From the Department of Microbiology and the Comprehensive Cancer Center, Ohio State University, Columbus, Ohio 43210
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ABSTRACT |
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We examined activation of the serine/threonine
kinase Akt in the murine B cell line A20. Akt is activated in a
phosphoinositide 3-kinase (PtdIns 3-kinase)-dependent
manner upon stimulation of the antigen receptor, surface immunoglobulin
(sIg). In contrast, Akt induction is reduced upon co-clustering of sIg
with the B cell IgG receptor, Fc Cellular survival in many systems is regulated by the
serine/threonine kinase Akt, also known as protein kinase B (reviewed in Ref. 1). Akt prevents apoptosis in growth factor-responsive cells
(1, 2) by phosphorylation of the protein Bad (3, 4), which causes the
latter to dissociate from Bcl-2 and Bcl-xL (5, 6). Akt
activation in most systems studied to date is dependent on the activity
of the lipid kinase phosphoinositide 3-kinase (PtdIns
3-kinase),1 which generates
3-phosphorylated inositol phospholipids. The PtdIns 3-kinase class IA
enzymes, which are regulated by tyrosine kinases, appear to be
responsible for Akt activation (7). This class of PtdIns 3-kinase
contains two distinct and constitutively associated subunits, an 85-kDa
regulatory protein, which contains two Src homology 2 (SH2) domains,
and a catalytic 110-kDa subunit.
Which PtdIns 3-kinase products are most important for Akt activity is
currently a matter of debate. Both phosphatidylinositol 3,4-bisphosphate (PtdIns 3,4-P2) and phosphatidylinositol
3,4,5-trisphosphate (PtdIns 3,4,5-P3) bind to the Akt
pleckstrin homology domain, but with different affinities, so that
PtdIns-3,4,5-P3 binding is considerably more avid (8). Both
3-phosphoinositide lipids were reported to promote Akt activation, but
whether this is a direct effect is unclear (9-12). Besides regulation
by 3-phosphoinositides, Akt must also be phosphorylated at conserved
serine and threonine residues for maximal activation (reviewed in Ref.
13). At least one kinase responsible for phosphorylating and activating
Akt has been isolated (9, 10). The activity of this kinase, PtdIns 3,4,5-P3-dependent kinase-1, is greatly
increased by PtdIns 3,4,5-P3 and to a lesser extent by
PtdIns 3,4-P2. Intriguingly, membrane localization of Akt
causes its maximal activation even in resting cells (14, 15). Based on
these findings, 3-phosphoinositides probably regulate Akt by a dual
mechanism, first by bringing Akt into close proximity with the
serine/threonine kinase PtdIns
3,4,5-P3-dependent kinase-1 and second by
stimulating PtdIns 3,4,5-P3-dependent kinase-1 activity (9, 10, 16). PtdIns 3,4,5-P3 is possibly a more effective mediator of Akt induction than PtdIns 3,4-P2;
however, there is no direct comparison of these two lipids in this regard.
Stimulation of B cells by the antigen receptor or surface
immunoglobulin (sIg) activates protein tyrosine kinases of the Src and
Syk family, which in turn induce several biochemical pathways that
cause B cell proliferation and Ig secretion (reviewed in Ref. 17).
These pathways proceed largely through tyrosine phosphorylation of the
conserved immunoreceptor tyrosine-based activation motifs within the
two proteins Ig Earlier experiments established that sIg stimulation of B lymphocytes
leads to PtdIns 3-kinase association with tyrosine-phosphorylated proteins (18) and the accumulation of 3-phosphoinositide products (19).
Tyrosine phosphorylation of the p85 and p110 subunits themselves varies
from system to system, but phosphorylation itself does not appear to
regulate PtdIns 3-kinase enzymatic activity (20). Recent studies
indicated that the B cell membrane protein CD19 engages the SH2 domains
of the p85 adapter subunit of PtdIns 3-kinase (21, 22). Presumably,
plasma membrane translocation of PtdIns 3-kinase by association of the
SH2 domains of the p85 subunit with surface receptors like CD19
promotes production of the 3-phosphoinositides, since membrane
targeting of the enzyme in other cells stimulated distal biological
effects of PtdIns 3-kinase (23-26).
In contrast to activation by sIg triggering, co-clustering of the B
cell IgG receptor Fc Recent experiments revealed that B cells exposed to negative signaling
conditions do not accumulate PtdIns 3,4,5-P3 and that the
induction of Btk, a PtdIns 3,4,5-P3-responsive tyrosine
kinase, was reduced (28). Inhibition of these signaling events may be due either to deficient PtdIns 3-kinase activation or to increased consumption of nascent PtdIns 3,4,5-P3. The former
possibility is raised in experiments examining the tyrosine
phosphorylation of CD19 and its subsequent engagement of the p85 SH2
domain; results indicated that both events are reduced under negative
signaling conditions (21, 22). The latter possibility was raised by recent studies indicating that negative signaling conditions promote tyrosine phosphorylation of the polyphosphoinositide 5-phosphatase, SHIP (29, 30), through SHIP recruitment to tyrosine-phosphorylated Fc We investigated the induction of Akt under positive and negative
signaling in the murine B cell line A20. We found that Akt was rapidly
induced under both conditions; however, the level of Akt activity under
negative signaling conditions was only half that under positive
signaling. The reduced activity was not due to deficient activation of
PtdIns 3-kinase itself and was dependent on the presence of the SHIP
docking protein Fc Antibodies, Cells, Stimulation, and Reagents--
The murine B
cell line A20 and its Fc
Anti-Akt used for both immunoprecipitation and immunoblotting was a
sheep polyclonal antibody from Upstate Biotechnology, Inc. (Lake
Placid, NY). Anti-p85 antiserum was generated to a glutathione
S-transferase fusion protein containing the N-terminal SH2
domain of p85, as described previously (31). Anti-HA was purchased from
Roche Molecular Biochemicals. Stimulating antibodies were from Pierce,
and 2.4G2 was purchased from Pharmingen (San Diego, CA); these were
used as described earlier (35). Mouse anti-chicken IgM monoclonal IgG
antibody M1 was kindly provided by Dr. Max Cooper (University of
Alabama, Birmingham, AL). DT40 transfectants were stimulated as
follows. 5 × 106 cells were stimulated with 2 µg/ml
M1 for 5 min at 37 °C. For most other experiments, 10 × 106 B cells were stimulated with 20-30 µg/ml anti-Ig
reagents as described (29). In some experiments, some samples were
preincubated for 10 min at 37 °C with 10 µg/ml (for DT40s) or 30 µg/ml (for A20s) rat anti-mouse Fc
Plasmids were obtained from the following sources. HA-Akt in pSG5 was
kindly provided by Dr. David Stokoe (University of California at San
Francisco) and has been described in Ref. 9. pcDNA3.1/His B vector
was purchased from Invitrogen (Carlsbad, CA). Wild type SHIP cDNA
was obtained from Dr. G. Krystal (Terry Fox Laboratory, British
Columbia Cancer Research Center, Vancouver, Canada) and subcloned into
pcDNA3.1/His B. The SHIP D672A mutant was made using a mutagenic
primer and a kit from Stratagene (La Jolla, CA). This mutant is
catalytically inactive (37).
Immunoprecipitation and Immunoblotting--
Akt was
immunoprecipitated as described below; other proteins (p85,
phosphotyrosine, HA-Akt) were obtained by overnight incubation with
1-5 µg of purified antibodies and processed and analyzed for
immunoblotting as described (31). Akt migrates at 59 kDa, very close to
the heavy chain of the immunoprecipitating antibody (~55 kDa).
However, the two proteins could be distinguished by running in parallel
an anti-sheep immunoglobulin immunoprecipitate (to detect IgH) and A20
murine B cell whole cell lysate (to detect Akt). For CD19
immunodepletion, samples were incubated with 25 µg of anti-CD19
followed by protein A-Sepharose. The supernatants were found to contain
no detectable CD19 (not shown) and were then subjected to
immunoprecipitation with anti-phosphotyrosine. Western blots were
developed by chemiluminescence and quantitated using a Roche Molecular
Biochemicals Lumi-Imager with LumiAnalyst software supplied by the
manufacturer or by NIH Image software.
Akt in Vitro Kinase Assay--
Lysates of resting or
anti-Ig-activated B cells were immunoprecipitated with anti-Akt
antibody and protein G-Sepharose. After washing extensively in 1%
Triton lysis buffer, the pellets were subjected to in vitro
kinase assays using histone H2B, essentially as described elsewhere
(38). The reaction was stopped with 5× SDS sample buffer (0.6 M Tris, pH 6.8, 50% glycerol, 12% SDS), separated by
SDS-PAGE, and transferred to polyvinylidene difluoride membranes.
Phosphorylation of histone H2B was quantitated either by a Molecular
Dynamics Storm system using ImageQuant software or by liquid
scintillation counting. Values are expressed as -fold increase over the
nonstimulated samples after subtracting background. Filters were
stained with Coomassie Blue stain (0.2% Coomassie Blue in 30%
methanol, 10% glacial acetic acid, 60% water) to show equal amounts
of substrate.
Isolation of Membranes and PI 3-Kinase Assay--
B cells were
stimulated and lysed by sonication in TES (20 mM Tris, pH
7.4, 5 mM EDTA, 250 mM sucrose, 3 mM sodium orthovanadate, 1 µg each of aprotinin and
leupeptin, and 1 mM phenylmethyl sulfonyl fluoride). Cell
fractions were prepared as described previously (39), with minor
modifications. Cytosol was defined as the supernatant of the initial
13,000 × g, 20-min centrifugation. The resulting pellet was washed, resuspended a second time, and centrifuged for
1 h at 30,000 × g onto a 1.12 M
sucrose cushion. The material above the sucrose cushion contained
plasma membrane markers and no detectable cytoplasmic markers (not
shown) and was resuspended in TES buffer. The membrane fractions from
each sample were normalized for protein content, and PtdIns 3-kinase
was immunoprecipitated with 5 µl of rabbit polyclonal anti-p85
antiserum. The resulting immunoprecipitates were applied to a PtdIns
3-kinase assay (40). The PtdIns 3-phosphate spots were quantitated with
a Molecular Dynamics Storm system, and the amount of radioactivity in
each case is taken to represent the amount of PtdIns 3-kinase present.
Transient Transfections--
COS-7 fibroblasts were transfected
using a protocol kindly supplied by Dr. David Stokoe. In brief, cells
were seeded into 10-cm culture dishes and grown at 37 °C until they
were 60-70% confluent (1 or 2 days) and then incubated for 3.5 h
with premixed DNA and lipofectamine added in serum-free DMEM. 4 µg of
HA-Akt DNA was added with 20 µg of wild-type SHIP or SHIP D672A or
equal moles of the vector pcDNA3.1/His B. The cells were grown in
DMEM with 10% FBS for 48 h and then starved in serum-free DMEM
for 6 h prior to harvesting. Stimulation was done by the addition of 20% FBS for 10 min. The Akt assay was performed on anti-HA immunoprecipitates as described above.
Reduced Activation of the PtdIns 3,4,5-P3-responsive
Enzyme Akt under Negative Signaling Conditions--
We examined the
induction of Akt in the murine B cell line A20 pretreated with the
PtdIns 3-kinase inhibitor wortmannin or an equal volume of
Me2SO and then stimulated under positive or negative
signaling conditions using intact anti-Ig antibody in the presence or
absence, respectively, of the Fc Similar Association and Activation of PtdIns 3-Kinase with
Tyrosine-phosphorylated Proteins and with the Membrane Fraction under
Positive and Negative Signaling--
The reduced induction of Akt
could conceivably result from lower PtdIns 3-kinase activity, since it
has been demonstrated that PtdIns 3,4,5-P3 levels are much
lower under negative signaling (28) and the association of the p85
subunit of PtdIns 3-kinase with CD19 is reduced due to decreased
tyrosine phosphorylation of CD19 (21, 22).
To compare the activity of PtdIns 3-kinase under positive and negative
signaling, we measured the amount of PtdIns 3-kinase associated with
tyrosine-phosphorylated proteins in lysates of A20 B cells stimulated
under either condition. First, anti-phosphotyrosine immunoprecipitates
were analyzed by immunoblotting with antibody to the p85 subunit of
PtdIns 3-kinase. Results, shown in Fig. 3A, indicated that the amount
of p85 associated with the phosphotyrosine fraction was equivalent at
all time points examined under both signaling conditions. However, in
confirmation of the earlier reports (21, 22), we observed that the
amount of p85 associated with CD19 was reduced by approximately 60% in
B cells after stimulation with intact anti-Ig compared with stimulation
with F(ab')2 fragments (Fig. 3B). Nevertheless,
anti-phosphotyrosine immunoprecipitates from samples immunodepleted
with anti-CD19 still contained the p85 subunit (Fig. 3C),
indicating that p85 was associated with phosphotyrosine-containing
proteins other than CD19.
The above procedures measure the amount of the p85 regulatory subunit
associated with tyrosine-phosphorylated proteins. Conceivably, association of p85 and the catalytic p110 subunit might be reduced under negative signaling conditions, and thus measurements of p85
association may not reflect PtdIns 3-kinase activity. To test this
possibility, we measured PtdIns 3-kinase enzymatic activity in
anti-phosphotyrosine immunoprecipitates. These measurements (Fig.
3D) consistently revealed approximately 2-fold greater
PtdIns 3-kinase activity derived from B cells stimulated under negative signaling conditions.
Membrane translocation of PtdIns 3-kinase is perhaps most relevant to
the activation of the enzyme, since genetic manipulations of either
PtdIns 3-kinase subunit that induce membrane targeting stimulate distal
biochemical and biological activation events (23-26). To examine
membrane translocation of PtdIns 3-kinase, we generated membrane and
cytosolic fractions by ultracentrifugation of B cell lysates following
stimulation under positive or negative signaling conditions. PtdIns
3-kinase activity in anti-p85 immunoprecipitates from the membrane
fractions was expressed as a -fold increase over the activity in the
nonstimulated sample, as shown in Fig. 4.
The results revealed a transient 4-fold increase of PtdIns 3-kinase
activity in the plasma membrane upon stimulation with anti-Ig
antibodies, either in the presence or absence of Fc The Role of SHIP in the Negative Signaling-induced Reduction in Akt
Activation--
Previous experiments in our laboratory indicated that
Fc
To directly test the effect of SHIP on Akt in vivo, COS-7
fibroblasts were transfected with HA-tagged Akt and co-transfected with
excess wild-type SHIP or a catalytically deficient SHIP mutant (SHIP
D672A) or the empty vector (pcDNA3.1/ His B). After 40 h to
permit expression of the transfected genes, Akt activity was examined
in serum-starved cells upon serum stimulation, which potently activates
Akt (42, 43). The results, shown in Fig. 6, indicate that co-transfection of
wild-type SHIP reduces the basal level of Akt activity by 70% and the
serum-stimulated level by 64%, compared with the activity in the cells
co-transfected with vector alone. In contrast, co-transfection of
catalytically deficient SHIP caused a reduction of only 23% of
serum-stimulated Akt activity.
Finally, the activation of Akt under positive and negative signaling
was examined in wild-type and SHIP ( The serine/threonine kinase Akt operates distal to PtdIns 3-kinase
in growth factor receptor-stimulated fibroblasts (2, 23, 25, 44-46)
and acts to protect cells from apoptosis (47-50). While neither
induction of Akt activity nor its relationship to apoptosis has been
investigated in a B cell model, studies have indicated that negative
signaling conditions promote apoptotic cell death in B cells (51,
52).
Results in Figs. 1 and 2 indicate that sIg stimulation induces Akt, and
this induction is completely blocked by the PtdIns 3-kinase inhibitor
wortmannin. Since PtdIns 3-kinase is known to be recruited and
activated under after sIg triggering (18, 19), Akt activation in this
system, like others, is downstream of PtdIns 3-kinase. It is worthwhile
noting that the kinetics of Akt activation (Fig. 2) closely parallel
the reported formation of PtdIns 3,4-P2 and PtdIns
3,4,5-P3 synthesis, which accumulate to high levels in less
than 1 min after sIg triggering (19, 28).
While Akt induction depends on activation of PtdIns 3-kinase and the
formation of its 3-phosphoinositide products, it is unclear which
3-phosphoinositide stimulates Akt. Some findings indicate that Akt
activity is directly stimulated by the SHIP product, PtdIns
3,4-P2 (12), while others show that Akt activity is
indirectly induced by the SHIP substrate, PtdIns 3,4,5-P3,
via PtdIns 3,4,5-P3-dependent kinase-1 (9, 10).
Our model of positive and negative signaling in B cells may be ideal to
resolve this issue, since the influence of SHIP can be turned on or off
by small changes in the stimulating reagent (29) or by known genetic
manipulations of the SHIP docking protein Fc The decreased Akt activation under negative signaling does not appear
to be due to decreased PtdIns 3-kinase activity, since equivalent
amounts of the enzyme are translocated to the membrane and associated
with the phosphotyrosine fraction under both signaling conditions. This
finding, together with the observation that p85 is present in
CD19-depleted lysates (Fig. 3), suggests that PtdIns 3-kinase is
translocated to the membrane by other tyrosine-phosphorylated membrane
proteins in addition to CD19, such that the reduced association of the
enzyme with CD19 under negative signaling (21, 22) does not
significantly affect the amount of PtdIns 3-kinase at the membrane. The
equivalent stimulation of PtdIns 3-kinase activity but deficient
generation of PtdIns 3,4,5-P3 (reported in Ref. 28) under
negative signaling conditions suggests a role for SHIP-mediated
consumption of nascent PtdIns 3,4,5-P3. It has been shown
that the induction of Btk, a protein-tyrosine kinase dependent on
formation of 3-phosphoinositides (54), is blocked by overexpression of
SHIP (28).
The reduction but not elimination of Akt activity under negative
signaling conditions may be due to the reported ability of PtdIns
3,4-P2 to directly activate the enzyme (11, 12). Negative signaling conditions would promote a large increase in PtdIns 3,4-P2 through the combined action of PtdIns 3-kinase and
SHIP. The PtdIns 3,4-P2 would then cause Akt activation,
albeit with less efficiency than PtdIns 3,4,5-P3, which
also activates Akt indirectly through PtdIns
3,4,5-P3-dependent kinase-1. An additional possibility is that PtdIns 3,4-P2 promotes inefficient
translocation of Akt, since it has less affinity than PtdIns
3,4,5-P3 for the pleckstrin homology domain of Akt (8).
Thus, reduced Akt membrane translocation would result in reduced Akt
activation under negative signaling conditions. We conclude that in the
B cell model, both PtdIns 3,4-P2 and PtdIns
3,4,5-P3 are capable of Akt activation but that PtdIns
3,4,5-P3 is a more potent inducer and hence SHIP behaves as
a negative regulator for Akt induction.
These findings in antigen receptor-stimulated B lymphocytes are similar
to earlier reports of monocyte colony-stimulating factor-induced FD-fms
monocytoid cells (33) and of interleukin-3-induced DA-ER cells (55),
which undergo accelerated apoptosis upon transfection with
wild-type but not catalytic deficient SHIP. Likewise, recent experiments investigating survival or apoptosis in cells stimulated with granulocyte colony-stimulating factor revealed SHIP recruitment and tyrosine phosphorylation to the granulocyte colony-stimulating factor receptor (56). Cells expressing granulocyte colony-stimulating factor receptor mutants lacking the fourth cytoplasmic tyrosine failed
to recruit and phosphorylate SHIP upon granulocyte colony-stimulating factor stimulation, and the cells displayed a 3-fold increase in cell
numbers, compared with those expressing the wild-type receptor. These
findings suggest that SHIP recruitment and phosphorylation in response
to cytokine stimulation tempers the biological responses to cytokines.
There is no molecular or biochemical explanation for the enhanced
apoptotic cell death seen in these cases. Based on our findings
reported here, we propose that SHIP recruitment and phosphorylation in
response to cytokines leads to a reduction in Akt activation, thereby
reducing cell survival.
RIIb. Co-clustering of sIg-Fc
RIIb
transmits a dominant negative signal and is associated with reduced
accumulation of the PtdIns 3-kinase product phosphatidylinositol
3,4,5-trisphosphate (PtdIns 3,4,5-P3), known to be a
potent activator of Akt. PtdIns 3-kinase is activated to the same
extent with and without Fc
RIIb co-ligation, indicating conditions
supporting the generation of PtdIns 3,4,5-P3. We
hypothesized that the decreased Akt activity arises from the
consumption of PtdIns 3,4,5-P3 by the
inositol-5-phosphatase Src homology 2-containing inositol 5-phosphatase
(SHIP), which has been shown by us to be tyrosine-phosphorylated
and associated with Fc
RIIb when the latter is co-ligated. In direct
support of this hypothesis, we report here that Akt induction is
greatly reduced in fibroblasts expressing catalytically active but not inactive SHIP. Likewise, the reduction in Akt activity upon
sIg-Fc
RIIb co-clustering is absent from avian B cells lacking
expression of SHIP. These findings indicate that SHIP acts as a
negative regulator of Akt activation.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and Ig
, which are associated with sIg.
Immunoreceptor tyrosine-based activation motif phosphorylation promotes
recruitment of proteins and enzymes via their SH2 domains.
RIIb with sIg provokes an dominant-inhibitory or
negative signal that abolishes the proliferative effect of sIg
stimulation. Co-clustering of sIg and Fc
RIIb (negative signaling) probably occurs in vivo in the later stages of the immune
response, when antigen is coated with specific IgG (reviewed in Ref.
27). Such complexes engage both sIg and Fc
RIIb on naïve B
cells by binding uncoated antigenic epitopes through sIg and the Fc
portions of the coating antibodies through Fc
RIIb.
RIIb (31). SHIP recognizes only 3-phosphoinositides (including PtdIns 3,4,5-P3) and hydrolyzes at the D5-position of the
inositol ring (32, 33). How these various events regulate SHIP activity is not clear; nevertheless, there is evidence that the catalytic activity of this enzyme is enhanced under conditions of negative signaling in B cells (28).
RIIb. Furthermore, COS-7 fibroblasts
co-transfected with Akt and wild-type SHIP exhibited reduced Akt
induction upon serum stimulation, whereas those co-transfected with Akt
and catalytically deficient SHIP did not. Last, the avian B cell line
DT40 stably transfected with murine Fc
RII exhibited a similar
decrease in Akt induction upon sIg-Fc
RII co-clustering, and this
effect was absent in the SHIP (
/
) DT40 derivative, previously
described (34). The evidence provided here suggests a model of Akt
regulation whereby both PtdIns 3,4-P2 and PtdIns 3,4,5-P3 contribute to Akt activation, PtdIns
3,4,5-P3 being more potent in this regard. SHIP negatively
regulates Akt by reducing the available PtdIns
3,4,5-P3.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RIIb-deficient derivative IIA1.6 were
maintained at 37 °C in RPMI with 10% fetal bovine serum (FBS; both
from Life Technologies, Inc.). COS-7 fibroblasts were grown in DMEM
with 10% FBS (both from Life Technologies, Inc.). Wild-type and SHIP
(
/
) DT40 chicken B cells stably transfected with murine Fc
RIIb
were kindly provided by Dr. Jeffrey V. Ravetch (Rockefeller University,
New York) and have been described (34).
RII/III monoclonal antibody
2.4G2 to block binding to Fc
RIIb or for 10 min at 37 °C with 100 nM wortmannin (a concentration known to specifically
inhibit PtdIns 3-kinase (36)) or with an equal volume of the wortmannin
solvent, Me2SO. For pervanadate stimulation, a solution of
1:1:48 of 100 nM activated sodium orthovanadate:30%
H2O2:deionized water was left at room temperature for 20 min; 5% by volume of this solution was added to
cells at 37 °C for 5 min.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RIIb-blocking monoclonal antibody
2.4G2, as described earlier (29, 31, 35, 41). Anti-Akt
immunoprecipitates were subjected to an in vitro kinase
assay with histone H2B as an in vitro substrate, as
described earlier (38). As a positive control, B cells were stimulated with pervanadate, previously shown to induce Akt activation through induction of PtdIns 3-kinase (42, 43). The results (Fig.
1, A and B)
demonstrate potent Akt induction by pervanadate (lane 6) and positive signaling conditions of sIg triggering alone
(lane 2). Akt induction under positive signaling
conditions was blocked by wortmannin (lane 5),
indicating that, as in other systems, stimulation of Akt by sIg is
dependent on PtdIns 3-kinase activity. However, the induction of Akt by
stimulation with intact anti-Ig (lane 3) was 50%
of that triggered by sIg stimulation alone. In a total of four similar
experiments, Akt activity under negative signaling was 40.1 ± 4.4% less than Akt activity under positive signaling. We examined the
kinetics of Akt induction in lysates derived from A20 B cells activated
under positive or negative signaling conditions, using
F(ab')2 or intact anti-Ig antibodies. As shown in Fig.
2, A and B, the
induction of Akt is rapid, peaking less than 1 min after stimulation.
However, at all time points, Akt induction under negative signaling
conditions was reduced to about half the levels seen under positive
signaling conditions.
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Fig. 1.
Akt activation by B cell antigen receptor
stimulation is downstream of PtdIns 3-kinase and reduced under negative
signaling. 5 × 106 A20 B cells were stimulated
with the indicated agents, and anti-Akt immunoprecipitates were assayed
for Akt activity. A, autoradiogram of a representative
experiment; the position of the Akt in vitro substrate,
histone H2B, is shown. B, quantitation of the same
experiment. Numbers above the bars
represent -fold increase over the activity in the unstimulated sample.
NS, no stimulus; I + 2.4G2, preincubated with
2.4G2 and stimulated with intact anti-Ig for 1 min; I,
stimulated for 1 min with intact anti-Ig; 2.4G2, incubated
with 2.4G2 alone; I + 2.4G2 + Wt, preincubated with 100 nM wortmannin and then treated exactly as in
lane 2; Pdt, stimulated with
pervanadate as described under "Experimental Procedures";
NSIg, treated as in lane 2 and
immunoprecipitated with normal sheep immunoglobulin. These results are
representative of four separate experiments.
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Fig. 2.
Kinetics of Akt activation under positive and
negative signaling conditions. Anti-Akt immunoprecipitates from
5 × 106 A20 B cells treated with either
F(ab')2 or intact rabbit anti-mouse Ig for the indicated
times were assayed for Akt activity using histone H2B as a substrate.
In A, the upper image shows the
autoradiograph, and the lower image shows a
Coomassie Blue stain; the position of histone H2B is shown in both. In
B, the labeled histone H2B bands were quantitated and are
graphed according to the time and condition of stimulation. These
results are representative of three separate experiments.
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Fig. 3.
Association of PtdIns 3-kinase with
tyrosine-phosphorylated proteins under positive and negative
signaling. A, amount of p85 in anti-phosphotyrosine
immunoprecipitates under positive and negative signaling. Lysates from
A20 B cells, resting (NS) or stimulated with
F(ab')2 or intact rabbit anti-mouse IgG for the indicated
times were immunoprecipitated with anti-phosphotyrosine antibody, and
the samples were probed with anti-p85 antibody after SDS-PAGE
separation and transfer to nitrocellulose. The position of p85 is
shown; -fold increase, shown below each lane, was
calculated using NIH Image software. These results are representative
of four separate experiments. B, amount of p85 associated
with CD19 under positive and negative signaling. A20 B cells were left
unstimulated (NS) or stimulated with F(ab')2
(F) or intact (I) rabbit anti-mouse IgG. Samples
were lysed and immunoprecipitated with anti-CD19 antibody. After
SDS-PAGE and transfer to nitrocellulose, the samples were probed with
anti-p85 antibody. NRS, immunoprecipitated with nonspecific
immunoglobulin; lysate, whole lysate of resting B cells. The
position of p85 is indicated, and quantitation as -fold increase is
shown below each lane. These results are
representative of four separate experiments. C, presence of
p85 in CD-19-depleted phosphotyrosine fraction under positive and
negative signaling. A20 B cells were stimulated as above, lysed, and
immunodepleted with 25 mg of anti-CD19 followed by Protein G-agarose
beads. The resulting supernatants were immunoprecipitated with
anti-phosphotyrosine antibody, and samples were analyzed by SDS-PAGE
and transfer to nitrocellulose and probed with anti-p85 antibody.
NS, nonstimulated; F, stimulated with
F(ab')2 anti-Ig; I, stimulated with intact
anti-Ig; lysate, A20 whole cell lysate. The position of p85
is indicated, and quantitation as -fold increase is shown
below each lane. These results are representative
of two similar experiments. D, amount of PtdIns 3-kinase activity
associated with the phosphotyrosine fraction under positive and
negative signaling. 4 × 106 A20 B cells were
stimulated for the indicated times with either F(ab')2
(open symbols) or intact (closed
symbols) anti-Ig, lysed, and immunoprecipitated with
anti-phosphotyrosine antibodies. The PtdIns 3-kinase activity in the
immunoprecipitates was assayed using phosphatidylinositol and
[ -32P]ATP as a substrate; the reaction products were
analyzed by thin layer chromatography. The radioactivity of the PtdIns
3-phosphate product was quantitated with a Storm imager, and expressed
as a multiple of the amount in the nonstimulated sample. The averages
of two duplicate sets are plotted; error bars are
plus or minus one S.D. These results are representative of three
similar experiments.
RIIb-blocking 2.4G2 after 1 min of stimulation and a decline to basal levels at 3 min
of stimulation. Thus, PtdIns 3-kinase translocates to the B cell plasma
membrane after stimulation under either positive or negative signaling
condition and does so despite the reduced phosphorylation and
association to CD19. Together, these findings indicate that by these
definitions PtdIns 3-kinase activation does not appear to be
deficient under negative signaling in B cells. Therefore, negative
signaling conditions are probably suitable for the formation of PtdIns
3,4,5-P3, and the reported reduction (28) of PtdIns
3,4,5-P3 upon exposure of B cells to negative signaling
conditions is probably due to SHIP recruitment.
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Fig. 4.
PtdIns 3-kinase activity in membrane
fractions of B cells under positive and negative signaling.
18-25 × 106 A20 B cells were stimulated with 20 µg/ml rabbit anti-mouse Ig, either with or without preincubation with
2.4G2 blocking anti-Fc RIIb antibodies, as indicated. The cells were
lysed by sonication, and the membrane and cytosol fractions were
isolated by ultracentrifugation. PtdIns 3-kinase was immunoprecipitated
with anti-p85 antibodies, and the immunoprecipitates were assayed for
PtdIns 3-kinase activity using phosphatidylinositol and
[
-32P]ATP as substrate. The radioactivity of the
product phosphatidylinositol 3-phosphate (PtdIns 3-P) was
measured with a Molecular Dynamics Storm imager. C, cytosol
fraction; PM, plasma membrane fraction. Amounts of
radioactive PtdIns 3-P in the plasma membrane fractions relative to the
nonstimulated sample are shown above the respective
lanes. The results are representative of two separate
experiments.
RIIb-deficient B cells are incapable of promoting SHIP tyrosine
phosphorylation (31). We examined the extent of Akt stimulation using
the same B cell lines to more rigorously test the role of SHIP in
blocking Akt induction under negative signaling conditions. Since SHIP tyrosine phosphorylation and/or Fc
RIIb recruitment of SHIP is associated with reduced PtdIns 3,4,5-P3, we predicted that
the Fc
RIIb-deficient B cells would not display the reduction in Akt activity after exposure to negative signaling conditions. Accordingly, Akt immunoprecipitates derived from resting or activated A20 B cells
(Fc
RIIb+) or from the A20 derivative IIA1.6
(Fc
RIIb
) were measured for Akt activity as described
above. Consistent with the notion that SHIP negatively influences Akt
activation, Fc
RIIb+ B cells (Fig.
5) displayed reduced Akt activity when
stimulated under negative signaling conditions (57.4 ± 4.7% that
under positive signaling in two similar experiments). In contrast, the
Fc
RIIb- B cells displayed similar induction of Akt
activity under both signaling conditions; in the same two experiments,
activity under negative signaling was 114.6 ± 27.4% that under
positive signaling.
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Fig. 5.
Akt activation in
Fc RIIb-negative IIA1.6 cells is similar under
positive and negative signaling. A, 1 × 107 A20 (Fc
RIIb+) or IIA1.6
(Fc
RIIb
) B cells were treated with intact or
F(ab')2 fragments of anti-Ig for 60 s. Anti-Akt
immunoprecipitates were assayed for Akt activity by in vitro
phosphorylation of histone H2B. The autoradiograph of a representative
experiment is shown; the position of the histone H2B substrate band is
indicated. The sample in the last lane on the
right labeled Control was derived from A20 B
cells stimulated with F(ab')2 anti-Ig and
immunoprecipitated with normal sheep immunoglobulin. These results are
representative of two separate experiments. B, anti-Akt
immunoblot of the same membrane; the 59-kDa Akt band migrates slightly
slower than the 55-kDa heavy chain of anti-Akt (IgH), shown clearly in
the normal sheep immunoglobulin lane (Control).
Lanes in the Akt immunoblot are aligned with the
corresponding lanes in the Akt autoradiograph.
A20 and IIA1.6 WCL, whole lysate from 2 × 106 nonstimulated A20 or IIA1.6 B cells, used to indicate
the position of Akt relative to IgH in the Control
lane. C, the phosphorylation of histone H2B in a
similar experiment was quantitated and graphed according to the
cellular source and condition of stimulation.
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Fig. 6.
Serum-stimulated Akt activity is reduced by
transiently co-transfected wild-type SHIP. COS-7 fibroblasts were
transfected with lipofectamine alone (Mock) or combinations
of the following: HA-tagged wild-type Akt (HA-Akt),
pcDNA3.1/His B (pcDNA); wild-type SHIP (wt
SHIP); and catalytically deficient SHIP (SHIP D672A),
as indicated above the lanes. Cells were either
stimulated with 20% FBS (+serum) or not (NS),
and anti-HA immunoprecipitates were assayed for activity by in
vitro phosphorylation of histone H2B. A, autoradiogram
of radioactive histone H2B bands. B, quantitation of histone
phosphorylation in A. The radioactivity was quantitated by
cutting the H2B band from the gel and measured by liquid scintillation
counting. These results are representative of two separate
experiments.
/
) DT40 chicken B cells
transfected with murine Fc
RIIb and described earlier (34). The cells
were stimulated with the mouse anti-chicken IgM monoclonal M1 (IgG2b
isotype) in the presence or absence of Fc
RII-blocking antibody
2.4G2. As shown in Fig. 7, the activation of Akt in the wild-type DT40 transfectants is inhibited under negative
signaling, whereas it is actually slightly enhanced under negative
signaling in the SHIP (
/
) B cells.
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Fig. 7.
Akt activation is not depressed under
negative signaling in SHIP ( /
) DT40 cells. 5 × 106 wild-type or SHIP (
/
) DT40 chicken B cells stably
transfected with murine Fc
RIIb were stimulated for 5 min with the
mouse anti-chicken Ig monoclonal antibody M1 either without (M1) or
with (2.4G2 + M1) preincubation with 2.4G2 for 10 min, as described
under "Experimental Procedures." The cells were lysed, Akt was
immunoprecipitated, and Akt activity was assayed as before. Results are
expressed as -fold increase over the nonstimulated samples and are
representative of two similar experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RIIb (31). Our findings
demonstrate that Akt induction in B cells inversely correlates with
SHIP induction, in that positive signaling conditions are more
efficacious in the stimulation of Akt than negative signaling
conditions. Furthermore, we found that the reduction in Akt activation
under negative signaling conditions required expression of Fc
RIIb,
since Fc
RIIb-deficient A20 derivative IIA1.6 responds equally upon
stimulation with F(ab')2 or intact anti-Ig reagents (Fig.
5). Last, we specifically tested the effect of SHIP on Akt in
transiently co-transfected COS-7 cells. Transfection of Akt with
wild-type SHIP inhibited Akt activation, whereas catalytically inactive
D672ASHIP did not. These findings directly indicate that SHIP
5-phosphatase activity has a negative effect on Akt induction and
suggest that the reduced Akt activity seen after sIg-Fc
RIIb
co-clustering is due to the recruitment of SHIP to Fc
RIIb (described
in Refs. 30 and 31). The observations in the wild type and SHIP (
/
)
DT40 Fc
RII transfectants (Fig. 7) demonstrate that in the absence of
SHIP, the inhibition of Akt activation under negative signaling is
relieved, confirming that SHIP is indeed responsible for
down-regulating Akt activation under negative signaling.
Similar results of Akt suppression have been recently reported in
studies of the tumor suppressor gene and inositol polyphosphate
phosphatase, PTEN (53).
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ACKNOWLEDGEMENTS |
---|
We thank Dr. David Stokoe of the University of California at San Francisco for the gift of HA-Akt DNA and Dr. Clark Anderson of Ohio State University for many helpful suggestions and discussions.
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Note Added in Proof |
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While this manuscript was in review, two
groups (Aman Lamkin, T. D., Okada, H., Kurosaki, T. and Ravichandran,
S. (1998) J. Biol. Chem. 273, 33922-33928 and Gupta
Scharenberg, A. M., Fruman, D. A., Cantley, L. C., Kinet, J.-P., and
Long, E. O. (1999) J. Biol. Chem. 274, 7489-7494)
independently reported Akt activation in B cells under sIg triggering
and its partial inhibition under co-ligation of sIg with FRIIb.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health (NIH) Grants CA64268 and AI41447 and by NCI, NIH, Grant P30CA16058.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.
Present address: Dept. of Internal Medicine, 2 East Means Hall,
1654 Upham Dr., Columbus OH 43210.
§ Present address: Division of Pulmonary and Critical Care Medicine, N32S Means Hall, Columbus, OH 43210.
¶ A scholar of the Leukemia Society of America. To whom correspondence should be addressed: Dept. of Microbiology, Ohio State University, 484 W. 12th. Ave., Columbus, OH 43210. Tel.: 614-292-5394; Fax: 614-292-8120; E-mail: coggeshall.1{at}osu.edu.
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ABBREVIATIONS |
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The abbreviations used are:
PtdIns 3-kinase, phosphoinositide 3-kinase;
SH2, Src homology 2;
PtdIns
3, 4-P2, phosphatidylinositol-3,4- bisphosphate;
PtdIns
3, 4,5-P3, phosphatidylinositol 3,4,5-trisphosphate;
sIg, surface immunoglobulin;
FcRIIb, IIb isoform of the B cell
low-affinity IgG Fc receptor;
SHIP, SH2-containing
inositol-5-phosphatase;
FBS, fetal bovine serum;
DMEM, Dulbecco's
modified Eagle's medium;
HA, peptide fragment from hemagglutinin;
IgH, immunoglobulin heavy chain;
PAGE, polyacrylamide gel
electrophoresis.
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REFERENCES |
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