atrice Conne,† Marcus Thelen,# Je´roˆme Delon,‡§¶ʈ Uta Ferch,†† Tak W. Mak,‡‡ Ju¨rgen Ruland,†† Ju¨rg Schwaller,4† and Margot Thome5*
Bcl10 plays an essential role in the adaptive immune response, because Bcl10-deficient lymphocytes show impaired Ag receptor- induced NF-B activation and cytokine production. Bcl10 is a phosphoprotein, but the physiological relevance of this posttrans- lational modification remains poorly defined. In this study, we report that Bcl10 is rapidly phosphorylated upon activation of human T cells by PMA/ionomycin- or anti-CD3 treatment, and identify Ser138 as a key residue necessary for Bcl10 phosphory- lation. We also show that a phosphorylation-deficient Ser138/Ala mutant specifically inhibits TCR-induced actin polymerization yet
does not affect NF-B activation. Moreover, silencing of Bcl10, but not of caspase recruitment domain-containing MAGUK Downloaded from protein-1 (Carma1) induces a clear defect in TCR-induced F-actin formation, cell spreading, and conjugate formation. Remark- ably, Bcl10 silencing also impairs Fc␥R-induced actin polymerization and phagocytosis in human monocytes. These results point to a key role of Bcl10 in F-actin-dependent immune responses of T cells and monocytes/macrophages. The Journal of Immu- nology, 2007, 178: 4373–4384.
6
he protein B cell lymphoma/leukemia-10 (Bcl10) plays a associated lymphoid tissue (MALT) lymphomas and in NK/T cell http://www.jimmunol.org/ key role in immune responses triggered by multisubunit lymphomas (4, 5). Moreover, activated B cell-type diffuse large B T immune recognition receptors. Indeed, Bcl10-deficient cell lymphomas were recently shown to critically depend on Bcl10 mice are severely immunodeficient, due to impaired Ag receptor- expression for their survival (6). and FcR-induced NF-B activation and cytokine secretion (1–3). Despite these recent insights into the biological relevance of Bcl10 may also be important for the progression of particular Bcl10, the molecular function of Bcl10 is only partly understood. forms of B cell lymphoma. Overexpression and nuclear transloca- Upon TCR triggering, Bcl10 is recruited to the TCR/CD3 complex tion of Bcl10 has been reported to occur frequently in mucosa- by a caspase recruitment domain (CARD)-dependent interaction with its binding partner CARD-containing MAGUK protein-1 (Carma1), and activates the NF-B-regulating IB kinase (IKK) by guest on September 29, 2021 *Department of Biochemistry, University of Lausanne, BIL Biomedical Research complex through the paracaspase protein Malt1 (MALT lym- Center, Epalinges, Switzerland; †Department of Clinical Pathology, Centre Medical Universitaire, Geneva, Switzerland; ‡Institut Cochin, De´partement de Biologie Cel- phoma translocation protein-1) (7–9). The C-terminal portion of lulaire, Paris, France; §Institut National de la Sante´et de la Recherche Me´dicale Unite´ Bcl10, which shares no obvious sequence homology to any other ¶ 567, Paris, France; Centre National de la Recherche Scientifique, Unite´Mixte de known protein, is rich in Ser and Thr residues and is subject to Recherche, Paris, France; ʈUniversite´Paris 5, Faculte´deMe´decine Rene´Descartes, Paris, France; #Institute for Research in Biomedicine, Bellinzona, Switzerland, phosphorylation events that are observed upon Bcl10 overexpres- ††Third Medical Department, Technical University of Munich, Klinikum Rechts der sion, but also specifically triggered upon Ag receptor engagement ‡‡ Isar, Munich, Germany; and The Campbell Family Institute for Breast Cancer or TNF-␣ stimulation (10–17). Recently, Wegener et al. (18) Research and Ontario Cancer Institute, University Health Network, University of Toronto, Toronto, Ontario, Canada showed that IKK-mediated phosphorylation of Bcl10 plays a Received for publication September 18, 2006. Accepted for publication January negative regulatory role in T cell activation by interfering with the 15, 2007. Bcl10-Malt1 interaction. It remains unclear, however, whether The costs of publication of this article were defrayed in part by the payment of page Bcl10 phosphorylation specifically regulates NF-B activation or charges. This article must therefore be hereby marked advertisement in accordance also as yet undefined biological functions of Bcl10. with 18 U.S.C. Section 1734 solely to indicate this fact. Stimulation of surface receptors of immune cells triggers not 1 This work was supported by grants from Swiss Cancer League (to M.Tho. and J.S.), Swiss National Science Foundation (to M.Tho. and J.S.), and Cancer and Solidarite´ only changes in gene expression but also morphological changes (to J.S.). F.N. is supported by grants from the Ville de Paris and Centre National de via the reorganization of the actin cytoskeleton, which contributes la Recherche Scientifique (Action The´matique d’Inte´reˆt Prioritaire Jeune Chercheur to efficient cellular activation. In immune cells, ligand-induced ac- Microbiologie). J.R. is supported by a Max-Eder-Program Grant from Deutsche Kreb- shilfe and by grants from Deutsche Forschungsgemeinschaft. O.G. was supported by tin polymerization is initiated by the activation of receptor-proxi- a M.D.-PhD fellowship from the Swiss Academy of Medical Sciences. D.R. was mal tyrosine kinases, which in turn control the activity of GDP/ supported by a postdoctoral fellowship from the Spanish Ministry of Education and GTP exchange factors (GEFs) that regulate the activity of Rho Science. S.H. was supported by a PhD fellowship from the Studienstiftung des Deut- schen Volkes. E.L. was supported by a Bourse de Docteur Inge´nieur du Centre Na- tional de la Recherche Scientifique. 2 O.G. and L.H. contributed equally to this study. 6 Abbreviations used in this paper: MALT, mucosa-associated lymphoid tissue; 3 Current address: University Medical Center, 1 rue Michel-Servet, 1211 Geneva 4, CARD, caspase recruitment domain; Carma1, CARD-containing MAGUK protein-1; Switzerland. IKK, IB kinase; Malt1, MALT lymphoma translocation protein-1; GEF, GDP/GTP 4 exchange factor; WASP, Wiskott-Aldrich syndrome protein; siRNA, small interfering Current address: Department of Research, University Hospital, Hebelstrasse 20, RNA; shRNA, short hairpin RNA; EGFP, enhanced GFP; IgG-SRBC, IgG-coated CH-4031 Basel, Switzerland. SRBC; wt, wild type; PKC, protein kinase C; DN, dominant negative; RIP, receptor- 5 Address correspondence and reprint requests to Dr. Margot Thome, Department interacting protein; SEE, staphylococcal enterotoxin E. of Biochemistry, University of Lausanne, Chemin des Boveresses 155, Epalinges, Switzerland. E-mail address: [email protected] Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 www.jimmunol.org 4374 Bcl10 PHOSPHORYLATION AND ACTIN POLYMERIZATION family GTPases (19). Among these, Cdc42 and Rac proteins are rose (Amersham Biosciences) were used for immunoprecipitation. Pri- proposed to control actin cytoskeletal changes via regulation of mary Abs used for Western blotting were anti-FLAG (M2; Sigma- Wiskott-Aldrich syndrome protein (WASP) and the Abi/Wave com- Aldrich), affinity-purified polyclonal rabbit Ab (AL114) recognizing the N terminus of mouse and human Bcl10 (14), rabbit anti-Bcl10 (H197; plex, respectively, which in turn regulate the activity of the Arp2/3 Santa Cruz Biotechnology), rabbit anti-Carma1 antiserum AL 220 (25), actin nucleation complex (20–24). To date, a possible role of Bcl10 in mAb anti-Malt1 (a gift from V. Dixit, Genentech), mAb anti-tubulin the regulation of the actin cytoskeleton has not been explored. (T-5168; Sigma-Aldrich), mAb anti-lamin A/C (SC-7292; Santa Cruz In this study, we reveal a novel function of Bcl10 in the control Biotechnology), mAb anti-phosphotyrosine (4G10; Upstate Biotechnol- ␥ ogy), rabbit anti-Vav (C-14; Santa Cruz Biotechnology), mAb anti- of TCR- and Fc R-induced actin polymerization. We show that phospho-ERK (Sigma-Aldrich), mAb anti-P-IB (5A5; Cell Signaling Bcl10 is phosphorylated rapidly upon TCR engagement, and iden- Technology), and rabbit anti-P-p38 (BioSource International). 138 tify Ser as a potential site of T cell activation-induced phos- 32 138 In vivo P labeling phorylation. Moreover, we show that Ser -dependent Bcl10 phosphorylation is critical for TCR-induced actin polymerization, For in vivo labeling experiments, 80% confluent 293T cells were trans- but not necessary for NF-B activation by Bcl10. Importantly, fected with PolyFect (Qiagen) following the manufacturer’s instructions. ϫ 5 Bcl10 silencing also affects Fc␥R-induced actin polymerization Transfected 293T cells (3 10 ) were washed twice with phosphate-free DMEM and incubated in this medium for1htofurther deplete the internal and phagocytosis. Together, these data identify a role for Bcl10 in phosphate store. Then, [32P]orthophosphate (100 Ci/ml; Amersham Bio- actin polymerization-dependent cellular immune responses. sciences) was added and the cells were incubated for 2 h. Materials and Methods Phospho-amino acid analysis
Culture and isolation of cells Radioactive bands corresponding to Bcl10 were excised from the gel, Downloaded from washed extensively with H2O, and dried in a Speed Vac (Heraeus). The 293T and Jurkat cells (J77 clone 20; a gift from O. Acuto, Pasteur Institute, proteins trapped in the gel slices were hydrolyzed in 1 ml of 6 M HCl for Paris, France) were grown as described previously (14, 25). The monocyte 2 h at 95°C under reduced pressure. Amino acids released from the gel cell line THP-1 (American Type Culture Collection) was grown in RPMI slices were recovered with the HCl and dried under vacuum. Samples were 1640 medium supplemented with 10% FCS at 37°C in 5% CO2. Human resuspended in 5 l of buffer 1 (8% acetic acid, 2% formic acid) supple- ϩ CD4 T cells were isolated from Sepacell RS-2000 filters (donated by the mented with 10 g each of phosphoserine, phosphothreonine, and phos- Centre de Transfusion Sanguine du Centre Hoˆpitalier Universitaire Vau- photyrosine and spotted on phosphocellulose TLC sheets (Sigma-Aldrich). dois) by Ficoll-Paque procedure (Pharmacia Biotech) and positive selec- Electrophoresis of the TLC sheets was performed in 8% acetic acid, 2% http://www.jimmunol.org/ tion with magnetic anti-human CD4 microbeads on miniMACS columns formic acid at 400 V for 105 min, and continued (same dimension) in 1% (Miltenyi Biotec). Mouse primary T cells were isolated from lymph nodes pyridine, 10% acetic acid at 400 V for 180 min. Phospho-amino acid stan- of Bcl10-deficient or littermate control mice (1) by negative selection using dards were visualized with ninhydrine. The radioactivity on the TLC sheet anti-B220-coated MACS beads (Miltenyi Biotec). Mice were bred in the was measured with a Phosphoimager (Storm; APB). animal facilities of the Technical University of Munich Medical School, and experiments with animals were conducted in accordance with the Two-dimesional analysis of phosphorylated Bcl10 German/institutional guidelines for animal care. Proteins were extracted from washed immunoprecipitates by 100 lof Activation of cells solubilization buffer S containing 9.0 M Urea, 4% (w/v) CHAPS (Sigma- Aldrich), 65 mM 1,4-dithio-DL-threitol, 0.8% (v/v) Resolytes 4–8 (BDH), For short-term activation, T cells were resuspended in RPMI 1640 at 107 4 mM Tris base (pH 10.5), and 0.001% (w/v) bromophenol blue and in- by guest on September 29, 2021 cells/200 l and stimulated during the indicated times using either PMA cubation at room temperature for 60 min, with vortexing every 10 min. The (10 ng/ml) and ionomycin (1 M), anti-human CD3 (10 g/ml OKT3; a beads were sedimented by centrifugation, and 60 l of the supernatant gift from S. Valitutti, Institut Claude de Pre´val, Toulouse, France), or anti- were loaded on 7-cm long immobilized pH gradient gel strips (pH 4–7) human CD3 (10 g/ml TR66; Apotech) together with anti-CD28 (10 (Amersham Biosciences) that had been rehydrated with buffer R (8.0 M g/ml CD28.2; Immunotech) followed by cross-linking with goat anti- Urea, 2% (w/v) CHAPS, 18 mM 1,4-dithio-DL-threitol, 0.8% (v/v) mouse IgG1 (5 g/ml; Southern Biotechnology Associates), or with the Resolytes 4–8, and 0.001% (w/v) bromophenol blue). In some experi- chemically synthesized chemokine CXCL12/SDF-1 (100 nM; a gift from I. ments, cells were directly lysed in buffer S (5 ϫ 106 cells/200 l) and 150 Clark-Lewis, Biomedical Research Center, University of British Columbia, l of sonicated and centrifuged extract was loaded on immobilized pH Vancouver, British Columbia, Canada). In some experiments, Jurkat cells gradient gel strips. Isoelectric focusing was performed at a maximum volt- -hour count of 35,000 was reached. Equilibraءwere preincubated with 500 nM GFX (bisindoleylmaleimide hydrochlo- age of 3,500 V, until a volt ride; Alexis) or solvent control (DMSO) in RPMI 1640 for 60 min before tion and transfer to the second dimension were done as described previ- stimulation. Murine T cells were stimulated with anti-mouse CD3 (10 ously (26). SDS-PAGE for the second dimension was performed on 11% g/ml 145-2C11; BD Pharmingen). polyacrylamide gels. For Fc␥R stimulation, THP-1 cells were resuspended in RPMI 1640 without serum at 106 cells/50 l. Upon addition of 50 l of IgG-SRBC at NF-B reporter assays (5 ϫ 106 cells/50 l), the cell mixture was pulse-centrifuged to favor For luciferase assays, Jurkat cells were cotransfected with 4 g of expres- cellular interactions and incubated for the indicated times at 37°C before sion vectors and 1 g of small interfering RNA (siRNA), as indicated, fixation and analysis of F-actin content (see below). together with 3 gofanNF-B luciferase reporter plasmid (NF-Bluc; a Expression vectors gift from V. Jongeneel, Institut Suisse de Bioinformatique, Lausanne, Switzerland) and the phRLTK Renilla luciferase reporter plasmid (1 g; Expression vectors for Bcl10, Carma1, and DN-IB␣ have been described Promega). Twenty-four hours after electroporation, cells were harvested previously (13, 14, 25). Ser/Ala point mutants of both human and murine and stimulated or not with PMA (10 ng/ml) and ionomycin (1 M) for 24 h Bcl10 were obtained by a standard double PCR approach and subcloning or with plate-bound anti-CD3 (OKT3, 10 g/ml) and anti-CD28 (CD28.2, of sequenced PCR products into expression vectors derived from pCR-3 10 g/ml) Ab for 10 h. After stimulation, cells were washed twice with (Invitrogen Life Technologies) to yield expression constructs with an N- PBS and lysed in 50 l of passive lysis buffer (Promega). Aliquots of cell terminal FLAG- or vesicular stomatitis virus glycoprotein tag. For lenti- lysates (10 l) were mixed with 50 l of dual luciferase assay reagent viral expression of Bcl10, constructs were subcloned into the lentiviral (Promega), and the luciferase activity was determined using a TD 20/20 vector pRDI_292 (a gift from R. Iggo, University of St. Andrews, Scot- luminometer (Turner Designs). land) that allows expression of constructs under the EF1 promoter. Transfection and transduction of Jurkat cells Immunoprecipitation and Western blotting Transient transfection of Jurkat cells (5 ϫ 106 cells/cuvette) with expres- Cell lysis, immunoprecipitation, phosphatase treatment, and Western blot- sion plasmids and/or synthetic siRNA was performed with the Nucleofec- ting were performed as described previously (14, 25). Five microliters of tor system (Amaxa; program O17) according to the manufacturer’s instruc- mouse anti-FLAG agarose (Sigma-Aldrich), 1 g of goat anti-Bcl10 Ab tions, yielding transfection efficiencies that were routinely between 60 and (N-20; Santa Cruz Biotechnology), or 1 g of anti-phosphotyrosine 70%. For transient gene silencing, siRNA specific for human Bcl10 (sc- (4G10; Upstate Biotechnology) previously bound to protein G Sepha- 29793; Santa Cruz Biotechnology) or negative control (nonsilencing) The Journal of Immunology 4375 siRNA (1022076; Qiagen) were used (1 g/cuvette). For stable siRNA expression, oligonucleotides encoding short hairpin RNAs (shRNAs) tar- geting nt 143–161 (GTAGAGAAGACACTGAAGA) of the human Bcl10 coding sequence, nt 457–475 of the human Carma1 5ЈUTR (GCTAT GATTTCTCTTGCAT), and nt 129–147 (GCAAACTTTCAGGACTT TGA) of the human Malt1 3ЈUTR were inserted into pSUPER. The Pol III promoter-shRNA cassettes from these vectors and from a lamin A/C-spe- cific pSUPER control construct were inserted into the lentiviral vector pAB286.1, a derivative of pHR (27) that contains a SV40-puromycin acetyl transferase cassette for antibiotic selection (lamin A/C-pSUPER and pAB286.1 were gifts from R. Iggo, University of St. Andrews, Scotland). Second generation packaging plasmids pMD2-VSVG and pCMV-R8.91 (27) were used for lentivirus production and infection as described else- where (www.tronolab.unige.ch). The lentiviral vector pRDI_292 (a gift from R. Iggo) was used to achieve stable expression of FLAG-tagged Bcl10 constructs in Jurkat cells. IL-2 assay Jurkat and Raji cells (0.75 ϫ 106 cells/0.5 ml each) were mixed at a ratio of 1:1 in the absence or presence of staphylococcal enterotoxin E. (SEE) (0.2 g/ml; Toxin Technology) in 24-well plates for 14 h at 37°C. The IL-2 concentration in the supernatants was determined by ELISA (R&D Sys- tems) according to the manufacturer’s instructions. Downloaded from Determination of cellular F-actin content Mouse primary T cells or Jurkat T cells (1 ϫ 106/100 l RPMI 1640) were incubated with or without 10 g/ml anti-CD3 (145-2C11 or OKT3) for the indicated times at 37°C. The reaction was terminated by addition of 400 l of 5% paraformaldehyde in PBS. Cells were fixed for 10 min at room temperature, blocked with 1% BSA in buffer A (PBS, 10 mM HEPES (pH http://www.jimmunol.org/ 7.3), and 0.5 mM EDTA), permeabilized with buffer B (0.1% saponin in buffer A containing 0.2% BSA), and stained with 2 g/ml FITC- or TRITC-conjugated phalloidin (Sigma-Aldrich) in buffer B. Cellular F-actin content was determined using a FACScan flow cytometer by gating on living cells based on their forward and side scatter. The relative F-actin content of the cells is proportional to the relative mean fluorescence intensity, which was determined as the ratio of the mean fluorescence in- tensity of each sample relative to the fluorescence intensity of unstimulated cells. In some experiments, actin polymerization was induced by transfec-
tion of enhanced GFP (EGFP)-tagged constitutively active mutants of Rac1 by guest on September 29, 2021 (Rac1.L61) and Cdc42 (Cdc42.L61) (28) into Jurkat cells using the Amaxa FIGURE 1. Ser138 and Ser170/171 are relevant for Bcl10 phosphorylation system, and relative F-actin content was determined 48 h after transfection ϫ by gating on EGFP-positive cells. in 293T cells. A, FLAG-tagged Bcl10 was immunoprecipitated from 3 105 in vivo 32P-labeled 293T cells, and the sample was analyzed by SDS- Spreading assay and quantification of T cell spreading PAGE and Western blot or autoradiography of the dried gel (left panels). Spreading assays were performed essentially as described by Bunnell et al. Radioactive protein eluted from the two P-low and P-high bands were (29). Briefly, 15-mm glass coverslips placed on 12-well plastic culture subjected to phospho-amino acid analysis (right panel). Samples were plates were treated with 0.01% poly-L-lysine solution (Sigma-Aldrich) for loaded at the bottom (Orig). Circles correspond to the positions where the 5 min at room temperature and then coated overnight at 4°C with PBS standards phosphoserine (pSer), phosphothreonine (pThr) and phosphoty- alone or with anti-CD3 Ab (OKT3) at 20 g/l. Wells were washed with rosine (pTyr) migrate. B, Schematic overview of the Ser residues present PBS before use to remove any excess of Ab, then 200 l of complete within the C-terminal portion of Bcl10 and of the clustered Ser to Ala medium was added to each well and equilibrated at 37°C with 5% CO2. mutations used in C. C, FLAG-tagged wt and indicated clustered Ser to Ala ϫ 6 Two hundred microliters of Jurkat cell suspension (at 2 10 cells/ml) point mutants of Bcl10 were immunoprecipitated from in vivo 32P-labeled were added into medium-containing plates. To increase the number of cells 293T cells, and immunoprecipitates were analyzed by SDS-PAGE fol- at the coverslip surface at initial time points, plates with cells were pulse- centrifuged up to 800 rpm, in a 37°C preheated centrifuge with plate adap- lowed by autoradiography or anti-FLAG Western blot. Arrowheads indi- tors. At time zero, just after pulse centrifugation, no spreading is observed, cate the positions of unphosphorylated and phosphorylated P-low and P- but enough cells attached to the coverslip for microscopy analysis. To high species of Bcl10 in the immunoprecipitates. The presence of an induce spreading, plates were incubated at 37°C for 3 min and fixed by additional phospho-Bcl10 species with intermediate migration behavior (P- removing 300 l of medium and gently adding 500 l of cold, freshly int) in the mutant B is indicated. Black lines indicate where irrelevant lanes prepared 5% paraformaldehyde in PBS. After 30 min, plates with cover- have been removed. D, Individual Ser to Ala point mutations were ana- slips were blocked with 1% BSA in buffer A (PBS, 10 mM HEPES (pH lyzed as in C. E, Cell lysates of Jurkat cells, stimulated as indicated, and of 7.3), and 0.5 mM EDTA), permeabilized with buffer B (0.1% saponin in FLAG-Bcl10-transfected 293T cells were analyzed by anti-Bcl10 Western buffer A containing 0.2% BSA), and stained with 2 g/ml FITC- or blot to allow a direct comparison of the apparent m.w. of Bcl10 phosphor- TRITC-conjugated phalloidin (Sigma-Aldrich) in buffer B to visualize the actin cytoskeleton. The coverslips were mounted in Fluorsafe (Calbio- ylation isoforms in T cells and Bcl10-overexpressing 293T cells. Efficient chem). Samples were examined under an Olympus IX70 inverted micro- T cell activation was monitored using anti-phospho-ERK. scope, and random fields were collected using a U-PlanApo objective (ϫ40/1.00 Oil Iris Ph3) and a cooled Sensi Cam 12 Bits camera (PCO- CCD Imaging). Images were acquired using Metamorph software and an- Conjugate formation alyzed using ImageJ National Institutes of Health freeware to measure the surface area of plate contact (spreading) of individual cells. Cells that had Jurkat and Raji cells were labeled with CFSE cell tracker (25 nM; Sigma- at least doubled the area of plate contact compared with the average of Aldrich) and CMTMR cell tracker (500 nM; Molecular Probes), respec- unstimulated cells were by morphological features always unequivocally tively, for 15 min at 37°C. Cells were then washed and incubated for 30 positive for spreading, so specific spreading (percentage) was defined as min at 37°C at a density of 106 cells/500 l. During this incubation, Raji 100 ϫ (number of cells with at least twice the average surface size of plate cells were incubated in the absence or presence of SEE superantigen (5 contact of control cells/number of total cells in the field). g/ml; Toxin Technology). Jurkat and Raji cells were then mixed at a ratio 4376 Bcl10 PHOSPHORYLATION AND ACTIN POLYMERIZATION Downloaded from http://www.jimmunol.org/
FIGURE 2. Bcl10 is phosphorylated on Ser138 in vivo after T cell activation. A, Bcl10 is modified upon T cell activation. Jurkat cells were stimulated with PMA and ionomycin for the indicated time, and postnuclear cell lysates were analyzed by SDS-PAGE and anti-Bcl10 Western blot. Within the first minutes of stimulation, Bcl10 isoforms appear that show a minimal retardation in migration. Prolonged stimulation (15 min and more) is necessary to induce a major shift in apparent m.w. that concerns a small fraction of Bcl10 and is thus visible only upon strong exposure of the blot. Cell lysates were blotted for phospho-ERK1/2 as a control for activation. B, Bcl10 is phosphorylated on two sites upon T cell activation. Bcl10 was immunoprecipitated from Jurkat cells stimulated for 15 min with or without PMA and ionomycin or cross-linked anti-CD3 and anti-CD28 Abs, respectively. Washed Bcl10 immunopre- cipitates were incubated with or without -phosphatase, as indicated, and analyzed by two-dimensional gel electrophoresis and anti-Bcl10 Western blot. by guest on September 29, 2021 C, Kinetics of Bcl10 phosphorylation. Jurkat cells were stimulated with anti-CD3 (OKT3) for the indicated time, and postnuclear cell lysates were analyzed by two-dimensional gel electrophoresis and anti-Bcl10 Western blot. D, Bcl10 phosphorylation occurs in primary T cells. Purified primary human CD4ϩ T cells (10 ϫ 106) were incubated for 15 min in the presence or absence of PMA and ionomycin or cross-linked anti-CD3 and anti-CD28 Abs, respectively, and cells were lysed and analyzed as in C. E, Mutation of Ser138 into Ala abolishes Bcl10 phosphorylation. Jurkat cells transfected with the indicated FLAG-Bcl10 constructs were stimulated with PMA and ionomycin for 20 min, and anti-FLAG immunoprecipitates were analyzed as in B. F, The pan-PKC inhibitor GFX partially inhibits Bcl10 phosphorylation. Jurkat cells were preincubated for 60 min in the presence of 500 nM GFX or solvent control, stimulated for 15 min with PMA and ionomycin, and phosphorylation of endogenous Bcl10 was assessed as in C. In all parts of the figure, arrowheads indicate the position of unphosphorylated Bcl10 (black arrowhead) and phosphorylated forms of Bcl10 (open arrowheads). of 1:1 (total volume 1 ml), centrifuged for 5 min at 1,000 ϫ g, and 850 l and parental nontransduced THP-1 cells showed similar phagocytosis ef- of medium was removed. Cell pellets were resuspended by vortexing for ficiencies (data not shown). 2 s and incubated for 15 min at 37°C. After gentle resuspension, cell con- jugates were fixed by adding 400 l of paraformaldehyde 5%. Conjugate Immunofluorescence microscopy of THP-1 cells formation was analyzed with a FACScan flow cytometer (BD Biosciences). The percentage of conjugates was determined as the number of Immunofluorescence, image acquisition, and deconvolution was performed (CFSEϩCMTMRϩ events/total number of CFSEϩ events) ϫ 100. as previously described (Braun et al. (30)), except that the samples were examined under an inverted wide-field microscope (Leica DMB) equipped Phagocytosis assay with an oil immersion objective (ϫ100 PL APO HCX, 1.4 NA) and a cooled CCD camera (MicroMAX; Princeton Instruments). Z-series of im- Preparation of IgG-coated SRBC (IgG-SRBC) and phagocytosis assays ages were taken at 0.2-m increments, and deconvolution was performed were performed as described previously (30), except that THP-1 cells were by the three-dimensional deconvolution module from Metamorph Software plated onto poly-L-lysine-coated coverslips before the incubation with op- (Universal Imaging). Three-dimensional reconstructions were obtained us- sonized SRBCs. To quantitate phagocytosis, the number of internalized ing the Surpass function of Imaris Software (Bitplane). SRBCs was counted in 50 cells randomly chosen on the coverslips, and the phagocytic index, i.e., the mean number of phagocytosed SRBCs per cell, was calculated. The index obtained was divided by the index obtained for Statistics control lamin-depleted cells and expressed as a percentage of control cells. The statistical significance of the data was tested with an unpaired Stu- We also counted the number of cell-associated (bound plus internalized) dent’s t test, and the calculated goodness-of-fit value ( p value) is indicated SRBCs, calculated the association index (mean number of associated in the figures. SRBCs per cell), and expressed it as percentage of control lamin-depleted cells. To quantitate polymerized actin recruitment, we scored the presence or absence of F-actin accumulations under the particles in 50 cells ran- Results domly chosen on the coverslips and calculated an accumulation index, i.e., Bcl10 is phosphorylated on Ser138 in activated T cells the mean number of accumulations per cell. The index obtained was di- vided by the index obtained for control (lamin-depleted) cells and ex- We had previously observed that ectopic expression of Bcl10 in pressed as a percentage of the latter. We checked that lamin-depleted cells 293T cells yielded a 30-kDa nonphosphorylated Bcl10 species and The Journal of Immunology 4377 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021
FIGURE 3. Impaired Ser138 phosphorylation of Bcl10 does not affect NF-B activation. A, Mutation of Ser138 does not affect PMA/ionomycin-induced T cell activation. Jurkat cells were transfected with a NF-B luciferase reporter plasmid, a TK-Renilla luciferase reporter plasmid, siRNA specific for human Bcl10 (Santa Cruz Biotechnology) or negative control (nonsilencing) siRNA (Qiagen), and the indicated mock (empty vector) or murine Bcl10 (mBcl10) expression constructs. Twenty-four hours after transfection, cells were treated or not with PMA/ionomycin for 24 h, and lysates were analyzed for NF-B-dependent luciferase activity using the dual luciferase assay system (Promega). Data shown are mean values Ϯ SEM from three independent experiments. The anti-Bcl10 Western blot is from one representative experiment. A nonspecific band (n.s.) that appeared on the same blot served as a loading control. Note that the Western blot assesses Bcl10 expression in the whole cell extract (comprising both transfected and untransfected cells), whereas luciferase activity is assessed only in the transfected cells. The partial reduction in Bcl10 expression in the total lysate is thus likely to underestimate the degree of Bcl10 silencing in the cells that were cotransfected with the reporter plasmids. B, Mutation of Ser138 does not affect anti-CD3/CD28-induced T cell activation. Jurkat cells were transfected as in A, stimulated with plate-bound anti-CD3 and anti-CD28 for 10 h, and lysates were analyzed for NF-B-dependent luciferase activity. Data shown are mean values Ϯ SEM from three independent experiments. C, Mutation of Ser138 does not affect PMA/iono-induced Bcl10 degradation. Lysates from Jurkat cells stably transduced with the indicated constructs were analyzed for protein expression by anti-Bcl10 (left panel) and for PMA/iono-induced Bcl10 degradation by anti-FLAG Western blot. Data shown are representative of three independent experiments. D, The Bcl10 S138A mutant does not affect IB phosphorylation and Bcl10 degradation. Jurkat cells stably transduced with the indicated FLAG-tagged Bcl10 constructs were stimulated with anti-CD3/CD28 for the indicated times, and lysates were analyzed for FLAG-Bcl10, IB phosphorylation (P-IB), and presence of activated ERK (P-ERK) and p38 (P-p38). A nonspecific band served as a loading control (n.s.). Data shown are representative of two independent experiments. E, Early Bcl10 phosphorylation is not affected by Carma1 silencing. Jurkat cells stably transduced with the indicated shRNA constructs were stimulated with PMA and ionomycin for the indicated times, and cell lysates were analyzed by Western blotting as indicated. Data shown are representative of three independent experiments. F, The Bcl10 S138A mutant is less efficient than wt Bcl10 in increasing superantigen-induced IL-2 production. Jurkat cells stably transduced with the indicated constructs were stimulated for 14 h with SEE-pulsed or unpulsed Raji cells, and the IL-2 concentration in the supernatant was determined by ELISA. Data shown are mean values Ϯ SEM of two independent experiments performed in duplicate. 4378 Bcl10 PHOSPHORYLATION AND ACTIN POLYMERIZATION
two major, more slowly migrating isoforms of Bcl10 that disap- peared upon phosphatase treatment (13, 14). Phosphorylation of Bcl10 was detectable by in vivo 32P-labeling of FLAG-Bcl10- transfected 293T cells and occurred exclusively on Ser residues (Fig. 1A). Analysis of various C-terminally truncated forms re- vealed that the observed Ser phosphorylation sites were present within aas 127 to 207 of the C-terminal portion of Bcl10 (data not shown). Clustered Ser to Ala point mutations within the C-terminal portion of Bcl10 revealed that Ser residues within a cluster com- prising Ser134, -136, and -138, and a second cluster comprising Ser167, -170, and -171 most strongly affected Bcl10 phosphorylation in the 293T cell system (Fig. 1, B and C). Ser to Ala point mutation of individual residues within these two clusters demonstrated that Ser138 is critical for the formation of the faster migrating phospho- Bcl10 species, whereas Ser170 and -171 contribute to the formation of the slower migrating species (Fig. 1D). Bcl10 is essential in the signaling pathway leading from TCR triggering to the activation of the transcription factor NF-B (8).
We therefore analyzed whether T cell activation induced phos- Downloaded from phorylation of endogenous Bcl10. Stimulation of Jurkat cells with PMA and ionomycin resulted in a minimal shift in the electro- phoretic mobility of endogenous Bcl10 within the first minutes of stimulation (Figs. 1E and 2A, see also Fig. 3, D and E). This min- imal shift in electrophoretic mobility was different from the mi-
gration pattern of Bcl10 obtained under conditions of overexpres- http://www.jimmunol.org/ sion in 293T cells, which induced a substantial shift in the apparent m.w. of Bcl10 (Fig. 1E). However, additional Bcl10 modifications that resulted in a substantial shift in electrophoretic mobility also became apparent upon prolonged stimulation of T cells (15 min FIGURE 4. Phosphorylation of Bcl10 on Ser138 is critical for TCR- and more) even though this concerned a very small fraction of total induced actin polymerization. A, Bcl10-deficient T cells show impaired Bcl10 (see Figs. 2A and 3E, strong exposures). The relation be- actin polymerization. Purified T cells from Bcl10-deficient mice and tween the altered migration of Bcl10 seen upon prolonged T cell control littermates were stimulated for 2 or 5 min with anti-CD3, and stimulation and the phosphorylated forms of Bcl10 observed in fixed and permeabilized cells were stained using FITC-conjugated phal- 293T cells is not fully understood. It is likely that in T cells, these loidin (2 g/ml) and analyzed for F-actin content by flow cytometry. by guest on September 29, 2021 later modifications correspond to IKK-dependent Bcl10 phos- The left panel shows the FACS profile from one experiment in which phorylation events that have been proposed recently to be associ- cells were stimulated for 2 min. Data shown in the graph (right panel) ated with a negative feedback regulation of the NF-B pathway are mean values Ϯ SEM from two independent experiments performed (18). Similar modifications resulting in substantial m.w. shifts have in duplicate. B, Bcl10 but not Carma1 is required for TCR-induced actin polymerization. Jurkat cells were stably transduced with lentiviral vec- also been observed by others (16, 18, 31, 32). tors expressing shRNA specific for human Bcl10, human Carma1, hu- A more detailed analysis of samples was performed by two- man Malt1, or human lamin A/C (control). Cells were stimulated with dimensional electrophoresis, which revealed the presence of two anti-CD3 for 1.5 min or 5 min and analyzed for F-actin content as in A. major Bcl10 isoforms with more acidic isoelectric point, but only Data shown are mean values Ϯ SEM of six (control cells), four (Bcl10- minimal changes in apparent m.w., upon stimulation with PMA/ silenced cells) and two (Carma1- and Malt1-silenced cells) independent ionomycin or anti-CD3/CD28 treatment (Fig. 2B, left panels). experiments performed in duplicate. C, Bcl10 is not required for che- These more acidic Bcl10 isoforms were strongly reduced by phos- mokine-induced actin polymerization. Jurkat cells stably transduced phatase treatment and therefore corresponded to phosphorylation with the indicated lentiviral vectors were stimulated with CXCL12/ isoforms of Bcl10 (Fig. 2B). Bcl10 phosphorylation could also be SDF-1 for 1.5 min and analyzed for F-actin content as in A. Data shown Ϯ induced by stimulation with anti-CD3 alone; it was detectable rap- are mean values SEM of two independent experiments performed in duplicate. Differences between cells transduced with Bcl10- and lamin- idly after the onset of stimulation and persisted for at least 60 min specific shRNA vectors were not significant (n.s.; p Ͼ 0.05). (Fig. 2C). Bcl10 phosphorylation was also observed in purified primary human T cells, upon either PMA/ionomycin or anti-CD3/ CD28 treatment (Fig. 2D). To examine which Ser residues were phosphorylated, Jurkat cells were transfected with FLAG-tagged ylation is critical for the subsequent phosphorylation of an addi- versions of wild-type (wt) Bcl10 or various Ser/Ala point mutants. tional residue. Preincubation of the cells with the pan-protein Analysis of FLAG-Bcl10 immunoprecipitates by two-dimensional kinase C (PKC) inhibitor GFX (bis-indolyl-maleimide) prevented electrophoresis and Western blotting revealed that PMA/ionomy- Bcl10 phosphorylation on this second, as yet unidentified site, cin treatment induced a shift in the electrophoretic mobility of whereas it did not affect the initial phosphorylation of Bcl10 on FLAG-Bcl10 that was similar to that observed for endogenous the site that is dependent on the intactness of the Ser residue Bcl10 (compare Fig. 2, B and E). Mutation of Ser138 to Ala abol- 138 (Fig. 2F).
ished formation of the most prominent phosphospecies, whereas 138 the corresponding mutation of Ser170 or -171 did not alter the PMA/ Ser -dependent Bcl10 phosphorylation is not required for ionomycin-induced Bcl10 phosphorylation pattern (Fig. 2E). NF- B activation These data suggest that Ser138 is a target of PMA/ionomycin-in- Bcl10 plays a key role in the Ag receptor-induced activation of the duced Bcl10 phosphorylation in T cells, and that Ser138 phosphor- transcription factor NF-B (1). Therefore, we tested the effect of The Journal of Immunology 4379
the Bcl10 Ser138 phosphorylation mutant on PMA/ionomycin or CD3/CD28-induced NF-B activation in Jurkat cells in a NF-B luciferase reporter gene assay. Transfection of the Ser138/Ala (S138A) mutant did not inhibit NF-B activation induced by PMA/ionomycin-treatment or Carma1 overexpression in Jurkat cells, whereas a dominant-negative (DN), nonphosphorylatable form of IB␣ impaired it under the same conditions (data not shown). To rule out the possibility that the Bcl10 S138A mutant had a recessive inhibitory effect on NF-B activation, we next analyzed its effect in Jurkat cells in which the expression of en- dogenous Bcl10 was silenced by the transfection of siRNA specific for human Bcl10 (Fig. 3, A and B). Reconstitution of the cells with either wt or S138A-mutated murine Bcl10 constructs (that are not targeted by the siRNA) restored PMA/ionomycin or CD3/CD28- induced NF-B activation to similar extents, suggesting that Ser138-dependent phosphorylation of Bcl10 was not necessary for NF-B activation (Fig. 3, A and B). Similar results were obtained using an IL-2 reporter construct (data not shown). Next, in Jurkat
cells stably transduced with the wt or S138A form of Bcl10 (Fig. Downloaded from 3C, left panel), we tested whether mutation of Ser138 either af- fected Bcl10’s stability or the cells’ capacity to induce IB phos- phorylation, an early event of NF-B activation. Using these cells, we found no difference in the PMA/ionomycin- or anti- CD3/CD28-induced degradation of the wt and the S138A con-
structs (Fig. 3, C, right panel, and D). Moreover, the cells showed http://www.jimmunol.org/ no difference in anti-CD3/CD28- or PMA/ionomycin-induced IB phosphorylation (Fig. 3D and data not shown). Other parameters of cellular activation, such as activation of the MAPKs ERK and p38, were also comparable in cells overexpressing the wt or S138A form of Bcl10 (Fig. 3D). Thus, phosphorylation on Ser138 is not
FIGURE 5. The Bcl10 Ser residue 138 is crucial for anti-CD3-induced actin polymerization and T cell spreading. A, Mutation of Ser138 affects by guest on September 29, 2021 TCR-induced actin polymerization. Jurkat cells were cotransfected with siRNA specific for human Bcl10 or negative control (nonsilencing) siRNA, together with the indicated murine Bcl10 expression constructs, and cells were analyzed for actin polymerization upon stimulation for 1.5 min with or without anti-CD3, as in Fig. 4B. Data shown are mean values Ϯ SEM of two independent experiments performed in duplicate. B, The pan-PKC inhibitor GFX does not affect TCR-induced actin polymerization. Jurkat cells were preincubated for 60 min with 500 nM GFX or solvent control, and anti-CD3-induced actin polymerization was analyzed upon stimulation for 1.5 min as in Fig. 4B. Data are representative of two independent experiments. C, Bcl10 is crucial for T cell spreading. Jurkat cells stably transduced with the indicated lentiviral shRNA constructs were plated on poly-L-lysine (a and c) or anti-CD3 Ab (OKT3)-coated coverslips (b and d), respectively, and fluorescence microscopy images were acquired after 3 min. Images are representative of three independent experiments. Bar, 10 m. D, Bcl10 Ser138 is crucial for T cell spreading. Jurkat cells stably transduced with the indicated expression constructs were analyzed as in C. Images are representative of two independent experiments. Bar, 10 m. E, Cell spreading was quantified using Image-J. Specific spreading (%) was determined as the percentage of cells with a plate contact surface area at time 3 min that is at least twice the average surface area of control cells (3 min on poly-L-Lys-coated plates), as indicated in Materials and Methods. For each experiment, three to five fields containing 30–80 cells were counted (a total of at least 200 cells/experiment). Data shown are mean values Ϯ SEM of two to four independent experiments. F, Bcl10 silencing affects conjugate formation. Jurkat cells lentivirally transduced with con- trol (lamin) shRNA, Bcl10-, or Carma1-specific shRNA were incubated for 5 min with SEE-pulsed Raji cells, and the formation of conjugates was determined by flow cytometry as described in Materials and Methods. FACS dot plot profiles are from one representative experiment of three performed. G, Quantification of conjugate formation. Shown are mean per- centages of T cells present in conjugates Ϯ SEM from two independent experiments. Similar results were obtained in one additional experiment. 4380 Bcl10 PHOSPHORYLATION AND ACTIN POLYMERIZATION required for Bcl10-mediated NF-B activation. These findings are consistent with the observation that a Bcl10 construct comprising aas 1–116 (comprising only the CARD and the subsequent Malt1 binding region) acts like full-length Bcl10 with respect to its abil- ity to activate NF-B and to synergize with PMA in NF-B in- duction (33). Moreover, this idea is consistent with the observation that Carma1 silencing did not affect early Bcl10 phosphorylation, whereas it clearly affected IB phosphorylation induced by PMA and ionomycin, as well as later Bcl10 modifications resulting in a more substantial shift in the apparent m.w. of Bcl10 (Fig. 3E). Finally, we also analyzed the effect of the Bcl10 phosphoryla- tion mutant on superantigen-induced IL-2 production. Jurkat cells stably transduced with the wt form of Bcl10 showed a strongly increased capacity to produce IL-2, whereas the Bcl10 S138A mu- tant was significantly less potent in increasing IL-2 production than the wt form of Bcl10 (Fig. 3F). Together, these findings sug- gest that the S138A mutant of Bcl10 affects functional T cell ac- tivation in a NF-B-independent manner. Downloaded from Bcl10 is essential for TCR-induced actin polymerization Bcl10 phosphorylation occurs rapidly after TCR triggering, sug- gesting that it may affect an early signaling event other than the activation of NF-B. One of the earliest events upon engagement of the TCR is the reorganization of the cytoskeleton, including the
rapid induction of actin polymerization (34). To test whether http://www.jimmunol.org/ Bcl10, and in particular its phosphorylation, plays a role in actin polymerization, primary purified T cells from Bcl10-deficient and control mice were stimulated with anti-CD3, and the resulting in- crease in F-actin was measured using fluorescently labeled phal- FIGURE 6. Bcl10 silencing does not affect Vav activation nor F- loidin. In the Bcl10ϩ/ϩ control cells, CD3 stimulation induced a actin induction by constitutively active forms of Cdc42 and Rac1. A, Vav phosphorylation is unaffected by Bcl10 silencing or by expression transient increase in the levels of F-actin, which was absent in the 138 Ϫ/Ϫ of a Ser /Ala mutant of Bcl10. Jurkat cells were incubated in the Bcl10 T cells (Fig. 4A). Similar results were obtained in Jurkat absence or presence of anti-CD3 Ab for the indicated times, and the cells in which Bcl10 expression was silenced by a lentiviral amount of Vav present in anti-phosphotyrosine immunoprecipitates or by guest on September 29, 2021 shRNA approach, whereas silencing of Carma1 or Malt1 expres- cell extracts was analyzed by Western blotting using anti-Vav Ab. Ef- sion had no significant effect on anti-CD3-induced actin polymer- ficient cellular activation was monitored by Western blotting of ex- ization (Fig. 4B). Silencing of Bcl10 expression did not reflect a tracts, using anti-phosphotyrosine and anti-phospho-ERK Ab, and the general defect in the generation of actin filaments, because actin expression level of endogenous and transfected Bcl10 was monitored polymerization induced by the chemokine CXCL12/SDF-1 via the using anti-Bcl10. B, Actin polymerization by constitutively active G protein-coupled receptor CXCR4 was not significantly affected forms of Cdc42 or Rac1 does not require Bcl10. Constitutively active by impaired Bcl10 expression (Fig. 4C). (CA) EGFP fusion constructs of Cdc42 and Rac1 were transfected into Jurkat cells. After 48 h, cells were fixed, labeled with TRITC-phalloi- Together, these results suggest that Bcl10 plays a key role in din, and F-actin levels were analyzed by flow cytometry, gating on TCR-induced actin polymerization, and that this function of Bcl10 EGFP-positive cells. Data are mean Ϯ SEM from three independent is independent of its association with Carma1 and Malt1. experiments. Differences between cells transduced with Bcl10- and lamin-specific shRNA vectors were not significant (n.s.; p Ͼ 0.05). Ser138 is crucial for Bcl10’s capacity to control F-actin formation and T cell spreading Next, we tested the relevance of Ser138 phosphorylation on TCR- countering the APC (36, 37). To this purpose, we used Jurkat cells induced F-actin formation. In contrast to the wt form of Bcl10, the lentivirally transduced with a Bcl10- or lamin-specific shRNA (see Ser138/Ala mutant was unable to restore anti-CD3-induced F-actin Fig. 4B). Although control cells showed strong anti-CD3- formation in cells with silenced Bcl10 expression (Fig. 5A). More- dependent spreading (Fig. 5C, compare panels a and b), almost no over, the mutant had a DN effect on F-actin generation (Fig. 5A). We spreading was observed for cells transduced with Bcl10 shRNA also tested the effect of the pan-PKC inhibitor GFX on TCR-induced (Fig. 5C, panels c and d). Quantification of the relative anti-CD3- actin polymerization. Consistent with the observation that the inhib- induced changes in cell spreading by measuring the plate surface itor did not affect Ser138-dependent Bcl10 phosphorylation (Fig. 2F), covered within the F-actin-positive cell boundary (see Materials we did not observe an inhibitory effect on F-actin formation (Fig. 5B). and Methods), showed that 3 min after onset of cell plating, 23% TCR-induced actin polymerization is important for various as- of the control cells showed an at least a 2-fold increase of their pects of T cell activation, including the generation of actin-rich spreading surface, compared with 1% of the cells transduced with plasma membrane protrusions and the formation of stable conju- Bcl10 shRNA (Fig. 5E). Ser138-dependent phosphorylation of gates with APCs (21, 35). To further address the functional rele- Bcl10 was relevant for this function, because transduction of the vance of Bcl10-dependent actin polymerization for these pro- cells with the S138A mutant, but not the wt form of Bcl10, inhib- cesses, we first tested whether Bcl10, and in particular the intact ited the cells’ capacity to spread (Fig. 5D, compare panels a and b Ser138 phosphorylation site, was necessary for the spreading of to c and d, and E). Finally, we also tested the effect of Bcl10 Jurkat T cells on anti-CD3-coated coverslips, a process that is silencing on conjugate formation between Jurkat cells and SEE- thought to mimic the cell surface expansion of T cells upon en- loaded Raji cells. Down-regulation of Bcl10, but not of Carma1, The Journal of Immunology 4381 Downloaded from http://www.jimmunol.org/
FIGURE 7. Bcl10 is critical for Fc␥R-mediated phagocytosis and normal actin cup formation. A, THP-1 cells transduced with the indicated shRNA constructs were lysed, and cell lysates or anti-Carma1 immunoprecipitates were analyzed by Western blotting. B, THP-1 cells transduced with the indicated shRNA constructs were left unstimulated (control) or incubated with IgG-SRBCs for the indicated times, cells were fixed, permeabilized, and stained using FITC-labeled phalloidin, and F-actin content was determined by flow cytometry. The mean Ϯ SEM of two independent experiments is plotted. Incubation of THP-1 cells with noncoated SRBCs did not induce an increase in F-actin content (data not shown).
C, THP-1 cells with silenced expression of lamin, Bcl10, or Carma1 were incubated with IgG-SRBCs for 3 min at 37°C. After fixation and staining by guest on September 29, 2021 of external IgG-SRBC with Cy3-anti-rabbit IgG Abs, cells were permeabilized and labeled with Alexa350-coupled phalloidin. Fifty cells were scored for association efficiency, and results are expressed as a percentage of control (lamin shRNA transduced) cells. The mean Ϯ SEM of four independent experiments is plotted. D, Cells were treated as described in C, except that phagocytosis was allowed to proceed for 60 min. The efficiency of phagocytosis was calculated on 50 cells. Results are expressed as a percentage of lamin shRNA-transduced control cells. Means Ϯ SEM of four independent experiments are plotted. E, Cells were treated as described in C, except that incubation with IgG-SRBC was performed for 10 min. Fifty cells were scored for the presence or absence of F-actin accumulation under bound particles. Results are expressed as a percentage of control lamin-depleted cells. Means Ϯ SEM of three independent experiments are plotted. F, Cells were treated as in D. Labeled cells were analyzed by wide-field microscopy with deconvolution. A projection of five (upper panels)orsix(lower panels) medial sections is shown. The stacks of images collected are presented as three-dimensional reconstructions in the right panels. F-actin was present at the site of particle ingestion in both lamin- and Bcl10-depleted cells, but actin cups were deeper in control lamin-depleted cells. Bar, 5 m. significantly impaired the cells’ capacity to form conjugates (Fig. 5, F rylated proteins were immunoprecipitated from lysates of control and G). Together, these results suggest that Bcl10, and in particular its cells and Bcl10-silenced cells, and analyzed by Western blotting Ser138-dependent phosphorylation, play critical roles for actin-depen- for Vav. In control cells, anti-CD3 stimulation induced a rapid and dent processes such as T cell spreading and conjugate formation. transient tyrosine phosphorylation of Vav, which was unaltered in Bcl10-silenced cells (Fig. 6A, left panel). Transduction of the cells Bcl10 depletion does not affect activation of Vav nor actin with the wt or Ser138/Ala mutant did not affect Vav phosphoryla- polymerization induced by Cdc42 or Rac1 tion either (Fig. 6A, right panel). Moreover, overexpression of The molecular control of actin-filament assembly and disassembly constitutively active forms of Cdc42 or Rac1 induced an increase upon receptor triggering is highly regulated and coordinated. The in F-actin content that was not significantly different in control Arp2/3 complex is a major regulator of actin filament nucleation cells and Bcl10-silenced cells (Fig. 6B). Together, these data sug- (38). The activity of the Arp2/3 complex is regulated by members gest that Bcl10 controls actin polymerization downstream or in- of the SCAR/WASP family that are in turn controlled by small dependently of Vav and upstream or independently of the Rho GTPases such as Cdc42 and Rac1 (24, 39). In T cells, the activa- family GTPases Cdc42 and Rac1. tion of Rac1 is controlled by the TCR-induced tyrosine phosphor- ␥ ylation of Vav, resulting in the stimulation of its GEF activity (34, Fc R-induced actin polymerization and phagocytosis depend 40, 41). Bcl10-deficient cells show a normal profile of total protein on Bcl10 tyrosine phosphorylation (1), but the phosphorylation of Vav has Recently, Bcl10 has been reported to play a key role in FcR- not been specifically addressed. To address the effect of Bcl10 induced NF-B activation (3). FcRs share a number of common silencing on the phosphorylation status of Vav, tyrosine-phospho- signaling features with the TCR, due to the presence of ITAMs that 4382 Bcl10 PHOSPHORYLATION AND ACTIN POLYMERIZATION
become phosphorylated by tyrosine kinases of the Src family, and The signaling cascades leading to actin polymerization down- allow the subsequent recruitment of Syk family kinases to activate stream of the TCR and the Fc␥R show striking similarities. Indeed, downstream signaling events via the phosphorylation of adaptor both involve Src- and Syk-family tyrosine kinases and small proteins and enzymes with various catalytic activities (42–45). GTPases such as Cdc42 and Rac that regulate Arp2/3-dependent Triggering of Fc␥R by immune complexes induces phagocytosis actin polymerization via protein complexes containing members of that depends on actin polymerization (43). To test whether Bcl10 the WASP/WAVE family (21, 34, 42–44). In T cells, the activity was essential for this process, lamin A/C, Bcl10, or Carma1 were of Rac GTPases is controlled by tyrosine phosphorylation and ac- silenced in the human THP-1 monocytes (Fig. 7A), and F-actin tivation of Vav family GEFs (34, 40–44, 48, 49). Whether Vav formation or phagocytosis was measured upon incubation of the plays a similar role downstream of the Fc␥R is controversial (50, cells with Ab-coated SRBC (IgG-SRBC) (Fig. 7, B–F). Silencing 51). In our hands, Bcl10 silencing had no effect on TCR-induced of Bcl10 strongly impaired the IgG-SRBC-induced increase in to- Vav phosphorylation, indicating that Bcl10 controls actin poly- tal F-actin content (Fig. 7B). Moreover, Bcl10- but not Carma1- merization downstream or independently of Vav. Moreover, the silenced monocytes showed an important decrease in phagocyto- lack of an effect of Bcl10 silencing on Cdc42 and Rac1-induced sis, whereas their capacity to associate with the IgG-SRBC was actin polymerization suggests that Bcl10 acts by targeting an un- comparable to control (lamin-silenced) cells (Fig. 7, C and D). known component of the signaling pathway that is upstream or When we analyzed early steps of phagocytosis, we observed that, independent of these small GTPases and common to both TCR- whereas Bcl10-silenced cells still formed F-actin cups at sites of and Fc␥R-induced actin polymerization. Another possibility is that particle attachment (Fig. 7E), the cells showed an incomplete en- Bcl10 phosphorylation affects the stability or stabilization of F- gulfment of the IgG-SRBC that was correlated with reduced size actin filaments rather than their initial formation. Indeed, a phys- Downloaded from of F-actin cups as illustrated on the three-dimensional reconstruc- ical association of Bcl10 with cytochalasin D-sensitive filaments tions (compare control and Bcl10-silenced cells in Fig. 7F). Thus, has been observed in HeLa cells when Bcl10 was overexpressed ␥ Bcl10 is crucial for Fc R-induced F-actin increase and the result- (52), a condition that favors constitutive Bcl10 phosphorylation. ing F-actin cup formation and phagocytosis. Moreover, in yeast two hybrid assays Bcl10 binds to ␣-actinin (52), an actin-binding protein that is thought to cross-link actin Discussion filaments and to link the actin fibrils to the cytoplasmic tail of http://www.jimmunol.org/ Receptor-induced remodeling of the actin cytoskeleton is a hall- certain transmembrane receptors (53). Although our data collec- mark of both engagement of lymphocyte Ag receptors and of tively suggest that direct phosphorylation of Ser138 is critical for its Fc␥Rs on phagocytes. In this study, we present several lines of role in the signaling pathway controlling actin polymerization, we evidence that support a previously unsuspected and physiologi- cannot formally exclude the possibility that mutation of Ser138 cally relevant role for Bcl10 in TCR- and Fc␥R-induced actin po- indirectly affects phosphorylation on another site by interfering lymerization. First, T cells with silenced Bcl10 expression showed with the recruitment of a kinase, or that the mutation has addi- impaired TCR-induced actin polymerization, which could be res- tional, phosphorylation-independent effects on the conformation cued by reconstitution with wt Bcl10. Second, Ser138-dependent
and molecular function of Bcl10 that may affect its interaction with by guest on September 29, 2021 phosphorylation of Bcl10 was critical for its capacity to control other components of the pathway. The functional connection of the actin polymerization, but did not affect its function in the NF-B Ser138-dependent phosphorylation of Bcl10 and the molecular ma- pathway. Third, a S138A mutant of Bcl10 was less potent than the chinery that polymerizes and stabilizes actin is currently under wt form in increasing superantigen-induced IL-2 production. Fourth, T cells with silenced expression of Carma1 or Malt1 investigation. showed no defect in TCR-induced actin polymerization, and si- A highly interesting question about the nature of the kinase- lencing of Carma1 did not affect initial Bcl10 phosphorylation, targeting Bcl10 remains open. The similarity of the phenotypes of suggesting that this function of Bcl10 is fully independent of its PKC and Bcl10-deficient mice (1, 54) together with the proposed binding partners in the NF-B pathway. Furthermore, Bcl10-si- role for PKC in the regulation of TCR-induced actin polymer- lenced T cells showed an impaired capacity to spread and to form ization (55), suggest a potential role for PKC in Bcl10 phosphor- conjugates with APCs. Finally, Bcl10 silencing led to an impaired ylation. However, the amino acid sequence of Bcl10 surrounding 138 Fc␥R-induced increase in F-actin content and altered formation of Ser does not match the described consensus motif for PKC- F-actin-dependent protrusions around the opsonized particle, re- dependent phosphorylation (56), and we and others were unable to sulting in defective phagocytosis. demonstrate a direct PKC -dependent phosphorylation of Bcl10 in The role of Bcl10 in immune receptor-induced actin polymeriza- in vitro kinase assays (Ref. 57 and our unpublished data). More- 138 tion has important functional implications. Bcl10-deficient mice show over, the pan-PKC inhibitor GFX did not affect the Ser -depen- impaired TCR-induced immune responses that have so far been at- dent initial phosphorylation of Bcl10 nor the TCR-induced F-actin tributed mainly to a lack of functional NF-B activation and IL-2 increase, but showed an inhibitory effect on a second, as yet un- production (1). Our findings suggest that the impaired T cell-depen- identified site of Bcl10 phosphorylation that is thus unlikely to be dent responses may, at least in part, be due to the impaired capacity of relevant for actin polymerization. The receptor-interacting protein the Bcl10-deficient cells to polymerize actin. This idea is consistent (RIP) family kinase RIP2 has been shown to associate with Bcl10 with our observation that T cell spreading and conjugate formation, and to induce its phosphorylation upon TCR stimulation in the both known to depend on TCR-induced actin polymerization (21, 34, context of NF-B activation (15). Additional experiments are 35, 46, 47), were affected in Bcl10-silenced cells. required to identify the RIP2-dependent phosphorylation site(s) Fc␥R-induced actin remodeling is essential for the internaliza- in Bcl10 and to assess whether RIP2-mediated Bcl10 phosphor- tion of opsonized micro-organisms or particles, and is thus crucial ylation contributes to the effect on the actin cytoskeleton for Ag capture and presentation by APCs (42–44). Our data iden- described here. tify a new role for Bcl10 as a key regulator of Fc␥R-induced Interestingly, Bcl10 appears to undergo two types of sequential phagocytosis in monocytes, and thus suggest that Bcl10 may also phosphorylation events that regulate distinct molecular functions be important for the activation of the adaptive immune system via of Bcl10. We propose that initial, rapidly occurring and Ser138- phagocytic cells. dependent phosphorylation of Bcl10 is crucial for its capacity to The Journal of Immunology 4383
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