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Int. J. Cancer: 70, 208–213 (1997)
r 1997 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
Publication de l’Union Internationale Contre le Cancer
MULTIPLE Grb2–PROTEIN COMPLEXES IN HUMAN CANCER CELLS
Lakshmi SASTRY*, Tin CAO and C. Richter KING
Department of Biochemistry, Lombardi Cancer Center, Georgetown University Medical Center, Washington DC 20007, USA
Grb2 is an SH2/SH3 domain-containing adaptor protein
that links receptor tyrosine kinases to the ras signaling
pathway. The Grb2-SH2 domain binds phosphotyrosine sequences on activated tyrosine kinases, and one target of the
SH3 domains is the ras-nucleotide-exchange factor Sos1. We
have examined Grb2-protein interactions in human cancer
cells that over-express the receptor tyrosine kinase erbB2.
Our results show that the 2 Grb2-SH3 domains complex with
Sos1, dynamin and at least 4 other proteins (p228, p140, p55,
p28) in these cells. The 2 Grb2-SH3 domains bind these
proteins differently, with the N-terminal SH3 domain interacting preferentially with p228, Sos1, p140 and dynamin. The
C-terminal SH3 domain has higher affinity toward p28. The
Grb2-SH3 domain interactions appear to be similar in erbB2
over-expressing breast, ovarian and lung cancer cells. Also,
the major tyrosine-phosphorylated proteins that associate
with Grb2 in erbB2 over-expressing cancer cells appear to be
erbB2 and Shc. The multiple Grb2-SH3 domain interactions
in these cells may mediate novel cellular functions. Int. J.
Cancer, 70:208–213, 1997.
r 1997 Wiley-Liss, Inc.
The adaptor protein Grb2 plays an important role in signaling by
receptor tyrosine kinases. Grb2 is a ubiquitously expressed 25 kDa
protein composed of a single SH2 domain flanked by aminoterminal and carboxy-terminal SH3 domains (NSH3 and CSH3,
respectively) (Lowenstein et al., 1992). One function of Grb2 is to
link receptor tyrosine kinases to the ras signaling pathway (Gale et
al., 1993; Egan et al., 1993; Buday and Downward, 1993;
Rozakis-Adcock et al., 1993). The Grb2-SH2 domain binds several
tyrosine-phosphorylated receptor type molecules, including erbB2
(Janes et al., 1994), EGFR (Lowenstein et al., 1992) and PDGFR
(Arvidsson et al., 1994), as well as other phosphotyrosinecontaining proteins like Shc (Rozakis-Adcock et al., 1992), insulin
receptor substrate 1 (Skolnik et al., 1993) and focal adhesion kinase
(Schlaepfer et al., 1994). Grb2 binds the ras-guanine nucleotide
exchange factor Sos1 constitutively through its SH3 domains
(Chardin et al., 1993; Li et al., 1993). The Grb2-SH3 domainbinding sequences have been localized to a proline-rich region in
the C-terminus of Sos1 (Chardin et al., 1993; Li et al., 1993). Upon
growth factor stimulation, the Grb2-Sos1 complex binds to activated receptors (Gale et al., 1993; Egan et al., 1993; Buday and
Downward, 1993; Rozakis-Adcock et al., 1993; Lowenstein et al.,
1992) or to an intermediate protein, Shc (Rozakis-Adcock et al.,
1992). It is proposed that binding of the Grb2-Sos1 complex to
phosphorylated receptors activates the ras pathway by bringing
Sos1 to the proximity of the membrane-bound ras (McCormick,
1993). This is supported by the observation that targeting Sos1 to
the plasma membrane is sufficient for the activation of the ras
pathway (Quilliam et al., 1994). Alternatively, Grb2 may activate
Sos1 by relieving the inhibition imposed by the C-terminus of Sos1
on its catalytic activity (Wang, W., et al., 1995).
Grb2 may have other cellular functions as well. Micro-injection
studies with anti-Grb2 antibodies in rat kidney cells suggest that
Grb2 may play a role in signaling from receptor tyrosine kinases to
the small GTP-binding protein Rac (Matuoka et al., 1993). Also,
the Grb2-SH3 domains have been shown to bind several proteins
besides Sos1, including dynamin (Miki et al., 1994), synapsin
(McPherson et al., 1994), p85a (Wang, J., et al., 1995), Sos2 (Yang
et al., 1995b), Cbl (Odai et al., 1995), Vav (Hanazono et al., 1995;
Ye and Baltimore, 1994; Ramos-Morales et al., 1995) and dystroglycan (Yang et al., 1995a). The exact cellular functions of these
Grb2/Grb2 SH3 domain-binding protein (GSBP) complexes are
not known. However, Cbl is implicated in the signal transduction of
erythropoietin and granulocyte macrophage colony-stimulating
factor in hematopoietic cells (Odai et al., 1995), and the GTPase
dynamin has been shown to be important in receptor endocytosis
(van der Bliek et al., 1993). It is therefore likely that Grb2
participates in signaling pathways other than ras.
Grb2 plays an important role in human cancers over-expressing
the receptor tyrosine kinase p185erbB2. In breast cancer cells
over-expressing the erbB2 tyrosine kinase, the ras signaling
pathway is activated, and a higher amount of the Grb2–Sos1
complex is associated with erbB2 (Janes et al., 1994). Moreover,
Grb2 is over-expressed in breast cancer cells and over-expression
correlates with high expression of erbB2 (Daly et al., 1994). These
findings suggest an important role for Grb2 in erbB2-induced
malignancies.
We are interested in investigating the signals carried by Grb2protein complexes in erbB2 over-expressing cancer cells. For this
purpose, we examined Grb2-protein complexes in these cells by
immunoprecipitation of the complexes followed by far-Western
blot analysis using isolated Grb2-SH3 domains. Our results show
that Grb2 can associate with Sos1, dynamin and at least 4 other
proteins (p228, p140, p55, p28) through SH3 domain interactions.
These proteins are bound differentially by the N-terminal and
C-terminal SH3 domains. Also, the Grb2-SH3 domain interactions
are similar in erbB2 over-expressing breast, ovarian and lung
cancer cells and in erbB2-transformed and -untransformed fibroblasts. Our findings support the view that Grb2 may have multiple
functions mediated by distinct SH3-binding proteins.
MATERIAL AND METHODS
Antibodies
Polyclonal antibodies against Grb2 (C-23) and monoclonal
antibodies (MAbs) against GST (SC-138) were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Shc (S14620),
anti-Sos1 (S15520) and anti-dynamin (D25520) MAbs were obtained from Transduction Labs (Lexington, KY). Anti-erbB2 MAbs
(OP15) were from Oncogene Science (Uniondale, NY).
Expression and purification of GST-Grb2 fusion proteins
GST fusion constructs of whole Grb2, N-terminal SH3 (aa 1–59)
and C-terminal SH3 (aa 159–217) domains were expressed in a
modified pGEX 2T-2 expression vector (Pharmacia, Uppsala,
Sweden). For protein expression analysis, fresh transformants were
grown in 3 ml LB with 100 µg/ml ampicillin at 37°C to an OD600 of
0.6–1.0. Expression was induced by adding IPTG to a final
concentration of 1 mM for 1 hr; protein was analyzed on an 8-16%
SDS-PAGE gel. GST-fusion proteins were purified using glutathione beads according to the manufacturer’s instructions (Glutathione Sepharose 4B Instructions, Pharmacia). Purified proteins were
dialyzed against PBS and stored in aliquots at 270°C.
Cell lines
MDA-MB-453, SK-BR-3, MCF-7, SK-OV-3, Calu-3, NIH 3T3
and NIH 3T3/erbB2 cells were grown in IMEM supplemented with
10% FBS (Biofluids, Gaithersburg, MD) and 1 mM glutamine
(GIBCO, Gaithersburg, MD) in 150 3 25 mm dishes. Twenty-four
*Correspondence to: E508, Research Building, 3970 Reservoir Rd., NW,
Washington, DC 20007, USA. Fax: (202) 687-7505.
Received 19 June 1996; revised 4 October 1996.
Grb2-BINDING PROTEINS
209
hours before harvesting, cells were washed twice with sterile PBS
(GIBCO) and grown in IMEM supplemented with 10% FBS and 1
mM glutamine or IMEM supplemented with 1% FBS and 1 mM
glutamine (for starving cells) at 37°C, in a 5% CO2 incubator.
Immunoprecipitation, far-Western blotting and Western blotting
For immunoprecipitation experiments, harvested cells were
rinsed twice with cold PBS and lysed with the addition of 0.5 ml
cold immunoprecipitation buffer (1% Triton X-100, 150 mM NaCl,
50 mM Hepes [pH 7.5], 10% glycerol, 1.5 mM MgCl2, 1 mM
EGTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM PMSF, 200
µM NaVO4 and 100 mM NaF) for 10 min at 4°C. Lysed cells were
centrifuged at 4°C for 10 min at 4,850g, and the supernatant, the
total native cell lysate, was collected. Total protein concentration of
the cell lysate was determined by the BCA method (BioRad,
Hercules, CA).
For immunoprecipitations, 12.5 µg of rabbit polyclonal IgG
against Grb2 or a control rabbit IgG were added to 5 mg of total
native cell lysate and incubated with rotation at 4°C for 2 hr.
Protein A-agarose (100 µl; Santa Cruz Biotechnologies) was then
added and the mixture incubated with rotation at 4°C overnight.
Protein A beads were recovered by centrifugation at 1,485g for 1
min. After 4 washes with cold immunoprecipitation buffer, 60 µl of
23 sample buffer (ISS, Natico, MA) were added to the agarose
beads, boiled for 8 min and stored at 220°C.
For far-Western blots, immunoprecipitated material was resolved on SDS-PAGE gels; blotted onto nitrocellulose filters; and
analyzed using 0.5 µg/ml of GST, GST-NSH3 or GST-CSH3
proteins. Blots were blocked in 5% BSA in TBST (20 mM Tris-HCl
[pH 7.5], 150 mM NaCl, 0.1% Tween 20) overnight and then in 3
mM reduced glutathione (Sigma, St. Louis, MO) in 5% BSA-TBST
for 1 hr at room temperature (RT). Prior to incubation of the blots in
GST, GST-NSH3 or GST-CSH3 proteins, proteins were preincubated in 3 mM reduced glutathione for 1 hr at RT. Following
this, blots were incubated for 1 hr at RT with the appropriate fusion
protein, washed 43 with TBST and incubated for 1 hr at RT with
anti-GST MAbs in 5% BSA-TBST at 0.5 µg/ml. Blots were then
washed with TBST and incubated with sheep anti-mouse horseradish peroxidase (Amersham, Arlington Heights, IL) at 1:5,000
dilution in 5% BSA-TBST for 1 hr at RT. After extensive washing
in TBST, blots were visualized with enhanced chemiluminescence
(ECL; Amersham).
For direct Western blotting, Grb2 or control immunoprecipitates
were electrophoresed, transferred to nitrocellulose membranes,
probed with MAbs against Sos1 (1/250 dilution), dynamin (1/1,000
dilution), Shc (1/1,000 dilution) or erbB2 (1/1,000 dilution) and
detected using sheep anti-mouse IgG-HRP conjugates and chemiluminescence. In co-precipitation experiments, Grb2 immunoprecipitates were probed with anti-Grb2 MAbs (1/1,000 dilution; Transduction Labs).
RESULTS
Grb2-protein complexes in erbB2 over-expressing human
cancer cells
To investigate Grb2-SH3 domain specificities in cancer cells, we
first immunoprecipitated Grb2 under conditions where the SH3
domain complexes are intact and detected co-precipitating proteins
by far-Western analysis using the GST fusion proteins of Grb2-SH3
domains (IP-far-Western). Proteins detected in this analysis most
likely bind Grb2 through the SH3 domains. For this analysis, it was
important to choose an antibody that specifically immunoprecipitated Grb2 and had minimal cross-reactivity with other SH2/SH3
domain-containing proteins. The specificity of the antibody used in
these studies was determined by first immunoprecipitating Grb2
and blotting the precipitated material with the same immunoprecipitating antibody (Fig. 1). These results show that the antibody is
specific for Grb2 and has little cross-reactivity with other cellular
proteins under our experimental conditions.
FIGURE 1 – Specificity of the anti-Grb2 antibody. Lysates from
erbB2-over-expressing (MDA-MB-453, SK-BR-3) and non-overexpressing (MCF-7) breast cancer cells were prepared, and Grb2 was
immunoprecipitated from each cell lysate (5 mg) using anti-Grb2
antibodies. Immunoprecipitated material was resolved on 8–16%
SDS-PAGE gels and analyzed by Western blotting with anti-Grb2
antibodies. Anti-Grb2 antibodies used for immunoblotting were the
same as those used for immunoprecipitation. Blots were detected with
ECL. Arrows indicate Grb2, heavy chain (HC) and light chain (LC);
m.w. markers are shown in kDa.
The Grb2-SH3 domain complexes were first examined in human
breast cancer cells over-expressing the erbB2 receptor tyrosine
kinase (SK-BR-3, MDA-MB-453) and non-over-expressing cells
(MCF-7) under serum-treated and serum-starved conditions. IP-farWestern analysis shows that the Grb2-SH3 domains bind 6 proteins
(p228, p170, p140, p125, p55, p28) that co-precipitate with Grb2 in
these cells (Fig. 2). This binding is specific for SH3 domains as
GST shows no reactivity when used to probe the blots of Grb2
immunoprecipitates (Fig. 2, GST). The SH3 domains show minimal reactivity with proteins immunoprecipitated with control
antibodies, indicating that far-Western blot analysis detects proteins associating with Grb2 specifically (Fig. 2, lane NS). The
specificity for Grb2-binding proteins is further supported by the
fact that far-Western blot analysis of Sos1 immunoprecipitates with
Grb2-SH3 domains detects only Sos1 (data not shown). Interestingly, the Grb2-NSH3 domain shows higher affinity toward most
proteins detected in the far-Western analysis, including p228, p170,
p140 and p125. The CSH3 domain appears to have preferential
interaction with p28. Both domains seem to interact with the 55
kDa protein.
The Grb2-binding proteins detected in this assay are similar in
erbB2-over-expressing (MDA-MB-453, SK-BR-3) and -non-
210
SASTRY ET AL.
FIGURE 2 – Grb2-protein complexes in breast cancer cells. Lysates were prepared from serum-treated and serum-starved erbB2-over-expressing
(MDA-MB-453, SK-BR-3) and non-overexpressing (MCF-7) breast cancer cells. Grb2-protein complexes were immunoprecipitated from each
lysate (5 mg) with anti-Grb2 antibodies. Immunoprecipitation was also carried out with a control antibody from MDA-MB-453 cell lysates (NS).
Immunoprecipitated material was resolved on 8–16% SDS-PAGE gels and analyzed by far-Western blotting with GST, GST-NSH3 or GST-CSH3
proteins. Grb2-SH3 domain-binding proteins (p228, p170, p140, p125, p55, p28) are shown by arrows. Sos1-c, the purified C-terminal region of
Sos1, binds the Grb2-NSH3 domain (Sastry et al., 1995) and is included as a positive control for far-Western blotting; m.w. markers are shown in
kDa.
FIGURE 3 – Grb2-protein complexes in erbB2-over-expressing cancer cells. Lysates were prepared from serum-treated erbB2-over-expressing
ovarian (SK-OV-3), lung (Calu-3) and breast (MDA-MB-453, SK-BR-3) cancer cells and erbB2-transformed (N/erbB2) and untransformed
fibroblasts (NIH 3T3). Grb2-protein complexes were immunoprecipitated from each lysate (5 mg) with anti-Grb2 antibodies and analyzed by
far-Western blotting with GST, GST-NSH3 or GST-CSH3 proteins. Grb2-SH3 domain-binding proteins (p228, p170, p140, p125, p55, p28) are
shown by arrows; m.w. markers are shown in kDa. NS is the immunoprecipitation with a control antibody from MDA-MB-453 cell lysates, and
Sos1-c is the positive control for far-Western blotting as described in Figure 2.
overexpressing (MCF-7) cells (Fig. 2). The assay also shows that
Grb2 binds similar m.w. proteins in starved and serum-treated cells,
though there was an increase in the amount of Grb2-binding
proteins in serum-treated MDA-MB-453 and MCF-7 cells (Fig. 2).
No quantitative differences are seen in Grb2-binding proteins from
starved or serum-treated SK-BR-3 cells. The quantitative increase
in Grb2-binding proteins in MDA-MB-453 and MCF-7 cells is
most likely due to a higher amount of Grb2 in these cells upon
serum treatment (data not shown).
The receptor tyrosine kinase erbB2 is also over-expressed in
certain lung and ovarian cancer cell lines and primary tumors. To
investigate if there are any tissue-specific GSBPs, we compared
Grb2-associating proteins in erbB2 over-expressing breast (SKBR-3, MDA-MB 453), ovarian (SK-OV-3) and lung (Calu-3)
cancer cells by IP-far-Western analysis (Fig. 3). This comparison
shows that the major proteins associated with Grb2-SH3 domains
(p228, p170, p140, p125, p55, p28) are similar in the breast, lung
and ovarian cancer cells tested (Fig. 3). A minor band detected at
approx. 160 kDa in Grb2 immunoprecipitates from all cells may be
a degradation product as this is not seen reproducibly. A 46 kDa
band seen in all cells does not appear to be specifically associated
with Grb2 as this is present in the control antibody immunoprecipitates as well (Fig. 3, lane NS). Also, untransformed and erbB2transformed NIH 3T3 (N/erbB2) cells show similar Grb2-SH3
domain-associating proteins (Fig. 3). In these cells, a protein
slightly smaller than 228 kDa is observed. It is likely that this is the
p228 protein observed in human cancer cells, and the slightly
smaller size may be due to a species difference.
The identity of Grb2-binding proteins
To examine whether any of the Grb2-SH3 domain-associating
proteins in our study correspond to previously characterized
Grb2-binding proteins, we carried out co-precipitation experiments. For these experiments, Grb2 immunoprecipitates from
erbB2-over-expressing SK-BR-3 cells were probed with antibodies
against known GSBPs. As shown in Figure 4, probing with the
anti-Sos1 antibodies indicates that the 170 kDa band detected in the
far-Western blot analysis is Sos1. The 125 kDa protein was
identified as dynamin by similar co-precipitation experiments using
anti-dynamin antibodies (Fig. 4). The identity of the 140 kDa
Grb2-BINDING PROTEINS
211
MCF7 cells that do not over-express erbB2, the predominant
phosphoproteins in Grb2 immunoprecipitates appear to be Shc and
a 80 kDa protein that does not react with Shc antibodies (Fig. 5b).
The 80 kDa protein also associates with Grb2 in MDA-MB-453
cells.
DISCUSSION
FIGURE 4 – Identity of Grb2-binding proteins. Grb2 was immunoprecipitated from lysates of serum-treated SK-BR-3 cells with anti-Grb2
antibodies (I) or control antibodies (NA), electrophoresed and blotted
onto nitrocellulose filters. Blots were probed with anti-Grb2 (a),
anti-Sos1 (b) or anti-dynamin (c) antibodies and detected with sheepanti-mouse-HRP conjugates using ECL. Grb2, Sos1 and dynamin are
indicated by arrows. The 28 kDa protein detected in the far-Western
studies has a different electrophoretic mobility than Grb2 and is
unlikely to be Grb2 itself; m.w. markers are shown in kDa.
protein is unknown, though a similar m.w. protein was found to
complex with Grb2 in hematopoietic cells (Pelicci et al., 1995) and
in synaptic fractions (McPherson et al., 1994). A 145 kDa protein
capable of binding Grb2-SH3 domains has been identified as
synaptojanin in brain tissue (McPherson et al., 1996). However,
probing Grb2 immunoprecipitates with anti-synaptojanin antibodies showed no reactivity, indicating that the 140 kDa protein seen in
our studies is unlikely to be synaptojanin (McPherson, personal
communication). Previous studies indicate that Grb2 can associate
with Cbl (Odai et al., 1995), Vav (Hanazono et al., 1995;
Ramos-Morales et al., 1994; Ramos-Morales et al., 1995) and
p85a (Wang, J., et al., 1995) constitutively in hematopoietic cells
and in mouse fibroblasts, respectively. Antibodies against Cbl and
p85a showed no reactivity with Grb2 IPs, though both were
expressed in all of the cells that we tested (data not shown). Vav is
expressed exclusively in cells of hematopoietic origin and is not
expected to associate with Grb2 in the cells being examined. The
identity of p228, p55 and p28 is currently not known, and they have
previously not been shown to associate with Grb2-SH3 domains.
The 28 kDa protein is unlikely to be Grb2 itself as it has a slightly
different electrophoretic mobility than Grb2 and is recognized by
Grb2-SH3 domains.
Phosphotyrosine proteins associated with Grb2 in
erbB2-over-expressing cancer cells
To investigate the major tyrosine-phosphorylated proteins associated with Grb2 in erbB2-over-expressing cancer cells, we conducted direct Western blotting of Grb2 immunoprecipitates with
anti-phosphotyrosine antibodies (Fig. 5a). This analysis shows that
there are 3 major phosphoproteins (p200, p52, p46) associated with
Grb2 in erbB2-over-expressing breast and ovarian cancer cells.
Longer exposure shows the presence of these bands in erbB2-overexpressing lung cancer cells (Calu-3) as well. Immunoblotting with
anti-erbB2 and anti-Shc antibodies identified the p200 and p52/p47
proteins as erbB2 and Shc, respectively (Fig. 5b). The slightly
higher m.w. of the erbB2 protein in these experiments is due to the
use of gradient gels. This study suggests that in erbB2-overexpressing cancer cells, Grb2 can associate with both erbB2 and
Shc (Fig. 5b). This association is most likely through the SH2
domain, as previously described (Rozakis-Adcock et al., 1992;
Daly et al., 1994). None of the GSBPs observed in our study were
tyrosine-phosphorylated under these experimental conditions. In
The major function of Grb2-SH3 domains is thought to be
association with the ras nucleotide exchange factor Sos1, thus
linking tyrosine kinase activation to the ras signaling pathway
(Lowenstein et al., 1992; Gale et al., 1993; Egan et al., 1993;
Buday and Downward, 1993; Rozakis-Adcock et al., 1993).
However, Grb2 may have other cellular functions as it has been
shown to bind a number of proteins, including dynamin, synapsin,
Cbl and Vav. Our results show that Grb2 can associate with at least
4 other proteins (p228, p140, p55, p28) besides Sos1 and dynamin,
through SH3 domain interactions (Fig. 6). These proteins coprecipitate with Grb2 and interact directly with the Grb2-SH3
domains, indicating the existence of at least 6 different pools of
Grb2-protein complexes in cells, including Grb2–Sos1 and Grb2–
dynamin complexes. Each of these Grb2 complexes may mediate a
different function. Our results, therefore, strongly support the
suggestion that Grb2 has other cellular functions in addition to
linking receptor tyrosine kinases to the ras signaling pathway
(McPherson et al., 1994; Miki et al., 1994; Odai et al., 1995).
The Grb2–GSBP complexes detected in our study are constitutively formed as they are similar in serum-starved and serumtreated cells. This is expected of Grb2–Sos1 (Rozakis-Adcock et
al., 1993; Egan et al., 1993; Buday and Downward, 1993) and
Grb2–dynamin (Miki et al., 1994) interactions and is consistent
with the fact that most reported Grb2–SH3 domain interactions are
constitutive and are not altered in response to cell growth conditions (Miki et al., 1994; Odai et al., 1995; Ramos-Morales et al.,
1994). In addition, near identical Grb2–GSBP complexes are seen
in erbB2-transformed and untransformed NIH 3T3 cells, indicating
that a powerful transforming signal does not alter the state of the
complexes. Similar Grb2–GSBP complexes are seen in fibroblasts
and in tumor epithelial cells derived from different tissues,
suggesting that the complexes may have important cellular functions and that they are not unique to transformed cells.
Our results show that the 2 Grb2-SH3 domains have different
binding specificities. The NSH3 domain shows higher affinity
toward most proteins seen in the far-Western blot analysis,
including p228, Sos1, p140 and dynamin. The CSH3 domain
shows minimal interaction with all of these proteins and preferentially interacts with p28. Only one protein, p55, appears to have
similar affinity toward both SH3 domains. These results suggest
that the NSH3 and CSH3 domains of Grb2 may have different
functions. This is supported by studies on genetic mutations in
these domains in the Caenorhabditis elegans Grb2 homologue
(Sem-5), which lead to different phenotypes (Clark et al., 1992).
We and others have previously shown that the Grb2–Sos1 interaction is primarily mediated by the NSH3 domain of Grb2 (Reif et al.,
1994; Sastry et al., 1995). The CSH3 domain has minimal
reactivity toward Sos1, and its cellular function is not completely
clear. Dominant negative mutants of Grb2 lacking the NSH3 or
CSH3 domain have different effects on the transformed phenotype
caused by mutation-activated neu and on p185-mediated ras
activation. The NSH3 domain deletion has a much stronger effect
than the CSH3 deletion (Xie et al., 1995). These results are
consistent with the fact that Grb2-Sos1 binding is mediated by the
NSH3 domain in cells. The authors also suggest the existence of
multiple routes to ras activation based on the observation that the
Grb2 dominant negative mutants do not completely suppress ras
activation. Our results identify proteins that may participate in such
pathways.
212
SASTRY ET AL.
FIGURE 5 – Phosphotyrosine proteins associated with Grb2 in erbB2-over-expressing cancer cells. Lysates were prepared from serum-treated
erbB2-over-expressing ovarian (SK-OV-3), lung (Calu-3) and breast (MDA-MB-453, SK-BR-3) cancer cells and non-overexpressing breast
cancer cells (MCF-7). Grb2 and Grb2-binding proteins were immunoprecipitated from each lysate (5 mg) with anti-Grb2 antibodies and analyzed
by Western blotting with (a) anti-phosphotyrosine antibodies and (b) anti-erbB2 or anti-Shc antibodies. In (a), the major phosphotyrosine proteins
in Grb2 immunoprecipitates are indicated by arrows; m.w. markers are shown in kDa. In (b), erbB2 (top panel) and the 2 isoforms of Shc (bottom
panel) are indicated. Longer exposures clearly show the p46 isoform of Shc in MCF-7 cells. In (b), NS represents an immunoprecipitation with
control antibodies from the 453 cell lysate.
FIGURE 6 – Schematic representation of Grb2-protein complexes in
erbB2-over-expressing human cancer cells. The Grb2-SH3 domainbinding proteins and the major SH2 domain-binding proteins in
erbB2-over-expressing human cancer cells are shown.
v-src-transformed or fibronectin-treated NIH 3T3 cells (Schlaepfer
et al., 1994) and Gab1 (Holgado-Madruga et al., 1996) in
insulin/EGF-stimulated A431 cells. However, under our experimental conditions, Grb2 seems to associate mostly with erbB2 and Shc
in erbB2-over-expressing cancer cells. This strongly suggests that
in these cells Grb2 is primarily involved in transmitting signals
from erbB2 and Shc. Similarly, in non-erbB2-over-expressing
breast cancer cells (MCF-7), Shc is a major up-stream target of
Grb2. Phosphorylation of Shc in response to growth factors and
subsequent recruitment of the Grb2–Sos1 complex is a major
mechanism for the activation of ras signaling (Rozakis-Adcock et
al., 1992). Our results add to the evidence for the importance of the
association of Grb2 with erbB2 or Shc in cancer cells.
Our findings reveal the existence of multiple Grb2-protein
interactions in human cancer cells and substantiate the idea that
Grb2 may have several cellular functions. The elucidation of novel
Grb2 functions may require the identification and characterization
of the Grb2-SH3-binding proteins described.
ACKNOWLEDGEMENTS
The major tyrosine-phosphorylated proteins associated with
Grb2 in erbB2-over-expressing cancer cells appear to be erbB2 and
Shc (Fig. 6). In these cells, none of the GSBPs are tyrosinephosphorylated, indicating that they are not substrates for tyrosine
kinases. Grb2 has previously been reported to bind other tyrosinephosphorylated proteins, including focal adhesion kinase (FAK) in
We thank Dr. P. McPherson (McGill University, Canada) for
probing Grb2 immunoprecipitation blots with anti-synaptojanin
antibodies. We also thank Drs. M. Lippman, D. Goldstein and A.
Dent for critically reviewing the manuscript and Drs. K. Rosenberg
and M. Hizaji for helpful suggestions and comments. This research
was supported in part by a generous gift from Mr. William Baker.
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