From the Departments of Microbiology and Immunology
and ¶ Zoology, University of British Columbia, Vancouver, British
Columbia V6T 1Z3, Canada, the ** Kimmel Cancer Institute, Thomas
Jefferson University, Philadelphia, Pennsylvania 19107, and the
§§ Molecular Oncology Group, Department of
Medicine, McGill University Hospital Centre, Montreal,
Quebec H3A 1A1, Canada
Received for publication, November 22, 2000, and in revised form, January 17, 2001
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ABSTRACT |
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B cell antigen receptor (BCR) signaling causes
tyrosine phosphorylation of the Gab1 docking protein. This allows
phosphatidylinositol 3-kinase (PI3K) and the SHP2 tyrosine phosphatase
to bind to Gab1. In this report, we tested the hypothesis that Gab1
acts as an amplifier of PI3K- and SHP2-dependent signaling
in B lymphocytes. By overexpressing Gab1 in the WEHI-231 B cell line,
we found that Gab1 can potentiate BCR-induced phosphorylation of Akt, a
PI3K-dependent response. Gab1 expression also increased
BCR-induced tyrosine phosphorylation of SHP2 as well as the binding of
Grb2 to SHP2. We show that the pleckstrin homology (PH) domain of Gab1
is required for BCR-induced phosphorylation of Gab1 and for Gab1
participation in BCR signaling. Moreover, using confocal microscopy, we
show that BCR ligation can induce the translocation of Gab1 from the cytosol to the plasma membrane and that this requires the Gab1 PH
domain as well as PI3K activity. These findings are consistent with a
model in which the binding of the Gab1 PH domain to PI3K-derived lipids
brings Gab1 to the plasma membrane, where it can be
tyrosine-phosphorylated and then act as an amplifier of BCR signaling.
Engagement of the B cell antigen receptor
(BCR)1 activates multiple
signaling pathways that regulate B cell development, survival, activation, and proliferation (1-5). The initiation of many BCR signaling pathways involves the recruitment of cytoplasmic signaling enzymes to the plasma membrane, where they can act on
membrane-associated substrates. For example, phosphatidylinositol
3-kinase (PI3K) and phospholipase C There are multiple ways in which receptors can recruit cytoplasmic
signaling enzymes to the plasma membrane. One important mechanism
involves the binding of SH2 domains in signaling proteins to
phosphotyrosine-containing sequences on membrane-associated docking/scaffolding proteins. Since the SH2 domains of different proteins bind different phosphotyrosine-containing sequences (6, 7),
the set of signaling enzymes a receptor recruits depends on the
spectrum of phosphotyrosine-containing sequences it generates on
membrane-associated proteins. For example, tyrosine phosphorylation of
its Ig- Signaling enzymes can also be recruited to the plasma membrane via
their pleckstrin homology (PH) domains (12). PH domains bind membrane
phospholipids, in particular the PI3K products phosphatidylinositol 3,4,5-trisphosphate (PIP3) and phosphatidylinositol
3,4-bisphosphate. PH domain-mediated recruitment to the plasma membrane
is important for the BCR to activate the Akt and Btk kinases (13-17).
In addition to promoting their membrane localization, PIP3
can also contribute to the activation of PH domain-containing signaling
enzymes such as phospholipase C The BCR uses multiple docking/scaffolding proteins to recruit PI3K to
the plasma membrane. BCR engagement results in tyrosine phosphorylation
of the cytoplasmic domain of CD19, a transmembrane protein; and this
creates binding sites for the SH2 domains of PI3K (20). Cytosolic
docking proteins that are recruited to the plasma membrane also
contribute to the ability of the BCR to direct PI3K to the plasma
membrane. Cbl, which uses its SH2-like domain to bind to phosphorylated
Syk (21), is tyrosine-phosphorylated after BCR engagement and binds
PI3K (22, 23). The BCR also uses the Gab1 docking protein to recruit
PI3K as well as other signaling enzymes to the plasma membrane (24). We
have previously shown that Gab1 is tyrosine-phosphorylated after BCR
ligation and that this allows the SH2 domains of PI3K, the SHP2
tyrosine phosphatase, and the Shc adaptor protein to bind directly to
Gab1 (24). Grb2 can then bind via its SH2 domain to the
tyrosine-phosphorylated SHP2 and Shc that are bound to Gab1.
Gab1 belongs to a family of docking/scaffolding proteins that includes
the closely related Gab2 protein as well as insulin receptor
substrate-1 and -2 and the Drosophila DOS
(daughter of sevenless) protein
(25-27). All of these proteins contain a PH domain, a cluster of
binding sites for the PI3K SH2 domain, and one or more binding sites
for the SH2 domain of SHP2. The presence of a PH domain in these
docking proteins suggests that they are recruited to the plasma
membrane when receptor signaling stimulates the production of
PI3K-derived lipids. Once at the plasma membrane, these docking
proteins can be tyrosine-phosphorylated, allowing them to recruit
additional PI3K complexes as well as other signaling enzymes. In this
report, we tested the hypothesis that Gab1 functions in this way as a
PI3K-dependent amplifier of BCR signaling. We found that
overexpressing Gab1 increased the ability of the BCR to signal via PI3K
and SHP2. We also show that the BCR recruits Gab1 to the plasma
membrane in a PI3K-dependent manner and that the PH domain
of Gab1 is required for membrane recruitment of Gab1 and for its
ability to amplify BCR signaling.
Antibodies and Other Reagents--
Goat anti-mouse IgM
antibodies were obtained from Bio-Can (Mississauga, Ontario, Canada).
Anti-Gab1 antibodies and the anti-phosphotyrosine monoclonal antibody
4G10 were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY).
Antibodies to SHP2, Grb2, and the p85 subunit of PI3K were from Santa
Cruz Biotechnology Inc. (Santa Cruz, CA). Antibodies against Akt and
antibodies that specifically recognize Akt phosphorylated on serine 473 (anti-phospho-Akt) were purchased from New England Biolabs
(Mississauga). LY294002 and wortmannin were from BIOMOL Research Labs
Inc. (Plymouth Meeting, PA).
Cell Culture--
WEHI-231 cells were grown in RPMI 1640 medium
supplemented with 10% heat-inactivated fetal calf serum, 50 µM 2-mercaptoethanol, 1 mM pyruvate, and 2 mM glutamine. AtT20/BCR/Syk cells (28) were maintained in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum.
cDNAs Encoding Mutant Forms of Gab1--
Polymerase chain
reaction overlap extension (29) was used to generate cDNAs encoding
a mutant form of murine Gab1 that cannot bind PI3K (Gab1 Expression of Gab1 cDNAs in WEHI-231 Cells--
cDNAs
encoding wild-type Gab1, Gab1 Expression of Gab1-EGFP Fusion Proteins in AtT20/BCR/Syk
Cells--
cDNAs encoding wild-type Gab1 and Gab1 Preparation of Cell Lysates--
WEHI-231 cells were resuspended
to 2.5 × 107/ml in modified HEPES-buffered saline (25 mM sodium HEPES, pH 7.2, 125 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM Na2HPO4, 0.5 mM
MgSO4, 1 mg/ml glucose, 2 mM glutamine, 1 mM sodium pyruvate, and 50 µM
2-mercaptoethanol) and stimulated with goat anti-mouse IgM antibodies
at a final concentration of 100 µg/ml. Reactions were stopped by
adding cold phosphate-buffered saline (PBS) to the cells. After
washing, the cells were solubilized in Triton X-100 lysis buffer (1%
Triton X-100, 20 mM Tris-HCl, pH 8, 137 mM
NaCl, 10% glycerol, 2 mM EDTA, 1 mM
Na3VO4, 1 mM phenylmethylsulfonyl
fluoride, 10 µg/ml leupeptin, and 1 µg/ml aprotinin).
Detergent-insoluble material was removed by centrifugation, and protein
concentrations were determined using the bicinchoninic acid assay (Pierce).
Prior to being stimulated, AtT20/BCR/Syk cells were washed with PBS and
cultured overnight in Dulbecco's modified Eagle's medium with 0.2%
fetal calf serum. The cells were then washed with PBS and incubated for
30 min at 37 °C in modified HEPES-buffered saline (see above) to
further reduce signaling due to serum growth factors. The cells were
washed again with PBS, and 10 ml of 37 °C modified HEPES-buffered
saline was added to each dish. BCR signaling was initiated by adding
goat anti-mouse IgM antibodies to a final concentration of 20 µg/ml.
Reactions were terminated by aspirating the medium, washing the cells
twice with cold PBS, and then solubilizing the cells with Triton X-100
lysis buffer containing protease and phosphatase inhibitors.
Immunoprecipitation and Immunoblotting--
For
immunoprecipitations, extracts from 1-2 × 107 cells
(0.5-1 mg of protein) were mixed with 1-2 µg of specific antibodies plus 10 µl of protein A-Sepharose for 1 h at 4 °C. The beads
were pelleted and washed three times with Triton X-100 lysis buffer before bound proteins were eluted with SDS-polyacrylamide gel electrophoresis sample buffer containing 0.1 M
dithiothreitol. Immunoprecipitated proteins were separated by
SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose,
and analyzed by immunoblotting as described (24). Immunoreactive bands
were visualized using enhanced chemiluminescence detection. Akt
activation was analyzed by immunoblotting total cell lysates (60 µg
of protein) with anti-phospho-Akt antibodies. The filters were then
stripped and reprobed with anti-Akt antibodies. The relative levels of Akt phosphorylation were determined by densitometry using an Alpha Innotech gel documentation system (Canberra Packard, Mississauga).
Immunofluorescence Studies on Gab1-EGFP-expressing AtT20/BCR/Syk
Cells--
The cells were grown to near confluency on 10-mm
poly-D-lysine-coated glass coverslips. After culturing
overnight in Dulbecco's modified Eagle's medium plus 0.2% fetal calf
serum, the cells were stimulated with anti-IgM antibodies as described
above. The reaction was stopped by washing the cells twice with cold
PBS. The coverslips were then prepared for fluorescence microscopy as
described previously (36). Images were collected using a Bio-Rad
Radiance Plus confocal microscope and analyzed using NIH Image Version
1.62 software.
Gab1 Links the BCR to the PI3K/Akt Signaling Pathway--
We have
previously shown that BCR ligation results in tyrosine phosphorylation
of Gab1 and that this allows PI3K to bind via its SH2 domains to Gab1
(24). Since Gab1·PI3K complexes are found in the membrane-enriched
particulate fraction of the RAMOS human B cell line after BCR ligation
(24), this led us to propose that Gab1 acts as a docking protein that
recruits PI3K to the plasma membrane after BCR engagement. This would
presumably result in increased production of PI3K-derived lipids that
can activate downstream targets such as the Akt protein kinase. To test
this idea that Gab1 functionally links the BCR to the PI3K/Akt
signaling pathway, we expressed Gab1 in the WEHI-231 murine B lymphoma
cell line. WEHI-231 cells normally express the closely related Gab2 protein (Ref. 25 and data not shown), but express little or no Gab1
(Fig. 1). Fig. 1 shows that this
transfected Gab1 protein was tyrosine-phosphorylated after BCR ligation
in the WEHI-231 cells and that it associated with a number of other
tyrosine-phosphorylated proteins. As in RAMOS cells (24), the 72-kDa
tyrosine-phosphorylated protein that coprecipitated with Gab1 after BCR
stimulation in WEHI-231 cells is the SHP2 tyrosine phosphatase (see
Fig. 9B), whereas the 46- and 52-kDa tyrosine-phosphorylated
proteins are two isoforms of the Shc adaptor protein (data not shown).
Thus, the transfected Gab1 expressed in the WEHI-231 cells appears to function similarly to the endogenous Gab1 present in other B cell lines
such as RAMOS.
We then asked whether expressing Gab1 in WEHI-231 cells could
potentiate the ability of the BCR to activate the PI3K/Akt signaling pathway. We used phosphorylation of Akt on serine 473 as a readout since we had previously shown that this response is dependent on PI3K
activity (37). Fig. 2 shows that
overexpressing Gab1 in WEHI-231 cells increased the ability of the BCR
to stimulate Akt phosphorylation. Densitometric analysis showed that
Gab1 expression increased anti-IgM antibody-stimulated Akt
phosphorylation by ~2-fold at all time points between 1 and 30 min.
Thus, Gab1 expression increased the magnitude of BCR-induced Akt
phosphorylation as opposed to merely changing the kinetics of this
response.
The ability of Gab1 to potentiate BCR-induced Akt phosphorylation
depended on its ability to bind PI3K (Fig.
3). This was shown by expressing in
WEHI-231 cells a mutant form of Gab1 (Gab1 Gab1 Requires Its PH Domain to Link the BCR to the PI3K/Akt
Signaling Pathway--
PI3K must be recruited to the plasma membrane
to convert PIP2 to PIP3 and to promote the
activation of Akt. The ability of Gab1 to potentiate BCR-induced Akt
activation presumably reflects the ability of Gab1 to recruit PI3K to
the plasma membrane. This implies that Gab1 is localized at the plasma
membrane in anti-IgM antibody-stimulated B cells and suggests that BCR
signaling may recruit Gab1 to the plasma membrane. Two distinct
mechanisms by which receptors can recruit Gab1 to the plasma membrane
have been described (32, 38). One involves the binding of
PIP3 to the PH domain of Gab1, whereas the second is a
PI3K-independent mechanism in which Gab1 binds either directly or via
Grb2 to tyrosine-phosphorylated membrane proteins. Since BCR engagement
leads to the rapid production of PIP3 (39), we postulated
that it is the binding of PIP3 to the PH domain of Gab1
that recruits Gab1 to the plasma membrane of B cells. This would
presumably allow Gab1 to be tyrosine-phosphorylated so that it could
then amplify PI3K signaling by recruiting additional PI3K molecules to
the plasma membrane.
To test this model, we expressed in WEHI-231 cells a truncated form of
Gab1 that lacks the PH domain (Gab1 BCR Signaling Can Cause Translocation of Gab1 to the Plasma
Membrane--
The requirement that Gab1 have a PH domain to
participate in BCR signaling is consistent with the idea that Gab1 is
recruited to the plasma membrane in response to the production of
PIP3 by PI3K. Since it is difficult to analyze the
translocation of cytoplasmic proteins to the plasma membrane in B
cells, we tested this hypothesis by expressing a Gab1-EGFP fusion
protein in a derivative of the AtT20 endocrine cell line that had been
transfected with DNAs encoding all four chains of the BCR as well as
the Syk tyrosine kinase (28). We have previously shown that many
aspects of BCR signaling, including PI3K-dependent Akt
activation (28, 37), can occur in these AtT20/BCR/Syk cells.
EGFP fusion proteins containing either full-length Gab1 (Gab1-EGFP) or
Gab1 lacking its PH domain (Gab1
The subcellular localization of the Gab1-EGFP and Gab1
To test whether the BCR-induced recruitment of Gab1 to the plasma
membrane is dependent upon the PH domain of Gab1, we analyzed the
subcellular distribution of the Gab1
To further test our model that BCR-induced recruitment of Gab1 to the
plasma membrane depends on the binding of the Gab1 PH domain to
PI3K-derived lipids, we investigated whether it could be blocked by
PI3K inhibitors. We found that pretreating AtT20/BCR/Syk cells with
LY294002 caused significant, although not complete, inhibition of
BCR-induced translocation of Gab1-EGFP to the plasma membrane (Fig.
8). Similar results were obtained when
wortmannin was used to inhibit PI3K activity (data not shown). The
partial inhibition of BCR-induced translocation of Gab1 to the plasma membrane may be due to small amounts of PIP3 being produced
even in the presence of LY294002 or wortmannin. This might be
sufficient to cause some membrane translocation of Gab1 since the Gab1
PH domain binds PIP3 with high affinity (41-43).
Gab1 Regulates the Function of the SHP2 Tyrosine
Phosphatase--
We have previously shown that the SHP2 tyrosine
phosphatase also binds to Gab1 after BCR ligation (24). Far Western
analysis indicated that this interaction is mediated by the binding of the SHP2 SH2 domains to tyrosine-phosphorylated Gab1 (24). We also
showed that the SHP2 that binds to Gab1 after BCR ligation is strongly
phosphorylated on tyrosine residues (24). Since both tyrosine
phosphorylation of SHP2 and engagement of its SH2 domains increase its
phosphatase activity (44, 45), Gab1 may be an important regulator of
both the enzymatic activity and subcellular localization of SHP2.
Tyrosine phosphorylation of SHP2 also allows it to act as an adaptor
protein that can bind the SH2 domain of Grb2 (46). We have shown that
BCR ligation causes Grb2 to bind to the tyrosine-phosphorylated SHP2
associated with Gab1 (24). Together, these findings suggest that Gab1
regulates the ability of SHP2 to function both as a phosphatase and as
an adaptor protein. To test this model, we investigated whether
expressing Gab1 in WEHI-231 cells would enhance both the tyrosine
phosphorylation of SHP2 and its ability to bind Grb2.
We found that expressing Gab1 in WEHI-231 cells greatly potentiated
both the tyrosine phosphorylation of SHP2 and its ability to bind Grb2.
Although BCR ligation caused modest tyrosine phosphorylation of SHP2 in
vector control cells, very strong tyrosine phosphorylation of SHP2 was
seen in Gab1-expressing WEHI-231 cells (Fig.
9A). The ability of Gab1 to
potentiate BCR-induced tyrosine phosphorylation of SHP2 required the
Gab1 PH domain. Expressing Gab1
Since expressing wild-type Gab1 increased BCR-induced tyrosine
phosphorylation of SHP2, we investigated whether this correlated with
an increase in the ability of Grb2 to bind to SHP2. Fig. 10 shows that expressing Gab1 in
WEHI-231 cells greatly increased the anti-IgM antibody-induced binding
of Grb2 to SHP2. This effect was dependent on the binding of SHP2 to
Gab1. Expressing a mutant form of Gab1 that lacks the major site of
SHP2 binding (Gab1 The Gab1 and Gab2 docking/scaffolding proteins participate in
signaling by a variety of tyrosine kinase-linked receptors, including
the T and B cell antigen receptors, a number of cytokine receptors, and
the receptors for growth factors such as epidermal growth factor,
hepatocyte growth factor, nerve growth factor, and insulin (24, 25, 30,
32, 38, 41, 47-53). In response to signaling by these receptors, the
Gab1 and/or Gab2 protein becomes tyrosine-phosphorylated and then binds
SH2 domain-containing signaling proteins, including PI3K (24, 25, 30,
32, 48, 49, 51); the SHP2 tyrosine phosphatase (24, 25, 30, 32,
48-54); and the Shc (24, 25, 50), Grb2 (24, 25, 49, 50, 53, 54), and
CrkL (55) adaptor proteins. In this report, we have shown that Gab1 can
functionally link the BCR to signaling events involving PI3K and SHP2.
We also showed that the PH domain of Gab1 is required for BCR-induced
recruitment of Gab1 to the plasma membrane, for tyrosine
phosphorylation of Gab1, and for the binding of SH2 domain-containing
signaling proteins to Gab1. This suggests that BCR-induced recruitment
of Gab1 to the plasma membrane allows Gab1 to act as a
docking/scaffolding protein that recruits PI3K and SHP2 to the plasma
membrane, where they can contribute to BCR signaling.
Receptor-induced recruitment of Gab1 to the plasma membrane can
occur by at least two different mechanisms (32). The first mechanism
involves the binding of the Gab1 PH domain to PIP3 and is
therefore dependent on prior activation of PI3K. The PH domain of Gab1
selectively binds PIP3 (41), and PI3K activation is sufficient to recruit either full-length Gab1 or the isolated Gab1 PH
domain to the plasma membrane (32, 42). Moreover, both epidermal growth
factor- and serum-induced membrane translocation of Gab1 can be blocked
by PI3K inhibitors (32, 41). In contrast, hepatocyte growth
factor-induced recruitment of Gab1 to the plasma membrane does not
require the PH domain of Gab1 and is independent of PI3K (32, 43).
Instead, Gab1 binds directly to the cytoplasmic domain of the activated
hepatocyte growth factor receptor, c-Met. This is mediated by a novel
phosphotyrosine-binding domain within Gab1 termed the Met-binding
domain (54). Gab1 can also be bridged to c-Met by Grb2, with the Grb2
SH3 domains binding Gab1 and the Grb2 SH2 domain binding
tyrosine-phosphorylated c-Met (54). It is possible that Grb2 also links
Gab1 to other receptors either by directly binding phosphorylated
receptors or by binding to tyrosine-phosphorylated Shc that is
associated with a receptor. Thus, recruitment of Gab1 to the plasma
membrane can be mediated either by the N-terminal PH domain of Gab1 or
by the Met-binding domain and/or Grb2-binding sites of Gab1, both of
which are located in the C-terminal portion of the protein (49, 54).
The PH domain-mediated membrane recruitment is dependent upon
production of PIP3 by PI3K, whereas membrane recruitment
mediated by the Met-binding domain or Grb2 is
phosphotyrosine-dependent, but independent of
PIP3 production.
Our microscopy data (Figs. 7 and 8) suggest that the BCR-induced
translocation of Gab1 to the plasma membrane is mediated by the binding
of PIP3 to the PH domain of Gab1. First, a truncated form
of Gab1-EGFP that lacks the PH domain was not recruited to the plasma
membrane of AtT20/BCR/Syk cells after BCR engagement, whereas plasma
membrane recruitment was readily observed for the full-length Gab1-EGFP
protein. In addition, we found that pretreating AtT20/BCR/Syk cells
with either LY294002 (Fig. 8) or wortmannin (data not shown)
significantly reduced BCR-induced translocation of Gab1-EGFP to the
plasma membrane. The use of two different PI3K inhibitors minimizes the
possibility that this inhibition was due to effects of these compounds
on cellular processes other than the production of PIP3 by
PI3K. Taken together, these data indicate that BCR-induced
translocation of Gab1 to the plasma membrane is a
PI3K-dependent event.
Since the ability of Gab1 to bind PI3K and to potentiate BCR-induced
phosphorylation of Akt also depends on the Gab1 PH domain, it suggests
that Gab1 is a PI3K-dependent amplifier of BCR signaling. We propose that Gab1 does not initiate BCR-induced PI3K signaling, but
instead increases the amount of PI3K that the BCR recruits to the
plasma membrane, an event that is necessary for PI3K to phosphorylate
its lipid substrates. The initial recruitment of PI3K to the plasma
membrane after BCR engagement in B cells is most likely mediated by
CD19, a transmembrane protein. BCR signaling results in tyrosine
phosphorylation of CD19, and this allows CD19 to bind the SH2 domains
of PI3K (20). PI3K may also be recruited to the plasma membrane by Cbl.
Cbl is strongly phosphorylated on tyrosine residues after BCR
engagement and binds significant amounts of PI3K (22, 23). Once PI3K is
recruited to the plasma membrane by binding via its SH2 domains to CD19
or Cbl, it can phosphorylate PIP2. The resulting
PIP3 can then bind the PH domain of Gab1 and recruit Gab1
to the plasma membrane. Our experiments using the truncated form of
Gab1 that lacks the PH domain suggest that membrane recruitment of Gab1
is required for tyrosine kinases to phosphorylate Gab1. Tyrosine
phosphorylation of Gab1 creates binding sites for the SH2 domains of
PI3K, allowing Gab1 to recruit additional PI3K molecules to the plasma
membrane. In this way, Gab1 can amplify the ability of the BCR to
signal via PI3K. The PI3K that is bound to Gab1 may further amplify
PI3K signaling by producing PIP3, which can bind to the
Gab1 PH domain and stabilize the association of Gab1 with the plasma membrane.
In this report, we also show that Gab1 can link the BCR to an important
PI3K-dependent signaling event, the phosphorylation of Akt
on serine 473. Phosphorylation of Akt at this site is required for its
full activation and is strongly correlated with an increase in the
enzymatic activity of Akt (14, 56, 57). Akt is a multifunctional kinase
that regulates many important processes. In particular, Akt is the
primary mediator of the anti-apoptotic/pro-survival functions of PI3K
(15). This appears to be true in B cells as well. Pogue et
al. (58) have recently shown that overexpressing Akt prevents
BCR-induced apoptosis in the DT40 chicken B cell line. The
anti-apoptotic/pro-survival functions of Akt may reflect its ability to
regulate the function of a variety of proteins involved in cell
survival and apoptosis, including NF- In addition to amplifying BCR signaling via PI3K, we found that Gab1
can also amplify the ability of the BCR to signal via the SHP2 tyrosine
phosphatase. We have previously shown that SHP2 binds via its SH2
domains to Gab1 in anti-Ig antibody-stimulated RAMOS B cells (24). The
binding of tyrosine-phosphorylated peptides to the SH2 domains of SHP2
has been shown to increase the phosphatase activity of SHP2 (45). The
phosphatase activity of SHP2 is also increased upon tyrosine
phosphorylation of SHP2 (44); and in this report, we have shown that
overexpression of Gab1 greatly increases BCR-induced tyrosine
phosphorylation of SHP2. In contrast, the Gab1 Although few substrates of SHP2 have been identified, recent reports
have shown that Gab1 and Gab2 can be dephosphorylated by SHP2 in
vitro (48). This suggests that SHP2 could negatively regulate the
ability of Gab1/2 to bind PI3K or other SH2 domain-containing signaling
proteins. This form of regulation may be common to all members of the
Gab1/2 family of docking/scaffolding proteins. Both insulin receptor
substrate-1 and DOS can be dephosphorylated by SHP2 (27, 60), and SHP2
has been shown to negatively regulate the binding of PI3K to insulin
receptor substrate-1 (61).
In addition to being a phosphatase, tyrosine-phosphorylated SHP2 can
also act as an adaptor protein that binds the SH2 domain of Grb2 (46).
This presumably allows SHP2 to recruit Grb2·mSOS complexes that can
activate Ras. Consistent with this idea, in many cell types, SHP2 is a
positive regulator of the ERK mitogen-activated protein kinase (25, 59,
62), a downstream target of Ras. Moreover, it has been shown that
overexpression of Gab1 can potentiate receptor-induced activation of
ERK and that this depends on the ability of Gab1 to bind SHP2 (48, 52,
53, 63). A physiological role for Gab1 in ERK activation has been
confirmed by studies showing that ERK activation is depressed in
embryos from Gab1 knockout mice (64). To date, we have not observed an
effect of Gab1 overexpression on BCR-induced ERK activation. This may reflect the possibility that activation of Ras by the BCR is mediated primarily by RasGRP (65), a diacylglycerol-regulated Ras exchange factor, and not by mSOS. Gab1-mediated recruitment of Grb2·mSOS complexes may therefore contribute to the activation of other signaling
pathways. mSOS has been shown to activate the Rac1 GTPase (18), and we
are currently testing whether overexpression of Gab1 potentiates
BCR-induced activation of JNK, a downstream target of Rac1 signaling
(66). Gab1 overexpression has been reported to potentiate activation of
JNK by the epidermal growth factor and hepatocyte growth factor
receptors (41, 67).
Both Gab1 and Gab2 are expressed in B cells (24, 25, 48), with Gab1
being more highly expressed in some cell lines (e.g. the
RAMOS human B cell line) and Gab2 being more highly expressed in other
cell lines (e.g. the WEHI-231 murine B cell line). An important question therefore is whether Gab1 and Gab2 are functionally equivalent. Both Gab1 and Gab2 have N-terminal PH domains that are 73%
identical (90% similar) at the amino acid level. Moreover, the
organization of the potential binding sites for the SH2 domains of PI3K
and SHP2 is highly conserved between Gab1 and Gab2. This suggests that
the BCR could use either Gab1 or Gab2 to recruit the same signaling
proteins. Tyrosine-phosphorylated Gab2 has been shown to bind PI3K and
SHP2 in other cell types (25, 48). Although BCR engagement results in
tyrosine phosphorylation of Gab2 (25, 48), the ability of Gab2 to bind
PI3K and SHP2 in anti-Ig antibody-stimulated B cells has not been
evaluated. We are currently analyzing whether Gab2 binds PI3K and SHP2
after BCR signaling and whether overexpression of Gab2 increases
BCR-induced phosphorylation of Akt. These experiments will reveal
whether Gab1 and Gab2 play equivalent roles in BCR signaling.
In summary, our data suggest the following model for how Gab1 and
perhaps the closely related Gab2 protein participate in BCR signaling.
In response to BCR engagement, PI3K is recruited to CD19 or other
membrane-associated docking proteins and produces PIP3.
This PIP3 can bind to the PH domain of Gab1 and recruit Gab1 to the plasma membrane. Once at the plasma membrane, Gab1 can be
tyrosine-phosphorylated by BCR-regulated tyrosine kinases. Phosphorylation of the appropriate tyrosine residues on Gab1 allows it
to bind the SH2 domains of Shc, PI3K, and SHP2, thereby recruiting these proteins to the plasma membrane. Recruitment of PI3K to the
plasma membrane allows it to convert PIP2 to
PIP3, resulting in further activation of
PI3K-dependent signaling events such as the activation of
Akt. Recruitment of SHP2 to the plasma membrane would allow it to be
phosphorylated by BCR-regulated tyrosine kinases, thereby increasing
its specific activity. SHP2 may then dephosphorylate
membrane-associated substrates including Gab1 itself. Although this
might limit Gab1-mediated signaling, tyrosine phosphorylation of the
Gab1-associated SHP2 and Shc would allow Grb2·mSOS complexes and
Grb2·SHIP complexes to be recruited to the plasma membrane. Since the
ability of mSOS to act as an exchange factor that activates Rac1 is
increased by the binding of PIP3 to its PH domain,
Gab1-mediated colocalization of mSOS and PI3K may facilitate the
activation of Rac1 by the BCR. Similarly, colocalization of PI3K and
SHIP by Gab1 may allow for efficient production of phosphatidylinositol
3,4-bisphosphate, a lipid that binds to a subset of PH
domain-containing proteins. Thus, Gab1 and perhaps Gab2 may act as
PI3K-dependent amplifiers of multiple BCR signaling pathways.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
must be recruited to the plasma
membrane to act on their substrate, the membrane lipid
phosphatidylinositol 4,5-bisphosphate (PIP2).
and Ig-
subunits allows the BCR to recruit the Syk tyrosine kinase (8, 9). However, the absence of other
phosphotyrosine-containing sequences in Ig-
and Ig-
prevents the
direct recruitment of other SH2 domain-containing proteins to the BCR.
The BCR overcomes this problem by using its associated tyrosine kinases
to phosphorylate appropriate tyrosine residues on other
membrane-associated docking proteins (e.g. CD19). In
addition, adaptor proteins (e.g. BLNK and Shc) that are
tyrosine-phosphorylated after BCR signaling can couple SH2
domain-containing signaling proteins with membrane-associated docking
proteins (10, 11). These SH2 domain-phosphotyrosine interactions are
essential for the BCR to recruit PI3K, phospholipase C
, mSOS
(son of sevenless homolog), and
other signaling enzymes to the plasma membrane.
, mSOS, and Vav (18, 19). Thus,
PI3K-derived lipids play a role in the activation of many different BCR
signaling pathways. The recruitment of PI3K to the plasma membrane,
where its substrates are located, is therefore a key event in BCR signaling.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
PI3K) (30)
as well as a mutant form of Gab1 that cannot bind SHP2 (Gab1
SHP2)
(31). The Gab1
PI3K protein has tyrosine-to-phenylalanine
substitutions at amino acids 448, 473, and 590 that eliminate the three
YXXM sequences that the PI3K SH2 domains can bind to. The
Gab1
SHP2 protein has a tyrosine-to-phenylalanine substitution at
amino acid 628 that eliminates the YXDL sequence that the
SHP2 SH2 domains can bind to. A cDNA encoding a truncated form of
Gab1 that includes amino acids 116-695 but lacks the N-terminal PH
domain (Gab1
PH) has been described previously (32).
PI3K, Gab1
SHP2, and Gab1
PH were
excised from the pLTR2 vector using BamHI and
NotI and subcloned into the pMX retroviral expression vector
(33). The resulting plasmids, as well as pMX with no insert, were
transfected into the BOSC23 packaging cell line (34). The retroviral
particles released into the culture supernatant were then used to
infect WEHI-231 cells as described (35). After retroviral infection, the WEHI-231 cells were cultured in the presence of 0.25 µg/ml puromycin for 2 days to select for infected cells. The resulting bulk
populations of infected WEHI-231 cells were maintained in culture
medium with 0.25 µg/ml puromycin. Expression of the various Gab1
proteins in these bulk populations was confirmed by immunoblotting. Previous analyses using enhanced green fluorescent protein (EGFP) cDNA cloned into pMX showed that >95% of the puromycin-resistant WEHI-231 cells obtained after retroviral infection express the transduced gene (35).
PH were
subcloned into the pEGFP-C2 vector (CLONTECH), as
described previously (32), to generate fusion proteins with EGFP at the
N terminus. AtT20/BCR/Syk cells were transfected as described (36) with
50 µg of the plasmid encoding either Gab1-EGFP or
Gab1
PH-EGFP in addition to 20 µg of pWZLBlast (a gift from Dr.
Steven Robbins, University of Calgary), a plasmid encoding resistance
to blasticidin. After culturing the cells for 10-14 days in the
presence of 2 µg/ml blasticidin (Invitrogen, Carlsbad, CA),
individual colonies of blasticidin-resistant cells were isolated as
described (28).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Gab1 expressed in WEHI-231 cells is inducibly
tyrosine-phosphorylated and binds tyrosine-phosphorylated signaling
proteins after BCR engagement. A stable population of WEHI-231
cells expressing murine Gab1 was produced by retroviral infection.
Vector control cells were infected with retroviruses containing the
empty pMX vector. The resulting cells were incubated for 2 min with or
without anti-IgM antibodies. Cell lysates were immunoprecipitated
(ippt) with anti-Gab1 antibodies, and the precipitated
proteins were analyzed by immunoblotting with the anti-Tyr(P)
monoclonal antibody 4G10. The filters were then stripped and reprobed
with anti-Gab1 antibodies. Tyrosine-phosphorylated Gab1, SHP2, and Shc
are indicated by the arrows. Molecular mass standards (in
kilodaltons) are indicated to the left. The data represent one of three
independent experiments that yielded similar results.
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Fig. 2.
Expression of Gab1 in WEHI-231 cells
potentiates BCR-induced phosphorylation of Akt. Gab1-expressing
WEHI-231 cells (G in the lower panel) and vector
control cells (V in the lower panel) were
incubated with anti-IgM antibodies for the indicated times. Cell
lysates were separated by SDS-polyacrylamide gel electrophoresis and
then analyzed by immunoblotting with an antibody that specifically
recognizes the activated form of Akt that is phosphorylated on serine
473 (P-Akt). The filters were then stripped and reprobed
with an anti-Akt antibody to show that equivalent amounts of Akt were
present in each lane. Molecular mass standards (in kilodaltons) are
indicated to the left. Two independent experiments are shown. The
potentiation of BCR-induced Akt phosphorylation by Gab1 expression was
observed in 10 independent experiments.
PI3K) in which all three
potential binding sites for the PI3K SH2 domains had been ablated by
tyrosine-to-phenylalanine mutations. Fig. 3 shows that Gab1
PI3K did
not bind PI3K after BCR ligation and that its ability to potentiate
BCR-induced Akt phosphorylation was much less than that of wild-type
Gab1. Compared with WEHI-231 cells infected with retroviruses
containing the empty pMX vector, Akt phosphorylation at 2 min after
anti-IgM antibody addition was 97 ± 21% (n = 10)
higher in cells expressing wild-type Gab1, but only 27 ± 16%
(n = 3) higher in cells expressing Gab1
PI3K. Thus,
the ability of Gab1 to bind PI3K allows it to link the BCR to
PI3K-dependent signaling events such as Akt
phosphorylation.
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Fig. 3.
Ability of Gab1 to potentiate BCR-induced Akt
phosphorylation depends on its ability to bind PI3K. Vector
control cells and WEHI-231 cells expressing either wild-type
(wt) Gab1 or a mutant form of Gab1 (Gab1 PI3K) that lacks
the three PI3K-binding sites were incubated with or without anti-IgM
antibodies for 2 min. Akt phosphorylation was analyzed by
immunoblotting cell lysates with the anti-phospho-Akt antibody. The
filter was then reprobed with anti-Akt antibodies to show that equal
amounts of Akt were present in each lane. The binding of PI3K to Gab1
was assessed by immunoprecipitating (ippt) cell lysates with
anti-Gab1 antibodies, followed by immunoblotting with antibodies
against the p85 subunit of PI3K. These filters were then reprobed with
anti-Gab1 antibodies to show that similar amounts of wild-type Gab1 and
Gab1
PI3K had been precipitated. Molecular mass standards (in
kilodaltons) are indicated to the left. The data represent one of four
independent experiments that yielded similar results.
PH). We found that the Gab1
PH
protein was very poorly phosphorylated in response to BCR engagement
and that, consequently, it bound other tyrosine-phosphorylated proteins
to a much lesser extent than the wild-type Gab1 protein (Fig.
4A). In particular, the
Gab1
PH protein did not bind significant amounts of PI3K after BCR
ligation (Fig. 4B), and this correlated with its inability
to significantly increase BCR-induced Akt phosphorylation (Fig.
4C). Anti-IgM antibody-stimulated Akt phosphorylation was only 11 ± 8% (n = 2) higher in the cells
expressing Gab1
PH than in the vector control cells. Taken together,
these data suggest that Gab1 uses its PH domain to come to the plasma
membrane after BCR engagement and that this is required for Gab1 to be
tyrosine-phosphorylated, to bind PI3K, and to contribute to Akt
activation.
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Fig. 4.
The PH domain of Gab1 is required for Gab1 to
be tyrosine-phosphorylated, bind PI3K, and potentiate Akt
phosphorylation after BCR engagement. Vector control cells and
WEHI-231 cells expressing either wild-type (wt) Gab1 or a
mutant form of Gab1 that lacks the PH domain (Gab1 PH) were incubated
with or without anti-IgM antibodies for 2 min. A, cell
lysates were immunoprecipitated (ippt) with anti-Gab1
antibodies, and the precipitated proteins were analyzed by
immunoblotting with the anti-Tyr(P) monoclonal antibody 4G10. The
filters were then stripped and reprobed with anti-Gab1 antibodies to
show that similar amounts of wild-type Gab1 and Gab1
PH had been
precipitated. Tyrosine-phosphorylated Gab1, SHP2, and Shc are indicated
by the arrows. B, the binding of PI3K to Gab1 was
assessed by immunoprecipitating cell lysates with anti-Gab1 antibodies,
followed by immunoblotting with antibodies against the p85 subunit of
PI3K. The filters were then reprobed with anti-Gab1 antibodies to show
that similar amounts of wild-type Gab1 and Gab1
PH had been
precipitated. C, Akt phosphorylation was analyzed by
immunoblotting cell lysates with the anti-phospho-Akt antibody. The
filter was then reprobed with anti-Akt antibodies to show that equal
amounts of Akt were present in each lane. Molecular mass standards (in
kilodaltons) are indicated to the left. All of the experiments were
performed on the same set of cell lysates. One of three independent
experiments that yielded similar results is shown.
PH-EGFP) were expressed in the
AtT20/BCR/Syk cells (Fig. 5A).
We then assessed whether these proteins behaved similarly to the
wild-type Gab1 and truncated Gab1
PH proteins that were expressed in
the WEHI-231 B cell line. When the BCR on the AtT20/BCR/Syk cells was
cross-linked with anti-IgM antibodies, we found that the Gab1-EGFP
protein became phosphorylated on tyrosine residues and associated with
tyrosine-phosphorylated proteins (Fig. 5B). Reprobing these
blots showed that these Gab1-associated proteins were SHP2 (Fig.
5B) and the p46 and p52 forms of Shc (data not shown). In
contrast to the wild-type Gab1-EGFP fusion protein, the Gab1
PH-EGFP
protein showed very little BCR-induced tyrosine phosphorylation, and
its ability to bind SHP2 and Shc did not increase after BCR engagement
(Fig. 5B). This is consistent with our finding that the PH
domain of Gab1 is required for its BCR-induced tyrosine phosphorylation
in WEHI-231 cells (Fig. 4). We went on to show that expressing the
wild-type Gab1-EGFP protein in AtT20/BCR/Syk cells increased
BCR-induced Akt phosphorylation by ~2-fold at all time points,
whereas expressing Gab1
PH-EGFP had a much smaller effect (Fig.
6). Thus, the wild-type Gab1-EGFP and
Gab1
PH-EGFP proteins expressed in AtT20/BCR/Syk cells appear to
function identically to the wild-type Gab1 and Gab1
PH proteins, respectively, that were expressed in the WEHI-231 B cell line. In
response to BCR engagement, wild-type Gab1-EGFP was
tyrosine-phosphorylated, bound SHP2 and Shc, and linked the BCR to Akt
activation. As in WEHI-231 cells, all of these responses were dependent
upon the PH domain of Gab1. Thus, the expression of Gab1-EGFP fusion
proteins in AtT20/BCR/Syk cells can be used as a model system to
investigate whether BCR engagement results in translocation of Gab1 to
the plasma membrane.
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Fig. 5.
BCR-induced tyrosine phosphorylation of a
Gab1-EGFP fusion protein expressed in AtT20/BCR/Syk cells.
A, expression of transfected Gab1-EGFP and Gab1 PH-EGFP
fusion proteins in AtT20/BCR/Syk cells was analyzed by immunoblotting
with anti-Gab1 antibodies. B, AtT20/BCR/Syk cells expressing
either Gab1-EGFP or Gab1
PH-EGFP were incubated with or without
anti-IgM antibodies for 15 min. Cell lysates were immunoprecipitated
(ippt) with anti-Gab1 antibodies, and the precipitated
proteins were analyzed by immunoblotting with an anti-Tyr(P) monoclonal
antibody. The filters were reprobed with anti-Gab1 antibodies to show
that similar amounts of Gab1-EGFP and Gab1
PH-EGFP were precipitated.
Tyrosine-phosphorylated Gab1 and Gab1-associated
tyrosine-phosphorylated proteins are indicated by the
arrows. The filter was also reprobed with antibodies to SHP2
(lower panel) and to Shc (not shown) to identify the
Gab1-associated phosphoproteins. Molecular mass standards (in
kilodaltons) are indicated to the left. The data represent one of three
independent experiments that yielded similar results.
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Fig. 6.
Wild-type Gab1-EGFP, but not
Gab1 PH-EGFP, potentiates BCR-induced Akt
phosphorylation in AtT20/BCR/Syk cells. The cells were stimulated
with anti-IgM antibodies for the indicated times. Cell lysates were
separated by SDS-polyacrylamide gel electrophoresis, and Akt
phosphorylation was analyzed by immunoblotting with the
anti-phospho-Akt antibody. The filters were then reprobed with anti-Akt
antibodies to show that equal amounts of Akt were present in each lane.
Molecular mass standards (in kilodaltons) are indicated to the left.
The data represent one of four independent experiments that yielded
similar results.
PH-EGFP
fusion proteins in AtT20/BCR/Syk cells was analyzed by confocal microscopy. In unstimulated AtT20/BCR/Syk cells, the Gab1-EGFP protein
was mostly cytoplasmic, with very little of the protein accumulating at
the margins of the cells that would correspond to the plasma membrane
(Figs. 7, a-c; and
8a). After BCR engagement, however, there was a significant
increase in the amount of Gab1-EGFP at the margins of the cells (Figs.
7, d-f; and 8, b and c). This likely
represents translocation of Gab1-EGFP to the plasma membrane of the
cells since staining nonpermeabilized AtT20/BCR/Syk cells with
antibodies to the cell-surface BCR yields a similar pattern of
fluorescence (40). The BCR-induced translocation of Gab1-EGFP to the
plasma membrane was most evident at 15 min after adding anti-IgM
antibodies to the cells (Figs. 7, d-f; and 8, b
and c), but could also be observed at 3, 5, and 10 min after
initiating BCR signaling (data not shown).
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Fig. 7.
BCR-induced recruitment of Gab1-EGFP to the
plasma membranes of AtT20/BCR/Syk cells. AtT20/BCR/Syk cells
expressing either Gab1-EGFP or Gab1 PH-EGFP were incubated with or
without anti-IgM antibodies for 15 min. The cells were then fixed and
analyzed by confocal microscopy. Two different representative confocal
sections are shown for each sample. In addition, flattened projections
(c, f, i, and l) that
represent a composite of the 90-110 sections analyzed for each sample
are shown. The scale bars in k and l
represent 10 µm. The scale is the same for all panels. One of three
experiments that yielded similar results is shown.
PH-EGFP protein. We found that
the Gab1
PH-EGFP protein was mostly cytoplasmic in unstimulated cells
(Fig. 7, g-i) and that its subcellular distribution did not
change significantly upon BCR engagement (j-l). The
Gab1
PH-EGFP protein did not accumulate at the margins of the cells
after BCR engagement. Thus, BCR signaling can recruit Gab1 to the
plasma membrane, and this membrane translocation requires that Gab1
have a PH domain. Our finding that the PH domain of Gab1 is required both for its recruitment to the plasma membrane and for its ability to
potentiate Akt phosphorylation is consistent with the idea that Gab1
must localize to the plasma membrane to amplify BCR signaling.
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Fig. 8.
BCR-induced recruitment of Gab1-EGFP to the
plasma membrane of AtT20/BCR/Syk cells is inhibited by LY294002.
AtT20/BCR/Syk cells expressing Gab1-EGFP were incubated with
buffer alone (a-c) or with 25 µM LY294002
(d-f). The cells were then either left unstimulated or were
incubated with anti-IgM antibodies for 15 min. The cells were fixed and
analyzed by confocal microscopy. Two different representative confocal
sections are shown for the anti-IgM antibody-treated samples, whereas a
single representative section is shown for the unstimulated samples.
The scale bar in f represents 10 µm. The scale
is the same for all panels. One of two experiments with similar results
is shown.
PH did not increase SHP2 tyrosine
phosphorylation (Fig. 9A). This presumably reflects the fact
that the Gab1
PH protein is not tyrosine-phosphorylated to a
significant extent after BCR ligation (Fig. 4A) and
therefore does not bind SHP2 (Fig. 9B).
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Fig. 9.
Gab1 expression potentiates BCR-induced
tyrosine phosphorylation of SHP2; dependence on the PH domain of
Gab1. Vector control cells and WEHI-231 cells expressing either
wild-type (wt) Gab1 or a mutant form of Gab1 that lacks the
PH domain (Gab1 PH) were incubated with or without anti-IgM
antibodies for 2 min. A, cell lysates were
immunoprecipitated (ippt) with anti-SHP2 antibodies, and the
precipitated proteins were analyzed by immunoblotting with an
anti-Tyr(P) monoclonal antibody. The filters were then reprobed with
anti-SHP2 antibodies to show that similar amounts of SHP2 had been
precipitated. Immunoblotting total cell lysates with anti-Gab1
antibodies (lower panel) showed that the cells expressed
similar amounts of wild-type Gab1 and Gab1
PH. One of two independent
experiments that yielded similar results is shown. B, the
binding of SHP2 to wild-type Gab1 and Gab1
PH was assessed by
immunoprecipitating cell lysates with anti-Gab1 antibodies, followed by
immunoblotting with anti-SHP2 antibodies. The filters were then
reprobed with anti-Gab1 antibodies to show that similar amounts of
wild-type Gab1 and Gab1
PH had been precipitated. Molecular mass
standards (in kilodaltons) are indicated to the left. One of four
independent experiments that yielded similar results is shown. All of
the experiments were performed on the same set of cell lysates.
SHP2) and therefore does not bind SHP2 after BCR
ligation (Fig. 10, lower panels) caused only a small
increase in the binding of Grb2 to SHP2 (upper panels).
Thus, the ability of Gab1 to bind SHP2, and to promote its tyrosine
phosphorylation, facilitates the binding of Grb2 to SHP2. This may be
an important mechanism by which Grb2 and Grb2-associated proteins such
as mSOS and SHIP are recruited to the plasma membrane by the BCR.
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Fig. 10.
Binding of SHP2 to Gab1 increases its
ability to bind Grb2 after BCR ligation. Vector control cells and
WEHI-231 cells expressing either wild-type (wt) Gab1 or a
mutant form of Gab1 that lacks the SHP2-binding site (Gab1 SHP2) were
incubated with or without anti-IgM antibodies for 2 min. Upper
panels, cell lysates were immunoprecipitated (ippt)
with anti-SHP2 antibodies, and the precipitated proteins were analyzed
by immunoblotting with anti-Grb2 antibodies. The filters were then
reprobed with anti-SHP2 antibodies to show that similar amounts of SHP2
had been precipitated. Lower panels, the binding of SHP2 to
wild-type Gab1 and Gab1
SHP2 was assessed by immunoprecipitating cell
lysates with anti-Gab1 antibodies, followed by immunoblotting with
anti-SHP2 antibodies. The filters were then reprobed with anti-Gab1
antibodies to show that similar amounts of wild-type Gab1 and
Gab1
SHP2 had been precipitated. Molecular mass standards (in
kilodaltons) are indicated to the left. All of the experiments were
performed on the same set of cell lysates. One of two independent
experiments that yielded similar results is shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B, forkhead family
transcription factors, and the Bcl-2 family member Bad (15). Akt
activation may also increase protein synthesis, an important aspect of
cell growth and activation, by either directly or indirectly regulating
the activity of glycogen synthase kinase-3, the mammalian
TOR/FRAP kinase, and p70 S6 kinase (15, 56). Thus, Gab1 may play an
important role in BCR signaling by amplifying the ability of the BCR to
activate Akt.
PH protein, which
cannot localize to the plasma membrane, did not bind SHP2 or potentiate
BCR-induced phosphorylation of SHP2. This suggests that the activated,
tyrosine-phosphorylated form of SHP2 that binds to Gab1 is associated
with the plasma membrane. Consistent with this idea, we have previously
shown that Gab1·SHP2 complexes are present in the membrane-enriched particulate fraction of anti-Ig antibody-stimulated RAMOS B cells (24).
Thus, in addition to increasing the enzymatic activity of SHP2, Gab1
appears to recruit SHP2 to the plasma membrane. Cell fractionation
studies by Frearson and Alexander (59) have shown that the
majority of SHP2 substrates in T cells are in the membrane fraction.
Gab1 may therefore play an important role in directing SHP2 to where
its major substrates are located.
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ACKNOWLEDGEMENT |
---|
We thank Dr. Elaine Humphrey (Electron Microscopy Facility at the University of British Columbia) for confocal microscope training and for assistance with data collection and analysis.
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FOOTNOTES |
---|
* This work was supported in part by grants from the Canadian Institutes of Health Research (to M. R. G. and L. M.) and by grants from the National Institutes of Health and the American Cancer Society (to A. J. W.).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.
§ Research student of the National Cancer Institute of Canada. Supported with funds provided by the Terry Fox run. Present address: Samuel Lunenfeld Research Inst., Mt. Sinai Hospital, Toronto, Ontario, Canada M5G 1X5.
Recipient of a graduate studentship from the Natural Sciences
and Engineering Research Council of Canada.
Recipient of a postdoctoral fellowship from the Ministerio de
Educacion y Ciencia de Espana.
¶¶ To whom correspondence should be addressed: Dept. of Microbiology and Immunology, University of British Columbia, 6174 University Blvd., Vancouver, British Columbia V6T 1Z3, Canada. Tel.: 604-822-4070; Fax: 604-822-6041; E-mail: mgold@interchange.ubc.ca.
Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M010590200
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ABBREVIATIONS |
---|
The abbreviations used are: BCR, B cell antigen receptor; PI3K, phosphatidylinositol 3-kinase; PIP2, phosphatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol 3,4,5-trisphosphate; mSOS, mammalian SOS; PH, pleckstrin homology; EGFP, enhanced green fluorescent protein; PBS, phosphate-buffered saline; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase.
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