 |
INTRODUCTION |
Ligation of the antigen receptor
(BCR)1 can lead to clonal
expansion, differentiation, or abortive activation-induced
apoptosis of B lymphocytes. Several early signaling events induced
by ligation of BCR have been described including the rapid activation
of protein tyrosine kinases, calcium mobilization, and activation of
downstream serine/threonine kinases (1-8). These early events are
followed by cytoskeletal reorganization, induced gene expression, and
increased mRNA and protein synthesis (9). The relationship between
the defined early signal transduction events, intermediate signaling events, and the fate of B cells is not clear.
We have been investigating the protein tyrosine
kinase-dependent regulation of BCR-mediated activation of
serine/threonine kinases. We demonstrated that ligation of BCR on the
avian B cell line DT40 results in activation of mitogen-activated
protein kinase (MAPK) and members of two families of ribosomal S6
kinases, p90Rsk and p70S6 kinase (p70S6k) (10).
Ribosomal S6 kinases are highly conserved proteins that are critical
for translational regulation particularly of genes containing
poly-pyrimidine tracts in their 5'-untranslated regions that encode
essential components of the protein synthesis apparatus (11-15). These
kinases can regulate mRNA translation through phosphorylation of
ribosomal S6 protein. Although both p90Rsk and
p70S6k can phosphorylate S6 in vitro,
p70S6k likely is responsible for in vivo
phosphorylation of S6 (16-20).
Several lines of evidence suggest that p70S6k is also
involved in cell cycle regulation (18, 21-26). First, microinjection
of antibody against p70S6k abolished PDGF-induced cell
cycle progression in rat embryo fibroblasts (21-23). Many other
studies of the function of p70S6k have relied on selective
inhibition by the immunosuppressant drug rapamycin that inactivates
p70S6 kinase without affecting mitogen-induced activation of
p90Rsk or MAPK (18, 24). Based on inhibition of
G1 progression by rapamycin, it has been suggested that
p70S6k plays an important role in the growth of
hematopoietic cells (18, 24-27). Recent evidence obtained with
embryonic stem cells in which p70S6k was depleted by
targeted gene disruption supports its role in ribosomal protein
synthesis and cell growth (20). Although mitogens that activate
p70S6k can simultaneously activate mitogen-activated
protein kinases, there is evidence that MAPK and p70S6k lie
on discrete pathways (16) and that p70S6k and MAPK may play
distinct roles in regulating cell growth (21-23, 28, 29). We recently
demonstrated that BCR-mediated activation of p70S6k can
occur independently of the pathways that mediate MAPK or p90Rsk (10). However, the role of either MAPK or
p70S6k in B cell proliferation has not been determined.
Mitogen-induced activation of p70S6k is initiated by ligand
binding to receptor tyrosine kinases in the case of PDGF and insulin stimulation, for example, or through activation of non-receptor tyrosine kinases as in the case of T cell stimulation by IL-2 (30-32).
Activation of these protein tyrosine kinases leads to the activation of
PI3-kinase-dependent as well as PI3-kinase-independent pathways that lead to the activation of p70S6 kinase (33-37). The activation of protein tyrosine kinases is an essential step in antigen
receptor-mediated activation of B cells. Lyn, one of several Src family
kinases expressed in B cells, Syk, and Btk are three of the major
tyrosine kinases involved in BCR-mediated activation (38-42). The
essential role of these tyrosine kinases in the regulation of some
early signaling events such as the activation of phospholipase-C
has
been established (5, 43, 44). However, the pathways leading to the
activation of the serine/threonine kinases such as MAPK,
p90Rsk, and p70S6k that may be required for the
reorganization of the actin cytoskeleton, cell cycle progression, gene
transcription, and protein translation associated with antigen
receptor-mediated activation of B lymphocytes have not been delineated.
In this report, we define the BCR-mediated protein tyrosine
kinase-dependent signaling cascades that lead to activation
of p70S6k. We futher demonstrate that the activation of
p70S6k results in the phosphorylation of S6 protein that is
important for ribosomal protein synthesis and may be coupled to
BCR-induced DNA synthesis in primary murine B cells.
 |
MATERIALS AND METHODS |
Isolation of Primary B Cells--
Splenic B cells were isolated
from 6- to 8-week-old CD2F1 mice as described (45). Freshly isolated
splenocytes were depleted of red blood cells using hypotonic red blood
cell lysis buffer (Sigma). T cells were depleted by antibody
(monoclonal anti-CD4 Ab (GK1.5), anti-CD8 Ab (3.168.8), and anti-Thy1.2
Ab (J1j))-dependent complement-mediated cytolysis using
Low-Tox® guinea pig sera as a source of complement (Accurate
Chemicals). The enrichment of B cells was over 85% as measured by
reactivity with anti-B220 and fluorescence-activated flow cytometry.
The purified B cells were incubated in serum-free medium for 3 h
prior to stimulation.
Fluorescence-activated Flow Cytometry--
Primary B cells and
DT40 cells were incubated with FITC-conjugated anti-B220 mAb and mouse
anti-chicken IgM mAb (M4), respectively, for 30 min at 4 °C. DT40
cells were then reacted with FITC-conjugated F(ab')2 goat
anti-mouse Ab for an additional 30 min at 4 °C. These cells were
then subjected to flow cytometric analysis on a FACScan and analyzed
using Cell Quest software (Becton Dickinson).
Cell Culture and DNA Transfection--
The chicken B cell line,
DT40, and mutant DT40 cells deficient in both Syk and Lyn
(DT40Syk
Lyn
) were cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 1% chicken
serum, 100 µg/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine. DT40Lyn
and
DT40Syk
cells were cultured in the same medium containing
2 mg/ml G418 (43). Wild-type and an enzymatically inactive mutant of
human Syk, SykK402R, were cloned into the expression vector pApuro (10) and transfected into DT40Syk
cells by electroporation
using a gene pulser apparatus (Bio-Rad) at 330 V, 250 microfarads and
selected in the presence of 0.5 µg/ml puromycin. Puromycin-resistant
clones were initially screened by Southern blotting of genomic DNA and
further selected for expression of comparable levels of surface
immunoglobulin by flow cytometry and for expression of comparable
amounts of wild-type Syk or SykK402R based on immunoblotting. All
results are representative of at least three experiments using a
minimum of two independent clones expressing wild-type Syk or SykK402R.
Immunoprecipitation and Immunoblotting--
For activation of
p70S6k, cells in an exponential growth phase were
serum-starved for 12 h prior to stimulation. DT40 cells were untreated or treated with rapamycin (Calbiochem) or 0.5 µM staurosporine (Sigma) for 15 min or 3 h,
respectively. Cells (2 × 107 cells/ml) were then
stimulated with indicated concentrations of mouse anti-chicken IgM mAb
M4 or 100 nM phorbol ester (phorbol 12,13-dibutyrate
(PdBu), Sigma) for 30 min at 37 °C. Cells were quickly chilled on
ice and pelleted by centrifugation immediately after stimulation. Cell
pellets were lysed in 400 µl of ice-cold lysis buffer (1×
phosphate-buffered saline, 1 mM phenylmethylsulfonyl fluoride, 100 µg/ml soybean trypsin inhibitor, 20 µg/ml aprotinin, 100 µg/ml leupeptin, 1% Nonidet P-40, 0.1% deoxycholate, 10 mM NaF, 10 mM sodium pyrophosphate, and 100 mM sodium orthovanadate) for 15 min on ice. Cell lysates
were clarified by centrifugation at 15,000 rpm for 10 min. Clarified
cell lysates were normalized based on protein concentration as
determined using the BCA® kit (Pierce), and equal amounts of protein
were subjected to immunoprecipitation or immunoblotting for each
lysate. The post-nuclear extracts (1 mg) were incubated directly with
polyclonal anti-p70S6k (Santa Cruz Biotechnology) at
4 °C overnight. Immune complexes were precipitated with 30 µl of
protein A-agarose beads (Life Technologies, Inc.) for an additional
3 h incubation at 4 °C. The immune precipitates were washed
twice with lysis buffer followed by two washes with phosphate-buffered
saline. Bound proteins were eluted by boiling in Laemmli buffer
containing 0.4% dithiothreitol and resolved on 8% SDS-polyacrylamide
gel electrophoresis (National Diagnostics Inc.). Proteins less than 45 kDa were run off gels to obtain better resolution of the
hypophosphorylated and hyperphosphorylated forms of the proteins of
interest and to maximize separation from the antibody used for
immunoprecipitation. The proteins were transferred to polyvinylidene
difluoride membrane and subjected to immunoblotting. The blots were
blocked with TNB buffer (30 mM Tris, pH 7.6, 75 mM NaCl, 3% bovine serum albumin) overnight at 4 °C.
The blots were then incubated with polyclonal anti-p7056k Ab followed
by 1 µCi/ml iodinated protein A for 45 min at room temperature
followed by autoradiography.
For detection of in vivo phosphorylation of endogenous S6
protein, 100 µg of cell lysates were resolved on 10%
SDS-polyacrylamide gels and transferred to polyvinylidene difluoride
membranes as described above. Membranes were immunoblotted with a
polyclonal antibody against the phosphorylated S6 protein (a generous
gift from Dr. Morris Birnbaum, University of Pennsylvania) followed by
incubation with horseradish peroxidase-conjugated goat anti-rabbit Ig.
Reactivity was detected using an ECL kit (Amersham Pharmacia Biotech).
In Vitro Kinase Assays--
Anti-p70S6k
immunoprecipitates were prepared as described above and washed once
with kinase buffer (50 mM MOPS, pH 7.2, 1 mM dithiothreitol, 30 µM ATP, 5 mM
MgCl2). The immune complexes were then resuspended in 12.5 µl of kinase buffer containing 0.3 mg/ml S6 peptide (Santa Cruz
Biotechnology) and 5 µCi of [32P-
]ATP (6000 Ci/mmol)
and incubated at 30 °C for 15 min. The reactions were terminated by
boiling in 7.5 µl of 3× Laemmli buffer for 5 min. Samples were
resolved on an 18.5% polyacrylamide gel which was dried and subjected
to autoradiography.
Quantitation of DNA Synthesis--
Splenic B cells (1 × 106 cells/ml) were incubated with the indicated dose of
rapamycin 15 min prior to and throughout the stimulation period. Cells
(200 µl) were cultured in 96-well microtiter plates and stimulated
with the indicated concentrations of F(ab')2 goat anti-mouse IgM in the presence or absence of baculovirus-derived murine
IL-4 and soluble recombinant CD40 ligand (CD40L)/CD8 fusion protein (a
generous gift from Dr. Jan Erikson, Wistar Institute) for 48 h.
Cells were pulsed with 1 µCi of [3H]thymidine per well
during the final 12 h of stimulation. [3H]Thymidine
incorporation was measured using a Harvester 96® (Tomtec, OR) and
analyzed using Matrix 96 software (Packard).
 |
RESULTS |
BCR-induced Activation of p70S6k in Splenic B Cells Is
Transient and Dose-dependent--
Activation of
p70S6k has been implicated in the regulation of several
pivotal functions including translation, transcription, and cell growth
in several cell types in response to growth factor (e.g.
PDGF), cytokine (e.g. interleukin 2), or hormone
(e.g. insulin) stimulation (24, 37, 46-50). However, the
role of p70S6k in antigen receptor-mediated signaling is
not known. We previously demonstrated that ligation of BCR can induce
activation of p70S6k in the DT40 avian B cell line (10). In
this study, we extended this finding to primary splenic B cells and
found that engagement of BCR can lead to activation of
p70S6k as measured by shift in mobility of
p70S6k on SDS-polyacrylamide gel electrophoresis and
in vitro kinase assays. Ligation of BCR with an optimal dose
of anti-Ig induced maximal phosphorylation and activation of
p70S6k in primary splenic B cells at 30 min after
stimulation (Fig. 1A). The
BCR-mediated activation of p70S6k decreased to basal levels
at 2 h after stimulation. The phosphorylation of
p70S6k was also dependent on the dose of anti-Ig (Fig.
1B). Importantly, in vivo phosphorylation of S6
protein was also induced in a dose-dependent manner (Fig.
1B). Together these data indicate that the degree to which
p70S6k is activated is determined by the extent of receptor
cross-linking.

View larger version (48K):
[in this window]
[in a new window]
|
Fig. 1.
Activation of p70S6k following
BCR cross-linking in primary B cells is transient and
dose-dependent. A, the kinetics of
BCR-induced activation of p70S6k. Splenic B cells were
isolated and stimulated with 10 µg/ml F(ab')2 goat
anti-mouse IgM Ab for the indicated times. Equal amounts of cell
lysates (500 µg) were immunoprecipitated with anti-p70S6k
Ab. Half of the immune complexes were immunoblotted with
anti-p70S6k Ab to measure the mobility shift of
p70S6k (top panel). The second half of the
immune complexes were subjected to an in vitro kinase assay
using S6 peptide as an exogenous substrate (bottom panel).
These data represent at least four independent experiments.
B, dose-dependent anti-Ig-induced activation of
p70S6k. Splenic B cells were stimulated with the indicated
doses of anti-Ig for 30 min. The mobility shift of p70S6k
was measured as described in A (top panel). For
in vivo phosphorylation of S6 protein, equal amounts of cell
lysates were immunoblotted with anti-phospho-S6 protein Ab
(bottom panel).
|
|
Activation of p70S6k Is Rapamycin-sensitive and
Associated with BCR-induced Phosphorylation of S6 Protein and DNA
Synthesis--
The immunosuppressive drug rapamycin selectively
inhibits growth factor and IL-2-induced activation of
p70S6k in fibroblasts and T cells, respectively (15, 18,
24, 51-54). Inhibition of p70S6k by rapamycin also
correlated with the inhibition of cell cycle progression of fibroblasts
and T cells stimulated by mitogens (27, 49, 54) and the in
vivo phosphorylation of S6 protein (20). Furthermore, targeted
disruption of p70S6k established that p70S6k is
required for in vivo phosphorylation of S6 protein in
embryonic stem cells (20). To investigate the role of
p70S6k in downstream responses in B cells we examined the
dose dependence and rapamycin sensitivity of anti-Ig-induced activation
of p70S6k, S6 phosphorylation, and DNA synthesis.
As expected, anti-Ig induced DNA synthesis in a
dose-dependent fashion (Fig.
2A). Interestingly, the dose
responses of anti-Ig-induced activation of p70S6k (Fig.
1B), DNA synthesis (Fig. 2A), and S6
phosphorylation (Fig. 1B) were all similar. BCR-mediated
activation of p70S6k and in vivo S6
phosphorylation was inhibited by rapamycin (Fig. 2B).
Furthermore, the inhibition of p70S6k by rapamycin
correlated with the dose-dependent inhibition of BCR-mediated DNA synthesis by rapamycin (Fig. 2A). The
inhibition by rapamycin was not due to cytotoxicity since the
BCR-mediated activation of p90Rsk was not affected (data
not shown). Moreover, stimulation of rapamycin-treated B cells with
IL-4 and CD40L rescued DNA synthesis in rapamycin-treated anti-Ig-stimulated B cells (Fig. 2C). The effect of CD40L
and IL-4 was not due to inactivation of rapamycin since BCR-mediated activation and phosphorylation of p70S6k were inhibited by
rapamycin even in the presence of IL-4 and CD40L (data not shown).
Taken together, these results indicated that BCR-mediated activation of
p70S6k leads to the phosphorylation of endogenous S6
protein and may contribute to enhanced DNA synthesis in B cells. In
addition, IL-4 and CD40L may synergize with BCR by inducing alternate
signaling pathway(s) that are resistant to rapamycin and therefore do
not require p70S6k but lead to DNA synthesis.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 2.
Rapamycin inhibits BCR-induced
p70S6k activation and DNA synthesis. A, DNA
synthesis. Splenic B cells were incubated with the indicated doses of
rapamycin for 15 min prior to and during stimulation. Cells were then
cultured in the presence or absence of various doses of
F(ab')2 goat anti-mouse IgM Ab for 48 h; 1 µCi of
[3H]thymidine was added for the last 12 h of the
incubation period. The amounts of incorporated
[3H]thymidine were measured, and the results are
presented as the average ± S.E. of triplicates for each
condition. These data are representative of at least three independent
experiments. B, mobility shift of p70S6k and
in vivo phosphorylation of S6 protein. B cells were
pretreated with rapamycin for 15 min and stimulated with 10 µg/ml
F(ab')2 goat anti-mouse IgM Ab in the presence of rapamycin
for an additional 30 min. For mobility shift of p70S6k,
equal amounts of cell lysates were immunoprecipitated followed by
immunoblotting with anti-p70S6k Ab. For in vivo
phosphorylation of S6 protein, equal amounts of cell lysates were
immunoblotted with anti-phospho-S6 protein. C, IL-4 and CD40
ligand rescue the inhibition of BCR-mediated DNA synthesis by
rapamycin. Rapamycin pretreated B cells were stimulated with 50 µg/ml
F(ab')2 goat anti-mouse IgM Ab in the presence or absence
of IL-4 and CD40L for 36 h and then pulsed with
[3H]thymidine for 12 h, and the amount of
incorporated [3H]thymidine was measured. The results are
presented as percent of inhibition of DNA synthesis by rapamycin in
anti-Ig-stimulated cells as compared with that in stimulated cells
without rapamycin. All data are representative of at least three
independent experiments.
|
|
Syk and Lyn Mediate Activation of p70S6k following
Suboptimal Cross-linking of the BCR--
Mitogen-induced activation of
p70S6 kinase in other cell types is mediated either by receptor
tyrosine kinases (e.g. insulin receptor and PDGF receptor)
or through cytoplasmic tyrosine kinases (IL-2 receptor). In addition,
the activation of cytoplasmic tyrosine kinases is critical for antigen
receptor-mediated B cell activation (1-8). Therefore, in an effort to
define BCR-coupled signaling pathways that lead to the activation of
p70S6 kinase, we first investigated the role of tyrosine kinases.
Treatment with genistein, a tyrosine kinase inhibitor, blocked both
basal and anti-Ig-induced hyperphosphorylation of p70S6k in
primary B cells and DT40 B cells (Fig.
3A). These data suggest that
both basal and BCR-mediated activation of p70S6k is
dependent on upstream tyrosine kinases. To define the role of Syk and
Lyn we compared the activation of p70S6k in DT40 cells
deficient in either Syk or Lyn to that in parental DT40 cells. We
previously reported that the activation of p70S6k was
comparable in DT40Syk
, DT40Lyn
, and
parental DT40 cells following stimulation with an optimal dose of
anti-Ig (10). These results suggested either that Lyn and Syk were each
sufficient to mediate BCR-induced activation of p70S6k in
DT40 cells or that neither of these tyrosine kinases were required at
least following optimal B cell receptor cross-linking. We have
addressed the role of Syk and Lyn further by comparing the activation
of p70S6k in DT40 cells deficient in both Syk and Lyn
(DT40Syk
Lyn
) and by examining whether Syk
or Lyn plays an important role in the activation of p70S6k
following stimulation with suboptimal doses of anti-Ig that induce a
degree of receptor cross-linking more likely consistent with stimulation by antigen.

View larger version (58K):
[in this window]
[in a new window]
|
Fig. 3.
A, tyrosine kinases are required for
both basal and anti-Ig-induced phosphorylation of p70S6k.
Splenic B cells (left panel) and DT40 B cells (right
panel) were pretreated with 50 µg/ml genistein for 30 min prior
to stimulation with 10 µg/ml F(ab')2 goat anti-mouse IgM
and mouse anti-chicken IgM for 30 min, respectively. Equal amounts of
cell lysates (500 µg) were immunoprecipitated and immunoblotted with
anti-p70S6k Ab. B, the requirement for Syk and
Lyn for activation of p70S6k are dependent on the extent of
receptor cross-linking. Parental and mutant DT40 cells were stimulated
with the indicated doses of mouse anti-chicken IgM Ab (M4) for 30 min.
Equal amounts of cell lysates were immunoprecipitated with anti-
p70S6k Ab. The immune complexes were divided for
immunoblotting with anti-p70S6k Ab (top panel)
and for in vitro kinase assays (bottom panel).
C, parental DT40 and mutant DT40 cells express comparable
amounts of B cell receptors. DT40 cells and mutant DT40 cells were
incubated with mouse anti-chicken IgM mAb M4 at 4 °C for 30 min
followed by staining with FITC-conjugated F(ab')2 goat
anti-mouse IgG at 4 °C for 30 min. Cells were analyzed by flow
cytometry. D and E, Syk is required for
BCR-induced activation of p70S6k in response to low dose
anti-Ig. DT40 cells were stimulated with indicated doses of M4 Ab at
37 °C for 30 min. Equal amounts of cell lysates were
immunoprecipitated with anti-p70S6k Ab. The immune
complexes were immunoblotted with anti-p70S6k Ab
(D) or subjected to in vitro kinase assays
(E, top panel). The results of the kinase assays were
quantitated using a PhosphorImager and ImageQuant software (Molecular
Dynamics) and expressed as arbitrary PhosphorImager units (E,
bottom panel).
|
|
The basal phosphorylation of p70S6k in
DT40Syk
Lyn
cells was decreased compared
with that in parental cells (Fig. 3B). Thus, Syk and Lyn
appear to be involved in the regulation of basal phosphorylation of
p70S6k. Furthermore, compared with the activation of
p70S6k seen in parental DT40 cells, activation of
p70S6k was abolished in
DT40Syk
Lyn
cells following stimulation with
low doses of anti-Ig (~1-3 µg/ml mAb M4) (Fig. 3B).
This phenotype is not due to lower levels of BCR expression in
DT40Syk
Lyn
cells compared with that in
parental cells since DT40Syk
Lyn
cells
express comparable amounts of BCR to that in parental DT40 cells by
FACS analysis (Fig. 3C). These data suggest Syk or Lyn are
required for activation of p70S6 kinase in response to suboptimal cross-linking of the BCR but that optimal doses of anti-Ig (100 µg/ml
mAb M4) promote sufficient cross-linking of BCR to initiate alternate
pathway(s) with higher threshold(s) of activation that also lead to
albeit reduced activation of p70S6 kinase independent of both Syk and Lyn.
To investigate further the roles of Syk and Lyn, we compared
anti-Ig-induced phosphorylation/activation of p70S6k in
DT40, DT40Syk
, DT40Lyn
, and
DT40Syk
Lyn
cells in response to stimulation
by different doses of anti-Ig. In response to low dose of anti-Ig (1 µg/ml mAb M4), BCR-induced activation of p70S6k was
comparable in DT40Lyn
but ablated in
DT40Syk
and DT40Syk
Lyn
cells
compared with that in DT40 cells (Fig. 3D, top panel, and E). Interestingly, the fold stimulation over basal
activity of p70S6k in DT40Lyn
cells is higher
than that in DT40 cells in response to BCR cross-linking as a result of
a decrease in the basal activity of p70S6k in the mutant
DT40 cells compared with parental DT40 cells (Fig. 3E). In
response to increasing concentrations of anti-Ig, suboptimal activation of p70S6k was evident starting at 3 µg/ml M4
in DT40Syk
cells but reduced compared with parental
cells, whereas DT40Syk
Lyn
cells were
unresponsive to this intermediate dose (Fig. 3D, middle panel). Thus, the dose response of Syk-deficient cells is shifted compared with parental DT40 cells and Lyn-deficient DT40 cells indicating that BCR-induced activation of p70S6 kinase is primarily mediated by a Syk-dependent pathway at low doses of anti-Ig
cross-linking. The more marked shift in the dose response of
DT40Syk
Lyn
cells compared with
DT40Syk
cells indicates that Lyn can also mediate
BCR-induced activation of p70S6 kinase in the absence of Syk. Taken
together, these data demonstrate that Syk is required for activation of
p70S6 kinase and that Lyn can also contribute to its activation in
response to suboptimal cross-linking of BCR but at higher doses of
anti-Ig (>10 µg/ml M4) a threshold of BCR cross-linking in DT40
cells is reached that leads to the activation of p70S6 kinase via an alternative pathway(s) that does not require either Syk or Lyn (Fig. 3,
B and D, bottom panel, and E).
Enzymatic Activity of Syk Is Required for BCR-mediated Activation
of p70S6 Kinase--
To test whether the failure of low dose anti-Ig
to result in activation of p70S6k in DT40Syk
cells was due to the absence of Syk, we determined if expression of
human Syk could revert the phenotype to that of parental DT40 cells.
DT40Syk
cells were transfected with wild-type human Syk
and an enzymatically inactive mutant K402R (10). Stable transfectants
were established and selected based on expression of comparable levels
of Syk and membrane immunoglobulin by immunoblotting and flow cytometry
analysis, respectively. Expression of wild-type human Syk in
DT40Syk
cells reverted both basal and suboptimal
anti-Ig-induced activation of p70S6 kinase to the parental phenotype,
whereas expression of the catalytically inactive mutant, K402R, did not
(Fig. 4). Thus the defect in
DT40Syk
cells can be attributed to the loss of Syk.
Furthermore, the enzymatic activity of Syk is required for the
BCR-induced activation of p70S6k in response to low doses
of anti-Ig cross-linking.

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 4.
The catalytic activity of Syk is required for
BCR-mediated activation of p70S6k.
DT40Syk cells stably transfected with wild-type human Syk
cDNA or a catalytically inactive mutant form of Syk, SykK402R, were
stimulated with 2 µg/ml M4 Ab for 30 min. Equal amounts of cell
lysates were immunoprecipitated with anti-p70S6k Ab. The
immune complexes were divided for immunoblotting with
anti-p70S6k Ab (top panel) and an in
vitro kinase assay using S6 peptide as a substrate (bottom
panel).
|
|
BCR-mediated Activation of p70S6 Kinase Can Occur via Both
PKC-dependent and -independent Pathways--
We next
sought to define the downstream effector molecules that can mediate the
Syk-dependent activation of p70S6 kinase. PI3-kinase and
protein kinase C (PKC) have both been implicated upstream of
p70S6k in growth factor and cytokine-induced signaling
(35-37, 46, 55). Syk was previously shown to be required for
anti-Ig-induced activation of phospholipase-C
and thereby generation
of phosphoinositide triphosphate and diacylglycerol followed by
Ca2+ mobilization. Both diacylglycerol and Ca2+
are involved in activation of several PKC isoforms, conventional PKC,
and novel PKC (56). We therefore tested the hypothesis that PKC
transduces the signals from Syk to p70S6k in B cells in
response to low dose anti-Ig cross-linking. To determine the role of
PKC in the Syk-dependent activation of p70S6 kinase, we
tested the effect of PKC inhibition either by treatment with a PKC
inhibitor, staurosporine, or by chronic treatment with PdBu on the
response of DT40 and DT40Lyn
cells stimulated with low
dose anti-Ig. Consistent with our previous report, short term treatment
with phorbol ester, PdBu (10), or low dose anti-Ig induced optimal
activation of p70S6k in both DT40 and DT40Lyn
cells (Fig. 5A). Inhibition of
PKC by staurosporine resulted in a complete abrogation of
p70S6k in both DT40 and DT40Lyn
cells in
response to PdBu or anti-Ig stimulation (Fig. 5A). To define
further the role of specific PKC isoforms in BCR-mediated activation of
p70S6k, we also measured the effects of chronic treatment
with PdBu on p70S6k activation. In contrast to short term
treatment with phorbol esters (56), prolonged treatment with phorbol
esters induces ubiquitination and proteolytic degradation of phorbol
ester responsive PKC isoforms (57). Interestingly, the BCR-induced
activation of p70S6k was also completely abolished in
PKC-depleted DT40 cells but was only partially inhibited in
DT40Lyn
cells (Fig. 5B, compare lanes marked
by asterisks). The partial inhibition in
DT40Lyn
was not due to residual PdBu-sensitive PKC since
these cells were unresponsive to restimulation of PdBu (Fig.
5B). The effect of pre-exposure to PdBu was not due to
cytotoxicity since BCR-induced tyrosine phosphorylation was unaffected
by PKC depletion (Fig. 5C). These data suggest that
Syk-dependent activation of p70S6k requires PKC
in both DT40 and DT40Lyn
cells. Moreover, phorbol
ester-responsive PKC isoforms, in particular, are essential for
anti-Ig-induced activation of p70S6k in DT40 cells but not
in DT40Lyn
cells. These data also suggested that
non-PdBu-sensitive PKC isoforms might be hyperactivated and sufficient
for the partial activation of p70S6k in
DT40Lyn
cells depleted of PdBu-sensitive PKC
isoforms.

View larger version (66K):
[in this window]
[in a new window]
|
Fig. 5.
Protein kinase C is involved in
anti-Ig-induced activation of p70S6k. A,
cells were treated with 0.5 µM staurosporine for 3 h
prior to stimulation. Cells were then stimulated with 2 µg/ml M4 Ab
for 30 min. The phosphorylation states of p70S6k were
determined by mobility shift as described in Fig. 4. B,
cells were cultured in serum-free media in the presence or absence of 5 µM PdBu for 12 h before stimulation with anti-Ig.
The PKC-depleted cells were then stimulated with 2 µg/ml M4 or 50 nM PdBu at 37 °C for 30 min. The phosphorylation states
of p70S6k were compared by mobility shift as described
above. C, the same PKC-depleted cells were stimulated with 2 µg/ml M4 at 37 °C for 3 min. Equal amounts of cell lysates (50 µg) were immunoblotted with anti-phosphotyrosine Ab.
|
|
Role of PI3-kinase in BCR-mediated Activation of
p70S6k--
PI3-kinase has been implicated in activation
of p70S6k (26, 31, 36, 37, 55). In addition, several
non-PdBu-sensitive PKC isoforms were shown to be activated by
PI3-kinase. PI3-kinase was therefore also a likely candidate as a
mediator of low dose anti-Ig-induced activation of p70S6 kinase in
DT40Lyn
cells (26, 31, 36, 37, 55). To address the role
of PI3-kinase, we tested the sensitivity of anti-Ig-induced activation of p70S6 kinase to the selective inhibitors of PI3-kinase, wortmannin, and Ly294002. We found that both basal and low dose anti-Ig-induced activation of p70S6k in DT40 and DT40Lyn
cells were partially sensitive to 50 nM wortmannin and 4 µM Ly294002 with the response in DT40Lyn
cells being somewhat more sensitive (Fig.
6, A and B).
Furthermore, treatment of PKC-depleted DT40Lyn
cells with
Ly294002 completely abrogated anti-Ig-induced activation of
p70S6k (Fig. 6C). Thus, together, PKC and
PI3-kinase account for the transduction of the
Syk-dependent activation of p70S6k in DT40 and
DT40Lyn
cells. The partial inhibition of
p70S6k activation by wortmannin and Ly294002 was not likely
due to incomplete inhibition of PI3-kinase since the BCR-induced
activation of Akt, another downstream effector of PI3-kinase, was
completely ablated under these
conditions.2 Furthermore,
BCR-induced activation of p90Rsk was not affected
demonstrating the specificity and lack of toxicity of the inhibitors
(data not shown). Taken together, these data demonstrate that, in
addition to PKC, a PI3-kinase-dependent pathway can also
mediate Syk-dependent activation of p70S6k in
DT40 and DT40Lyn
cells. However, the PI3-kinase pathway
does not appear to be sufficient for the activation of
p70S6k in parental DT40 cells in the absence of phorbol
ester-sensitive PKC isoforms, whereas this pathway can be sufficiently
activated to mediate activation of p70S6 kinase in Lyn-deficient cells
under the same condition.

View larger version (42K):
[in this window]
[in a new window]
|
Fig. 6.
The role of PI3-kinase in BCR-mediated
activation of p70S6k. Cells were treated with 50 nM wortmannin (A) or 4 µM Ly294002
(B, bottom panel) for 15 min prior to stimulation with 2 µg/ml mouse anti-chicken IgM Ab (M4) for 30 min. Equal amounts of
cell lysates were immunoprecipitated with anti-p70S6k Ab.
The immune complexes were divided for immunoblotting with
anti-p70S6k Ab (top panel) and in
vitro kinase assays (bottom panel) using S6 peptides as
exogenous substrates. C, DT40Lyn cells were
treated with PdBu for 12 h followed by incubation with 4 µM Ly294002 for an additional 15 min prior to
stimulation. Cells were stimulated with 2 µg/ml M4 or 50 nM PdBu for 30 min. The phosphorylation states of
p70S6k were determined as described in Fig. 4.
|
|
 |
DISCUSSION |
Antigen receptor-mediated signaling plays a pivotal role in
determining the fate of lymphocytes. BCR-mediated cellular responses are based on the nature of the ligand and depend on the integration of
additional signals provided, for example by CD40/CD40L-mediated interactions with T cells and T cell-derived cytokines such as IL-4.
The state of B cell maturation as well as the dose and nature (e.g. valency, affinity) of the ligand-receptor interaction
determine the degree to which receptor-coupled signal transduction
pathways are activated. In a primary immune response, unprimed B cells can be stimulated even by limiting concentrations of nominal antigen and undergo clonal expansion and affinity maturation. In contrast, ligand binding to immature autoreactive B cells typically results in
clonal deletion or anergy. Although anergic self-reactive B cells in
the periphery are non-responsive to low doses of antigen, high receptor
occupancy and appropriate T cell help can break tolerance in
autoreactive B cells (58, 59). Thus, defining the mechanisms underlying
B cell activation in response to varying degrees of receptor engagement
is important in understanding B cell activation and tolerance.
We recently demonstrated that ligation of BCR on the avian B cell line
DT40 results in activation of MAPK and members of two families of
ribosomal S6 kinases, p90Rsk and p70S6k (10).
In this report, we extended our previous findings by demonstrating that
ligation of BCR can induce activation of p70S6k in murine
splenic B cells. BCR-induced activation of p70S6k in
primary murine B cells was associated with the in vivo
phosphorylation of endogenous S6 protein, which is important for
ribosomal biogenesis, and the induction of DNA synthesis. BCR-induced
activation of p70S6k was mediated by multiple pathways, but
the degree to which the receptor must be cross-linked to activate these
pathways, is apparently distinct. Thus, Syk was required for
BCR-mediated activation of p70S6k in response to
stimulation with low dose anti-Ig. However, as we reported earlier (10)
activation of p70S6k in response to high dose anti-Ig was
not Syk-dependent, indicating that under optimal
stimulatory conditions other pathways can compensate for the absence of
Syk. Finally, we demonstrated that Syk-dependent activation
of p70S6k is mediated by signal transduction pathway(s)
involving PKC and PI3-kinase.
Multiple hierarchical phosphorylation events are involved in the
regulation of p70S6k (60-64). At least eight different
phosphorylation sites have been implicated; of these, Thr-229, Ser-371,
and Thr-389 are essential for p70S6k activation (52,
65-67). Ser-411, Thr-421, and Ser-424 lie in the pseudosubstrate
domain and are sensitive to rapamycin treatment (55, 68).
Phosphorylation of these three sites appears to occur first and
facilitates phosphorylation of Thr-389 via a
PI3-kinase-dependent pathway (55). This renders Thr-229
accessible for phosphorylation by the recently identified kinase, PDK1
(49, 55, 60, 69-72). Under appropriate conditions p70S6 kinase can
also be activated via a PI3-kinase-independent pathway(s) through PKC,
Raf-1, and small G proteins such as Rac1 and Cdc42 (10, 33, 34). In this study, we showed that both PKC and PI3-kinase pathways contribute to the BCR-mediated activation of p70S6k. However it is not
yet clear whether these pathways act in parallel or sequentially to
activate p70S6k.
The role of p70S6k in B cell activation is not yet clear.
However, the similar dose-response curves and rapamycin sensitivity observed for anti-Ig-induced activation of p70S6k, the
in vivo phosphorylation of endogenous S6 protein, and
induction of DNA synthesis in primary murine B cells suggest that
p70S6k may play an important role in BCR-induced protein
synthesis and DNA synthesis in B cells. Rapamycin inhibits cell cycle
progression in many cell types by binding to its cellular receptor,
FK506-binding protein. This complex then associates with the direct
target of rapamycin (mTOR·FRAP·RAFT) and inhibits activation of
several downstream effectors, including the transcription factor CREM, the translation initiation factor 4E-BP1, cyclin-dependent
kinase, and p70S6k (13, 46, 73-75). Thus, the effect of
rapamycin on p70S6k is indirect. Additionally, rapamycin
also targets other signaling pathways that do not involve
p70S6k. Therefore, inhibition of S6 phosphorylation and DNA
synthesis by rapamycin does not provide definitive evidence of a role
for p70S6k in BCR-induced protein synthesis or cell cycle
regulation. Kawasome et al. (20) recently generated
p70S6k-deficient embryonic stem cells by targeted gene
disruption. The mitogen-induced proliferation of these mutant cells
remained partially sensitive to rapamycin. However, the absence of
p70S6k in embryonic stem cells resulted in decreased
proliferation in response to growth factor stimulation, directly
indicating the role of p70S6k in cell cycle progression.
Furthermore, ribosomal S6 phosphorylation was ablated in the mutant
cells, and translation of mRNA encoding ribosomal proteins was not
increased in response to serum indicating that p70S6k plays
a unique role in ribosomal protein synthesis. The generation of
p70S6k-deficient mice in the future should provide further
insight into the role of p70S6k in BCR-induced protein and
DNA synthesis as well.
We demonstrated that Syk is required for activation of MAPK even in
response to high doses of anti-Ig (10) and as demonstrated in this
study, for low dose anti-Ig induced activation of p70S6k.
These data suggest that Syk may be essential for antigen-induced clonal
expansion of B cells when challenged with physiologic doses of ligand.
Consistent with this hypothesis, the differentiation of B-lineage cells
in Syk-deficient mice exhibits a block at the pro-B to pre-B transition
due to defects in signaling through the pre-B cell receptor that
prevent clonal expansion and maturation of pre-B cells (39, 77). In
contrast to low dose anti-Ig stimulation, another protein tyrosine
kinase-dependent signaling pathway that is inhibited by
genistein, but that does not require either Syk or Lyn, can apparently
be engaged in p70S6k activation following optimal receptor
cross-linking and lead to activation of p70S6k. One likely
candidate upstream protein tyrosine kinase that may mediate activation
of this alternative pathway is Btk which can activate
phospholipase-C
and hence PKC (5).
Other signaling pathways have also been implicated in BCR-induced
proliferation responses, in particular the
Ras/Mek/MAPK/p90Rsk pathway. However, it is noteworthy that
BCR ligation in anergized B cells induced activation of
MAPK/p90Rsk but failed to induce proliferation suggesting
that this pathway is not sufficient for BCR-induced proliferation (76).
If a p70S6k-dependent pathway(s) is primarily
responsible, or is required in addition to activation of MAPK to
promote BCR-induced growth, then down-regulation of p70S6k
is a potential mechanism underlying the unresponsiveness of anergic B
cells. Theoretically, regulation of BCR-mediated signaling in anergic
cells may be achieved by several mechanisms including uncoupling of the
receptor from downstream signaling cascades, down-regulation of
receptor expression, or increased activity of negative regulatory
mechanisms. With regard to the pathways described in this study, low
doses of autoantigen may fail to engage the
Syk/Lyn-dependent signaling pathway leading to
p70S6k activation. Interestingly, autoreactive B cells in
both anti-HEL and anti-DNA Ig transgenic mice express reduced levels of
surface Ig (78, 79) and conceivably the threshold doses of antigen may
be greater to activate the Syk/Lyn-mediated pathway of
p70S6k. Alternatively, the pathway could be completely
inactivated in anergic B cells, but at high doses of autoantigen the
requirement for this pathway may be overcome by activation of the
Syk/Lyn-independent pathway which we demonstrated could also be
activated in normal B cells following optimal receptor cross-linking.
Although further studies will be required to determine the mechanism(s)
underlying the unresponsiveness of anergic B cells, it is interesting
to note that the phosphorylation of Syk in response to optimal doses of
anti-Ig is defective in anergic B cells (79).
In summary, our results suggest that the activation of
p70S6k may be an important intermediate step in antigen
receptor-induced activation of B lymphocytes. Future studies will
therefore be focused on the role of p70S6k and its
regulation in determining the fate of mature B cells challenged by
foreign antigen as well as the fate of antigen-challenged autoreactive
B cells.