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Co-Recruitment Analysis of the CBL and CBLB Signalosomes in Primary

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Co-recruitment analysis of the CBL and CBLB signalosomes in primary T cells identifies CD5 as a key regulator of TCR-induced ubiquitylation Guillaume Voisinne, Antonio García-Blesa, Karima Chaoui, Frédéric Fiore, Elise Bergot, Laura Girard, Marie Malissen, Odile Burlet-Schiltz, Anne Gonzalez de Peredo, Bernard Malissen, et al. To cite this version: Guillaume Voisinne, Antonio García-Blesa, Karima Chaoui, Frédéric Fiore, Elise Bergot, et al.. Co- recruitment analysis of the CBL and CBLB signalosomes in primary T cells identifies CD5 as a key regulator of TCR-induced ubiquitylation. Molecular Systems Biology, EMBO Press, 2016, 12, pp.876. 10.15252/msb.20166837. inserm-01368500 HAL Id: inserm-01368500 https://www.hal.inserm.fr/inserm-01368500 Submitted on 19 Sep 2016 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Published online: July 29, 2016 Article Co-recruitment analysis of the CBL and CBLB signalosomes in primary T cells identifies CD5 as a key regulator of TCR-induced ubiquitylation Guillaume Voisinne1,†, Antonio García-Blesa1,†, Karima Chaoui2, Frédéric Fiore3, Elise Bergot1, Laura Girard1,3, Marie Malissen1,3, Odile Burlet-Schiltz2, Anne Gonzalez de Peredo2, Bernard Malissen1,3,* & Romain Roncagalli1,** Abstract (interactomes) and of signaling complexes (signalosomes) in their physiological cellular context (Gingras et al, 2007; Hein et al, T-cell receptor (TCR) signaling is essential for the function of T cells 2015). Although such large-scale studies have resulted in compre- and negatively regulated by the E3 ubiquitin–protein ligases CBL hensive views of several signalosomes, they provided limited and CBLB. Here, we combined mouse genetics and affinity purifica- information on their internal organization and dynamics of assem- tion coupled to quantitative mass spectrometry to monitor the bly. Such information is, however, particularly valuable to under- dynamics of the CBL and CBLB signaling complexes that assemble stand how the flow of information resulting from engagement of a in normal T cells over 600 seconds of TCR stimulation. We identify given cell surface receptor propagates over time (Collins et al, most previously known CBL and CBLB interacting partners, as well 2013; Zheng et al, 2013). The T-cell antigen receptors (TCRs) as a majority of proteins that have not yet been implicated in found at the surface of T cells carry out the formidable task of those signaling complexes. We exploit correlations in protein asso- identifying minute number of antigenic peptides. Despite a wealth ciation with CBL and CBLB as a function of time of TCR stimulation of information concerning the dozens of molecules involved in for predicting the occurrence of direct physical association TCR signaling, the general principle of information processing between them. By combining co-recruitment analysis with by the TCR signal transduction network remains incompletely biochemical analysis, we demonstrated that the CD5 transmem- understood (Chakraborty & Weiss, 2014; Malissen & Bongrand, 2015). brane receptor constitutes a key scaffold for CBL- and CBLB- TCR signals propagate through the cytosol and the nucleus via a mediated ubiquitylation following TCR engagement. Our results multilayered signal transduction network involving PPI, phosphory- offer an integrated view of the CBL and CBLB signaling complexes lation, and ubiquitylation. Regulatory mechanisms limit and termi- induced by TCR stimulation and provide a molecular basis for their nate TCR-driven signals allowing the immune system to return to a negative regulatory function in normal T cells. basal state after antigen has been cleared (Acuto et al, 2008). The E3 ubiquitin–protein ligases CBL and CBLB have been recognized as Keywords CBL; CBLB; CD5; ubiquitylation important negative regulators of T-cell activation (Huang & Gu, Subject Categories Immunology; Post-translational Modifications, 2008). Following TCR engagement, the protein tyrosine kinases (PTK) Proteolysis & Proteomics; Signal Transduction LCK and ZAP-70 are activated and phosphorylate several trans- DOI 10.15252/msb.20166837 | Received 26 January 2016 | Revised 14 June membrane and cytosolic proteins, leading in turn to the recruitment of 2016 | Accepted 23 June 2016 CBL and CBLB via their tyrosine kinase binding (TKB) domains or Mol Syst Biol. (2016) 12: 876 indirectly via the GRB2 adaptor. As a result, CBL and CBLB are rapidly phosphorylated on tyrosine residues, causing conformational changes allowing the recruitment of E2 ubiquitin-conjugating enzymes and Introduction their juxtaposition to substrates that include the TCR itself, scaffold proteins (also known as adaptors), cytosolic PTK, and phosphatases Affinity purification followed by mass spectrometry (AP-MS) (Schmidt & Dikic, 2005). Modification of the TCR and of its signaling enables analysis of protein–protein interaction (PPI) networks partners with ubiquitin promotes their sorting to multivesicular 1 Centre d’Immunologie de Marseille-Luminy, Aix Marseille Université, Inserm, CNRS, Marseille, France 2 Institut de Pharmacologie et de Biologie Structurale, Département Biologie Structural Biophysique, Protéomique Génopole Toulouse Midi Pyrénées, CNRS UMR 5089, Toulouse Cedex, France 3 Centre d’Immunophénomique, Aix Marseille Université UM2, Inserm US012, CNRS UMS3367, Marseille, France *Corresponding author. Tel: +33 491269478; Fax: +33 491269430; E-mail: [email protected] **Corresponding author. Tel: +33 491269478; Fax: +33 491269430; E-mail: [email protected] †These authors contributed equally to this work ª 2016 The Authors. Published under the terms of the CC BY 4.0 license Molecular Systems Biology 12: 876 | 2016 1 Published online: July 29, 2016 Molecular Systems Biology Assembly dynamics of the CBL and CBLB signalosomes Guillaume Voisinne et al bodies and lysosomal degradation, thereby ensuring termination of we generated two lines of gene-targeted mice expressing One- TCR signaling (Vardhana et al, 2010). CBL and CBLB are more than STrEP-tag (OST) (Junttila et al, 2005) at the carboxyl-terminus of just E3 ubiquitin–protein ligases and possess numerous interactive endogenous CBL and CBLB proteins (Fig EV1A and B). Analysis of elements that nucleate the formation of complexes thought to mice homozygous for the CblOST allele (also known as B6-Cbltm1Mal; regulate cytoskeletal rearrangement (Lee & Tsygankov, 2013). These called “CBLOST” mice here) and for the CblbOST allele (also known interactive elements comprise a proline-rich region that binds SH3 as B6-Cblbtm1Ciphe; called “CBLBOST” mice here) showed that their T domain-containing proteins, multiple tyrosine phosphorylation sites cells developed properly and that they contained normal numbers enabling interactions with SH2 domain-containing proteins, and a of CD4+ and CD8+ T cells (Fig EV1C–F). CD4+ T cells purified ubiquitin association (UBA) domain. from wild-type, CBLOST, and CBLBOST mice and stimulated with Although the presence of several conserved structural domains anti-CD3 antibodies in the presence or absence of anti-CD28 anti- in CBL and CBLB suggests that they exert redundant functions, the bodies showed that expression of the CBL-OST and CBLB-OST mature T cells of mice deprived of CBL or CBLB showed distinct proteins had no detrimental effect on their proliferation (Fig EV1G phenotypes. Mature T cells from CBL-deficient mice are less respon- and H) or production of interleukin 2 (Fig EV1I and J). Immunoblot sive to antigenic stimulation (Naramura et al, 2002). In contrast, analysis showed that before and after activation with anti-CD3 and CBLB deficiency lowers the threshold that needs to be overcome by anti-CD4 antibodies, CBL-OST and CBLB-OST proteins can be puri- antigenic stimulation to trigger T-cell responses (Bachmaier et al, fied from lysates of CBLOST and CBLBOST CD4+ T cells, respectively, 2000; Chiang et al, 2000). As a result, CBLB-deficient T cells prolif- using Sepharose beads coupled to Strep-Tactin, a streptavidin erate and synthesize cytokines following stimulation with subopti- derivative that binds with high affinity to the OST sequence mal concentration of anti-TCR antibody or in the absence of CD28 (Fig 1A). As expected, no detectable material was recovered from costimulation, and in turn, CBLB-deficient mice are prone to wild-type CD4+ T cells. Therefore, primary CD4+ T cells from autoimmunity (Gronski et al, 2004; Chiang et al, 2007; Teh et al, CBLOST and CBLBOST mice are normal and can be used in AP-MS 2010; Zhou et al, 2014). CBLB-deficient T cells have increased analysis. anti-tumor efficacy, suggesting that CBLB might be exploited as a drug target for the purpose of boosting T-cell responses against tumors (Hinterleitner et al, 2012). A Pull-Down B Immature thymocytes Here, we combined mouse genetics and time-resolved, quantita- WT CBLOST CBLBOST tive proteomics analysis to compare the mode of action of CBL and CD3+CD4: -+ -+-+ WT CBLB following TCR-mediated activation of mature T cells. Consid- kDa CBLBOST ering that the transformed T-cell lines
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