(Received for publication, November 18, 1994; and in revised form, December 7, 1994)
From the
Insulin receptor signaling acutely stimulates GTP loading of
p21, apparently by mobilizing complexes of Grb2
and the guanine nucleotide exchangers Son-of-sevenless (Sos) 1 and 2 to
associate with tyrosine-phosphorylated proteins in the plasma membrane.
Here we show that in
P-labeled 3T3-L1 adipocytes the
elevated cellular concentrations of [
P]GTP-bound
p21
in response to insulin return to near basal
levels after 20-30 min of hormone stimulation, while insulin
receptors remain activated. Lysates of such desensitized cells were
quantitatively immunoprecipitated with an antiserum recognizing both
Sos1 and Sos2 proteins or a specific anti-Sos2 antiserum. Immunoblot
analysis of these precipitates revealed that insulin causes a marked
hyperphosphorylation of Sos1 and a 50% decrease in Grb2 associated with
Sos proteins under these conditions. Similarly, anti-Grb2
immunoprecipitates of such lysates revealed the presence of decreased
Sos1 protein due to insulin action. The disassembly of Grb2 from Sos
proteins slightly precedes the time course of p21
deactivation in response to insulin. These data are
consistent with the hypothesis that the dissociation of Grb2 from Sos
proteins caused by insulin in 3T3-L1 cells mediates
p21
deactivation and desensitization.
The small GTPase p21 is a key
intermediate in the signaling pathways of insulin and numerous other
hormones and growth
factors(1, 2, 3, 4, 5, 6, 7, 8) .
In the biologically active GTP-bound state, p21
functions to initiate cellular reactions catalyzed by
multiple serine protein kinases, including the mitogen-activated
protein
kinases(9, 10, 11, 12, 13) .
These enzymes in turn regulate many cellular components such as
phospholipase A
(14) and nuclear transcription
factors(15, 16, 17, 18) . The
increased GTP binding to p21
caused by growth
factors is apparently mediated by complexes containing guanine
nucleotide exchange factors such as the murine Son-of-sevenless
proteins (mSos1 and mSos2) and substrates of receptor tyrosine kinases
at the plasma membrane(19, 20, 21) . Such
substrates appear to include the protein Shc, which is
tyrosine-phosphorylated in response to insulin and many stimuli (22, 23, 24) , as well as the insulin and
insulin-like growth factor-1 receptor substrate IRS-1 (insulin receptor
substrate-1)(25, 26) . Assembly of these complexes are
promoted by the adaptor Grb2, which binds proline-rich motifs in the
COOH terminus of Sos through its Src homology 3 domains, and tyrosine
phosphate sites on receptor substrates through its Src homology 2
domain(19, 27, 28, 29, 30, 31, 32, 33, 34) .
Enhancement of GTP binding to p21 by
extracellular stimuli is transient in virtually all cells and organisms
studied. The deactivation phase generally ensues after 5 min of cell
stimulation at 37 °C, returning p21
GTP
content to near basal levels by about 30
min(2, 3, 35, 36) . Interestingly,
the stimulation of mitogen-activated protein kinases that occurs
secondary to p21
activation is also reversed to
near control levels within this time
frame(37, 38, 39, 40, 41) .
Although the response pattern of p21
activation
and deactivation is likely to be important in restraining and
coordinating downstream biological responses, little is known about its
molecular basis. In the case of insulin action, recent data indicated
that GTP loading of p21
did not significantly
decline with time(42) . However, these studies were conducted
in stably transfected cells expressing high levels of insulin
receptors. Preliminary experiments in our laboratory using 3T3-L1
adipocytes, a well established model system with insulin receptor
levels similar to those of primary fat cells, revealed rapid
p21
deactivation and desensitization following
stimulation by insulin. We document in this report that marked
dissociation of cellular complexes containing Grb2 and Sos proteins
occurs just prior to the observed decreases in GTP
p21
concentrations in these insulin-treated cells, suggesting a
molecular basis for the desensitization of p21
.
In order to probe the underlying mechanisms of
insulin-mediated p21 desensitization in 3T3-L1
adipocytes, we first corroborated previous data showing that insulin
receptors in the plasma membrane remain activated during prolonged
insulin treatment(47) . Plasma membranes prepared as described (43) from 3T3-L1 adipocytes treated without or with 100 nM insulin for up to 1 h at 37 °C were found to contain
continuous high levels of tyrosine-phosphorylated insulin receptors, as
assessed by immunoblotting with anti-receptor (CT-1 monoclonal) and
anti-tyrosine phosphate (4G10) antibodies (not illustrated). These data
indicate that receptor down-regulation cannot explain p21
desensitization which is complete by 30 min (for time course, see Fig. 4) in 3T3-L1 adipocytes. We then focused on cellular
components known to modulate GTP binding to p21
proteins.
Initial experiments tested whether Grb2 binding to
tyrosine-phosphorylated Shc proteins in response to insulin was
decreased with prolonged treatment. Insulin rapidly stimulated complex
formation between these proteins, as published previously(23) ,
but no diminution of this effect could be observed during the time
course of p21
desensitization (not illustrated).
Figure 4:
Time course of insulin-mediated Grb2
dissociation from mSos1 and p21 activation in
3T3-L1 adipocytes. The mSos1 and mSos2 proteins were immunoprecipitated
from cells that had been stimulated with 10
M insulin for various periods of time as described under
``Experimental Procedures.'' The inset shows
immunoblots of mSos immunoprecipitates using the mSos1 or Grb2
antibodies. The graph shows the amount of Grb2 associated with mSos
immunoprecipitates as determined by densitometry. The amount of Grb2
present in each of the immunoprecipitates was compared to the amount of
Grb2 in immunoprecipitates from unstimulated control cells (100%). Each
data point represents the average of four independent experiments, and
data points marked by * were determined to be statistically different
from the control (p
0.95) by the paired t test.
The graph also shows the stimulation of [
P]GTP
loading of p21
by 10
M insulin in 3T3-L1 adipocytes at 37 °C. Cells were labeled with
[
P]orthophosphate and the GTP content of
p21
was determined as described under
``Experimental Procedures.'' The calculated ratios of
[
P]GTP/([
P]GTP +
[
P]GDP) on p21
are
shown.
Next,
the association of Grb2 and Sos proteins was examined under these
conditions. Cultured 3T3-L1 adipocytes were incubated with or without
insulin to cause p21 activation and desensitization.
Lysates were immunoprecipitated with rabbit anti-mSos antibodies raised
against a peptide within the NH
-terminal region of murine
mSos1 that is identical to mSos2. Immunoblot analysis of such
immunoprecipitates from control cell lysates with anti-mSos antibody
revealed quantitative immunoprecipitation of both mSos1 and mSos2
proteins (Fig. 1). Furthermore, as shown in Fig. 2,
equivalent amounts of mSos1 was present in such immunoprecipitates from
control versus insulin-treated cells. Treatment of cells with
insulin caused a shift in electrophoretic migration of mSos1 proteins (Fig. 2), reflecting hyperphosphorylation on serine and
threonine residues(32) . Immunoblotting of the anti-mSos
precipitates with anti-Grb2 antibodies revealed Grb2 associated with
Sos proteins (Fig. 2). Comparison of the Sos-associated Grb2
with the amount of total Grb2 in lysates (with appropriate
normalization for volumes of samples used) indicates only a few percent
of cellular Grb2 is complexed with Sos. Importantly, insulin treatment
of cells for 10 min was associated with a marked decrease in Grb2
content in these Sos immunoprecipitates.
Figure 1: Immunoprecipitation of mSos1 and mSos2 from 3T3-L1 adipocytes with mSos antibody. Preimmune or immune serum was used to immunoprecipitate mSos from lysates of 150-mm plates of 3T3L1 adipocytes as described under ``Experimental Procedures.'' 5% of the immunoprecipitate supernatants (Sup) and 20% of the pellets were run on separate 6% SDS-PAGE, transferred to nitrocellulose and Western blotted with antibody specific for mSos1 or mSos2.
Figure 2:
Amounts of Grb2 associated with anti-mSos
immunoprecipitates from 3T3-L1 adipocytes treated with or without
insulin. 150-mm plates of 3T3-L1 adipocytes were starved for 24 h and
then stimulated for 10 min with 10M insulin. A and B show mSos immunoprecipitates
from untreated control cell and insulin-stimulated cells lysates (10 mg
of total protein) as described under ``Experimental
Procedures.'' Immunoprecipitates with nonimmune serum are also
shown. A, 17% of each immunoprecipitate pellet was separated
by reducing SDS-PAGE (6% gel), transferred to nitrocellulose, and
blotted with mSos1 antibody. B, 17% of the each
immunoprecipitate pellet and 0.5% of a lysate were separated by
reducing SDS-PAGE (12% gel), transferred to nitrocellulose, and blotted
with Grb2 antibody.
In order to determine
whether mSos2 dissociates from Grb2 under conditions of
insulin-mediated p21 desensitization, 3T3-L1 adipocyte
lysates were immunoprecipitated with antibodies specific for mSos2. Fig. 3shows that this antisera quantitatively precipitates mSos2
from such lysates whether or not cells were first incubated with
insulin. Immunoblotting the mSos2 immunoprecipitates with anti-Grb2
antisera revealed a marked decrease in the amount of Grb2 associated
with mSos2 in response to insulin treatment of the intact cells. It
should be noted that only a small percentage of total cellular Grb2
protein is associated with mSos2 whether or not insulin is present
because no detectable depletion of Grb2 from the lysates is seen after
immunoprecipitation of mSos2 (Fig. 3B).
Figure 3:
Amounts of Grb2 associated with anti-mSos2
immunoprecipitates from 3T3-L1 adipocytes treated with or without
insulin. A and B show immunoprecipitates from 150-mm
plates of 3T3-L1 untreated control cells and cells that were stimulated
for 10 min with 10M insulin. About
one-half of the total protein (4 mg) were used in each
immunoprecipitate. A, 17% of each immunoprecipitate and 0.5%
of each supernatant (Sup) were separated by reducing SDS-PAGE
(6% gel), transferred to nitrocellulose and blotted with antibody
specific for mSos2. Samples were also blotted with mSos1 specific
antibody revealing mSos1 present only in the supernatants and not in
the pellets (data not shown). B, 17% of each immunoprecipitate
pellet and 0.5% of each supernatant were separated by reducing SDS-PAGE
(12% gel), transferred to nitrocellulose, and blotted with Grb2
antibody.
The time
course of Grb2 dissociation from Sos proteins was compared to that for
p21 desensitization in response to insulin (Fig. 4). Significant decreases of immunoreactive Grb2 present
in anti-mSos precipitates were first detected after 5 min of insulin
treatment of 3T3-L1 adipocytes at 37 °C, with a maximal effect
observed after about 10 min of incubation with insulin. Decreases in
steady-state p21
GTP content from the peak levels
observed 5 min after addition of insulin correlated closely with this
time course (Fig. 4). It should be noted that this decay of
GTP
p21
levels with time represents a true
desensitization process because further addition of insulin at 30 min
fails to reactivate p21
. (
)Insulin-mediated
hyperphosphorylation of mSos1 proteins was stoichiometric under these
conditions, in that all detectable Sos protein shifted migration on
polyacrylamide gels, and was maximal by about 10 min (Fig. 4, inset). These data demonstrate that disassembly of complexes
containing Grb2 and Sos proteins by insulin action occurs with a time
course that precedes and can account for p21
deactivation
in response to the hormone.
In order to confirm this result with independent methodology, antisera was raised in rabbits against a Grb2-glutathione S-transferase fusion protein and used to immunoprecipitate Grb2 from 3T3-L1 adipocyte lysates. The antisera could deplete virtually all of the immunoreactive Grb2 from 3T3-L1 cell lysates (Fig. 5B). Immunoprecipitation of control cell lysates with anti-Grb2 antibody also depleted over half of the cellular mSos1 protein as evidenced by comparison of immunoreactive mSos1 in supernatants after precipitation with non-immune versus immune sera (Fig. 5). Thus, these data indicate that at least half of the cellular Sos1 proteins are associated with Grb2 under these conditions. Furthermore, insulin treatment of 3T3-L1 adipocytes for 20 min caused a marked decrease in the intensities of immunoreactive mSos1 protein associated with cellular Grb2 (Fig. 5). This conclusion is reinforced by the observation that the ratio of mSos1 in post-precipitation supernatants to mSos1 in the immunoprecipitates is higher when cells are incubated with insulin.
Figure 5:
Amounts of mSos1 associated with anti-Grb2
immunoprecipitates from 3T3-L1 adipocytes treated with or without
insulin. A and B show Grb2
immunoprecipitates from lysates of 100-mm plates of untreated control
cells and cells that were stimulated for 20 min with 10M insulin as described under ``Experimental
Procedures.'' 2.5 mg of total protein was used in each
immunoprecipitation. Immunoprecipitates with nonimmune serum are also
shown. A, 17% of each immunoprecipitate pellet and 0.5% of
each supernatant (Sup) were separated by reducing SDS-PAGE (6%
gel), transferred to nitrocellulose, and blotted with mSos1 antibody. B, 17% of each immunoprecipitate and 0.5% of each supernatant (Sup) were separated by reducing SDS-PAGE (12% gel),
transferred to nitrocellulose and blotted with Grb2 antibody. The
bottom band of the doublet seen in the gel is Grb2, and the top band is
a nonspecific band that cross-reacts with anti-mouse Ig
antibody.
The mechanism by which
Sos disassembly from Grb2 occurs in response to insulin is unknown but
may relate to Sos phosphorylation. Phosphorylation of the exchange
factor CDC25 in yeast by cAMP-dependent proteins kinases (48) has been reported to release it from its plasma membrane
localization with Ras, indicating a negative feedback regulation of the
nucleotide exchange reaction. The mSos1 and mSos2 proteins contain
numerous potential phosphorylation sites flanking proline-rich motifs
in these proteins that bind the NH-terminal Src homology 3
domain of Grb2. Sos protein phosphorylation in mammalian cells by
mitogen-activated protein kinases (49) may also reflect a
feedback mechanism for regulating mSos1 and mSos2 proteins. Consistent
with this hypothesis, our data show the mobility shift of Sos1 due to
insulin is greater in the supernatant than in the pellet after
immunoprecipitation with anti-Grb2 antibody (Fig. 5). However,
we cannot yet rule out other mechanisms of Sos regulation or the
modulation of Grb2 structure to account for the desensitization of
p21
caused by insulin.
A key question raised by the
present studies is whether disassembly of Sos proteins from Grb2
actually causes the p21 desensitization to insulin. Only
about half of the complexes containing Grb2 and Sos proteins are
dissociated by insulin treatment during the deactivation phase of
p21
modulation by the hormone. These data suggest the
hypothesis that a specific pool of Grb2-associated Sos is related to
p21
activation by insulin and is specifically
disassembled after prolonged incubation with insulin. Since Grb2
remains bound to tyrosine-phosphorylated Shc proteins during this
entire time course (not shown), the remaining half of cellular Grb2/Sos
complexes may not be able to displace this Shc-associated Grb2. This
would prevent p21
reactivation until such displacement
could take place. These postulates require further testing to determine
the exact relationship between Sos dissociation from Grb2 and
p21
desensitization. Other mechanisms such as engagement
of GTPase-activating proteins may also be induced. In any case, the
data presented here emphasize the need to further understand the
cellular localizations and activities of Grb2/Sos complexes in relation
to p21
modulation.