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INTRODUCTION |
Coengagement of Fc
RIIb1 with the B cell receptor
(BCR)1 by an immune complex
consisting of antigen and a specific antibody provides a feedback
mechanism for the down-regulation of B cell activation (1, 2). A
distinct effect of BCR/Fc
RIIb1 coligation is the loss of sustained
calcium influx and a selective reduction in the tyrosine
phosphorylation of certain proteins (3-7). The molecular events
responsible for this phenotype are not clearly understood.
Cocross-linking of BCR with Fc
RIIb1 results in recruitment of SHIP
(SH2 domain-containing inositol-polyphosphate
5'-phosphatase) to the immunoreceptor tyrosine-based
inhibition motif present in the cytoplasmic tail of Fc
RIIb1 (8). Two
approaches provided evidence for a functional requirement for SHIP
during Fc
RIIb1-mediated inhibitory signaling. Ectopic expression of
a chimeric KIR/Fc
RIIb1 protein, containing the extracellular and
transmembrane regions of KIR and the cytoplasmic tail of Fc
RIIb1, in
natural killer cells inhibited the lysis of target cells bearing the
HLA class I ligand for the extracellular KIR portion of the chimeric
receptor (9). Coexpression of a dominant-negative mutant of SHIP, but not the tyrosine phosphatase Shp-1, reverted the inhibitory signal delivered by Fc
RIIb1 in natural killer cells. Conversely,
dominant-negative Shp-1, but not SHIP, reverted the negative signal
mediated by KIR (9). The second approach made use of chicken DT40 B
cells in which the SHIP or Shp-1 genes had been
deleted by targeted homologous recombination (10).
Fc
RIIb1dependent inhibition was lost in the absence of SHIP,
but remained intact in cells lacking Shp-1 (10).
SHIP is a 145-kDa cytosolic protein that contains a single SH2 domain,
a catalytic region that bears significant homology to inositol
5'-phosphatases, and several binding sites for other signaling proteins
in its C-terminal region (11-13). SHIP interacts with Shc (14), which
couples proximal signaling to the Grb2/Sos/Ras activation pathway. SHIP
tyrosine phosphorylation and association with Shc increases upon
BCR/Fc
RIIb1 coligation (14). It was proposed that SHIP inhibits the
BCR activation signal by competing with Grb2 for binding to Shc,
thereby breaking the Ras signaling pathway (15).
BCR ligation leads to phosphorylation of the tyrosines at positions 484 and 515 in CD19, which then recruit and activate phosphoinositide 3-kinase (PI3K) (16, 17). Coligation of BCR with Fc
RIIb1 leads to
initial phosphorylation of CD19, followed by its rapid dephosphorylation (6, 7). One model proposes that a tyrosine phosphatase, such as Shp-1, dephosphorylates CD19, thereby blocking BCR-mediated activation by preventing PI3K activation (7). However,
CD19 dephosphorylation cannot always account for Fc
RIIb1-mediated negative signaling because such signaling operates in mast cells and
natural killer cells that do not express CD19 (8, 9). Furthermore,
dephosphorylation by Shp-1 is not required for Fc
RIIb1-mediated inhibition (8-10, 18).
In vitro, SHIP cleaves the 5'-phosphate from
phosphatidylinositol 3,4,5-trisphosphate (PIP3) and
inositol 1,3,4,5-tetrakisphosphate to give rise to phosphatidylinositol
3,4-bisphosphate (PIP2) and inositol 1,3,4-trisphosphate,
respectively (12). Unlike other 5'-phosphatases, SHIP preferentially
utilizes substrates that are phosphorylated on the D3 position of the
inositol ring, thereby linking its activity to the PI3K pathway.
Coengagement of Fc
RIIb1 with BCR leads to a drastic reduction of
cellular PIP3 at any time point of cross-linking as
detected by thin-layer chromatography (19). PIP3 may either
not be produced because of inactivation of PI3K, as proposed in the
CD19 dephosphorylation model, or be rapidly turned over. Recruitment of
SHIP by Fc
RIIb1 may serve to achieve a rapid conversion of
PIP3 to PIP2. Therefore, the possibility of a
physical association between SHIP and PI3K was investigated.
Coengagement of BCR with Fc
RIIb1 resulted in a tyrosine
phosphorylation-dependent recruitment of the p85 subunit of
PI3K to SHIP. This interaction is mediated by direct binding of the SH2
domain of PI3K to a signature motif in the C-terminal region of SHIP.
In addition, production of PIP2 and activation of Akt (also
called protein kinase B) were observed during BCR/Fc
RIIb1 coengagement.
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EXPERIMENTAL PROCEDURES |
Cells, Antibodies, and Other Reagents--
The B cell line A20
was maintained in RPMI 1640 medium with 10% fetal bovine serum, 2 mM glutamine, and 50 µM
-mercaptoethanol. NIH 3T3 cells were grown in Dulbecco's modified Eagle's medium with
10% calf serum and 2 mM glutamine. F(ab')2,
intact rabbit anti-mouse IgG, and peroxidase-conjugated goat
anti-rabbit IgG were purchased from Jackson ImmunoResearch
Laboratories, Inc. (West Grove, PA). Anti-PI3K p85 and p110 subunit
antibodies, unconjugated and biotin-conjugated anti-phosphotyrosine
4G10 antibodies, and a glutathione S-transferase (GST)
fusion protein of the PI3K p85 C-terminal SH2 domain were purchased
from Upstate Biotechnology, Inc. (Lake Placid, NY). Recombinant GST
protein and wortmannin were obtained from Sigma. Antibodies against Akt
and phospho-Akt (specific for phosphoserine 473) were from New England
Biolabs Inc. (Beverly, MA), and anti-Flag antibody (M2) was from
Eastman Kodak Co. A rabbit antiserum against the peptide sequence
VPACGVSSLNEMINP in the C-terminal region of SHIP was generated
(Research Genetics, Huntsville, AL). Peroxidase conjugates of
streptavidin and sheep anti-mouse IgG were from Amersham Pharmacia Biotech.
Deletion Mutants of SHIP and Recombinant Vaccinia Virus
Production--
Different deletion mutants of SHIP were obtained
from M. Lioubin and L. Rohrschneider (Fred Hutchinson Cancer
Research Center, Seattle, WA). The N-terminal SH2 domain is designated
as n, the catalytic domain as cat, and the C-terminal region following
the catalytic domain as c. Thus, the truncated mutants contain
different combinations of n, cat, and c regions, i.e. ncat,
nc, and catc. SHIPncat has amino acids 5-866; SHIPnc has a deletion in
the catalytic domain corresponding to amino acids 500-809 and a
replacement with amino acids EF arising from an EcoRI site
located at the site of deletion; and SHIPcatc contains amino acids
174-1190. All constructs have a Flag tag followed by a NotI
site at the amino terminus, which adds amino acids MGDYKDDDDKRPH onto
the amino terminus of each. The cDNAs were cloned into plasmid
pSCF4, a modified pSC65 plasmid (a gift of B. Moss), which contains a Kozak sequence and a Flag sequence followed by a multiple cloning site.
Recombinant vaccinia viruses were generated and amplified as described
(20).
A20 Cell Stimulation, Lysis, and Immunoprecipitation--
A20
cells (2 × 107) were washed twice with serum-free
Iscove's medium, resuspended, and incubated with F(ab')2
fragment or intact rabbit anti-mouse IgG for the indicated times at
37 °C. Stimulation was stopped by addition of cold Dulbecco's
phosphate-buffered saline (DPBS) and by rapid centrifugation of the
cells in a Picofuge (Stratagene, La Jolla, CA). The cells were lysed in
Tris-buffered saline, pH 8.0, containing 0.5% Triton X-100, 5 mM EDTA, 2 mM iodoacetamide, 5 µg/ml
pepstatin A, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium metavanadate, and 10 mM sodium
fluoride. Lysates were immunoprecipitated with the indicated antibodies and protein G-agarose beads.
Vaccinia Virus Infection of NIH 3T3 Cells and
Stimulation--
Recombinant vaccinia viruses encoding SHIPncat,
SHIPnc, or SHIPcatc were used to infect NIH 3T3 cells as described
(21). Briefly, NIH 3T3 cells (5 × 106) were infected
in suspension at 5 plaque-forming units/cell with the indicated
recombinant viruses in 2 ml of infection medium consisting of
Dulbecco's modified Eagle's medium, 2 mM glutamine, 10 mM HEPES, and 0.5% bovine serum albumin for 3.5 h at
37 °C. The cells were washed once with DPBS and incubated in 1 ml of DPBS or pervanadate solution (10 mM
H2O2 + 0.1 mM sodium metavanadate in DPBS) for 15 min at 37 °C. Subsequently, the cells were washed with cold DPBS and lysed, and the lysates were used for
immunoprecipitation as described above.
Synthetic Peptides and Agarose Beads--
Synthetic peptides
corresponding to amino acid sequences in the C-terminal region of SHIP
and in the PI3K-binding motif in CD19 (SLGSQS(pY)EDMRG) were purchased
from Quality Controlled Biochemicals (Hopkinton, MA). The SHIP peptides
used were EMINPNYIGMGP, EMINPN(pY)IGMGP, and EMINPN(pY)IGRGP. All
peptides were synthesized with an N-terminal biotin tag for coupling
with streptavidin-agarose beads. The peptides were dissolved at 0.1 mg/ml in PBS, pH 7.4, and incubated with streptavidin-agarose beads
(1-ml packed volume) overnight at 4 °C. The beads were washed four
times with PBS, pH 7.4, and suspended in 1 ml of PBS. Lysates of
unstimulated A20 cells were prepared as described above and incubated
with 100 µl of the above peptide-streptavidin-agarose conjugate
overnight at 4 °C. Beads were washed and boiled with SDS-PAGE sample
buffer, and the bound material was separated by SDS-PAGE and subjected to silver staining or immunoblotting.
Western and Far Western Blotting--
Immunoprecipitates were
separated on SDS-polyacrylamide gels and transferred to Immobilon P
membranes. The blots were probed with the indicated antibodies and
developed using the ECL detection reagents from Amersham Pharmacia
Biotech. In the far Western blotting procedure, membranes were overlaid
with 4 µg/ml recombinant GST protein or GST fused to the C-terminal
SH2 domain of PI3K p85 in phosphate-buffered saline containing 5%
bovine serum albumin, 0.1% Tween 20, and 1 mM
dithiothreitol. The membranes were washed with buffer without
dithiothreitol, reblocked, and incubated with rabbit polyclonal
anti-GST antibodies. After washing, the membranes were incubated with
peroxidase-conjugated goat anti-rabbit IgG and developed with ECL reagents.
Phosphoinositide Analysis--
A20 cells were labeled with
32P and stimulated as described above. This was followed by
extraction and deacylation of lipids and high performance liquid
chromatography (HPLC) analysis of the glycerophosphoinositol head
groups (22, 23).
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RESULTS |
Recruitment of PI3K to Tyrosine-phosphorylated SHIP upon Coligation
of the B Cell Receptor with Fc
RIIb1--
A20 cells were stimulated
with F(ab')2 or intact anti-IgG antibodies, and
immunoprecipitates of PI3K were resolved by SDS-PAGE and probed for
associated phosphotyrosine-containing proteins by Western blotting. A
distinct phosphoprotein band migrating at ~145 kDa
coimmunoprecipitated with PI3K as early as 5 s after stimulation
with intact antibody, but not with the F(ab')2 antibody (Fig. 1A). Probing with
anti-SHIP antiserum revealed the presence of SHIP at that position
(Fig. 1B). To test whether SHIP was directly associated with
PI3K or whether it was immunoprecipitated as part of the receptor
complex by the stimulating intact anti-Ig antibody, the lysates were
incubated with protein G-agarose beads alone prior to SDS-PAGE and
Western blotting with anti-SHIP antibodies. Under those conditions, no
145-kDa band was seen in the protein G precipitates (data not shown).
Cocross-linking of BCR with Fc
RIIb1 through an intact IgG also
enhanced the level of p85 in immunoprecipitates of SHIP (data not
shown). Thus, coligation of BCR with Fc
RIIb1 leads to the
recruitment of PI3K to SHIP.

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Fig. 1.
Association of PI3K with
tyrosine-phosphorylated SHIP upon coligation of the B cell receptor
with Fc RIIb1. A20 cells were unstimulated
(N) or stimulated for 5, 15, 30, 60, or 180 s with
F(ab')2 (F) or intact (I) anti-mouse
IgG (Anti-mIgG). Lysates were immunoprecipitated with
anti-p85 antibodies. Immunoprecipitates were fractionated on a 7.5%
SDS-polyacrylamide gel and Western-blotted with monoclonal
anti-phosphotyrosine antibody 4G10 (A) or rabbit polyclonal
anti-SHIP antiserum (B).
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Tyrosine phosphorylation of SHIP upon BCR/Fc
RIIb1 coligation exceeds
that obtained by cross-linking BCR alone (8, 14). Therefore, PI3K
association with SHIP observed during coligation could be mediated by
the binding of PI3K SH2 domains to phosphorylated tyrosine residues in
SHIP. The p85 subunit of PI3K has two SH2 domains, one each at the N
and C termini. The phosphotyrosine-containing motif recognized by these
two domains includes a pYXXM sequence for the C-terminal SH2
domain and a more stringent pY(I/V/L)XM sequence for the N-terminal SH2
domain (24). A GST fusion protein of the C-terminal SH2 domain was used
to test for direct binding to SHIP. A20 cells were stimulated with
F(ab')2 or intact antibodies, and either SHIP or
phosphotyrosine-containing proteins were immunoprecipitated from the
lysates. In both cases, the GST-p85 SH2 fusion protein bound in a far
Western blot to a protein of 145 kDa present in lysates of A20
cells stimulated with intact antibody (Fig.
2A, Expts. 1 and
2). Thus, p85 can bind directly to SHIP and to a tyrosine-phosphorylated protein that comigrated with SHIP on SDS-PAGE. GST alone did not bind SHIP under the same conditions (Fig.
2B), but it reacted with two nonspecific bands migrating at
~135 and 140 kDa in anti-SHIP immunoprecipitates of both unstimulated
and F(ab')2- and intact anti-Ig-stimulated cell lysates.
The presence of SHIP in the anti-phosphotyrosine and anti-SHIP
immunoprecipitates is shown in Fig. 2C. Increased tyrosine
phosphorylation of SHIP under conditions of BCR and Fc
RIIb1
coligation is evident. Direct binding of the PI3K SH2 domain to SHIP by
far Western blotting was also greater after receptor coligation than
after cross-linking BCR alone.

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Fig. 2.
The SH2 domain of PI3K directly binds to
tyrosine-phosphorylated SHIP upon Fc RIIb1
engagement. A20 cells were either unstimulated (N) or
stimulated with F(ab')2 (F) or intact
(I) anti-mouse IgG (Anti-mIgG) for 5 s, and
lysates were immunoprecipitated with anti-phosphotyrosine
( pY) or anti-SHIP ( SHIP) antibodies.
Immunoprecipitates (IP) were subjected to 7.5% SDS-PAGE and
far Western blotting with GST-PI3K p85 C-terminal SH2 domain fusion
protein (A) and recombinant GST protein (B) or to
Western blotting with anti-SHIP antiserum (C).
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The SH2 Domain of PI3K Binds to the C-terminal Region of
SHIP--
The SHIP cDNA was broadly divided into three regions
encoding the SH2 domain designated as n, the central catalytic region containing the sequences conserved in several 5'-phosphatases designated as cat, and the C-terminal region designated as c (which contains sites for interaction with the PTB domains of Shc (12, 25) and
multiple prolines that interact with Grb2 (11)). Deletion mutants
containing different combinations of these three domains (Fig.
3), namely ncat (~105 kDa), nc (~110
kDa), and catc (~120 kDa), were inserted into recombinant vaccinia
viruses and tested for their ability to bind PI3K.

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Fig. 3.
Schematic representation of different
deletion mutants of SHIP. See "Experimental Procedures" for
details.
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The deletion mutants were expressed in NIH 3T3 fibroblasts,
immunoprecipitated following a stimulation with pervanadate, and subjected to far Western blotting with the GST-p85 SH2 fusion protein.
All three mutants were tyrosine-phosphorylated upon pervanadate treatment (Fig. 4A), but only
the nc and catc mutants bound the SH2 domain of PI3K (Fig.
4B). As these two molecules share only the C-terminal
sequence of SHIP, the binding site must be in that region. The level of
expression of all three deletion mutants was comparable (Fig.
4C). The deletion mutants ncat and nc also coimmunoprecipitated a protein at 52 kDa upon pervanadate stimulation (Fig. 4A), which could be the Shc adaptor protein associated
with the N-terminal SH2 domain of SHIP.

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Fig. 4.
The SH2 domain of PI3K binds to the C
terminus of SHIP. NIH 3T3 cells were either uninfected
(first and second lanes) or infected
with 5 plaque-forming units/cell of Vac-SHIPncat (third and
fourth lanes), Vac-SHIPnc (fifth and
sixth lanes), or Vac-SHIPcatc (seventh
and eighth lanes) for 4 h at 37 °C. The
cells were washed once with DPBS, stimulated (+) or not ( ) with
pervanadate for 15 min at 37 °C, washed twice with DPBS, and lysed.
Immunoprecipitates of Flag-tagged SHIP deletion mutants (A
and B) and total lysates (C) were separated by
7.5% SDS-PAGE and subjected to Western blotting with
anti-phosphotyrosine antibody 4G10 (A) and to far Western
blotting with GST-PI3K SH2 domain fusion protein (B) and
anti-Flag antibody (C).
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A SHIP Phosphotyrosine Peptide Binds to the p85/p110 Subunits of
PI3K in Lysates of A20 Cells--
Amino acids 917-920 (YIGM) in the
C-terminal region of SHIP correspond to a perfect motif for binding the
N- and C-terminal SH2 domains of PI3K (24). The tyrosine at position
917 is phosphorylated upon BCR/Fc
RIIb1 coligation (25). Twelve-amino
acid-long peptides containing SHIP sequence 917-920 were synthesized
with either unphosphorylated (YIGM) or phosphorylated (pYIGM) Tyr-917.
Another phosphorylated peptide carrying the substitution M920R (pYIGR) and a phosphopeptide corresponding in sequence to the C-terminal PI3K-binding motif in CD19 (SLGSQSYEDMRG) were also synthesized for
negative and positive controls, respectively. All biotinylated peptides
were coupled to streptavidin-agarose beads and used to pull down
proteins in A20 lysates. Two proteins of ~85 and 110 kDa bound only
to the CD19 peptide and the pYIGM peptide and not to the
streptavidin-agarose beads alone or to pYIGR and unphosphorylated YIGM
peptides (Fig. 5A).
Immunoblotting with anti-PI3K antibodies revealed that these proteins
comigrated with the p85 (Fig. 5B) and p110 (Fig.
5C) subunits of PI3K, respectively. Thus, the in vitro data shown in Figs. 4 and 5 suggest a possible mechanism by
which SHIP binds PI3K upon B cell stimulation with intact anti-Ig antibodies or immune complexes.

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Fig. 5.
A phosphopeptide corresponding to 12 amino
acids in the C-terminal region of SHIP binds to the p85 and p110
subunits of PI3K in A20 lysates. A, A20 lysates
(~80 × 106 cells) were incubated with
streptavidin-agarose beads (lane 1) or with beads coupled to
CD19 phosphopeptide (lane 2), YIGM peptide (lane
3), pYIGM peptide (lane 4), or pYIGR peptide
(lane 5) overnight at 4 °C with end-to-end mixing. Bound
proteins were separated by 7.5% SDS-PAGE and silver-stained.
Lane M refers to molecular mass markers (in kilodaltons).
B and C, A20 lysates (~10 × 106 cells) were treated similarly as described for
A, but the separated proteins were sequentially
immunoblotted using anti-p85 (B) and anti-p110
(C) antibodies.
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Production of PIP2 and Akt Activation during Coligation
of BCR with Fc
RIIb1--
A potential outcome of the association of
SHIP with PI3K in A20 cells stimulated with intact anti-Ig is the
efficient production of PIP2, provided that SHIP and PI3K
retain their catalytic activities. To test this possibility, A20 cells
were stimulated with F(ab')2 or intact antibodies for
different times, and the total cellular levels of PIP2 and
PIP3 were determined using a sensitive HPLC assay.
Production of PIP2 upon Fc
RIIb1 coligation was
approximately two-thirds of that upon BCR stimulation alone (Fig.
6, upper panel). In contrast,
there was a marked inhibition of the PI3K product PIP3 at
early time points and complete loss at sustained time points (Fig. 6,
lower panel).

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Fig. 6.
Generation of PIP2 and
PIP3 upon BCR ligation and BCR coligation with
Fc RIIb1. A20 cells were either
unstimulated or stimulated with F(ab')2 ( ) or intact
anti-mouse IgG ( ) for 1, 2, 5, or 10 min. Lipids were extracted,
deacylated, and analyzed by HPLC. PIP2
(PI-3,4-P2; upper panel) and
PIP3 (PI-3,4,5-P3; lower
panel) levels are expressed as percentage of total
phosphoinositide. The PIP2 data are presented as an average
of n = 2 experiments.
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Activation of the serine/threonine kinase Akt requires binding of its
pleckstrin homology domain to membrane-bound phosphatidylinositides (26, 27). In particular, binding to PIP2 results in
activation of Akt in vitro (28, 29). Full Akt activation
requires sequential phosphorylation by two kinases, the second of which
phosphorylates serine 473 in Akt after binding to PIP3
(30). We used phosphorylation of serine 473 as an indicator of Akt
activation after signaling via BCR. A20 cells were stimulated with
F(ab')2 or intact antibodies for 2, 5, or 10 min, and
active Akt was immunoprecipitated and immunoblotted using antibodies
specific for phosphoserine 473. Fig.
7A reveals a large increase in
the activity of Akt, which was clearly diminished during coligation of
BCR with Fc
RIIb1. To test whether PI3K activity is required for Akt
activation upon B cell stimulation, two inhibitors, wortmannin and
LY294002, were used. At low concentrations, these inhibitors block PI3K
activity without affecting phosphoinositide 4-kinases (31). A20 cells were pretreated with wortmannin (Fig. 7B) or LY294002 (data
not shown) and then stimulated with F(ab')2 or intact
antibodies for 2 min. Both BCR- and BCR/Fc
RIIb1-induced Akt
activities were completely lost upon inhibition of PI3K (Fig.
7B). Thus, PI3K activity persists during BCR/Fc
RIIb1
coligation and is required for Akt activation.

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Fig. 7.
PI3K-dependent activation of the
Ser/Thr kinase Akt upon B cell stimulation. A, A20
cells were either unstimulated (N) or stimulated with
F(ab')2 (F) or intact (I) anti-mouse
IgG (Anti-mIgG) for 2, 5, or 10 min. Cell lysates were
immunoprecipitated with an anti-Ser(P)473 Akt antibody.
Immunoprecipitates were subjected to 7.5% SDS-PAGE and Western
blotting with the same antibody. B, A20 cells were either
untreated ( ) or pretreated with 100 nM wortmannin for 10 min (+) before stimulation with F(ab')2 (F) or
intact (I) anti-mouse IgG for 2 min. Immunoprecipitates were
analyzed as described for A. C, equal loading of protein was
confirmed by blotting the lysates with an anti-Akt antibody. The data
shown are representative of five different experiments.
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DISCUSSION |
Coengagement of BCR with Fc
RIIb1 results in a diminished
transient calcium flux and a loss of sustained calcium flux (3-5). The
sustained calcium flux in BCR-triggered B cells requires activation of
Btk, a member of the Tec kinase family that, in turn, activates phospholipase C
(19, 32-34). Activation of Btk is dependent on the
binding of its pleckstrin homology domain to PIP3 (19). A
noticeable effect of Fc
RIIb1 coligation is a drastic reduction of
PIP3, otherwise produced very rapidly upon BCR triggering
(19). The loss of PIP3 could be due to a reduced PI3K
activity and conversion to PIP2 by a non-rate-limiting
SHIP, to an increased SHIP activity, or to a complete loss of PI3K
activity. However, the production of the SHIP metabolite
PIP2 suggests that PI3K remains active during
Fc
RIIb1-mediated inhibition of the BCR activation signal. In
addition, the direct association of PI3K with SHIP, demonstrated here,
may serve to enhance the conversion of PIP3 to
PIP2. Far Western blotting with the C-terminal SH2 domain
of the p85 subunit of PI3K mapped the site of interaction to the
C-terminal region of SHIP. Synthetic phosphopeptides that included
sequences flanking tyrosine 917 of SHIP bound PI3K in cell lysates.
The inducible association of PI3K with tyrosine-phosphorylated SHIP
described here is different from the constitutive association of PI3K
with an unidentified PIP3 5-phosphatase activity in human platelets (35). The novel 5-phosphatase reported in that study is
distinct from SHIP since its catalytic activity in vitro was limited to the substrate PIP3.
Production of PIP2 during BCR/Fc
RIIb1 coligation was
consistently less than during BCR-mediated activation. This is probably due, in part, to a lower activity of PI3K and hence lower production of
the SHIP substrate PIP3. As CD19 is dephosphorylated
rapidly after BCR/Fc
RIIb1 coligation (6, 7), a major source of PI3K
activation is lost. Recruitment of PI3K by tyrosine-phosphorylated SHIP
may serve to compensate for this loss. However, SHIP is not absolutely
required for PI3K activation in avian DT40 B cells because a sustained
calcium signal was observed after BCR/Fc
RIIb1 coengagement in a
SHIP-negative DT40 mutant cell (10). It is also possible that the rapid
conversion of PIP3 by SHIP affects PI3K activation
directly, or indirectly through a diminished
PIP3-dependent activation of Ras (via Sos) (36,
37). To clearly address whether the catalytic activity of SHIP and/or
PI3K is responsible for the observed pattern of PIP3 and
PIP2 production, an inhibitor of SHIP phosphatase activity
would be necessary.
PIP2 and PIP3 control the activation of Akt by
recruiting the pleckstrin homology domains of Akt and of another
serine/threonine kinase that phosphorylates Akt (26-30). Akt delivers
an anti-apoptotic signal by phosphorylating the pro-apoptotic molecule
BAD, a member of the Bcl-2 protein family (38, 39). Our data show
residual activation of Akt during BCR/Fc
RIIb1 coligation as measured
by Akt phosphorylation on serine 473. This remaining Akt activation is
in contrast to the complete loss of the sustained calcium flux mediated
by the PIP3-dependent Tec kinase Btk during
BCR/Fc
RIIb1 coligation (19). The wortmannin sensitivity of Akt
activation strongly suggests that PI3K activity is also retained.
Although apoptosis of B cells after BCR/Fc
RIIb1 coligation can occur
and may even exceed that observed after BCR-mediated activation (40), the SHIP/PI3K/Akt pathway described here may lead to at least some
anti-apoptotic signal. An anti-apoptotic effect of SHIP after BCR/Fc
RIIb1 coligation has been suggested by the observation of
increased apoptosis of DT40 cells deficient in SHIP and of DT40 cells
expressing a mutant Fc
RIIb1 that fails to bind SHIP (10). A
pro-apoptotic mediator that binds to Fc
RIIb1 was proposed to explain
these observations (10). On the other hand, the reduced survival of
DT40 cells expressing the mutated Fc
RIIb1 that fails to recruit SHIP
may have been caused by the lack of SHIP-mediated PIP2
production and, in turn, by a reduced Akt-mediated survival signal.
In conclusion, this study demonstrates an association of the p85
subunit of PI3K with the inositol phosphatase SHIP in response to
coligation of BCR with the inhibitory receptor Fc
RIIb1. PI3K activity and PIP2 production were not abrogated by
Fc
RIIb1 ligation to BCR. We suggest that the physical association of
SHIP and PI3K may provide a novel mode of PI3K activation and an
enhanced conversion of PIP3 to PIP2.