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Proteomic Analysis of the SH2Domain-containing Leukocyte Protein of 76 kDa (SLP76) Interactome

2013; Elsevier BV; Volume: 12; Issue: 10 Linguagem: Inglês

10.1074/mcp.m112.025908

1535-9484

Yacine Bounab, Anne-Marie Hesse, Bruno Iannascoli, Luca Grieco, Yohann Couté, Anna Niarakis, Romain Roncagalli, Eunkyung Lie, Kong‐Peng Lam, Caroline Demangel, Denis Thieffry, Jérôme Garin, Bernard Malissen, Marc Daëron,

Cellular transport and secretion

We report the first proteomic analysis of the SLP76 interactome in resting and activated primary mouse mast cells. This was made possible by a novel genetic approach used for the first time here. It consists in generating knock-in mice that express signaling molecules bearing a C-terminal tag that has a high affinity for a streptavidin analog. Tagged molecules can be used as molecular baits to affinity-purify the molecular complex in which they are engaged, which can then be studied by mass spectrometry. We examined first SLP76 because, although this cytosolic adapter is critical for both T cell and mast cell activation, its role is well known in T cells but not in mast cells. Tagged SLP76 was expressed in physiological amounts and fully functional in mast cells. We unexpectedly found that SLP76 is exquisitely sensitive to mast cell granular proteases, that Zn2+-dependent metalloproteases are especially abundant in mast cells and that they were responsible for SLP76 degradation. Adding a Zn2+ chelator fully protected SLP76 in mast cell lysates, thereby enabling an efficient affinity-purification of this adapter with its partners. Label-free quantitative mass spectrometry analysis of affinity-purified SLP76 interactomes uncovered both partners already described in T cells and novel partners seen in mast cells only. Noticeably, molecules inducibly recruited in both cell types primarily concur to activation signals, whereas molecules recruited in activated mast cells only are mostly associated with inhibition signals. The transmembrane adapter LAT2, and the serine/threonine kinase with an exchange factor activity Bcr were the most recruited molecules. Biochemical and functional validations established the unexpected finding that Bcr is recruited by SLP76 and positively regulates antigen-induced mast cell activation. Knock-in mice expressing tagged molecules with a normal tissue distribution and expression therefore provide potent novel tools to investigate signalosomes and to uncover novel signaling molecules in mast cells. We report the first proteomic analysis of the SLP76 interactome in resting and activated primary mouse mast cells. This was made possible by a novel genetic approach used for the first time here. It consists in generating knock-in mice that express signaling molecules bearing a C-terminal tag that has a high affinity for a streptavidin analog. Tagged molecules can be used as molecular baits to affinity-purify the molecular complex in which they are engaged, which can then be studied by mass spectrometry. We examined first SLP76 because, although this cytosolic adapter is critical for both T cell and mast cell activation, its role is well known in T cells but not in mast cells. Tagged SLP76 was expressed in physiological amounts and fully functional in mast cells. We unexpectedly found that SLP76 is exquisitely sensitive to mast cell granular proteases, that Zn2+-dependent metalloproteases are especially abundant in mast cells and that they were responsible for SLP76 degradation. Adding a Zn2+ chelator fully protected SLP76 in mast cell lysates, thereby enabling an efficient affinity-purification of this adapter with its partners. Label-free quantitative mass spectrometry analysis of affinity-purified SLP76 interactomes uncovered both partners already described in T cells and novel partners seen in mast cells only. Noticeably, molecules inducibly recruited in both cell types primarily concur to activation signals, whereas molecules recruited in activated mast cells only are mostly associated with inhibition signals. The transmembrane adapter LAT2, and the serine/threonine kinase with an exchange factor activity Bcr were the most recruited molecules. Biochemical and functional validations established the unexpected finding that Bcr is recruited by SLP76 and positively regulates antigen-induced mast cell activation. Knock-in mice expressing tagged molecules with a normal tissue distribution and expression therefore provide potent novel tools to investigate signalosomes and to uncover novel signaling molecules in mast cells. Proteomic Analysis of the SH2 Domain-containing Leukocyte Protein of 76 kDa (SLP76) Interactome in Resting and Activated Primary Mast CellsMolecular & Cellular ProteomicsVol. 13Issue 2PreviewVol. 12 (2013) (2874–2889) Full-Text PDF Open Access Mast cells play critical roles in the initiation of IgE-dependent allergic inflammation (1Kinet J.P. The high-affinity IgE receptor (Fc epsilon RI): from physiology to pathology.Annu. Rev. Immunol. 1999; 17: 931-972Crossref PubMed Scopus (854) Google Scholar). They express high-affinity receptors for the Fc portion of IgE (FcεRI) 1The abbreviations used are:FcεRIhigh-affinity receptors for the Fc portion of IgEADAPadhesion- and degranulation-promoting adapter proteinBcrbreakpoint cluster region proteinBMMCBone marrow-derived mast cellsDAGdiacyl-glycerolDNPDinitrophenylESEmbryonic stemFDRfalse discovery rateFybFyn-binding proteinGrap2Grb2-related adapter protein 2Grb2growth factor receptor-bound protein 2HSAhuman serum albuminiBAQintensity-based absolute quantificationIP3inositol tris-phosphateITAMimmunoreceptor tyrosine-based activation motifItkIL-2-inducible T-cell kinaseKIknock-inLATlinker of activation of T cellsLCP2lymphocyte cytosolic protein 2LMlaurylmaltosidemAbmonoclonal antibodyMAPmitogen-activated proteinMS/MStandem mass spectrometrynanoLCnano liquid chromatographyNcknoncatalytic region of tyrosine kinase adapter proteinNTALnon-T cell activation linkerOSTone-strep-tagPAGphosphoprotein associated with glycosphingolipid-enriched microdomainsPLCphospholipase CSHSrc homologySHIPSH2 domain-containing inositol 5′-phosphataseSKAPSrc kinase-associated phopshoproteinsSLP76SH2 domain-containing leukocyte protein of 76 kDaSDSsodium dodecyl sulfateTCAtrichloroacetic acidTCRT cell receptorWTwild-typeZAP70zeta-associated protein of 70 kDa. 1The abbreviations used are:FcεRIhigh-affinity receptors for the Fc portion of IgEADAPadhesion- and degranulation-promoting adapter proteinBcrbreakpoint cluster region proteinBMMCBone marrow-derived mast cellsDAGdiacyl-glycerolDNPDinitrophenylESEmbryonic stemFDRfalse discovery rateFybFyn-binding proteinGrap2Grb2-related adapter protein 2Grb2growth factor receptor-bound protein 2HSAhuman serum albuminiBAQintensity-based absolute quantificationIP3inositol tris-phosphateITAMimmunoreceptor tyrosine-based activation motifItkIL-2-inducible T-cell kinaseKIknock-inLATlinker of activation of T cellsLCP2lymphocyte cytosolic protein 2LMlaurylmaltosidemAbmonoclonal antibodyMAPmitogen-activated proteinMS/MStandem mass spectrometrynanoLCnano liquid chromatographyNcknoncatalytic region of tyrosine kinase adapter proteinNTALnon-T cell activation linkerOSTone-strep-tagPAGphosphoprotein associated with glycosphingolipid-enriched microdomainsPLCphospholipase CSHSrc homologySHIPSH2 domain-containing inositol 5′-phosphataseSKAPSrc kinase-associated phopshoproteinsSLP76SH2 domain-containing leukocyte protein of 76 kDaSDSsodium dodecyl sulfateTCAtrichloroacetic acidTCRT cell receptorWTwild-typeZAP70zeta-associated protein of 70 kDa., which are prototypic immunoreceptors (2Reth M. Antigen receptor tail clue.Nature. 1989; 338: 383-384Crossref PubMed Scopus (1164) Google Scholar). Mast cell FcεRI are composed of an IgE-binding subunit, FcεRIα, and of two Immunoreceptor Tyrosine-based Activation Motif (ITAM)-containing subunits, FcRβ and FcRγ (3Blank U. Ra C. Miller L. White K. Metzger H. Kinet J.P. Complete structure and expression in transfected cells of high affinity IgE receptor.Nature. 1989; 337: 187-189Crossref PubMed Scopus (396) Google Scholar). FcεRI activate mast cells when receptor-bound IgE antibodies are aggregated by a multivalent specific antigen (4Turner H. Kinet J.P. Signalling through the high-affinity IgE receptor Fc epsilonRI.Nature. 1999; 402: B24-B30Crossref PubMed Scopus (619) Google Scholar). FcεRI aggregation triggers the constitution of signalosomes in which positive and negative signals are generated, the integration of which determines quantitatively and qualitatively the biological responses of the mast cell. The composition of signalosomes is likely to evolve rapidly, as molecules are sequentially recruited and as enzymes act on their substrates. Determining the composition and describing the dynamics of FcεRI signalosomes is a major challenge for who aims at understanding fundamental mechanisms of allergy and at developing therapeutic tools for controlling allergic reactions. high-affinity receptors for the Fc portion of IgE adhesion- and degranulation-promoting adapter protein breakpoint cluster region protein Bone marrow-derived mast cells diacyl-glycerol Dinitrophenyl Embryonic stem false discovery rate Fyn-binding protein Grb2-related adapter protein 2 growth factor receptor-bound protein 2 human serum albumin intensity-based absolute quantification inositol tris-phosphate immunoreceptor tyrosine-based activation motif IL-2-inducible T-cell kinase knock-in linker of activation of T cells lymphocyte cytosolic protein 2 laurylmaltoside monoclonal antibody mitogen-activated protein tandem mass spectrometry nano liquid chromatography noncatalytic region of tyrosine kinase adapter protein non-T cell activation linker one-strep-tag phosphoprotein associated with glycosphingolipid-enriched microdomains phospholipase C Src homology SH2 domain-containing inositol 5′-phosphatase Src kinase-associated phopshoproteins SH2 domain-containing leukocyte protein of 76 kDa sodium dodecyl sulfate trichloroacetic acid T cell receptor wild-type zeta-associated protein of 70 kDa. high-affinity receptors for the Fc portion of IgE adhesion- and degranulation-promoting adapter protein breakpoint cluster region protein Bone marrow-derived mast cells diacyl-glycerol Dinitrophenyl Embryonic stem false discovery rate Fyn-binding protein Grb2-related adapter protein 2 growth factor receptor-bound protein 2 human serum albumin intensity-based absolute quantification inositol tris-phosphate immunoreceptor tyrosine-based activation motif IL-2-inducible T-cell kinase knock-in linker of activation of T cells lymphocyte cytosolic protein 2 laurylmaltoside monoclonal antibody mitogen-activated protein tandem mass spectrometry nano liquid chromatography noncatalytic region of tyrosine kinase adapter protein non-T cell activation linker one-strep-tag phosphoprotein associated with glycosphingolipid-enriched microdomains phospholipase C Src homology SH2 domain-containing inositol 5′-phosphatase Src kinase-associated phopshoproteins SH2 domain-containing leukocyte protein of 76 kDa sodium dodecyl sulfate trichloroacetic acid T cell receptor wild-type zeta-associated protein of 70 kDa. Mass spectrometry (MS)-based proteomics has emerged as a powerful tool to study signaling networks. Indeed, it enables large-scale analysis of stimulus-induced post-translational modifications (5Sardiu M.E. Washburn M.P. Building protein-protein interaction networks with proteomics and informatics tools.J. Biol. Chem. 2011; 286: 23645-23651Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 6Bensimon A. Heck A.J. Aebersold R. Mass spectrometry-based proteomics and network biology.Ann. Rev. Biochem. 2012; 81: 379-405Crossref PubMed Scopus (317) Google Scholar, 7Choudhary C. Mann M. Decoding signalling networks by mass spectrometry-based proteomics.Nat. Rev. 2010; 11: 427-439Crossref Scopus (479) Google Scholar). MS-based proteomics has also been used to identify molecular partners that, at any given time, are associated with a molecular bait of interest (8Collins M.O. Choudhary J.S. Mapping multiprotein complexes by affinity purification and mass spectrometry.Curr. Opin. Biotechnol. 2008; 19: 324-330Crossref PubMed Scopus (102) Google Scholar). This bait carries an affinity-purification tag that markedly enhances the efficiency with which it can be purified from a cell lysate (9Rigaut G. Shevchenko A. Rutz B. Wilm M. Mann M. Seraphin B. A generic protein purification method for protein complex characterization and proteome exploration.Nat. Biotechnol. 1999; 17: 1030-1032Crossref PubMed Scopus (2285) Google Scholar). The experimental conditions used may, however, limit the significance of the result. Classically, baits are over-expressed in cells that already express an untagged endogenous version of the bait. Unbalanced expression of corresponding molecules may profoundly alter biological responses. In some cases, baits are expressed in cells that do not normally express the molecule, where they may generate artifactual signalosomes. Finally, baits are often expressed in transformed cells that can be grown in high numbers, so that affinity-purified molecules are obtained in amounts amenable to MS analysis (10Daulat A.M. Maurice P. Froment C. Guillaume J.L. Broussard C. Monsarrat B. Delagrange P. Jockers R. Purification and identification of G protein-coupled receptor protein complexes under native conditions.Mol. Cell. Proteomics. 2007; 6: 835-844Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 11Oellerich T. Bremes V. Neumann K. Bohnenberger H. Dittmann K. Hsiao H.H. Engelke M. Schnyder T. Batista F.D. Urlaub H. Wienands J. The B-cell antigen receptor signals through a preformed transducer module of SLP65 and CIN85.EMBO J. 2011; 30: 3620-3634Crossref PubMed Scopus (53) Google Scholar). Signaling pathways can be constitutively activated in these cells, because of the expression of transforming oncogenes. To overcome these problems, we generated a series of knock-in (KI) mice expressing each a key signaling molecule with a C-terminal one-strep-tag (OST) (12Schmidt T.G. Skerra A. The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins.Nat Protoc. 2007; 2: 1528-1535Crossref PubMed Scopus (454) Google Scholar). OST-tagged molecules, as well as the molecules with which they interact can be affinity-purified using beads coated with Strep-Tactin. Strep-Tactin is a streptavidin derivative that has a high affinity for OST (12Schmidt T.G. Skerra A. The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins.Nat Protoc. 2007; 2: 1528-1535Crossref PubMed Scopus (454) Google Scholar). Affinity-purified molecules can then be identified by MS. As documented here, this novel approach enables one to study the interactome of endogenous OST-tagged molecules that are present in physiological amounts, in nontransformed cells by which they are normally expressed. Combining the interactomes of each tagged molecule analyzed before and at different times after FcεRI engagement should ultimately make it possible to obtain a comprehensive dynamic functional map of the FcεRI signalosome. As a proof-of-concept, we studied the interactome of the Src Homology (SH)2 domain-containing leukocyte protein of 76 kDa (SLP76) (a.k.a. Lymphocyte cytosolic protein 2 or LCP2) in primary mouse mast cells. SLP76 is a cytosolic adapter that nucleates signaling complexes generated by immunoreceptors (13Jackman J.K. Motto D.G. Sun Q. Tanemoto M. Turck C.W. Peltz G.A. Koretzky G.A. Findell P.R. Molecular cloning of SLP-76, a 76-kDa tyrosine phosphoprotein associated with Grb2 in T cells.J. Biol. Chem. 1995; 270: 7029-7032Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). It contains an N-terminal leucine Zip motif, a tyrosine-rich domain, a proline-rich domain and a C-terminal SH2 domain. It is constitutively associated with the growth factor receptor-bound protein 2 (Grb2)-related adapter protein 2 (Grap2, a.k.a. GADS) through the interaction of its proline residues with one of the SH3 domains of Grap2 (14Liu S.K. Fang N. Koretzky G.A. McGlade C.J. The hematopoietic-specific adaptor protein gads functions in T-cell signaling via interactions with the SLP-76 and LAT adaptors.Curr. Biol. 1999; 9: 67-75Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). SLP76 was mostly studied in T lymphocytes. Following T-cell receptor (TCR) engagement, Grap2 binds to phosphorylated residues in the raft-associated transmembrane adapter linker of activation of T cells LAT1. SLP76-Grap2 complexes bind to phospholipase C (PLC)γ-1 (15Yablonski D. Kadlecek T. Weiss A. Identification of a phospholipase C-gamma1 (PLC-gamma1) SH3 domain-binding site in SLP-76 required for T-cell receptor-mediated activation of PLC-gamma1 and NFAT.Mol. Cell. Biol. 2001; 21: 4208-4218Crossref PubMed Scopus (168) Google Scholar), which stabilizes the recruitment of this enzyme by LAT1. SLP76 tyrosines are phosphorylated by the cytosolic kinase zeta-associated protein of 70 kDa (ZAP70) (16Bubeck Wardenburg J. Fu C. Jackman J.K. Flotow H. Wilkinson S.E. Williams D.H. Johnson R. Kong G. Chan A.C. Findell P.R. Phosphorylation of SLP-76 by the ZAP-70 protein-tyrosine kinase is required for T-cell receptor function.J. Biol. Chem. 1996; 271: 19641-19644Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 17Raab M. da Silva A.J. Findell P.R. Rudd C.E. Regulation of Vav-SLP-76 binding by ZAP-70 and its relevance to TCR zeta/CD3 induction of interleukin-2.Immunity. 1997; 6: 155-164Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar), which provides binding sites for the SH2 domains of Tec kinases such as the IL-2-inducible T-cell kinase (Itk)(18Su Y.W. Zhang Y. Schweikert J. Koretzky G.A. Reth M. Wienands J. Interaction of SLP adaptors with the SH2 domain of Tec family kinases.Eur. J. Immunol. 1999; 29: 3702-3711Crossref PubMed Scopus (199) Google Scholar), for the guanine nucleotide exchange factors Vav-1 (17Raab M. da Silva A.J. Findell P.R. Rudd C.E. Regulation of Vav-SLP-76 binding by ZAP-70 and its relevance to TCR zeta/CD3 induction of interleukin-2.Immunity. 1997; 6: 155-164Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar) and for adapters such as the noncatalytic region of tyrosine kinase adapter protein (Nck) (19Wunderlich L. Farago A. Downward J. Buday L. Association of Nck with tyrosine-phosphorylated SLP-76 in activated T lymphocytes.Eur. J. Immunol. 1999; 29: 1068-1075Crossref PubMed Google Scholar). Molecules recruited by SLP76 launch the intracellular propagation of signals leading to calcium mobilization, the activation of Mitogen-activated protein (MAP) kinases and, ultimately, full-blown T-cell responses. SLP76 is also expressed by mast cells, and mast cell activation was markedly impaired in SLP76-deficient mice (20Pivniouk V.I. Martin T.R. Lu-Kuo J.M. Katz H.R. Oettgen H.C. Geha R.S. SLP-76 deficiency impairs signaling via the high-affinity IgE receptor in mast cells.J. Clin. Invest. 1999; 103: 1737-1743PubMed Google Scholar). Surprisingly, signaling molecules that interact with SLP76 in mast cells are not well characterized, and only a few papers have been published on the subject. It was however shown that the function of SLP76 in mast cells may not depend entirely on LAT1, as it does in T cells. Indeed, LAT1 deficiency could be, at least in part, compensated by the presence of LAT2 in mast cells (21Kambayashi T. Okumura M. Baker R.G. Hsu C.J. Baumgart T. Zhang W. Koretzky G.A. Independent and cooperative roles of adaptor molecules in proximal signaling during FcepsilonRI-mediated mast cell activation.Mol Cell. Biol. 2010; 30: 4188-4196Crossref PubMed Scopus (15) Google Scholar). LAT2 (a.k.a. non-T-cell activation linker or NTAL) is a raft-associated tyrosine-rich transmembrane adapter related to LAT1. LAT2 is absent in resting T cells, but present in B cells where it fulfills some of the functions exerted by LAT1 in T cells. In mast cells, LAT1 and LAT2 function as a pair of antagonistic molecules (22Roget K. Malissen M. Malbec O. Malissen B. Daeron M. Non-T cell activation linker promotes mast cell survival by dampening the recruitment of SHIP1 by linker for activation of T cells.J. Immunol. 2008; 180: 3689-3698Crossref PubMed Scopus (26) Google Scholar). The role of SLP76 in mast cells cannot therefore be deduced from that of SLP76 in T cells, and the mechanism(s) by which it contributes to mast cell activation needs to be established. We report here the first description of the SLP76 interactome in resting and activated primary cultured mast cells. This was done by combining high-affinity purification and label-free quantitative proteomics in mast cells derived from KI mice expressing SLP-76 with a C-terminal OST (Slp76OST/OST mice). To reach this goal, we first unraveled and solved an unexpected problem because of proteases contained in high amounts in mast cell granules. We found that, compared with the T-cell SLP76 interactome, the mast cell SLP76 interactome is enriched in molecules involved in negative signaling. We also found that the Breakpoint Cluster Region protein Bcr is inducibly recruited by SLP76 and we could validate this unexpected finding by showing that mast cells from Bcr-deficient mice released less granular mediators than mast cells from wt mice. This is the first evidence that Bcr contributes to FcεRI signaling. A 6.2-kb genomic fragment containing exons 19–21 of the Slp76 gene was isolated from a BAC clone (clone # RP23–216O16A; http://www.lifesciences.sourcebioscience) of C57BL/6J origin. A OST-(Stop)2loxP-tACE-CRE-PGK-gb2-neo-loxP cassette (23Mingueneau M. Roncagalli R. Gregoire C. Kissenpfennig A. Miazek A. Archambaud C. Wang Y. Perrin P. Bertosio E. Sansoni A. Richelme S. Locksley R.M. Aguado E. Malissen M. Malissen B. Loss of the LAT adaptor converts antigen-responsive T cells into pathogenic effectors that function independently of the T cell receptor.Immunity. 2009; 31: 197-208Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) was introduced at the 3′ end of the Slp76 coding sequence found in exon 21 using homologous recombination (24Zhang Y. Muyrers J.P. Testa G. Stewart A.F. DNA cloning by homologous recombination in Escherichia coli.Nat. Biotechnol. 2000; 18: 1314-1317Crossref PubMed Scopus (212) Google Scholar). The targeting construct was then abutted to a thymidine kinase expression cassette and linearized with FseI. JM8.F6 C57BL/6N ES cells (25Pettitt S.J. Liang Q. Rairdan X.Y. Moran J.L. Prosser H.M. Beier D.R. Lloyd K.C. Bradley A. Skarnes W.C. Agouti C57BL/6N embryonic stem cells for mouse genetic resources.Nat. Methods. 2009; 6: 493-495Crossref PubMed Scopus (267) Google Scholar) were electroporated with the Slp76OST targeting vector and selected in G418 and gancyclovir. Colonies were screened for homologous recombination by Southern blot. A neomycin-specific probe was used to ensure that adventitious non-homologous recombination events had not occurred in the selected ES clones. Appropriately targeted ES cells were injected into FVB blastocysts. After germline transmission, screening for the deletion of the neo cassette and the presence of the intended modification was performed by PCR and Southern blot. All experiments were done in accordance with French and European guidelines for animal care. Bone marrow-derived mast cells (BMMC): BMMC were generated from femoral bone marrow harvested from Slp76OST/OST and C57BL/6 (Slp76+/+) control mice (Charles River, France), from Bcr−/− and littermate control (Bcr+/+) mice (a gift from Dr. Eunjoon Kim, Korea Advanced Institute of Science and Technology, Daejeon, South Korea), and from Dok-3−/− and littermate control (Dok-3+/+) mice. BMMC were propagated and cultured in OptiMEM (Invitrogen, St Aubin, France) supplemented with 10% FBS (PAA Laboratories, Austria), 50 μm 2-mercaptoethanol (Invitrogen), 100 μg/ml Penicillin-Streptomycin (Invitrogen), and 2 ng/ml recombinant murine IL-3 (Biolegend, San Diego, CA). Homogeneous populations of FcεRI+, Kit+ cells were obtained after 3 weeks of culture. Thymocytes: Thymi were collected from 6-week old C57BL/6 mice. Red cells were lysed in 10 mm Tris-HCL pH 7.5 and 142 mm NH4Cl. Three independent cultures were seeded from the bone marrow of three individual mice and BMMC were generated as described. Total RNA was extracted from resting BMMC from the three replicate cultures using TRIzol reagent (Invitrogen, Invitrogen), and RNeasy mini kit from Qiagen (Courtaboeuf, France). Purified total RNA was amplified by RT-PCR, and transcripts were analyzed using the Applied Biosystems AB1700 microarrays (Carlsbad, California). We estimated the number of proteases expressed in BMMC, according to the procedure described in Supplementary Materials. A total of 539 out of the 685 annotated protease-coding transcripts were found to be represented in the AB1700 microarrays. Among them, 400 genes were found expressed in BMMC with a confidence level greater than 90% (Fig. 3A). Supplemental Table S2 reports the results for the expression of the 539 protease-coding genes covered by the AB1700 microarrays. BMMC were sensitized overnight with IgE anti-DNP (mAb 2682-I) supernatant in complete culture medium (100 ng/ml IgE, final concentration). Cells were extensively washed with HEPES-containing RPMI (Invitrogen), and challenged for various periods of time at 37 °C with 100 ng/ml DNP-HSA (Sigma-Aldrich, St. Louis, MO) in the same medium. IgE anti-DNP-sensitized cells were challenged for 20 min at 37 °C with the indicated concentrations of DNP-HSA. β-hexosaminidase released in cell supernatants was measured using an enzymatic assay (26Malbec O. Malissen M. Isnardi I. Lesourne R. Mura A.M. Fridman W.H. Malissen B. Daeron M. Linker for activation of T cells integrates positive and negative signaling in mast cells.J. Immunol. 2004; 173: 5086-5094Crossref PubMed Scopus (44) Google Scholar). Laurylmaltoside (LM) lysates: BMMC or thymocytes were solubilized on ice for 10 min in lysis buffer consisting of 100 mm Tris-HCL, pH 7.4 or pH 8.0, 150 mm NaCl, 2 mm MgCl2, 5% glycerol, 0.2% LM (Thermo Scientific, Courtaboeuf, France), and 25 U/ml Benzonase (Merck, Darmstadt, Germany), supplemented with specific protease inhibitors (supplemental Table S4) or without. Protease inhibitor cocktails were from Roche (Mannheim, Germany, Cat. #11 697 498 001), and from Sigma-Aldrich (Cat. #P8340. Phosphoramidon (Cat. #R7385), EDTA and EGTA were from Sigma-Aldrich. SDS lysates: BMMC or thymocytes were lysed by boiling at 90 °C for 5 min in 1% SDS and 100 mm Tris-HCL pH 8.0. 25 U/ml Benzonase and 2 mm MgCl2 was added to SDS lysate that was left on ice for 10 min. LM and SDS cell lysates were centrifuged at 14,000 rpm for 15 min at 4 °C and supernatants were electrophoresed on 4–12% Criterion XT precast gel (Bio-Rad, Marnes-la-Coquette, France) using XT MOPS (Bio-Rad) running buffer, and transferred onto nitrocellulose Hybond-P membranes (Amersham Biosciences, Velizy-Villacoublay, France). Membranes were saturated either with 5% BSA (Sigma-Aldrich) or 5% skimmed milk (Régilait, Saint-Martin-Belle-Roche, France) for 1 h at room temperature and blotted overnight at 4 °C with the indicated antibodies. Rabbit anti-SLP76, anti-Akt, anti-Erk1/2, anti-phospho-Erk1/2, anti-phospho-p38 and mouse anti-phospho-Tyrosine (P-Tyr-100) antibodies were from Cell Signaling Technology (St-Quentin-en-Yvelines, France). Rabbit anti-LAT1 and anti-phospho-LAT1, mouse anti-Vav, rabbit anti-Sos antibodies were from Upstate Biotechnology (Lake placid, NY). Goat anti-actin, mouse anti-PLCγ-1, anti-Dok-3 and anti-SHIP1, Rabbit anti-Fyn, anti-Bcr, anti-phopsho-Bcr (pY177), anti-Lyn and anti-phospho-PLCγ-1 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse anti-LAT2 antibodies were from Alexis Biochemical (Lausanne, Switzerland). Rabbit anti-PAG antibodies were from ExBio (Praha, Czech Republic). Mouse anti-Grb2 and anti-phospho SLP76 antibodies were from BD Bioscience (Le-Pont-de-Claix, France). Goat anti-Dok-3 antibody was from Abcam (Paris, France). HRP-conjugated secondary antibodies were from Santa Cruz Biotechnology. Mouse anti-phosphotyrosine antibodies (mAb 4G10) were a gift from Dr. S. Latour (Inserm U.768, Hôpital Necker, Paris, France). HRP was detected using an ECL kit (Thermo Scientific). Four independent affinity purifications were performed on each set of samples (Slp76+/+ and Slp76OST/OST BMMC challenged with antigen or without). One × 108 Slp76+/+ or Slp76OST/OST BMMC were lysed for 10 min at 0 °C with buffer containing 100 mm Tris-HCL, pH 8, 150 mm NaCl, 2 mm MgCl2, 5% glycerol, 0.2% LM, 1 mm sodium orthovanadate, 5 mm sodium floride, 25 U/ml Benzonase and mast cells complete inhibitor mixture (supplemental Table S4). Cell extracts were centrifuged at 14,000 rpm for 15 min at 4 °C. Cleared lysates were incubated with 200 μl prewashed Strep-Tactin Sepharose beads (Iba BioTagnologies, Goettingen, Germany) on a wheel for 1 h at 4 °C. Beads were washed twice in 1 ml LM lysis buffer containing protease and phosphatase inhibitors and three times with 1 ml LM buffer without inhibitors. Beads were eluted 4 times with 150 μl of 2.5 mm d-Biotin (Sigma-Aldrich). Eluates were pooled and precipitated with TCA/Acetone. LM lysates of Slp76+/+ BMMC were incubated for 2 h at 4 °C with protein-A Sepharose beads (GE Healthcare BioScience, Sweden) coated with anti-Bcr or anti-Dok-3 antibodies. After washing with lysis buffer, the beads were boiled at 90 °C with loading buffer for 5 min. Eluates were electrophoresed, and Western blotted with anti-SLP76, anti-Bcr, anti-Dok-3, or anti-pY antibodies. (1) TCA precipitation. After elution, 125 μl of ice-cold TCA (Sigma-Aldrich) was added (final concentration 20%) to each sample and the mixture was incubated 30 min on ice and centrifuged at 20,000 × g for 20 min at 4 °C. The supernatant was removed and 600 μl of TCA 10% was added to wash the pellet. The samples were centrifuged as above. The supernatant was removed and 1 ml of ice-cold acetone (Sigma-Aldrich) was added to wash the pellet. The samples were centrifuged as above. The acetone-containing supernatan