From the G. W. Hooper Foundation and the Departments of Biochemistry and Biophysics and Microbiology and Immunology, University of California, San Francisco, California 94143
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The inositol phosphatase SHIP has been implicated
in signaling events downstream of a variety of receptors and is thought to play an inhibitory role in stimulated B cells. We and others have
reported that SHIP is rapidly tyrosine phosphorylated upon B cell
antigen receptor (BCR) cross-linking and forms a complex with the
adapter protein Shc. Here, we report that cross-linking of the BCR
induces association between Grb2 and SHIP as well as association
between Shc and SHIP. We made use of a Grb2-deficient B cell line to
demonstrate both in vitro and in vivo that Grb2 expression is required for the efficient association between Shc and
SHIP. The results indicate that SHIP, Shc, and Grb2 form a ternary
complex in stimulated B cells, with Grb2 stabilizing the interaction
between Shc and SHIP. The interactions between Shc, Grb2, and SHIP are
therefore analogous to the interactions between Shc, Grb2, and SOS. Shc
and Grb2 may help to localize SHIP to the cell membrane, regulating
SHIP's inhibitory function following BCR stimulation.
The appropriate development and functioning of B lymphocytes is
dependent upon signal transduction through the B cell antigen receptor
(BCR).1 BCR stimulation may
cause B cells to differentiate, proliferate, arrest growth, or undergo
apoptosis. The actual response of the cell to BCR cross-linking is
determined by its developmental state and the context in which the
signal is received (1). The biochemical changes evoked by stimulation
of the BCR include many of the well described events observed upon
growth factor stimulation of cells, such as the rapid activation of
tyrosine kinases and the subsequent activation of multiple signaling
enzymes including phospholipase C The Ras signaling pathway regulates cell growth and differentiation in
many systems (8), and has recently been found to be essential for
BCR-induced proliferation of mature B
cells.2 The mechanism by
which BCR cross-linking activates Ras is not established, although
tyrosine kinase inhibitors block Ras activation in B cells (9). Part of
this Ras activation is dependent upon protein kinase C (PKC) activity,
while some is PKC-independent (10). The adapter protein Shc, which has
been implicated in Ras activation in other systems, becomes tyrosine
phosphorylated following BCR stimulation and has been suggested to play
a role in Ras activation in B cells (11-13). Shc contains an SH2
domain and a PTB domain, allowing it to bind to proteins containing
phosphotyrosines (14). It also contains two tyrosines, residues 239 and
313, that when phosphorylated act as binding sites for the SH2 domain of a second adapter protein, Grb2 (15-17). Grb2 possesses two SH3 domains that permit it to bind to proline-rich sequences in the C
terminus of the Ras guanine nucleotide exchange factor SOS (18). Since
Shc translocates to the plasma membrane of B cells after BCR
stimulation (12), it is possible that it also recruits Grb2 and SOS to
the membrane and thus mediates Ras activation by bringing SOS near its
substrate Ras. Indeed, Shc forms a complex with both Grb2 and SOS after
BCR stimulation (12, 15).
We and others have observed that another signaling protein, variously
called "p145" or SHIP (for SH2-containing inositol phosphatase), also interacts with Shc after BCR cross-linking (12, 13, 19, 20). SHIP
is an inositol polyphosphate 5-phosphatase which selectively removes
the 5-phosphate from inositol 1,3,4,5-tetrakisphosphate and
phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) (21, 22), suggesting that it may act downstream of or counter to PI
3-kinase. Indeed, overexpression of SHIP can antagonize PI 3-kinase
function in Xenopus oocytes (23). Both
PI(3,4,5)P3 and phosphatidylinositol 3,4-bisphosphate
(PI(3,4)P2) levels increase transiently in B cells
stimulated through the BCR (24), consistent with the possibility that
SHIP may play a role in modulating the levels of these lipids.
PI(3,4,5)P3 and PI(3,4)P2 have been shown to
bind to the SH2 and pleckstrin homology domains of a variety of
proteins, regulating their enzymatic activity and presumably their
intracellular localization (25-28).
Overexpression of SHIP inhibits cellular proliferation and/or induces
apoptosis, suggesting that SHIP acts as a negative regulator of
hematopoietic cells (29, 30). In addition, SHIP has been implicated in
inhibitory signaling induced by Fc It has been proposed that SHIP may indirectly inhibit Ras activation by
competing with SOS for binding to Shc (20). To test this idea, we have
examined the mechanism by which Shc and SHIP interact in stimulated B
cells. We report that SHIP forms complexes with both Shc and Grb2 in
BCR-stimulated cells and that mutation of the Grb2-binding sites on Shc
perturbs both of these interactions. Significantly, SHIP and Shc
associate poorly in Grb2-deficient cells and this defect can be
complemented in vitro and in vivo by the addition
of Grb2. These results demonstrate that Grb2 is important for the
association between Shc and SHIP. We also found that a phosphopeptide
that binds to the SH2 domain of SHIP brings down the intact
SHIP·Shc·Grb2 complex, indicating that the SH2 domain of SHIP does
not directly bind Shc or Grb2. We therefore suggest that SHIP, Shc, and
Grb2 form a ternary complex similar to the Shc·Grb2·SOS complex in
B cells stimulated through the BCR.
Cell Lines and Culture Conditions--
The murine B cell line
WEHI-231 was originally obtained from Dr. L. Lanier (DNAX Research
Institute, Palo Alto, CA). The WEHI-231 cells transfected with HA-Shc
were previously described (15). The two BAL17 B cell lines were
originally obtained from Dr. R. Asofsky (National Institutes of
Health). It is not known when these cell lines diverged and BAL17.TR
lost Grb2 expression. The human B cell line Ramos was obtained from the
American Type Culture Collection (Rockville, MD), the murine pre-B cell
line 70Z/3 was a gift from Dr. C. Sibley (University of Washington,
Seattle, WA), the human B cell line J/Y was obtained from Dr. F. Brodsky (University of California, San Francisco, CA), and the murine B
cell line A20 was provided by Dr. R. Asofsky (National Institutes of
Health). All cell lines were cultured in RPMI 1640 medium supplemented with 5% fetal calf serum, 2 mM sodium pyruvate, 1 mM glutamine, and 50 µM 2-mercaptoethanol. To
induce maximal expression of transfected HA-Shc genes in WEHI-231,
cells were incubated for 48 h in media plus 5 mM
isopropyl- Cell Stimulation and Lysate Preparation--
B lymphocytes
(5 × 106 cells/ml in culture medium) were treated at
37 °C for 3 min with medium alone, with goat anti-mouse IgM
antibodies, rabbit anti-mouse IgM antibodies, or rabbit anti-mouse IgM
F(ab')2 fragments (all antibodies from Jackson
Immunological Research Laboratories, West Grove, PA) at 20 µg/ml
unless otherwise indicated. Alternatively, cells were stimulated with
150 µM pervanadate (a 50 mM pervanadate stock
was prepared by adding equal volumes of 100 mM
Na3VO4 and 2% H2O2 and
incubating at 25 °C for 30 min prior to use) for 4 min at 37 °C.
After stimulation, cell suspensions were diluted with cold
phosphate-buffered saline containing 1 mM
Na3VO4, pelleted by centrifugation, and
resuspended in ice-cold lysis buffer consisting of 20 mM
Tris (pH 8.0), 90 mM NaCl, 10% glycerol, 2 mM
EDTA, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride,
1 mM Na3VO4, and 1 mg/ml aprotinin.
Detergent-insoluble material was removed by centrifugation and the
protein concentration of the soluble fraction was determined using the
bicinchoninic acid assay (Pierce Chemical Co., Rockford, IL).
Immunoprecipitation and Immunoblotting--
Immunoprecipitation
and immunoblotting were carried out as described previously (38).
Lysates were immunoprecipitated with anti-Grb2 antiserum (Santa Cruz
Biotechnology, Santa Cruz, CA; used at 3 µg/40 × 106 cell equivalents), with anti-HA monoclonal antibody
(12CA5; from BAbCO, Richmond, CA; used at 2 µg/50 × 106 cell equivalents), with anti-Shc antibody
(affinity-purified rabbit antibody generated against glutathione
S-transferase (GST) fused to amino acids 359-473 of human
Shc; used at 1.5 µg/45 × 106 cell equivalents
(39)), or with anti-SHIP antibody (Santa Cruz Biotechnology; used at 6 µg/40 × 106 cell equivalents) and 40 µl of 50%
protein-A Sepharose solution (Zymed Laboratories Inc.
Laboratories, South San Francisco, CA) for 2 h at 4 °C. The
beads were then washed three times in lysis buffer, analyzed on
SDS-polyacrylamide gels, and transferred to nitrocellulose. Blots were
probed with anti-Grb2 monoclonal antibody (clone 81, Transduction
Laboratories, Lexington, KY), anti-Shc monoclonal antibody (PG-797,
Santa Cruz Biotechnology), anti-SHIP antiserum (kind gift of Drs. M. Lioubin and L. Rohrschneider; serum 5369 (29)), anti-Grap antiserum
(kind gift of Dr. G. Feng (49)), anti-Active MAPK antibody (Promega,
Madison, WI), anti-ERK2 antibody (C-14) (Santa Cruz Biotechnology),
anti- GST-Grb2 Fusion Proteins--
The GST-Grb2 and GST-Grb2R86K
expression vectors were generously provided by Dr. G. Koretzky,
University of Iowa (50). Unwanted alterations in the coding sequences
of these clones were converted to the published sequence via
site-directed mutagenesis. The fusion proteins were expressed in
Escherichia coli by standard methods (40). Expression of the
fusion proteins in bacteria was induced with 0.1 mM
isopropyl- Synthetic Peptides--
The ITIM peptide corresponds to amino
acids 303-315 of the murine Fc Vaccinia Virus--
Grb2 and Grb2R86K cDNAs (described
above) were cloned into the vaccinia expression plasmid pSC65 (gift of
Dr. B. Moss, National Institutes of Health, Bethesda, MD) and
recombinant viruses generated using standard techniques (41). BAL17.TR
B cells were infected at an multiplicity of infection of 20 and
harvested 24 h after infection, before cytopathic effects were
visible. Cells were stimulated and lysed as described above.
Association of the Inositol Polyphosphatase SHIP with the Adapter
Proteins Shc and Grb2 in Stimulated B Lymphocytes--
We and others
have previously noted that the inositol polyphosphate 5-phosphatase
SHIP forms complexes with the adapter protein Shc in hematopoietic
cells upon stimulation with antigen or cytokines (19-21, 29, 42). This
complex has been variously reported to include (42, 43) and exclude
(21) the adapter protein Grb2. To assess whether SHIP associates with
Grb2 as well as Shc after cross-linking of the BCR, we
immunoprecipitated SHIP, Shc, or Grb2 from unstimulated and stimulated
B cells and then probed immunoblots with anti-SHIP, -Shc, or -Grb2
antibodies. As is shown in Fig.
1A, when any one of these
three proteins was immunoprecipitated from stimulated cells, the other
two were also precipitated. Thus anti-SHIP immunoprecipitates contained
both Shc and Grb2, anti-Shc immunoprecipitates contained Grb2 and SHIP,
and anti-Grb2 immunoprecipitates contained SHIP and Shc. These
interactions were primarily stimulation dependent, although low levels
of association were seen in unstimulated cells.
It was not clear from these results whether these associations
represented the existence of separate Shc·Grb2, Shc·SHIP, and SHIP·Grb2 complexes or the existence of a ternary SHIP·Shc·Grb2 complex. To address this point, we examined whether depletion of Grb2
from stimulated cells also led to the depletion of the Shc·SHIP
complex. Grb2 was removed from stimulated B cell lysates by serial
immunoprecipitation, as was the tyrosine kinase Lyn as a control. Next,
Shc was immunoprecipitated from these depleted lysates and association
of SHIP and Grb2 assessed (Fig. 1B). Depletion of Grb2 led
to a roughly 2-fold decrease in the amount of Shc immunoprecipitated
from the lysate, indicating that at least half of the Shc in the
stimulated lysate was associated with Grb2. SHIP could barely be
detected in association with the Shc that did remain. SHIP therefore
associates preferentially with the population of Shc that is bound to
Grb2, indicating that SHIP, Shc, and Grb2 form a ternary complex upon
BCR stimulation.
The Grb2-binding Sites of Shc Are Required for Its Efficient
Association with SHIP--
It has previously been shown that Shc
contains two tyrosines that when phosphorylated act as binding sites
for the SH2 domain of Grb2 (15-17) and that the PTB domain of Shc
binds to phosphorylated tyrosines in the C terminus of SHIP (22, 30,
44, 45). In addition, SHIP contains several proline-rich regions in its C terminus that can bind to the SH3 domains of Grb2 in vitro
(22, 42). Therefore a SHIP·Shc·Grb2 complex could be stabilized by SH2, SH3, and/or PTB interactions in a manner analogous to the SOS·Shc·Grb2 complex (15, 17). The tyrosine phosphorylation of Shc
and SHIP that occurs after BCR cross-linking (11-13, 46) may trigger
complex formation. To examine the importance of phosphorylation of the
Grb2-binding sites for Shc association with SHIP, we examined the
interactions of SHIP with mutated Shc proteins in which two of the
three known tyrosine phosphorylation sites (15, 16, 47) had been
changed to phenylalanine. These experiments were performed in the
immature B cell line WEHI-231 which had been stably transfected with
wild type and mutant Shc proteins tagged with the influenza virus
hemagglutinin (HA) epitope (15). HA-Shc was immunoprecipitated from
unstimulated or BCR-stimulated cells and associated SHIP protein was
detected by immunoblotting with anti-SHIP antibodies. SHIP was readily
detected in association with wild type HA-Shc isolated from stimulated
cells (Fig. 2A, top panel).
Less SHIP was found associated with HA-Shc in which tyrosine 313 was
mutated to phenylalanine (HA-Shc Y313F), and little or no SHIP
associated with HA-Shc in which the other Grb2-binding site,
Tyr239, was mutated to phenylalanine (HA-Shc Y239F), or in
which both Grb2-binding sites were mutated (HA-Shc Y239F/Y313F). The
SHIP associated with Shc was also phosphorylated on tyrosine, as could be seen by reprobing these immunoblots with anti-phosphotyrosine antibodies (Fig. 2A, middle panel). Detection of associated
SHIP by this method appeared to be more sensitive, but otherwise gave similar results.
To confirm these results, the reciprocal experiment was performed, in
which SHIP was immunoprecipitated from unstimulated and stimulated
cells and Shc was detected by immunoblotting (Fig. 2B).
HA-Shc can be distinguished from endogenous Shc protein by its slower
migration on SDS-PAGE gels, due to the epitope tag addition. In
BCR-stimulated cells, SHIP associated well with wild type HA-Shc,
whereas mutation of Tyr239, Tyr313, or both led
to a dramatic decrease in the amount of HA-Shc associated with SHIP.
Similar amounts of endogenous Shc were associated with SHIP in each
cell line, providing an internal control. These results indicate that
the two Grb2-binding sites of Shc (15, 16) are both required for the
efficient formation of Shc·SHIP complexes, although a low level of
association occurs in the absence of these sites.
Requirement of Grb2 for Formation of SHIP·Shc
Complexes--
Since Grb2 co-immunoprecipitated with Shc and with SHIP
and mutation of the Grb2-binding sites on Shc decreased its ability to
form complexes with SHIP, it seemed likely that Grb2 helps hold the
Shc·SHIP complex together. An alternative explanation of the latter
results would be that the SH2 domain of SHIP binds directly to the
phosphorylated Tyr239 and/or Tyr313 regions of
Shc. One way to test these possibilities is to examine the interaction
between wild type Shc and SHIP in the absence of Grb2. This approach
was made possible by our serendipitous finding that a variant of the
mature B cell line BAL17, BAL17.TR, does not express Grb2. Expression
of the proteins SHIP, Shc, and Grb2 was examined in the B cell lines
BAL17 and BAL17.TR. Although both cell lines expressed equivalent
amounts of SHIP and Shc, Grb2 could not be detected in BAL17.TR either
by immunoblotting of whole cell lysates or immunoblotting of anti-Grb2
immunoprecipitates (Fig. 3A
and data not shown). By other criteria, these cell lines were similar
to one another. They responded similarly to BCR stimulation, as judged
by blotting whole cell lysates with anti-phosphotyrosine antibodies and
Ca2+ flux assays, and they expressed similar amounts of
other signaling proteins such as Syk and Fc
Another adapter protein called Grap may act similarly to Grb2, as they
are 59% identical, have highly related structures and can bind the
same proteins (48, 49). However, since the BAL17 and BAL17.TR cells do
not express detectable levels of Grap, it is unlikely to play a
significant role in these cells (Fig. 3B).
BAL17 and BAL17.TR cells were stimulated via the BCR and the amount of
SHIP in anti-Shc immunoprecipitations was examined. As expected, SHIP
was easily detected with both anti-SHIP antibodies and
anti-phosphotyrosine antibodies in Shc immunoprecipitates from
stimulated BAL17 (Grb2+) cells (Fig. 3C, top two
panels). In contrast, SHIP was not detected with anti-SHIP
antibodies in association with Shc in the stimulated BAL17.TR
(Grb2
The interpretation of these results is complicated by the poor tyrosine
phosphorylation of Shc in the BAL17.TR cells following BCR stimulation
(Fig. 3C, fourth panel; also visible by shift of Shc
mobility in third panel). In contrast, SHIP was tyrosine phosphorylated
to a similar extent in both the Grb2 Grb2 Strongly Enhances the Association between Shc and SHIP, Both
in Vitro and in Vivo--
To test more directly whether Grb2
stabilizes complexes of Shc and SHIP, we added recombinant, purified
Grb2 to lysates made from Grb2
To assess the importance of Grb2 for Shc/SHIP association in intact
cells, BAL17.TR cells were infected with recombinant vaccinia viruses
containing no insert (vector), wild type Grb2 (Grb2), or Grb2 with a
mutated SH2 domain (Grb2R86K) and association between Shc and SHIP was
examined. We found that expression of wild type Grb2, but not Grb2R86K,
caused a robust association between Shc and SHIP upon BCR stimulation
(Fig. 5A). This Shc/SHIP
association correlated with the association between Shc and Grb2.
Similar amounts of Grb2 and Grb2R86K were expressed in this experiment (Fig. 5B). Interestingly, the tyrosine phosphorylation of
Shc upon BCR stimulation was strikingly enhanced in the cells
expressing wild type Grb2 but not in cells expressing the SH2 domain
mutant Grb2R86K (Fig. 5C), perhaps because binding of the
Grb2 SH2 domain to Shc protected phosphorylated tyrosines 239 and 313 from intracellular phosphatases. These results indicate that Grb2
expression is essential for the formation of complexes between Shc and
SHIP.
Grb2 has been implicated in the activation of the Ras/MAPK cascade in a
number of systems (51). We therefore examined whether vaccinia
virus-mediated expression of Grb2 in BAL17.TR cells increased activation of MAPK in response to BCR cross-linking. To our surprise, we found that expression of either wild type Grb2 or Grb2R86K caused
little or no change in the extent of MAPK activation as judged by
immunoblotting whole cell lysates with antibodies that recognize only
phosphorylated, activated MAPK (Fig. 5D). Since a
substantial component of MAPK activation in BCR-stimulated B cells is
PKC-dependent (10), we also stimulated these cells in the
presence and absence of the PKC inhibitor compound 3. Even in compound
3-pretreated cells, we saw little or no difference in MAPK activation
in BAL17.TR cells infected with the different Grb2 viruses (data not shown).
Interactions of SHIP, Shc, and Grb2 with Synthetic
Peptides--
The above data strongly suggested that SHIP, Shc, and
Grb2 form a ternary complex upon B cell stimulation. To test this model further, we assessed the ability of synthetic peptides designed to
interact with the SH2, SH3, and PTB domains of Shc, Grb2, and SHIP to
precipitate the complex of these proteins. We first used a
phosphopeptide corresponding to the phosphorylated ITIM sequences of
the Fc
We performed similar experiments with peptides known to bind to Grb2
and Shc. A proline-rich peptide (VPV PPP VPP RRR PE), derived from the
C-terminal sequences of SOS and which binds to the SH3 domains of Grb2
(53) purified Grb2 from unstimulated and stimulated BAL17 lysates (Fig.
6, lanes 5-8). We found that a small amount of Shc
co-purified with Grb2 upon cell stimulation, probably due to binding of
the Grb2 SH2 domain to phosphorylated Tyr239 and
Tyr313 of Shc. A small amount of SHIP also co-purified with
Grb2 upon cell stimulation, perhaps reflecting a Grb2-independent
interaction between the Shc PTB domain and phosphorylated SHIP,
analogous to the low level of Shc/SHIP association seen in the absence
of Grb2 (Fig. 3C). In any case, most of the
Shc·Grb2·SHIP complex was not pulled down by this peptide,
indicating a relative lack of availability of the Grb2 SH3 domains in
this complex.
We next used a phosphopeptide (EMI NPN Y(p)IG MG) derived from the
C-terminal region of murine SHIP that was previously shown to bind the
PTB domain of Shc (30, 45) and that contains the consensus PTB-binding
motif NPxY(p). This peptide indeed affinity-purified Shc from BAL17 and
BAL17.TR cells (Fig. 6, lanes 9-12). More Shc was isolated
from unstimulated BAL17 cells than from stimulated BAL17 cells,
suggesting that in the stimulated cells the PTB domains of many Shc
proteins were not accessible for peptide binding. No such decrease in
association upon cell stimulation was seen in the Grb2-deficient
BAL17.TR cells. Interestingly, no SHIP or Grb2 was detected in
association with this peptide, indicating that the Shc PTB is occupied
in the Shc·Grb2·SHIP complex. Finally, we used a phosphopeptide
(LFD DPS Y(p)VN IQN) corresponding to a region of Shc known to bind
Grb2 and containing the consensus motif (Y(p)xNx) known to bind the
Grb2 SH2 domain (17). This peptide did indeed purify Grb2 from
unstimulated and stimulated BAL17 cell lysates (Fig. 6, lanes
13-16). Surprisingly, this peptide also precipitated Shc from
stimulated and unstimulated BAL17 and BAL17.TR cells, indicating that
Shc binds to this peptide in a Grb2-independent manner. Inspection of
this peptide's sequence revealed that it contains an isoleucine at the
Y(p) + 3 position, and thus also contains a consensus binding site for
the SH2 domain of Shc (54). Shc, as well as Grb2, may therefore bind
directly to this phosphopeptide. Shc association with the peptide
decreased upon cell stimulation in the BAL17 cells, suggesting that the SH2 domain of Shc, like the PTB domain, is less accessible to peptides
after stimulation of these cells. SHIP association with this peptide
was detected in the BAL17 cells but not in the BAL17.TR cells,
suggesting that SHIP was co-purified via its interaction with the SH3
domains of Grb2.
In related experiments, we used these synthetic peptides as competitive
inhibitors to disrupt Shc·Grb2·SHIP association in lysates made
from stimulated BAL17 cells. We found that the addition of the ITIM
phosphopeptide, which binds to the SH2 domain of SHIP, had little or no
effect on association between Shc, SHIP, and Grb2 (Fig.
7). This suggests that the SH2 domain
of SHIP is not involved in stabilization of this complex. In contrast,
the NPxY(p) peptide (that binds to the PTB domain of Shc), the Y(p)xNx
peptide (that binds to the SH2 domain of Grb2), and the proline-rich
peptide (that binds to the SH3 domains of Grb2) all disrupted the
association between SHIP and Shc. The Y(p)xNx and proline-rich peptides
caused the greatest decrease in SHIP/Shc association, correlating with a decreased association between Shc and Grb2. The NPx(p)Y peptide had
no observable effect on Shc/Grb2 association, perhaps because it would
not be expected to disrupt the Shc·Grb2·SOS complex whereas the
proline-rich and Y(p)xNx peptides would probably disrupt both Shc·Grb2·SHIP and Shc·Grb2·SOS complexes. However, all three
peptides disrupted Shc·Grb2·SHIP association, implicating the PTB
domain of Shc and the SH2 and SH3 domains of Grb2 in stabilization of this complex.
To better understand how the inositol phosphatase SHIP influences
B cell activation, we have studied its mechanism of interaction with
the adapter protein Shc. Four lines of evidence suggest that SHIP forms
a ternary complex with Shc and Grb2 analogous to the SOS·Shc·Grb2
complex. First, SHIP co-immunoprecipitates with Grb2 and
immunodepletion of Grb2 removes almost all of the Shc·SHIP complex
from the lysate. Second, mutation of the Grb2-binding sites on Shc lead
to loss of association with Grb2 and a commensurate loss of association
with SHIP. Third, SHIP associates poorly with Shc in Grb2-deficient
cells and this defect can be complemented in vitro and
in vivo by exogenous Grb2. Finally, affinity purification and competitive inhibition experiments with synthetic peptides suggest
that the SH2 and SH3 domains of Grb2 and the PTB domain of Shc, but not
the SH2 domain of SHIP, act together to promote association between
SHIP, Shc, and Grb2. Thus, the complex between Shc and SHIP exists as a
ternary complex with Grb2.
It has previously been suggested that Shc and SHIP interact in a
"bi-dentate" complex in which Grb2 plays no role (30, 45) (Fig.
8A). In this model, the SH2
domain of SHIP binds to phosphorylated Shc and the PTB domain of Shc
binds to phosphorylated SHIP. Although this model explains the failure
of tyrosine-mutated forms of Shc to bind to SHIP (Fig. 2), it cannot
explain the requirement for Grb2 in the formation of this complex
(Figs. 4 and 5) or the ability of the ITIM peptide, which binds to the
SH2 domain of SHIP (35, 52), to pull down the intact SHIP·Shc·Grb2
complex (Fig. 6). Moreover, it has been reported that the SH2 domain of
Grb2 binds to the Tyr313 region of Shc with a 10-fold
higher affinity than does the SH2 domain of SHIP (30), suggesting that
the former interaction is more likely to be relevant in
vivo. Although one study has reported that the SH2 domain of SHIP
is essential for its association with Shc (30), this may be an indirect
effect as mutation of the SH2 domain of SHIP abrogated SHIP tyrosine
phosphorylation in these experiments and thus would preclude binding of
the Shc PTB domain to SHIP. This interpretation is supported by the
previous observation that the SH2 domain of SHIP is dispensable for
Shc-SHIP interaction when SHIP is tyrosine phosphorylated (44).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, phosphatidylinositol 3-kinase (PI
3-kinase), and Ras (2-7).
RIIB, the B cell receptor for the
Fc portion of soluble IgG. Co-cross-linking of the Fc
RIIB with the
BCR inhibits B cell proliferation, Ras activation, and the influx of
calcium across the cell membrane normally observed after BCR
stimulation (31-34). SHIP binds to the cytoplasmic tail of the
Fc
RIIB after its co-ligation with the BCR (35, 36) and has been
shown to inhibit sustained influx of calcium into the cell (37).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactoside (Research Products
International Corp., Mt. Prospect, IL). The cell lines used for
vaccinia virus production (CV-1, 143B, and HeLa) were obtained from
American Type Culture Collection (Manassas, VA) and maintained as directed.
-galactosidase antibody (Promega), or anti-phosphotyrosine
monoclonal antibody (4G10 hybridoma). Next, the immunoblots were
incubated with horseradish peroxidase-conjugated secondary antibody
(horseradish peroxidase-conjugated sheep anti-rabbit IgG was from Roche
Molecular Biochemicals (Indianapolis, IN) and horseradish
peroxidase-conjugated sheep anti-mouse IgG was from Amersham Corp.) and
developed using the Renaissance chemiluminescence detection system (NEN
Life Science Products Inc., Boston, MA).
-D-thiogalactoside treatment for 3 h and the fusion proteins were affinity purified using glutathione-agarose beads (Sigma). Ten units of thrombin (Novagen, Madison, WI) were added
per 500 µg of GST-Grb2 fusion protein and proteolysis allowed to
proceed for 3-4 h at 25 °C. The Grb2-containing supernatant was
removed and passed over a p-aminobenzamidine column (Sigma) to remove thrombin. Purified Grb2 was analyzed and quantitated by
SDS-PAGE followed by Coomassie staining. Three to four µg of recombinant wild type Grb2 or SH2-mutant Grb2 (R86K) was added to
stimulated B cell lysates and incubated overnight at 4 °C. Anti-Shc
immunoprecipitations were then performed as described above.
receptor IIb (AEN TIT Y(p)SL LKH P),
the Y(p)xNx peptide corresponds to amino acids 307-318 of murine
p52Shc (LFD DPS Y(p)VN IQN), the NPxY(p) peptide
corresponds to residues 911-921 of murine SHIP (EMI NPN Y(p)IG MG),
and the proline-rich peptide to amino acids 1149-1162 of murine SOS1
(VPV PPP VPP RRR PE). All peptides were obtained from Quality
Controlled Biochemicals, Inc. (Hopkinton, MA) and were biotinylated at
the N terminus. For affinity purifications, 30 nmol of each peptide was
pre-bound to 50 µl of NeutrAvidin (Pierce) and then incubated with
40 × 106 cell equivalents (stimulated and lysed as
described above) for several hours at 4 °C. The beads were then
treated as described for immunoprecipitations. For peptide competition
assays, 25 × 106 cell equivalents were incubated with
100 µM of each peptide for 3 h at 4 °C. Anti-Shc
immunoprecipitations were then carried out as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (36K):
[in a new window]
Fig. 1.
Association of SHIP with the adapter proteins
Shc and Grb2 in BCR-stimulated B lymphocytes. A, BAL17
cells were left unstimulated or incubated with 20 µg/ml goat anti-IgM
antibody for 3 min ( Ig) and then lysed. Lysates (40 × 106 cell equivalents per sample) were immunoprecipitated
with anti-SHIP antibody, anti-Grb2 antibody, or anti-Shc antiserum,
separated on an 8% gel by SDS-PAGE and then transferred to
nitrocellulose. The blot was cut into thirds and probed with anti-SHIP
antiserum, anti-Shc antibody, or anti-Grb2 antibody. B,
BAL17 cells were stimulated with goat anti-mouse IgM antibody for three
minutes and then lysed. Four serial anti-Grb2 immunoprecipitations and
four serial anti-Lyn immunoprecipitations were performed, each on
40 × 106 cell equivalents. Shc, Lyn, and Grb2 were
then immunoprecipitated from the Grb2-depleted lysate and from the
control, Lyn-depleted lysate. These immunoprecipitations were separated
by SDS-PAGE and immunoblotted as described above. The positions of
marker proteins and their molecular masses (in kDa) are indicated on
the side of each panel.
View larger version (33K):
[in a new window]
Fig. 2.
Requirement of the two Grb2-binding sites on
Shc for efficient formation of SHIP·Shc complexes. WEHI-231 B
cells were stably transfected with cDNAs encoding wild-type or
mutant forms of HA-tagged p52Shc. The indicated
transfectants were incubated with or without goat anti-IgM antibodies
for 3 min and then lysed. A, the transfected HA-Shc proteins
were immunoprecipitated from 40 × 106 cell
equivalents of lysate using anti-HA antibody, separated on an 8% gel
by SDS-PAGE, and transferred to nitrocellulose. The top portion of the
blot was probed with anti-SHIP antiserum (top panel), and
then stripped and probed with anti-phosphotyrosine antibody
(middle panel). The middle portion of the blot was probed
with anti-Shc antibody to verify equivalent amounts of protein were
immunoprecipitated (lower panel). B, SHIP was
immunoprecipitated from 40 × 106 cell equivalents of
lysate using anti-SHIP antibody, separated on an 8% gel by SDS-PAGE,
and transferred to nitrocellulose. The top portion of the blot was
probed with anti-SHIP antiserum (top panel) to verify
equivalent amounts of protein were immunoprecipitated. The middle
portion of the blot was probed with anti-Shc antibody.
R (data not shown).
View larger version (43K):
[in a new window]
Fig. 3.
Shc and SHIP associate poorly in
Grb2-deficient cells. A, lysates were made from
unstimulated BAL17 and BAL17.TR cells. Approximately 1 × 106 cell equivalents were separated on an 8% gel by
SDS-PAGE, and transferred to nitrocellulose. The top portion
of the blot was probed with anti-SHIP antiserum, the middle
portion of the blot was probed with anti-Shc antibody, and the
bottom portion with anti-Grb2 antibody. B,
lysates were made from the human and murine B cell lines WEHI-231,
BAL17, BAL17.TR, Ramos, A20, J/Y, and 70Z/3 and approximately 1 × 106 cell equivalents of each was analyzed by immunoblotting
with anti-Grap antiserum. C, BAL17 and BAL17.TR cells were
left unstimulated, or were treated for 3 min with 13.3 µg/ml goat
anti-mouse IgM F(ab')2 fragments, 20 µg/ml goat
anti-mouse IgM antibody, or 20 µg/ml rabbit anti-mouse IgM antibody
and then lysed. Anti-Shc immunoprecipitations were performed on 30 × 106 cell equivalents of lysate and the samples were
analyzed by immunoblotting as described above. D, anti-SHIP
immunoprecipitations were performed on 35 × 106 cell
equivalents of lysate and then analyzed by immunoblotting.
) cells (Fig. 3C, first panel). The more
sensitive anti-phosphotyrosine antibodies did reveal that a small
amount of tyrosine-phosphorylated SHIP was associated with Shc in these
Grb2
cells (Fig. 3C, second panel). However,
we estimated that Shc bound 20-fold less SHIP in the Grb2
cells than in the Grb2+ cells. Therefore, Grb2 expression
seemed to be crucial for efficient association between SHIP and Shc
following BCR stimulation.
and the
Grb2+ cells (Fig. 3D). The defect in SHIP/Shc
association in the Grb2
cells might therefore have been
due to a lack of Shc tyrosine phosphorylation rather than a lack of
Grb2 expression per se. Shc tyrosine phosphorylation in
BAL17.TR cells could be strongly induced by treatment with pervanadate,
an inhibitor of tyrosine phosphatases (data not shown). Pervanadate
stimulation of the Grb2
cells did induce a low level of
association between SHIP and Shc, but much less than was observed in
the Grb2+ cells (Fig. 4,
top panel, compare lanes 7 and 10).
Thus, in the absence of Grb2, tyrosine phosphorylation of Shc and SHIP
was not sufficient for the efficient association of these two
proteins.
View larger version (40K):
[in a new window]
Fig. 4.
Enhancement of the association between SHIP
and Shc in BAL17.TR (Grb2 ) lysates by recombinant
Grb2. BAL17 and BAL17.TR cells were left unstimulated or were
treated with 150 µM pervanadate for 4 min and then lysed.
Three to four µg of recombinant wild type Grb2, SH2-mutant Grb2
(R86K), or no protein was added to tubes containing 30 × 106 cell equivalents of lysate. The lysates were blotted
with anti-Grb2 antibodies to confirm that similar amounts of wild type
Grb2 and Grb2R86K were added (data not shown). The lysates were
incubated overnight at 4 °C, after which anti-Shc antibody and
protein A-Sepharose was added. Immunoprecipitations were performed for
2 h, and the samples analyzed by immunoblotting as described
above.
cells and examined its
effect on this association. We hypothesized that the complex between
Shc and SHIP would require phosphorylation of Shc tyrosines 239 and
313. As Shc from BCR-stimulated BAL17.TR cells was poorly
phosphorylated on tyrosine (Fig. 3C), we stimulated these
Grb2
cells and the Grb2+ BAL17 cells with
pervanadate to induce strong tyrosine phosphorylation of Shc (data not
shown) and used these lysates as a source of Shc and SHIP. After
addition of either wild type Grb2 (WT), or Grb2 in which the SH2 domain
had been inactivated by the mutation of arginine 86 to lysine (R86K)
(50) to these lysates, Shc was immunoprecipitated and the amount of
associated SHIP was assessed by immunoblotting (Fig. 4). The addition
of wild type Grb2 clearly enhanced the association between SHIP and Shc
in lysates from pervanadate-stimulated BAL17.TR cells but not in
lysates from unstimulated cells (Fig. 4, compare lanes 10, 11, and 12). This effect was apparently dependent upon
the tyrosine phosphorylation of Shc, as the addition of wild type Grb2
did not enhance the association of SHIP and Shc derived from BAL17.TR
cells that were stimulated through the BCR (data not shown). In
contrast to wild type Grb2, the Grb2 mutant (R86K) that lacks a
functional SH2 domain was unable to promote the formation of a complex
between Shc and SHIP. The Grb2 SH2 domain was thus required for the
formation of this complex, as was phosphorylation of Shc (presumably at its Grb2-binding sites). Interestingly, the addition of Grb2 did not
increase the amount of SHIP associated with Shc in lysates of BAL17
cells, indicating that the amount of Grb2 present in those cells was
not limiting for the formation of this complex.
View larger version (28K):
[in a new window]
Fig. 5.
Expression of Grb2 in BAL17.TR cells rescues
Shc/SHIP association. BAL17.TR cells were infected with
recombinant vaccinia viruses containing vector only, wild type Grb2, or
Grb2 in which the SH2 domain had been mutated (Grb2R86K). Each batch of
cells was then split in half; one-half was left unstimulated while the
other was treated with 20 µg/ml goat anti-µ (stim. Ab)
for 3 min. A, 30 × 106 cell equivalents
were immunoprecipitated with anti-Shc antibodies and subject to
immunoblotting as described above. B, 5 × 105 cell equivalents of whole cell lysates (WCL)
were blotted with anti-Grb2 antibody to demonstrate similar expression
levels of Grb2 and Grb2R86K. The last two lanes in the panel were
prepared from one batch of cells split in half just before harvesting,
suggesting that the lighter Grb2 band in the last lane is due to the
poor transfer of protein at the edge of the gel rather than differences
in Grb2R86K expression. C, 30 × 106 cell
equivalents were immunoprecipitated with anti-Shc antibodies and
immunoblots analyzed with anti-phosphotyrosine and anti-Shc antibodies.
D, 5 × 105 cell equivalents of whole cell
lysates (WCL) were blotted with anti-Active MAPK antibody to
assess levels of MAPK activation (second panel). This blot
was then stripped and probed with anti-MAPK antibody to determine total
level of MAPK present (third panel). Since all three
vaccinia viruses express -galactosidase, the blot was also probed
with an anti-
-galactosidase antibody to demonstrate that levels of
vaccinia virus-mediated gene expression were similar in all cells
(first panel).
RIIB (AEN TIT Y(p)SL LKH P), which has previously been reported to bind to the SH2 domain of SHIP (35, 52), to affinity purify
SHIP from unstimulated and stimulated BAL17 and BAL17.TR cell lysates
(Fig. 6, lanes 1-4). Upon
cell stimulation, Shc and Grb2 co-purified with SHIP isolated from the
BAL17 cells. This required Grb2 expression, as Shc did not co-purify
with SHIP in BAL17.TR cells. It therefore appears that the SH2 domain
of SHIP is free to bind to the phosphorylated Fc
RIIB even when SHIP
is bound to Shc.
View larger version (29K):
[in a new window]
Fig. 6.
Binding of SHIP, Shc, and Grb2 to synthetic
peptides. 30 nmol of the indicated peptides were pre-bound to
NeutrAvidin-agarose. Beads were washed, and 40 × 106
cell equivalents of BAL17 or BAL17.TR cells lysates (cells were
stimulated for 3 min with 20 µg/ml goat anti-mouse IgM) were added to
each tube. Samples were incubated at 4 °C for 7 h and then
analyzed by immunoblotting as described above.
View larger version (39K):
[in a new window]
Fig. 7.
Various peptides competitively inhibit the
Shc-Grb2-SHIP interactions. 30 × 106 cell
equivalents of BAL17 lysates stimulated with goat anti-mouse IgM as
described above were incubated with 100 µM of the
indicated peptides for 3 h at 4 °C. Anti-Shc antibodies and
protein A-Sepharose were then added and the immunoprecipitations
allowed to proceed overnight at 4 °C. Samples were then analyzed by
immunoblotting as described above.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (47K):
[in a new window]
Fig. 8.
Two models for Shc-SHIP interactions.
A, the bi-dentate model of Shc-SHIP interaction, in which
the SH2 domain of SHIP binds to phosphorylated Shc and the PTB domain
of Shc binds to phosphorylated SHIP. B, the ternary complex
model of Shc-SHIP interaction. In this model as in the bi-dentate
model, the PTB domain of Shc binds to phosphorylated SHIP. However, we
suggest that two Grb2 molecules bind to Shc with their SH2 domains and
then to SHIP with their SH3 domains. According to this model, the SH2
domain of SHIP plays no role in stabilization of this complex.
"PPP" represents proline-rich regions in SHIP while the
encircled P's represent phosphorylated tyrosine
residues.
We have combined our results summarized above with previously reported features of the association of SHIP with Shc to derive a new model for the mechanism by which these proteins associate (Fig. 8B). According to this ternary complex model, BCR stimulation leads to phosphorylation of tyrosines 239 and 313 of Shc, which are bound by two Grb2 molecules via their SH2 domains (15, 16, 44). The SH3 domains of Grb2 then bind to the three proline-rich regions of SHIP, as previously reported with in vitro binding assays (22, 42). The formation of the complex between SHIP and Shc also appears to require association between the PTB domain of Shc and tyrosine phosphorylated sites on SHIP (30, 44, 55) (Fig. 7). This is a major difference between the Shc·Grb2·SHIP complex and the Shc·Grb2·SOS complex, as SOS does not become phosphorylated on tyrosine residues. The formation or maintenance of the Shc·Grb2·SHIP complex appears to require all of these protein-protein interactions. We observed association between Shc, Grb2, and SHIP upon cell stimulation in all three murine B cell lines that we investigated (BAL17, WEHI-231, and A20, this report and data not shown), suggesting that this is a general mechanism in BCR-stimulated B cells. Other means of association may occur in other cell types, since it has been reported that SHIP does not associate with Grb2 in a hematopoietic cell line stimulated with IL-3 (21).
Association between Shc and SHIP in B cells has been reported to be
optimal when the inhibitory receptor FcRIIB is co-cross-linked with
the BCR (20, 36, 43, 46). However, a substantial amount of Shc·SHIP
complex forms in B cells stimulated only through the BCR (19). In BAL17
cells, we found only modest differences in the amount of SHIP
associated with Shc when the cells were stimulated with anti-IgM
antibodies that differed in their ability to engage the Fc
R (Fig.
3C and data not shown). Similarly, a small decrease in
SHIP-Shc association was observed when the Fc
RIIB was blocked with a
monoclonal antibody prior to stimulation with intact anti-IgM
antibodies (data not shown). We therefore postulate that there are two
mechanisms by which BCR stimulation causes tyrosine phosphorylation of
SHIP, resulting in its association with Shc and Grb2. The first
mechanism involves the binding of SHIP to the ITIM of the Fc
RIIB,
while the second is Fc
RIIB-independent and may involve the
association of SHIP with the BCR-activated tyrosine kinase Syk (19).
Perhaps SHIP acts as a tonic negative regulator even in positively
stimulated cells and negative stimulation, i.e.
co-clustering of the BCR and the Fc
RIIB, enhances SHIP-Shc-Grb2 interaction (and perhaps SHIP function) further. There are now several
precedents for tonic negative regulators in tyrosine kinase receptor
and cytokine receptor signaling (56-59).
The effect that association of SHIP with Shc and Grb2 has on SHIP
function is not yet clear. It has been suggested that SHIP binding to
Shc inhibits Ras activation indirectly by preventing Shc association
with SOS (20). In support of this model, Shc uses primarily the same
protein-protein interaction sites to interact with SOS and to interact
with SHIP, indicating that Shc is unlikely to bind both of these
molecules simultaneously. Indeed, we have never detected any
association between SHIP and SOS (data not shown). However, we have not
detected any decrease in association between Shc and SOS1 or Shc and
SOS2 when cells were stimulated through the BCR and the FcRIIB
instead of stimulated through the BCR alone (data not shown). Also, the
addition of recombinant SOS1 protein to stimulated B cell lysates did
not lead to any decrease in association between Shc and SHIP (data not
shown). These results suggest that the amount of Grb2 and
tyrosine-phosphorylated Shc in stimulated B cells is not limiting and
that Ras inhibition may occur via a mechanism other than the
sequestration of Shc by SHIP. In support of this finding, it has
recently been reported that ERK activation is unaffected in a
SHIP-deficient mutant of the chicken B cell line DT-40, suggesting that
SHIP does not modulate Ras activation in BCR-stimulated B cells (60).
It should also be noted that there is currently no direct evidence
showing that Shc and Grb2 play an important role in Ras activation in
BCR-stimulated B cells. Indeed, we noted little difference in MAPK
activation when comparing either the BAL17 and BAL17.TR cells or
BAL17.TR cells that had been infected with Grb2-expressing vaccinia
viruses or with control viruses (Fig. 5D). In addition, vaccinia
virus-mediated overexpression of either wild type Shc or mutant Shc
that no longer bound Grb2 (Shc Y239F/Y313F) had no obvious effect on
MAPK activation in stimulated B cells (data not shown). These results
suggest that BCR-induced Ras activation is not primarily mediated by
Shc·Grb2·SOS complexes in BAL17 cells.
Another possibility is that formation of the Shc·Grb2·SHIP complex serves to regulate SHIP activity by controlling its intracellular localization. It has previously been reported that a Shc-associated, tyrosine-phosphorylated protein of 145 kDa translocated from the cytosol to the membrane fraction upon BCR stimulation (12). We also have found that complexes of Shc and a tyrosine-phosphorylated protein of 145 kDa are primarily membrane-bound in BCR-stimulated B cells (19). This 145-kDa protein is very likely to be SHIP, as tryptic fragments obtained from it match the published SHIP protein sequence.3 Recruitment of SHIP to the plasma membrane by Shc and Grb2 might control its activity by bringing it near its substrate PI(3,4,5)P3.
SHIP activity has been shown to antagonize PI 3-kinase function in
Xenopus oocytes and to inhibit calcium influx in stimulated B cells (23, 37, 60). Both of these effects could be mediated by the
hydrolysis of PI(3,4,5)P3 by SHIP, as
PI(3,4,5)P3 is thought to enhance the phospholipase
C-catalyzed breakdown of PI(4,5)P2 to inositol
1,4,5-trisphosphate and diacylglycerol (61). SHIP activity would
therefore prevent the prolonged influx of calcium mediated by inositol
1,4,5-trisphosphate and might inhibit calcium influx through other
mechanisms as well (60). We therefore prefer the hypothesis that SHIP
inhibits BCR signaling by its ability to hydrolyze
PI(3,4,5)P3 and that its association with Shc and Grb2
leads to SHIP localization at the plasma membrane, promoting its
subsequent inhibitory actions.
![]() |
FOOTNOTES |
---|
* 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.
Current address: Dept. of Cell Biology, BCC-265, The Scripps
Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037.
§ To whom correspondence should be addressed. Tel.: 415-476-5488; Fax: 415-476-6185; E-mail: defranco{at}socrates.ucsf.edu.
2 J. D. Richards and A. L. DeFranco, submitted for publication.
3 M. T. Crowley, R. Aebersold, and A. L. DeFranco, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: BCR, B cell antigen receptor; PI 3-kinase, phosphatidylinositol 3-kinase; SHIP, SH2-containing inositol phosphatase; PI(3, 4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PI(3, 4)P2, phosphatidylinositol 3,4-bisphosphate; SH2, Src homology domain 2; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin; MAPK, mitogen-activated protein kinase.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|