(Received for publication, July 10, 1995; and in revised form, August 31, 1995)
From the
Insulin and epidermal growth factor receptors transmit signals
for cell proliferation and gene regulation through formation of active
GTP-bound p21 mediated by the guanine nucleotide
exchange factor Sos. Sos is constitutively bound to the adaptor protein
Grb2 and growth factor stimulation induces association of the Grb2/Sos
complex with Shc and movement of Sos to the plasma membrane location of
p21
. Insulin or epidermal growth factor
stimulation induces a rapid increase in p21
levels, but after several minutes levels decline toward
basal despite ongoing hormone stimulation. Here we show that
deactivation of p21
correlates closely with
phosphorylation of Sos and dissociation of Sos from Grb2, and that
inhibition of mitogen-activated protein (MAP) kinase kinase (also known
as extracellular signal-related kinase (ERK) kinase, or MEK) blocks
both events, resulting in prolonged p21
activation. These data suggest that a negative feedback loop
exists whereby activation of the Raf/MEK/MAP kinase cascade by
p21
causes Sos phosphorylation and, therefore,
Sos/Grb2 dissociation, limiting the duration of p21
activation by growth factors. A serine/threonine kinase
downstream of MEK (probably MAP kinase) mediates this desensitization
feedback pathway.
The tyrosine kinase receptors for insulin, EGF, ()and
other growth factors mediate cell proliferation and gene regulation,
and it has been demonstrated that activation of p21
is essential for these effects(1, 2) .
p21
is one of a family of closely related
membrane-bound guanine nucleotide-binding proteins, which bind GTP and
catalyze its hydrolysis to GDP. The GTP-bound protein is active, while
the GDP-bound form is not. The GTPase-activating protein GAP promotes
inactivation of p21
by stimulating the
hydrolysis of GTP (3) , whereas the guanine nucleotide exchange
factor (GEF) Sos (homologue of the Drosophila Son-of-sevenless
protein) mediates activation by inducing the release of
GDP(4, 5, 6, 7, 8) . In the
case of insulin and EGF, activation of p21
has
been shown to be mediated by Sos rather than by
GAP(8, 9) .
In the basal state, Sos is a
cytoplasmic protein constitutively bound to the adaptor protein Grb2.
This interaction is mediated by the two Src homology 3 (SH3) domains of
Grb2, which bind to proline-rich regions in the C terminus of
Sos(6, 10, 11, 12) . Insulin or EGF
stimulation results in the translocation of Sos to the plasma membrane,
where activation of p21 occurs(5). Growth factor
stimulation also results in the tyrosine phosphorylation of the
intermediary signaling molecule Shc on a tyrosine residue in a
consensus binding motif for the Grb2 SH2 domain, resulting in the
binding of the Grb2/Sos complex to Shc(6, 11) . In the
case of the EGF receptor the mechanism for targeting Sos to the plasma
membrane is binding of the SH2 domain or phosphotyrosine binding domain
of Shc to phosphotyrosine residues of the EGF
receptor(5, 13, 14, 15) ; direct
binding of the Grb2 SH2 domain to the EGF receptor most likely plays a
lesser role(16) . There is evidence that Shc plays a key role
upstream of p21
in the mitogenic response to
both insulin and EGF, suggesting that in fact the trimeric Shc/Grb2/Sos
complex is important for activation of
p21
(16, 17, 18, 19) .
The pattern of p21 activation after growth
factor stimulation is one of a rapid increase in GTP binding, peaking
in the first several minutes and then falling rather rapidly toward
basal levels despite ongoing ligand
stimulation(10, 20, 21, 22, 23, 24) .
The mechanism of this down-regulation is unknown. However, it has
recently been demonstrated that Sos undergoes serine/threonine
phosphorylation following growth factor
stimulation(24, 25, 26) , and dissociation of
the Grb2/Sos complex appears to correlate temporally with Sos
phosphorylation(24, 25) . The purpose of this study
was to elucidate the serine kinase pathway leading to Sos
phosphorylation and to determine whether Grb2/Sos dissociation is
causally related. In addition, we sought to learn whether this
dissociation is a negative feedback mechanism resulting in the
desensitization of p21
to prolonged activation
by growth factor stimulation.
Growth factor-induced serine/threonine phosphorylation of Sos
is mediated by kinases downstream of Raf1 in the Raf1/MEK/MAP kinase
cascade activated by p21(28) . To examine the
role of Sos phosphorylation in insulin signaling, an inhibitor of MEK
activity, PD098059, was used. This compound is relatively specific for
MEK with no inhibitory activity against a number of other
serine/threonine and tyrosine kinases(29) . Insulin stimulation
of HIRcB cells, a Rat1 fibroblast line expressing the human insulin
receptor, resulted in a significant retardation of mobility of Sos on
SDS-PAGE, reflecting Sos phosphorylation (Fig. 1A, lanes 1-6). This occurred first between 1 and 5 min of
stimulation and was maximal by 10 min, decreasing somewhat by 120 min.
The breadth of the Sos signal at 5 and 120 min may reflect a mix of
more and less highly phosphorylated Sos phosphoprotein species, as Sos
has at least two phosphorylation sites(30) .
Figure 1: Effects of insulin with or without MEK inhibition on Sos phosphorylation and binding between Sos, Grb2, and Shc. Serum-starved cells were treated with either the MEK inhibitor PD098059 or vehicle alone for 1 h, then stimulated with insulin for the indicated times. Cells lysates were divided for analysis by immunoprecipitation and immunoblotting with the indicated antibodies. A, Sos phosphorylation and mobility shift; B, Sos coprecipitation with Grb2; C, Sos coprecipitation with Shc; D, Grb2 coprecipitation with Shc; E, tyrosine phosphorylation of Shc. Results are representative of three separate experiments. IP, immunoprecipitation; IB, immunoblotting.
Grb2 has two Src homology 3 (SH3) domains, which bind to proline-rich regions in the C terminus of Sos(11, 12) . Coimmunoprecipitation studies of Grb2 and Sos showed that Grb2/Sos complexes were present in the basal state, but after insulin treatment these complexes rapidly dissociated such that they were undetectable (Fig. 1B, lanes 1-6). Some reassociation was seen at the later time points as Sos became dephosphorylated. Pretreatment of the cells with the MEK inhibitor prevented Sos phosphorylation (Fig. 1A, lanes 7-12) and Grb2/Sos dissociation (Fig. 1B, lanes 7-12). This strongly suggests that Sos phosphorylation causes dissociation of Sos from Grb2. This is further supported by the observation that the Sos which was complexed with Grb2 at 5 and 40 min showed only minimal mobility shift compared to total cellular Sos, suggesting that the Sos which remained bound to Grb2 represented a subpopulation of Sos that was less heavily phosphorylated. MAP kinase has been shown to phosphorylate Sos in vitro(30) , and there are several MAP kinase consensus phosphorylation sequences in the C terminus of Sos(31, 32) , suggesting that phosphorylation of Sos by MAP kinase may disrupt binding of Sos to the SH3 domains of Grb2.
Insulin stimulation leads to binding of the Grb2 SH2 domain to
tyrosine-phosphorylated Shc, which plays a key role in
insulin-stimulated mitogenesis, presumably by targeting Sos to the
plasma membrane where it activates p21(19, 33, 34). Thus,
the importance of Grb2/Sos dissociation may be that it decreases
linkage of Sos to Shc. Consistent with this notion, Shc/Sos
coprecipitation studies showed only a transient association, seen at 1
and 5 min and gone by 10 min (Fig. 1C, lanes
1-6), while in cells treated with PD098059, the Sos/Shc
interaction persisted for 120 min (Fig. 1C, lanes
7-12). Shc/Grb2 coimmunoprecipitation analysis revealed
minimal or no basal coprecipitation; insulin treatment induced
significant Shc/Grb2 association by 1 min, which was maximal by 5 min
and unchanged thereafter, and was unaffected by the MEK inhibitor (Fig. 1D). As expected, Shc/Grb2 association correlated
closely with the time course of Shc tyrosine phosphorylation (Fig. 1E), which was also unaffected by the inhibitor.
Thus, initial formation of the Shc/Grb2/Sos complex is caused by
binding of preformed Grb2/Sos complexes to tyrosine-phosphorylated Shc.
However, disassembly of the Shc/Grb2/Sos trimer is caused by rapid
phosphorylation of Sos and dissociation of Sos from Grb2, leaving the
Shc/Grb2 complex intact, while Sos becomes free (and presumably unable
to activate p21
). As Shc remains bound to Grb2, it is
unavailable to bind to any residual free Grb2/Sos complexes, which
might otherwise contribute to p21
activation. MEK
inhibition blocks Sos phosphorylation and Grb2/Sos dissociation,
resulting in Shc/Grb2/Sos complexes persisting throughout insulin
treatment.
p21 activation, as reflected by the
percentage of the protein in the GTP-bound state, is transient, despite
ongoing growth factor
stimulation(10, 20, 21, 22, 23, 24) .
p21
activation by insulin in HIRcB cells was transient,
with peak p21
-GTP levels by 7 min and a subsequent rapid
decline (Fig. 2). This correlates closely with the time course
of Shc/Grb2/SOS association (Fig. 1C), which peaked at
5 min and fell by 10 min. In contrast, MEK inhibition resulted in
prolonged stimulation of p21
-GTP, with peak levels not
seen until 15 min, and only a gradual decline thereafter. Taken
together, the data strongly suggest that dissociation of SOS from Grb2
plays a key role in limiting the duration of activation of p21
in response to ongoing insulin stimulation. This would be
analogous to a negative feedback loop described in Saccharomyces
cerevisiae, in which glucose induced elevated levels of cAMP via a
Ras/adenylyl cyclase pathway activated by the GEF Cdc25. The resultant
rapid activation of cAMP-dependent protein kinase then caused
phosphorylation of Cdc25 and caused it to dissociate from Ras,
resulting in Ras deactivation(35) .
Figure 2:
Time course of p21 activation by insulin with or without MEK inhibition.
Quiescent HIRcB cells labeled with
[
P]orthophosphate were treated with 20
µM PD098059 (
) or vehicle only (
) for 1 h and
then stimulated with insulin for the indicated times. p21
was isolated by immunoprecipitation, and the
p21
-bound labeled guanine nucleotides were
eluted and analyzed by TLC. Results were quantitated directly by
PhosphorImager and are expressed as GTP/(GTP + GDP)
100%.
Data are the mean values of two separate
experiments.
Rat1 fibroblasts express
10
EGF receptors/cell, and a similar analysis of the
association patterns of Shc, Grb2, and Sos upon EGF stimulation was
performed. EGF also caused Sos phosphorylation (Fig. 3A), and this was correlated with dissociation
from Grb2 and from Shc (Fig. 3, B and C). The
MEK inhibitor PD098059 prevented Sos phosphorylation, as well as
dissociation of Sos from Grb2 and Shc ((Fig. 3, A-C). Interestingly, the association of Grb2 with Shc,
illustrated in Fig. 3D, occurred rapidly but was not as
sustained as it was with insulin stimulation (Fig. 1D).
This may reflect down-regulation of the low number of endogenous EGFRs,
and could explain why at 60 and 120 min there was only a minimal Sos
mobility shift (Fig. 3A, lanes 5 and 6), and significant Grb2/Sos reassociation (Fig. 3B, lanes 5 and 6).
Furthermore, it explains why, in the presence of PD098059, Sos
coprecipitation with Shc declines by 30 min (i.e. although Sos
and Grb2 remain in a complex, much of Grb2 has dissociated from Shc).
Interestingly, while some Grb2/Sos reassociation was seen at 40 and 120
min, there was no concomitant coprecipitation of Sos with Shc (Fig. 3C, lanes 5 and 6), suggesting
that only Grb2 already dissociated from Shc reassociated with Sos.
Thus, the phenomenon of Sos phosphorylation, with Grb2/Sos complex
dissociation reducing the duration of existence of the active
Shc/Grb2/Sos complex, is also observed with signaling through the EGF
receptor.
Figure 3: Effects of EGF with or without MEK inhibition on Sos phosphorylation and binding between Sos, Grb2 and Shc. Serum-starved cells were treated with either the MEK inhibitor PD098059 or vehicle alone for 1 h, then stimulated with EGF for the indicated times. Cells lysates were divided for analysis by immunoprecipitation and immunoblotting with the indicated antibodies. A, Sos phosphorylation and mobility shift; B, Sos coprecipitation with Grb2; C, Sos coprecipitation with Shc; D, Grb2 coprecipitation with Shc. Results are representative of two separate experiments. IP, immunoprecipitation; IB, immunoblotting.
Prolonged p21 activation, either by
oncogenic forms of p21
(36) or by activation of
endogenous p21
by the introduction of upstream activators
such as membrane-targeted Sos (34) , is a transforming event
that plays a significant role in many malignancies(37) . Thus,
tight control of p21
activation is important in growth
regulation. Growth factors typically cause a transient rise in
p21
-GTP formation and the subsequent fall back to
base-line values serves to attenuate the hormonal signal, and may make
cells refractory to subsequent growth factor stimulation. The current
studies indicate that the mechanism underlying this attenuation process
involves hyperphosphorylation of Sos on serine/threonine residues with
subsequent dissociation of Grb2/Sos complexes; p21
activates the MAP kinase pathway, and this feedback signal is
generated from MEK or a serine/threonine kinase downstream of MEK.