Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M314273200 on February 17, 2004

J. Biol. Chem., Vol. 279, Issue 19, 19523-19530, May 7, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/19/19523    most recent
M314273200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Guilbault, B.
Right arrow Articles by Kay, R. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Guilbault, B.
Right arrow Articles by Kay, R. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

RasGRP1 Sensitizes an Immature B Cell Line to Antigen Receptor-induced Apoptosis*

Benoit Guilbault{ddagger} and Robert J. Kay§

From the Terry Fox Laboratory, British Columbia Cancer Agency, and the Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia V5Z 4E6, Canada

Received for publication, December 29, 2003 , and in revised form, February 12, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
RasGRP1 is a guanine nucleotide exchange factor that activates Ras GTPases and is activated downstream of antigen receptors on both T and B lymphocytes. Ras-GRP1 provides signals to immature T cells that confer survival and proliferation, but RasGRP1 also promotes T cell receptor-mediated deletion of mature T cells. We used the WEHI-231 cell line as an experimental system to determine whether RasGRP1 can serve as a quantitative modifier of B cell receptor-induced deletion of immature B cells. A 2-fold elevation in RasGRP1 expression markedly increased apoptosis of WEHI-231 cells following B cell receptor ligation, whereas a dominant negative mutant of RasGRP1 suppressed B cell receptor-induced apoptosis. Activation of ERK1 or ERK2 kinases was not required for RasGRP1-mediated apoptosis. Instead, elevated RasGRP1 expression caused down-regulation of NF-{kappa}B and Bcl-xL, which provide survival signals counter-acting apoptosis induction by B cell receptor. Inhibition of NF-{kappa}B was sufficient to enhance B cell receptor-induced apoptosis of WEHI-231 cells, and ligation of co-stimulatory receptors that activate NF-{kappa}B suppressed the ability of RasGRP1 to promote B cell receptor-induced apoptosis. These experiments define a novel apoptosis-promoting pathway leading from B cell receptor to the inhibition of NF-{kappa}B and demonstrate that differential expression of RasGRP1 has the potential to modulate the sensitivities of B cells to negative selection following antigen encounter.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
RasGRP1 is a guanine nucleotide exchange factor that transduces intracellular signaling by converting Ras GTPases to their active GTP-bound states (1–6). RasGRP1 is expressed most prominently in thymocytes and mature T cells but is also expressed in some immature B cell lines (2, 3, 5, 6). RasGRP1 is positively regulated by membrane translocation, which is driven by binding of its C1 domain to diacylglycerol (3, 4, 6–9). This provides a mechanism for RasGRP1 activation by any receptor that couples to diacylglycerol-generating phospholipase Cs, including the T cell receptor (TCR)1 (4) and the B cell receptor (BCR) (8).

Recent work with genetically modified mice has demonstrated that RasGRP1 provides signals that enhance the survival and proliferation of T cells at several stage during their development and immunological selection. RasGRP1 is transcriptionally induced by pre-TCR signaling, and the resulting increase in RasGRP1 expression is sufficient to confer survival and proliferation in the absence of other pre-TCR-derived signals (10). RasGRP1 expression augments the effectiveness with which {alpha}{beta}TCR/major histocompatibility complex interactions confer survival and maturation through the process of positive selection (11) and increases the proliferative responses of thymic T cells to TCR ligation (10–12). RasGRP1 deficiency does not noticeably alter the efficiency of TCR-dependent deletion (negative selection) of thymic T cells by a self-antigen (11) but impedes apoptosis induction in mature splenic T cells following TCR ligation (13). These experiments portray RasGRP1 as a promoter of survival and proliferation responses to pre-TCR or TCR during intrathymic T cell development but with the converse role of mediating TCR-induced deletion in mature peripheral T cells.

B cell development in the bone marrow is also governed by a series of selections for immunologically competent but not self-reactive antigen receptors. To avoid auto-immunity, immature B cells carrying BCR that recognize self-antigen need to be suppressed either by anergization, by respecification through receptor editing, or by deletion through BCR-induced apoptosis (14–16). Much of our knowledge of the signal transduction pathways that mediate BCR-induced deletion of immature B cells has been obtained from experiments with WEHI-231 cells, a B lymphoma-derived murine cell line that resembles immature B cells both phenotypically and functionally (17). WEHI-231 cells express surface IgM of unknown antigen specificity, but cross-linking with anti-IgM mimics the interaction of this BCR with a high affinity antigen. Anti-IgM-initiated signaling imposes cell cycle arrest on WEHI-231 cells within a few hours, and maintenance of BCR ligation beyond 1 day results in apoptosis. BCR-induced deletion of WEHI-231 cells shares many of the regulatory mechanisms relevant to immunological negative selection of primary B cells, including suppression by the co-stimulators CD40 ligand or lipopolysaccharide (LPS) (18, 19). The regulatory roles of Ras GTPases and Ras-specific exchange factors in BCR-induced deletion of WEHI-231 cells are unknown.

We used WEHI-231 cells to test the potential of differential expression of RasGRP1 to modify cell cycle arrest and apoptosis responses to BCR signaling. A doubling of RasGRP1 protein levels conferred hypersensitivity to deletion following BCR ligation. The cells with elevated RasGRP1 expression became apoptotic more rapidly and were severely depleted from mixed populations after a 2-day exposure to anti-IgM. Expression of a dominant negative form of RasGRP1 suppressed apoptosis induction following BCR ligation. The WEHI-231 cells with elevated RasGRP1 expression had reduced NF-{kappa}B activity, and inhibition of NF-{kappa}B was sufficient to hypersensitize WEHI-231 cells to apoptosis induction following BCR ligation. Our experiments demonstrate that differential expression of RasGRP1 has the potential to acutely determine B cell responses to BCR ligation. But in contrast to what has been found in immature T cells undergoing pre-TCR or TCR-mediated selection, Ras-GRP1 promotes deletion rather than survival in the WEHI-231 model for BCR-mediated selection of immature B cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and Reagents—WEHI-231 cells were obtained from Mike Gold (University of British Columbia) and cultured in RPMI medium supplemented with 10% fetal bovine serum and 50 µM {beta}-mercaptoethanol (complete medium). LPS was from Sigma. Bay 11-7082 as well as the MEK inhibitors PD98059 and U0126 were from Calbiochem. Anti-mouse CD40 and anti-human Thy-1 were from Pharmingen, goat anti-mouse IgM was from Jackson Immunoresearch, and anti-HA was from BABCO/Covance. Antibodies detecting extracellular signal-regulated kinase (ERK)-1/2 and phosphorylated ERK1/2 were from Cell Signaling Technology. The anti-RasGRP1 antibody (199) was from Santa Cruz. The specificity of this antibody for RasGRP1 has been demonstrated in RasGRP1-deficient thymocytes (11). Antibodies against I{kappa}B{alpha} (C-21) and Bcl-xL were from Santa Cruz and BD Transduction Laboratories, respectively, and their specificities have also been confirmed by analyses of genetically deficient cells (20, 21). CFSE was from Molecular Probes.

cDNA Constructs and Retroviral Transduction of WEHI-231 Cells— The full-length HA-tagged RasGRP1 cDNA is identical to the XFL construct previously described (6). The full-length, HA-tagged R271E, EF hand-deleted, and C1 domain-deleted forms of RasGRP1 were derived from the GEFµ, {Delta}C1+, and EF{Delta} mutants (6). HA-tagged, wild-type, and G12V mutants of Ras GTPases were constructed in our lab. The wild-type Ras GTPases had a GFP N-terminal fusion, which suppresses cellular responses to Ras activation downstream signaling while retaining the ability to bind RafRBD when GTP-bound.2 M-Ras Q71L was a generous gift of L. Quilliam (Indiana University). The Bcl-xL cDNA was a generous gift of C. Thompson (University of Pennsylvania). Retroviral vectors were derivatives of CTV vectors (22), with or without IRES-mediated expression of GFP or of the extracellular domain of human Thy-1. Transfection of ecotropic packaging cells and viral infection was performed as described (23, 24). After infection, transduced cells were first selected for drug resistance and then further selected for co-expression of human Thy-1 or GFP by sorting on a FacsVantage flow cytometer (BD Biosciences).

Stimulation of Cells with anti-IgM and Reversal of Apoptosis Induction by Trypsin—WEHI-231 cells in complete medium were stimulated through BCR by the addition of anti-IgM at the concentrations indicated. To reverse the apoptosis induction signal provided by anti-IgM, the cells were rinsed in PBS, treated with 0.025% trypsin in PBS for 10 min at 20 °C, rinsed with PBS, and returned to culture in complete medium. The mechanism by which trypsin treatment reverses the induction of apoptosis that is initiated by anti-IgM treatment is unknown. Trypsinization does not result in the immediate removal of anti-IgM from the cell surface but could cause peptide cleavages that reduce the extent of cross-linking within surface IgM-anti-IgM complexes.

Apoptosis Detection Using Propidium Iodide (PI) Staining of Fixed Nuclei—WEHI-231 cells stimulated as indicated were rinsed in PBS containing 0.1% glucose and fixed in 70% ethanol at 4 °C for at least 24 h. The cells were then rinsed in PBS and stained with PBS containing 5 µg/ml propidium iodide and 200 µg/ml RNaseA overnight in the dark at 20 °C. The cells were then analyzed for PI content by flow cytometry using a FacsCalibur cytometer and Cellquest software (BD Biosciences).

Cell Division Analysis Using CFSE Labeling—WEHI-231 cells at 105 cells/ml were cultured with or without anti-IgM for 24 h. The cells were then treated with 0.025% trypsin as described above to reverse anti-IgM treatment and plated in fresh medium for 30 min. Up to 2 x 107cells were incubated in 2 ml of PBS containing 9 µM CFSE for 10 min at 37 °C, rinsed twice in ice-cold PBS, and incubated overnight at 37 °C. The cells were then sorted, cultured for the indicated time and analyzed by flow cytometry.

Preparation of Protein Lysates—WEHI-231 cells were cultured in serum-containing medium at 5 to 10 x105 cells/ml, treated as indicated, rinsed with PBS, and lysed in ice-cold Mg+ lysis buffer (25 mM HEPES, pH 7.2, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 10% glycerol, 25 mM NaF, 10 mM MgCl2, 1 mM EDTA, 1 mM sodium vanadate, 1 mM sodium molybdate, plus protease inhibitors). The cytoplasmic fractions were isolated by preparing lysates in 50 mM HEPES, 100 mM NaF, 10 mM sodium pyrophosphate, 2 mM sodium vanadate, 4 mM EDTA, 0.2% Nonidet P-40, 10 mM MgCl2 containing protease inhibitors (25). Total or cytoplasmic lysates were then centrifuged at 13,000 rpm for 5 min, and protein content was measured prior to Western blot analysis.

Affinity Precipitation of Active Ras Proteins—The cells were incubated at 1 x107 cells/ml in activation buffer (25 mM HEPES, 125 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM Na2HPO4, 0.5 mM MgSO4, 2 mM sodium pyruvate, 0.1% glucose, 50 µM {beta}-mercaptoethanol) for 15 min at 37 °C. Anti-IgM was then added to 20 µg/ml for the indicated times. The stimulations were interrupted by the addition of ice-cold, Mg+ lysis buffer, and the lysate protein content was measured. Affinity precipitation of activated Ras proteins by binding to RafRBD was performed as described (26, 27).

Western Blots—The cell lysates were combined with 1 volume of loading buffer (8% SDS, 40% glycerol, 20% {beta}-mercaptoethanol), separated by electrophoresis on 12.5% acrylamide gels containing SDS, and transferred to polyvinylidene difluoride membranes (Millipore). The membranes were blocked overnight with 5% bovine serum albumin in TBST (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20). The membranes were then rinsed twice in TBST incubated overnight at 4 °C or 90 min at room temperature with primary antibody diluted in TBST with 3% bovine serum albumin. The membranes were then rinsed four times for 10 min at room temperature and incubated with horseradish peroxidase-conjugated secondary antibody (Jackson Immunoresearch). The ECL chemiluminescence system (Santa Cruz) was used to visualize immunoreactive bands either by film or using the Versadoc 5000 imaging system (Bio-Rad). Band volume analysis was performed using Quantity One software (Bio-Rad).

NF-{kappa}B Luciferase Assay—5 x 106 cells/ml were suspended in medium containing a reporter gene construct (10 µg/ml, pNF-{kappa}B-LUC; Stratagene) in combination with 2 µg/ml of thymidine kinase promoter-dependent Renilla luciferase construct to assess transfection efficiency (pRL-TK; Promega, Madison, WI), placed on ice for 20 min, and electroporated at 270 V and 950 microfarads in 4-mm cuvettes (Gene Pulser; Bio-Rad). The cells were then rinsed twice with medium, and 106 cells/ml were plated in fresh medium and incubated at 37°C for 4 h. The cells were then rinsed with PBS and lysed for luciferase assays performed according to the manufacturer's instructions (Dual Luciferase Reporter Assay; Promega).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
RasGRP1 Enhances Apoptosis of WEHI-231 Cells Following BCR Ligation—WEHI-231 cells express RasGRP1, as determined by Northern blot (6) and Western blot (Fig. 1). A cDNA encoding full-length, N-terminally HA epitope-tagged murine RasGRP1 was stably expressed in WEHI-231 cells by retroviral transduction. The cDNA-encoded RasGRP1 was expressed in transduced cells at about 1.5-fold higher levels than the endogenous RasGRP1 in control (empty vector-transduced) WEHI-231 cells (Fig. 1). However, endogenous RasGRP1 levels were reduced in the RasGRP1-transduced cells, indicating negative feedback regulation of RasGRP1 translation or turnover. The net result was a 2-fold increase in total RasGRP1 in the cDNA-transduced versus control cells. We will refer to WEHI-231 cells transduced with the RasGRP1 cDNA as RasGRP1high cells, whereas WEHI-231 cells that were concurrently transduced with empty retroviral vector and selected will be referred to as control cells. Two or more independently derived polyclonal sets of control and RasGRP1high cells were used for our experiments, to ensure that variation in phenotypes were a function of RasGRP1 transduction and not due to variation within the cell populations prior to transduction.



View larger version (37K):
[in this window]
[in a new window]
  		FIG. 1.
Expression of endogenous and transduced RasGRP1 in WEHI-231 cells. Lysates of WEHI-231 cells transduced with empty vector (control) or with vector encoding HA-tagged RasGRP1 (RasGRP1high) were analyzed by Western blot, using anti-RasGRP (top panel) or anti-HA (bottom panel) antibodies for detection. Transduced HA-RasGRP1 bands are indicated by arrowheads. The numbers indicate relative quantities of each band. The main endogenous band has an estimated molecular mass of 89 kDa, and the main transduced (HA+) band has an estimated molecular mass of 91 kDa.

 
To test the functionality of the transduced RasGRP1, we compared levels of Ras GTP loading in RasGRP1high cells versus control WEHI-231 cells. There was a marginal increase in GTP-bound K-Ras in RasGRP1high cells prior to BCR ligation, whereas the transient increase in GTP-bound K-Ras that occurs in response to BCR ligation in control WEHI-231 cells was exaggerated and prolonged in RasGRP1high cells (Fig. 2). Activation of N-Ras and H-Ras was also increased in RasGRP1high cells, although with somewhat different kinetics and dependences on BCR ligation (Fig. 2).



View larger version (34K):
[in this window]
[in a new window]
  		FIG. 2.
Elevated RasGRP1 expression causes increased GTP loading of Ras GTPases. Control or RasGRP1high cells expressing K-Ras, N-Ras, or H-Ras tagged with both HA and GFP were stimulated for the indicated times with 20 µg/ml anti-IgM. GTP-bound Ras GTPases were purified by binding to GST-RafRBD and quantified by Western blot using anti-HA for detection. The numbers indicate relative quantities of each band, normalized to control cells at 0 min. The results are representative of three experiments.

 
In the absence of BCR ligation, elevated RasGRP1 expression had no effect on the incidence of apoptosis, as measured by subdiploid DNA content after cell permeabilization (Fig. 3A), and slightly lowered the rate of cell division as measured by dilution of labeling by CFSE (Fig. 3B). Ligation of BCR with anti-IgM for 48 h imposed cell cycle arrest in RasGRP1high and control cells with similar efficiency, but the apoptotic response to BCR ligation was enhanced about 3-fold in the RasGRP1high cells (Fig. 3A). Elevated expression of RasGRP1 was particularly effective at sensitizing WEHI-231 cells to deletion following transient exposure to anti-IgM. When control WEHI-231 cells were treated with anti-IgM for 24 h and then treated with trypsin (see "Experimental Procedures"), they evaded apoptosis and re-entered the cell cycle within 24 h (Fig. 3A), and most of the cells had passed through two cell divisions by 48 h (Fig. 3B). In contrast, a 24-h exposure to anti-IgM followed by trypsin treatment induced apoptosis in about half of the RasGRP1high cells over the next 24 h (Fig. 3A), and the surviving RasGRP1high cells had delayed re-entry into the cell cycle (Fig. 3, A and B).



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 3.
Elevated RasGRP1 expression enhances BCR-induced apoptosis and delays cell cycle re-entry after reversal of BCR-induced apoptosis. A, cell cycle and apoptosis analysis using propidium iodide staining of permeabilized cells. Control or RasGRP1high cells were untreated (top panels), treated with 10 µg/ml anti-IgM for 48 h (middle panels), or treated with 10 µg/ml anti-IgM for 24 h followed by trypsinization and culture for a further 24 h (bottom panels). After permeabilization and staining with PI, the cells were analyzed by flow cytometry. The percentages of cells with subdiploid (apoptotic) or greater than diploid (in S or G phases of cell cycle) quantities of DNA are indicated. The results are 2representative of three experiments. B, cell division analysis. Control or RasGRP1high cells were untreated (top panels) or treated with 10 µg/ml anti-IgM for 24 h followed by trypsinization (bottom panels). The cells were then stained with CFSE, and after a further 48 h of culture the progress of cell division was quantified by measuring dilution of CFSE fluorescence. Nonviable cells (PI+) were gated out of the analysis. CFSE fluorescence intensity is displayed on a log scale, and peaks corresponding to 2-fold reductions in fluorescence intensity represent sequential cell divisions. The fluorescence intensity of undivided cells was determined by analysis of cells after CFSE labeling but prior to culturing. The percentages of cells in each peak are indicated. The results are representative of three experiments. C, differential depletion of RasGRP1high cells by BCR ligation. Control cells or RasGRP1high cells co-expressing signaling-defective human Thy-1 were mixed with control cells expressing GFP. The mixed populations were cultured for 48 h without or with 10 µg/ml anti-IgM, stained with an anti-human Thy-1 antibody and then analyzed by flow cytometry. Nonviable cells (PI+) were gated out of the analysis. The results are expressed as the ratio of the percentage of hThy-1+ cells in the culture at 48 h to the percentage of hThy-1+ cells at the start of culture, with error bars indicating standard deviations of duplicate cultures. A ratio of less than 1 indicates that the hThy-1-expressing cells (control or RasGRP1high) had been depleted relative to GFP-expressing control cells. The data are representative of three experiments.

 
To directly assess differential sensitivities of cells to BCR-induced deletion, control and RasGRP1high cells were co-cultured in anti-IgM for 48 h, after which viable cells of each type were quantified by flow cytometry. There was a 5-fold reduction in viable RasGRP1high cells relative to co-cultured control cells following exposure to anti-IgM (Fig. 3C), demonstrating that a doubling of RasGRP1 expression greatly increased the sensitivity of WEHI-231 cells to deletion by BCR ligation. This experiment also showed that RasGRP1-mediated deletion was autonomous to the RasGRP1high cells.

The potential of endogenous RasGRP1 to contribute to BCR-induced apoptosis was addressed by expression of RasGRP1 with a R271E point mutation in the nucleotide exchange domain. This mutation is predicted to prevent binding of Ras GTPases (28) and eliminates Ras activation by RasGRP1 (6, 9). In Jurkat T cells, the R271E mutant suppresses TCR-induced Ras activation specifically at cellular sites where RasGRP1 is activated (9) and therefore acts as a dominant negative for RasGRP1, presumably by competing for positive regulators of RasGRP1 but failing to provide downstream signaling through Ras GTPases. When expressed in WEHI-231 cells at nine times the level of endogenous RasGRP1, the R271E mutant almost completely suppressed BCR-induced apoptosis (R271E2+; Fig. 4), whereas lower expression of R271E resulted in partial inhibition of BCR-induced apoptosis (R271E+; Fig. 4). Assuming that the R271E dominant negative is specific for RasGRP1 in WEHI-231 cells, this apoptosis-suppressing phenotype indicates that endogenous RasGRP1 is essential for efficient induction of apoptosis following BCR ligation.



View larger version (25K):
[in this window]
[in a new window]
  		FIG. 4.
Suppression of BCR-induced apoptosis by a dominant negative mutant of RasGRP1. A, expression levels of R271E Ras-GRP1 relative to endogenous RasGRP1. In two different sets of transductions, WEHI-231 cells were infected with empty vector (control) or with vectors encoding wild-type (wt) or R271E forms of RasGRP1. The cell lysates were analyzed for total and HA-tagged RasGRP1 by Western blot with detection by anti-RasGRP1 (for control and wild type) or anti-HA antibodies (for wild type and R271E). The numbers indicate quantities in each band, normalized to the endogenous RasGRP1 in control cells. B, the cells described in A above were untreated (nil) or treated with 10 µg/ml of anti-IgM for 48 h. Apoptosis was quantified by PI staining of permeabilized cells. The bars are the means of five (control and RasGRP1 wt+), three (RasGRP1 R271E2+), or two experiments (RasGRP1 R271E+) with duplicate cultures, with error bars indicating standard deviations.

 
RasGRP1 Contributes to BCR-induced Deletion via the Activation of Ras Family Members—Because the only mechanistic defect of the R271E mutant is an inability to bind to and activate Ras GTPases, the suppression rather than enhancement of BCR-induced apoptosis by R271E implies that enhancement of BCR-induced apoptosis by RasGRP1 occurs through activated Ras GTPases. The ability of Ras activation to trigger apoptosis of WEHI-231 cells was tested by expression of G12V mutants of K-Ras, N-Ras, or H-Ras, which are GTPase-defective and thus constitutively activated. After infection with retroviral vectors expressing the G12V Ras mutants, transduced cells were selected by a combination of drug resistance and sorting for expression of a co-transduced cell surface marker. The expression of each of the G12V Ras GTPases was very low in the selected WEHI-231 cells (data not shown), but despite this the G12V Ras-transduced cells that could be recovered following selection had enhanced sensitivity to BCR-induced apoptosis (Fig. 5), albeit with considerable variation among experiments. These G12V Ras GTPases can be expressed at high levels when transduced by the same vectors into other cell types (data not shown), so the inability to recover highly expressing WEHI-231 cells even after vigorous selection implies that the WEHI-231 cells that acquired proviruses conferring high G12V expression were deleted during the selection, in the absence of BCR ligation. The weakly expressing cells that were selectable had Ras signaling below a threshold required for BCR-independent deletion but sufficient to synergize with BCR in inducing apoptosis. M-Ras Q71L is an activated Ras GTPase that, in comparison with H-Ras G12V, provides lower signaling intensity through Ras-activated signaling pathways (29). M-Ras Q71L-transduced WEHI-231 cells could be readily selected and were consistently more sensitive to BCR-induced apoptosis than control cells (Fig. 5). Combined, these results indicate that Ras signaling at low intensity (via M-Ras Q71L or via proviral insertions providing very low expression of G12V Ras mutants) synergizes with BCR signaling to induce apoptosis of WEHI-231 cells, whereas high intensity signaling from Ras GTPases is not tolerated in these cells, presumably because this causes deletion in the absence of BCR signaling.



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 5.
Activated Ras GTPases enhance BCR-induced apoptosis. WEHI-231 cells were transduced with retroviral vectors expressing the indicated proteins, selected, and treated with 10 µg/ml anti-IgM for 48 h. Apoptosis was quantified by PI staining of permeabilized cells, as shown in Fig. 3A. To control for variation between experiments in the extent to which anti-IgM treatment causes apoptosis, the data are presented as the percentage of apoptotic cells in transduced cells divided by the percentage of apoptotic cells in control cells. Each circle indicates the mean of a single experiment with duplicate cultures, and the lines indicate the means of the five experiments.

 
Role of BCR in Apoptosis Induction by RasGRP1—BCR ligation is necessary for apoptosis induction via RasGRP1 (Fig. 3A). This could be due to synergy between RasGRP1-mediated Ras activation and parallel BCR-initiated signaling pathways not involving Ras activation. Alternatively, the requirement for BCR ligation could reflect activation of RasGRP1 by BCR-initiated signaling pathways. Previous work has defined an activation mechanism for RasGRP1 that requires membrane translocation via binding of the lipid second messenger diacylglycerol to the C1 domain of RasGRP1 (3, 4, 6, 8). Diacylglycerol is generated via the BCR-coupled lipase PLC{gamma}2, and BCR ligation triggers translocation of RasGRP1 to the plasma membrane in the DT40 B cell line (8, 30). We used phorbol myristate acetate (PMA), a lipophilic phorbol ester that binds the C1 domain of RasGRP1 and thus confers membrane localization (3, 6), as a manipulatible but BCR-independent means of activating RasGRP1. PMA treatment of WEHI-231 cells was sufficient to induce apoptosis, and elevated expression of Ras-GRP1 strongly enhanced PMA-induced apoptosis (Fig. 6A). Deletion of the C1 domain eliminated the ability of RasGRP1 to enhance apoptosis in response to either PMA treatment or BCR ligation (Fig. 6). These results demonstrate that direct C1 domain-dependent activation of RasGRP1 by PMA is sufficient for apoptosis induction in WEHI-231 cells and indicate that the contribution of BCR-initiated signaling to apoptosis induction may be predominantly achieved by activation of RasGRP1.



View larger version (16K):
[in this window]
[in a new window]
  		FIG. 6.
Enhancement of PMA- or BCR-induced apoptosis requires the C1 domain and EF hands of RasGRP1. Control or RasGRP1high cells or WEHI-231 cells transduced with EF hand-deleted or C1 domain-deleted forms of RasGRP1 were untreated (nil) or treated with 1.6 µM PMA (A) or 10 µg/ml anti-IgM (B) for 48 h. Apoptosis was quantified by PI staining of permeabilized cells. The bars indicate the means of two experiments each with duplicate cultures, with standard deviations indicated by error bars. wt, wild type.

 
RasGRP1 contains a pair of calcium-binding EF hands (3), but the roles of the EF hands in receptor-mediated activation of RasGRP1 are not well characterized. Kawasaki et al. (2) found that calcium ionophore induced a minor increase in Ras activation via RasGRP1 but did not link this to the EF hands. We have previously shown that the EF hands are not required for either serum- or PMA-induced activation of RasGRP1 in fibroblasts, as measured by a transformation assay (6). However, in WEHI-231 cells the EF hands proved to be essential for Ras-GRP1-mediated enhancement of either BCR-induced or PMA-induced apoptosis (Fig. 6). This unexpected result indicates that the EF hands positively cooperate with the C1 domain in achieving full activation of RasGRP1 in WEHI-231 cells, even when direct, C1 domain-dependent activation of RasGRP1 is provided by PMA.

Activation of ERK1 or ERK2 Is Not Required for RasGRP1-mediated Enhancement of BCR-induced Apoptosis—The mitogen-activated protein kinases ERK1 and particularly ERK2 are activated in WEHI-231 cells in response to BCR ligation (31). There are conflicting reports about whether or not ERK activation contributes to BCR-induced apoptosis in WEHI-231 cells (32–34). A predominant mechanism for ERK1 and ERK2 activation is phosphorylation via the Ras-Raf-MEK1 signaling cascade, raising the possibility that elevated expression of Ras-GRP1 enhances apoptosis by increasing signaling through ERK1 or ERK2. Relative to control cells, RasGRP1high cells had more prolonged ERK2 phosphorylation following anti-IgM treatment (Fig. 7A). However, when ERK phosphorylation was partially or completely suppressed by the MEK1 inhibitors PD98059 or U0126 (Fig. 7B), BCR-induced apoptosis of control cells was unaffected or marginally increased, and RasGRP1high cells remained hypersensitive to BCR-induced apoptosis (Fig. 7C). Therefore, activation of ERK1 or ERK2 does not contribute to BCR-mediated apoptosis in control WEHI-231 cells, and the higher level of ERK2 activation that results from elevated RasGRP1 expression does not contribute to the enhancement of BCR-induced apoptosis by RasGRP1.



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 7.
ERK1 and ERK2 activation is not required for enhancement of BCR-induced apoptosis by RasGRP1. A, control or RasGRP1high cells were treated for the indicated time with 10 µg/ml of anti-IgM. The cell lysates were quantified for Thr202/Tyr204-phosphorylated ERK1/ERK2 by Western blot. The numbers indicate relative quantities of each ERK2 band, normalized to control cells at 0 min. The arrowheads indicate the positions of the pERK1 and pERK2 bands, which had estimated molecular masses of 44 and 42 kDa, respectively. The results are representative of three experiments. B, control or RasGRP1high cells were treated for the indicated time with 10 µg/ml of anti-IgM. As indicated, the MEK1 inhibitors PD98059 (20 µM) or U0126 (10 µM) were added 60 min prior to and during treatment with anti-IgM. The cell lysates were quantified for phosphorylated ERK1/ERK2 by Western blot. The results are representative of two independent experiments. C, control or RasGRP1high cells were treated with 10 µg/ml anti-IgM for 48 h in the presence or absence of MEK inhibitors as indicated. Apoptosis was quantified by PI staining of permeabilized cells. The bars indicate means of duplicate cultures from one experiment, with standard deviations indicated by lines. The results are representative of two experiments.

 
Inhibition of NF-{kappa}B Provides a Mechanism for Apoptosis Induction by RasGRP1—Transcription factors of the NF-{kappa}B family contribute to the development, survival, and proliferation of B cells (35) and provide survival signals to WEHI-231 cells (36–38). NF-{kappa}B activity was reduced about 3-fold in RasGRP1high cells relative to control cells (Fig. 8A), raising the possibility that RasGRP1 sensitizes WEHI-231 cells to apoptosis by lowering NF-{kappa}B activity. NF-{kappa}B subunits are sequestered in the cytoplasm by I{kappa}B proteins, and transcriptional activation by NF-{kappa}B is dependent on phosphorylation-induced degradation of the I{kappa}B proteins (35, 39). Elevated RasGRP1 expression caused a 2–3-fold increase in cytoplasmic I{kappa}B{alpha} levels prior to and for 1 h following BCR ligation, whereas the large increase in cytoplasmic I{kappa}B{alpha} that occurs by 4 h post-BCR ligation was only marginally enhanced by RasGRP1 (Fig. 8B). Cytoplasmic I{kappa}B{beta} levels were not altered by RasGRP1 expression (data not shown).



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 8.
Elevated RasGRP1 expression reduces NF-{kappa}B activity and increases cytoplasmic I{kappa}B{alpha}. A, control or RasGRP1high cells were transfected with a firefly luciferase-expressing reporter gene for NF-{kappa}B. Firefly luciferase activity in cell lysates were normalized to Renilla luciferase activity expressed from a co-transfected plasmid, to control for variation in transfection efficiency. The bars indicate means of triplicate cultures from one experiment, with standard deviations indicated by error bars. The results are representative of four experiments. B, control or RasGRP1high cells were treated with 10 µg/ml anti-IgM for the indicated time. Cytoplasmic extracts were prepared and quantified for I{kappa}B{alpha} by Western blot. The arrowhead indicates the position of the I{kappa}B{alpha} band, which had an estimated molecular mass of 35 kDa. The numbers indicate the relative quantities of each band, normalized to control cells at 0 min. The results are representative of three experiments.

 
The causative role of NF-{kappa}B inhibition in BCR-induced apoptosis was investigated with stimulators or inhibitors of I{kappa}B{alpha} degradation. Ligation of either CD40 or the LPS receptor on WEHI-231 cells suppresses BCR-induced apoptosis by inducing I{kappa}B{alpha} degradation and NF-{kappa}B activation (36–38, 40). RasGRP1high cells were fully susceptible to suppression of BCR-induced apoptosis by either anti-CD40 or LPS (Fig. 9A), indicating that NF-{kappa}B inhibition could be essential for enhancement of BCR-induced apoptosis by RasGRP1. To test the sufficiency of NF-{kappa}B inhibition for enhancing BCR-induced apoptosis, we used BAY 11-7082, an inhibitor of I{kappa}B{alpha} phosphorylation that blocks NF-{kappa}B nuclear translocation in WEHI-231 cells (37). At 5 µM, BAY 11-7082 increased cytoplasmic levels of I{kappa}B{alpha} and induced apoptosis of WEHI-231 cells even in the absence of BCR ligation, whereas 1 µM BAY 11-7082 had no effect on apoptosis in the absence of BCR ligation but increased the incidence of apoptosis following BCR ligation (Fig. 9B and data not shown). Thus, the survival of WEHI-231 cells requires negative regulation of I{kappa}B{alpha} and signals that modulate I{kappa}B{alpha} levels can dramatically shift the sensitivity of WEHI-231 cells to deletion via BCR ligation.



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 9.
RasGRP1-mediated enhancement of BCR-induced apoptosis is blocked by NF-{kappa}B activators and mimicked by stabilization of cytoplasmic I{kappa}B{alpha}. A, control or RasGRP1high cells were untreated (nil) or treated with 10 µg/ml anti-IgM with or without 0.1 µg/ml anti-CD40 or 5 µg/ml LPS for 48 h. After permeabilization and staining with PI, the cells were analyzed by flow cytometry to determine the percentages of apoptotic cells. The bars indicate mean values from three experiments each with duplicate cultures, with standard deviations indicated by error bars. B, WEHI-231 cells were treated with Bay 11-7082 and/or 10 µg/ml anti-IgM for 48 h as indicated. After permeabilization and staining with PI, the cells were analyzed by flow cytometry. The percentages of apoptotic cells are indicated. The results are representative of three experiments.

 
Bcl-xL is a direct target gene of NF-{kappa}B (41). Bcl-xL plays an important role in maintaining survival of WEHI-231 cells and is down-regulated in response to BCR ligation (42–44). Relative to control cells, Bcl-xL protein levels were reduced in RasGRP1high cells before and particularly after BCR ligation (Fig. 10A). Constitutive expression of Bcl-xL by retroviral transduction completely blocked BCR-induced apoptosis in both control and RasGRP1high WEHI-231 cells (Fig. 10B), demonstrating that RasGRP1-mediated apoptosis is suppressible by Bcl-xL. Down-regulation of Bcl-xL as a result of NF-{kappa}B inhibition may therefore be at least partially responsible for the hypersensitivity of RasGRP1high cells to BCR-induced apoptosis.



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 10.
Elevated RasGRP1 expression causes a decrease in Bcl-xL levels, and enforced expression of Bcl-xL suppresses RasGRP1-induced apoptosis. A, control or RasGRP1high cells were treated with 10 µg/ml anti-IgM for the indicated time. The cell extracts were quantified for Bcl-xL by Western blot. The arrowhead indicates the position of the Bcl-xL band, which had an estimated molecular mass of 26 kDa. The numbers indicate relative quantities of each band, normalized to control cells at 0 min. The results are representative of three experiments. B, control or RasGRP1high cells were transduced with empty vector or retroviral vector expressing Bcl-xL and treated with 10 µg/ml anti-IgM for 48 h. Apoptosis was quantified by PI staining of permeabilized cells. The bars indicate means of duplicate cultures from two experiments, with standard deviations indicated by error bars. The results are representative of three experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
RasGRP1 has been shown to enhance pre-TCR or TCR-directed survival during intrathymic T cell development (10, 11) but conversely is required for TCR-induced deletion of peripheral T cells (13). Using the WEHI-231 experimental system, we have investigated the effect of differential expression of Ras-GRP1 on BCR-induced deletion of immature B cells. A moderate elevation in RasGRP1 expression dramatically increased the sensitivity of WEHI-231 cells to BCR-induced deletion by accelerating the onset of apoptosis. The response-modifying effect of differential RasGRP1 expression was particularly evident when WEHI-231 cells were exposed to anti-IgM for 24 h and then cultured in the absence of anti-IgM. After this transient ligation of BCR, WEHI-231 cells with low RasGRP1 expression evaded apoptosis and re-entered the cell cycle within 1 day, whereas most WEHI-231 cells with high RasGRP1 expression apoptosed, and the survivors had delayed entry into the cell cycle. The result was a severe depletion of RasGRP1high cells relative to control WEHI-231 cells.

Expression of a dominant negative form of RasGRP1 strongly suppressed apoptosis induction following BCR ligation. This occurred in a dose-dependent manner when the amount of the dominant negative form was in excess of endogenous RasGRP1, as expected for a signaling-defective dominant negative that acts by competing with the wild-type protein for upstream activators. The same dominant negative mutant was as effective as full extinction of RasGRP1 expression by siRNA at blocking Ras activation via RasGRP1 in Jurkat T cells (9). The mechanism of action of the R271E mutant as a dominant negative is not known, and therefore its strict specificity for RasGRP1 has not been fully established. Nonetheless, the reverse phenotype of the R271E dominant negative form of RasGRP1 is compatible with endogenous Ras-GRP1 playing an essential role in the efficient induction of apoptosis following BCR ligation.

RasGRP1 is thought to mediate its effects on TCR-mediated positive selection of double positive thymocytes via the activation of signaling through the ERK1 and/or ERK2 kinases (4, 11, 12). We found that activating phosphorylation of ERK2 was moderately increased and prolonged in RasGRP1high WEHI-231 cells following BCR ligation. However, ERK2 and ERK1 phosphorylation could be blocked by pharmacological inhibitors without affecting the ability of RasGRP1 to enhance BCR-induced apoptosis. Therefore, attention to other signaling pathways downstream of Ras is warranted for understanding how RasGRP1 modulates lymphocyte selection. Survival of WEHI-231 cells is known to be dependent on signaling through phosphatidylinositol 3-kinase (PI3-K) (45) and NF-{kappa}B (36–38). Ras signaling typically activates PI3-K (46) and NF-{kappa}B (47), and therefore the inhibition of these survival pathways was not predicted to result from RasGRP1 activation. Pharmacological inhibition of PI3-K promoted rather than impeded apoptosis in both control and RasGRP1high WEHI-231 cells.2 But contrary to expectations, elevated RasGRP1 expression in WEHI-231 cells resulted in reduced NF-{kappa}B activity. RasGRP1 decreased expression of the NF-{kappa}B target gene Bcl-xL, and constitutive expression of Bcl-xL was sufficient to suppress BCR-induced apoptosis. Levels of I{kappa}B{alpha}, a repressor of NF-{kappa}B, were raised in WEHI-231 cells transduced with RasGRP1. Using a pharmacological inhibitor of I{kappa}B{alpha} degradation, we demonstrated that stabilization of I{kappa}B{alpha} was sufficient to enhance BCR-induced apoptosis. These results point to an unanticipated mechanism for regulating negative selection of B cells, involving inhibition of NF-{kappa}B by RasGRP1-initiated signaling. The expression of activated mutants of H-Ras in murine embryonic fibroblasts (48) or Raf-1 in a rat kidney fibroblast line (49) inhibit the I{kappa}B{alpha} degradation and NF-{kappa}B activation, which is normally induced by TNF-{alpha}. However, Raf-mediated inhibition of TNF-{alpha}-induced NF-{kappa}B activation in kidney fibroblasts was completely dependent on ERK1 or ERK2 (49), whereas Ras-mediated inhibition of TNF-{alpha}-induced NF-{kappa}B activation in embryonic fibroblasts was not mimicked by an activated Raf mutant and was dependent on PI3-K (48). This contrasts with our findings in WEHI-231 cells, where RasGRP1-mediated enhancement of BCR-induced apoptosis occurred independently of both ERK or PI3-K activation and was mimicked by expression of an activated mutant of Raf-1.2 There appear to be multiple mechanisms by which signaling downstream of a Ras exchange factor can affect NF-{kappa}B activation, with the outcome being highly variable among distinct cell types.

Within the limitations of the WEHI-231 model of immature B cell deletion, our results extend the concept that minor variations in RasGRP1 expression levels within a lymphocyte population could be the source of variability in antigen receptor-mediated selection of that population. In particular, our studies highlight the potential of RasGRP1 to promote the deletion of immature B cells that are exposed to high affinity antigen if this occurs in the absence of NF-{kappa}B-activating co-stimulatory signals (e.g. CD40 ligand or LPS) that divert BCR signaling away from deletion and toward the amplification of an immune response. Transgenic mice expressing an activated mutant of Raf-1 have a 2–3-fold depletion of immature B cells relative to their pre-B cell progenitors (50). Depletion of immature B cells, in part by hypersensitization to BCR-induced deletion, also results from loss of negative regulation of BCR signaling, i.e. SHP-1 or Lyn deficiency or truncation of the cytoplasmic domain of Ig-{alpha} (51–54). Immature B cells in these mutant mice have increased and prolonged signaling from the BCR and in particular have increased calcium mobilization (51–53). The latter is indicative of increased phospholipase C activation, and therefore deletion of immature B cells may be associated with increased signaling from BCR to RasGRP1 or to other members of the RasGRP family that are activated by diacylglycerol (55–59). By modulating the signal flux from BCR through Ras GTPases to NF-{kappa}B, differential expression of RasGRPs may shift the signal intensity thresholds that determine whether self-antigen induces anergy versus receptor editing versus deletion in the tolerizing responses of B cells to self-antigens.


   FOOTNOTES
 
* This work was supported by a grant from the Canadian Institutes for Health Research (to R. J. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Recipient of a Studentship from the Natural Sciences and Engineering Research Council of Canada. Back

§ To whom correspondence should be addressed: Terry Fox Laboratory, British Columbia Cancer Agency, 600 West 10th Ave., Vancouver, BC V5Z 4E6, Canada. Tel.: 604-877-6070; Fax: 604-877-0712; E-mail: rkay{at}bccrc.ca.

1 The abbreviations used are: TCR, T cell receptor; BCR, B cell receptor; CFSE, carboxyfluorescein succinimidyl diester; ERK, extracellular signal-regulated kinase; LPS, lipopolysaccharide; Ig, immunoglobulin; PI3-K, phosphatidylinositol 3-kinase; PMA, phorbol myristate acetate; PI, propidium iodide; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; HA, hemagglutinin; PBS, phosphate-buffered saline. Back

2 B. Guilbault and R. J. Kay, unpublished results. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Matallanas, D., Arozarena, I., Berciano, M. T., Aaronson, D. S., Pellicer, A., Lafarga, M., and Crespo, P. (2003) J. Biol. Chem. 278, 4572–4581[Abstract/Free Full Text]
  2. Kawasaki, H., Springett, G. M., Toki, S., Canales, J. J., Harlan, P., Blumenstiel, J. P., Chen, E. J., Bany, I. A., Mochizuki, N., Ashbacher, A., Matsuda, M., Housman, D. E., and Graybiel, A. M. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 13278–13283[Abstract/Free Full Text]
  3. Ebinu, J. O., Bottorff, D. A., Chan, E. Y., Stang, S. L., Dunn, R. J., and Stone, J. C. (1998) Science 280, 1082–1086[Abstract/Free Full Text]
  4. Ebinu, J. O., Stang, S. L., Teixeira, C., Bottorff, D. A., Hooton, J., Blumberg, P. M., Barry, M., Bleakley, R. C., Ostergaard, H. L., and Stone, J. C. (2000) Blood 95, 3199–3203[Abstract/Free Full Text]
  5. Ohba, Y., Mochizuki, N., Yamashita, S., Chan, A. M., Schrader, J. W., Hattori, S., Nagashima, K., and Matsuda, M. (2000) J. Biol. Chem. 275, 20020–20026[Abstract/Free Full Text]
  6. Tognon, C. E., Kirk, H. E., Passmore, L. A., Whitehead, I. P., Der, C. J., and Kay, R. J. (1998) Mol. Cell. Biol. 18, 6995–7008[Abstract/Free Full Text]
  7. Caloca, M. J., Zugaza, J. L., and Bustelo, X. R. (2003) J. Biol. Chem. 278, 33465–33473[Abstract/Free Full Text]
  8. Caloca, M. J., Zugaza, J. L., Matallanas, D., Crespo, P., and Bustelo, X. R. (2003) EMBO J. 22, 3326–3336[CrossRef][Medline] [Order article via Infotrieve]
  9. Bivona, T. G., Perez De Castro, I., Ahearn, I. M., Grana, T. M., Chiu, V. K., Lockyer, P. J., Cullen, P. J., Pellicer, A., Cox, A. D., and Philips, M. R. (2003) Nature 424, 694–698[CrossRef][Medline] [Order article via Infotrieve]
  10. Norment, A. M., Bogatzki, L. Y., Klinger, M., Ojala, E. W., Bevan, M. J., and Kay, R. J. (2003) J. Immunol. 170, 1141–1149[Abstract/Free Full Text]
  11. Priatel, J. J., Teh, S. J., Dower, N. A., Stone, J. C., and Teh, H. S. (2002) Immunity 17, 617–627[CrossRef][Medline] [Order article via Infotrieve]
  12. Dower, N. A., Stang, S. L., Botorff, D. A., Ebinu, J. O., Dickie, P., Ostergaard, H. L., and Stone, J. C. (2000) Nat. Immunol. 1, 317–321[CrossRef][Medline] [Order article via Infotrieve]
  13. Layer, K., Lin, G., Nencioni, A., Hu, W., Schmucker, A., Antov, A. N., Li, X., Takamatsu, S., Chevassut, T., Dower, N. A., Stang, S. L., Beier, D., Buhlmann, J., Bronson, R. T., Elkon, K. B., Stone, J. C., Van Parijs, L., and Lim, B. (2003) Immunity 19, 243–255[CrossRef][Medline] [Order article via Infotrieve]
  14. Goodnow, C. C. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 2264–2271[Abstract/Free Full Text]
  15. Monroe, J. G. (1996) J. Immunol. 156, 2657–2660[Abstract]
  16. Nemazee, D., and Hogquist, K. A. (2003) Curr. Opin. Immunol. 15, 182–189[CrossRef][Medline] [Order article via Infotrieve]
  17. Gottschalk, A. R., and Quintans, J. (1995) Immunol. Cell Biol. 73, 8–16[Medline] [Order article via Infotrieve]
  18. Jakway, J. P., Usinger, W. R., Gold, M. R., Mishell, R. I., and DeFranco, A. L. (1986) J. Immunol. 137, 2225–2231[Abstract]
  19. Tsubata, T., Wu, J., and Honjo, T. (1993) Nature 364, 645–648[CrossRef][Medline] [Order article via Infotrieve]
  20. Goudeau, B., Huetz, F., Samson, S., Di Santo, J. P., Cumano, A., Beg, A., Israel, A., and Memet, S. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 15800–15805[Abstract/Free Full Text]
  21. Umeda, J., Sano, S., Kogawa, K., Motoyama, N., Yoshikawa, K., Itami, S., Kondoh, G., Watanabe, T., and Takeda, J. (2003) FASEB J. 17, 610–620[Abstract/Free Full Text]
  22. Whitehead, I., Kirk, H., and Kay, R. (1995) Mol. Cell. Biol. 15, 704–710[Abstract]
  23. Krebs, D. L., Yang, Y., Dang, M., Haussmann, J., and Gold, M. R. (1999) Methods Cell Sci. 21, 57–68[CrossRef][Medline] [Order article via Infotrieve]
  24. Pear, W. S., Nolan, G. P., Scott, M. L., and Baltimore, D. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8392–8396[Abstract/Free Full Text]
  25. Mui, A. L., Wakao, H., O'Farrell, A. M., Harada, N., and Miyajima, A. (1995) EMBO J. 14, 1166–1175[Medline] [Order article via Infotrieve]
  26. Taylor, S. J., Resnick, R. J., and Shalloway, D. (2001) Methods Enzymol. 333, 333–342[CrossRef][Medline] [Order article via Infotrieve]
  27. Taylor, S. J., and Shalloway, D. (1996) Curr. Biol. 6, 1621–1627[CrossRef][Medline] [Order article via Infotrieve]
  28. Park, W., Mosteller, R. D., and Broek, D. (1994) Mol. Cell. Biol. 14, 8117–8122[Abstract/Free Full Text]
  29. Quilliam, L. A., Castro, A. F., Rogers-Graham, K. S., Martin, C. B., Der, C. J., and Bi, C. (1999) J. Biol. Chem. 274, 23850–23857[Abstract/Free Full Text]
  30. Kurosaki, T. (2002) Nat. Rev. Immunol. 2, 354–363[CrossRef][Medline] [Order article via Infotrieve]
  31. Sutherland, C. L., Heath, A. W., Pelech, S. L., Young, P. R., and Gold, M. R. (1996) J. Immunol. 157, 3381–3390[Abstract]
  32. Lee, J. R., and Koretzky, G. A. (1998) J. Immunol. 161, 1637–1644[Abstract/Free Full Text]
  33. Gauld, S. B., Blair, D., Moss, C. A., Reid, S. D., and Harnett, M. M. (2002) J. Immunol. 168, 3855–3864[Abstract/Free Full Text]
  34. Richards, J. D., Dave, S. H., Chou, C. H., Mamchak, A. A., and DeFranco, A. L. (2001) J. Immunol. 166, 3855–3864[Abstract/Free Full Text]
  35. Li, Q., and Verma, I. M. (2002) Nat. Rev. Immunol. 2, 725–734[CrossRef][Medline] [Order article via Infotrieve]
  36. Schauer, S. L., Bellas, R. E., and Sonenshein, G. E. (1998) J. Immunol. 160, 4398–4405[Abstract/Free Full Text]
  37. Zhu, N., Ramirez, L. M., Lee, R. L., Magnuson, N. S., Bishop, G. A., and Gold, M. R. (2002) J. Immunol. 168, 744–754[Abstract/Free Full Text]
  38. Wu, M., Lee, H., Bellas, R. E., Schauer, S. L., Arsura, M., Katz, D., FitzGerald, M. J., Rothstein, T. L., Sherr, D. H., and Sonenshein, G. E. (1996) EMBO J. 15, 4682–4690[Medline] [Order article via Infotrieve]
  39. Karin, M., and Ben-Neriah, Y. (2000) Annu. Rev. Immunol. 18, 621–663[CrossRef][Medline] [Order article via Infotrieve]
  40. Schauer, S. L., Wang, Z., Sonenshein, G. E., and Rothstein, T. L. (1996) J. Immunol. 157, 81–86[Abstract]
  41. Lee, H. H., Dadgostar, H., Cheng, Q., Shu, J., and Cheng, G. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 9136–9141[Abstract/Free Full Text]
  42. Merino, R., Grillot, D. A., Simonian, P. L., Muthukkumar, S., Fanslow, W. C., Bondada, S., and Nunez, G. (1995) J. Immunol. 155, 3830–3838[Abstract]
  43. Wang, Z., Karras, J. G., Howard, R. G., and Rothstein, T. L. (1995) J. Immunol. 155, 3722–3725[Abstract]
  44. Ishida, T., Kobayashi, N., Tojo, T., Ishida, S., Yamamoto, T., and Inoue, J. (1995) J. Immunol. 155, 5527–5535[Abstract]
  45. Banerji, L., Glassford, J., Lea, N. C., Thomas, N. S., Klaus, G. G., and Lam, E. W. (2001) Oncogene 20, 7352–7367[CrossRef][Medline] [Order article via Infotrieve]
  46. Campbell, S. L., Khosravi-Far, R., Rossman, K. L., Clark, G. J., and Der, C. J. (1998) Oncogene 17, 1395–1413[CrossRef][Medline] [Order article via Infotrieve]
  47. Mayo, M. W., and Baldwin, A. S. (2000) Biochim. Biophys. Acta 1470, 55–62
  48. Hanson, J. L., Anest, V., Reuther-Madrid, J., and Baldwin, A. S. (2003) J. Biol. Chem. 278, 34910–34917[Abstract/Free Full Text]
  49. Liu, Q., Fan, J., McMahon, M., Prince, A. M., and Zhang, P. (2001) Mol. Cell. Biol. Res. Commun. 4, 381–389[CrossRef][Medline] [Order article via Infotrieve]
  50. Iritani, B. M., Alberola-Ila, J., Forbush, K. A., and Perimutter, R. M. (1999) Immunity 10, 713–722[CrossRef][Medline] [Order article via Infotrieve]
  51. Cornall, R. J., Cyster, J. G., Hibbs, M. L., Dunn, A. R., Otipoby, K. L., Clark, E. A., and Goodnow, C. C. (1998) Immunity 8, 497–508[CrossRef][Medline] [Order article via Infotrieve]
  52. Cyster, J. G., and Goodnow, C. C. (1995) Immunity 2, 13–24[CrossRef][Medline] [Order article via Infotrieve]
  53. Kraus, M., Saijo, K., Torres, R. M., and Rajewsky, K. (1999) Immunity 11, 537–545[CrossRef][Medline] [Order article via Infotrieve]
  54. Torres, R. M., and Hafen, K. (1999) Immunity 11, 527–536[CrossRef][Medline] [Order article via Infotrieve]
  55. Lorenzo, P. S., Kung, J. W., Bottorff, D. A., Garfield, S. H., Stone, J. C., and Blumberg, P. M. (2001) Cancer Res. 61, 943–949[Abstract/Free Full Text]
  56. Teixeira, C., Stang, S. L., Zheng, Y., Beswick, N. S., and Stone, J. C. (2003) Blood 102, 1414–1420[Abstract/Free Full Text]
  57. Reuther, G. W., Lambert, Q. T., Rebhun, J. F., Caligiuri, M. A., Quilliam, L. A., and Der, C. J. (2002) J. Biol. Chem. 277, 30508–30514[Abstract/Free Full Text]
  58. Yang, Y., Li, L., Wong, G. W., Krilis, S. A., Madhusudhan, M. S., Sali, A., and Stevens, R. L. (2002) J. Biol. Chem. 277, 25756–25774[Abstract/Free Full Text]
  59. Oh-Hora, M., Johmura, S., Hashimoto, A., Hikida, M., and Kurosaki, T. (2003) J. Exp. Med. 198, 1841–1851[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Mol. Biol. CellHome page
N. Beaulieu, B. Zahedi, R. E. Goulding, G. Tazmini, K. V. Anthony, S. L. Omeis, D. R. de Jong, and R. J. Kay
Regulation of RasGRP1 by B Cell Antigen Receptor Requires Cooperativity between Three Domains Controlling Translocation to the Plasma Membrane
Mol. Biol. Cell, August 1, 2007; 18(8): 3156 - 3168.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/19/19523    most recent
M314273200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Guilbault, B.
Right arrow Articles by Kay, R. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Guilbault, B.
Right arrow Articles by Kay, R. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement