Molecular Cell, Vol. 17, 205–214, January 21, 2005, Copyright ©2005 by Elsevier Inc.DOI 10.1016/j.molcel.2004.12.012
RhoA GTPase Regulates B Cell Receptor Signaling
1997; Schuebel et al., 1998; Zeng et al., 2000) and are
required for immune receptor signaling, suggesting that
signals dependent on Rho GTPases are also necessary.
Splenic B cells from mice lacking expression of either
Vav1 or Vav2 have subtle defects in development and
BCR signaling. B cells from mice lacking expression of
both Vav1 and Vav2, or all three Vav family members,
however, have profound defects in B cell development
and BCR signaling, including absence of BCR-stimu-
lated calcium flux (Zhang et al., 1995; Tedford et al.,
2001; Doody et al., 2001; Fujikawa et al., 2003). The
function of Rho family members in B cells is not under-
stood, nor is it even known whether RhoA is required
for signaling in B cells.
Signals generated by the BCR are crucial for B cell
survival, proliferation, development, differentiation, and
apoptosis (Kurosaki et al., 2000; Turner, 2002). The sig-
contain immunoreceptor tyrosine-based activation mo-
bind to the protein tyrosine kinase Syk, leading to its
phosphorylation and activation. Subsequently, Btk, Vav
proteins, and phosphoinositide 3-kinase (PI3K) are acti-
vated, resulting in PLC?2-dependent IP3production and
Although many of the components of BCR-regulated
pathways have been identified, there still are significant
gaps in our understanding. The functions of PI3K and
the Vav proteins required for BCR signaling are not es-
tablished, and the regulation of PLC?2 is incompletely
Here, we investigated the role of RhoA in BCR signal-
ing and provide evidence for its involvement in the regu-
lation of BCR-dependent calcium flux and proliferation.
RhoA is activated upon BCR engagement, downstream
of PI3K activation, in both A20 and primary B cells. We
used both a dominant-negative (DN) mutant of RhoA
and a RhoA-specific inhibitor, C3 toxin, to show that
RhoA is important for BCR-dependent calcium flux. IP3
production is inhibited by DN RhoA mutant, but PLC?2
tyrosine phosphorylation is unaffected. Inhibition of RhoA
function blocks PtdIns-4,5-P2, PA, and PtdIns-4-P syn-
thesis. RhoA associates with phosphatidylinositol-4-
phosphate 5-kinase and phosphatidylinositol 4-kinase,
the enzymes that synthesize PtdIns-4,5-P2 and PtdIns-
4-P, respectively. Providing B cells with exogenous
PtdIns-4,5-P2rescued BCR-dependent calcium flux in
cells expressing the N19RhoA mutant, confirming the
requirement for substrate provision for PLC?2 activity.
We showed that RhoA is necessary for BCR-mediated
cell proliferation by using C3 toxin in primary B cells.
These data support a function for RhoA in the regulation
of B cell receptor signaling.
Abdelhafid Saci and Christopher L. Carpenter*
Division of Signal Transduction
Department of Medicine
Beth Israel Deaconess Medical Center
Boston, Massachusetts 02115
The RhoA GTPase controls many cellular functions,
including gene transcription and actin polymerization.
Several lines of evidence suggest that Rho GTPases
are required for B cell receptor (BCR) signaling, but
whether RhoA is necessary has not been investigated.
Here, we show that RhoA is activated, downstream of
PI3K, in response to BCR stimulation and is important
for BCR-dependent calcium flux and cell proliferation.
A RhoA dominant-negative mutant strongly inhibited
BCR-dependent calcium mobilization. The RhoA-spe-
cific inhibitor, C3 toxin, inhibited both BCR-dependent
calcium flux and cell proliferation. RhoA is important
for BCR-dependent synthesis of IP3by PLC?2, but is
not required for tyrosine phosphorylation of PLC?2.
BCR-dependent synthesis of phosphatidylinositol-
4,5-bisphosphate (PtdIns-4,5-P2) is inhibited in the ab-
sence of RhoA function. Providing exogenous PtdIns-
4,5-P2restores BCR-dependent calcium flux in cells
lacking functional RhoA. Our findings support a func-
tion for RhoA in BCR-dependent PtdIns-4,5-P2synthe-
sis, PLC?2 activation, calcium mobilization, and cell
RhoA is a member of the Rho family of small GTPases
and regulates several cellular functions, including actin
rearrangement, polarity, transcription, and cell cycle
progression. RhoA, like other GTPases, transmits sig-
nals by binding to effector proteins. GTPases undergo
a conformational change when bound to GTP, and most
effectors associate with this form. Binding to GTPases
results in effector activation and/or specific localization.
GTP loading is catalyzed by guanine nucleotide ex-
change factors (GEFs). GTPase-activating proteins
(GAPs) accelerate hydrolysis of GTP and the return to
the inactive GDP-bound state (Schmidt and Hall, 2002).
Lymphocytes require Rho family members for normal
development and function. Expression of RhoA mutants
or the RhoA-specific inhibitor, C3 toxin, as transgenes
in T cells affects T cell development and survival by an
unknown mechanism (Henning et al., 1997; Costello et
al., 2000; Corre et al., 2001). B cell development and
BCR signaling leading to proliferation and survival re-
quire Rac1 and Rac2 (Walmsley et al., 2003). Lym-
phocytes from mice lacking RhoG, however, develop
normally, despite a slight increase in some immune re-
sponses (Vigorito et al., 2004). Vav proteins are GEFs
for Rho family GTPases (Crespo et al., 1997; Han et al.,
RhoA Is Activated upon B Cell
Since activation of RhoA is a prerequisite for its partici-
pation in BCR-dependent signaling, we initially investi-
was confirmed by Western blotting (Figure 2A, inset).
Expression of N19RhoA dramatically inhibited calcium
flux upon BCR engagement compared to control in-
regulates BCR-stimulated calcium flux.
Dominant-negative GTPases function by binding to
exchange factors and thereby preventing activation of
activate more than one GTPase (Scita et al., 2000), a
dominant-negative effect may not be specific for the
GTPase used. For example, if Vav2 activates both Rac1
and RhoA, DN RhoA could also function by inhibiting
Rac1, rather than RhoA, activation. To address this is-
sue, wedetermined whetherother DNGTPases blocked
BCR-dependent calcium flux in A20 cells. Expression
of N17Cdc42 had no effect on BCR-stimulated calcium
flux (Figure 2B), suggesting that RhoA is important for
BCR-stimulated calcium flux, but other Rho GTPases
could at least in theory be inhibited by N19RhoA.
To determine whether RhoA specifically regulates
BCR-dependent calcium flux, we used the RhoA-spe-
cific inhibitor, C3 toxin. A cell membrane-permeable
peptide complexed to C3 toxin was used to transduce
A20 cells (Morris et al., 2001). About 50% of the cells
were transduced, based on transduction of ?-galactosi-
dase assessed by staining. Calcium flux in A20 cells
treated with C3 toxin was inhibited in a dose-dependent
manner (Figure 2C). C3 toxin-dependent inhibition
would likely have been even greater if the transduction
efficiency were higher. These results indicate that RhoA
specifically regulates BCR-dependent calcium flux.
Dominant-active (DA) GTPase mutants sometimes
potentiate signals in pathways for which they are re-
quired. In the case of V14RhoA, this effect would likely
be specific, since RhoA has distinct downstream ef-
fectors. Expression of DA V14RhoA minimally potenti-
ated BCR-mediated calcium flux in A20 cells stimulated
expression of V14RhoA significantly increased calcium
flux in A20 cells stimulated with a submaximal concen-
tration of F(ab)?2 (Figure 2D).
flux in primary B cells. We first identified conditions that
would allow primary B cells to be infected by vaccinia
virus and used this method to express RhoA mutants
(Figure 2F). Control and RhoA mutant-expressing cells
were treated with F(ab)?2, and calcium flux was mea-
sured. Infection of primary splenic B cells with vaccinia
virus expressing N19RhoA inhibited BCR-dependent
calcium flux, and expression of V14RhoA potentiated
BCR-mediated calcium flux (Figure 2E). Although the
had similar effects in primary B cells, supporting a role
for RhoA in the pathway by which the BCR activates
Figure 1. RhoA Is Activated following BCR Stimulation
A20 (A) or primary splenic B cells (B) were starved and then stimu-
lated via the BCR for 2, 5, or 10 min with F(ab)?2, in the presence
and then GST-RBD bound to GSH beads were incubated with the
soluble fraction. Samples were subjected to SDS-PAGE and West-
ern blotting using an anti-RhoA antibody (upper panels, GST-RBD).
Aliquots from each lysate were also Western blotted to verify the
amount of RhoA in each condition (lower panels, lysate). These
results are representative of three independent experiments.
gated whether RhoA is activated in B cells in response
to stimulation of the BCR. Either A20 or primary B cells
were stimulated with F(ab)?2 fragments to activate the
BCR. At the indicated time points the cells were lysed,
and RhoA activation was measured using a GST fusion
of the RhoA binding domain of Rhotekin (GST-RBD)
(Sander et al., 1999). BCR stimulation activated RhoA
in both A20 (Figure 1A) and primary B cells (Figure 1B).
at 5 and 10 min following BCR engagement (lanes 3 and
4). The total cell lysates were also Western blotted for
RhoA to be certain that the RhoA levels were similar
before the GST pull-down experiments (Figures 1A and
1B, lower panels). Phosphoinositide 3 kinase (PI3K) is
necessary for BCR signaling and is required for activa-
tion of Rho GTPases in some pathways and may also
regulate the function of Vav proteins (Grill and Schrader,
2002; Han et al., 1998). Therefore, we determined
whether PI3K is necessary for the activation of RhoA by
the BCR. We treated primary or A20 B cells with the
PI3K inhibitor wortmannin and then measured the acti-
vation of RhoA upon BCR engagement. Wortmannin
inhibited BCR-dependent RhoA activation in both A20
and primary B cells (Figures 1A and 1B, lanes 5–7),
indicating that BCR-dependent stimulation of RhoA oc-
curs downstream of PI3K activation.
RhoA Regulates BCR-Dependent Calcium Flux
suggested that it could be required for BCR-dependent
signals. Vav proteins can activate RhoA (Movilla and
Bustelo, 1999; Schuebel et al., 1998), and since Vav1
and Vav2 are essential for BCR-dependent calcium flux
whether RhoA is required. We used both dominant-neg-
ative (DN) and active RhoA constructs and C3 toxin, a
RhoA-specific inhibitor. A DN mutant of RhoA, N19RhoA,
was expressed in A20 cells using a vaccinia virus vector
RhoA Regulates the Release of Calcium
from Intracellular Stores
BCR-dependent calcium flux is a multistep process. IP3
derived from hydrolysis of PtdIns-4,5-P2by PLC?2, acti-
intracellular stores and opening of the CRAC channels
RhoA GTPase Regulates B Cell Receptor Signaling
Figure 2. RhoA Regulates BCR-Dependent
Ca2?Flux in A20 Cells
(A) A20 cells were infected with either wild-
type vaccinia virus (pSC65) or a virus ex-
pressing V14RhoA or N19RhoA. Cells were
then loaded with Fura-2, the BCR was stimu-
lated with 15 ?g/ml F(ab)?2, and the Fura-2
fluorescence ratio was determined.
(B) A20 cells were infected by pSC65 or by
N17Cdc42 recombinant vaccinia virus, and
then calcium flux was measured as in (A).
The insets represent the expression of RhoA
mutants (A) and N17Cdc42 (B) tested with
Western blotting before adding Fura-2.
(C) A20 cells were treated or not with C3 toxin
before loading with Fura-2, and calcium flux
was measured as in (A).
(D) A20 cells were infected and treated as in
(A) and then stimulated with a submaximal
concentration (2 ?g/ml) of F(ab)?2.
(E) Infected primary B cells were loaded with
Fura-2 and stimulated with 20 ?g/ml F(ab)?2,
and calcium flux was measured.
cated viruses and then lysed and subjected
to SDS-PAGE and Western blot analysis with
anti-myc antibody to verify RhoA mutant ex-
pression. These results are representative of
three independent experiments.
(calcium release-activated calcium channels). To place
RhoA in this pathway, we investigated whether it was
necessary for release of calcium from intracellular
stores. A20 cells were infected with vaccinia virus ex-
pressing N19RhoA, and the effect on calcium flux in the
absence of extracellular calcium and the presence of
EGTA was determined. Expression of N19RhoA mark-
of extracellularcalcium (Figure3A), indicatingthat RhoA
To affect the release of calcium from intracellular
stores, RhoA could be necessary for the production of
IP3or could act directly on the IP3receptor, as reported
recently in endothelial cells (Mehta et al., 2003). To dis-
tinguish between these possibilities, we determined the
effect of N19RhoA expression on BCR-dependent IP3
production in both A20 and primary B cells. Cells were
infected with vaccinia viruses expressing the indicated
were measured. As shown in Figure 3B, expression of
duction in primary B cells, whereas expression of
Figure 3. RhoA Regulates BCR-Dependent
IP3Production, but Not PLC?2 or Akt Phos-
(A) A20 cells were infected with the indicated
vaccinia viruses and then loaded with Fura-2
and resuspended in calcium-free buffer con-
lated, and calcium flux was measured.
(B) A20 cells infected with the indicated vi-
ruses were stimulated with F(ab)?2, and after
2 min the cells were lysed and IP3production
was measured as described in the Experi-
(C and D) Infected A20 cells were stimulated
lysed, and phosphorylation of PLC?2 (C) or
PLC?2 and Akt were also determined (lower
panels). Results are representative of three
obtained in A20 cells (data not shown), indicating that
RhoA regulates PLC?2-dependent hydrolysis of PtdIns-
PLC?2 activity is regulated by tyrosine phosphoryla-
tion (Watanabe et al., 2001; Rodriguez et al., 2001), so
we next investigated whether RhoA was required for
thisstep. Weusedaphosphospecific antibodytoY1217
to determine whether expression of N19RhoA affected
BCR-dependent tyrosine phosphorylation of PLC?2.
A20 cells were infected with a control virus or a virus
expressing N19RhoA and were stimulated with F(ab)?2
blotted for phospho-PLC?2 and total PLC?2. As shown
in Figure 3C, expression of N19RhoA had no effect on
BCR-stimulated phosphorylation of Y1217, suggesting
that phosphorylation of PLC?2 is not dependent on
RhoA. Similarly, expression of N19RhoA did not affect
BCR-stimulated tyrosine phosphorylation of PLC?2, as-
sessed using a general anti-phosphotyrosine antibody
(data not shown).
Since PI3K is essential for BCR-stimulated calcium
mobilization, and its product PtdIns-3,4,5-P3may help
to localize and activate PLC?2 (Bae et al., 1998; Rameh
et al., 1998), we determined whether RhoA was neces-
sary for BCR-dependent activation of PI3K. Akt phos-
infected with either a control virus or a virus expressing
N19RhoA were stimulated with F(ab)?2 fragments, the
cells were lysed, and the lysates were Western blotted
for total and phospho-Akt. As shown in Figure 3D, BCR-
dependent Akt phosphorylation was not affected by ex-
pression of N19RhoA. BCR-dependent ERK activation
was also unaffected by expression of N19RhoA (data
not shown). RhoA is not necessary for BCR-dependent
regulation of PI3K activity or PLC?2 phosphorylation.
indicating that the other ATP-requiring BCR-dependent
pathways are activated normally in cells expressing
To verify that RhoA was a specific regulator of de
novo phospholipid synthesis, we measured phospho-
lipid synthesis in the presence of C3 toxin in A20 B
BCR-stimulated PtdIns-4,5-P2and PA synthesis (p ?
0.001 and p ? 0.01, respectively), but not that of PtdIns-
4-P (p ? 0.18), confirming a specific role for RhoA in
regulating BCR-dependentphospholipid synthesis (Fig-
ures 5A and 5B). Because the transduction efficiency of
C3 toxin was about 50%, these results are not as dra-
matic as expression of N19RhoA, which affects all of
RhoA Binds PI4K and PI4P5K Activities in B Cells
We next investigated the mechanism by which RhoA
might directly regulate PtdIns-4,5-P2synthesis. Several
studies have shown that RhoA can regulate PIP5Ks and
increase PtdIns-4,5-P2 synthesis (Chong et al., 1994;
Oude Weernink et al., 2000). The RhoA-activated kinase
ing activation of PIP5Ks in some cells, such as HEK-
293 (Oude Weernink et al., 2000). To determine whether
RhoA function in B cells requires ROK, we measured
BCR-stimulated calcium flux in cells treated with ROK
inhibitors. Neither ROK inhibitor (HA-1077 and Y-27632
at concentrations of 1–10 ?M and 10–100 ?M, respec-
tively) had a significant effect on BCR-stimulated cal-
cium flux (data not shown). N19RhoA could also block
Rac1 activation and thereby PtdIns-4,5-P2 synthesis,
since Rac1stimulates PIP5K activity(Tolias etal., 2000).
Therefore, we measured Rac1 activation upon BCR
stimulation in A20 cells expressing N19RhoA, using a
GST fusion of the p21 binding domain of PAK (Benard
et al., 1999). Expression of N19RhoA did not affect the
activation of Rac1 by the BCR (data not shown). Treat-
ment of B cells with cytochalasin D had no effect on
BCR-dependent calcium flux, indicating that the RhoA
effect is not mediated through the actin cytoskeleton.
RhoA has been reported to associate with and regu-
latePIP5Ks. Wehavealsodetected associationofRhoA
with PIP5Ks in fibroblasts, although RhoA binds less
well than Rac1 (Tolias et al., 1995). RhoA could bind to
and recruit PIP5Ks to sites of BCR activation to provide
ciates withphosphoinositide kinases in Bcells, we incu-
GTP?S or GDP?S and measured associated PI4K and
PIP5K activities. RhoA associated with both PI4K and
PIP5K activities (Figure 5C, left panel). The association
with PI4K and PIPK was slightly, but significantly, in-
creased for GTP-bound RhoA (p ? 0.005 and p ? 0.05,
respectively). To determine whether endogenous RhoA
associates with PI4K and PIP5K activities, we assayed
RhoA immunoprecipitates from quiescent and stimu-
lated B cells. Both PI4K and PIP5K activities were also
present in RhoA immunoprecipitates (Figure 5C, right
panel). RhoA associates with the enzymes that synthe-
size PtdIns-4-P and PtdIns-4,5-P2and likely regulates
RhoA Regulates PIPK, PI4K, and DGK Activities
Downstream of the BCR Engagement
The other mechanism by which PLC?2 might be regu-
lated is the availability of its substrate, PtdIns-4,5-P2.
To investigate whether RhoA regulates the synthesis of
PtdIns-4,5-P2, we expressed N19RhoA in either A20 or
primary B cells and measured BCR-dependent PtdIns-
(necessary to detect new synthesis of PtdIns-4,5-P2)
with32P-PO4, and then the BCR was stimulated. Lipids
were extracted, separated by TLC, and quantified with
a molecular imager. N19RhoA significantly (p ? 0.005)
inhibited PtdIns-4,5-P2synthesis in both primary B cells
(Figure 4A, right panel) and A20 cells (Figure 4A, left
panel, and Figure 4B). Expression of N19RhoA also in-
hibited PA and PtdIns-4-P synthesis (Figure 4; p ? 0.01
and p ? 0.005, respectively). BCR-dependent synthesis
of PtdIns-4,5-P2, PA, and PtdIns-4-P was also slightly
stimulated by expression of V14RhoA (Figures 4A and
4B). In the short term, nonequilibrium labeling the spe-
cific activity of the precursor pool (in this case ATP)
determines the labeling efficiency. We measured the
effect of expression of RhoA mutants on the incorpora-
tion of32P-PO4into ATP to be certain that expression of
N19RhoA did not affect the labeling of ATP, and we
found no effect (data not shown). Furthermore, BCR-
RhoA GTPase Regulates B Cell Receptor Signaling
Figure 4. RhoA Regulates BCR-Dependent
(A) A20 B cells (left) or primary splenic B cells
(right) were infected with recombinant vac-
cinia viruses and then were incubated in
phosphate- and serum-free medium for 1–2
hr. Cells were then labeled with32P-PO4for 5
min at 37?C and stimulated for 3 min. The
reaction was stopped with 1 N HCl, and lipids
were extracted with methanol/chloroform (1:1)
and separated by TLC (thin-layer chromatog-
raphy). Phospholipid synthesis (PtdIns-4,5-P2,
PtdIns-4-P and PA) in A20 cells was quanti-
fied using a PhosphorImager (B). These re-
sults are representative of four experiments.
the activity and/or location of the complex to promote
PtdIns-4,5-P2synthesis at sites near PLC?2.
Pretreatment of A20 cells expressing N19RhoA with
histone/PtdIns-4,5-P2 resulted in an almost complete
recovery of calcium flux (Figure 6B) following BCR stim-
ulation, indicating that RhoA functions to provide
PtdIns-4,5-P2 as substrate for PLC?2. To determine
whether the PtdIns-4,5-P2effect was specific, we mea-
sured calciumflux in A20cells expressingN19RhoA and
pretreated with PtdIns-3,5-P2, an isomer that is not a
substrate for PLC?2. Histone/PtdIns-3,5-P2complexes
did not rescue calcium flux in cells expressing N19RhoA
(Figure6C). PretreatmentwithPAor PtdIns-4-Presulted
in only a small increase in calcium flux compared to
untreated cells (data not shown). These results indicate
that RhoA is important for providing PtdIns-4,5-P2as
substrate for PLC?2 to be active in response to BCR
Exogenous PtdIns-4,5-P2Rescues Calcium Flux
in Cells Expressing N19RhoA
The effect of N19RhoA and C3 toxin on BCR-dependent
phospholipid synthesis could reflect direct inhibition of
phospholipid synthesis or could result from inhibition of
PLC?2 activity. In the latter case, the lack of PtdIns-4,5-
P2consumption and DAG production by PLC?2 could
affect PtdIns-4,5-P2and PA synthesis. To distinguish
between these possibilities, we investigated whether
cium flux in cells expressing N19RhoA. If N19RhoA
blocks BCR-dependent IP3production and calcium flux
by inhibiting synthesis of PtdIns-4,5-P2, the substrate
for PLC?2, providing exogenous PtdIns-4,5-P2should
sary for some other aspect of PLC?2 activation, such
as localization or phosphorylation, providing PtdIns-
4,5-P2would not rescue the defect. Phosphoinositides
bound to histone H1 are cell permeable (Ozaki et al.,
sitides to B cells. PtdIns-4,5-P2 or PtdIns-3,5-P2 was
bound to histone H1 and incubated with A20 cells that
had previously been infected with either pSC65 or
N19RhoA-expressing vaccinia virus. Addition of the his-
(before BCR stimulation) resulted in a small increase in
intracellular calcium (Figures 6B and 6C). BCR stimula-
and treated with histone/PtdIns-4,5-P2 had a slightly
higher peak and was more sustained compared to un-
treated cells (compare Figures 6B and 6C with Figure 6A).
RhoA Regulates BCR-Dependent Cell Proliferation
To investigate the role of RhoA in B cell function, we
determined whether RhoA is necessary for BCR-stimu-
lated mitogenesis in primary B cells. The cells were
transduced with C3 toxin to inhibit RhoA, the BCR was
dine incorporation. As shown in Figure 7A, BCR-medi-
ated cell proliferation increased 3-fold in the absence
of C3 toxin. In the presence of 5 ?g/ml C3 toxin, BCR-
mediated cell proliferation was significantly inhibited
(p ? 0.0019). In cells stimulated with LPS, proliferation
a specific role for RhoA in BCR-stimulated cell prolifera-
tion. As expected, treatment with C3 toxin inhibited
BCR-dependent calcium flux in primary B cells (Figure
7B). These results argue for an important role of RhoA
in the regulation of BCR-dependent cell signaling and
by Kurosaki, 2000). PLC?2 activity requires tyrosine
phosphorylation of specific sites (Rodriguez et al., 2001;
Watanabe et al., 2001). We showed that RhoA was not
required for phosphorylation of one of these sites
(Y1217), nor was total BCR-stimulated tyrosine phos-
phorylation of PLC?2 affected by expression of DN
RhoA. Based on these results, we conclude that RhoA
is not necessary for direct activation of PLC?2. The
RhoA-specific inhibitor, C3 toxin, inhibited both BCR-
mediated calcium flux and proliferation in primary B
cells, emphasizing the importance of RhoA in this path-
way. These results are similar to the phenotype of B
cells from Vav1 and Vav2 null mice. Calcium flux and
cell proliferation in these cells are markedly reduced,
but BCR-stimulated PLC?2 phosphorylation is normal
(Tedford et al., 2001).
BCR-stimulated calcium flux, we investigated pathways
known to be activated by RhoA. Disruption of the actin
cytoskeleton had no effect on BCR-stimulated calcium
The protein kinase ROK, a RhoA effector, was not re-
quired for BCR-dependent calcium flux. Phospholipase
D, another downstream target of RhoA (Kuribara et al.,
1995), was not activated by BCR engagement, and its
basal activity was not affected by the expression of
RhoA mutants (data not shown). We then investigated
whether RhoA was necessary for BCR-stimulated syn-
ment of cells with C3 toxin blocked BCR-stimulated
synthesis of PtdIns-4,5-P2, PtdIns-4-P, and PA. RhoA
associates with PIP5K and PI4K in both GST pull-downs
and immunoprecipitates of endogenous RhoA. These
findings suggest that RhoA regulates the enzymes that
provide PtdIns-4,5-P2, which is necessary for PLC?2 ac-
kinases are part of the B cell signalosome. The associa-
tion of RhoA with PI4K and PIP5K activity suggests
that RhoA could either directly activate the enzymes or
mediate their localization. This complex could provide
for efficient de novo synthesis of PtdIns-4,5-P2to be
used as substrate by PLC?2. The best illustration of
this mechanism is the rescue by histone H1-conjugated
PtdIns-4,5-P2of BCR-dependent calcium flux inhibited
by N19RhoA. This result argues strongly for a crucial
role of substrate provision for PLC?2 activity upon
RhoA has been reported to stimulate PtdIns-4,5-P2
synthesis in several other systems, but the mechanism
by which RhoA regulates PtdIns-4,5-P2synthesis is not
certain. Altun-Gultekin et al. (1998) showed that ROK is
required for RhoA-dependent PtdIns-4,5-P2synthesis.
In contrast, Matsui et al. (1999) found that RhoA-depen-
dent PtdIns-4,5-P2synthesis did not require ROK, con-
sistent with our finding that ROK is not necessary for
BCR-dependent calcium flux. O’Rourke et al. (1998) ex-
amined PtdIns-4,5-P2synthesis in permeabilized B cells
labeled with [?-32P]ATP and found that PtdIns-4,5-P2
ited in Vav1 null primary B cells. C3 toxin had no effect,
indicating that RhoA was not required (O’Rourke et al.,
1998). There are several reasons why our results may
differ. The BCR may activate different pathways than
Figure 5. C3 Toxin Inhibits BCR-Dependent Phospholipid Synthe-
sis, and RhoA Associates with Lipid Kinases
(A) A20 cells were grown in a six-well plate at 50% confluence on
the day of the experiment. C3 toxin was introduced using a cell-
permeable peptide, as described in the Experimental Procedures.
Cells were then loaded with32P-PO4for 5 min at 37?C in phosphate-
and serum-free medium and stimulated for 3 min. Phospholipids
were extracted and analyzed as in Figure 4.
(B) Phospholipid synthesis was quantified using a PhosphorImager.
(C) A20 cell lysates were incubated with GST or GST-RhoA (loaded
with GDP?S or GTP?S) bound to GSH beads for 1 hr at 4?C. The
beads were washed and assayed for associated phospholipid ki-
nase activities. RhoA or nonimmune immunoprecipitates from qui-
escent or stimulated A20 cells were also assayed for associated
phosholipid kinase activities. Results are representative of four ex-
RhoA affects transcription and cell cycle progression,
often coordinated with changes in the actin cytoskele-
ton. In B cells, RhoA is activated by the BCR and has
a function independent of the actin cytoskeleton. RhoA
activation was sensitive to wortmannin, indicating that
PI3K is required for its activation. A DN mutant of RhoA
and the RhoA-specific C3 toxin both blocked BCR-
dependent release of calcium from intracellular stores.
Since calcium release is stimulated by IP3production,
we investigated whether RhoA was necessary for BCR-
dependent IP3synthesis. A DN RhoA mutant inhibited
BCR-stimulated IP3 synthesis. Production of IP3 in B
cells is dependent on the activity of PLC?2, which
RhoA GTPase Regulates B Cell Receptor Signaling
Figure 6. PtdIns-4,5-P2
Flux Inhibited by N19RhoA Expression
(A) A20 cells were infected with vaccinia virus
expressing N19RhoA mutant or empty vector
(pSC65). Cells were then loaded with Fura-2,
the BCR was stimulated [15 ?g/ml F(ab)?2],
and calcium flux was measured.
(B) A20 cells were infected and loaded with
Fura-2 as above. PtdIns-4,5-P2-histone H1
complexes were added 1 min before BCR
stimulation, and calcium flux was measured.
(C) A20 cells were infected and loaded with
H1 or PtdIns-3,5-P2-histone H1 complexes
were added 1 min before BCR stimulation,
and calcium flux was measured. Upward
arrows indicate BCR stimulation, and down-
ward arrows indicate where lipids were added.
Results are representative of four experi-
and/or enzyme localization (PIP5Ks or PLC?2) so that
RhoA is not required for PtdIns-4,5-P2synthesis under
We recently described the association of PIP5Ks with
Btk and showed that the association was important for
(Saito et al., 2003). RhoA appears to have a related
function. There are several possible explanations for
this apparent redundancy. RhoA and Btk could be in
separate complexes with distinct functions, or they
can provide PtdIns-4,5-P2to stimulate PtdIns-3,4,5-P3
synthesis by PI3K, but DN RhoA does not block PI3K
activation by the BCR. B cells from Btk null mice do not
have a defect in PI3K activation, but they also express
Tec. Like Btk, Tec associates with PIP5Ks (Saito et al.,
2003) and could substitute for Btk in a complex that
provides PtdIns-4,5-P2 for PLC?2 and PI3K. Btk and
RhoA could also be in a common complex following
BCR activation. Btk and RhoA might interact with the
same PIP5K molecule to stabilize its presence in the
complex. Alternatively, PIP5Ks might be brought to
the complex by both Btk and RhoA, providing a way to
integrate signal strength by providing PtdIns-4,5-P2as
substrate for PLC?2. RhoA has been reported to associ-
ate with Bmx, another Tec family member, but we have
not detected an association between RhoA and Btk in
B cells (unpublished data).
RhoA associates constitutively with PI4K (although
PI4K activity associated with GTP?S-loaded GST-RhoA
was increased) and PIP5K activity. Based on the effects
ing is required for RhoA stimulation of phosphoinositide
of the enzymes in the complex or their localization in a
using C3 toxin showed that RhoA mediates PI4K activa-
tion by mastoparan in adrenal chromaffin cells.
In summary, we demonstrated that RhoA regulates
BCR-mediated cell signaling. RhoA is activated down-
stream of BCR engagement and PI3K activation, and
regulates IP3production by PLC?2 and thereby calcium
mobilization and cell proliferation. RhoA regulates
PtdIns-4,5-P2synthesis, which is shown here to rescue
Figure 7. RhoA Regulates BCR-Dependent Cell Proliferation
alone or transduced with 5 ?g/ml C3 toxin for 3 hr using the peptide.
Cells were stimulated in duplicate with either rabbit anti-mouse IgM
F(ab)?2 antibody (10 ?g/ml) or LPS (10 ?g/ml) for 40 hr in the pres-
ence of [3H]thymidine for the last 18 hr.
(B) After C3 toxin transduction by Chariot, cells were loaded with
Fura-2 and subjected to calcium flux analysis upon stimulation with
10 ?g/mlanti-IgM F(ab)?2antibody. Theseresults arerepresentative
of four experiments.
stimulated or not with F(ab)?2 fragments for 2 min. The incubation
was stopped with 1/5 volume of 100% TCA. After centrifugation at
10,000 ? g for 1 min, the supernatants were used for IP3measure-
ments with the reagents supplied by the manufacturer.
calcium flux from inhibition by N19RhoA, reflecting a
requirement for stimulated PtdIns-4,5-P2synthesis for
PLC?2 activity. The increase of cytoplasmic calcium is
also crucial for geneexpression, motility, adhesion (Dol-
metsch et al., 1997), and apoptosis (Takata et al., 1995)
in B cells. In addition to cell proliferation, RhoA may be
downstream of BCR activation.
Immunoprecipitation, GST Pull-Down, and Immunoblotting
Resting or stimulated A20 cells were lysed in a buffer (20 mM Tris-
HCl [pH 7.5], 150 mM NaCl, 1% NP-40, 1 mM Na3VO4, 1 mM PMSF,
10 ?g/ml leupeptin, 10 ?g/ml aprotinin, and 5 mg/ml MgCl2). Lysates
were clarified by centrifugation at 15,000 ? g for 10 min at 4?C.
Immunoprecipitation was performed at 4?C by adding the appro-
complexes were washed, separated by SDS-PAGE, and transferred
to a nitrocellulose membrane. After blocking with 5% fat-free milk
in TBS-Tween, the membranes were incubated with the appropriate
primary antibodies, followed by a secondary HRP-linked antibody.
Enhanced chemiluminescence (ECL) was used to detect the signal.
Antibodies directed against phospho-Akt, Akt, PLC?2, and phos-
pho-PLC?2 were purchased from Cell Signaling Technology (Bev-
erly, MA). Mouse anti-RhoA and rabbit anti-RhoA and Cdc42 anti-
CA). Rabbit F(ab?)2anti-IgG and anti-IgM used to crosslink the BCR
were purchased from Jackson ImmunoResearch (West Grove, PA).
Phospholipids were purchased from Avanti Polar Lipids (Alabaster,
AL). Mice were purchased from Charles River (Wilmington, MA).
Recombinant vaccinia viruses expressing myc-tagged wild-type,
dominant-negative, and dominant-active RhoA were provided by
Dr. E. Hong-Geller (Los Alamos, NM). The expression vector for
GST-C3 was obtained from Prof. Larry A. Feig (Tufts University
School of Medicine, Boston, MA).
Measurement of Lipid Kinase Activities
A20 cells were starved overnight and either left unstimulated or
were stimulated by 30 ?g/ml F(ab)?2 rabbit anti-mouse IgG for 2
min. The cells were then lysed and centrifuged at 4?C. The superna-
tants were immunoprecipitated with rabbit anti-RhoA antibody or
subjected to a GST pull-down assay. Following extensive washing,
the kinase activities were assayed as previously described (Tolias
et al., 1998). PI4P 5-kinase and PI 4-kinase activities associated
with RhoA were measured by adding PI4P and PI, respectively,
as substrates. Reactions were performed during 10 min at room
temperature in the presence of 1 ?Ci of [?-32P]ATP per reaction.
Individual radioactive spots were quantified using a Phosphor-
Imager (Molecular Dynamics).
Cell Culture and Viral Infection
A20 cells (mouse leukemia cell line) were grown in RPMI 1640 sup-
plemented with 10% fetal bovineserum (FBS), 1% sodium pyruvate,
0.1% penicillin (100 U/ml), streptomycin (100 ?g/ml), and 50 ?M
2-mercaptoethanol. Cells were starved overnight in 0.1% FBS be-
fore activation and analysis. Cell infection by recombinant vaccinia
viruses (20 pfu/cell) was performed in 2.5% FBS for about 14 hr
prior to analysis.
Primary B cells were isolated from the spleens of ?10-week-old
C57Bl/6N mice using MACS CD43 (Ly-48) microBeads as described
in the manufacturer’s protocol (Miltenyl Biotech). They were grown
for 24 or 48 hr before infection with vaccinia virus, in the same
culture medium as A20 cells supplemented with 1–2 ?g/ml lipopoly-
saccharide. Infection of primary splenic B cells with vaccinia virus
was performed in the same culture medium with 2.5 % FBS without
LPS for 14 hr. The cell concentration was 5 ? 106to 107cells per
10 cm plate, and the viral concentration was 20 pfu/cell, as for
In Vivo Labeling of Novel Phospholipid Synthesis
Phospholipids were metabolically labeled by incubating A20 or pri-
mary B cells (1–2 ? 106cells per sample) with 300 ?Ci of carrier-
of phosphate- and serum-free RPMI for 5 min at 37?C. The cells
for 3 min. After extraction and separation by TLC, phospholipids
were analyzed by autoradiography and quantified using a Phos-
32P-PO4(inorganic phosphate)/106cells in a volume of 100 ?l
Introduction of C3 Toxin into B Cells
In order to inhibit RhoA by C3 toxin in A20 cells, a cell-permeable
peptide was used as described in the manufacturer’s protocol (Ac-
confluence)and incubatedwith premixedpeptide-C3 toxincomplex
for 1 hr in serum-free medium, followed by a 2 hr incubation in the
labeling experiments, the cells were incubated in phosphate-free
andserum-free mediumfor1–2 hrat37?C beforelabeling andstimu-
A20 B cells were starved overnight, and primary splenic B cells
were starved for 2 hr. The cells were then left unstimulated or were
stimulated through the BCR for 2, 5, or 10 min by F(ab)?2 rabbit
anti-mouse IgG (30 ?g/ml) or IgM (40 ?g/ml), respectively. Cells
were then lysed in 50 mM Tris (pH 7.5), 0.5% Triton X-100, 0.5%
sodium deoxycholate, 150 mM NaCl, 10 mM MgCl2, 1 mM phenyl-
methylsulfonyl fluoride, 10 ?g/ml aprotinin, and 10 ?g/ml leupeptin.
protein of the RhoA binding domain (GST-RBD) to the cell lysates
for 1 hr. Precipitated Rho-GTP was detected by Western blot analy-
sis using a monoclonal anti-RhoA antibody.
Preparation of Phospholipid-Histone H1 Conjugates
Preparation of lipids and their delivery into the cells were performed
as described (Weiner et al., 2002). Briefly, phospholipids (300 ?M)
were freshly prepared in a buffer containing 150 mM NaCl, 4 mM
KCl, and 20 mM HEPES (pH 7.2). In parallel, histone H1 was freshly
prepared at 100 ?M. 300 ?M lipids were mixed with 100 ?M histone
H1 and incubated 5 min at room temperature for lipid-histone com-
plex formation. The complex was diluted 1:10 with HBSS buffer
before addition to the cells. The lipid-histone H1 complexes were
added 1 min before BCR stimulation.
Analysis of Calcium Flux and IP3Production
A20 cells were infected at 5 ? 106/plate by recombinant vaccinia
viruses (20 pfu/cell) expressing either the vector alone (pSC65),
N19RhoA, or V14RhoA for 14 hr. Cells were then loaded with 2
?M Fura-2AM for 20–30 min at 37?C in Hanks’ balanced salt (HBS)
solution containing 1 mM Ca2?. After centrifugation, cells were
washed and resuspended in HBS solution containing 1 mM Ca2?.
Cells were stimulated with 15 ?g/ml F(ab)?2 anti-IgG, and intracellu-
lar Ca2?was measured at 37?C in a SPEX FluoroMax-2 spectrofluor-
ometer.Calcium fluxwasquantified fromthefluorometric ratioemit-
ted at 510 nm from dual-wavelength excitation at 340 nm and
IP3production was measured using an3H-IP3competitive radiore-
facturer’s instructions. Briefly, 2 ? 106A20 or primary B cells were
Splenic B cells were isolated and cultured as described above. After
24 hr, the cells were washed twice with PBS and treated or not with
C3 toxin (5 ?g/ml) in six-well plates (2 ? 106per well) using the
Chariot peptide for transduction, as described above and by the
manufacturer (Active Motif). After 3 hr of treatment, the cells were
RhoA GTPase Regulates B Cell Receptor Signaling
FBS and transferred to 24-well plates (3 ? 105per well). Cells were
treated or not with either 10 ?g/ml F(ab)?2 anti-IgM or 10 ?g/ml LPS
for 40 hr. [3H]thymidine was added for the last 18 hr at 0.8 ?Ci/ml.
The incorporation of [3H]thymidine was measured on a radioactive
scintillation counter (Beckman LS6000SC).
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icance was determined by Student’s t test (paired-data analysis).
*p ? 0.05 was considered to be statistically significant.
We thank Elizabeth Hong-Geller, Lewis L. Cantley, Thomas Roberts,
Benjamin Neel, Larry A. Feig, Gudula Schmidt, Michel R. Popoff,
Matt Topham, Helen Yin, Moti Liscovitch, and Sylvain Bourgoin for
their help and for providing reagents. This work was supported by
NIH grant GM54389 to C.L.C.
Received: May 6, 2004
Revised: September 27, 2004
Accepted: November 24, 2004
Published: January 20, 2005
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