From the Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146
Received for publication, October 6, 2000
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ABSTRACT |
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Mutations in the gene encoding Bruton's tyrosine
kinase (BTK) interfere with B cell proliferation and lead to an
X-linked immunodeficiency in mice characterized by reduced B cell
numbers. Recent studies have established that BTK transmits signals
from the B cell antigen receptor (BCR) to transcription factor NF- The generation and survival of B lymphocyte subpopulations is
contingent upon the expression of a functional B cell antigen receptor
complex (BCR)1 (1, 2). BCR
engagement directs B cell biological responses by initiating
biochemical signaling cascades involving the cytoplasmic protein
tyrosine kinases Lyn, Syk, and BTK (3-5). BTK plays an integral role
in transducing BCR-directed signals, because mutations in the
btk gene result in the B cell deficiencies X-linked
agammaglobulinemia (XLA) in man and X-linked immunodeficiency (xid) in
mice (6-10). B cells from xid mice are defective in
survival and proliferation, implicating BTK in these biological
processes (10-12). However, the molecular mechanisms by which BTK
effects B cell proliferation and survival are not well understood.
Like BTK, transcription factor NF- BTK, in concert with the protein tyrosine kinase Syk and the adaptor
protein BLNK, has recently been demonstrated to phosphorylate and
activate PLC- In this report, we provide two lines of evidence indicating that
BCR-initiated activation of NF- Cells and Reagents--
The chicken B cell line DT40, DT40 cells
deficient for either BTK or PLC-
Splenocytes and primary B lymphocytes were isolated from spleens of
C57Bl6 mice. For phospho-I
All pharmacological reagents were purchased from Calbiochem. For
inhibition of BCR signaling, cells were incubated with EGTA (5 mM), BAPTA-AM (20 nM), cyclosporin A (20 µg/ml), bisindolylmaleimide I (20 µM), or U-73122 (5 µM) for 30 min prior to and during stimulation. Except
where indicated in the figure legends, DT40 B cells were either left
unstimulated or stimulated with a 1:2 dilution of hybridoma
supernatants containing anti-chicken IgM monoclonal antibody (M4) or
PMA and ionomycin, 1 µM each. Purified B cells (3-5 × 106 cells per sample) were incubated with 10 µg/ml
polyclonal goat anti-mouse IgM F(ab')2 fragments (Jackson
ImmunoResearch), 10 µg/ml anti-mouse CD40 (PharMingen), or with PMA
and ionomycin (1 µM each) at a cellular density of 2 × 106/ml in culture media (RPMI 1640 supplemented with
10% serum). To monitor any effects of serum on the activation of
NF- Electrophoretic Mobility Shift Assays (EMSAs)--
Nuclear
extracts were prepared and used in DNA-binding reactions as described
previously (18). For EMSAs, an [ Western Blot Analyses--
For Western blot analysis of
RelA and c-Rel, nuclear extracts equivalent to 2 × 107 cells were denatured in Laemmli reducing buffer by
boiling at 95 °C for 3 min, and the proteins were resolved by
SDS-PAGE. Proteins were electrotransferred onto nitrocellulose
membranes and subjected to immunoblotting with rabbit polyclonal
antibodies against RelA, c-Rel, or SP1 as described previously
(18). For I Plasmid Constructs and Luciferase Assays--
The
The indicated DT40 cell lines were each cotransfected by
electroporation (250 V, 960 microfarads, Bio-Rad Gene Pulser) with 5 µg of the 6 In Vitro Kinase Assays--
In vitro kinase assays
were performed on the cytosolic fraction of 5 × 106 B
cells as described previously (18). Briefly, cell extracts from
0.5 × 106 cell equivalents were removed for Western
blot analysis, and the remaining cell extract was subjected to
immunoprecipitation with anti-IKK In prior studies, we established that BTK is required for nuclear
translocation of NF- The DT40 B cell system is amenable to genetic manipulation and has thus
proven invaluable for biochemical analysis of BCR-signaling events
(27). To determine whether calcium and calcineurin play a role in
BCR-responsive activation of NF-B, which in turn reprograms a set of genes required for normal B cell
growth. We now demonstrate that induction of NF-
B via this pathway
requires the intermediate action of the -
2 isoform of phospholipase
C (PLC-
2), a potential phosphorylation substrate of BTK.
Specifically, pharmacologic agents that block the action of either
PLC-
2 or its second messengers prevent BCR-induced activation
of I
B kinase. Moreover, activation of NF-
B in response to BCR
signaling is completely abolished in B cells deficient for PLC-
2.
Taken together, these findings strongly suggest that PLC-
2 functions
as an integral component of the BTK/NF-
B axis following BCR
ligation. Interference with this NF-
B cascade may account for some
of the B cell defects reported for
plc-
2
/
mice,
which develop an X-linked immunodeficiency-like phenotype.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
B has been implicated in the
regulation of genes essential for B cell responses including proliferation and survival (13-15). In resting cells, NF-
B is sequestered in the cytoplasmic compartment via its association with a
family of inhibitory proteins, termed I
Bs (16). Recent studies have
identified a cytokine-inducible I
B kinase complex (IKK) consisting
of two catalytic (IKK
and IKK
) and one regulatory subunit
(IKK
) (17). In response to NF-
B activating signals, IKK
phosphorylates and targets I
B for degradation (17). We and others
(18, 19) have recently shown that BTK couples the BCR to IKK and
NF-
B. However, the biochemical mechanism by which BTK activates
NF-
B remains largely undefined.
2 (22-24). In response to BCR signals, PLC-
2 catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate, generating inositol 1,4,5-trisphosphate and diacylglycerol. Inositol 1,4,5-trisphosphate induces the release of Ca2+ from
intracellular stores, and diacylglycerol facilitates the activation of
PKC isoenzymes (20, 21). Thus, BTK-dependent activation of
PLC-
2 is essential for BCR-initiated calcium fluxes (22). However,
the functional consequences of PLC-
2 signaling in the activation of
nuclear factors that direct B cell responses are not known.
B is mediated by PLC-
2. First,
DT40 chicken B cells deficient for PLC-
2 fail to translocate NF-
B
to the nucleus upon BCR activation. Second, pharmacologic inhibition of
PLC-
2 or its second messengers prevents BCR-responsive activation of
IKK and phosphorylation of I
B
in primary B cells. These
biochemical findings provide a potential molecular explanation for the
B cell defects recently reported for
plc-
2
/
mice, which
display an xid-like phenotype reminiscent of animals lacking
functional BTK (10).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
2 (DT40.BTK, DT40.PLC-
2), or
mutant DT40 cells reconstituted with either human BTK or PLC-
2
(DT40.BTKR, DT40.PLC-
2R) were a kind gift of Dr. Tomohiro Kurosaki,
Riken Cell Bank, Japan (23, 24). DT40 cells were maintained as
described previously and were cultured in low serum media (RPMI with
0.5% FCS, 0.05% chicken serum) for 8-12 h prior to stimulation
(18).
B
Western analyses, RBC-depleted splenocytes were cultured and stimulated as indicated. For IKK in
vitro kinase assays, B cells were purified by a process of negative selection on an affinity chromatography column (Cedarlane, Ontario, Canada). The purity of B cells isolated in this manner was
~90-95% as verified by fluorescence-activated cell sorter analysis
using anti-B220 and anti-IgM antibodies (PharMingen). All purifications
were performed at 4 °C, and primary cells were used immediately upon purification.
B, cells that were not stimulated were also incubated in medium
containing 10% serum for the duration of stimulation.
-32P]CTP- and
[
-32P]ATP-labeled double-stranded oligonucleotide
probe derived from the
B enhancer element of the IL-2R
receptor
promoter (5'-CAACGGCAGGGGAATTCCCCTCTCCTT-3') was used. To
verify equal amounts and integrity of proteins in the nuclear extracts,
a control oligonucleotide for NF-Y was used. DNA-binding reactions were
resolved by PAGE and visualized by autoradiography.
B
degradation assays, 4 × 106
cells/sample were preincubated for 30 min in medium containing 50 µM cycloheximide and then stimulated as indicated. Cell
extracts were resolved by SDS-PAGE, transferred onto nitrocellulose
membranes, probed with antibodies against chicken I
B
(pp40; gift
of C. Chen) and p38 MAPK (Santa Cruz Biotechnology), and detected using the ECL system. Western blot analyses of I
B
phosphorylation were
performed as above and probed with antibodies against mouse I
B
(Santa Cruz Biotechnology) or phosphorylated Ser-32/Ser-36 I
B
(Santa Cruz Biotechnology).
B reporter
plasmid encoding firefly luciferase under the control of a promoter
containing six consensus NF-
B binding sites (6
B) and a control
vector containing a Renilla luciferase gene fused to a
thymidine kinase promoter have been described previously (25).
B reporter construct and 1 µg of the
Renilla construct. 18 h post-transfection, cells were
stimulated for 6 h with anti-IgM. Cells were harvested, and levels
of both firefly and Renilla luciferase were determined using
a Dual Luciferase Reporter Assay System (Promega). Levels of firefly
luciferase expression were normalized against Renilla as a
control for transfection efficiency.
plus anti-IKK
antibodies (Santa
Cruz Biotechnology). The immunocomplexes were then resuspended in 20 µl of kinase buffer (20 mM HEPES, pH 7.2, MgCl2 (2 mM), MnCl2 (2 mM), dithiothreitol (1 mM), ATP (20 µM)) containing 1.0 µCi of [
-32P]ATP
and 50 µg/ml wild type GST-I
B
substrate. The reaction was
allowed to continue for 30 min at 30 °C under agitation and then was
terminated by the addition of 4× SDS sample buffer. The samples were
resolved by 8% SDS-PAGE and stained with Coomassie Brilliant Blue to
visualize the GST-I
B
substrate. The gels were dried and exposed
to x-ray film to visualize
-32P-phosphorylated
GST-I
B
.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
B in BCR-stimulated B cells (18). However, the
molecular mechanism by which BTK facilitates NF-
B activation is
poorly defined. Recent findings suggest that BCR-directed nuclear
translocation of NF-
B requires the activation of the calcium-responsive phosphatase calcineurin (26). To define further the
mechanism employed by BTK to effect NF-
B activation, we investigated a role for calcium and calcineurin in BCR-responsive nuclear
translocation of NF-
B in DT40 B cells.
B in this cellular background, EMSA
analyses were performed on nuclear extracts prepared from DT40 cells
preincubated with pharmacological inhibitors of calcium, calcineurin,
and PKCs. We used BAPTA-AM/EGTA to chelate intra- and extracellular
calcium and cyclosporin A (CsA) to inhibit the calcium-responsive
phosphatase calcineurin or bisindolylmaleimide (Bis I), a broad
spectrum inhibitor of PKC isoenzymes (Fig.
1). We also treated DT40 cells with PMA
and ionomycin, pharmacological agents known to activate NF-
B via
IKK, as a positive control (28). As expected, BCR cross-linking or
PMA/ionomycin treatment resulted in the rapid nuclear accumulation of
NF-
B (compare lane 1 with 2 and
10). However, BCR-directed nuclear translocation of NF-
B
was inhibited by treatment with BAPTA-AM/EGTA or CsA (lanes
3 and 4). Bis I treatment significantly, although not
completely, inhibited this response (lane 5). However,
preincubation with Bis in combination with either BAPTA-AM/EGTA or CsA
resulted in a complete block in NF-
B nuclear translocation upon BCR
activation (lanes 6 and 7). This
result demonstrates that inhibition of either calcium or calcineurin
and PKC completely abolishes BCR-directed activation of NF-
B in DT40
B cells.
View larger version (68K):
[in a new window]
Fig. 1.
A requirement for Ca2+,
calcineurin, and PKC in BCR-directed activation of
NF- B. Pharmacological inhibition of
BCR-responsive second messengers prevents BCR-directed nuclear
translocation of NF-
B. DT40 cells were pretreated with
EGTA/BAPTA-AM, cyclosporin A, and bisindolylmaleimide I and then were
left unstimulated or stimulated as indicated. Nuclear extracts were
used in EMSA. Autoradiograms were quantitated with Image Gauge software
(Koshin graphics system), and levels of nuclear NF-
B were normalized
against NF-Y. Quantitative results for each sample are reported as the
percentage of NF-
B nucleoprotein complexes present relative to
anti-IgM-treated B cells (lane 2).
Upon BCR ligation, BTK activates a distinct set of signal transducers
to initiate downstream signaling events (3, 29). Of these, Akt, MAPK,
and PLC-2 have the capability to activate NF-
B via IKK. Although
both Akt and MAPK have been directly linked to IKK activation (30, 31),
such a role has not been demonstrated for PLC-
2. However, our
finding that calcium and PKC are essential for nuclear translocation of
NF-
B in BCR-stimulated DT40 cells implicates PLC-
2 in this
response (Fig. 1). Therefore, we next explored an involvement of
PLC-
2 in NF-
B nuclear translocation in B cells stimulated via the
BCR.
To determine whether PLC-2 is critical for BCR-directed nuclear
translocation of NF-
B, we used mutant chicken DT40 B cells lacking
PLC-
2 (DT40.PLC-
2) along with BTK-deficient (DT40.BTK) and
parental DT40 B cells. Cells were induced via the BCR, and their
nuclear NF-
B content was assessed by EMSA (Fig.
2A). Although BCR
cross-linking leads to a marked increase in nuclear NF-
B in DT40
cells (Fig. 2A, compare lanes 1 and
4), both DT40.PLC-
2 and DT40.BTK B cells failed to
demonstrate this response (Fig. 2A, compare lanes
2 and 5 and 3 and 6). However,
PMA and ionomycin mobilized similar levels of nuclear NF-
B in all
three cell types (Fig. 2A, lanes 7-9). These results
strongly suggest that like BTK, PLC-
2 plays an essential role in the
transmission of BCR signals to activate NF-
B.
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To ascertain whether the observed defect was due to delayed kinetics of
NF-B activation, we compared BCR-responsive nuclear translocation of
NF-
B in DT40.PLC-
2 cells with that in DT40 B cells over a period
of 4 h (Fig. 2B). Upon BCR cross-linking, DT40 B cells
rapidly translocated NF-
B to the nucleus and maintained elevated
levels up to 4 h after activation. In contrast, nuclear levels of
NF-
B did not increase in DT40.PLC-
2 B cells at any time point
within that period (Fig. 2B, compare lanes, 1, 3, 5, 7, and 9 with 2, 4, 6, and 8). To
verify further that the NF-
B activation defect in DT40.PLC-
2 B
cells was due to PLC-
2 deficiency, reconstitution experiments were
performed. In response to BCR engagement, DT40.PLC-
2 B cells
expressing wild type human PLC-
2 (DT40.PLC-
2R (23)) were capable
of NF-
B nuclear translocation as determined by EMSA and a NF-
B
responsive luciferase reporter assay (Fig. 2, C and
D). These data strongly suggest that PLC-
2 is critical
for transmission of BCR-dependent signals that lead to the
nuclear translocation of NF-
B.
Members of the NF-B/Rel family of proteins include p50/NF-
B1,
p52/NF-
B2, RelA, c-Rel, and RelB, which have the capacity to form
either homo- or heterodimers (16). NF-
B dimers containing the Rel
family proteins RelA or c-Rel have been demonstrated to be the
principal transactivating species activated in response to BCR
engagement in B cells (35). We previously demonstrated that RelA and
c-Rel fail to undergo nuclear translocation upon BCR stimulation in
BTK-deficient B cells. To test whether BTK-mediated RelA and c-Rel
nuclear translocation requires PLC-
2, we compared the ability of
DT40.PLC-
2, DT40.BTK, and DT40 B cells to translocate these subunits
to the nucleus upon BCR-cross-linking (Fig.
3, A and B).
Immunoblotting of nuclear extracts from unactivated (lanes
1-3), anti-IgM stimulated (lanes 4-6), and
PMA/ionomycin treated (lanes 7-9) cells with Rel
subunit-specific antibodies revealed that nuclear accumulation of both
RelA and c-Rel occurs in DT40 B cells following BCR stimulation (Fig.
3, A and B, lanes 1 and 4).
In contrast, BCR-responsive nuclear translocation of RelA and c-Rel is
not observed in either DT40.PLC-
2 or DT40.BTK B cells. Treatment
with PMA/ionomycin induced nuclear translocation of both Rel species
(Fig. 3, A and B, lanes 7-9) in all
three cell lines. Furthermore, the observed differences in Rel subunit translocation are not attributable to either variation in total protein
content of the nuclear extracts or their integrity, because similar
amounts of the constitutively expressed transcription factor SP1 are
detectable in all samples (Fig. 3, A and B, lower panels). Thus, BCR-directed nuclear translocation of RelA and c-Rel is PLC-
2-dependent.
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NF-B dimers are found in the cytoplasm of quiescent cells, bound to
members of a family of inhibitory molecules termed I
Bs. BCR-induced
nuclear translocation of NF-
B is contingent upon the phosphorylation
and proteolytic degradation of I
B
, a process that requires BTK.
We compared the ability of DT40.PLC-
2 B cells with DT40.BTK and DT40
B cells to degrade I
B
in response to BCR activation. Cells were
incubated with anti-IgM antibodies or PMA and ionomycin for indicated
periods, and cytoplasmic extracts were immunoblotted for chicken
I
B
(Fig. 4, upper
panel). As expected, DT40 B cells rapidly degraded I
B
upon
BCR activation. Consistent with the results shown in Fig. 2,
DT40.PLC-
2 B cells failed to degrade I
B
in response to BCR
stimulation (Fig. 4, compare lanes 1-4 with 6-9
and 11-14). All three cell lines efficiently degraded
I
B
in response to treatment with PMA and ionomycin. Therefore,
loss of PLC-
2 does not affect the downstream components necessary
for I
B
degradation. These results demonstrate that BCR-directed
degradation of I
B
specifically requires PLC-
2.
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Prior biochemical studies have identified several NF-B agonists that
converge on IKK
and IKK
including TNF and IL-1 (17). Additionally, we have recently established that BCR-initiated activation of NF-
B by BTK proceeds via IKK (18). To extend our
finding that PLC-
2 is required for BCR-directed nuclear
translocation of NF-
B, we explored a role for PLC-
2 in IKK
activation. We tested whether pharmacological agents that block
PLC-
2 and its second messengers could prevent BCR-induced activation
of IKK in primary B cells (Fig.
5A). In response to activation
signals via the BCR or CD40, or treatment with PMA, IKK enzymatic
activity was significantly increased as determined by in
vitro kinase assays using recombinant GST-I
B
as the
substrate (Fig. 5A, compare lane 1 with 2, 7, and 8). In contrast, incubation of B cells with either the PLC-
-specific inhibitor (U-73122) or inhibitors of its
second messengers (BAPTA-AM/EGTA, CsA, or Bis I) prior to BCR
stimulation abolished this activity (Fig. 5A, lanes 3-6). These data implicate PLC-
2, calcium, calcineurin, and PKC in IKK
activation upon BCR ligation. Moreover, they verify the role of these
signaling molecules in BCR-responsive activation of IKK in a
physiologically relevant background.
|
To confirm this observation, we performed Western blot analyses of
cytosolic fractions from BCR-, CD40-, or PMA-stimulated splenocytes
using an antibody directed against Ser-32/Ser-36-phosphorylated IB
(Fig. 5B, upper panel). Stimulation via either the
BCR or CD40 induced phosphorylation of I
B
(Fig. 5B,
compare lanes 1, 2, and 6). BCR-responsive
I
B
phosphorylation was blocked by pretreatment with either
BAPTA-AM/EGTA, CsA, or Bis I (Fig. 5B, lanes 3-5). Also,
PMA stimulation resulted in I
B
phosphorylation that was abrogated
by pretreatment with the PKC inhibitor Bis I (Fig. 5B, lanes
7 and 8). These observations implicate PLC-
2, calcium, and PKC in BCR-responsive activation of IKK and
phosphorylation of I
B
. Moreover, the observation that cells
pretreated with CsA fail to activate IKK upon BCR cross-linking
identifies calcineurin as a critical mediator of this response. This
observation is consistent with the recent finding that calcineurin and
PKCs synergize to induce IKK activation in T cells (28). Collectively,
these data suggest that PLC-
2 is likely to mediate BCR-responsive
activation of IKK, phosphorylation of I
B
, and nuclear
translocation of NF-
B.
We have found that PLC-2 and its downstream signals are essential
for BCR-directed activation of IKK and NF-
B. Prior studies in
BCR-stimulated B cells have revealed that PLC-
2 is activated via the
concerted actions of BTK, Syk, and BLNK (3, 32). Therefore, it is
likely that PLC-
2 is the principal BTK signal transducer for
BCR-directed activation of IKK and NF-
B. Further investigation is
required to determine whether additional BTK targets, including Akt and
MAPK, synergize with PLC-
2 to effect nuclear translocation of
NF-
B in BCR-stimulated B cells. However, the placement of PLC-
2
in the BCR/BTK/NF-
B signaling pathway provides the first potential
molecular explanation for the similar xid-like B cell
deficiencies displayed by
plc-
2
/
and
btk
/
mice (10, 33).
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ACKNOWLEDGEMENTS |
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We thank David Strayhorn for technical advice and Drs. Dean Ballard, Eugene Oltz, Sebastian Joyce, and Jacek Hawiger for helpful discussions in the preparation of this manuscript.
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FOOTNOTES |
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* 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.
To whom correspondence should be addressed: Dept. of Microbiology
and Immunology, Vanderbilt University school of Medicine, Nashville, TN
37232-0146. Tel.: 615-343-5632; Fax: 615-343-7392; E-mail:
Khanwn@ctrvax.vanderbilt.edu.
Published, JBC Papers in Press, October 19, 2000, DOI 10.1074/jbc.M009137200
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ABBREVIATIONS |
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The abbreviations used are:
BCR, B cell antigen
receptor;
xid, X-linked immunodeficiency;
BTK, Bruton's tyrosine
kinase;
PLC-2, phospholipase C-
2;
NF-
B, nuclear factor-
B;
IKK, I
B kinase;
PKC, protein kinase C;
PMA, phorbol 12-myristate
13-acetate;
Bis I, bisindolylmaleimide I;
CsA, cyclosporin A;
BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid acetoxymethyl ester;
GST, glutathione S-transferase;
EMSA, electrophoretic mobility shift assays;
PAGE, polyacrylamide gel
electrophoresis;
MAPK, mitogen-activated protein kinase.
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