Haploinsufficiency of B cell linker protein enhances B cell signaling defects in mice expressing a limiting dosage of Brutons tyrosine kinase
Lindsey R. Whyburn1,
Kristina E. Halcomb1,
Cristina M. Contreras1,
Rajita Pappu2,
Owen N. Witte3,
Andrew C. Chan2 and
Anne B. Satterthwaite1
1 Simmons Arthritis Research Center, Department of Internal Medicine and Center for Immunology, UT Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA 2 Division of Rheumatology, Department of Internal Medicine, Washington University School of Medicine, St Louis, MO 63110, USA 3 Howard Hughes Medical Institute, and Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90095, USA
Correspondence to: A. B. Satterthwaite; E-mail: anne.satterthwaite{at}utsouthwestern.edu
Transmitting editor: T. Tedder
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Abstract
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Current models of lymphocyte activation suggest that formation of a signaling complex, or signalosome, composed of Syk, Brutons tyrosine kinase (Btk), phospholipase
2 and the adaptor protein B cell linker protein (BLNK) is critical for transmission of signals from the BCR. However, impaired B cell development in mice lacking each individual signalosome component has made it difficult to study the functional consequences of the formation of this complex in mature B cells. Sensitized genetic systems, commonly used in Drosophila, define signaling pathways by combining partial loss of function mutations in the components of interest. This allows genetic interactions to be observed in the absence of pleiotropic or lethal effects of complete deficiency of either gene. We used this approach to demonstrate that Btk and BLNK are limiting components of a common signaling pathway that mediates the mitogenic response of mature B cells to antigen. B cells from transgenic mice expressing a limiting dosage of Btk (Btklo) have normal numbers of mature B cells that have reduced, but measurable, responses to BCR cross-linking. Haploinsufficiency of BLNK did not affect the development of Btklo B cells. However, it exacerbated their defects in BCR-induced Ca2+ flux, I
B degradation, and up-regulation of cyclin D2, bcl-xL and A1 leading to dramatic impairment of B cell mitogenic responses. In contrast, no effect of reduced Btk and BLNK dosage was observed on extracellular signal-regulated kinase activation. These results suggest that the signals regulating the maintenance and activation of mature B cells are differentially sensitive to the strength of the signal emanating from the signalosome.
Keywords: B lymphocyte, BCR, immunodeficiency diseases, knockout, signal transduction, transgenic
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Introduction
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Signals from the BCR are critical at multiple stages of the development and function of a self-tolerant, diverse repertoire of B lymphocytes. The outcome of these signals varies and can include proliferation, differentiation, survival, apoptosis, anergy or receptor editing. Factors affecting the response to BCR stimulation include the maturity of the recipient cell, the strength and duration of the signal, and the presence or absence of additional signals from co-receptors, cytokines or T cell help (1).
BCR signals are initiated by the activation of three families of tyrosine kinases: Src family kinases including Lyn, Fyn and Blk, the Tec family kinase Brutons tyrosine kinase (Btk), and Syk (25). The formation of a signaling complex, or signalosome, in which the adaptor protein B cell linker protein (BLNK) allows tyrosine kinases access to critical substrates has recently been proposed as a major step in BCR signal transduction (6,7). According to this model, BLNK is phosphorylated by Syk in response to BCR cross-linking (8,9). The Src homology 2 (SH2) domains of Btk and phospholipase C (PLC)
2 are then recruited to phosphorylated BLNK, allowing Syk and Btk to phosphorylate and activate PLC
2 (811). Activated PLC
2 cleaves phosphatidylinositol-4,5-bisphosphate into inositol-1,4,5-trisphosphate and diacyglycerol, leading to Ca2+ mobilization, activation of protein kinase C (PKC) ß, and eventual changes in gene expression mediated by transcription factors such as NF-
B and NF-AT (25). Among the genes up-regulated in response to BCR cross-linking include those controlling cell cycle entry, such as cyclin D2 (12), and cell survival, including bcl-xL (13) and A1 (14).
Biochemical studies support the signalosome model but suggest that Btk and BLNK may also have independent functions. Physical interactions between BLNK, PLC
2 (8) and Btk (11) have been demonstrated in vitro. Overexpression experiments in 293 cells have shown that optimal phosphorylation of PLC
2 by Syk and Btk requires the presence of BLNK (11). This approach has also suggested that BLNK mediates the activation of Btk by Syk (15), although BCR-induced tyrosine phosphorylation of Btk is normal in BLNK/ murine B cells (16). However, BLNK can also physically associate with a number of other signaling molecules, including growth factor receptor-binding protein (Grb2), Vav and Nck (8) that are not necessarily involved in the regulation of the Btk/PLC
2 pathway. Btk has been shown to interact with or phosphorylate a number of transcription factors, including transcription factor II-I (17), STAT-5a (18) and BRIGHT (19). None of these interactions have been shown to require BLNK.
B cells deficient in signalosome components also reveal both overlapping and separate functions for these molecules. BCR-stimulated PLC
2 activation and Ca2+ flux are defective in BLNK/ (9), Syk/ (20) and Btk/ (21,22) chicken DT40 cells, and reduced in B cells from BLNK/ (16,23,24) and X-linked immunodeficiency (xid) (25) mice or Btk-deficient humans (26). However, in the chicken system BCR-mediated activation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 requires BLNK (9), while Btk is absolutely necessary only for JNK activation (27).
Finally, in vivo genetic analysis also demonstrates a complex interaction between Btk and BLNK. xid mice, which have a naturally occurring point mutation in Btk (28,29), and mice deficient in Btk (3032), PLC
2 (33,34) or BLNK (23,24,35,36) all have a block in B cell development at the immature to mature transition in the periphery. Impaired responses to T-independent type II antigens, reduced numbers of CD5+ B-1 cells and failure of B cells to proliferate in response to BCR cross-linking are also shared features of these mice (6). Humans with mutations in BLNK have a severe B cell immunodeficiency (37) resembling X-linked agammaglobulinemia (XLA), which results from loss of Btk function (38,39). The striking similarity of these phenotypes suggests that these molecules regulate similar pathways and processes in B cell development and function. However, unlike Btk/ mice, BLNK/ mice also have a block in B lymphopoiesis at the pro-B to pre-B stage (23,24,35,36). This is also observed in Syk/ mice (40,41), suggesting that at least some B cell signalosome components mediate pre-BCR signals as well (42). Formal genetic proof of independent roles for Btk and BLNK comes from the demonstration that mice lacking both genes have more profound defects than either Btk/ or BLNK/ mice alone (43).
The developmental block in mice lacking Btk, BLNK or both has precluded analysis of whether these molecules function in concert or independently to mediate the activation of mature primary B cells. The well-documented differences in the consequences of BCR stimulation between immature and mature mammalian B cells (44,45), and the differential effects of BLNK deficiency on BCR signaling in chicken and murine B cells (9,16) limit the extrapolation of results in these systems to mature B lymphocytes. We therefore used a sensitized genetic system analogous to those commonly used in Drosophila (46) to assess the functional consequences of the interaction between Btk and BLNK. This approach defines signaling pathways by combining partial loss of function mutations in the components of interest, allowing genetic interactions to be observed in the absence of pleiotropic or lethal effects of complete deficiency of either gene.
We have previously employed this strategy to define functional interactions between Btk and its upstream regulators (47). Unlike xid or Btk/ mice, mice expressing a limiting dosage of Btk (Btklo) have normal numbers of phenotypically mature B cells (48). These cells display an intermediate proliferative response to BCR cross-linking between that of wild-type and Btk/ B cells (48). The mitogenic response of Btklo B cells was shifted towards wild-type by mutations in Lyn, SH2-containing inositol phosphatase or phosphatase and tensin homolog, defining these genes as negative regulators of Btk signaling pathways (47). In this report, we demonstrate that haploinsufficiency of BLNK enhances the signaling defects of Btklo B cells without affecting their development. Thus, Btk and BLNK interact functionally to mediate the responses of mature B cells to BCR stimulation.
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Methods
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Mice
Btklo mice are Btk/ mice carrying a wild-type Btk transgene driven by the Ig heavy chain promoter and enhancer (48). BLNK+/ (23) mice were mated to Btklo mice to generate progeny of the appropriate genotypes. Since Btk is X-linked and the mutant allele is inactivated in mature B cells of carrier females (49), Btk+/ and Btk+/+ mice were used interchangeably. All mice were between 6 and 12 weeks of age. Since mice were of mixed genetic background (C57BL/6 x 129 x BALB/c), littermates were compared directly when possible and experiments were repeated with multiple litters. Mice were genotyped by PCR.
Flow cytometry
Splenocytes were depleted of red blood cells by 5 min incubation in 0.15 M NH4Cl, 1 mM KHCO3 and 0.1 mM Na2EDTA, and stained with combinations of anti-IgDFITC (PharMingen, San Diego, CA), anti-CD21FITC (PharMingen), anti-IgMphycoerythrin (PE) (PharMingen), anti-B220TriColor (Caltag, Burlingame, CA), anti-B220PerCP (PharMingen) and anti-CD23biotin (PharMingen) plus streptavidinallophycocyanin (Caltag). Samples were run on a Becton Dickinson (San Jose, CA) FACSCalibur and analyzed with CellQuest software.
B cell purification
Splenic B cells were purified by negative selection with anti-CD43 magnetic beads using the Miltenyi (Auburn, CA) MidiMACS system according to the manufacturers instructions. The purified cells were typically >90% B220+ as measured by flow cytometry as described above.
Proliferation assays
Purified B cells were plated in 96-well plates at 106/ml in RPMI + 10% FCS. Triplicate wells were used for each stimulation condition. Stimulation conditions included media alone, 20 µg/ml goat anti-mouse IgM F(ab')2 fragments (Jackson ImmunoResearch, West Grove, PA), and 10 ng/ml phorbol myristate acetate (Sigma, St Louis, MO) + 1 µg/ml ionomycin (Calbiochem, San Diego, CA). Cells were incubated at 37% in a humidified incubator for 48 h. 1 µCi [3H]thymidine (Amersham, Piscataway, NJ) was added per well for the final 8 h. Cells were harvested onto Beckman Ready Filters and [3H]thymidine incorporation measured with a scintillation counter.
Cell cycle analysis
Purified B cells were plated in 48-well plates at 106/ml in RPMI + 10% FCS and stimulated with 20 µg/ml goat anti-mouse IgM F(ab')2 fragments (Jackson ImmunoResearch) for 48 h. Cells were fixed in 70% EtOH for 24 h and subsequently stained with 80 µg/ml propidium iodide in the presence of 250 µg/ml RNase A. Samples were run on a FACScan (Becton Dickinson) and analyzed with CellQuest software (Becton Dickinson).
Immunoblot analysis
For BLNK and Btk expression and cyclin D2 and bcl-xL up-regulation, purified B cells were harvested at time 0 or stimulated with 10 µg/ml goat anti-mouse IgM F(ab')2 fragments (Jackson ImmunoResearch) for 18 h. Cells were washed in PBS and resuspended in 50 µl ice-cold lysis buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100 and 0.5 mM PMSF) per 107 cells. Extracts were spun at maximum speed in a refrigerated microfuge to remove insoluble material. For I
B degradation studies, purified B cells were treated with 50 nM cycloheximide (Sigma) for 30 min at 37°C. Cells were then stimulated for 4 h at 37°C in the continued presence of cycloheximide with either no additional treatment or 10 µg/ml goat anti-mouse IgM F(ab')2 fragments (Jackson Immuno Research). Cytoplasmic extracts were prepared by washing cells in PBS and resuspending them in 100 µl Buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.2 mM PMSF, 10 mM iodoacetamide, 1 µg/ml pepstatin A and 1 µg/ml aprotinin) per 24 x 107 cells. Samples were incubated on ice for 10 min, vortexed for 10 s and spun at maximum speed for 10 s in a microfuge to remove nuclei. The supernatant was collected as the cytoplasmic fraction. For ERK activation, purified B cells were resuspended at 108/ml in serum-free media and stimulated with 0 or 10 µg/ml goat anti-mouse IgM F(ab')2 fragments (Jackson ImmunoResearch) for 5 min. Cells were lysed by boiling in SDS sample buffer for 10 min.
Total cell extracts (6 µg, 20 µg or 2 x 106 cell equivalents) were electrophoresed by 10 or 12% SDSPAGE gels and blotted to nitrocellulose. Blots were blocked in 5% milk in 10 mM Tris, pH 7.5 and 150 mM NaCl, and subsequently probed with anti-BLNK (9), anti-Btk (38), anti-cyclin D2 (Santa Cruz Biotechnologies, Santa Cruz, CA), anti-bcl-xL (Santa Cruz Biotechnologies), anti-I
B
(Santa Cruz Biotechnologies), anti-pERK (Cell Signaling Technologies, Beverley, MA), anti-ERK (Cell Signaling Technologies) or anti-actin (Sigma) diluted in 10 mM Tris, pH 7.5, 250 mM NaCl, 0.05% Tween 20 and 0.2% sodium azide. Blots were washed in 10 mM Tris, pH 7.5, 500 mM NaCl and 0.05% Tween 20, and then incubated with horseradish peroxidase-conjugated goat anti-mouse Ig (bcl-xL, actin and BLNK) (Bio-Rad, Hercules, CA) or goat anti-rabbit Ig (Btk, cyclin D2, I
B
, pERK and ERK) (Bio-Rad) diluted in 10 mM Tris, pH 7.5, 250 mM NaCl and 0.05% Tween 20. Blots were washed in 10 mM Tris, pH 7.5, 500 mM NaCl and 0.05% Tween 20, and horseradish peroxidase subsequently visualized using an ECL kit (Amersham).
Real time quantitative RT-PCR
Purified B cells were harvested at time 0 or stimulated with 10 µg/ml goat anti-mouse IgM F(ab')2 fragments (Jackson ImmunoResearch) for 1 h. Total RNA was obtained using TRIzol (Gibco/BRL, Carlsbad, CA) according to the manufacturers instructions. Contaminating genomic DNA was removed by treatment with RNase-free DNase (Gibco/BRL). cDNA was prepared using Superscript II reverse transcriptase (Gibco/BRL) and random hexamers. Real-time PCR was performed in an Applied Biosystems (Foster City, CA) Gene Amp 5700 Sequence Detection System using ABI Prism optical tubes. Reactions were performed in triplicate in a total volume of 20 µl that included 10 ng cDNA, 10 µl SyBR Green master mix (Applied Biosystems) and 200 nM of each primer. Primer sequences are as follows. A1: 5'-CAA ATC TGG CTG GCT GAC TTT TC-3' and 5'-CAA GTG CTG ATA ACC ATT CTC GTC-3' (50); actin: 5'-GAG GCC CAG AGC AAG AGA G-3' and 5'-GTC ATC TTT TCA CGG TTG G-3'. Standard curves were generated for each primer set using serial dilutions of cDNA from stimulated wild-type cells. The relative amount of A1 cDNA in each sample was calculated based on the standard curve, then normalized to the amount of actin to control for cDNA loading.
Ca2+ flux
Purified B cells were resuspended in loading buffer (HBSS + 10 mM HEPES, pH 7.2, 2mM CaCl2, 1 mM MgCl2 and 1% BSA) at 5 x 106/ml and loaded with 5 µM Fluo-4-AM (Molecular Probes, Eugene, OR) in the presence of 0.2 mg/ml Pluronic F-127 (Molecular Probes) for 30 min at 37°C. Cells were washed and resuspended in loading buffer at 5 x 106/ml. Cells were prewarmed to 37°C for 5 min. and maintained at 37°C throughout the analysis. Cells were plated at 106 cells/well in a black 96-well plate with a clear bottom (Corning, Corning, NY), with triplicate wells per sample. Baseline Fluo-4 fluorescence (a measure of intracellular Ca2+ levels) was read at 10-s intervals for 1 min in an FLx800 microplate fluorescence reader (BioTek, Winooski, VT). Goat anti-mouse IgM F(ab')2 fragments (Jackson ImmunoResearch) were then added to a final concentration of 20 µg/ml. Fluo-4 fluorescence was measured at 10-s intervals for an additional 5 min.
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Results
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To obtain B cells with reduced dosage of Btk, BLNK or both, we crossed Btklo mice to BLNK+/ mice. This resulted in progeny of the following genotypes: wild-type, Btklo, Btk/, BLNK+/, BLNK+/Btklo and BLNK+/Btk/. Purified splenic B cells from each type of mouse were subjected to immunoblot analysis to ensure that Btk and BLNK were expressed at the expected levels (Fig. 1). BLNK+/ B cells had about half of the amount of BLNK as BLNK+/+ mice regardless of the amount of Btk they expressed. Similarly, expression of Btk from either the endogenous gene or the transgene was independent of BLNK.

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Fig. 1. Expression of Btk and BLNK are independent of each other. Whole-cell extracts (20 µg) prepared from purified splenic B cells were electrophoresed on a 10% SDSPAGE gel, blotted to nitrocellulose, and probed sequentially with anti-Btk, anti-BLNK and anti-actin antibodies.
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Both BLNK and Btk are required for the immature to mature B transition in the periphery (23,24,3032,35,36). We therefore asked whether partial loss of function of either or both genes would also impair this developmental process. Mature IgMloIgDhi B cells were present at similar relative frequencies and absolute numbers in BLNK+/, Btklo and BLNK+/Btklo mice (Fig. 2A and B) (48). We also did not observe any differences in the frequency of marginal zone, T1, T2 or mature B cells between wild-type and BLNK+/Btklo mice as measured by CD21, CD23 and IgM expression (Fig. 2C).
Both BLNK+/ and Btklo B cells can proliferate upon BCR cross-linking, although their response is reduced relative to that of wild-type B cells (Fig. 3) (48). To determine whether Btk and BLNK work together to transmit this mitogenic signal, the proliferative response of B cells with reduced dosage of both genes was assessed. BLNK+/Btklo B cells did not respond to BCR cross-linking, thus resembling Btk/ B cells. This was not due a general inability to enter the cell cycle, as BLNK+/Btklo B cells responded as well as or better than wild-type cells to phorbol myristate acetate + ionomycin (Fig. 3A). To assess whether the failure of BLNK+/Btklo B cells to proliferate upon BCR stimulation was due to defects in cell survival, entry into S phase or both, we performed cell cycle analysis on B cells stimulated for 48 h with 20 µg/ml anti-IgM (Fig. 3B). Btklo and BLNK+/ B cells each displayed a reduced frequency of cycling cells and an increased percentage of cells with a <2n DNA content indicative of increased cell death. Both defects were further exacerbated in BLNK+/ Btklo B cells. Thus, Btk and BLNK work together to regulate both survival and proliferation of BCR-stimulated B cells. These results are consistent with observations made in the relatively immature B cells from Btk/ (Fig. 3B), xid (51,52) and BLNK/ (16) mice.
This type of genetic experiment alone cannot determine whether Btk and BLNK are components of the same pathway or parallel, independent pathways mediating BCR-induced proliferation. The former possibility predicts that downstream signaling events partially impaired in Btklo B cells would be further decreased in BLNK+/Btklo B cells, while the latter implies that events sensitive to reduced Btk dosage would not be affected by the heterozygous mutation in BLNK. Previous reports have shown that B cells from xid, Btk/ and BLNK/ mice fail to up-regulate bcl-xL and cyclin D2 upon BCR cross-linking (16,5153). It is not clear, however, whether the impaired activation of these pathways results from the immaturity of Btk- or BLNK-deficient B cells or a requirement for Btk and BLNK per se to transmit the BCR signal. We therefore assessed the expression of these genes in Btklo, BLNK+/ and BLNK+/Btklo B cells, which are phenotypically mature (Fig. 2) (48). BCR stimulated expression of bcl-xL and cyclin D2 occurred in Btklo and BLNK+/ B cells, but to a lesser degree than was observed in wild-type B cells (Fig. 4A). This is consistent with the partial defects in proliferative response and survival of these cells (Fig. 3). BLNK+/Btklo B cells resembled Btk/ B cells in their failure to induce either of these genes in response to BCR cross-linking. The increased level of bcl-xL in unstimulated Btk/ B cells is consistent with their immature phenotype (13).


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Fig. 4. BCR-stimulated up-regulation of cyclin D2, bcl-xL and A1 expression is mediated by BLNK and Btk. (A) Whole-cell extracts were prepared from splenic B cells purified from pools of two or three mice per genotype and stimulated with 10 µg/ml anti-IgM F(ab')2 fragments for 18 h. Then 20 µg of extract was electrophoresed on a 12% SDSPAGE gel, blotted to nitrocellulose, and probed consecutively with anti-cyclin D2, anti-bcl-xL and anti-actin antibodies. These results are representative of three independent experiments. (B) cDNA was prepared from splenic B cells purified from pools of two or three mice per genotype and stimulated with 10 µg/ml anti-IgM F(ab')2 fragments for 0 or 1 h. Real-time PCR was performed in triplicate with primers for either A1 or actin. Standard curves were generated for each primer set using serial dilutions of cDNA from stimulated wild-type cells. The relative amount of A1 cDNA in each sample was calculated based on the standard curve, then normalized to the amount of actin to control for cDNA loading. Data are the mean of triplicate samples ± SD and are representative of two independent experiments.
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Both cyclin D2 and bcl-xL are up-regulated relatively late after BCR stimulation. To assess the effect of the B cell signalosome on the regulation of early response genes, we chose the bcl-2 family member A1 as a representative example. This gene was of particular interest for several reasons. A1 can protect B cells from BCR-induced apoptosis (14), which is increased in xid and BLNK/ B cells (16,51). Its expression is regulated by c-rel (14) and calcium-dependent signals from the BCR (54), both of which are impaired in xid (25,55,56) and BLNK/ (16,23,24) B cells. Therefore we used real-time quantitative RT-PCR to assess the dependence of BCR-induced up-regulation of A1 on Btk and BLNK. Like cyclin D2 and bcl-xL, A1 induction was attenuated in Btklo and BLNK+/ B cells, and almost completely impaired by reduced dosage of both genes (Fig. 4b).
A major function of the B cell signalosome is to regulate BCR-induced Ca2+ flux (6,7). In primary mouse B cells, influx of extracellular Ca2+ is required for BCR-stimulated up-regulation of A1 (54) and cyclin D2 (57). We therefore assessed the effect of reduced Btk and/or BLNK levels on BCR-stimulated Ca2+ flux (Fig. 5A). BLNK+/ B cells had relatively normal responses, while reduced dosage of Btk led to a small decrease in the peak Ca2+ level during the initial flux. The latter result is consistent with observations made in B cells from Btk/ mice (58). A more pronounced effect was observed in BLNK+/Btklo cells, with lower than normal responses during both the early and late phases of Ca2+ mobilization.
Activation of the NF-
B pathway is downstream of both major signals regulated by the B cell signalosome, Ca2+ flux (59) and PKCß activation (60). c-rel, a member of the NF-
B family, is required for A1 (14) and bcl-xL transcription (6163). NF-
B activation requires degradation of I
B, which is impaired in BCR-stimulated immature B cells from both Btk/ (55,56) and BLNK/ (16) mice. Taken together, these observations predict that BLNK+/Btklo B cells will demonstrate decreased I
B degradation in response to BCR cross-linking compared to Btklo or BLNK+/ B cells. Indeed, a partial inhibition of I
B
degradation was observed in both Btklo and BLNK+/ B cells (Fig. 5B). This impairment was more severe when the dosage of both Btk and BLNK was reduced.
BCR-induced proliferation requires activation of the ERK pathway (64). The role of the B cell signalosome in this process is controversial. While BCR-stimulated ERK activation is normal in B cells from both Btk/ (58) and BLNK/ mice (16), it is reduced in Btk/ DT-40 cells (27) and completely impaired in BLNK/ DT-40 cells (9). To determine whether these contradictory results stem from the relative immaturity of B cells from Btk/ and BLNK/ mice and/or a redundant function of BLNK and Btk in this pathway in murine cells, we assessed ERK activation in wild-type and BLNK+/Btklo B cells (Fig. 5C). BCR-stimulated ERK phosphorylation was unimpaired in BLNK+/Btklo B cells. Thus, the B cell signalosome does not contribute significantly to the activation of this pathway in mature mouse B cells.
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Discussion
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The present study demonstrates that Btk and BLNK are limiting components of a common signaling pathway that mediates mitogenic signals from the BCR in mature B cells. The functional consequences of their interaction can be observed at both the cellular and molecular level. These studies emphasize the utility of gene dosage/compound heterozygote analysis to delineate mammalian signaling pathways both genetically and biochemically. A similar strategy has recently been used by others to demonstrate interactions among signaling molecules in other B cell processes, including the regulation of early B lymphopoiesis by helix-loop-helix transcription factors (65,66), and the inhibition of BCR signals by CD22, Lyn and SH2 domain-containing tyrosine phosphatase 1 (67).
Reduced dosage of BLNK alone had significant effects on many of the aspects of the B cell response to BCR cross-linking measured here. Recently, Gong et al. demonstrated that a heterozygous mutation in the adaptor protein Grb2 leads to attenuated TCR-induced activation of JNK and p38, but not ERK (68). This results in impaired negative selection during thymocyte development. Clearly, even subtle changes in the stoichiometry of linker proteins relative to other components of the signaling complexes they nucleate can have significant effects on the consequences of those signals. Regulated expression of such limiting signaling molecules may be a major mechanism for modulating signal outcome (69). Indeed, increased expression of several signalosome components is correlated with a reduced threshold for BCR stimulation and a unique pattern of BCR-induced gene expression in immature B cells (45,70).
Failure to induce expression of A1, bcl-xL and cyclin D2, which regulate cell survival and cell cycle entry, would be sufficient to explain the impaired proliferation and survival of BLNK+/Btklo cells upon antigen stimulation. A1 and cyclin D2 are likely critical components of Btk signaling pathways as the xid phenotype is rescued only partially by transgenes expressing bcl-xL (51) or bcl-2 (71). It is probable that the B cell signalosome regulates A1, bcl-xL and cyclin D2 expression via its effects on BCR-induced Ca2+ flux and/or NF-
B activation (14, 54,57,5963) (Fig. 5). Interestingly, the effect of reducing Btk and BLNK levels on BCR-induced Ca2+ flux (Fig. 5) was subtler than that observed for expression of A1, bcl-xL and cyclin D2 (Fig. 4). It is possible that small changes in early signaling events, such as Ca2+ flux, are amplified into larger effects on more downstream processes, such as transcriptional responses. Alternatively, Btk and/or BLNK may regulate gene expression by Ca2+-independent mechanisms as well. These are unlikely to involve the Ras/ERK pathway, however, as we saw no difference between wild-type and BLNK+/Btklo B cells with respect to ERK activation (Fig. 5). The conflicting results regarding the requirement for B cell signalosome components in BCR-stimulated ERK activation in primary murine cells (16,58) versus chicken DT-40 cells (9,27) may be due to species-specific effects, differences between primary resting cells and continuously proliferating cell lines or differences in the maturation state of the cells examined.
A tonic BCR signal is required for the maintenance of the mature peripheral B cell population (72). The presence of normal numbers of IgMloIgDhi mature B cells indicates that this signal is intact and exceeds a minimal threshold in BLNK+/Btklo mice. BCR-initiated activation and proliferation of mature B cells is thus significantly more sensitive to subtle changes in Btk/BLNK signal strength than is BCR-mediated B cell development and homeostasis. Alternatively, Btk and BLNK may function in concert to control the activation of mature B cells but independently to regulate their development and/or survival. These studies also demonstrate that signals regulating the maintenance of mature B cell numbers are independent of the ability of the BCR to induce A1, bcl-xL and cyclin D2. Consistent with these observations, mice lacking either c-rel (73), which is required for bcl-xL (61) and A1 (14) expression, or cyclin D2 (74,75) have normal B cell development but, impaired mitogenic responses to BCR cross-linking. Further studies are warranted to define the Btk/BLNK mediated signaling events governing the immature to mature B cell transition.
Loss of Btk function results in the B cell immunodeficiency XLA in humans (38,39). The severity of disease in XLA differs significantly with genetic background (76). More than one-third of the alterations in Btk associated with XLA are missense or other in-frame mutations (77). Many of these are likely to have some residual function, behaving as weak alleles analogous to the Btklo transgene. Polymorphisms resulting in subtle changes in the expression level or activity of components of Btk signaling pathways may be clinically insignificant on their own, but lead to severe XLA in individuals carrying partial loss of function mutations in Btk.
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Acknowledgements
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We thank Charles Nguyen for assistance with real time RT-PCR and Dr David Karp for critical reading of the manuscript. A. B. S. was supported in part by a Special Fellowship from the Leukemia and Lymphoma Society (formerly the Leukemia Society of America). A. B. S is the Southwestern Medical Foundation Scholar in Biomedical Research and an Investigator of the Simmons Arthritis Research Center. A. C. C. was supported by NIH R01AI42787 and was an Associate Investigator of the Howard Hughes Medical Institute. O. N. W. is an Investigator of the Howard Hughes Medical Institute.
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Abbreviations
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BLNKB cell linker protein
BtkBrutons tyrosine kinase
Btklomice expressing 25% of endogenous Btk levels in B cells
ERKextracellular signal-regulated kinase
Grb2growth factor receptor-binding protein
JNKc-Jun N-terminal kinase
PEphycoerythrin
PKCprotein kinase C
PLCphospholipase C
SH2Src homology 2
xidX-linked immunodeficiency
XLAX-linked agammaglobulinemia
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References
|
---|
- Goodnow, C. C. 1996. Balancing immunity and tolerance: deleting and tuning lymphocyte repertoires. Proc. Natl Acad. Sci. USA 93:2264.[Abstract/Free Full Text]
- Reth, M. and Wienands, J. 1997. Initiation and processing of signals from the B cell antigen receptor. Annu. Rev. Immunol. 15:453.[CrossRef][ISI][Medline]
- DeFranco, A. L. 1997. The complexity of signaling pathways activated by the BCR. Curr. Opin. Immunol. 9:296.[CrossRef][ISI][Medline]
- Kurosaki, T. 1999. Genetic analysis of B cell antigen receptor signaling. Annu. Rev. Immunol. 17:555.[CrossRef][ISI][Medline]
- Cambell, K. S. 1999. Signal transduction from the B cell antigen receptor. Curr. Opin. Immunol. 11:256.[CrossRef][ISI][Medline]
- Fruman, D. A., Satterthwaite, A. B. and Witte, O. N. 2000. Xid-like phenotypes: a B cell signalosome takes shape. Immunity 13:1.[ISI][Medline]
- Kurosaki, T. and Tsukada, S. 2000. BLNK: connecting Syk and Btk to calcium signals. Immunity 12:1.[ISI][Medline]
- Fu, C., Turck, C. W., Kurosaki, T. and Chan, A. C. 1998. BLNK: a central linker protein in B cell activation. Immunity 9:93.[ISI][Medline]
- Ishiai, M., Kurosaki, M., Pappu, R., Okawa, K., Ronko, I., Fu, C., Shibata, M., Iwamatsu, A., Chan, A. C. and Kurosaki, T. 1999. BLNK required for coupling Syk to PLC gamma 2 and Rac1-JNK in B cells. Immunity 10:117.[ISI][Medline]
- Ishiai, M., Sugawara, H., Kurosaki, M. and Kurosaki, T. 1999. Cutting edge: association of phospholipase C-gamma 2 Src homology 2 domains with BLNK is critical for B cell antigen receptor signaling. J. Immunol. 163:1746.[Abstract/Free Full Text]
- Hashimoto, S., Iwamatsu, A., Ishiai, M., Okawa, K., Yamadori, T., Matsushita, M., Baba, Y., Kishimoto, T., Kurosaki, T. and Tsukada, S. 1999. Identification of the SH2 domain binding protein of Brutons tyrosine kinase as BLNKfunctional significance of Btk-SH2 domain in B-cell antigen receptor-coupled calcium signaling. Blood 94:2357.[Abstract/Free Full Text]
- Solvason, N., Wu, W. W., Kabra, N., Wu, X., Lees, E. and Howard, M. C. 1996. Induction of cell cycle regulatory proteins in anti-immunoglobulin-stimulated mature B lymphocytes. J. Exp. Med. 184:407.[Abstract]
- Grillot, D. A., Merino, R., Pena, J. C., Fanslow, W. C., Finkelman, F. D., Thompson, C. B. and Nunez, G. 1996. bcl-x exhibits regulated expression during B cell development and activation and modulates lymphocyte survival in transgenic mice. J. Exp. Med. 183:381.[Abstract]
- Grumont R. J., Rourke, I. J. and Gerondakis, S. 1999. Rel-dependent induction of A1 transcription is required to protect B cells from antigen receptor ligation-induced apoptosis. Genes Dev. 13:400.[Abstract/Free Full Text]
- Baba, Y., Hashimoto, S., Matsushita, M., Watanabe, D., Kishimoto, T., Kurosaki, T. and Tsukada, S. 2001. BLNK mediates Syk-dependent Btk activation. Proc. Natl Acad. Sci. USA 98:2582.[Abstract/Free Full Text]
- Tan, J. E., Wong, S. C., Gan, S. K., Xu, S. and Lam, K. P. 2001. The adaptor protein BLNK is required for B cell antigen receptor-induced activation of nuclear factor-kappa B and cell cycle entry and survival of B lymphocytes. J. Biol. Chem. 276:20055.[Abstract/Free Full Text]
- Novina, C. D., Kumar, S., Bajpai, U., Cheriyath, V., Zhang, K., Pillai, S., Wortis, H. H. and Roy, A. L. 1999. Regulation of nuclear localization and transcriptional activity of TFII-I by Brutons tyrosine kinase. Mol. Cell. Biol. 19:5014.[Abstract/Free Full Text]
- Mahajan, S., Vassilev, A., Sun, N., Ozer, Z., Mao, C. and Uckun, F. M. 2001. Transcription factor STAT5A is a substrate of Brutons tyrosine kinase in B cells. J. Biol. Chem. 276:31216.[Abstract/Free Full Text]
- Webb, C. F., Yamashita, Y., Ayers, N., Evetts, S., Paulin, Y., Conley, M. E. and Smith, E. A. 2000. The transcription factor Bright associates with Brutons tyrosine kinase, the defective protein in immunodeficiency disease. J. Immunol. 165:6956.[Abstract/Free Full Text]
- Takata, M., Sabe, H., Hata, A., Inazu, T., Homma, Y., Nukada, T., Yamamura, H. and Kurosaki, T. 1994. Tyrosine kinases Lyn and Syk regulate B cell receptor-coupled Ca2+ mobilization through distinct pathways. EMBO J. 13:1341.[Abstract]
- Takata, M. and Kurosaki, T. 1996. A role for Brutons tyrosine kinase in B cell antigen receptor-mediated activation of phospholipase C-gamma 2. J. Exp. Med. 184:31.[Abstract]
- Kurosaki, T. and Kurosaki, M. 1997. Transphosphorylation of Brutons tyrosine kinase on tyrosine 551 is critical for B cell antigen receptor function. J. Biol. Chem. 272:15595.[Abstract/Free Full Text]
- Pappu, R., Cheng, A. M., Li, B., Gong, Q., Chiu, C., Griffin, N., White, M., Sleckman, B. P. and Chan, A. C. 1999. Requirement for B cell linker protein (BLNK) in B cell development. Science 286:1949.[Abstract/Free Full Text]
- Jumaa, H., Wollscheid, B., Mitterer, M., Wienands, J., Reth, M. and Nielsen, P. J. 1999. Abnormal development and function of B lymphocytes in mice deficient for the signaling adaptor protein SLP-65. Immunity 11:547.[ISI][Medline]
- Rigley, K. P., Harnett, M. M., Phillips, R. J. and Klaus, G. G. 1989. Analysis of signaling via surface immunoglobulin receptors on B cells from CBA/N mice. Eur. J. Immunol. 19:2081.[ISI][Medline]
- Fluckiger, A. C., Li, Z., Kato, R. M., Wahl, M. I., Ochs H. D., Longnecker, R., Kinet, J. P., Witte, O. N., Scharenberg, A. M. and Rawlings, D. J. 1998. Btk/Tec kinases regulate sustained increases in intracellular Ca2+ following B-cell receptor activation. EMBO J. 17:1973.[Free Full Text]
- Jiang, A., Craxton, A., Kurosaki, T. and Clark, E. A. 1998. Different protein tyrosine kinases are required for B cell antigen receptor-mediated activation of extracellular signal-regulated kinase, c-Jun NH2-terminal kinase 1, and p38 mitogen-activated protein kinase. J. Exp. Med. 188:1297.[Abstract/Free Full Text]
- Rawlings, D. J., Saffran, D. C., Tsukada, S., Largaespada, D. A., Grimaldi, J. C., Cohen, L., Mohr, R. N., Bazan, J. F., Howard, M., Copeland, N. G., et al. 1993. Mutation of unique region of Brutons tyrosine kinase in immunodeficient XID mice. Science 261:358.[ISI][Medline]
- Thomas, J. D., Sideras, P., Smith, C. I., Vorechovsky, I., Chapman, V. and Paul, W. E. 1993. Colocalization of X-linked agammaglobulinemia and X-linked immunodeficiency genes. Science 261:355.[ISI][Medline]
- Khan, W. N., Alt, F. W., Gerstein, R. M., Malynn, B. A., Larsson, I., Rathbun, G., Davidson, L., Muller, S., Kantor, A. B., Herzenberg, L. A., et al. 1995. Defective B cell development and function in Btk-deficient mice. Immunity 3:283.[ISI][Medline]
- Kerner, J. D., Appleby, M. W., Mohr, R. N., Chien, S., Rawlings, D. J., Maliszewski, C. R., Witte, O. N. and Perlmutter R. M. 1995. Impaired expansion of mouse B cell progenitors lacking Btk. Immunity 3:301.[ISI][Medline]
- Hendriks, R. W., de Bruijn, M. F., Maas, A., Dingjan, G. M., Karis, A. and Grosveld, F. 1996. Inactivation of Btk by insertion of lacZ reveals defects in B cell development only past the pre-B cell stage. EMBO J. 15:4862.[Abstract]
- Wang, D., Feng, J., Wen, R., Marine, J. C., Sangster, M. Y., Parganas, E., Hoffmeyer, A., Jackson, C. W., Cleveland, J. L., Murray, P. J. and Ihle, J. N. 2000. Phospholipase Cgamma2 is essential in the functions of B cell and several Fc receptors. Immunity 13:25.[ISI][Medline]
- Hashimoto, A., Takeda, K., Inaba, M., Sekimata, M., Kaisho, T., Ikehara, S., Homma, Y., Akira, S. and Kurosaki, T. 2000. Cutting edge: essential role of phospholipase C-gamma 2 in B cell development and function. J. Immunol. 165:1738.[Abstract/Free Full Text]
- Xu, S., Tan, J. E., Wong, E. P., Manickam, A., Ponniah, S. and Lam, K. P. 2000. B cell development and activation defects resulting in xid-like immunodeficiency in BLNK/SLP-65-deficient mice. Int. Immunol. 12:397.[Abstract/Free Full Text]
- Hayashi, K., Nittono, R., Okamoto, N., Tsuji, S., Hara, Y., Goitsuka, R. and Kitamura, D. 2000. The B cell-restricted adaptor BASH is required for normal development and antigen receptor-mediated activation of B cells. Proc. Natl Acad. Sci. USA 97:2755.[Abstract/Free Full Text]
- Minegishi, Y., Rohrer, J., Coustan-Smith, E., Lederman, H. M., Pappu, R., Campana, D., Chan, A. C. and Conley, M. E. 1999. An essential role for BLNK in human B cell development. Science 286:1954.[Abstract/Free Full Text]
- Tsukada, S., Saffran, D. C., Rawlings, D. J., Parolini, O., Allen, R. C., Klisak, I., Sparkes, R. S., Kubagawa, H., Mohandas, T., Quan, S., et al. 1993. Deficient expression of a B cell cytoplasmic tyrosine kinase in human X-linked agammaglobulinemia. Cell 72:279.[ISI][Medline]
- Vetrie, D., Vorechovsky, I., Sideras, P., Holland, J., Davies, A., Flinter, F., Hammarstrom, L., Kinnon, C., Levinsky, R., Bobrow, M., et al. 1993. The gene involved in X-linked agammaglobulinaemia is a member of the src family of protein-tyrosine kinases. Nature 361:226.[CrossRef][ISI][Medline]
- Cheng A. M., Rowley, B., Pao, W., Hayday, A., Bolen, J. B. and Pawson, T. 1995. Syk tyrosine kinase required for mouse viability and B-cell development. Nature 378:303.[CrossRef][ISI][Medline]
- Turner, M., Mee. P. J., Costello, P. S., Williams, O., Price, A. A., Duddy, L. P., Furlong, M. T., Geahlen, R. L. and Tybulewicz, V. L. 1995. Perinatal lethality and blocked B-cell development in mice lacking the tyrosine kinase Syk. Nature 378:298.[CrossRef][ISI][Medline]
- Guo, B., Kato, R. M., Garcia-Lloret, M., Wahl, M. I. and Rawlings, D. J. 2000. Engagement of the human pre-B cell receptor generates a lipid raft-dependent calcium signaling complex. Immunity 13:243.[ISI][Medline]
- Jumaa, H., Mitterer, M., Reth, M. and Nielsen, P. J. 2001. The absence of SLP65 and Btk blocks B cell development at the preB cell receptor-positive stage. Eur. J. Immunol. 31:2164.[CrossRef][ISI][Medline]
- Yellen, A. J., Glenn, W., Sukhatme, V. P., Cao, X. M. and Monroe, J. G. 1991. Signaling through surface IgM in tolerance-susceptible immature murine B lymphocytes. Developmentally regulated differences in transmembrane signaling in splenic B cells from adult and neonatal mice. J. Immunol. 146:1446.[Abstract/Free Full Text]
- Benschop R. J., Melamed, D., Nemazee, D. and Cambier, J. C. 1999. Distinct signal thresholds for the unique antigen receptor-linked gene expression programs in mature and immature B cells. J. Exp. Med. 190:749.[Abstract/Free Full Text]
- Simon, M. A., Bowtell, D. D., Dodson, G. S., Laverty, T. R. and Rubin, G. M. 1991. Ras1 and a putative guanine nucleotide exchange factor perform crucial steps in signaling by the sevenless protein tyrosine kinase. Cell 67:701.[ISI][Medline]
- Satterthwaite A. B., Willis, F., Kanchanastit, P., Fruman, D., Cantley, L. C., Helgason, C. D., Humphries, R. K., Lowell, C. A., Simon, M., Leitges, M., Tarakhovsky, A., Tedder, T. F., Lesche, R., Wu, H. and Witte, O. N. 2000. A sensitized genetic system for the analysis of murine B lymphocyte signal transduction pathways dependent on Brutons tyrosine kinase. Proc. Natl Acad. Sci. USA 97:6687.[Abstract/Free Full Text]
- Satterthwaite, A. B., Cheroutre, H., Khan, W. N., Sideras, P. and Witte, O. N. 1997. Btk dosage determines sensitivity to B cell antigen receptor cross-linking. Proc. Natl Acad. Sci. USA 94:13152.[Abstract/Free Full Text]
- Forrester, L. M., Ansell, J. D. and Micklem, H. S. 1987. Development of B lymphocytes in mice heterozygous for the X-linked immunodeficiency (xid) mutation. xid inhibits development of all splenic and lymph node B cells at a stage subsequent to their initial formation in bone marrow. J. Exp. Med. 165:949.[Abstract]
- Tomayko, M. M. and Cancro, M. P. 1998. Long-lived B cells are distinguished by elevated expression of A1. J. Immunol. 160:107.[Abstract/Free Full Text]
- Solvason, N., Wu, W. W., Kabra, N., Lund-Johansen, F., Roncarolo, M. G., Behrens, T. W., Grillot, D. A., Nunez, G., Lees, E. and Howard, M. 1998. Transgene expression of bcl-xL permits anti-immunoglobulin (Ig)-induced proliferation in xid B cells. J. Exp. Med. 187:1081.[Abstract/Free Full Text]
- Anderson, J. S., Teutsch, M., Dong, Z. and Wortis, H. H.. 1996. An essential role for Brutons tyrosine kinase in the regulation of B-cell apoptosis. Proc. Natl Acad. Sci. USA 93:10966.[Abstract/Free Full Text]
- Brorson, K., Brunswick, M., Ezhevsky, S., Wei, D. G., Berg, R., Scott, D. and Stein, K. E. 1997. xid affects events leading to B cell cycle entry. J. Immunol. 159:135.[Abstract]
- Glynne, R., Akkaraju, S., Healy, J. I., Rayner, J., Goodnow, C. C. and Mack, D. H. 2000. How self-tolerance and the immunosuppressive drug FK506 prevent B-cell mitogenesis. Nature 403:672.[CrossRef][ISI][Medline]
- Bajpai, U. D., Zhang, K., Teutsch, M., Sen, R. and Wortis, H. H. 2000. Brutons tyrosine kinase links the B cell receptor to nuclear factor kappaB activation. J. Exp. Med. 191:1735.[Abstract/Free Full Text]
- Petro, J. B., Rahman, S. M., Ballard, D. W. and Khan, W. N. 2000. Brutons tyrosine kinase is required for activation of IkappaB kinase and nuclear factor kappaB in response to B cell receptor engagement. J. Exp. Med. 191:1745.[Abstract/Free Full Text]
- Glassford, J., Holman, M., Banerji, L., Clayton, E., Klaus, G. G., Turner, M. and Lam, E. W. 2001. Vav is required for cyclin D2 induction and proliferation of mouse B lymphocytes activated via the antigen receptor. J. Biol. Chem. 276:41040.[Abstract/Free Full Text]
- Forssell, J., Nilsson, A. and Sideras, P. 2000. Reduced formation of phosphatidic acid upon B-cell receptor triggering of mouse B-lymphocytes lacking Brutons tyrosine kinase. Scand. J. Immunol. 52:30.[CrossRef][ISI][Medline]
- Petro, J. B. and Khan, W. N. 2001. Phospholipase C-gamma 2 couples Brutons tyrosine kinase to the NF-kappaB signaling pathway in B lymphocytes. J. Biol. Chem. 276:1715.[Abstract/Free Full Text]
- Su, T. T., Guo, B., Kawakami, Y., Sommer, K., Chae, K., Humphries, L. A., Kato, R. M., Kang, S., Patrone, L., Wall, R., Teitell, M., Leitges, M., Kawakami, T. and Rawlings D. J. 2002. PKC-beta controls I kappa B kinase lipid raft recruitment and activation in response to BCR signaling. Nat. Immunol. 3:780.[ISI][Medline]
- Lee, H. H., Dadgostar, H., Cheng, Q., Shu, J. and Cheng, G. 1999. NF-kappaB-mediated up-regulation of Bcl-x and Bfl-1/A1 is required for CD40 survival signaling in B lymphocytes. Proc. Natl Acad. Sci. USA 96:9136.[Abstract/Free Full Text]
- Chen, C, Edelstein, L. C. and Gelinas, C. 2000. The Rel/NF-kappaB family directly activates expression of the apoptosis inhibitor Bcl-x(L). Mol. Cell. Biol. 20:2687.[Abstract/Free Full Text]
- Owyang, A. M., Tumang, J. R., Schram, B. R., Hsia, C. Y., Behrens, T. W., Rothstein, T. L. and Liou, H. C. 2001. c-Rel is required for the protection of B cells from antigen receptor-mediated, but not Fas-mediated, apoptosis. J. Immunol. 167:4948.[Abstract/Free Full Text]
- Richards, J. D., Dave, S. H., Chou, C. H., Mamchak, A. A. and DeFranco, A. L. 2001. Inhibition of the MEK/ERK signaling pathway blocks a subset of B cell responses to antigen. J. Immunol. 166:3855.[Abstract/Free Full Text]
- Zhuang, Y., Cheng, P. and Weintraub, H. 1996. B-lymphocyte development is regulated by the combined dosage of three basic helix-loop-helix genes, E2A, E2-2, and HEB. Mol. Cell. Biol. 16:2898.[Abstract]
- ORiordan, M. and Grosschedl, R. 1999. Coordinate regulation of B cell differentiation by the transcription factors EBF and E2A. Immunity 11:21.[ISI][Medline]
- Cornall, R. J., Cyster, J. G., Hibbs, M. L., Dunn, A. R., Otipoby, K. L., Clark, E. A, and Goodnow, C. C. 1998. Polygenic autoimmune traits: Lyn, CD22, and SHP-1 are limiting elements of a biochemical pathway regulating BCR signaling and selection. Immunity 8:497.[ISI][Medline]
- Gong, Q., Cheng, A. M., Akk, A. M., Alberola-Ila, J., Gong, G., Pawson, T. and Chan, A. C. 2001. Disruption of T cell signaling networks and development by Grb2 haploid insufficiency. Nat. Immunol. 2:29.[CrossRef][ISI][Medline]
- Kelley, M. E. and Chan, A. C. 2000. Regulation of B cell function by linker proteins. Curr. Opin. Immunol. 12:267.[CrossRef][ISI][Medline]
- Benschop, R. J., Brandl, E., Chan, A. C. and Cambier, J. C. 2001. Unique signaling properties of B cell antigen receptor in mature and immature B cells: implications for tolerance and activation. J. Immunol. 167:4172.[Abstract/Free Full Text]
- Woodland, R. T., Schmidt, M. R., Korsmeyer, S. J. and Gravel, K. A. 1996. Regulation of B cell survival in xid mice by the proto-oncogene bcl-2. J. Immunol. 156:2143.[Abstract]
- Lam K. P., Kuhn, R. and Rajewsky, K. 1997. In vivo ablation of surface immunoglobulin on mature B cells by inducible gene targeting results in rapid cell death. Cell 90:1073.[ISI][Medline]
- Kontgen, F., Grumont, R. J., Strasser, A., Metcalf, D., Li, R., Tarlinton, D. and Gerondakis, S. 1995. Mice lacking the c-rel proto-oncogene exhibit defects in lymphocyte proliferation, humoral immunity, and interleukin-2 expression. Genes Dev. 9:1965.[Abstract]
- Solvason, N., Wu, W. W., Parry, D., Mahony, D., Lam, E. W., Glassford, J., Klaus, G. G., Sicinski, P., Weinberg, R., Liu, Y. J., Howard, M. and Lees, E. 2000. Cyclin D2 is essential for BCR-mediated proliferation and CD5 B cell development. Int. Immunol. 12:631.[Abstract/Free Full Text]
- Lam, E. W., Glassford J., Banerji, L., Thomas, N. S., Sicinski, P. and Klaus, G. G. 2000. Cyclin D3 compensates for loss of cyclin D2 in mouse B-lymphocytes activated via the antigen receptor and CD40. J. Biol. Chem. 275:3479.[Abstract/Free Full Text]
- Bykowsky, M. J., Haire, R. N., Ohta, Y., Tang, H., Sung, S. S., Veksler, E. S., Greene, J. M., Fu, S. M., Litman, G. W. and Sullivan, K. E. 1996. Discordant phenotype in siblings with X-linked agammaglobulinemia. Am. J. Hum. Genet. 58:477.[ISI][Medline]
- Vihinen, M., Kwan, S. P., Lester, T., Ochs, H. D., Resnick, I., Valiaho, J., Conley, M. E. and Smith, C. I. 1999. Mutations of the human BTK gene coding for bruton tyrosine kinase in X-linked agammaglobulinemia. Hum. Mutat. 13:280.[CrossRef][ISI][Medline]