(Received for publication, December 22, 1995; and in revised form, February 5, 1996)
From the
Insulin stimulates the Ras/Raf/MEK/ERK pathway leading to feedback phosphorylation of the Ras guanylnucleotide exchange protein SOS and dissociation of Grb2 from SOS. Even though epidermal growth factor (EGF) also stimulates ERK activity and phosphorylation of SOS similar to insulin, EGF induces a dissociation of the Grb2-SOS complex from Shc. To determine the molecular basis for this difference, we examined the signaling properties of a mutant EGF receptor lacking the five major autophosphorylation sites. Although EGF stimulation of the mutant EGF receptor activates ERK and phosphorylation of both Shc and SOS, it fails to directly associate with either Shc or Grb2. However, under these conditions EGF induces a dissociation of the Grb2-SOS complex suggesting a role for receptor and/or plasma membrane targeting in the stabilization of Grb2-SOS interaction. Consistent with this hypothesis, expression of an SH2 domain Grb2 mutant which is unable to mediate plasma membrane targeting of the Grb2-SOS complex results in both insulin- and EGF-stimulated uncoupling of Grb2 from SOS. Furthermore, a plasma membrane-bound Grb2 fusion protein remains constitutively associated with SOS. Together, these data demonstrate that EGF stimulation prevents the feedback uncoupling of Grb2 from SOS by inducing a persistent plasma membrane receptor targeting of the Grb2-SOS complex.
Recently, substantial progress has been made in defining the
molecular mechanisms by which tyrosine kinase receptors activate
Ras-dependent signaling events. For example, insulin stimulation of the
insulin receptor kinase results in the tyrosine phosphorylation of
several substrates including the 185-kDa insulin receptor substrates 1
and 2 (IRS1/2) and isoforms of the src homology 2 (SH2)
domain-containing 2 collagen-related proteins, termed Shc (1, 2, 3, 4) . Tyrosine
phosphorylation of IRS1 on Tyr-895 and Shc on Tyr-317 generates
specific docking sites for the SH2 domain of Grb2, a 23-kDa adapter
protein containing a single SH2 domain flanked by two SH3
domains(5, 6, 7, 8) . Numerous
studies have established an association between the Grb2 SH3 domains
and the carboxyl-terminal proline-rich domain of SOS, a 170-kDa
guanylnucleotide exchange factor for
Ras(5, 9, 10, 11, 12, 13) .
Recently it has been demonstrated that the expression of plasma
membrane targeted forms of SOS result in the constitutive activation of
Ras(14, 15) . Based upon these data, it has been
suggested that plasma membrane targeting of the Grb2-SOS complex, by a
Grb2-mediated association with tyrosine phosphorylated Shc and/or IRS1,
accounts for the increased conversion of Ras from the inactive
GDP-bound state to the active GTP-bound form(16, 17) .
Alternatively, several studies have suggested that the
carboxyl-terminal domain of SOS functions as an auto-inhibitory domain
which may be derepressed by the binding of Grb2 (18, 19, 20) . In either case, the
interaction of SOS with Grb2 plays an important role in regulating the
activation state of Ras.
Once in the GTP-bound state, Ras associates with and activates members of the Raf family of serine/threonine kinases(21, 22, 23, 24, 25) . In turn, activated Raf phosphorylates and activates the dual functional protein kinase MEK which then phosphorylates the ERK family of mitogen-activated protein kinases on both threonine and tyrosine residues(12, 26) . These phosphorylation events activate ERK and are required for the phosphorylation-dependent regulation of various cytosolic proteins and nuclear DNA binding transcription factors(27, 28, 29) . Thus, the mitogenic actions of growth factors can be directly linked to transcriptional regulatory events utilizing Ras as a molecular switch converting upstream tyrosine kinase signals into a serine/threonine kinase cascade.
Although these data have provided a mechanism
accounting for the activation and positive signaling role of Ras, Ras
activation is transient and rapidly returns to its basal GDP-bound
state(30, 31, 32) . We and others have
observed that insulin activation of the Ras/Raf/MEK/ERK pathway results
in the serine/threonine phosphorylation of SOS, followed by
dissociation of the Grb2-SOS
complex(33, 34, 35) . Furthermore, blockade
of ERK activation by either expression of a dominant-interfering MEK
mutant or with a specific MEK inhibitor prevents SOS phosphorylation,
dissociation of the Grb2-SOS complex, and prolongs the time Ras remains
GTP-bound(36, 37) . These data suggest that an
insulin-stimulated feedback uncoupling of Grb2 from SOS may contribute
to Ras desensitization. In this study we demonstrate, however, that SOS
phosphorylation following EGF ()stimulation does not result
in dissociation of Grb2 from SOS but instead induces the dissociation
of the Grb2-SOS complex from Shc. This results from an EGF-stimulated
persistent plasma membrane receptor targeting of the Shc-Grb2-SOS
complex which does not occur following insulin stimulation.
NIH-3T3 fibroblasts were engineered to express the human wild type EGF receptor (EGFR-WT) and an EGF receptor mutant (EGFR-5F) in which the five major tyrosine autophosphorylation sites (Tyr-992, -1068, -1086, -1148, and -1173) were changed to phenylalanine as described previously (39) . These cells were grown in Dulbecco's modified Eagle's medium supplemented with nucleosides, 500 µg/ml neomycin, and 5% calf serum.
Figure 1: Insulin and EGF stimulate ERK and SOS phosphorylation but only insulin induces the dissociation of the Grb2-SOS complex. A, Chinese hamster ovary cells genetically engineered to express the human insulin and EGF receptors (CHO/IR/EGFR) were incubated in the absence (lane 1) or presence of 100 nM insulin (lane 2) or 20 nM EGF (lane 3) for 5 min. Whole cell detergent extracts were subjected to Western blotting using an ERK antibody as described under ``Materials and Methods.'' B, CHO/IR/EGFR cells were incubated in the absence (lane 1) or presence of 100 nM insulin (lane 2) or 20 nM EGF (lane 3) for 15 min. Whole cell detergent extracts were subjected to Western blotting using a SOS antibody. C, the cell extracts prepared in panel B were immunoprecipitated with a Grb2 antibody and the resultant immunoprecipitates were subjected to Western blotting using a SOS antibody. D, the Grb2 immunoprecipitates obtained in panel C were subjected to Western blotting using a Grb2 antibody.
Figure 3: Expression of an autophosphorylation defective EGF receptor mutant does not impair ERK activation and results in the dissociation of the Grb2-SOS complex. A, 3T3 fibroblasts expressing the wild type (ER-WT) and mutant (ER-5F) EGF receptors were incubated in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 20 nM EGF for 5 min as described under ``Materials and Methods.'' Whole cell detergent extracts were subjected to Western blotting using an ERK antibody. B, EGFR-WT and EGFR-5F cells were incubated in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 20 nM EGF for 15 min. Whole cell detergent extracts were subjected to Western blotting using a SOS antibody. C, the cell extracts prepared in panel B were immunoprecipitated with a Grb2 antibody and the resultant immunoprecipitates were subjected to Western blotting using a SOS antibody. D, the Grb2 immunoprecipitates obtained in panel C were subjected to Western blotting using a Grb2 antibody.
Figure 2:
EGF stimulation results in a
time-dependent association, followed by a dissociation of SOS from Shc.
CHO/IR/EGFR cells were incubated in the absence (lane 1) or
presence of 20 nM EGF for 1 (lane 2), 15 (lane
3), and 30 (lane 4) min. Whole cell detergent extracts
were prepared and immunoprecipitated with a Shc antibody as described
under ``Materials and Methods.'' The resultant
immunoprecipitates were then subjected to Western blotting using a SOS
antibody (A), a Shc antibody (B), or the PY20
phosphotyrosine antibody (C). IgG indicates the heavy chain of the Shc
antibody.
Consistent with the normal mitogenic signaling properties of the EGFR-5F receptor(39) , EGF-dependent phosphorylation of ERK was identical to that observed in cells expressing the wild type EGF receptor (Fig. 3A, lanes 1-4). Similarly, EGF stimulation of both the EGFR-WT and EGFR-5F cells resulted in a comparable reduction of SOS mobility (Fig. 3B, lanes 1-4). However, in contrast to the EGFR-WT receptor, activation of the EGFR-5F receptor resulted in a marked EGF-dependent decrease in the amount of Grb2 bound to SOS (Fig. 3C, lanes 1-4). The ability of the EGFR-5F receptor to mediate dissociation of the SOS-Grb2 complex occurred without any significant changes in the amount of immunoprecipitated Grb2 protein (Fig. 3D, lanes 1-4). These data suggest that the inability of the wild type EGF receptor to signal dissociation of the Grb2-SOS complex may be an intrinsic property of the tyrosine-phosphorylated EGF receptor itself.
We next determined whether the EGFR-5F receptor could tyrosine phosphorylate Shc despite the absence of the high affinity binding sites normally provided by receptor autophosphorylation (Fig. 4). Incubation of the EGFR-WT cells with EGF resulted in the binding of the EGFR-WT receptor with Grb2 (Fig. 4A, lanes 1 and 2) as well as the association of Shc with Grb2 (Fig. 4B, lanes 1 and 2). As expected, stimulation of the EGFR-5F cells failed to induce an association of Grb2 with the mutant EGF receptor (Fig. 4A, lanes 3 and 4). Nevertheless, EGF activation of the EGFR-5F receptor resulted in association of Grb2 with Shc (Fig. 4B, lanes 3 and 4). Phosphotyrosine immunoblots of Grb2 immunoprecipitates demonstrated that the association of Grb2 with Shc correlated with tyrosine phosphorylation of Shc by the EGFR-5F receptor (Fig. 4C, lanes 1-4). These results are consistent with the mutant EGF receptor utilizing Shc as a phosphotyrosine acceptor substrate which then binds to the Grb2 SH2 domain. These data suggest further that the inability of the wild type EGF receptor to induce dissociation of the Grb2-SOS complex resulted from the targeting and/or localization of Grb2 and Shc to the EGF receptor. Disruption of this interaction, by deletion of the EGF receptor autophosphorylation sites, resulted in an EGF receptor that functions in a manner analogous to the insulin receptor with regard to Grb2/Shc binding and Grb2-SOS dissociation.
Figure 4: Activation of the autophosphorylation defective EGF receptor induces Shc phosphorylation but does not induce the direct interactions with Shc and/or Grb2. The EGFR-WT (ER-WT) and EGFR-5F (ER-5F) cells were incubated in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 20 nM EGF for 15 min. Whole cell detergent extracts were prepared and immunoprecipitated with a Grb2 antibody as described under ``Materials and Methods.'' The resultant immunoprecipitates were then subjected to Western blotting using an (A) EGF receptor antibody, (B) Shc antibody, or (C) the PY20 phosphotyrosine antibody.
Figure 5: Expression of mutant Grb2 proteins results in either a gain of function or loss of function for insulin and EGF-stimulated dissociation of the Grb2-SOS complex. CHO/IR/EGFR cells were transfected with either the wild type myc epitope-tagged Grb2 (A, Grb2-myc), a Grb2 SH2 domain mutant (B, Grb2/R86K-myc), or a constitutive plasma membrane-bound Grb2 fusion protein (C, A2/Grb2-myc) as described under ``Materials and Methods.'' The cells were then incubated in the absence (lanes 1 and 4) or presence of 100 nM insulin (lanes 2 and 5) or 20 nM EGF (lanes 3 and 6) for 15 min. Whole cell detergent extracts were prepared and immunoprecipitated with the myc epitope antibody, 9E10. The resultant immunoprecipitates were then subjected to Western blotting using either a SOS antibody (lanes 1-3) or a Grb2 antibody (lanes 4-6).
The differences in Grb2-SOS targeting induced by insulin and EGF receptor activation could reflect either a direct physical association of the Grb2-SOS complex with the EGF receptor itself or via a general association of the complex with the plasma membrane. To distinguish between these possibilities, we prepared a plasma membrane-associated form of Grb2 by constructing a cDNA to encode a fusion protein containing the extracellular and transmembrane domains of the human A2 MHC class I protein in-frame with wild type Grb2-myc(42) . Similar to the cytosolic forms of Grb2, the expressed membrane-bound form of Grb2 (A2/Grb2-myc) also associated with SOS in unstimulated cells (Fig. 5C, lane 1). However, neither insulin nor EGF induced dissociation of this Grb2 fusion protein from SOS (Fig. 5C, lanes 2 and 3). Thus, under these conditions the insulin regulation of Grb2-SOS interaction was essentially identical to that observed for EGF.
Figure 6: EGF but not insulin induces a persistent membrane targeting of Grb2. CHO/IR/EGFR cells were transfected with either the wild type myc epitope-tagged Grb2 (Am Grb2-myc), a Grb2 SH2 domain mutant (B, Grb2/R86K-myc), or a constitutive plasma membrane-bound Grb2 fusion protein (C, A2/Grb2-myc). The cells were then incubated in the absence (lanes 1 and 2) or presence of 100 nM insulin (lanes 3 and 4) or 20 nM EGF (lanes 5 and 6) for 15 min. The cells were lysed and separated into cytosolic (S, lanes 2, 4, and 6) and particulate (P, lanes 1, 3, and 5) fractions as described under ``Materials and Methods.'' The fractions were then subjected to Western blotting using the myc epitope antibody, 9E10.
Figure 7: Insulin and EGF-stimulated dissociation of the SOS-Grb2 complex determined by precipitation with a GST-Grb2 fusion protein. CHO/IR/EGFR cell extracts were prepared from unstimulated cells (lanes 1, 4, 7, 10, 13, and 16) and cells incubated with 100 nM insulin (lanes 2, 5, 8, 11, 14, and 17) or 20 nM EGF for 20 min (lanes 3, 6, 9, 12, 15, and 18). The cell extracts were incubated for 1 h with increasing amounts (1, 3, 9, 30, 60, and 100 µg) of the GST-Grb2 fusion protein as indicated in the figure. The total amount of resin in each sample was maintained at a constant amount by the addition of Sepharose beads. The samples were then precipitated, subjected to SDS-polyacrylamide gel electrophoresis, and immunoblotted with a SOS antibody as described under ``Materials and Methods.''
Surprisingly, cell extracts from EGF-stimulated cells also displayed a reduced association of SOS with Grb2 when incubated with low concentrations of the GST-Grb2 fusion protein in an identical manner to extracts from insulin-stimulated cells (Fig. 7, lanes 3, 6, and 9). Incubation with higher GST-Grb2 concentrations (30-100 µg) also resulted in an apparent saturation of SOS binding with no significant difference in association between unstimulated and EGF-stimulated cell extracts (Fig. 7, compare lanes 13 with 15 and lanes 16 with 18). These data demonstrate that the functional differences between insulin and EGF-stimulated dissociation of the Grb2-SOS complex is not intrinsic to the SOS protein itself and thus cannot be accounted for by differences in receptor-mediated post-translational modifications of SOS.
Recent studies have begun to define a pathway directly linking tyrosine kinase growth factor signaling to Ras activation. In the basal state, the guanylnucleotide exchange protein SOS is bound to the SH3 domains of the small adapter protein Grb2. Following receptor kinase activation, either through receptor autophosphorylation and/or tyrosine phosphorylation of Shc, the SH2 domain of Grb2 targets the cytosolic Grb2-SOS complex to the plasma membrane location of Ras(14, 15) . In addition, several studies have suggested that the SOS carboxyl-terminal Grb2 binding domain functions as an autoinhibitory region(14, 18, 19, 20) . In either case, the targeting and/or allosteric regulation of SOS provides a mechanism for the interaction of SOS with its substrate Ras, thus allowing for the productive exchange of GTP for GDP. Although these mechanisms account for the rapid growth factor-mediated activation of Ras, even in the continuous presence of growth factors Ras-GTP binding is transient and returns to the inactive GDP-bound state within 30 to 60 min(30, 31, 32) . This desensitization of Ras activation is an important event, as disruption of normal Ras regulation can lead to oncogenesis with nearly 15% of all human tumors containing mutant forms of Ras that maintain the protein in its active GTP-bound state(47) .
In addition to the GTP for GDP exchange activity of SOS, Ras is also regulated by the GTPase activating protein (Ras-GAP) which hydrolyzes GTP-bound Ras to GDP(48) . Since Ras itself has relatively low intrinsic guanylnucleotide exchange or GTPase activities, the dynamic equilibrium between SOS and Ras-GAP activities within a cell defines the relative activation state of Ras. Thus, modulation of either of these two effector proteins could be responsible for the inactivation of Ras. However, insulin does not have any significant effect on Ras-GAP phosphorylation, activity, and/or localization, whereas EGF has been reported to inhibit Ras-GAP activity(49) . Based upon these previous findings, it is unlikely that Ras-GAP plays a role in growth factor-stimulated desensitization of Ras activation.
To address the mechanism of Ras desensitization, we and others have observed recently that following insulin-mediated Ras activation there is a Raf/MEK/ERK pathway feedback phosphorylation of SOS(33, 35, 36, 37) . SOS phosphorylation temporally correlates with dissociation of the Grb2-SOS complex which also parallels the return of Ras back to the inactive GDP-bound state. Furthermore, prevention of the Grb2-SOS dissociation by inhibition of MEK activity results in a prolongation of GTP-bound Ras(36, 37) . Thus, these data support a model in which a feedback uncoupling of the Grb2-SOS complex removes the Ras activation signal and thereby limits the duration of Ras activation by insulin.
Similar to insulin, various agents which activate the ERK pathway (serum, platelet-derived growth factor, v-Ras, v-Raf) induce phosphorylation of SOS and result in dissociation of the Grb2-SOS complex(35) . However, although several studies have also observed an EGF-induced phosphorylation of SOS, there are marked discrepancies with regard to Shc-Grb2-SOS interactions. In one study, EGF was observed to induce the dissociation of Grb2 from SOS in an identical fashion to that of insulin(36) . In contrast, other studies have not observed any effect of EGF on the association state of the Grb2-SOS complex(45, 50, 51) . In two of these studies, SOS phosphorylation resulted in an uncoupling of the Grb2-SOS complex from Shc due to a reduction in affinity of the Grb2 SH2 domain without any effect on the Shc tyrosine phosphorylation state(45, 51) . Alternatively, another report indicated that the uncoupling of the Grb2-SOS complex from Shc correlated with the tyrosine dephosphorylation of Shc(50) . Although the basis for these differences in EGF action are not readily apparent, the data presented in this article are consistent with a rapid insulin-stimulated formation of a Shc-Grb2-SOS ternary complex followed by an insulin-induced dissociation of Grb2 from SOS, thereby decreasing the amount of SOS indirectly bound to Shc. However, in the case of EGF stimulation the net result of uncoupling Shc from SOS also occurred but was a consequence of an EGF-induced dissociation of Shc from the Grb2-SOS binary complex without any significant effect on Grb2-SOS interactions. The inability of EGF to induce a dissociation of Grb2-SOS was surprising since EGF also stimulates ERK activation and SOS phosphorylation in an apparently identical manner to that of numerous agents which result in Grb2-SOS dissociation(33, 34, 35, 36, 37) .
To explore these apparent differences between insulin and EGF signaling, we considered that one unique feature of the EGF receptor pathway compared to the insulin receptor is the direct association of the EGF receptor itself with Grb2-SOS(5, 6, 9, 11) . In contrast, insulin stimulation does not result in a stable association of the Shc-Grb2-SOS and/or Grb2-SOS complexes with the insulin receptor(16, 43, 44, 52, 53) . We therefore speculated that EGF would induce dissociation of the Grb2-SOS complex if membrane EGF receptor targeting was disrupted. This was initially accomplished by expression of an autophosphorylation defective but kinase active EGF receptor mutant in which the five major tyrosine acceptor sites were mutated to phenylalanine(39) . Interestingly, despite the lack of detectable autophosphorylation and/or association with Grb2, this EGF receptor mutant functions to phosphorylate Shc and induces both ERK and SOS phosphorylation. It is unlikely that these signaling properties are due to dimerization of the mutant EGF receptor with either ErbB3 or ErbB4 receptors since the only tyrosine-phosphorylated proteins detected in Grb2 immunoprecipitates was Shc.
The effect of EGF receptor binding on Grb2-SOS interaction was further substantiated by expression of a targeting defective Grb2 mutant which does not associate with either the tyrosine-phosphorylated EGF receptor or Shc. Under these conditions, EGF stimulated dissociation of Grb2 from SOS to the same degree as insulin. Furthermore, a membrane localized chimeric Grb2 protein remained bound to SOS following insulin or EGF stimulation. Thus, in terms of Grb2-SOS interaction, expression of a targeting defective Grb2 mutant confers an insulin-like response to EGF stimulation, whereas a membrane bound form of Grb2 prevents the insulin stimulated dissociation of Grb2 from SOS. In any case, persistent membrane association of the Shc-Grb2-SOS and/or Grb2-SOS complexes via the EGF receptor apparently overrides the effect of SOS phosphorylation to induce its uncoupling from Grb2. Instead, association of the EGF receptor with the Shc-Grb2-SOS ternary complex seems to influence the interaction of Shc with the Grb2-SOS complex, without any significant effect on Grb2-SOS binding. Thus, these data suggest that the binding interaction between Shc, Grb2, and SOS are dependent upon the intracellular location of this complex following growth factor stimulation.
One additional possibility to account for
the ability of insulin but not EGF to induce dissociation of the
Grb2-SOS complex is a difference in site-specific phosphorylation of
SOS. However, this is unlikely since in vitro reconstitution
of Grb2-SOS binding in extracts from both insulin and EGF-stimulated
cells demonstrated an equivalent reduction in the apparent binding
affinity of SOS for Grb2. Despite this reduction in apparent affinity,
it is possible that the effective concentration of Grb2 and SOS might
be sufficient to overcome this lower binding interaction following EGF
but not insulin stimulation. This would be consistent with the
targeting of the Shc-Grb2-SOS and/or Grb2-SOS complexes to the EGF
receptor but not to the insulin receptor. Alternatively, it is also
possible that EGF receptor targeting of the Grb2-SOS complex induces
the association of an additional accessory factor(s) that might
influence the binding interaction between Shc, Grb2, and SOS. For
example, the tyrosine-phosphorylated EGF receptor associates with the
GTPase activating protein and phospholipase C which are not bound
by the insulin receptor.
In either case, it is important to
recognize that even though EGF does not uncouple Grb2 from SOS, Ras
activation is transient and recovers to the basal GDP-bound state
within 30 min following both insulin and EGF stimulation in numerous
cell types including the CHO/IR/EGFR cells. ()We and others
have recently reported that the insulin-stimulated dissociation of the
Grb2-SOS complex is an important mechanism involved in the Ras
inactivation process(36, 37) . Thus, the essential
feature of uncoupling SOS from Shc appears to be the necessary event in
both the insulin- and EGF-dependent recovery of activated GTP-bound Ras
to the inactive GDP-bound state. Currently, we are attempting to
determine the molecular basis for the EGF receptor-targeted
dissociation of Shc from the Grb2-SOS complex versus the
insulin induced dissociation of Grb2-SOS interactions.