(Received for publication, July 28, 1995; and in revised form, October 3, 1995)
From the
To clarify the role of the Shc-Grb2-Sos trimer in the oncogenic
signaling of the point mutation-activated HER-2/neu receptor tyrosine
kinase (named p185), we interfered with the protein-protein
interactions in the ShcGrb2
Sos complex by introducing Grb2
mutants with deletions in either amino- (
N-Grb2) or carboxyl-
(
C-Grb2) terminal SH3 domains into B104-1-1 cells derived from
NIH3T3 cells expressing the point mutation-activated HER-2/neu. We found that the transformed phenotypes of the
B104-1-1 cells were largely reversed by the
N-Grb2. The effect of
the
C-Grb2 was much weaker. Biochemical analysis showed that the
N-Grb2 was able to associate Shc but not p185 or Sos, while the
C-Grb2 bound to Shc, p185, and Sos. The p185-mediated Ras
activation was severely inhibited by the
N-Grb2 but not the
C-Grb2. Taken together, these data demonstrate that interruption
of the interaction between Shc and the endogenous Grb2 by the
N-Grb2 impairs the oncogenic signaling of the activated p185,
indicating that (i) the
N-Grb2 functions as a strong
dominant-negative mutant, and (ii) Shc/Grb2/Sos pathway plays a major
role in mediating the oncogenic signal of the activated p185. Unlike
the
N-Grb2,
C-Grb2 appears to be a relatively weak
dominant-negative mutant, probably due to its ability to largely
fulfill the biological functions of the wild-type Grb2.
The HER-2/neu (also known as erbB-2)
protooncogene encodes an M 185,000 transmembrane
glycoprotein with intrinsic tyrosine kinase activity homologous to the
epidermal growth factor (EGF) (
)receptor(1, 2, 3, 4, 5, 6) .
The transforming potential of the HER-2/neu receptor tyrosine kinase
(named p185) has been well documented in both clinical analysis and
experimental
studies(7, 8, 9, 10, 11) .
The mechanisms of aberrant activation of p185 have also been
extensively
investigated(12, 13, 14, 15, 16, 17, 18, 19) .
A carcinogen-induced point mutation replacing a valine residue with a
glutamic acid in the transmembrane domain confers transforming ability
on p185(12) . Alternatively, overexpression of the wild-type
p185 can also induce neoplasia
transformation(13, 14, 15) . Both mutation
and overexpression are believed to result in enhancing formation and
stabilization of receptor dimers, which allow the p185 tyrosine kinase
to maintain in its active
status(16, 17, 18, 19) . However,
the downstream signaling pathway relaying the oncogenic signal
triggered from the abnormally activated p185 is not well defined,
likely due to the absence of a consensus of its
ligand(20, 21, 22, 23, 24, 25, 26) .
Activation of Ras is an important convergence point in the mitogenic
signaling pathway of receptor tyrosine kinases(27) . A key
upstream pathway leading to Ras activation by receptor tyrosine kinases
has recently been established, primarily as a result of studies with
the receptors for EGF, platelet-derived growth factor, and insulin (28, 29, 30, 31, 32, 33) .
The most important components of this pathway include Shc, Grb2, and
Sos. Shc stands for SH2 domain-containing 2 collagen-related
proteins. The Shc family consists of three isoforms (34) . The
p46
and p52
isoforms come from the same
transcript with different translation initiation sites. The p66
species most likely arises from a distinct transcript. Tyrosine
phosphorylation of Shc provides a docking site for Grb2 which was
originally identified as a growth factor receptor-bound
protein(35) , a mammalian homolog of Caenorhabditis elegans Sem-5 and Drosophila Drk (36, 37) . Grb2
is a 24-kDa adaptor protein containing an SH2 domain flanked by two SH3
domains. Through the SH3 domains, Grb2 constitutively associates with
Sos (named for the Son of Sevenless gene), a 150-kDa
guanyl-nucleotide exchange factor for
Ras(38, 39, 40, 41) , by targeting
the proline-rich motif at its carboxyl terminus. Upon ligand
stimulation, most receptor tyrosine kinases examined to date have been
able to induce tyrosine phosphorylation of Shc, which subsequently
binds to the SH2 domain of Grb2. The formation of the
Shc
Grb2
Sos ternary complex has been proposed to play an
important role in activating
Ras(28, 29, 30, 31, 32, 33) .
Alternatively, the Grb2
Sos complex can be directly recruited to
the activated EGF receptor(43) . Activation of Ras leads to
stimulation of downstream kinase cascades, which at least include
Raf-1/MEK/MAPK and MEKK-1/JNKK/JNK pathways(44) .
Unlike EGF
receptor and other receptor tyrosine kinases, the mutation-activated
p185 tyrosine kinase is constitutively active in the absence of
exogenously added ligand. Although activation of Ras has been proposed
to play an important role in the oncogenic signaling of the
mutation-activated p185(45) , coupling of p185 to Ras via
ShcGrb2
Sos or Grb2
Sos or both has not been yet
determined. Our recent data and that of others indicated that tyrosine
phosphorylation of Shc and formation of the Shc
Grb2 complex
occurred in transformed NIH3T3 cells that express the
mutation-activated p185 and human breast cancer cells that overexpress
p185, which suggests that the Shc/Grb2/Sos/Ras pathway may be
responsible for transmitting the oncogenic signal from the activated
p185(46, 55) . One way to provide more direct evidence
to support this idea is to interfere with the protein-protein
interactions involved in this pathway by using dominant-negative
mutants and then examine whether these mutants can reverse the
transformed phenotypes caused by the activated p185. Grb2 is a central
component in this pathway. On the one hand, it binds tightly to Sos
through its SH3 domains; on the other hand, it can bind to Shc and
probably the activated p185 as well through its SH2 domain.
Interestingly, recent studies suggested that the SH2 and SH3 domains of
Grb2 functioned independently(47, 48) . Binding of
phosphopeptides to the SH2 domain of Grb2 does not appreciably affect
the association of its SH3 domains with proline-rich peptides.
Conversely, binding of excessive peptides derived from Sos to Grb2 does
not influence the interaction between its SH2 domain with
phosphopeptides. We, therefore, reasoned that deleting one of the SH3
domains of Grb2 might create dominant-negative mutants which compete
with the endogenous Grb2
Sos complex for Shc or activated p185 and
block the oncogenic signaling pathway of the activated p185. To test
this hypothesis we transfected either an amino-terminal or an
carboxyl-terminal SH3 domain deletion mutant of Grb2 into B104-1-1
cells which are transformed NIH3T3 cells expressing the
mutation-activated p185. We found that the transformed phenotypes of
B104-1-1 were largely reversed by the amino-terminal SH3 domain
deletion mutant of Grb2 (
N-Grb2). The effect of the
carboxyl-terminal SH3 domain deletion mutant (
C-Grb2) on
phenotypic reversion was much weaker. Biochemical analysis data
indicated that the
N-Grb2 functioned as a strong dominant-negative
mutant, whereas the
C-Grb2 seemed to be a weak one. These results
support the notion that the Shc/Grb2/Sos pathway plays an important
role in the oncogenic signaling of the mutation-activated p185.
Figure 1:
A, schematic representation of Grb2
deletion mutants. N-Grb2 represents the Grb2 mutant containing
only the SH2 and the carboxyl-terminal SH3 domains.
C-Grb2 stands
for the Grb2 mutant carrying only the SH2 and the amino-terminal SH3
domains. B, SH3 domain deletion Grb2 mutants suppress the
transforming ability of the mutation-activated neu. Focus
forming assays were performed as described under ``Experimental
Procedures.'' The resulting number of foci from each transfection
was corrected for transfection efficiency by dividing by the number of
G418-resistant colonies created by the same transfection. Results are
expressed as percent of foci in control transfection with cNeu-104
(100%). Data shown here are the average from three individual
experiments. Standard deviations are shown by error
bars.
Figure 2:
Morphologic reversion of the transformed
B104-1-1 cells by N-Grb2. A, immunoblot analysis of
expression of the Grb2 deletion mutants stably transfected into
B104-1-1 cells. B104-1-1 is a transformed cell line derived from NIH3T3
by stable transfection of the point mutation-activated HER-2/neu. Vector, Grb2
C-11, and Grb2
N-11 are stable
transfectants derived from B104-1-1 cells, expressing vector alone,
C-Grb2, and
N-Grb2, respectively. Fifty micrograms of cell
extracts from various cell lines as indicated were subject to 10%
SDS-PAGE. After transfer, the top portion of the nitrocellulose filter
was probed with c-Neu-Ab3, a monoclonal anti-p185 antibody, the lower
part was incubated with either 12CA5, a monoclonal antibody specific to
the HA1 tag (middle panel), or monoclonal anti-Grb2 antibody (bottom panel). Endogenous Grb2 is indicated by an arrowhead. B, morphology of various cell lines. a, NIH3T3; b, B104-1-1; c, Vector; d, Grb2
C-11; and e,
Grb2
N-11.
To
ascertain whether HER-2/neu was still expressed in the stable
transfectants, we performed immunoblot analysis with c-neu-Ab3, a
monoclonal antibody specific to p185. As shown in Fig. 2A, the expression level of p185 was comparable
between the parental B104-1-1 and its transfectants, indicating that
the morphologic reversion seen in Grb2N-11 cells was not due to
spontaneous loss of the HER-2/neu gene or down-regulation of HER-2/neu expression by the
N-Grb2 mutant. Similarly, the
levels of endogenous Grb2 were comparable between the parental B104-1-1
line and its transfectants along with NIH3T3 cells as shown in Western
analysis with anti-Grb2 monoclonal antibody (Fig. 2A).
Both
C-Grb2 and
N-Grb2 were also recognized by the same
anti-Grb2 monoclonal antibody (Fig. 2A).
To evaluate
more precisely the phenotypic reversion caused by the SH3 domain
deletion Grb2 mutants, we performed microfocus forming assays (52) and soft agar colony formation assays to compare the
transformed properties of the parental B104-1-1 cell line and its
stable transfectants. As shown in Table 1, the focus formation
efficiency of Grb2N-11 cells dramatically decreased. For example,
the number of foci formed by the Grb2
N-11 cells was less than 25%
that of the parental B104-1-1 cells. In addition, the size of foci
formed by the Grb2
N-11 cells were much smaller than the parental
B104-1-1 cells. As expected, the Vector control cell line displayed a
formation efficiency similar to the B104-1-1 cell line. The
Grb2
C-11 cell line showed only a 30% reduction in focus formation
when compared to the parental B104-1-1 line and the foci were slightly
smaller. Consistent with the data from the microfocus forming assays,
soft agar colony formation assays also indicated that the
transformation potency of the Grb2
N-11 cells was significantly
weakened, while the Grb2
C-11 cells were only moderately affected (Table 1). The phenotypic reversion observed in the microfocus
forming assays and soft agar colony assays was not due to the decreased
growth rates of Grb2
N-11 and Grb2
C-11 cells demonstrated in in vitro growth rate assays (data not shown), since extending
the culture time for these two cell lines in the above assays did not
result in extra numbers of foci or colonies (data not shown). Taken
together, our data indicated that the transformed phenotypes of
B104-1-1 cells could be largely reversed by stable transfection of the
N-Grb2 mutant. The
C-Grb2 mutant had a relatively weak
effect.
Figure 3: Association of Shc or p185 with the truncated Grb2 products. Two milligrams of cell extracts from different cell lines were used in immunoprecipitation with monoclonal anti-HA1 antibody. Immunocomplexes or 50 µg of lysates from B104-1-1 cells were separated on a 6-12% gradient SDS-PAGE. The filter was cut into three pieces after transfer. The top portion was probed with c-Neu-Ab3 for associated p185, the middle was incubated with polyclonal anti-Shc antibody for associated Shc, and the bottom portion was incubated with monoclonal anti-HA1 antibody, in order to evaluate equal loading. The band above the truncated Grb2 products was most likely from IgG light chain.
Detecting the
association between Shc and the truncated Grb2 proteins prompted us to
ask whether the interaction of Shc and the endogenous Grb2 is inhibited
in Grb2N-11 and Grb2
C-11 cells. To address this issue, we
used anti-Shc antibody to precipitate Shc and associated proteins,
followed by immunoblotting with anti-Grb2 or anti-Shc antibodies. As
shown in Fig. 4, the endogenous Grb2 co-precipitated by the
anti-Shc antibody dramatically decreased in both Grb2
N-11 and
Grb2
C-11 cell lines, as compared to that in the parental B104-1-1
and the vector control cell lines. Consistently, co-immunoprecipitation
of the
C-Grb2 and
N-Grb2 by anti-Shc antibody was detected by
the anti-Grb2 antibody. Equal loading was confirmed by Western analysis
with the anti-Shc antibody. These results are consistent with that seen
in Fig. 3, which showed that both
N-Grb2 and
C-Grb2
products bound to p52
. We, therefore, concluded that both
N-Grb2 and
C-Grb2 were able to compete with the endogenous
Grb2 for Shc.
Figure 4:
The association between Shc and the
endogenous Grb2 is impaired by both N-Grb2 and
C-Grb2. Two
milligrams of lysates from each cell line were immunoprecipitated with
a polyclonal anti-Shc antibody. Immunocomplexes were dissected by 10%
SDS-PAGE, followed by immunoblotting analysis with either monoclonal
anti-Grb2 antibody (top panel) or polyclonal anti-Shc antibody (lower panel). Immunoprecipitated Shc proteins and
co-immunoprecipitated endogenous Grb2 are
indicated.
Figure 5:
Endogenous Grb2Sos association is
interfered with the
C-Grb2 but not the
N-Grb2. One milligram
of lysate from each cell line as indicated was incubated with
polyclonal anti-Sos antibody. Immunocomplexes were separated by 10%
SDS-PAGE, followed by Western analysis with either monoclonal anti-Grb2 (upper panel) or polyclonal anti-Sos antibody (lower
panel).
Figure 6:
Effect of expression of SH3 domain
deletion mutants of Grb2 on Ras activation. Cells were labeled with P
in phosphate-free medium at 25,000
cells/cm
. Cell lysates were prepared and subjected to
immunoprecipitation with anti-Ras monoclonal antibody Y13-259. Guanine
nucleotides precipitated with Ras were separated with thin-layer
chromatography. The amount of Ras-GTP was expressed as a percentage of
the amount of Ras-GDP plus Ras-GTP. The GTP-Ras percentage in the
B104-1-1 cells was arbitrarily set at 100%. The percentage of Ras-GTP
in other cell lines were standardized against that of the B104-1-1.
Data shown here represent the average of two separate experiments.
Deviations are less than 10%.
Grb2 consists of a single SH2 domain and two SH3 domains.
Previous studies have indicated that Grb2 is a key component of the
pathway leading to Ras activation by receptor tyrosine
kinases(56) . In the present study we tested whether deletion
of the amino- or carboxyl-SH3 domain of Grb2 could create
dominant-negative mutants which are capable of binding to
tyrosine-phosphorylated Shc or mutation-activated p185 but are unable
to associate with Sos. We speculated that these mutants could interfere
with the recruitment of the SosGrb2 complex to Shc or p185,
leading to inhibition of Ras activation. Our data demonstrated here
that the
N-Grb2 functioned as a dominant-negative mutant that
suppressed by more than 65% the activation of Ras by the
mutation-activated p185 and largely reversed the transformed phenotypes
of B104-1-1 cells. The
C-Grb2 appears to be a weak
dominant-negative mutant. It down-regulated Ras activation, by only
25%, and slightly induced phenotypic reversion of the B104-1-1 cells.
Similar results have been recently obtained for the oncogenic Bcr-Abl
tyrosine kinase (57) . A
N-Grb2 mutant suppresses
Bcr-Abl-mediated Ras activation and reverses the transformed phenotype.
As shown here, the
C-Grb2 mutant is less effective in reversing
Bcr-Abl-induced transformation as compared to the
N-Grb2.
It is
of interest to note that the dominant-negative effect of the
C-Grb2 is much weaker than that of the
N-Grb2 even though the
C-Grb2 is able to compete with the endogenous Grb2 for Shc, p185,
and Sos while the
N-Grb2 can only bind to Shc. One possible model
to explain this phenomenon is shown in Fig. 7. Wild-type Grb2
(wt-Grb2) constitutively binds to Sos mainly through its amino-terminal
SH3 domain. The wt-Grb2
Sos complex is recruited to the
tyrosine-phosphorylated Shc, subsequently resulting in Ras activation.
The wt-Grb2
Sos may also be directly recruited to the activated
p185, which is not shown here in order to simplify the model
(discussion seen in the text). When introduced into the B104-1-1 cells,
the
N-Grb2 sequesters tyrosine-phosphorylated Shc. Therefore,
recruitment of the wt-Grb2
Sos complex to Shc is severely
impaired. Furthermore,
N-Grb2 cannot appreciably bind to Sos. Thus
the complex of Shc and
N-Grb2 cannot lead to Ras activation. Taken
together, the
N-Grb2 inhibits Ras activation mediated by the
Shc/wt-Grb2/Sos pathway. Unlike the
N-Grb2, the
C-Grb2 can
bind to both tyrosine-phosphorylated Shc and Sos. Deletion of the
carboxyl-terminal SH3 domain of Grb2 may not significantly affect its
biological functions. In other words, the exogenously expressed
C-Grb2 by itself may largely fulfill the functions of the wt-Grb2.
Therefore, Ras activation is not dramatically shut down and the
transformed phenotypes of the B104-1-1 cells are not obviously reversed
even though the endogenous Grb2 is largely competed out from the
Shc/Grb2/Sos pathway by the
C-Grb2. This model is indirectly
supported by previous studies on the C. elegans Sem-5. The
mutation in Sem-5 (sem-5 allele n2195) corresponding to the
G203R Grb2 mutant (C-terminal SH3) had a much weaker phenotypic effect
in C. elegans than the mutation (sem-5 allele n1619)
corresponding to the P49L Grb2 mutant (N-terminal SH3)(59) .
However, other studies suggest that both P49L and G203R Grb2 were
loss-of-function mutants(35) . For example, co-microinjection
of either P49L or G203R Grb2 protein together with the H-ras protein
did not stimulate DNA synthesis in quiescent rat embryo fibroblast
cells while coinjection of the wild-type Grb2 and H-ras proteins
enhanced DNA synthesis, suggesting that the two SH3 domains of Grb2
constitute an essential functional component of the protein. These
conflicting observations may be explained by the use of different
assays in different biological systems. The functional difference
between
C-Grb2 and G203R Grb2 is not known currently. We speculate
that the
C-Grb2 possesses, at least in part, the biological
functions of the wild-type Grb2 since it can bind to Shc, p185, and
Sos. The slight down-regulation of Ras activation and partial reversal
of transformed phenotypes in Grb2
C-11 cells may be due to the
relatively lower efficiency of recruitment of the Sos
C-Grb2
complex to Shc, as compared to the complex of Sos and the endogenous
Grb2. This is supported by the observation seen in Fig. 4in
which comparable amounts of the
C-Grb2 in the Grb2
C-11 cells
and the endogenous Grb2 in the B104-1-1 cells were co-precipitated by
the anti-Shc antibody even though the expression level of the
C-Grb2 was much higher in the Grb2
C-11 cells than that of the
endogenous Grb2 in B104-1-1 cells (Fig. 2A). This
finding suggests that the
C-Grb2 may have a lower affinity for Shc
than does the endogenous Grb2. Alternatively, the nucleotide exchange
activity of Sos may be relatively weaker in the Sos
C-Grb2
complex than in the Sos-endogenous Grb2 complex, probably because of an
unknown allosteric effect. Therefore, Ras activation in the
Grb2
C-11 cells is not as efficient as that in the B104-1-1 cells,
leading to slight reversal of the transformed phenotypes caused by the
mutation-activated p185.
Figure 7:
Hypothetic working modes of wild-type Grb2
and its SH3 domain deletion mutants. Wild-type Grb2 (wt-Grb2)
constitutively binds to Sos mainly through its amino-terminal SH3
domain. The Grb2Sos complex is recruited to the Shc that is
tyrosine phosphorylated, leading to Ras activation. After introduced
into the B104-1-1 cells, the
N-Grb2 can bind to Shc but not Sos.
The Shc
N-Grb2 complex by itself is unable to trigger Ras
activation. On the other hand,
N-Grb2 sequesters Shc. Therefore,
the endogenous wt-Grb2 cannot be recruited to Shc. Thus,
N-Grb2 is
a dominant-negative mutant of Grb2. In contrast, the
C-Grb2 binds
to Sos and Shc. The Shc
C-Grb2
Sos complex can largely
fulfill the functions of the Shc
wt-Grb2
Sos complex, leading
to Ras activation. Since the recruitment efficiency of
C-Grb2
Sos by Shc is relatively lower as compared to that of
the wt-Grb2
Sos, Ras activation is slightly reduced in B104-1-1
transfectant expressing the
C-Grb2. In order to simplify the
model, direct recruitment of wt-Grb2 or
C-Grb2 to the
mutation-activated p185 is not included in this model, which is
described in the text instead.
Previous studies on the EGF receptor
signaling pathway indicated that the Grb2Sos complex could be
recruited to tyrosine-phosphorylated Shc or directly to the activated
EGF receptor(38, 39, 40, 41) . The
data presented here imply that the Shc/Grb2/Sos pathway is most likely
the dominant one coupling the activated p185 to Ras since interference
of the interaction between Shc and Grb2 by
N-Grb2 leads to a
dramatic inhibition of Ras activation. This idea is consistent with our
previous observation that deletion of most of the autophosphorylation
sites, including the potential Grb2 binding site on the
mutation-activated p185 did not affect its transforming ability,
suggesting that direct binding of Grb2 to p185 is not essential for Ras
activation(46) . Similarly, recent studies using peptide
competition and immunodepletion approaches also demonstrated that
formation of a complex of EGF receptor with Grb2 was only responsible
for a minor part of EGF-stimulated Ras activation while the formation
of the Shc
Grb2
Sos complex played the major
role(28, 29) . Indirect evidence has been shown
suggesting that Grb2 binding to tyrosine-phosphorylated Shc is more
important than Grb2 binding to the insulin receptor substrate-1 in the
activation of Ras in response to insulin(30, 31) .
Our data, however, also suggest that the Shc/Grb2/Sos pathway may
not be the sole pathway that leads to the activation of Ras by the
mutation-activated p185. Disruption of the association between Shc and
Grb2 by N-Grb2 is unable to completely inhibit Ras activation or
to completely reverse the transformed phenotypes mediated by the
mutation-activated p185, suggesting the existence of multiple routes to
Ras, which may not be influenced by the
N-Grb2. One conceivable
pathway is the direct recruitment of Grb2
Sos to the activated
p185 since
N-Grb2 appears to be unable to compete with the
endogenous Grb2 for p185. Alternatively, the formation of complexes
containing Grb2 and phosphorylated proteins other than Shc, which can
stimulate the Ras pathway, may not be interfered with by the
N-Grb2. It has been shown that a complex of Syp/SH-PTP2 tyrosine
phosphatase and Grb2 can couple platelet-derived growth factor
receptors to Ras(60) . Recently, a Ras-GAP associated protein,
named p62, has been found to form a complex with Grb2 in v-src transformed NIH3T3 cells (61) . Interestingly, the
presence of the Grb2
p62 complex correlates with the
phosphorylation of p62 and cellular transformation, suggesting that the
Grb2
p62 complex may be able to lead to Ras activation. It will be
of interest to test whether these complexes exist in the B104-1-1 cells
and whether
N-Grb2 and
C-Grb2 are able to interfere with
these pathways. Furthermore, the existence of other potential pathways
to Ras, in which Grb2 or Sos is not involved, may also account for the
absence of complete Ras inhibition and incomplete phenotypic reversion
induced by the
N-Grb2. It is now known that mammalian cells
contain several Ras guanine nucleotide exchange factors (GEF) apart
from Sos. One of these, C3G (named for Crk SH3 binding GEF), can activate Ras in yeast(62) .
Intriguingly, via its proline-rich domain C3G binds the amino-terminal
domain of the adaptor protein Crk(62, 63) . The
Crk
C3G complex may thus, like the Grb2
Sos complex, couple
the oncogenic signal of the mutation-activated p185 to Ras. However, no
data exist showing that C3G is an exchange factor for Ras in mammalian
cells. On the other hand, an alternative explanation for the failure to
completely reverse the transformed phenotypes of B104-1-1 cells by the
N-Grb2 could be that additional, perhaps less efficient, signaling
pathways which do not involve Ras are not influenced by the
N-Grb2
and may culminate in cell transformation by the activated p185. Indeed,
recent studies have shown that Raf can be activated by the Drosophila torso receptor tyrosine kinase in a Ras-independent
pathway (58) . Our studies provided direct evidence to support
the hypothesis that the Shc/Grb2/Sos pathway plays a major role in the
oncogenic signaling of the mutation-activated p185 and may shed light
on developing therapeutic agents to block the oncogenic signaling
pathway of the p185 oncoprotein.