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• W2096592094 abstract "Article12 February 2009free access Signalling of the BCR is regulated by a lipid rafts-localised transcription factor, Bright Christian Schmidt Christian Schmidt Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, USA Search for more papers by this author Dongkyoon Kim Dongkyoon Kim Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, USA Search for more papers by this author Gregory C Ippolito Gregory C Ippolito Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, USA Search for more papers by this author Hassan R Naqvi Hassan R Naqvi Department of Molecular Cell and Developmental Biology, The University of Texas at Austin, Austin, TX, USA Search for more papers by this author Loren Probst Loren Probst Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, USA Search for more papers by this author Shawn Mathur Shawn Mathur Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, USA Search for more papers by this author German Rosas-Acosta German Rosas-Acosta Department of Microbial and Molecular Pathogenesis, Texas A&M Health Science Center, College Station, TX, USA Search for more papers by this author Van G Wilson Van G Wilson Department of Microbial and Molecular Pathogenesis, Texas A&M Health Science Center, College Station, TX, USA Search for more papers by this author Athenia L Oldham Athenia L Oldham Immunobiology and Cancer Program, Oklahoma Medical Research Foundation, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Search for more papers by this author Martin Poenie Martin Poenie Department of Molecular Cell and Developmental Biology, The University of Texas at Austin, Austin, TX, USA Search for more papers by this author Carol F Webb Carol F Webb Immunobiology and Cancer Program, Oklahoma Medical Research Foundation, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Search for more papers by this author Philip W Tucker Corresponding Author Philip W Tucker Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, USA Search for more papers by this author Christian Schmidt Christian Schmidt Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, USA Search for more papers by this author Dongkyoon Kim Dongkyoon Kim Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, USA Search for more papers by this author Gregory C Ippolito Gregory C Ippolito Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, USA Search for more papers by this author Hassan R Naqvi Hassan R Naqvi Department of Molecular Cell and Developmental Biology, The University of Texas at Austin, Austin, TX, USA Search for more papers by this author Loren Probst Loren Probst Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, USA Search for more papers by this author Shawn Mathur Shawn Mathur Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, USA Search for more papers by this author German Rosas-Acosta German Rosas-Acosta Department of Microbial and Molecular Pathogenesis, Texas A&M Health Science Center, College Station, TX, USA Search for more papers by this author Van G Wilson Van G Wilson Department of Microbial and Molecular Pathogenesis, Texas A&M Health Science Center, College Station, TX, USA Search for more papers by this author Athenia L Oldham Athenia L Oldham Immunobiology and Cancer Program, Oklahoma Medical Research Foundation, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Search for more papers by this author Martin Poenie Martin Poenie Department of Molecular Cell and Developmental Biology, The University of Texas at Austin, Austin, TX, USA Search for more papers by this author Carol F Webb Carol F Webb Immunobiology and Cancer Program, Oklahoma Medical Research Foundation, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Search for more papers by this author Philip W Tucker Corresponding Author Philip W Tucker Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, USA Search for more papers by this author Author Information Christian Schmidt1, Dongkyoon Kim1, Gregory C Ippolito1, Hassan R Naqvi2, Loren Probst1, Shawn Mathur1, German Rosas-Acosta3, Van G Wilson3, Athenia L Oldham4, Martin Poenie2, Carol F Webb4 and Philip W Tucker 1 1Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, USA 2Department of Molecular Cell and Developmental Biology, The University of Texas at Austin, Austin, TX, USA 3Department of Microbial and Molecular Pathogenesis, Texas A&M Health Science Center, College Station, TX, USA 4Immunobiology and Cancer Program, Oklahoma Medical Research Foundation, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA *Corresponding author. Institute for Cellular and Molecular Biology, Molecular Genetics and Microbiology, The University of Texas at Austin, 1 University Station A5000, Austin, TX 78712, USA. Tel.: +1 512 475 7705; Fax: +1 512 475 7707; E-mail: [email protected] The EMBO Journal (2009)28:711-724https://doi.org/10.1038/emboj.2009.20 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Regulation of BCR signalling strength is crucial for B-cell development and function. Bright is a B-cell-restricted factor that complexes with Bruton's tyrosine kinase (Btk) and its substrate, transcription initiation factor-I (TFII-I), to activate immunoglobulin heavy chain gene transcription in the nucleus. Here we show that a palmitoylated pool of Bright is diverted to lipid rafts of resting B cells where it associates with signalosome components. After BCR ligation, Bright transiently interacts with sumoylation enzymes, blocks calcium flux and phosphorylation of Btk and TFII-I and is then discharged from lipid rafts as a Sumo-I-modified form. The resulting lipid raft concentration of Bright contributes to the signalling threshold of B cells, as their sensitivity to BCR stimulation decreases as the levels of Bright increase. Bright regulates signalling independent of its role in IgH transcription, as shown by specific dominant-negative titration of rafts-specific forms. This study identifies a BCR tuning mechanism in lipid rafts that is regulated by differential post-translational modification of a transcription factor with implications for B-cell tolerance and autoimmunity. Introduction B-cell development and response to antigen depend on signalling through the B-cell antigen receptor (BCR) complex (Gauld et al, 2002; Meyer-Bahlburg et al, 2008). BCR signalling directs positive and negative selection of immature B cells and their progression through transitional (T) stages into mature B cells. Surface markers allow the resolution of three non-proliferative immature B-cell subpopulations: T1, T2 and T3 (Allman et al, 2001; Sims et al, 2005). The lineage origins and signalling requirements of these intermediate stages of B cells are the subject of considerable interest and debate (Matthias and Rolink, 2005; Teague et al, 2007; Welner et al, 2008). It is generally agreed that sequential progression requires an increasingly higher threshold level of BCR signalling; that is, low or ‘tonic’ threshold signals promote T1 to T2, whereas relatively higher levels of signalling are needed for T2 to progress to FO or MZB (Petro et al, 2002; Su and Rawlings, 2002; Hoek et al, 2006). Strong BCR signalling also is required to direct non-transitional, fetal progenitors to B-1 fate (Loder et al, 1999; Cariappa et al, 2001; Casola et al, 2004). The amplitude of BCR signalling is positively and negatively regulated by coreceptors (Carter and Fearon, 1992; Cherukuri et al, 2001; Ravetch and Bolland, 2001) and crosstalk between the antigen receptors and other pathways, particularly BAFF (Guo and Rothstein, 2005; Venkatesh et al, 2006). A spatially continuous but mobile unit of critical size within the plasma membrane is required for efficient initiation of BCR activation by multivalent antigen (Dintzis et al, 1976). Engagement of the antigen receptor yields ‘microclusters’ that can be found in highly ordered domains within the plasma membrane, known as lipid rafts (Dykstra et al, 2003; Saeki et al, 2003; Harwood and Batista, 2008). Size and composition of these platforms of BCR signalling are dynamic and responsive to signalling events mediated by the actin cytoskeleton through plasma membrane linker proteins, such as Ezrin (Stoddart et al, 2002; Gupta et al, 2006; Sohn et al, 2006). Bright (B-cell regulator of IgH transcription/Dril1/ARID3A) is the founder of the AT-rich interaction domain (ARID) super-family of DNA-binding proteins (Herrscher et al, 1995; Wilsker et al, 2005). Bright shuttles between the cytoplasm and the nucleus in a Crm1- and cell cycle-dependent fashion (Kim and Tucker, 2006). Bright transactivates the IgH intronic enhancer (Eμ) and certain IgH promoters by binding as a tetramer to ATC motifs within nuclear matrix associating regions (Webb et al, 1999; Kim et al, 2007; Lin et al, 2007). DNA binding and IgH transcriptional activities of Bright are stimulated by its interaction with Btk and transcription initiation factor-II (TFII-I), a direct substrate of Btk (Webb et al, 2000; Rajaiya et al, 2005, 2006). TFII-I also undergoes nucleocytoplasmic shuttling (Hakre et al, 2006), and, within the cytoplasm, it associates with PLCγ to inhibit Ca2+ mobilisation (Caraveo et al, 2006). Bright is lineage and stage-specifically expressed with high basal levels in immature B cells and in mitogen or cytokine-induced mature B cells (Webb et al, 1991a, 1991b, 1998; Nixon et al, 2004a, 2004b). Shankar et al (2007) recently demonstrated the pathological consequences of loss of this tight control. Transgenic (TG) mice that over-express wild-type (WT) Bright specifically within the B lineage display spontaneous autoimmunity. This intrinsic B-cell autoreactivity was not accompanied by global increase in serum Ig. Instead, a markedly expanded population of T1 and MZB cells was observed. These observations, along with the extranuclear expression of Bright, TFII-I and their functional association with Btk, prompted us to examine whether Bright is used in BCR signal transduction. We show here that a pool of Bright acts within lipid rafts as a ‘brake’ to set a signalling threshold on the BCR. Results Association of Bright with mIgM on B-cell membranes is reduced after antigen receptor stimulation Immunostaining of murine B splenocytes indicated that a fraction of the non-nuclear Bright pool colocalised with mIgM, suggesting cortical and/or membrane-associated localisation (Figure 1A and readdressed below). This observation was confirmed by computerised 3D reconstructions of the immunofluorescence data (Figure 1A′ and Supplementary Video 1). Figure 1.Bright accumulates within lipid rafts of resting but not stimulated B cells. (A) Association of Bright with mIgM on B-cell membranes is reduced after antigen receptor stimulation. CD43− B cells from spleens of BALB/c adult mice were fixed and stained for Bright (red), mIgM (green) and DNA (blue). Arrows point to areas (yellow) where Bright colocalises with membrane IgM. (A′, A″) Engagement of the antigen receptor reduces the colocalisation between Bright and mIgM. CD43− B cells (∼1 × 104) from spleens of BALB/c adult mice were left untreated (A′) or stimulated for 5 min (A″) with 10 pg α-μ, followed by immunostaining as described above. Deconvoluted images are shown with arrows pointing to areas (yellow) where Bright colocalises with mIgM. (B) BCR engagement leads to a discharge of Bright from lipid rafts. CD43− B cells (∼2 × 106) were stimulated with either 2 ng α-μ or 2 ng α-μ+2 ng α-CD19 for 5 min. Lipid rafts or whole cell lysates (WCL) were prepared from half of each sample. Proteins from each fraction were analysed by SDS–PAGE/western blot using the antibodies indicated. Download figure Download PowerPoint To determine whether this colocalisation remains intact after engagement of the BCR, cells were stimulated for 5 min with α-μ. Only modest colocalisation of Bright and IgM was retained, as assessed by computerised 3D reconstructions of the immunofluorescence data (Figure 1A″ and Supplementary Video 2). Inspection of these and additional images (data not shown) indicated that the observed redistribution of mIgM-associated Bright in stimulated B cells was not accompanied by significant alteration in either its nuclear or its cytoplasmic levels (data not shown). Bright accumulates within lipid rafts of resting but not stimulated B cells Because lipid rafts serve as platforms for BCR signalling, we assayed purified plasma membranes and lipid rafts (Supplementary Figure 1A) for the presence of Bright. A small pool of Bright resides in lipid rafts purified from unstimulated CD43− B cells (Figure 1B, upper panel). Consistent with the imaging results, Bright was not detected within lipid rafts after BCR engagement that was sufficient to elicit a phosphotyrosine (pY) response (Figure 1B, lower panel). This suggested that the presence or absence of Bright within lipid rafts might influence BCR signalling. Levels of Bright within lipid rafts determine BCR signalling threshold Normal B cells and mature B-cell lines were examined semi-quantitatively for lipid raft content of Bright using the B cell-specific lipid rafts component, Raflin (Saeki et al, 2003), as an internal control (Supplementary Figure 1B and data not shown). We estimated that raft-localised Bright accounted for 1–10% of total cellular Bright, consistent with percentages previously estimated for mIgM concentrations in lipid rafts (Sproul et al, 2000; Putnam et al, 2003). Lipid rafts of Raji and Daudi cells contained ∼10-fold less Bright than those of CL01 or Ramos (Figure 2A). However, no significant differences were observed in other subcellular fractions among these lines (Supplementary Figure 1B). Figure 2.Levels of Bright within lipid rafts determine BCR signalling threshold. (A) Bright levels within lipid rafts vary among B-cell lines. Lipid rafts were prepared from the indicated exponentially growing human B-cell lines (107 cells) and probed for Bright and (as loading control) Raftlin. (B, C) Antigen receptor engagement results in a discharge of Bright from lipid raft-localised BCR complexes. (B) The indicated cell lines (∼5 × 108) were stimulated for 5 min with 500 ng of α-μ, followed by preparation of lipid rafts using discontinuous gradient centrifugation. Aliquots of each fraction were analysed directly by western or (C) were extracted with RIPA buffer and subjected to co-IP/western using the antibodies indicated. (D) Raji, Daudi, Ramos and CL01 cells respond differentially to BCR stimulation. The indicated cells were stimulated (100 ng α-μ for 108 cells) for 5, 10 and 60 min (Raji and Ramos; left panel) or for 5 min (CL01 and Daudi; right panel), as indicated, and whole cell extracts were blotted with α-phosphotyrosine (pY). Equal loading was confirmed by staining of the filters with India Ink (data not shown). (E–G) BCR+CD19 stimulation leads to Bright discharge from lipid rafts and accumulation of Sumo-I-Bright in plasma membranes. Raji, Ramos, CL01 and Daudi (∼108 cells) were stimulated for 5 min with (E) 100 ng α-μ, (F) 100 ng α-CD19 or (G) 100 ng α-μ+100 ng α-CD19. Lipid rafts (Raft), plasma membranes (membrane) and whole cell lysates (WCL) were analysed by IP/western using the antibodies indicated. Download figure Download PowerPoint To achieve maximal BCR responses under minimal antibody concentrations and minimal receptor internalisation, an approach using an anti-IgM mAb in the absence of secondary cross-linking was optimised (Supplementary Figures 2B and 4B; Materials and methods; data not shown). Lipid rafts from resting and stimulated cell lines were purified on sucrose gradients, and fractions were analysed for Bright and other signalosome occupants (Figure 2B). In agreement with published reports (Saeki et al, 2003; Depoil et al, 2008), levels of mIgM, CD19, and the Bright-interacting partner Btk increased in lipid rafts after α-μ stimulation (Figure 2B). As observed for normal B cells, Bright moved in the opposite manner. However, its discharge from lipid rafts was complete only in cell lines in which its starting levels in lipid rafts were low (Daudi and Raji, Figure 2B, fractions 3 and 4; boxed in red). These differences in trafficking could reflect differences in composition of raft-localised complexes, or artifacts resulting from increased resistance to solubilisation, as BCR ligation is known to induce coalescence of lipid rafts (Gupta et al, 2006). Therefore, we compared profiles obtained from RIPA-solubilised versus non-soluble lipid rafts immunoprecipitated (IP) with α-μ, α-Raftlin and α-Btk (Supplementary Figure 2E). We observed that, as previously published (Saeki et al, 2003), a complex containing Raflin and IgM was seen only in non-solubilised rafts (Supplementary Figure 2E); this indicated that our solubilisation conditions were sufficient. However, unexpectedly, Raflin did IP with Btk under both conditions, suggesting that an IP complex containing Btk and Raflin is not disrupted by RIPA solubilisation of lipid rafts (readdressed below). Importantly, Bright remained in a complex with mIgM and Btk in solubilised lipid rafts of all unstimulated cells (Figure 2C) but was lost only in α-μ stimulated cells (Daudi and Raji) that contained lower starting levels in their lipid rafts (Figure 2C, lanes 6 and 12; boxed in red). These results suggested that B cells that contain more lipid rafts-associated Bright (Ramos and CL01) would be less sensitive (higher threshold) to BCR ligation. This was confirmed by the pY responses of these cell lines to α-μ stimulation (Figure 2D). Ramos and CL01 also responded less vigorously to pro-apoptotic signals shown previously (Chen et al, 1999) to result from long-term stimulation by α-μ (Supplementary Figure 2C). Ligation of the BCR coreceptor, CD19, is known to synergistically enhance antigen receptor-mediated signalling (Carter and Fearon, 1992; Cherukuri et al, 2001; Depoil et al, 2008). Accordingly, all cell lines responded to α-μ+α-CD19 costimulation with robust responses (Supplementary Figure 2B). BCR costimulation was required to expel Bright from lipid rafts of the less sensitive (higher threshold) cell lines Ramos and CL01 (Figure 2E–G). That Bright migrates as a doublet is apparent in these experiments (addressed below in the context of the sumoylation observations). Thus, engagement of the BCR results in a significant and specific reduction of the small pool of lipid rafts-localised Bright. This pool is lost from lipid rafts, as Btk and other signalosome components accumulate there, in proportion to BCR signalling strength. Entry of Bright into lipid rafts does not require interaction with Btk but does require palmitoylation Bright was readily detected in lipid rafts prepared from retrovirally transduced NIH/3T3 fibroblasts and other non-B cells (Figure 3B; data not shown). This indicated that even though Bright associates (at least transiently) with Btk and other signalsome components in α-μ stimulated lipid rafts (Figure 2C and addressed further below), these B-cell-restricted proteins are not required to designate or retain Bright in lipid rafts. Bright point mutants (Figure 3A; Kim and Tucker, 2006) that are retained either within the cytoplasm (K466A) or the nucleus (G532A) did not localise to rafts (Figure 3B). Thus, the rafts-localised pool of Bright is not directly diverted from the cytoplasmic pool, suggesting that nucleocytoplasmic shuttling (Kim and Tucker, 2006) is required. Figure 3.Entry and exit of Bright from lipid rafts requires nucleocytoplasmic shuttling, alternative post-translational modifications, but not association with Btk. (A) Schematic of Bright indicating domains and positions of substitution mutations. (B) Cytoplasmic-nuclear shuttling is a requirement for Bright's inclusion into lipid rafts. NIH/3T3 fibroblasts were transfected with constructs encoding wild type and nuclear export signal-defective (NES, G532A) and nuclear localisation signal-defective (NLS, K466A) Bright. Fractions from discontinuous (5–40%) sucrose gradient purification of lipid rafts were analysed by western for Bright. (C) Palmitoylation of Bright requires cysteine 342. Bright constructs (indicated at the top) were transfected into Cos-7 cells, and after 48 h, were incubated as indicated with 14C palmitic acid. Whole cell lysates were subjected to immunoprecipitation using antibodies against Bright and VSV as indicated (left). SDS–PAGE separated proteins were transferred onto nitrocellulose, and the metabolic incorporation of 14C palmitic acid was determined by autoradiography. Solvent control is indicated by −. (D) Specification of Bright to lipid rafts requires cysteine 342. NIH/3T3 fibroblasts were transfected with constructs indicated in the figure. Crude plasma membrane and lipid rafts, prepared as described in previous legends, were analysed by anti-Bright western blotting. (E) Sumo-I modification of Bright is lost after mutation of 401KIKK. Cos-7 cells were transfected with GFP-SUMO-I and GFP-Bright expression constructs, as indicated. Whole cell lysates were prepared using RIPA buffer and analysed by western using α-Bright anti-serum. An arrow points to a GFP-Sumo-I conjugated species of GFP-Bright. (F) Bright forms a transient, stimulation-specific complex with Sumo-I-conjugation enzymes PIAS1 and Ubc9 in lipid rafts. Indicated B cells (∼108) were stimulated mildly (30 s; 100 ng α-μ). Lipid rafts were collected on gradients, subjected to immunoprecipitation with anti-Bright antiserum, and then analysed by western using the antibodies indicated. Download figure Download PowerPoint Palmitoylation of cysteine residues is a feature shared by a number of lipid raft occupants (Simons and Toomre, 2000; Ashery et al, 2006). Bright contains a single cysteine (C342) in its ARID DNA-binding domain, which is conserved among all identified orthologues and paralogues (Figure 3A; Wilsker et al, 2005). After transfection into fibroblasts, WT Bright, but not point mutants (C342S and C342D), were palmitoylated (Figure 3C). Sumoylation of Bright regulates its discharge from lipid rafts into membranes after BCR stimulation Yeast 2-hybrid cDNA library screening and additional analyses (Supplementary Figure 3A; data not shown) detected strong and specific Bright interactions with Sumo-I conjugating enzymes Ubc9 and PIAS1. Further investigations indicated that Bright is conjugated to Sumo-I at a consensus motif (ΨKxE, Sampson et al, 2001; Gocke et al, 2005; Bossis and Melchior, 2006) 401KIKKE (Figure 3A) both in cultured cell lines and in vitro (Figure 3E; Supplementary Figure 3B). Sumo-I-Bright was readily detected in α-Bright IPs of whole cell lysates prepared from B-cell lines and normal B cells (Figures 2E–G; Supplementary Figures 2D, 3B and C). We found no Sumo-I-Bright in lipid rafts regardless of cell source and stimulation regime; only membranes prepared from stimulated B cells contained Sumo-I-Bright (Figure 2E–G; Supplementary Figure 2D). Yet Sumo-I-deficient (401KIKK/AIAA) Bright was capable of entering lipid rafts and membranes as efficiently as WT in transfected fibroblasts (Supplementary Figure 3D). These results prompted us to speculate that the Sumo-I-Bright pool within stimulated plasma membranes might derive from a sumoylation reaction initiated in B cell rafts immediately after BCR ligation. If so, a transient sumoylation initiation complex might be trapped in lipid rafts under much weaker BCR stimulation conditions. To test this, we isolated lipid rafts following conditions (30 s; α-μ). Consistent with our hypothesis, Bright was detected in these mildly stimulated rafts in an IP complex with sumolyation E2 and E3 components, Ubc-9 and PIAS-1 (Figure 3F; Schwarz et al, 1998; Kahyo et al, 2001). Dominant-negative, lipid rafts localisation-defective mutants modulate BCR signalling Because Bright exists primarily as a homo-tetramer, we established the basis for a dominant-negative approach by pulling down endogenous Sumo-I-Bright using V5 tagged 401KIKK/AIAA-Bright (Supplementary Figure 4A; Herrscher et al, 1995; Kim and Tucker, 2006). Thus, we reasoned that over-expression of palmitoylation-defective C342S or C342D Bright should titrate the small pool of palmitoylated Bright tetramers destined for lipid rafts while sparing tetramers destined for the nucleus. Conversely, the inability to sumoylate Bright should trap it in lipid rafts, leading to suppression of BCR signalling. As shown in Figure 4A, rafts prepared from WT transductants contained increased levels (relative to mock controls) of V5-tagged retroviral Bright, whereas those expressing C342 substitutions were virtually depleted. Over-expression of 401KIKK/AIAA-Bright in lipid rafts (Figure 4A) led to retention of both retroviral and endogenous Bright within rafts after BCR stimulation. Figure 4.Dominant-negative, lipid rafts localisation-defective mutants modulate BCR signalling. Raji and Ramos cells (5 × 108) were infected with retroviruses encoding wild type and mutant forms of Bright and were then stimulated for 5 min with 500 ng α-μ, 500 ng α-CD19 or 500 ng α-μ+500 ng α-CD19. (A) Levels of Bright in lipid rafts are altered by dominant-negative forms. Lipid rafts levels of total Bright (endogenous+ectopic) were measured by anti-Bright western. Levels of ectopic V5-tagged wild-type Bright, a palmitoylation-defective (C342S/D) form, which is unable to enter lipid rafts, and a Sumo-I-mutant form (401KIKK/AIAA), unable to be discharged from rafts, were detected by anti-V5 western. Raftlin was used as a loading control. (B) Intracellular free [Ca2+] is increased by palmitoylation-deficient and decreased by wild type or Sumo-I-deficient titration of endogenous Bright. Transduced Raji B cells (1 × 106 cells/ml) were loaded with 2 μM Indo-1 and stimulated with 1 ng or 40 μg of α-μ (indicated as low and high concentrations, respectively, by triangles). Fluorescence signal was plotted against time (scale bar=100 s). Internal calibration was performed as detailed in Materials and methods. Downward arrows indicate times of reagent addition. (C) Dominant-negative Bright mutants alter global phosphotyrosine responses. Whole cell lysates prepared from cell lines established and stimulated in (A) were analysed by anti-pY, Bright and tubulin (loading control) western. Download figure Download PowerPoint We next measured the effect of the dominant-negative titrations on BCR signalling. As shown in Figure 4B and C, signalling was markedly increased in Bright C342S and C342D-infected cells. Notably, the signalling threshold of the Raji BCR was converted from low to high, because weak stimulation (α-μ only) now resulted in significantly reduced signalling (Figure 4; Supplementary Figure 4B). Conversely, over-expression of WT Bright and the 401KIKK/AIAA dominant-negative mutant form virtually eliminated BCR-stimulated Ca2+ flux and pY activity. Yet, unstimulated 401KIKK/AIAA-transduced Ramos cells appeared to be constitutively ‘hyperactivated’ with respect to pY signals (Figure 4C, lane 7). This was a consistent result (please see Figure 7A and discussion below) that we suspect derived from Ramos-specific, off-target (i.e., non-BCR mediated) effects of this dominant negative. We conclude that a palmitoylated pool of Bright is dispatched to lipid rafts to dampen BCR signalling, and sumoylation-triggered discharge of Bright is essential for relieving this inhibition. Bright regulates signalling and IgH transcription independently As the small lipid rafts-localised pool of Bright is diverted from its nucleocytoplasmatic shuttling pool (Figure 3B; Kim and Tucker, 2006), we examined the potential nuclear consequences of the dominant-negative-mediated signalling perturbations. Neither nuclear-cytoplasmic ratios nor in vitro DNA binding of Bright to a target VH-associated promoter were significantly altered (Figure 5B and C). Figure 5.Wild type and dominant-negative mutant forms of Bright are indistinguishable in DNA-binding, subcellular localisation and transcriptional activity. (A) Schematic illustration of Bright amino acid substitution mutations. (B) Subcellular fractionation of Bright is unaltered by dominant-negative transduction. Raji (107 cells), expressing wild type and the indicated mutant forms of Bright, were subjected to fractionation into cytoplasm (CY), nucleoplasm (NP), chromatin (CH) and nuclear matrix (NM) and analysed by western using α-Bright anti-serum. (C) Substitution mutants bind indistinguishably to IgH promoter sites. The indicated forms of Bright were prepared by in vitro transcription/translation (IVT) and subjected to electrophoretic mobility shift assays using 32P labelled S107 VH1-MAR probe. Specificity of binding was demonstrated by α-Bright super-shift and cold probe competition (not shown) as previously described (Herrscher et al, 1995; Zong et al, 2000; Kaplan et al, 2001). (D) Bright transactivation of inducible IgH promoter activity is not changed by dominant-negative transduction. Exponentially growing Raji and Ramos cells, stably transduced with retrov" @default.
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• W2096592094 date "2009-02-12" @default.
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• W2096592094 title "Signalling of the BCR is regulated by a lipid rafts-localised transcription factor, Bright" @default.
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