(Received for publication, July 14, 1995; and in revised form, August 2, 1995)
From the
Previous work suggested that desensitization of p21 in response to growth factors such as epidermal growth
factor (EGF) results from receptor down-regulation. Here we show that
p21
is desensitized by insulin in 3T3-L1
adipocytes in the continued presence of activated insulin receptors,
while loss of epidermal growth factor and platelet-derived growth
factor (PDGF) receptors in response to their ligands correlates with
p21
desensitization. Furthermore, elevated
amounts of Grb2/Shc complexes persisted throughout p21
desensitization by insulin. However, immunoblotting of
anti-Son-of-sevenless (Sos) 1 and 2 immunoprecipitates with anti-Grb2
antisera revealed that p21
desensitization in
response to insulin and PDGF, but not EGF, is associated with a marked
decrease in cellular complexes containing Sos and Grb2 proteins.
Nonetheless, the desensitization of p21
in
response to these stimuli was homologous, in that each peptide could
reactivate [
P]GTP loading of p21
after desensitization by any of the others. Taken together,
these data indicate that insulin, EGF, and PDGF all cause disassembly
of Sos proteins from signaling complexes during p21
desensitization, but at least two mechanisms are involved.
Insulin elicits dissociation of Sos from Grb2 SH3 domains, whereas EGF
signaling is reversed by receptor down-regulation and Shc
dephosphorylation, releasing Grb2 SH2 domains. PDGF action triggers
both mechanisms of Grb2 disassembly, which probably operate in concert
with GAP to attenuate p21
signaling.
Peptide growth factors are key extracellular regulators that
modulate pathways of intermediary metabolism, protein synthesis, and
mRNA transcription. Growth factors also mediate critical steps in cell
cycle control and DNA synthesis. This remarkable multitude and
diversity of biological effects raises important questions about the
molecular signaling mechanisms involved in the actions of these
peptides. Recent work has revealed that several signaling pathways are
simultaneously stimulated by growth factors, including the
phosphoinositide cycle(1, 2, 3) ,
p21(4, 5, 6, 7, 8, 9, 10) ,
and the phosphatidylinositol 3-kinase
reaction(11, 12, 13, 14) . These
pathways initiate downstream events that must be highly coordinated,
controlled, and ultimately extinguished to elicit appropriate types and
duration of biological effects. Thus, understanding mechanisms that
restrain these signaling circuits is an important aspect of the
knowledge base required to fully describe them in molecular terms.
Cellular stimulation by peptide growth factors through small GTP
binding proteins exemplifies the highly regulated nature of
intermediary steps in signaling pathways. The p21 proteins cycle between the inactive, GDP-bound form and a
GTP-bound, biologically active state through the actions of guanosine
nucleotide exchange factors that catalyze release of GDP from
p21
, allowing GTP to bind, and GTPase activating
proteins which enhance p21
-bound GTP hydrolysis
to
GDP(4, 5, 6, 7, 8, 9, 10) .
Growth factors and insulin are thought to activate p21
by recruitment of guanosine nucleotide exchange factors such
as the Son-of-sevenless (Sos) (
)1 and 2 to
tyrosine-phosphorylated Shc proteins through the adaptor
Grb2(15, 16, 17, 18, 19) .
Grb2 binds to tyrosine phosphate on Shc (Tyr-317) through its src
homology SH2 domain and binds proline-rich regions on Sos proteins
through its SH3 domains(17, 20) . In the case of
insulin but not EGF or PDGF, another tyrosine-phosphorylated protein,
IRS-1, also binds Grb2 and may be involved in p21
activation(21, 22) . Rapid increases in GTP
loading of p21
proteins in response to growth
factors is followed by a deactivation phase whereby
GTP
p21
concentrations return to near basal
levels(23, 24, 25, 26, 27) .
Activated, GTP-bound p21
associates with the
N-terminal, regulatory domain of Raf protein kinases, leading to events
that elevate Raf kinase
activity(28, 29, 30) . The Raf kinases in
turn phosphorylate and activate MEK protein kinases, which further
activate a cascade of protein kinases, including the MAP
kinases(6, 28, 31, 32, 33, 34) .
Importantly, MAP kinase activation by growth factors through this
mechanism is also transient and returns to near basal levels with about
the same time course as p21
deactivation(35, 36, 37, 38) .
Thus, important feedback mechanisms operate to restrain this signaling
pathway and control the extent of cellular modulation.
Previous work
indicated that desensitization of p21 caused by
EGF is associated with rapid disappearance of EGF receptors from the
cell surface(39) , suggesting a simple mechanism for the
desensitization. Consistent with the concept that EGF-mediated
down-regulation of its specific receptors causes the loss of
p21
responsiveness to EGF, insulin was found to
reactivate p21
after desensitization by
EGF(39) . However, interpretation of those studies is difficult
because insulin itself did not elicit p21
desensitization under the conditions of the experiments.
This was perhaps due to the use of a unique cell line heterologously
expressing very high levels of human insulin receptors because we have
recently reported marked p21
desensitization in
response to insulin in 3T3-L1 adipocytes(40) . We and others
have also found that insulin caused partial dissociation of
Grb2
Sos complexes, suggesting an alternative mechanism of
deactivation(40, 41) . Furthermore, no detailed
studies have appeared which evaluate the basis for PDGF-induced
p21
desensitization. Thus, the aim of the
present investigation was to characterize the molecular nature of
p21
activation and deactivation in a well
established model system, the 3T3-L1 adipocyte, that responds to
insulin, EGF, and PDGF without overexpressed receptors.
We
demonstrate here that insulin, EGF, or PDGF treatment of 3T3-L1
adipocytes causes a rapid desensitization of p21 following the initial activation phase, but that
reactivation can be achieved by either of the two other peptides.
Importantly, we show that insulin-mediated p21
desensitization occurs without loss of activated cell
surface insulin receptors or Shc/Grb2 complexes, in contrast to
EGF-mediated p21
desensitization. Furthermore,
both insulin and PDGF cause disassembly of Grb2
Sos complexes in
these cells, while EGF does not. These data demonstrate that receptor
down-regulation cannot explain the homologous p21
desensitization caused by insulin and that regulation of Sos
function through its dissociation from Grb2 by both insulin and PDGF
may play an important role in this process.
Most studies on p21 regulation by insulin have
been performed on cells overexpressing insulin receptors rather than
primary fat or muscle cells. Cultured 3T3-L1 adipocytes were chosen for
the present studies because they have been extensively used as a model
system for insulin sensitive tissues and are highly responsive to the
hormone. In order to characterize the dynamics of GTP loading of
p21
in response to insulin, EGF, and PDGF in 3T3-L1
adipocytes, the GTP and GDP contents of p21
were
determined at various times after addition of insulin, EGF (Fig. 1) or PDGF (Fig. 2) to
P-labeled
cells. These measurements were conducted at room temperature because
greater responses to growth factors were observed compared to 37
°C. Nucleotides bound to p21
from
[
P]orthophosphate-labeled cells were analyzed by
thin layer chromatography. GTP accounted for about 10% of total labeled
GTP plus GDP in p21
immunoprecipitates from control cells
( Fig. 1and Fig. 2).
Figure 1:
Stimulation
and deactivation of GTP loading of p21 in
response to insulin and EGF. 3T3-L1 fibroblasts in 10-cm plates were
differentiated to adipocytes, labeled with
[
P]orthophosphate, and stimulated for various
times with either 10
M insulin or
10
M EGF at 22 °C. p21
was immunoprecipitated, and the bound nucleotides were
eluted and analyzed by chromatography on polyethyleneimine cellulose
plates. A, autoradiography of plates. B, calculated
ratio of [
P]GTP/([
P]GTP
+ [
P]GDP) as a function of
time.
Figure 2:
Stimulation and deactivation of GTP
loading of p21 in response to PDGF. 3T3-L1
fibroblasts in 10-cm plates were differentiated to adipocytes, labeled
with [
P]orthophosphate, and stimulated for
various times with 10
M PDGF. The relative
content of GTP and GDP was determined as described in the legend for Fig. 1. A, autoradiography of plates. B,
calculated ratio of
[
P]GTP/([
P]GTP +
[
P]GDP) as a function of
time.
Treatment of the cultured
adipocytes with insulin, EGF, or PDGF caused a 2-3-fold increase
in labeled GTP recovered from p21 immunoprecipitates
within 5 min. A gradual decay in this effect was observed in the
continued presence of these growth factors, resulting in the return of
GTP
p21
concentrations to near basal levels by
120-180 min ( Fig. 1and Fig. 2). Interestingly,
maximal [
P]GTP loading of p21
in
response to stimulation of 3T3-L1 adipocytes at 37 °C was also
observed by 5 min, but the deactivation phase was much more rapid at
the higher temperature (not illustrated). These data demonstrate that
insulin, EGF, and PDGF cause similar stimulatory effects on
steady-state GTP binding to endogenous p21
in these
cells, followed by a decay to GTP
p21
levels
approaching those observed in the basal state within 2-3 h at
room temperature and 20-30 min at 37 °C.
It has been
proposed (39) that receptor down-regulation accounts for
p21 desensitization in response to EGF, based on the
observed rapid loss of cell surface receptors that paralleled the
deactivation of p21
. However, certain receptor tyrosine
kinases known to desensitize p21
, such as the insulin
receptor, recycle to the plasma membrane in response to ligand-mediated
endocytosis, ensuring a high steady-state cell surface receptor content (46) . Experiments were designed to determine whether
tyrosine-phosphorylated receptors in the plasma membrane of 3T3-L1
adipocytes remain at high levels during prolonged insulin, EGF, or PDGF
treatment. As shown in Fig. 3, insulin receptor
subunit,
EGF, and PDGF receptors in plasma membranes were readily visualized by
immunoblotting with anti-tyrosine phosphate antibody 5 min after
incubation of cultured adipocytes with 100 nM insulin, 100
nM EGF or 10 nM PDGF, respectively. Little or no
receptor tyrosine phosphorylation could be detected in the absence of
these peptides. The level of tyrosine-phosphorylated insulin receptor
in the plasma membrane fraction of 3T3-L1 adipocytes remained elevated
throughout the 2-h incubation period during which p21
desensitization was observed (Fig. 3). In contrast, EGF or
PDGF receptor tyrosine phosphorylation in response to EGF, and PDGF,
respectively, markedly decreased during the 3-h p21
desensitization phase elicited by EGF or PDGF treatment (Fig. 3). These data indicate that EGF and PDGF receptor
down-regulation or dephosphorylation correlates with p21
desensitization, whereas insulin receptors in the plasma
membranes of cultured adipocytes remain tyrosine phosphorylated and
active throughout the course of p21
desensitization.
Figure 3:
Levels
of tyrosine- phosphorylated insulin, EGF, and PDGF receptors in plasma
membrane fractions of 3T3-L1 adipocytes. Plasma membranes from 3T3-L1
adipocytes were isolated after various times of stimulation with
10M insulin, 10
M EGF, or 10
M PDGF. The membrane
proteins (6 µg/lane) were separated by SDS-PAGE using 6% gels,
transferred to nitrocellulose, and blotted with anti-phosphotyrosine
antibody. A comparison of the intensities of the signals from the
insulin receptor by densitometry revealed no significant decrease from
5 min to 120 min of insulin stimulation (average of triplicate
determinations).
In order to extend the results in Fig. 3, we reasoned that
tyrosine phosphorylation of Shc proteins should correlate with the
presence of activated receptors in the plasma membrane. Thus, for
example, Shc tyrosine phosphorylation should be transient and decrease
with a time course similar to the loss of activated EGF receptors from
plasma membranes. This should be accompanied by dissociation of Grb2
from Shc. This was tested by immunoprecipitation of Shc proteins from
lysates of 3T3-L1 adipocytes treated with insulin or EGF for various
times. As shown in Fig. 4, immunoblotting such Shc
immunoprecipitates after SDS-PAGE with anti-Grb2 antiserum revealed
both insulin and EGF rapidly increased the amount of Grb2 associated
with Shc. In the case of insulin stimulation, the amount of Grb2 in Shc
complexes remain elevated for 2 h. In contrast, complexes containing
Shc and Grb2 formed in response to EGF receptor activation are mostly
dissociated during the time course of p21 desensitization. The time course of this disassembly is
consistent with the hypothesis that it constitutes, at least in part,
an underlying mechanism of p21
desensitization in
response to EGF.
Figure 4:
Association of Grb2 with Shc proteins in
cultured adipocytes. 3T3-L1 adipocytes were stimulated with either
10M insulin or 10
M EGF for the indicated times. The cells were then lysed, and 500
µg of total cell protein was immunoprecipitated with 2 µg of
Shc antibody. A quarter of the precipitates were separated by SDS-PAGE,
transferred to nitrocellulose, and blotted with antibodies to Grb2. A, autoradiography of the blots. NI is precipitation
of extracts from the 30 min time points with a non-immune serum. NE is mock precipitation using the anti-Shc antibody, but no extract. B, quantification of Grb2 in the immunoprecipitates by
densitometry. The data were normalized to the initial stimulated level
of Grb2 (10 min) and were compiled from two
experiments.
The results indicating that Grb2 proteins remain
associated with Shc during insulin-mediated p21 desensitization prompted us to examine the interaction of Grb2
and Sos proteins under these conditions. It was recently shown that
insulin causes disassembly of Sos from Grb2 during p21
desensitization(40, 41) . Cultured 3T3-L1
adipocytes were incubated at 37 °C with or without insulin, EGF, or
PDGF for 20 min to cause p21
desensitization. Lysates
were immunoprecipitated with rabbit anti-mSos antibodies raised against
a peptide corresponding to an N-terminal region of murine mSos1 that is
identical to mSos2. Immunoblot analysis of such immunoprecipitates with
anti-mSos1 antibodies specific to that isoform revealed equivalent
amounts of mSos1 present in the lysates under all experimental
conditions (Fig. 5). Treatment of cells with insulin, EGF, or
PDGF caused a shift in electrophoretic migration of mSos1 proteins,
reflecting hyperphosphorylation on serine and threonine
residues(23, 47) . Immunoblotting of the anti-mSos
precipitates with anti-Grb2 antibodies revealed Grb2 associated with
Sos proteins (Fig. 5B). Importantly, insulin- and
PDGF-mediated p21
desensitization was associated with a
marked decrease in Grb2 content in these Sos immunoprecipitates. PMA,
which also causes Sos hyperphosphorylation, mimicked the ability of
insulin or PDGF to dissociate Grb2 from Sos (Fig. 5). In
contrast, EGF action failed to cause a detectable reduction in
complexes containing Sos and Grb2 proteins. Densitometry of the
autoradiographs from several such experiments demonstrated a mean
inhibition by insulin, PDGF, and PMA of about 60% in the amount of Grb2
protein that is associated with mSos proteins (Fig. 5C).
Figure 5:
Disassembly of Grb2 from Sos in response
to treatment of 3T3-L1 adipocytes with insulin, PDGF, or PMA but not
EGF. A-C show immunoprecipitates from lysates of untreated
control cells (CON) and cells that were stimulated for 20 min
with 10M insulin (INS),
10
M EGF, 10
M PDGF, or 10
M PMA. A, equal
parts of the immunoprecipitates were separated by reducing SDS-PAGE (6%
gel), transferred to nitrocellulose, and blotted with mSos1 antibody. B, equal parts of the immunoprecipitates were separated by
reducing SDS-PAGE (12% gel), transferred to nitrocellulose, and blotted
with Grb2 antibody. C, quantification of the amounts of Grb2
in mSos immunoprecipitates by densitometry. Data from insulin- and
EGF-stimulated cells represent three independent experiments performed
in duplicate. Data for PMA and PDGF treatment represent two independent
experiments performed in duplicate. The results are normalized to the
amount of Grb2 in unstimulated cells.
The fact that p21 desensitization due to insulin and PDGF action appears associated
with the disassembly of cellular Grb2
Sos complexes suggested a
desensitization mechanism that may be general rather than selective. On
the other hand, EGF-mediated deactivation might be expected to block
the action of EGF specifically, based on the rapid down-regulation of
its specific receptors. These hypotheses were tested by prolonged
stimulation of
P-labeled 3T3-L1 adipocytes with either
EGF, PDGF, or insulin to cause activation and deactivation of
p21
proteins, followed by a further addition of either of
the three growth factors alone. Fig. 6shows, as expected, that
3T3-L1 cells treated with EGF for 3 h at 22 °C failed to display
increased [
P]GTP loading of p21
in
response to a second addition of EGF, but did respond to insulin
treatment. PDGF could also reactivate p21
after its
desensitization to EGF (Fig. 7). Surprisingly, the
desensitization of p21
mediated by insulin action was
also homologous. Thus, prior prolonged treatment of the cultured
adipocytes with insulin blocked p21
activation due to
further addition of insulin, while EGF (Fig. 6) or PDGF (Fig. 7) treatment of such cells caused reactivation of
p21
. The increase in [
P]GTP
binding to p21
proteins caused by insulin, EGF, or PDGF
in homologously desensitized cells was as great as in control
adipocytes ( Fig. 6and Fig. 7).
Figure 6:
Effects of insulin or EGF treatment of
3T3-L1 cells on GTP loading of p21 and their
responsiveness to a subsequent incubation with the peptides. Cells were
labeled with [
P]orthophosphate as in Fig. 1and were treated or not with 10
M EGF or 10
M insulin at 22 °C.
After 2 h (insulin-stimulated cells) or 3 h (EGF-stimulated cells), the
cells were treated again with the same concentrations of hormones for 5
min as indicated in the figure. The nucleotide load on p21
was then determined. A, autoradiography of plates. B, the measured [
P]GTP content in
p21
immunoprecipitates under various conditions.
The data are from two experiments and are normalized to the amount of
GTP in unstimulated cells. The values are means of triplicate or
quadruplicate determinations, and the error bars represent the
standard deviations.
Figure 7:
Effects of treatment of 3T3-L1 cells with
PDGF, EGF, or insulin on their responsiveness to a subsequent
incubation with peptides. Cells were labeled with
[P]orthophosphate as in Fig. 1and were
treated or not with 10
M PDGF,
10
M EGF, or 10
M insulin at 22 °C. After 2 h (insulin-stimulated cells) or 3 h
(EGF- and PDGF-stimulated cells), the cells were treated with the same
concentrations of peptides for 5 min as indicated in the figure. The
nucleotide load on p21
was then determined. A, autoradiography of plates. B, the measured
[
P]GTP content in p21
under various conditions. The values are means of four to
eight determinations from three separate experiments, and the error
bars represent the standard
deviations.
The data in Fig. 7show that desensitization of p21 by PDGF is
also readily reversed by EGF or insulin. The stimulation of
[
P]GTP binding to p21
by EGF or
insulin is similar in magnitude whether or not p21
desensitization to PDGF was first accomplished. Thus, for all
combinations of growth factors used in this study, p21
desensitization is homologous in that the initiating growth
factor is unable to maintain high cellular levels of
GTP
p21
, while all others can reactivate GTP binding
to the proto-oncogene. Furthermore, homologous desensitization of
p21
occurs whether or not activated receptors are
maintained in the cell surface membrane and whether or not a portion of
the cellular Grb2
Sos complexes are dissociated. To confirm this
latter point,
P-labeled 3T3-L1 adipocytes were treated
with PMA under conditions (see Fig. 5) where about 60% of the
Sos proteins were dissociated from Grb2 and the p21
activation by growth factors was assessed. As shown in Fig. 8, PDGF, EGF, or insulin added to such PMA-treated cells
were fully able to cause GTP loading of p21
. Thus,
disassembly of about 60% of the cellular Grb2
Sos complexes is not
sufficient to prevent acute activation of p21
by insulin,
EGF, or PDGF.
Figure 8:
Activation of p21 by growth factors after partial dissociation of
Grb2
Sos complexes induced by PMA. Adipocytes were treated with 1
µM PMA for 30 min, and then for 5 min with or without
10
M insulin, 10
M EGF, or 10
M PDGF as indicated. The
cells were then lysed, and the nucleotide load on p21
was determined. The values are means of four determinations
from two separate experiments, and error bars represent the
standard deviations.
Stimulated GTP loading of p21 proteins by
growth factors is now well established as a key signaling element in
their enhancement of cell
proliferation(4, 5, 6, 7, 8, 9, 10) .
The potent activation of protein kinases(28, 31, 32, 33, 34, 48) and
the strong biological responses elicited by cellular
p21
GTP complexes dictates the need for finely tuned
regulatory mechanisms eliciting reversal of these effects. Consistent
with results on the actions of growth factors in other cell
types(23, 24, 25, 26, 27) ,
we observe a marked deactivation phase of p21
proteins
after their enhanced GTP loading in response to EGF, PDGF, or insulin
in 3T3-L1 adipocytes ( Fig. 1and Fig. 2). EGF and PDGF
were more effective in stimulating levels of p21
GTP
(3-fold) than was insulin (2-fold) at 22 °C in these cells, and the
deactivation phase was somewhat slower during treatment with EGF or
PDGF (3 h) versus insulin (2 h). However, in all cases, the
cellular concentrations of p21
GTP fell to levels
approaching those measured under basal conditions ( Fig. 1and Fig. 2), indicating the operation of a strong desensitization
process. This was confirmed by the observation that readdition of the
growth factor that had initiated the p21
activation and
deactivation phases resulted in no further stimulation ( Fig. 6and Fig. 7).
Experiments with 3T3-L1 adipocytes
performed at 37 °C showed more rapid desensitization phases for
both insulin and EGF treatment, which were complete within 30 min of
initial incubation with peptide (not illustrated). Desensitization at
37 °C led to levels of p21GTP that were not
significantly different than those measured in untreated adipocytes.
Furthermore, this rapid time course of p21
activation and
deactivation observed at 37 °C paralleled that of the activation
and deactivation phases for MAP kinase activity in response to insulin
or EGF in 3T3-L1 cells (49 and data not shown). Taken together, these
data demonstrate that effective cellular mechanisms operate in this
cultured adipocyte model system to limit the duration of maximal
p21
signaling in response to insulin and growth factors.
A recent report suggested that little deactivation and
desensitization of p21 occurs in response to insulin
treatment of transfected cultured cells overexpressing insulin
receptors(39) . In contrast, our present results show
unequivocal p21
desensitization during prolonged
incubation of 3T3-L1 adipocytes with insulin ( Fig. 1and Fig. 6). Furthermore, more recent work in our laboratory has
demonstrated insulin-mediated p21
desensitization in
primary rat adipocytes as well (not illustrated). The failure to
observe p21
desensitization in response to insulin in the
previous study (39) probably reflects the different cell types
used. Cells expressing high levels of insulin receptors were employed
in those studies. Experiments in our laboratory show that Chinese
hamster ovary cells expressing human insulin receptors at high levels
also exhibit both activation and deactivation phases with respect to
GTP loading of p21
upon insulin treatment (not
illustrated). Thus it is unlikely that heterologous expression of
insulin receptors in cultured cells eliminates the desensitization
phenomenon, although perhaps extraordinarily high levels of insulin
receptors used in the previous study (39) may account for the
difference in results.
It has been suggested that p21 desensitization occurs through specific down-regulation of growth
factor receptors based on studies with EGF(39) . In those
experiments, EGF-mediated p21
desensitization was
accompanied by rapid disappearance of cell surface EGF receptors, as
detected by binding of cells to labeled EGF peptide. The present work
demonstrates that stimulated GTP loading of p21
can be
reversed and p21
desensitized even in the continued
presence of activated receptors ( Fig. 1and Fig. 3). Our
experimental approach took advantage of the fact that relatively pure
preparations of plasma membranes can be prepared from
adipocytes(40, 50) . Tyrosine-phosphorylated EGF,
PDGF, or insulin receptors are readily identified in these membranes
very rapidly after treatment of the intact cells with growth factors by
SDS-PAGE and immunoblotting with anti-tyrosine phosphate antibody (Fig. 3). Three hours after incubation of cultured adipocytes
with EGF or PDGF, tyrosine-phosphorylated EGF or PDGF receptors,
respectively, were greatly diminished, while tyrosine-phosphorylated
insulin receptors remained in these plasma membranes at high levels.
These data are consistent with extensive literature documenting the
rapid cellular internalization and degradation of EGF and PDGF
receptors in response to their respective
ligands(51, 52, 53, 54, 55, 56, 57, 58, 59) ,
and the rapid recycling of insulin receptors back to the cell surface
during insulin treatment (46, 60, 61) . The
present data, in combination with results of others, show that the
decay of EGF or PDGF action on p21
under the conditions
of our experiments could result from loss of their functional cell
surface receptors, but this mechanism cannot explain p21
deactivation in response to insulin.
A major pathway of
p21 activation appears to be phosphorylation of Shc
proteins at tyrosine 317, which in turn binds the SH2 domain of
Grb2(20, 47, 62, 63, 64, 65) .
Shc proteins, which contain an SH2 domain and a domain with sequence
similarity to collagen, are the products of a single gene that
apparently gives rise to multiply spliced mRNA
transcripts(63) . Upon its activation and autophosphorylation
in response to EGF, the EGF receptor binds the SH2 domain of Shc
directly through its phosphorylated tyrosines 1173 and 992. Activated
EGF receptors also bind Grb2 directly through phosphorylated tyrosines
1068 and 1086. Activated insulin and PDGF receptors do not appear to
directly bind Shc or Grb2, but initiate tyrosine phosphorylation of Shc
through an unknown mechanism(64, 65) . This same
alternate mechanism may be shared by EGF receptors because the latter
can cause Shc tyrosine phosphorylation even when the tyrosines on the
receptor are removed(66, 67) . Thus, EGF receptors can
apparently act to mobilize Sos proteins through direct binding of Grb2
or Shc, and by an independent pathway leading to Shc phosphorylation.
These considerations may explain the observation that GTP loading of
p21
is enhanced more markedly by EGF than by insulin (Fig. 1). Similarly, the time course of EGF-mediated p21
desensitization is longer than that for insulin (Fig. 1).
Consistent with this concept, the present studies show Shc association
with Grb2 is more pronounced in response to EGF than insulin at the
earliest time point measured in 3T3-L1 adipocytes (Fig. 4).
The time course of Shc/Grb2 association in response to EGF and
insulin (Fig. 4) further confirm the postulate that EGF
receptors rapidly down-regulate while insulin receptors remain active.
The extensive decline in Grb2 associated with Shc after EGF stimulation
apparently reflects a decrease in Shc tyrosine phosphorylation state.
Insulin action, on the other hand, is not associated with a decrease in
cellular Grb2/Shc complexes following the initial stimulation of
complex formation observed at 10 min. Tyrosine phosphatase activity is
presumably responsible for Shc dephosphorylation during loss of active
EGF receptors, although the tyrosine phosphatase involved is not known.
In any case, these results provide an independent confirmation that a
decay in relevant tyrosine phosphorylation (EGF receptors and Shc)
correlates with p21 desensitization caused by EGF, but
not insulin. Thus, as shown in Fig. 9, the activation complex
containing EGF receptor, Shc, and Grb2
Sos proteins is
hypothesized to disassemble during desensitization, yielding
dephosphorylated (and degraded) EGF receptor, dephosphorylated Shc, and
released Grb2
Sos complexes. According to this model (Fig. 9, bottom), all of these components are removed
from the plasma membrane, while p21
remains. PDGF
receptor down-regulation also occurs in response to PDGF, and thus
PDGF-mediated desensitization would be expected to be similar to the
EGF system in this respect. In contrast, our data indicate that Sos
proteins disengage from p21
in response to insulin while
Shc
Grb2 complexes remain present (Fig. 9, top).
PDGF is also able to cause disassembly of Sos
Grb2 complexes (Fig. 5), suggesting that disengagement of both SH2 and SH3
domains of Grb2 from Shc and Sos, respectively, occurs in response to
PDGF.
Figure 9:
Hypothetical protein complexes involved in
the activation and desensitization of p21 by
insulin and EGF. In this model, both insulin and EGF cause Sos to bind
to tyrosine-phosphorylated Shc, allowing it to interact with
p21
at the plasma membrane. In the case of
insulin action on 3T3-L1 adipocytes, the continued presence of
activated insulin receptors ensures that Shc is maintained in its
tyrosine-phosphorylated state, and desensitization hypothetically
occurs by Sos dissociation from Grb2 in the complexes. Prolonged EGF
treatment causes disassembly of Sos-containing complexes by a different
mechanism. Upon down-regulation of the EGF receptor, Shc is released
from the receptor. Shc is dephosphorylated, leading to dissociation of
Grb2-Sos from Shc in the membrane. Thus, EGF receptors, Shc, and
Grb2
Sos are removed from plasma membrane-bound
p21
. These mechanisms presumably operate in
conjunction with GAP activities to attenuate p21
activation.
The disassembly of Sos from Grb2 complexes during insulin- and
PDGF-mediated p21 desensitization may reflect an
important feedback mechanism elicited by a protein kinase or kinases.
Sos is known to be hyperphosphorylated in response to insulin (68) or EGF (69) , and PDGF also causes this effect as
indicated in Fig. 5A by the shift in electrophoretic
mobility of Sos on SDS-PAGE. We previously reported that at least two
of the sites phosphorylated in heterologously expressed Drosophila Sos protein in intact cells matched sites phosphorylated in
vitro by MAP kinase(70) . Phosphorylation of the yeast (Saccharomyces cerevisiae) CDC25 Ras exchange factor has also
been reported to correlate with release of this protein from the plasma
membrane and Ras deactivation(71) . Recently, it was proposed
that MAP kinase phosphorylation of Sos results in its dissociation from
Grb2. However, a key finding presented in this study is the failure of
EGF to cause Sos dissociation from Grb2 (Fig. 5), in spite of
its ability to cause Sos hyperphosphorylation. Both insulin and EGF
cause similar stimulations of MAP kinases in 3T3-L1 adipocytes (49 and
data not shown). These considerations indicate that phosphorylation of
Sos by MAP kinases is not sufficient to cause disassembly of
Grb2
Sos complexes. Perhaps a unique protein kinase or kinases are
stimulated by insulin and PDGF and cause Sos phosphorylation on a
unique site that regulates Grb2 binding. This hypothesis will require
further testing. Protein kinase C or protein kinases activated in
response to protein kinase C stimulation by PMA appear to catalyze this
response as well (Fig. 5).
The fact that only about 60% of
the cellular Grb2Sos complexes are dissociated during p21
desensitization in response to insulin raises interesting
questions about the role of this disassembly in p21
desensitization. Insulin increases GTP loading of p21
in 3T3-L1 adipocytes by only about 2-fold, followed by a return
to basal levels ( Fig. 1and Fig. 6). Thus, the magnitude
of the decrease in Sos
Grb2 complexes in response to prolonged
insulin treatment approximately coincides with the extent of decrease
in GTP
p21
levels during deactivation and
desensitization. The amount of Shc
Grb2
Sos complexes present
at the plasma membrane probably reflects an equilibrium involving the
concentration of tyrosine-phosphorylated Shc and concentration of
available Sos
Grb2 complexes. In the case of insulin action,
activated receptors and tyrosine-phosphorylated Shc, reflected by
Shc
Grb2 complexes (Fig. 4), remain elevated during
prolonged treatment, but Sos
Grb2 complexes are decreased by about
half. This results in an expected decrease in plasma membrane Sos that
matches the decreased GTP
p21
levels. Thus
insulin-mediated dissociation of Sos from Grb2 can account
quantitatively for p21
deactivation, and desensitization
could result because no further increase in insulin receptor
phosphorylation occurs upon adding more insulin.
If p21 is desensitized to insulin by decreased Grb2
Sos complexes,
how do EGF and PDGF reactivate p21
under these
conditions? One possibility is that these growth factors cause highly
elevated levels of tyrosine phosphorylated Shc that effectively
recruits Sos
Grb2 complexes from the remaining pool. This
explanation would be particularly satisfactory if EGF and PDGF normally
elicit much higher levels of tyrosine phosphorylated Shc than are
required for the 3-fold activation of p21
. This may be
the case because EGF treatment for 10 min is observed to cause much
greater recruitment of Grb2 to Shc than does insulin (Fig. 4).
Thus, EGF may be able to cause recruitment of sufficient
Shc
Grb2
Sos complexes even after depletion of about half of
the cellular Grb2
Sos complexes due to insulin action. It is also
possible that specific cellular pools of Sos/Grb2 are subject to
insulin-mediated dissociation, leaving other pools available for
reactivation of p21
by EGF or other external stimuli.
Alternatively, EGF and PDGF may recruit a different exchange factor or
factors such as the newly reported C3G protein(72) . Further
experiments are required to definitively link the Grb2/Sos disassembly
observed here to the p21
desensitization mechanism.
However, the data presented here and elsewhere (40) by our
laboratory strongly implicate a link between these two processes. It is
also likely that additional mechanisms, presumably involving GAP
func-tion, act in concert with receptor down-regulation and Sos/Grb2
disassembly to attenuate p21
signaling.