SLP-65 Phosphorylation Dynamics Reveals a Functional Basis for Signal Integration by Receptor-proximal Adaptor Proteins
2009; Elsevier BV; Volume: 8; Issue: 7 Linguagem: Inglês
10.1074/mcp.m800567-mcp200
ISSN1535-9484
AutoresThomas Oellerich, Mads Grønborg, Konstantin Neumann, He‐Hsuan Hsiao, Henning Urlaub, Jürgen Wienands,
Tópico(s)Neurobiology and Insect Physiology Research
ResumoUnderstanding intracellular signal transduction by cell surface receptors requires information about the precise order of relevant modifications on the early transducer elements. Here we introduce the B cell line DT40 and its genetically engineered variants as a model system to determine and functionally characterize post-translational protein modifications in general. This is accomplished by a customized strategy that combines mass spectrometric analyses of protein modifications with subsequent mutational studies. When applied to the B cell receptor (BCR)-proximal effector SLP-65, this approach uncovered a differential and highly dynamic engagement of numerous newly identified phospho-acceptor sites. Some of them serve as kinase substrates in resting cells and undergo rapid dephosphorylation upon BCR ligation. Stimulationinduced phosphorylation of SLP-65 can be early and transient, or early and sustained, or late. Functional elucidation of conspicuous phosphorylation at serine 170 in SLP-65 revealed a BCR-distal checkpoint for some but not all possible B cell responses. Our data show that SLP-65 phosphorylation acts upstream for signal initiation and also downstream during selective processing of the BCR signal. Such a phenomenon defines a receptor-specific signal integrator. Understanding intracellular signal transduction by cell surface receptors requires information about the precise order of relevant modifications on the early transducer elements. Here we introduce the B cell line DT40 and its genetically engineered variants as a model system to determine and functionally characterize post-translational protein modifications in general. This is accomplished by a customized strategy that combines mass spectrometric analyses of protein modifications with subsequent mutational studies. When applied to the B cell receptor (BCR)-proximal effector SLP-65, this approach uncovered a differential and highly dynamic engagement of numerous newly identified phospho-acceptor sites. Some of them serve as kinase substrates in resting cells and undergo rapid dephosphorylation upon BCR ligation. Stimulationinduced phosphorylation of SLP-65 can be early and transient, or early and sustained, or late. Functional elucidation of conspicuous phosphorylation at serine 170 in SLP-65 revealed a BCR-distal checkpoint for some but not all possible B cell responses. Our data show that SLP-65 phosphorylation acts upstream for signal initiation and also downstream during selective processing of the BCR signal. Such a phenomenon defines a receptor-specific signal integrator. Cell surface receptors regulate multiple and overlapping sets of intracellular signaling proteins. These effector molecules can be structurally organized into distinct signaling cascades, which act in concert to coordinate precise cellular responses following receptor engagement (1Pawson T. Dynamic control of signaling by modular adaptor proteins.Curr. Opin. Cell Biol. 2007; 19: 112-116Crossref PubMed Scopus (150) Google Scholar, 2Abram C.L. Lowell C.A. The expanding role for ITAM-based signaling pathways in immune cells.Sci. STKE. 2007; 2007: 377Crossref Scopus (97) Google Scholar). Immediate early reactions include reorganization of the actin cytoskeleton associated with changes in cell morphology and migration (3Harwood N.E. Batista F.D. New insights into the early molecular events underlying B cell activation.Immunity. 2008; 28: 609-619Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 4Le Clainche C. Carlier M.F. Regulation of actin assembly associated with protrusion and adhesion in cell migration.Physiol. 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As a consequence, intracellular signal transduction is not linear, i.e. one receptor-specific adaptor can simultaneously control different positive as well as negative signaling cascades. The molecular basis for the pleiotropic yet specific processing of signals is still poorly understood. The multimeric antigen receptors on B and T lymphocytes utilize adaptors called SLP 1The abbreviations used are:SLPSrc homology 2 domain-containing leukocyte proteinsSHSrc homologyBCRB cell receptorPLCphospholipase CMAPmitogen-activated proteinErkextracellular signal-regulated kinaseJNKc-Jun NH2-terminal kinaseAP1activator protein-1MSmass spectrometrySILACstable isotope labeling by amino acids in cell cultureGFPgreen fluorescent proteinwtwild-typeACNacetonitrileEGFPenhanced green fluorescent protein1Done-dimensionalFCSfetal calf serum1The abbreviations used are:SLPSrc homology 2 domain-containing leukocyte proteinsSHSrc homologyBCRB cell receptorPLCphospholipase CMAPmitogen-activated proteinErkextracellular signal-regulated kinaseJNKc-Jun NH2-terminal kinaseAP1activator protein-1MSmass spectrometrySILACstable isotope labeling by amino acids in cell cultureGFPgreen fluorescent proteinwtwild-typeACNacetonitrileEGFPenhanced green fluorescent protein1Done-dimensionalFCSfetal calf serum (Src homology (SH) 2 domain-containing leukocyte proteins) (11Wu J.N. Koretzky G.A. The SLP-76 family of adapter proteins.Semin Immunol. 2004; 16: 379-393Crossref PubMed Scopus (71) Google Scholar). B cells express the 65 kDa family member SLP-65 (12Wienands J. Schweikert J. Wollscheid B. Jumaa H. Nielsen P.J. Reth M. SLP-65: a new signaling component in B lymphocytes which requires expression of the antigen receptor for phosphorylation.J. Exp. Med. 1998; 188: 791-795Crossref PubMed Scopus (234) Google Scholar), (also named BLNK (13Fu C. Turck C.W. Kurosaki T. Chan A.C. BLNK: a central linker protein in B cell activation.Immunity. 1998; 9: 93-103Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar) or BASH (14Goitsuka R. Fujimura Y. Mamada H. Umeda A. Morimura T. Uetsuka K. Doi K. Tsuji S. Kitamura D. BASH, a novel signaling molecule preferentially expressed in B cells of the bursa of Fabricius.J. 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The dual role of SLP-65 for the development and activation of B cells demonstrates a remarkable plasticity of the BCR signaling machinery (20Engelke M. Engels N. Dittmann K. Stork B. Wienands J. Ca(2+) signaling in antigen receptor-activated B lymphocytes.Immunol. Rev. 2007; 218: 235-246Crossref PubMed Scopus (71) Google Scholar). The underlying molecular details, which allow BCR signal modulation in a differentiation stage-specific manner, are unknown. Src homology 2 domain-containing leukocyte proteins Src homology B cell receptor phospholipase C mitogen-activated protein extracellular signal-regulated kinase c-Jun NH2-terminal kinase activator protein-1 mass spectrometry stable isotope labeling by amino acids in cell culture green fluorescent protein wild-type acetonitrile enhanced green fluorescent protein one-dimensional fetal calf serum Src homology 2 domain-containing leukocyte proteins Src homology B cell receptor phospholipase C mitogen-activated protein extracellular signal-regulated kinase c-Jun NH2-terminal kinase activator protein-1 mass spectrometry stable isotope labeling by amino acids in cell culture green fluorescent protein wild-type acetonitrile enhanced green fluorescent protein one-dimensional fetal calf serum A key event for the activation of peripheral B cells is the BCR-induced tyrosine phosphorylation of SLP-65. This enables SLP-65 to nucleate the formation of a multiprotein complex by recruiting several SH2 domain-containing effector proteins such as phospholipase (PLC)-γ2 and Bruton's tyrosine kinase (21Chiu C.W. Dalton M. Ishiai M. Kurosaki T. Chan A.C. BLNK: molecular scaffolding through 'cis'-mediated organization of signaling proteins.EMBO J. 2002; 21: 6461-6472Crossref PubMed Scopus (103) Google Scholar). SLP-65 not only assembles this signalosome but is also critical for its stimulation-induced translocation from the cytosol to the plasma membrane (22Kohler F. Storch B. Kulathu Y. Herzog S. Kuppig S. Reth M. Jumaa H. A leucine zipper in the N terminus confers membrane association to SLP-65.Nat. Immunol. 2005; 6: 204-210Crossref PubMed Scopus (46) Google Scholar, 23Abudula A. Grabbe A. Brechmann M. Polaschegg C. Herrmann N. Goldbeck I. Dittmann K. Wienands J. 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Involvement of guanosine triphosphatases and phospholipase C-gamma2 in extracellular signal-regulated kinase, c-Jun NH2-terminal kinase, and p38 mitogen-activated protein kinase activation by the B cell antigen receptor.J. Exp. Med. 1998; 188: 1287-1295Crossref PubMed Scopus (183) Google Scholar). A prominent MAP kinase activation target is the transcription factor activator protein-1 (AP1), which is a heterodimer of c-Fos and c-Jun proto-oncoproteins (28Foletta V.C. Segal D.H. Cohen D.R. Transcriptional regulation in the immune system: all roads lead to AP-1.J. Leukoc. Biol. 1998; 63: 139-152Crossref PubMed Scopus (312) Google Scholar). Hence, tyrosine phosphorylation of SLP-65 provides a single trigger for a series of canonical and lymphocyte-specific signaling pathways. More complex levels of regulation must however exist to fulfill the different BCR signaling requirements during the development of B cells on the one hand and their activation upon antigen encounter in secondary lymph organs on the other hand. Indeed, recent studies showed that SLP-65 is capable of regulating MAP kinase activity in an inositol triphosphate/diacylglycerol-independent manner (25Ishiai M. Kurosaki M. Pappu R. Okawa K. Ronko I. Fu C. Shibata M. Iwamatsu A. Chan A.C. Kurosaki T. BLNK required for coupling Syk to PLC gamma 2 and Rac1-JNK in B cells.Immunity. 1999; 10: 117-125Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 29Grabbe A. Wienands J. Human SLP-65 isoforms contribute differently to activation and apoptosis of B lymphocytes.Blood. 2006; 108: 3761-3768Crossref PubMed Scopus (16) Google Scholar). These data suggest that phosphotyrosine-dependent and phosphotyrosine-independent processes cooperate to modulate SLP-65 signal output for early and late activation events. A likely mode of signal adjustment is phosphorylation of SLP-65 on serine and threonine residues as this has been reported to regulate adaptor protein function in several signaling pathways including T cell receptor-mediated signaling (30Di Bartolo V. Montagne B. Salek M. Jungwirth B. Carrette F. Fourtane J. Sol-Foulon N. Michel F. Schwartz O. Lehmann W.D. Acuto O. A novel pathway down-modulating T cell activation involves HPK-1-dependent recruitment of 14–3-3 proteins on SLP-76.J. Exp. Med. 2007; 204: 681-691Crossref PubMed Scopus (68) Google Scholar). Moreover, the majority of putative phospho-acceptor sites within SLP-65 are serine and threonine residues, but their role in BCR signaling has not yet been explored. A molecular understanding of the complete SLP-65 network thus requires a comprehensive and kinetic analysis of its phosphorylation states in resting and stimulated B cells by functional proteomic approaches. The qualitative and quantitative analyses of protein phosphorylation cycles has been greatly improved by the introduction of enrichment techniques for phosphorylated peptides such as titanium dioxide (TiO2)-based purification, which combined with subsequent state-of-the-art mass spectometry (MS) have paved the way for rapid and reliable phosphoproteome analyses (31Pinkse M.W. Uitto P.M. Hilhorst M.J. Ooms B. Heck A.J. Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns.Anal. Chem. 2004; 76: 3935-3943Crossref PubMed Scopus (827) Google Scholar, 32Larsen M.R. Thingholm T.E. Jensen O.N. Roepstorff P. Jørgensen T.J. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns.Mol. Cell. Proteomics. 2005; 4: 873-886Abstract Full Text Full Text PDF PubMed Scopus (1335) Google Scholar). The additional implementation of stable isotope labeling by amino acids in cell culture (SILAC) allowed a more detailed view on differential phosphorylation events and their time-dependent dynamics (33Ong S.E. Blagoev B. Kratchmarova I. Kristensen D.B. Steen H. Pandey A. Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.Mol. Cell. Proteomics. 2002; 1: 376-386Abstract Full Text Full Text PDF PubMed Scopus (4576) Google Scholar, 34Olsen J.V. Blagoev B. Gnad F. Macek B. Kumar C. Mortensen P. Mann M. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.Cell. 2006; 127: 635-648Abstract Full Text Full Text PDF PubMed Scopus (2822) Google Scholar). However, the functional phosphoproteomic analysis of a given effector protein, such as SLP-65, is hampered by two obstacles. First, signaling molecules, especially eukaryotic proteins, are often difficult to enrich because of limited physiological sources. Second, experimental systems to assess the functional relevance of post-translational protein modifications are almost lacking. We have now employed the DT40 B cell reconstitution system (35Buerstedde J.M. Takeda S. Increased ratio of targeted to random integration after transfection of chicken B cell lines.Cell. 1991; 67: 179-188Abstract Full Text PDF PubMed Scopus (483) Google Scholar) to purify wild-type SLP-65 in large amounts, to subject it to qualitative and quantitative MS-based phosphoproteomic analysis and lastly, to test in vivo the importance of individual phosphorylation sites for distinct signaling pathways by site-directed mutagenesis. This comprehensive approach has identified SLP-65 as one of the most phosphorylated proteins known so far. It has also revealed an unexpected complexity in the phosphorylation kinetics of distinct phospho-acceptor sites, which together with a functional crosstalk between tyrosine and serine/threonine phosphorylation events fine-tunes MAP kinase and AP1 activation. Our data help to explain how SLP-65 can act as a BCR-proximal master activator for many signaling pathways while retaining the possibility of signal modulation. Furthermore, these results demonstrate that the DT40 system is a useful tool for combined proteomic and functional analyses. Wild-type chicken DT40 cells and the SLP-65-deficient mutant (kindly provided by T. Kurosaki, Yokohama, Japan) were cultured in RPMI 1640 supplemented with 10% fetal calf serum, 1% chicken serum, 3 mm l-glutamine, and antibiotics. SLP-65-deficient DT40 cells are described by Ishiai et al. (25Ishiai M. Kurosaki M. Pappu R. Okawa K. Ronko I. Fu C. Shibata M. Iwamatsu A. Chan A.C. Kurosaki T. BLNK required for coupling Syk to PLC gamma 2 and Rac1-JNK in B cells.Immunity. 1999; 10: 117-125Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). BCR stimulation of the cells was performed with 2 µg/ml anti-chicken IgM (M4, Southern Biotechnology, BioMol, Hamburg, Germany). For cell lysis 1% Nonidet P-40 buffer containing 50 mm Tris/HCl, pH 7.8, 150 mm NaCl, 0.5 mm EDTA, 2 mm Na3VO4, 1 mm NaF, 10% glycerol, and protease inhibitors P2714 (Sigma-Aldrich) was used. SLP-65 was immunoprecipitated from lysates of 2 × 107 cells with either anti-chicken SLP-65 (kindly provided by T. Kurosaki) or anti-GFP (Roche) antibodies immobilized by protein A/G-Sepharose (Pierce) according to the manufacturer's instructions. Antibodies for immunoblotting are specific for phosphotyrosine (4G10; BioMol), Erk (BD Biosciences), p38 (Cell Signaling Technology), or actin (Sigma-Aldrich). Phosphosite-specific antibodies recognizing phospho-Erk, phospho-p38, phospho-JNK, or phospho-14–3-3-binding sites were purchased from Cell Signaling Technology. The cDNA encoding chicken SLP-65 with an N-terminal One-STrEP-Tag (Iba BioTAGnology, Göttingen, Germany) was cloned into pAbes-puro (kindly provided by M. Reth, Freiburg, Germany). The resulting expression plasmid was introduced into SLP-65-deficient DT40 B cells by electroporation (300 V, 975 µF). Transfectants were selected in the presence of 1 µg/ml puromycin, and protein expression was confirmed by immunoblot analysis. The cDNA encoding N-terminally EGFP-tagged chSLP-65 was inserted into pCRII-Topo vector. Site-directed mutagenesis (QuikChange) was used to generate the S170A and S173A mutants of SLP-65. cDNAs encoding EGFP fusion proteins encompassing either wild-type or mutant SLP-65 (EGFP-SLP-65-wt, EGFP-SLP-65-S170A, or -S173A, respectively) were inserted into pMSCV, and SLP-65-deficient B cells were retrovirally transduced as described (36Stork B. Engelke M. Frey J. Horejsí V. Hamm-Baarke A. Schraven B. Kurosaki T. Wienands J. Grb2 and the non-T cell activation linker NTAL constitute a Ca(2+)-regulating signal circuit in B lymphocytes.Immunity. 2004; 21: 681-691Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). SILAC was performed by culturing DT40 cells in RPMI 1640 medium devoid of arginine and lysine (Pierce) supplemented with 10% dialyzed FCS (Invitrogen), 1 mm pyruvate, and 4 mm glutamine. "Heavy", "intermediate", and "light" medium were prepared by adding 0.115 mm 13C615N4 l-arginine and 0.275 mm 13C615N2 l-lysine (Sigma Isotec), or equimolar levels of 13C614N4 l-arginine and l-lysine-4,4,5,5-D4 (Cambridge Isotopes), or the corresponding non-labeled amino acids (Sigma-Aldrich), respectively. For affinity purification of One-STrEP-tagged chSLP-65 labeled with either heavy, intermediate, or light isotopes, 108 DT40 B cell transfectants from the respective cultures were left untreated or BCR-stimulated and lysed. Following determination of protein concentrations (37Boeri Erba E. Matthiesen R. Bunkenborg J. Schulze W.X. Di Stefano P. Cabodi S. Tarone G. Defilippi P. Jensen O.N. Quantitation of multisite EGF receptor phosphorylation using mass spectrometry and a novel normalization approach.J. Proteome Res. 2007; 6: 2768-2785Crossref PubMed Scopus (27) Google Scholar) lysates were pooled at a 1:1:1 ratio and incubated with 200 µl of Strep-Tactin Superflow matrix (Iba BioTAGnology) for 1 h at 4 °C. The immobilized chSLP-65 was eluted at room temperature with 500 µl of D-Desthiobiotin elution buffer (Iba BioTAGnology) and concentrated with ultrafiltration spin columns (Sartorius, Göttingen, Germany). Phosphopeptides were enriched by TiO2 chromatography (GL Sciences Inc., Tokyo, Japan) as described by Larsen et al. (32Larsen M.R. Thingholm T.E. Jensen O.N. Roepstorff P. Jørgensen T.J. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns.Mol. Cell. Proteomics. 2005; 4: 873-886Abstract Full Text Full Text PDF PubMed Scopus (1335) Google Scholar). In brief, aliquots of peptides were dissolved with 20 µl of 200-mg 2,5-dihydroxybenzoic acid (DHB; Sigma-Aldrich) in 80% acetonitrile (ACN), 5% trifluoroacetic acid and loaded onto a TiO2 column. The column was washed three times with 20 µl of 200-mg DHB in 80% ACN, 5% trifluoroacetic acid and five times with 20 µl of 80% ACN, 5% trifluoroacetic acid. The column was then incubated three times with 20 µl of 0.3 normal (N) NH4OH, pH ≥ 10.5, and subsequently bound peptides were eluted and afterward evaporated with a SpeedVac for further MS analysis. Enriched tryptic peptides were first loaded at a flow rate of 10 µl/min onto a C18 trap column packed in-house (1.5 cm, 360-µm outer diameter, 150-µm inner diameter, Nucleosil 100-5 C18; MACHEREY-NAGEL, GmbH & Co. KG). The peptides retained were then eluted and separated on an analytical C18 capillary column (30 cm, 360-µm outer diameter, 75 µm-inner diameter, Nucleosil 100-5 C18) at a flow rate of 300 nL/min, with a gradient from 7.5 to 37.5% ACN in 0.1% (v/v) formic acid for 60 min using an Agilent 1100 nano-flow LC system (Agilent Technologies, Palo Alto, CA), coupling with Ultima™ API-Q-TOF mass spectrometer (Waters/Micromass, Milford, MA) or LTQ-Orbitrap XL hybrid mass spectrometer (Thermo Electron, Bremen, Germany). The API-Q-TOF was operated in data-dependent acquisition mode. Briefly, 1 s survey scans were run over the mass range m/z 350 to 1600. A maximum of three concurrent MS/MS acquisitions were triggered for doubly, triply, and quadruply charged precursors detected at intensities above 15 counts; after 3 s of acquisition, the system switched back to survey scan mode. A Q-TOF peaklist file was generated by MassLynx 4.0 SP4. All spectra were searched by using MASCOT v2.2.04 against the Swiss-Prot database (20080701/39069 entries) limited to chicken with the following criteria: peptide mass tolerance, 50 ppm; MS/MS ion mass tolerance, 0.25 Da; number of missed cleavages allowed, up to three; the variable modifications considered were phosphorylation of serine, threonine and tyrosine, methionine oxidation, and cysteine carboxyamidomethylation. The LTQ-Orbitrap was operated in the data-dependent mode. Briefly, survey full scan MS spectra were acquired in the orbitrap (m/z 350–1600) with the resolution set to 30,000 at m/z 400 and automatic gain control target at 106. The five most intense ions were sequentially isolated for collision-induced decay MS/MS fragmentation and detection in the linear ion trap with previously selected ions dynamically excluded for 90 s. Ions with single and unrecognized charge states were also excluded. To improve the fragmentation spectra of the phosphopeptides, "multistage activation" corresponding to a neutral loss of phosphoric acid from doubly and triply charged precursor ions was enabled in all MS/MS events. All the measurements in the orbitrap were performed with the lock mass option (lock mass: m/z 445,120025) for internal calibration. Orbitrap DTA files were generated by BioWorks version 3.3.1 SP1 and were merged and converted to MASCOT generic format files. All spectra were searched using MASCOT v2.2.04 against the Swiss-Prot database limited to chicken with the following criteria: peptide mass tolerance, 10 ppm; MS/MS ion mass tolerance, 0.8 Da; number of missed cleavages allowed, up to three. The variable modifications considered were phosphorylation of serine, threonine and tyrosine, methionine oxidation, and cysteine carboxyamidomethylation. All phosphorylated sites were examined manually by the presence of a mass difference of 69 Da between fragment ions for phosphoserine and a difference of 83 Da for phosphothreonine (see supplemental data). Quantification of phosphopeptides was performed by using MSQuant. Preselected peptides with a score greater 10 were validated. For quantification the combined peak intensities of each phosphorylated peptide were compared. All phosphorylated sites were examined manually (see supplemental data). For recording intracellular Ca2+ concentrations, 106 DT40 cells were loaded with the ratiometric Ca2+ chelator Indo-1 AM (Molecular Probes, BioMol) at a concentration of 1 µm in 1-ml RPMI 1640 10% FCS for 30 min at 30 °C. Subsequently, cells were washed twice with and resuspended in a Krebs Ringer solution composed of 10 mm HEPES (pH 7.0), 140 mm NaCl, 4 mm KCl, 1 mm MgCl2, 1 mm CaCl2, and 10 mm glucose. After monitoring basal Ca2+concentrations, cells were BCR-stimulated, and the ratio of Ca2+-bound to Ca2+-unbound Indo-1 (Indo-1 violet/Indo-1 blue) was monitored on an LSR II cytometer (Becton Dickinson) at wavelengths 440 and 510 nm. For analysis of BCR-induced AP1 activation, 2 × 107 DT40 cells were cotransfected by electroporation (300 V, 975 µF) with 10 µg of a β-galactosidase expression plasmid (pCMVβ; BD Biosciences - Clontech) and 20 µg of an AP1-driven luciferase reporter gene plasmid (3 × TRE/tk) as described in Ref. 38. Transfectants were cultured 48 h and subsequently starved for 12 h in RPMI 1640 1% FCS. Luciferase and β-galactosidase activities were assayed and standardized as described by Brummer et al. (39Brummer T. Shaw P.E. Reth M. Misawa Y. Inducible gene deletion reveals different roles for B-Raf and Raf-1 in B-cell antigen receptor signaling.EMBO J. 2002; 21: 5611-5622Crossref PubMed Scopus (68) Google Scholar) for resting DT40 B cells and cells stimulated through their BCR for 6 h with M4 monoclonal antibody at a concentration of 2 µg/ml. Where indicated, cells were pretreated one hour prior to BCR stimulation with SB202190 or SP600125 (Biomol) to inhibit p38 or JNK, respectively (29Grabbe A. Wienands J. Human SLP-65 isoforms contribute differently to activation and apoptosis of B lymphocytes.Blood. 2006; 108: 3761-3768Crossref PubMed Scopus (16) Google Scholar). The chicken B cell line DT40 is highly susceptible to homologous recombination of gene fragments (40Winding P. Berchtold M.W. The chicke
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