From the Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Singapore
Received for publication, November 30, 2000, and in revised form, March 1, 2001
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ABSTRACT |
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B lymphocytes lacking the adaptor protein B cell
linker (BLNK) do not proliferate in response to B cell antigen receptor
(BCR) engagement. We demonstrate here that BCR-activated
BLNK The B cell antigen receptor
(BCR)1 plays a pivotal role
in the generation and activation of B lymphocytes. Its signaling can lead to various distinct cellular responses including receptor editing,
anergy, and cell death of immature B cells and activation, proliferation, and differentiation of mature B lymphocytes (1). In
molecular terms, the engagement of the BCR is known to activate cytoplasmic protein tyrosine kinases (PTKs) of the Syk, Src, and Tec
families such as Syk, Lyn, Blk, and Bruton's tyrosine kinase (Btk).
The activation of these PTKs generally leads to calcium fluxes, the
phosphorylation of other signaling molecules, and, ultimately, to the
induction of transcription factors that result in new gene expression
(2). However, little is known about how these downstream signaling
events are being coordinated with the activation of the BCR-proximal
PTKs. In particular, it is still not clear how the biochemical events
initiated by BCR signaling could be translated into distinct cellular responses.
Recently, adaptor proteins have been shown to interface PTK activation
with selective downstream molecules and could therefore channel BCR
signaling to elicit distinct cellular outcomes (3). One such adaptor
molecule in B-lymphocyte is B cell linker (BLNK) (4), otherwise known
as SLP-65 (5) or BASH (6). BLNK is a Src homology 2 and 3 domain-containing protein that bears homology to another adaptor
molecule called SLP-76 that is found in T cells (7). BLNK is
phosphorylated upon BCR stimulation (5) and couples Syk activation to
Grb2, Vav, Nck, and phospholipase C (PLC)- We and others have previously generated mice lacking BLNK (18-21).
BLNK Given the similarities in the phenotypes of
Btk Mice--
The generation of BLNK Preparation of Cells and Extracts--
Single cell suspensions
were prepared from spleens of wild-type and
BLNK
Purified primary B cells were stimulated with LPS (Sigma), PMA (Sigma),
or goat anti-mouse IgM F(ab')2 fragment (Jackson
Immunoresearch Laboratories). For the NF-
For the preparation of nuclear extracts, cells were lysed for 20 min on
ice in a hypotonic buffer (5 mM Tris-Cl, pH 7.5, 5 mM KCl, 1.5 mM MgCl2, 0.1 mM EGTA, 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, and
5 µg/ml aprotinin). The nuclear extracts were spun through Microcon
centrifugal filter devices (Millipore) to concentrate and desalt the
samples. A Bio-Rad DC Protein Assay was employed to determine the
amount of nuclear proteins present using known concentrations of bovine serum albumin (BSA) as standards. For the preparation of total cell
lysate, cells were lysed for 20 min on ice in a phosphotyrosine lysis
buffer (1% Nonidet P-40, 10 mM Tris-Cl, pH 8, 150 mM NaCl, 1 mM EDTA, 0.2 mM
Na3VO4, 1 mM phenylmethylsulfonyl
fluoride, and 10 µg/ml aprotinin).
Cell Proliferation Assay--
A colorimetric MTT assay (Roche,
Germany) was used according to the manufacturer's instructions to
measure cell proliferation in vitro. Briefly, 5 × 105 B cells were stimulated with varying concentrations of
F(ab)'2 goat anti-mouse IgM (Jackson Immunoresearch
Laboratories) antibody or LPS (Sigma) for 48 h in a 96-well tissue
culture plate. Subsequently, the cells were incubated with the MTT
labeling reagent for 4 h and with the second step reagent
overnight. Cell proliferation was quantified using an enzyme-linked
immunosorbent assay reader at 570 nm wavelength.
Cell Cycle and Cell Death Analyses--
For cell cycle analysis,
wild-type and BLNK
For the analysis of cell death, ex vivo cells and cells that
were non-treated or stimulated with anti-IgM or LPS overnight were
harvested, washed once in PBS, and fixed overnight in ethanol at
Electrophoretic Mobility Shift Assays--
Nuclear extract (10 µg) isolated from B cells was incubated with an
[ Western Blot Analyses--
After stimulation with anti-IgM, LPS,
or PMA, the purified B cells were lysed on ice for 15 min in a 1%
Nonidet P-40 buffer (10 mM Tris-C1, pH 8, 150 mM NaCl, 1 mM EDTA, 0.2 mM
Na3VO4, 1 mM phenylmethylsulfonyl
fluoride, and 10 µg/ml aprotinin). Proteins corresponding to 5 × 106 cells or 5 µg of nuclear extracts were
electrophoresed in 9-12% SDS-polyacrylamide gels and transferred onto
immunoblot polyvinylidene difluoride membranes (Bio-Rad). The membranes
were blocked with 5% nonfat milk in Tris-buffered saline containing
0.1% Tween 20 for 1 h at room temperature and incubated
separately with the various antibodies that recognized the different
molecules being studied. Protein bands were visualized using
horseradish peroxidase-coupled secondary antibodies and the enhanced
chemiluminescence detection system (Amersham Pharmacia Biotech). The
following antibodies were used in this study: anti-cyclin D2 (M-20,
Santa Cruz), anti-cdk4 (SC-22, Santa Cruz), anti-Bcl-xL
(clone 44, Transduction Laboratories), anti-bcl-2 (Ab-4, Oncogene),
anti-tubulin (Ab-4, NeoMarkers), anti-NF- Immunoprecipitations of Btk and PLC- Anti-IgM-stimulated BLNK
Anti-IgM stimulation causes B cell to enter the cell cycle (25). The
failure of BLNK Impaired Induction of Cell Cycle Regulatory Proteins in
Anti-IgM-stimulated BLNK
As control, we examined the induction of cell cycle regulatory proteins
in BLNK BLNK
Normal B lymphocytes also express the anti-apoptotic protein, Bcl-2
(32). To examine if BLNK deficiency affects the expression of Bcl-2, as
is the case for Bcl-xL above, we examined the expression level of this protein in normal and BLNK BLNK Normal Activation of MAPKs and Akt in Mouse BLNK
As shown in Fig. 7A, the
phosphorylation of the p42 and p44 forms of ERK occurs within 30 s
and is sustained for as long as 10 min (data not shown) after BCR
cross-linking in wild-type B cells. ERK phosphorylation can also be
detected with the same kinetics after BCR stimulation in
BLNK
Other than the MAPKs, BCR engagement also activates the Akt signaling
pathway that is known to regulate cell survival (34). As such, we
examine if this pathway is compromised in
BLNK BLNK Is Required for the Activation of NF-
To investigate the role of BLNK in BCR-induced NF-
The immediate events regulating NF-
The lack of nuclear NF-
It is conceivable that BLNK
As mentioned above, NF-
Taken together, the data indicate that BLNK is required for BCR-induced
NF- The Activation of Bruton's Tyrosine Kinase Is Normal, but That of
PLC-
While the current work was in progress, Petro and Khan (39) showed that
the enzyme PLC- BLNK The defect in cell cycle progression and cell proliferation may explain
why, at the physiological level, BLNK Of the major signaling pathways that are known to regulate cell
proliferation and/or survival such as those of Akt (34), MAPKs (33) and
NF- Previous analyses of BLNK Taken together, a detailed model for the activation of NF-/
B cells fail to enter the cell
cycle, and this is due to their inability to induce the expression of
the cell cycle regulatory proteins such as cyclin D2 and
cyclin-dependent kinase 4. BCR-stimulated BLNK
/
B cells also do not up-regulate the
cell survival protein Bcl-xL, which may be necessary for
the cells to complete the cell cycle. In addition,
BLNK
/
B cells exhibit a high rate of
spontaneous apoptosis in culture. Examination of the various
BCR-activated signaling pathways in mouse
BLNK
/
B cells reveals the intact activation
of Akt and mitogen-activated protein kinases but the impaired
activation of nuclear factor (NF)-
B that is known to regulate genes
involved in cell proliferation and survival. The inability to activate
NF-
B in BCR-stimulated BLNK
/
B cells is
due to a failure to induce the degradation of the inhibitory
B
protein. In all these aspects, BLNK
/
B
cells resemble xid B cells that have a mutation in
Bruton's tyrosine kinase (Btk). Recently, phospholipase C (PLC)-
2
has also been demonstrated to be essential for NF-
B activation.
Since BLNK has been shown separately to interact with both Btk and
PLC-
2, our finding of normal Btk but impaired PLC-
2 activation in
BCR-stimulated BLNK
/
B cells strongly
suggests that BLNK orchestrates the formation of a Btk-PLC-
2
signaling axis that regulates NF-
B activation. Taken together, the
NF-
B activation defect may be sufficient to explain the similar
defects in BCR-induced B cell proliferation and T cell-independent
immune responses in BLNK
/
,
Btk
/
, and
PLC-
2
/
mice.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 that is intimately
associated with intracellular Ca2+ mobilization (8). In
addition, BLNK has been shown to play a role in the activation of all
three major classes of mitogen-activated protein kinases (MAPKs),
namely the extracellular signal-regulated kinase (ERK), the c-Jun
NH2-terminal kinase (JNK), and p38 MAPK in the DT40 chicken
B cell line (8). BLNK also interacts with Btk through the Src homology
2 domain of the latter (9, 10). Btk is a key molecule in the BCR
signaling and is known to regulate the survival (11) and cell cycle
status (12, 13) of activated B lymphocytes. Mutation in Btk causes
X-linked immunodeficiency (xid) in mice and X-linked
agammaglobulinemia in humans (14-17).
/
mice have a severe but incomplete
block in B cell development and lack specifically CD5+ B
cells due to a defect in the generation and maintenance of this B cell
subset (22). In addition, they do not respond to T cell-independent
antigen (18, 19). BLNK
/
B cells also do not
proliferate and up-regulate the expression of activation markers upon
BCR cross-linking (18, 19, 21). Overall, the phenotype of
BLNK
/
mice closely resembles that of
xid or Btk
/
mice (23, 24).
/
and BLNK
/
mice and the demonstrated interaction between Btk and BLNK (9, 10), we
now examine the BCR-initiated molecular events leading to cell cycle
entry and survival in BLNK
/
B cells. In
this report, we demonstrate that BCR-activated
BLNK
/
B cells fail to enter the cell cycle;
this is due to their inability to express the cell cycle regulatory and
cell survival proteins. Finally, we examine the BCR-activated signal
transduction pathways that may be perturbed in mouse primary B
lymphocytes lacking BLNK and show that these mutant cells specifically
do not activate the NF-
B signaling pathway that have been implicated
in the regulation of genes essential for B cell proliferation and survival.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice has been described previously (18). All mice were used at 6-12
weeks of age and in accordance with institutional guidelines.
/
mice and depleted of erythrocytes by
treatment with red blood cell lysis solution (0.15 M
NH4Cl, 0.1 mM Na2EDTA, pH 7.2).
Primary B lymphocytes were isolated by negative selection of splenic
cell samples using anti-CD43 monoclonal antibody-coupled MACS beads (Miltenyi, Germany). The purity of the B cells recovered was ~90%, as assessed by FACS analyses using anti-B220 and anti-IgM antibodies.
B assays, cells were
cultured initially at 5 × 106 cells/200 µl in
Opti-MEM I reduced serum medium containing HEPES buffer, 2400 mg/liter
sodium bicarbonate and L-glutamine (Life Technologies,
Inc.) to reduce the background levels of NF-
B activity. For the
I
B
assays, cells were pre-treated with 50 µM
cycloheximide (Sigma) for 30 min and subsequently stimulated in the
continued presence of the drug.
/
B cells that were not
treated or were stimulated with anti-IgM (20 µg/ml) or LPS (25 µg/ml) were cultured in the presence of 40 µM
5'-bromo-2'-deoxyuridine (BrdUrd; Sigma) at 4 × 106
cells/well in a 48-well tissue culture plate. After 48 h of
incubation, cells were harvested and washed once with 1% BSA in PBS.
The cell pellet was subsequently resuspended in 200 µl of PBS and
fixed overnight in 1 ml of 70% ethanol at
20 °C. After
centrifugation, the cells were incubated with 1 ml of 2 N
HCl/Triton X-100 at room temperature for 30 min to denature the DNA.
The cells were further washed with 0.5% Tween 20/BSA/PBS. Anti-BrdUrd
antibody (Becton Dickinson) was added to the cells for 30 min at room
temperature. Finally, propidium iodide (PI; Sigma) was added at 5 µg/ml to the fixed cells, and the analyses were carried out on a
FACScan using CellQuest software (Becton Dickinson).
20 °C. Subsequently, the cells were washed once in PBS,
resuspended in 100 µl of 100 µg/ml ribonuclease A (Roche Molecular
Biochemicals), and incubated for 5 min at room temperature. Finally,
propidium iodide was added at 50 µg/ml to the cells prior to their
analyses on a FACScan.
-32P]ATP end-labeled, double-stranded oligonucleotide
probe that contains two tandemly positioned NF-
B-binding sites,
5'-CATGGCCTGGGAAAGTCCCCTCAACT-3' and
5'-CATGAGTTGAGGGGACTTTCCCAGGC-3'. The reaction was performed in
20 µl of binding buffer (60 mM Hepes, pH 7.9, 20 mM Tris-HCl, pH 7.9, 300 mM KCl, 150 mM NaCl, 25 mM MgCl2, 25 mM dithiothreitol, 62.5% glycerol, and 1 µg of
poly(dI-dC)) for 30 min on ice. After incubation, the samples were
resolved in a 5% native gel for 2 h. The gel was dried for 1 h and exposed to an autoradiography film. Densitometry analysis was
carried out using a Bio-Rad imaging densitometer and multi-analysis
software (Bio-Rad).
B p50 (H-119, santa Cruz),
anti-NF-
B p65 (C-20, Santa Cruz), anti-I
B
(SC-371, Santa
Cruz), anti-p38 MAPK (SC-721, Santa Cruz), anti-phospho-p38 MAPK
(catalog no. 9211, Cell Signaling Technology (CST)), anti-JNK (catalog
no. 9252, CST), anti-phospho-SAPK/JNK (catalog no. 9251, CST),
anti-ERK2 (SC-154, Santa Cruz), anti-phospho-ERK (SC-7383, Santa Cruz),
anti-Akt (catalog no. 9272, CST), and anti-phospho-Akt (catalog no.
9271, CST). All antibodies were used according to the manufacturers' instructions.
2--
For
immunoprecipitation studies, anti-Btk (catalog no. 65251A, PharMingen)
or anti-PLC
2 (SC-407, Santa Cruz) antibodies were coupled to Protein
A/G Plus-Agarose (SC-2003, Santa Cruz) at 4 °C overnight. The beads
were washed twice in lysis buffer and incubated with pre-cleared total
cell lysates of untreated or goat anti-mouse IgM F(ab)'2
(10 µg/ml) stimulated B cells for 1 h at 4 °C. Subsequently,
the beads were boiled in loading buffer for 3 min, and the released
proteins were resolved in a 7% SDS-polyacrylamide gel electrophoresis
and blotted onto polyvinylidene difluoride membranes. The membranes
were first probed with horseradish peroxidase coupled
anti-phosphotyrosine antibody (P11625, Transduction Laboratories) and
subsequently with the immunoprecipitating antibodies.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
B Cells Fail to Enter the
Cell Cycle--
We have shown previously that anti-IgM stimulation
that induces BCR signaling does not lead to the proliferation of
BLNK
/
B cells (18). This is re-capitulated
and shown in Fig. 1A, where
BLNK
/
B cells are non-responsive to
increasing amount of anti-IgM stimulation. In comparison and as
expected, wild-type B cells undergo cellular proliferation in a manner
proportional to the extent of their BCR cross-linking. The inability of
BLNK
/
B cells to proliferate upon BCR
engagement is specific to this stimulus as lipopolysaccharides (LPS), a
potent mitogen, induces similar levels of proliferation in both the
wild-type and BLNK
/
B cells (Fig.
1B). This phenomenon whereby
BLNK
/
B cells respond to LPS but not
anti-IgM stimulations was observed regardless of the age of the mice in
which the cells were isolated (data not shown).
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Fig. 1.
Defective proliferation of
BLNK /
B
cells in response to anti-IgM but not LPS stimulation. Purified
splenic wild-type and BLNK
/
B cells were
stimulated for 48 h with increasing concentrations of goat
anti-mouse IgM F(ab')2 fragment (A) or LPS
(B). Cell proliferation was quantified in an MTT
colorimetric assay. Figure shown is representative of five independent
experiments and reproducible with B cells obtained from mice of
different ages. O.D., optical density.
/
B cells to proliferate
upon BCR engagement would suggest that these cells are defective in
their entry and/or completion of their cell cycle. To distinguish
between these possibilities, we performed cell cycle analysis on
anti-IgM-treated wild-type and BLNK
/
B
cells. As described previously (26), the combined use of BrdUrd
incorporation and PI staining of total cellular DNA content allows one
to discriminate cells in the various stages of the cell cycle. In
contrast to cells in the resting G0/G1
phase, cells in the S phase of the cell cycle are actively
synthesizing DNA and will incorporate BrdUrd. They will also exhibit a
higher level of PI staining due to their increased cellular DNA
content. As seen in Fig. 2, analysis of
anti-IgM-stimulated wild-type B cells clearly reveals a population of
cells in the S phase of the cell cycle. In contrast, such S phase
cycling cells are absent in anti-IgM-treated samples of
BLNK
/
B cells, suggesting that the mutant
cells are arrested at the G0/G1 phase. Again as
control, BLNK
/
B cells are able to enter
the cell cycle upon treatment with LPS, and they show a pattern of
BrdUrd incorporation indistinguishable from that of LPS-treated
wild-type B cells (Fig. 2). This is consistent with the proliferation
data shown in Fig. 1B, which indicate the ability of
BLNK
/
B cells to respond to LPS
stimulation. Thus, the data presented in Figs. 1 and 2 together show
that the failure of BLNK
/
B cells to
proliferate in respond to anti-IgM stimulation is due to their
inability to enter the cell cycle.
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Fig. 2.
Anti-IgM-stimulated
BLNK /
B
cells failed to enter the cell cycle. Purified splenic wild-type
and BLNK
/
B cells were cultured in the
presence of 40 µM BrdUrd (BrdU) for 48 h
without stimulus or treated with 20 µg/ml goat anti-mouse IgM
F(ab)'2 fragment or 25 µg/ml LPS. BrdUrd incorporation
and PI staining of total cellular DNA content are used to reveal cells
at various stages of the cell cycle. Arrows indicate
apoptotic cells (A) or cells in the
G0/G1 and S phase of the cell cycle.
/
B Cells--
The entry of
cells into the cell cycle is regulated by the activity of specific
proteins such as the cyclins and the cyclin-dependent kinases (cdks) (27). The D-type cyclins and their kinase partners, cdk4
and cdk6, are the earliest cell cycle regulatory protein complexes to
be expressed when cells leave quiescence and enter the cell cycle (28).
Normal proliferating B cells express cyclins D2 and D3 but not D1 (25).
Cyclin D2 and its kinase partner, cdk4, are up-regulated from mid to
late-G1 phase of the cell cycle and are readily detected in cycling B
cells (29). To determine if cyclin D2 and cdk4 can be induced in
BLNK
/
B cells, we treated the wild-type and
mutant cells with anti-IgM antibodies for various times. As shown in
Fig. 3, cyclin D2 is maximally induced in
wild-type B cells 24 h after BCR cross-linking and its expression
can be detected even after 48 h of treatment. Similarly, cdk4,
which is expressed at basal level in normal resting B cells, is also
up-regulated upon anti-IgM stimulation. In contrast, both cyclin D2 and
cdk4 are not expressed or up-regulated in
BLNK
/
B cells regardless of the duration of
their BCR cross-linking.
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Fig. 3.
Lack of induction of cell cycle regulatory
proteins in anti-IgM-stimulated
BLNK /
B
cells. Purified splenic wild-type and
BLNK
/
B cells were treated with goat
anti-mouse IgM F(ab)'2 fragment or LPS for various times
and examined for the expression of cell cycle regulatory proteins in
Western blot analyses. The anti-tubulin blot served as a control for
the loading of whole cell lysate. Figure shown is representative of
three separate analyses.
/
B cells after LPS treatment. As
expected, LPS induces the up-regulation of cyclin D2 and cdk4 in
wild-type B cells (Fig. 3). LPS is also able to induce the expression
of cyclin D2 and cdk4 in BLNK
/
B cells,
consistent with the fact that LPS-treated mutant B cells do proliferate
(Fig. 1), incorporate BrdUrd (Fig. 2), and enter the cell cycle. This
suggests that the failure to express the cell cycle regulatory proteins
in BLNK
/
B cells is specific to anti-IgM
stimulation. Thus, the failure of anti-IgM-stimulated
BLNK
/
B cells to proliferate may be due to
the inability of these cells to transduce the signal for the induction
of specific regulatory proteins that are critical for the entry into
cell cycle.
/
B Cells Do Not Express
Bcl-xL upon Anti-IgM Stimulation--
In
addition to the induction of cell cycle regulatory proteins, the
cross-linking of the BCR on normal B cells also leads to the expression
of the cell survival protein, Bcl-xL (30, 31). The
induction of Bcl-xL has been correlated with the ability of
activated B cells to undergo cellular proliferation (30) and may be
required for them to complete the cell cycle (11, 13). As shown in Fig.
4A, this protein is expressed
in anti-IgM-treated wild-type or BLNK+/
B cells within
24 h of stimulation and its continued expression can be detected
up to 72 h of culture (data not shown). In contrast, Bcl-xL is absent in BLNK
/
B
cells treated with anti-IgM for the same time duration (Fig. 4A and data not shown). Again as control, Bcl-xL
expression can be detected within 24 h in both LPS-treated
wild-type and BLNK
/
B cells (Fig.
4B). Thus, the data indicate that anti-IgM-treated BLNK
/
B cells may also abort the cell cycle
due to their inability to express the cell survival Bcl-xL
protein upon activation.
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Fig. 4.
Absence of Bcl-xL expression in
anti-IgM-stimulated
BLNK /
B
cells. Western blot analysis of Bcl-xL expression in
wild-type, BLNK+/
and BLNK
/
B
cells stimulated with 10 µg/ml goat anti-mouse IgM F(ab)'2 fragment
for 48 h (A) or 10 µg/ml LPS for 24-48 h
(B). The A431 cell line provides the specificity control for
the anti-Bcl-xL antibody. The anti-tubulin blot serves as a
control for the loading of whole cell lysate.
/
B
cells before and after various stimulations. As shown in Fig. 5 (A and B), Bcl-2
is expressed at equivalent level in wild-type and
BLNK
/
B cells ex vivo and its
expression level remains relatively unchanged upon stimulation with
anti-IgM antibodies or LPS. Thus, a BLNK deficiency specifically
affects the expression of Bcl-xL but not that of Bcl-2 in B
lymphocytes.
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Fig. 5.
The expression of Bcl-2 is not altered in
wild-type and
BLNK /
B
cells treated with various stimuli. Western blot analysis of Bcl-2
expression in wild-type and BLNK
/
B cells
stimulated for 24-48 h with either 10 µg/ml goat anti-mouse IgM
F(ab)'2 fragment (A) or 10 µg/ml LPS (B). The
anti-tubulin blot serves as a control for the loading of whole cell
lysate.
/
B Cells Exhibit a High Rate of Spontaneous
Apoptosis in Culture--
In the course of the cell cycle analysis
as shown in Fig. 2, it was noticed that
BLNK
/
B cells cultured for 48 h in
medium alone or in the presence of anti-IgM antibodies show a higher
degree of apoptosis compared with similarly treated wild-type B cells.
To determine if BLNK
/
B cells indeed
exhibit a higher propensity to undergo spontaneous apoptosis in
vitro, we examined these cells after overnight culture, either
non-stimulated or treated with the various stimuli. Apoptotic cells can
be distinguished in FACS analyses by their reduced cellular DNA
content, as revealed by PI staining, and by their smaller size, as
shown by a reduction in the forward and side scatter profiles. As shown
in Fig. 6,
BLNK
/
B cells are twice as likely
(50-65%) to undergo spontaneous apoptosis in medium after overnight
culture as compared with their normal counterparts (33-38%). Anti-IgM
treatment can reduce the fraction of dying wild-type (20-23%) but not
BLNK
/
(55-63%) B cells. This can be
explained by the ability of wild-type but not
BLNK
/
B cells to express Bcl-xL
upon activation (see Fig. 4). Finally, LPS treatment substantially
reduced the fraction of dying cells in both the wild-type and
BLNK
/
B cell samples, although the
population of apoptotic cells is still higher in the latter compared
with the former. This is consistent with the fact that LPS can induce
the expression of Bcl-xL in both the wild-type and
BLNK
/
B cells (see Fig. 4).
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Fig. 6.
High rate of spontaneous apoptosis of
BLNK /
B
cells in culture. Purified splenic wild-type and
BLNK
/
B cells were cultured overnight in
media alone or with 20 µg/ml goat anti-mouse IgM F(ab)'2
fragment or 25 µg/ml LPS. A, apoptotic cells with
sub-G0 amount of DNA as revealed by PI staining in FACS
analyses were marked and expressed as a percentage of total cells
analyzed. B, apoptotic cells as revealed by their reduced
forward and side scatter profiles in FACS analyses were gated and
expressed as a percentage of total cells examined.
/
B
Cells--
The various analyses above describe the cellular defects of
BLNK
/
B cells in response to BCR signaling.
Given the fact that BLNK is an adaptor protein involved in signal
transduction, it would be of interest to correlate the above cellular
defects with disruption of distinct signaling pathways. BCR engagement
is known to activate the three different classes of MAPKs: ERK, JNK,
and p38 MAPK, that have been shown to regulate proliferation, survival,
and differentiation in various cellular systems (33). In the DT40 chicken B cell line, BLNK is required for the activation of both JNK
and p38 MAPK and for the sustained activation of ERK in response to BCR
signaling (8). We therefore re-examined if the activation of MAPKs was
similarly affected in mouse primary B cells lacking BLNK and if these
signaling defects (if any) could plausibly explain the cellular defects
that we have observed above.
/
B cells, suggesting that the
activation of ERK is normal in these mutant B cells. Similarly, Western
blot analyses of whole cell lysates derived from non-treated and
anti-IgM-stimulated wild-type and BLNK
/
B
cells reveals that JNK (Fig. 7B) and p38 MAPK (Fig.
7C) are also phosphorylated with the same kinetics in both
the samples tested. The intact activation of the three classes of MAPKs
in BLNK
/
mouse primary B cells is in
contrast to the impaired activation of these kinases in the DT40
chicken B cell line (8).
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Fig. 7.
Normal activation of MAPKs and Akt in
BCR-stimulated
BLNK /
B
cells. Wild-type and BLNK
/
B cells
were treated with anti-IgM antibodies and the expression and activation
of ERK (A), JNK (B), p38 MAPK (C), and
Akt (D) were examined in Western blot analyses. The whole
cell lysates were first probed with an antibody that recognizes the
phosphorylated form and subsequently with an antibody that binds the
non-phosphorylated form of the protein that is being studied.
/
B cells. As shown in Fig.
7D, the kinetics of Akt activation is again similar in both
the wild-type and BLNK
/
B cells stimulated
by anti-IgM antibodies. Thus, the overall data suggest that BLNK plays
no role in transducing the BCR signal that activates the three
different classes of MAPKs and Akt in mouse primary B lymphocytes and
that these signaling pathways are not likely responsible for the
cellular defects described earlier.
B in Response to BCR
Engagement--
The engagement of the antigen receptor on B lymphocyte
also activates the transcription factor NF-
B that is known to
regulate genes involved in cell proliferation and survival (35).
Several groups have shown that the predominant form of NF-
B in B
cells is largely the p50-c-Rel heterodimer (36); in particular, c-Rel was shown to be essential for B cell proliferation after BCR engagement (35, 36).
B activation,
nuclear extracts were prepared from non-treated and anti-IgM-stimulated wild-type and BLNK
/
mouse primary B cells.
As shown in the EMSA in Fig.
8A, BCR stimulation of
wild-type B cells leads to a marked increase in nuclear NF-
B activity (lanes 1 and 2) as evidenced
by the binding of a radiolabeled probe that contains two consensus
NF-
B binding sequences. In contrast, there was no increase in
BCR-induced NF-
B activity above the background levels in the nuclear
extract obtained from BLNK
/
B cells
(lanes 4 and 5). As control, the
treatment of wild-type and BLNK
/
B cells
with phorbol ester (PMA) leads to a corresponding increase in nuclear
NF-
B activity in both the samples tested (compare lanes
1 and 3 and lanes 4 and
6). This suggests that BLNK
/
B
cells can activate NF-
B in response to other stimulus but not to
anti-IgM stimulation. Thus, the data indicate that BLNK is specifically
required for BCR-induced activation of NF-
B.
View larger version (52K):
[in a new window]
Fig. 8.
Impaired NF- B
activation in BCR-stimulated
BLNK
/
B
cells. A, lack of NF-
B binding activity in the
nuclear extract of BLNK
/
B cells. EMSA
analysis of nuclear NF-
B activity in wild-type (lanes
1-3) and BLNK
/
(lanes 4-6) splenic B cells that were either
non-treated or stimulated with PMA (2 µg/ml) or anti-IgM antibodies
(50 µg/ml) for 3 h. B, impaired nuclear translocation
of c-Rel and the p50 subunit in BCR-stimulated
BLNK
/
B cells. Western blot analysis of
nuclear extracts (5 µg) obtained from wild-type and
BLNK
/
B cells that were non-treated or
stimulated with PMA or anti-IgM antibodies. The blot was first probed
with anti-c-Rel antibody and subsequently re-probed with
anti-transcription factor IID (anti-TFIID) antibody to
control for the amount and integrity of the nuclear extracts used. In a
separate blot, the amount of nuclear p50 (bottom
panel) was examined. C, equivalent amount of
cytoplasmic c-Rel is found in wild-type and
BLNK
/
B cells. Western blot analysis of
whole cell lysates (5 × 106 cells) obtained from
wild-type and BLNK
/
B cells that were
treated as above. The blot was initially probed with the anti-c-Rel
antibody and re-probed with the anti-p38 MAPK antibody as control.
D, degradation of I
B
is impaired in BCR-stimulated
BLNK
/
B cells. Splenic wild-type and
BLNK
/
B cells were pre-cultured and later
stimulated with PMA or anti-IgM antibodies for 3 h in the
continuous presence of cycloheximide. Anti-I
B
antibody was used
to determine for the presence of the I
B
proteins, whereas
anti-p38 MAPK antibody was used as control for the equivalent loading
of whole cell lysates.
B activation have been
elucidated. In non-stimulated cells, NF-
B/Rel factors are
sequestered in the cytoplasm in complexes with the I
B family of
proteins (35, 36). Upon treatment of cells with specific stimulus, the
I
B kinases are activated and this results in the serine/threonine phosphorylation of I
B proteins and their subsequent degradation. The
NF-
B/Rel proteins are released from the inhibitory complexes and
localized to the nucleus to effect gene transcription (35, 36).
B binding activity in BCR-stimulated
BLNK
/
B cells could therefore be due to
reduced NF-
B proteins in the nucleus. To determine if this is indeed
the case, we examined the amount of c-Rel and the p50 subunit in the
nucleus of mutant cells. As seen in the Western blot analysis in Fig.
8B, PMA or anti-IgM stimulation led to an accumulation of
c-Rel in the nucleus of wild-type B cells (lanes
2 and 3). However, in contrast, the accumulation
of nuclear c-Rel was effected only with PMA but not with anti-IgM
treatment of BLNK
/
B cells
(lanes 5 and 6). These data are
consistent with the NF-
B binding assay shown in Fig. 8A.
The reduced level of nuclear c-Rel in the BCR-induced
BLNK
/
B cell sample was not due to
variation in the integrity of the nuclear extracts, as control Western
blot analysis revealed the presence of similar amount of nuclear
transcription factor IID in all the samples tested (Fig. 8B,
middle panel). Examination of the p50 subunit
also reveals that it is not translocated into the nucleus of
BLNK
/
B cells after anti-IgM stimulation
(Fig. 8B, lower panel) similar to the
situation found with c-Rel.
/
B cells may
produce reduced amount of total c-Rel/p50 proteins and this in turn
affects the amount found in the nucleus. To explore this possibility,
we examine the amount of c-Rel in the whole cell lysates of wild-type
and BLNK
/
B cells. Western blot analysis
shown in Fig. 8C reveals that untreated, PMA-stimulated, or
anti-IgM-stimulated BLNK
/
B cells produced
amounts of c-Rel equivalent to that produced by similarly treated
wild-type B cells. Thus, the data suggest that
BLNK
/
B cells have specific impairment in
the nuclear translocation of NF-
B factors in response to BCR engagement.
B/Rel transcription factors are sequestered
in the cytoplasm by I
B factors (35, 36). The failure of NF-
B/Rel
transcription factors to translocate into the nucleus of BCR-stimulated
BLNK
/
B cells could be due to impairment in
the degradation of the I
B proteins. To test this possibility, we
examine the degradation of I
B
in these cells in response to the
different stimuli. Both the wild-type and
BLNK
/
splenic B cells were cultured in the
presence of cycloheximide to prevent the de novo synthesis
of I
B
. As seen in Fig. 8D and consistent with the data
presented earlier, I
B
was degraded in wild-type B cells
stimulated with either PMA or anti-IgM antibodies (lanes
2 and 3). In comparison, the degradation of
I
B
was normal in BLNK
/
B cells
treated with PMA (lane 5) but impaired in the
mutant cells stimulated with anti-IgM antibodies (lane
6). This suggests that the failure of NF-
B to translocate
to the nucleus in BCR-stimulated BLNK
/
B
cells is due to a specific impairment in the degradation of I
B factors.
B activation in B cells and this occurs via a mechanism that
involves the degradation of the I
B subunits and the subsequent
nuclear translocation of the NF-
B/Rel transcription factors.
2 Is Impaired in BCR-stimulated BLNK
/
B
Cells--
Recently Btk has been shown to be essential for the
BCR-induced activation of NF-
B in B cells (37, 38), and this
involves a similar mechanism that requires the degradation of I
B
subunits. BLNK has been shown to associate with Btk (9, 10). Hence, it
is possible that the inactivation of BLNK may affect the expression and/or activation of Btk, which in turn leads to the impairment in
NF-
B activation in BLNK
/
B cells. We
therefore immunoprecipitated Btk from lysates of non-treated or
anti-IgM-stimulated wild-type and mutant B cells to explore this
possibility. As shown in Fig.
9A (lower
panel), Btk is expressed at equivalent levels in both the
wild-type and BLNK
/
B cells that were
either non-treated or stimulated with anti-IgM antibodies. In addition,
Btk is also activated with the same kinetics in both the wild-type and
mutant B cells, as revealed by the anti-phosphotyrosine antibody that
reveals the phosphorylation and activation status of Btk (Fig.
9A, upper panel). Thus, the inability
of BLNK
/
B cells to activate NF-
B in
response to BCR stimulation is not due to a defect in the expression or
activation of Btk per se.
View larger version (57K):
[in a new window]
Fig. 9.
Expression and activation of Btk and
PLC- 2 in anti-IgM-stimulated
BLNK
/
B
cells. Btk (A) and PLC-
2 (B) were
immunoprecipitated from whole cell lysates of wild-type and
BLNK
/
B cells that were treated with
anti-IgM antibodies for various times and probed with
anti-phosphotyrosine (pY) and the immunoprecipitating
antibodies in Western blot (WB) analyses. The figures shown
are representative of five separate experiments.
2 that catalyzes the hydrolysis of
phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate and diacylglycerol is also essential for the BCR-induced activation of
NF-
B. We thus immunoprecipitated PLC-
2 from
BLNK
/
B cells that were stimulated with
anti-IgM antibodies for various times to determine if the enzyme is
activated normally in these cells. As shown in Fig. 9B,
PLC-
2 is fully activated within 3 min, and its phosphorylation
persisted for at least 10 min after BCR engagement in wild-type B
cells. In contrast, PLC-
2 remains non-phosphorylated and therefore
not activated after BCR stimulation for the same time duration examined
in BLNK
/
B cells. Thus, the defect in
NF-
B activation in BLNK
/
B cells can be
correlated to a defect in PLC-
2 activation in response to BCR engagement.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
B cells do not proliferate upon
BCR stimulation (Fig. 1). Our data presented here indicate that this
defect is due to the failure of mutant B cells to enter the cell cycle
in response to this stimulus. In contrast to wild-type B cells,
BLNK
/
B cells fail to incorporate BrdUrd
and are arrested prior to the S phase of the cell cycle (Fig. 2).
Indeed, this specific impairment is correlated at the molecular level
to the inability of anti-IgM activated
BLNK
/
B cells to express the cell cycle
regulatory proteins such as cyclin D2 and cdk4 (Fig. 3) that are
necessary for the progression of cell cycle beyond the
G0/G1 phase (29). Anti-IgM treatment of
BLNK
/
B cells also fails to induce the
expression of the cell survival protein Bcl-xL (Fig. 4)
that has been postulated to be critical for the viability of
proliferating B cells (11, 13, 30). Thus,
BLNK
/
B cells may have a compound defect in
not being able to sustain cell survival for the progression of the cell
cycle after anti-IgM stimulation. Taken together, the data strongly
suggest that BLNK
/
B cells fail to enter
and abort the cell cycle upon BCR engagement.
/
mice
failed to mount an effective humoral immune response to T
cell-independent antigen that involved extensive BCR cross-linking (18,
19). Antigen-specific B cells are rare and undergo clonal expansion
when activated. It is likely that antigen-activated B cells are not
clonally expanded in BLNK
/
mutant mice due
to their inability to undergo the cell cycle and to their higher
propensity to undergo apoptosis. Interestingly, BLNK
/
mice are able to mount a normal T
cell-dependent immune response (18, 19). This would suggest
that T cell help in the form of co-stimulation or secreted cytokines
might overcome the cell cycle defect associated with the BCR signaling
resulting from a BLNK deficiency.
B (35, 36), we show here that only the latter is impaired in
BCR-activated BLNK
/
B cells (see Figs. 7
and 8). The finding that Bcl-xL, which is regulated by
NF-
B (40), is not induced in BLNK
/
B
cells in response to BCR stimulation is consistent with this signaling defect.
/
mice (18-21)
and our current biochemical study of BLNK
/
B cells suggest that the defects resulting from a BLNK deficiency mirror more closely to those resulting from a lack of Btk as in xid mice and Btk
/
B cells (23,
24). BLNK
/
and xid B cells are
both unable to up-regulate the cell cycle regulatory proteins and enter
the cell cycle upon BCR engagement (12), and they both show a
propensity to undergo a higher rate of spontaneous apoptosis in culture
(11, 12). In addition, BCR-stimulated Btk
/
B cells also fail to activate the NF-
B signaling pathway (37, 38).
We demonstrate here that the expression and activation of Btk is normal
in BLNK
/
B cells (Fig. 9). Hence, the
phenotypes of BLNK
/
mice and cells are not
likely due to defective expression and activation of Btk per
se. Recently, PLC-
2 was also shown to be essential for the
activation of NF-
B in response to BCR engagement (39). Our
demonstration that this enzyme is not activated in BCR-stimulated
BLNK
/
B cells suggests that this is the
likely defect that impairs NF-
B activation in these mutant cells.
B in
response to BCR stimulation is now emerging (see Fig.
10). Engagement of the BCR activates
the immediate downstream tyrosine kinases Syk and Btk (2). It is known
that both Syk and Btk are required for the full phosphorylation and
activation of PLC-
2 (41). BLNK is the adaptor molecule that couples
Syk to PLC-
2 (4). Since BLNK has also been shown separately to
associate with Btk (9, 10), it is likely to couple Btk to PLC-
2 as well, although this has not been directly proven. Our data of normal
Btk but impaired PLC-
2 activation in BCR-stimulated
BLNK
/
B cells are consistent with this
model of tri-molecular interaction. Thus, BLNK emerges as the key
adaptor molecule that couples Syk and Btk, either in concert or
sequentially to activate PLC-
2, which in turn activates NF-
B that
regulates genes involved in proliferation and survival such as
bcl-x. These signaling molecules Syk, Btk, BLNK, and
PLC-
2 together form a "signalosome" (42). Inactivation of BLNK
(this report) or Btk or PLC-
2 will disrupt this signalosome and lead
to a common NF-
B signaling defect. This may provide an explanation
for the similar B cell defects found in
BLNK
/
(18-21),
Btk
/
(23, 24), and
PLC-
2
/
(43, 44) mice. By extrapolation,
one would then expect that PLC-
2
/
B
cells would also not up-regulate cell cycle regulatory and cell
survival molecules upon BCR engagement, and this awaits further confirmation. Finally, it is worthy to note that B cells deficient in
the various components of NF-
B also exhibit proliferation defects
(35, 36, 45, 46), again consistent with the model that we presented
here.
View larger version (20K):
[in a new window]
Fig. 10.
A model for the BCR-induced activation of
NF- B. Engagement of the BCR activates Syk
and Btk (1). Syk phosphorylates BLNK (2). BLNK
couples Syk and Btk to PLC-
2 (3), and this results in its
activation and to the eventual activation of NF-
B that regulates
genes involved in cell survival and proliferation such as
bcl-x.
Our current analyses indicate that BLNK/
B
cells are able to proliferate upon LPS stimulation. This contrasts
with previous reports that have stated otherwise (19, 21). The
discrepancy in the response to LPS may be due to the relative
sensitivity of the various assays used to examine cellular
proliferation. It may be that the MTT assay used in our current study
is more easily saturated compared with the thymidine incorporation
assay used by others (19, 21) and thus failed to measure the reduction in the LPS-induced proliferation of BLNK
/
B
cells compared with the wild-type B cells. However, it is noted that,
in one of the previous published reports (19),
BLNK
/
B cells did respond to some extent to
LPS stimulation and thus did not contradict our current data
qualitatively. Indeed, the various experiments outlined in this paper
that examine the incorporation of BrdUrd (Fig. 2), the induction of
cell cycle regulatory proteins (Fig. 3), and cell survival protein,
Bcl-xL (Fig. 4), are all consistent with the fact that
BLNK
/
B cells are able to respond to LPS
stimulation and proliferate. Finally, examination of LPS-stimulated
BLNK
/
B cells (Fig. 6) indicates that they
are bigger in size (as reflected by the forward scatter profile)
compared with anti-IgM-stimulated or non-treated
BLNK
/
B cells, again consistent with the
notion that BLNK
/
B cells are activated by
LPS. LPS signals through CD14 and the Toll-like receptors (47), and,
although it activates NF-
B (48), it appears to do so via a pathway
that does not require BLNK. Incidentally, it was reported that
PLC-
2
/
B cells, which have defects
similar to those of BLNK
/
B cells, also
proliferate normally in response to LPS stimulation (43).
Much of the recent work in BCR signaling had been done using the
chicken DT40 B cell line. It was shown in this system that the
BCR-induced activation of ERK and JNK was impaired in the absence of
Syk and Btk (49). Given that BLNK interacts with both Syk and Btk (4,
9, 10), it would be reasonable to assume that some of the MAPKs might
also be affected by a BLNK deficiency. Indeed, it was shown recently
that the BCR-induced activation of ERK, JNK, and p38 MAPK was also
perturbed in the DT40 cell line lacking BLNK (8). However, our data
presented in this paper seem to contradict the latter observation. We
show here that the BCR-induced activation of all three classes of MAPKs remain intact in mouse primary B cells lacking BLNK (see Fig. 7). A
likely explanation in the discrepancy is the cellular context of the
systems used, namely the DT40 chicken B cell line used by others (4,
8-10) versus the mouse primary B lymphocytes used in this
report. Such a discrepancy has indeed been observed in several
instances previously. First of all, it was shown that the BCR-induced
Ca2+ response was reduced in human B cell lines and mouse
primary B lymphocytes lacking Btk (50) but totally abolished in the DT40 chicken B cells (41). Similarly, DT40 chicken B cells lacking BLNK
do not flux Ca2+ in response to BCR cross-linking (8),
whereas mouse B cells lacking BLNK do although the response was again
reduced (19). Thus, there may exist real signaling differences in mouse
primary B lymphocytes and the chicken DT40 cell line. Alternatively, it remains possible that the DT40 chicken B cell line is in a different state of maturation compared with the splenic B cells that we used in
this paper. It is interesting to note that mouse primary B cells
lacking BLNK (19) or Btk (50) still retain the ability to flux
Ca2+ to some extent, although the activation of PLC-2 is
impaired in these mutant cells. On the same note, mouse primary B cells lacking PLC-
2 (43) also retain the ability to flux Ca2+
in response to BCR engagement, although the magnitude is again much reduced.
Finally, it has been suggested that BCR specificity and hence signaling
may play a role in the development of CD5+ B cells (51).
Mice deficient in BLNK (18-21), Btk (23, 24), or PLC-2 (43, 44),
which are molecules involved in BCR signaling, all lack
CD5+ B cells. Recently, it was shown that cyclin D2
expression is also essential for CD5+ B cell development
(52). CD5+ B cells are thought to undergo self-renewal, a
process that is most likely induced through the recognition of an
antigen by their BCR (51). Our current data and those previously
published (37, 38) indicate that BLNK and Btk are part of a signalosome
that transduces the BCR signal that leads to the expression of cyclin D2 and the entry into cell cycle. Thus, in the absence of BLNK or Btk,
the BCR signal that leads to the expression of cell cycle regulatory
molecules is impaired and this will result in the subsequent defect in
BCR-induced cellular proliferation that may affect the self-renewal of
a population of CD5+ B cells.
![]() |
ACKNOWLEDGEMENT |
---|
We thank the Institute of Molecular and Cell Biology In Vivo Model Unit for the care and maintenance of mice.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the National Science and Technology Board of Singapore.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
These authors contributed equally to this work.
§ To whom correspondence should be addressed. Tel.: 65-874-3784; Fax: 65-779 -1117; E-mail: mcblamkp@imcb.nus.edu.sg.
Published, JBC Papers in Press, March 23, 2001, DOI 10.1074/jbc.M010800200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
BCR, B cell antigen
receptor;
BLNK, B cell linker protein;
BrdUrd, 5'-bromo-2'-deoxyuridine;
Btk, Bruton's tyrosine kinase;
cdk, cyclin-dependent kinase;
ERK, extracellular
signal-regulated kinase;
IgM, immunoglobulin M;
IB, inhibitory
B;
JNK, c-Jun NH2-terminal kinase;
LPS, lipopolysaccharide;
MAPK, mitogen-activated protein kinase;
NF-
B, nuclear factor
B;
PI, propidium iodide;
PLC, phospholipase C;
PMA, phorbol 12-myristate
13-acetate;
PTK, protein-tyrosine kinase;
xid, X-linked
immunodeficiency;
PBS, phosphate-buffered saline;
BSA, bovine serum
albumin;
MTT, (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl
tetrazolium bromid) labeling reagent;
FACS, fluorescence-activated cell
sorting.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | DeFranco, A. (1997) Curr. Opin. Immunol. 9, 296-308[CrossRef][Medline] [Order article via Infotrieve] |
2. | Campbell, K. (1999) Curr. Opin. Immunol. 11, 256-264[CrossRef][Medline] [Order article via Infotrieve] |
3. | Myung, P., Boerthe, N., and Koretzky, G. (2000) Curr. Opin. Immunol. 12, 256-266[CrossRef][Medline] [Order article via Infotrieve] |
4. | Fu, C., Turck, C., Kurosaki, T., and Chan, A. (1998) Immunity 9, 93-103[Medline] [Order article via Infotrieve] |
5. |
Wienands, J.,
Schweikert, J.,
Wollscheid, B.,
Jumaa, H.,
Nielsen, P.,
and Reth, M.
(1998)
J. Exp. Med.
188,
791-795 |
6. |
Goitsuka, R.,
Fujimura, Y.,
Mamada, H.,
Umeda, A.,
Morimura, T.,
Uetsuka, K.,
Doi, K.,
Tsuji, S.,
and Kitamura, D.
(1998)
J. Immunol.
161,
5804-5808 |
7. |
Jackman, J.,
Motto, D.,
Sun, Q.,
Tanemoto, M.,
Turck, C.,
Peltz, G.,
Koretzky, G.,
and Findell, P.
(1995)
J. Biol. Chem.
270,
7029-7032 |
8. | Ishiai, M., Kurosaki, M., Pappu, R., Okawa, K., Ronko, I., Fu, C., Shibata, M., Iwamatsu, A., Chan, A., and Kurosaki, T. (1999) Immunity 10, 117-125[Medline] [Order article via Infotrieve] |
9. |
Hashimoto, S.,
Iwamatsu, A.,
Ishiai, M.,
Okawa, K.,
Yamadori, T.,
Matsushita, M.,
Baba, Y.,
Kishimoto, T.,
Kurosaki, T.,
and Tsukada, S.
(1999)
Blood
94,
2357-2364 |
10. | Su, Y., Zhang, Y., Schweikert, J., Koretzky, G., Reth, M., and Wienands, J. (1999) Eur. J. Immunol. 29, 3702-3711[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Anderson, J.,
Teutsch, M.,
Dong, Z.,
and Wortis, H.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
10966-10971 |
12. | Brorson, K., Brunswick, M., Ezhevsky, S., Wei, D., Berg, R., Scott, D., and Stein, K. E. (1997) J. Immunol. 159, 135-143[Abstract] |
13. |
Solvason, N.,
Wu, W.,
Kabra, N.,
Lund-Johansen, F.,
Roncarolo, M.,
Behrens, T.,
Grillot, D.,
Nunez, G.,
Lees, E.,
and Howard, M.
(1998)
J. Exp. Med.
187,
1081-1091 |
14. | Tsukada, S., Saffran, D., Rawlings, D., Parolini, O., Allen, R., Klisak, I., Sparkes, R., Kubagawa, H., Mohandas, T., and Quan, S. (1993) Cell 72, 279-290[Medline] [Order article via Infotrieve] |
15. | Thomas, J., Sideras, P., Smith, C., Vorechovsky, I., Chapman, V., and Paul, W. (1993) Science 261, 355-358[Medline] [Order article via Infotrieve] |
16. | Vetrie, D., Vorechovsky, I., Sideras, P., Holland, J., Davies, A., Flinter, F., Hammarstrom, L., Kinnon, C., Levinsky, R., and Bobrow, M. (1993) Nature 361, 226-233[CrossRef][Medline] [Order article via Infotrieve] |
17. | Rawlings, D., Saffran, D., Tsukada, S., Largaespada, D., Grimaldi, J., Cohen, L., Mohr, R., Bazan, J., Howard, M., and Copeland, N. (1993) Science 261, 358-361[Medline] [Order article via Infotrieve] |
18. |
Xu, S.,
Tan, J.,
Wong, E.,
Manickam, A.,
Ponniah, S.,
and Lam, K. P.
(2000)
Int. Immunol.
12,
397-404 |
19. | Jumaa, H., Wollscheid, B., Mitterer, M., Wienands, J., Reth, M., and Nielsen, P. (1999) Immunity 11, 547-554[Medline] [Order article via Infotrieve] |
20. |
Pappu, R.,
Cheng, A.,
Li, B.,
Gong, Q.,
Chiu, C.,
Griffin, N.,
White, M.,
Sleckman, B.,
and Chan, A.
(1999)
Science
286,
1949-1954 |
21. |
Hayashi, K.,
Nittono, R.,
Okamoto, N.,
Tsuji, S.,
Hara, Y.,
Goitsuka, R.,
and Kitamura, D.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
2755-2760 |
22. |
Xu, S.,
Wong, S.,
and Lam, K. P.
(2000)
J. Immunol.
165,
4153-4157 |
23. | Khan, W., Alt, F., Gerstein, R., Malynn, B., Larsson, I., Rathbun, G., Davidson, L., Muller, S., Kantor, A., and Herzenberg, L. (1995) Immunity 3, 283-299[Medline] [Order article via Infotrieve] |
24. | Kerner, J., Appleby, M., Mohr, R., Chien, S., Rawlings, D., Maliszewski, C., Witte, O., and Perlmutter, R. (1995) Immunity 3, 301-312[Medline] [Order article via Infotrieve] |
25. | Solvason, N., Wu, W., Kabra, N., Wu, X., Lees, E., and Howard, M. (1996) J. Exp. Med. 184, 407-417[Abstract] |
26. | Carayon, P., and Bord, A. (1992) J. Immunol. Methods 147, 225-230[Medline] [Order article via Infotrieve] |
27. | Reed, S. (1997) Cancer Surv. 29, 7-23[Medline] [Order article via Infotrieve] |
28. | Sherr, C. (1993) Cell 73, 1059-1065[Medline] [Order article via Infotrieve] |
29. | Tanguay, D., and Chiles, T. (1996) J. Immunol. 156, 539-548[Abstract] |
30. | Choi, M., Holmann, M., Atkins, C., and Klaus, G. (1996) Eur. J. Immunol. 26, 676-682[Medline] [Order article via Infotrieve] |
31. | Grillot, D., Merino, R., Pena, J., Fanslow, W., Finkelman, F., Thompson, C., and Nunez, G. (1996) J. Exp. Med. 183, 381-391[Abstract] |
32. | Merino, R., Ding, L., Veis, D., Korsmeyer, S., and Nunez, G. (1994) EMBO J. 13, 683-691[Abstract] |
33. | Craxton, A., Otipoby, K. L., Jiang, A., and Clark, E. A. (1999) Adv. Immunol. 73, 79-152[Medline] [Order article via Infotrieve] |
34. | Downward, J. (1998) Curr. Opin. Cell Biol. 10, 262-267[CrossRef][Medline] [Order article via Infotrieve] |
35. | Gerondakis, S., Grumont, R., Rourke, I., and Grossmann, M. (1998) Curr. Opin. Immunol. 10, 353-359[CrossRef][Medline] [Order article via Infotrieve] |
36. | Baldwin, A. S., Jr. (1996) Annu. Rev. Immunol. 14, 649-681[CrossRef][Medline] [Order article via Infotrieve] |
37. |
Bajpai, U. D.,
Zhang, K.,
Teutsch, M.,
Sen, R.,
and Wortis, H.
(2000)
J. Exp. Med.
191,
1735-1744 |
38. |
Petro, J. B.,
Rahman, S. M. J.,
Ballard, D. W.,
and Khan, W. N.
(2000)
J. Exp. Med.
191,
1745-1753 |
39. |
Petro, J. B.,
and Khan, W. N.
(2001)
J. Biol. Chem.
276,
1715-1719 |
40. | Glasgow, J. N., Wood, T., and Perez-Polo, J. R. (2000) J. Neurochem. 75, 1377-1389[CrossRef][Medline] [Order article via Infotrieve] |
41. | Takata, M., and Kurosaki, T. (1996) J. Exp. Med. 184, 31-40[Abstract] |
42. | Fruman, D., Satterthwaite, A., and Witte, O. (2000) Immunity 13, 1-3[Medline] [Order article via Infotrieve] |
43. |
Hashimoto, A.,
Takeda, K.,
Inaba, M.,
Sekimata, M.,
Kaisho, T.,
Ikehara, S.,
Homma, Y.,
Akira, S.,
and Kurosaki, T.
(2000)
J. Immunol.
165,
1738-1742 |
44. | Wang, D., Feng, J., Wen, R., Marine, J., Sangster, M., Parganas, E., Hoffmeyer, A., Jackson, C., Cleveland, J., Murray, P., and Ihle, J. (2000) Immunity 13, 25-35[Medline] [Order article via Infotrieve] |
45. | Kontgen, F., Grumont, R. J., Strasser, A., Metcalf, D., Li, R., Tarlinton, D., and Gerondakis, S. (1995) Genes Dev. 9, 1965-1977[Abstract] |
46. | Snapper, C. M., Zelazowski, P., Rosas, F. R., Kehry, M. R., Tian, M., Baltimore, D., and Sha, W. (1996) J. Immunol. 156, 183-191[Abstract] |
47. |
Perera, P. Y.,
Mayadas, T. N.,
Takeuchi, O.,
Akira, S.,
Zaks-Zilberman, M.,
Goyert, S. M.,
and Vogel, S. N.
(2001)
J. Immunol.
166,
574-581 |
48. |
Faure, E.,
Equils, O.,
Sieling, P. A.,
Thomas, L.,
Zhang, F. X.,
Kirschning, C. J.,
Polentarutti, N.,
Muzio, M.,
and Arditi, M.
(2000)
J. Biol. Chem.
275,
11058-11063 |
49. |
Jiang, A.,
Craxton, A.,
Kurosaki, T.,
and Clark, E. A.
(1998)
J. Exp. Med.
188,
1297-1306 |
50. |
Buhl, A. M.,
and Cambier, J. C.
(1999)
J. Immunol.
162,
4438-4446 |
51. | Haughton, G., Arnold, L., Whitmore, A., and Clarke, S. (1993) Immunol. Today 14, 84-91[Medline] [Order article via Infotrieve] |
52. |
Solvason, N.,
Wu, W.,
Parry, D.,
Mahony, D.,
Lam, E.,
Glassford, J.,
Klaus, G.,
Sicinski, P.,
Weinberg, R.,
Liu, Y.,
Howard, M.,
and Lees, E.
(2000)
Int. Immunol.
12,
631-638 |