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INTRODUCTION |
After insulin binds to its receptor, it activates an intrinsic
tyrosine kinase activity that mediates the tyrosine phosphorylation of
a variety of endogenous substrates including insulin receptor substrates 1-4 (1). Binding of these tyrosine-phosphorylated substrates to the Src homology
(SH)1 domain-2 of the
regulatory subunit of the heterodimeric p85/p110 phosphatidylinositol (PI) 3-kinase leads to a 3-5-fold stimulation in its enzymatic activity and an increase in the PI 3,4-bisphosphate and 3,4,5-trisphosphate in the cell (2, 3). A variety of data from
several approaches has demonstrated a role for the production of these
lipid products in mediating many, if not all, of the subsequent actions
of insulin including for example the stimulation of glucose uptake,
activation of both the glycogen synthase and the 70-kDa S6 kinase (4).
The effects of PI 3,4,5-trisphosphate may in part be mediated through
the activation of the Ser/Thr kinases called Akt or one of the atypical
protein kinase Cs (4, 5).
Because of the critical role of the activation of this lipid kinase in
inducing subsequent biological responses, increasing interest has
focused on the regulation of this process. Indeed, several mechanisms
for regulating this process have been identified. First, long term
treatment of cells with insulin or the glucocorticoid dexamethasone has
been shown to induce the degradation of IRS-1 (6, 7). Second, increased
serine phosphorylation of IRS-1 has been observed after treatment of
cells with either activators of protein kinase C, Ser/Thr phosphatase
inhibitors like okadaic acid, platelet-derived growth factor (PDGF),
insulin, angiotensin II, or activation of cellular stress pathways by
tumor necrosis factor and other cytokines (8-15). This increased
serine phosphorylation of IRS-1 has been shown to inhibit the
subsequent ability of this substrate to be tyrosine-phosphorylated by
the insulin receptor and to bind and activate the PI 3-kinase (8-15).
In addition, serine phosphorylation of IRS-1 has been shown to
interfere with the ability of the IRS-1 to interact with the insulin
receptor (16).
In the case of cells treated with an activator of protein kinase C, an
increase in the serine phosphorylation of a particular serine, serine
612, in the IRS-1 molecule was found to play a prominent role in this
inhibition (12). This residue appeared to be phosphorylated by one of
the MAP kinases (also called extracellular signal-regulated kinases),
and an increase in MAP kinase activity was shown to inhibit subsequent
signaling by the insulin receptor kinase (17). However, as noted above,
a number of other stimuli also appear to negatively regulate the
ability of the insulin receptor to tyrosine-phosphorylate IRS-1 by
serine phosphorylation. In particular, PDGF has recently been shown to
stimulate the Ser/Thr phosphorylation of IRS-1 and to inhibit the
ability of insulin to stimulate the subsequent tyrosine phosphorylation
of IRS-1 and its association with PI 3-kinase (14, 15). Since PDGF can
activate multiple signaling pathways including the MAP kinase cascade
(18), the present studies were therefore designed to examine whether
PDGF was affecting IRS-1 via the same pathway as was observed after PKC
activation. In particular, we have tested whether the negative
regulation of insulin signaling by PDGF was also mediated via MAP
kinase and the phosphorylation of Ser-612 in the IRS-1 molecule. We
have found that treatment of cells with PDGF inhibits the insulin
signaling pathway via a distinct mechanism, possibly by activation of a
serine kinase modulated by the Akt pathway, which appears to require
the serine phosphorylation of distinct residues on the IRS-1 molecule.
In contrast, we find that endothelin-1, an activator of PKC (19),
inhibits the insulin signaling pathway via the MAP kinase pathway and
the serine phosphorylation of Ser-612 in IRS-1. These results indicate
that there are MAP kinase-dependent and -independent
pathways that regulate the insulin-stimulated association of IRS-1 with
PI 3-kinase.
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EXPERIMENTAL PROCEDURES |
Plasmid Construction--
A Myc-tagged wild type IRS-1 was
obtained by PCR using pCLDN/IRS-1 as the template and the same primers
described previously (12). The SalI-digested PCR product was
subcloned into the retroviral vector pWZL-neo (20). The single mutants
of IRS-1 were generated by using either the Transformation-based
mutagenesis kit from CLONTECH or a PCR-based
strategy. In order to make the triple mutant of IRS-1
(S632A/S662A/S731A), two initial PCR reactions were performed using
puc18/IRS-1/S632A and puc18/IRS-1/S731A as templates. One set of
primers consisted of a 5'-flanking primer and a 3'-internal S662A
mutagenesis primer; the other set consisted of a 5'-internal S662A
mutagenesis primer and a 3'-flanking primer. Both PCR products were
combined in a final PCR reaction with only the flanking 5' and 3'
primers. This amplified fragment was then subcloned into the pWZL-neo
vector. The quadruple IRS-1 mutant (S612A/S632A/S662A/S731A) was made
by introducing S612A single mutation into the IRS-1 triple mutant. The
sequence of the constructs were confirmed by restriction mapping and
DNA sequencing.
Generation of Stable Cell Lines Expressing the Wild Type and
Mutant IRS-1--
3T3-L1 preadipocytes were infected with the
pWZL-expressing IRS-1 constructs as described previously (17). In
brief, 70% confluent Phoenix packaging cells were transiently
transfected using the calcium phosphate precipitation method as
described (17). After the final media change, the cells were incubated for 3 days at 30 °C, 5% CO2, to generate the viral
supernatant. Fifty percent confluent 3T3-L1 cells were incubated with
the viral supernatant, centrifuged for 90 min at 2,500 rpm, and then
cultured until confluent. Cells were selected in complete media
containing 1 mg/ml G418. The total pool of selected cells was used in
all subsequent experiments. In the case of the IRS-1 pWZL infections of
the 3T3-L1 cells expressing MER-Akt (21), no additional drug selection
was used. In most experiments, the cells were utilized when they became
confluent. In the indicated experiments, cells were induced to
differentiate into adipocytes as described.
Cell Culture and Treatments--
3T3-L1 cells were grown in
Dulbecco's modified Eagle's medium containing 10% calf serum, at
37 °C, 5% CO2. The cells were serum-starved overnight
before performing each experiment. PDGF (the B/B isoform, from
Boehringer Mannheim), endothelin-1 (Sigma), and PD98059 and rapamycin
(Life Technologies, Inc.) were added as described in the figure legends.
Akt and PI 3-Kinase--
The Akt kinase assay of the expressed
HA-tagged MER-Akt was performed after immunoprecipitation with anti-HA
antibodies and using the GSK-3 peptide (sequence GRPRTSSFAEG) as
substrate as described (21). The phosphorylated peptide was separated
on a 40% urea gel, and the identified peptide band was cut out and counted. IRS-associated PI 3-kinase was assayed after
immunoprecipitation of the cell lysates with control antibodies,
anti-Myc antibodies (BabCo), anti-IRS-1 antibodies (1D6), or anti-IRS-2
antibodies. The precipitates were assayed for PI 3-kinase activity as
described (12) or visualized by immunoblotting with an antibody to the p85 subunit of the PI 3-kinase (Transduction Laboratories).
Assessing IRS-1 Expression and MAP Kinase
Activation--
Confluent cells were lysed in lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 10 mM
NaPPi, 2 mM sodium vanadate, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 1% Nonidet P-40).
The lysates were incubated with either anti-Myc antibodies or normal
mouse Ig prebound to protein G-agarose (Pharmacia), washed twice each
with lysis buffer and a low salt buffer (20 mM Tris, pH
7.4, 100 mM NaCl). The bound proteins were eluted by
boiling in sample buffer, analyzed on a 10% SDS-polyacrylamide gel
electrophoresis, and transferred to nitrocellulose. The blots were
developed with either anti-IRS-1 antibodies (Upstate Biotechnology
Inc.) or anti-phosphotyrosine antibodies (Transduction Laboratories).
Measurements of the formation of phospho-MAP kinase were performed by
immunoblotting total cell lysates with antibodies specific to the
activated phospho-MAP kinase (Promega).
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RESULTS |
PDGF and Endothelin-1 Modulate Insulin-stimulated IRS-1 Association
with PI 3-Kinase via Distinct Mechanisms--
As recently reported
(14, 15), PDGF treatment of 3T3-L1 cells rapidly inhibited the
subsequent ability of insulin to stimulate the association of an
expressed Myc-tagged IRS-1 with PI 3-kinase by about 80% (Fig.
1B). To test whether this
inhibition was mediated via MAP kinase, we utilized the MEK inhibitor
PD98059 (22). This compound had no significant effect on the inhibitory
response induced by PDGF (Fig. 1B). A control Western blot
verified that in these cells PDGF was stimulating the formation of the
activated, phosphorylated MAP kinase and this stimulation was
completely inhibited by the MEK-inhibitor, PD98059 (Fig.
1A).

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Fig. 1.
Effects of PD98059 and rapamycin
on PDGF or endothelin-1 inhibition of the insulin-induced IRS-1
association with PI 3-kinase. A, analysis of MAP kinase
activation. 3T3-L1 cells expressing the Myc-tagged wild type IRS-1 were
pretreated with or without 50 µM PD98059 for 30 min,
treated for an additional 20 min with or without 50 ng/ml PDGF, and
then stimulated with 100 nM insulin for 5 min as indicated.
Activated MAP kinase was analyzed by immunoblotting cell lysates with
an anti-phospho-MAP kinase antibody. B, modulation of PI
3-kinase activation by PDGF. The cell lysates from experiments like
those shown in panel A were adsorbed with antibodies to the
Myc tag, and the precipitated Myc-tagged IRS-1 was assayed for the
associated PI 3-kinase activity. Where indicated, rapamycin was added
to a final concentration of 200 nM. Results shown are
means ± S.E. from three experiments normalized to the amount of
activity present in the precipitates from cells treated with only
insulin (i.e. maximum activation). C, modulation
of PI 3-kinase activation by endothelin-1. The cell lysates from
experiments like those shown in panel A except
utilizing 1 µM endothelin-1 in place of PDGF were
processed as described in the legend to panel B.
D, effect of wortmannin on the PDGF and endothelin
inhibition of the insulin-stimulated association of IRS-1 and PI
3-kinase. 3T3-L1 cells were pretreated with 1 µM
wortmannin for 30 min as indicated, then 50 ng/ml PDGF or 1 µM endothelin were added and after an additional 20 min,
100 nM insulin was added. After 5 min, the cells were
lysed, the IRS-1 was immunoprecipitated and the precipitates were
immunoblotted with an antibody to the p85 subunit of PI 3-kinase
(upper panel). The same blot was stripped and
reprobed with an antibody to IRS-1 (lower panel).
Results shown are representative of three experiments.
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Since prior studies have indicated that endothelin-1, an
activator of the PKC and the MAP kinase pathways (19), could also induce insulin resistance in rat adipocytes (23), we tested whether
this hormone would inhibit the insulin-stimulated increase in
IRS-1-associated PI 3-kinase. Endothelin-1 was found to inhibit by 50%
the insulin-stimulated association of IRS-1 with PI 3-kinase in the
3T3-L1 cells (Fig. 1C). However, in contrast to the results with PDGF, this endothelin-1 response was completely blocked by the
MEK-inhibitor PD98059 (Fig. 1C).
These results indicated that the inhibition observed with PDGF is
unlikely to be due to MAP kinase. The other major signaling pathway
stimulated by PDGF is the PI 3-kinase/Akt/mTOR kinase pathway (18).
Prior studies had shown that the PI 3-kinase inhibitor, wortmannin,
blocked the PDGF-induced inhibition of the insulin response (14). To
see if the downstream members of this pathway were also involved,
rapamycin, an inhibitor of mTOR (also called FRAP), was tested for its
effect (24). Rapamycin was found to largely block the ability of PDGF
to inhibit the insulin-stimulated association of IRS-1 with PI 3-kinase
(Fig. 1). These results are consistent with a role for the PI
3-kinase/Akt/mTOR pathway being involved in the PDGF inhibition of the
insulin signaling cascade and suggest that different signaling cascades
are utilized by PDGF and endothelin to inhibit the subsequent ability
of insulin to stimulate a response.
To further test this hypothesis, we compared the ability of the PI
3-kinase inhibitor, wortmannin, to block the PDGF and endothelin-1 effects. To accomplish this, we measured the ability of wortmannin to
affect the ability of PDGF and endothelin to inhibit the
insulin-induced association of IRS-1 with the PI 3-kinase. In agreement
with the above studies, both PDGF and endothelin inhibited the
insulin-stimulated association of IRS-1 with PI 3-kinase, with PDGF
being more potent then endothelin-1 (Fig. 1D). Wortmannin
blocked the effect of PDGF but had no significant effect on the
inhibition observed with endothelin-1 (Fig. 1D). Controls
verified that the amount of IRS-1 precipitated in each case was the
same. These results support the hypothesis that the ability of PDGF to
interfere with the insulin pathway utilizes the PI 3-kinase cascade
whereas the endothelin-1 effect is via a distinct pathway.
Expression of Mutant IRS-1 Molecules in 3T3-L1 Cells and Their
Regulation by PDGF and Endothelin-1--
3T3-L1 cells were infected
with retroviral vectors encoding Myc-tagged wild type IRS-1 molecules
or various mutant IRS-1 molecules in which particular serine residues
have been changed to alanine. In particular, mutant IRS-1 molecules
were expressed in which either serine 612 (S612A), serines 632, 662, and 731 (S632A/S662A/S731A), or serines 612, 632, 662, and 731 (S612A/S631A/S662A/S731A) were changed to alanine. In each case,
expression of the mutant molecules was confirmed by Western blotting
with antibodies to the Myc epitope and insulin was found to stimulate
their tyrosine phosphorylation (Fig. 2
and data not shown). The levels of expressed IRS-1 were comparable to
those of the endogenous IRS-1, as demonstrated by the approximate
2-fold increase in immunoreactive IRS-1 observed in the blots of total
lysates of infected cells in comparison to the control cells (Fig. 2).
Of most interest was the finding that, although PDGF inhibited the
insulin-stimulated tyrosine phosphorylation of the wild type expressed
IRS-1, it did not inhibit the insulin-stimulated tyrosine
phosphorylation of the quadruple IRS-1 mutant (S612A/S631A/S662A/S731A)
(Fig. 2B). In addition, PDGF did not inhibit the subsequent
insulin-stimulated association of the quadruple mutant of IRS-1 with PI
3-kinase (Fig. 3A). To verify
that these transfected cells were still responsive to PDGF, the
supernatants from the anti-IRS-1 precipitates were adsorbed with
antibodies to IRS-2. The insulin-stimulated association of IRS-2 with
the PI 3-kinase was inhibited by PDGF in the cells expressing both the
wild type IRS-1 as well as the quadruple IRS-1 mutant (Fig.
3B), indicating that the cells expressing the mutant IRS-1
had not lost their PDGF responsiveness.

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Fig. 2.
Expression of Myc-tagged IRS-1 in 3T3-L1
cells and the effect of PDGF on insulin-stimulated tyrosine
phosphorylation. A, expression of Myc-tagged wild type
(wt) and a quadruple mutant (quad mut)
IRS-1. 3T3-L1 cells were infected with a retrovirus encoding either the
wild type or a quadruple mutant IRS-1 (S612A/S632A/S662A/S731A),
drug-selected, and the resulting cell pools were lysed; either the
total cell lysates were analyzed, or the lysates were
immunoprecipitated with anti-Myc antibodies or normal mouse Ig
(NIg). The proteins were separated on 10%
SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose,
and blotted with anti-IRS-1 antibodies. Control (con)
samples of parental cells were also analyzed. B, tyrosine
phosphorylation of the Myc-tagged IRS-1. Cells expressing either the
wild type or quadruple mutant IRS-1 were pretreated with or without 50 ng/ml PDGF for 20 min, then stimulated with 100 nM insulin
for 5 min, lysed and the Myc-tagged IRS-1 was immunoprecipitated and
blotted with anti-phosphotyrosine antibodies. The figure shown is
representative of three experiments.
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Fig. 3.
Effect of PDGF on the insulin-stimulated
association of PI 3-kinase and the quadruple mutant (quad
mut) IRS-1. 3T3-L1 cells expressing either the
wild type or quadruple mutant IRS-1 were pretreated with or without 50 ng/ml PDGF for 20 min, then stimulated with 100 nM insulin
for 5 min and lysed, and the Myc-tagged IRS-1 was immunoprecipitated
and tested for its associated PI 3-kinase activity (A). The
supernatants from these precipitations were adsorbed with anti-IRS-2
antibodies, and the amount of IRS-2-associated PI 3-kinase activity was
then analyzed (B). To directly measure the amount of
associated PI 3-kinase, the IRS-1 immunoprecipitates were also
analyzed by immunoblotting with an antibody to the p85 subunit of PI
3-kinase (C). The panels shown are representative of three
experiments.
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The quadruple mutation in the IRS-1 molecule could affect the
activation of the PI 3-kinase independently of its association. To test
this possibility, we also directly examined the insulin-stimulated association of PI 3-kinase with the wild type and quadruple mutant IRS-1 molecules. As observed for the PI 3-kinase enzymatic activity, the insulin-stimulated association of PI 3-kinase with the wild type
IRS-1 was inhibited by PDGF whereas the insulin-stimulated association of PI 3-kinase with the quadruple mutant IRS-1 was not
affected by PDGF (Fig. 3C).
To further characterize the role of particular serines in the ability
of PDGF to regulate the insulin-stimulated association of PI 3-kinase
with the mutant IRS-1 molecules, cells expressing the single, triple,
or quadruple mutation were examined. PDGF treatment was found to
inhibit the insulin-stimulated association of the single mutant IRS-1
(S612A) with PI 3-kinase by about 80%, a level comparable to that
observed with the wild type IRS-1 (Fig. 4). In contrast, the endothelin-1
inhibition of the insulin-stimulated association of IRS-1 with PI
3-kinase was essentially abolished with the same single mutation (data
not shown). However, the mutation of three other serines (Ser-631,
Ser-662, and Ser-731) dramatically reduced the inhibition with PDGF
treatment (Fig. 4).

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Fig. 4.
Effect of PDGF on different IRS-1
mutants. Cells expressing either the wild type (Wt)
IRS-1, a single mutation (S612A) IRS-1, a triple mutant
(trip mut) (S632A/S662A/S731A) IRS-1, or quadruple mutant
(quad mut) S612A/S632A/S662A/S731A IRS-1 were
treated with or without 50 ng/ml PDGF for 20 min, then stimulated with
100 nM insulin for 5 min and lysed, and the Myc-tagged
IRS-1 was immunoprecipitated and tested for its associated PI 3-kinase
activity. Results shown are means ± S.E. from three experiments
normalized to the amount of activity present in the precipitates from
cells treated with only insulin (i.e. maximum
activation).
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Regulation of the Insulin-stimulated Association of IRS-1 with PI
3-Kinase by Akt--
Since PDGF can stimulate the PI 3-kinase/Akt
pathway as well as the MAP kinase cascade (18), we tested whether Akt
could also modulate the insulin-stimulated association of IRS-1 with PI
3-kinase. 3T3-L1 cells expressing a hydroxytamoxifen-regulatable form
of Akt (called MER-Akt) (21) were stimulated with insulin either with
or without a prior treatment with hydroxytamoxifen and lysed, and the
amount of IRS-1-associated PI 3-kinase was measured. Treatment of cells
expressing the MER-Akt with hydroxytamoxifen caused a 75% inhibition
in the insulin-stimulated increase in IRS-1-associated PI 3-kinase
(Fig. 5). In contrast, no significant inhibition was observed in the control 3T3-L1 cells, which do not
express MER-Akt after a prior pretreatment with hydroxytamoxifen. The
inhibition in the MER-Akt-expressing cells was dependent upon the dose
of hydroxytamoxifen used to stimulate the cells and paralleled the
increase in Akt activity in these cells (Fig.
6).

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Fig. 5.
Activation of Akt inhibits the
insulin-stimulated IRS-1 association with PI 3-kinase. 3T3-L1
cells either expressing MER-Akt or the control, parental cells, were
pretreated with 1 µM HT for 4 h and then stimulated
with 100 nM insulin for 5 min as indicated. The cells were
lysed, and the IRS-1-associated PI 3-kinase activity was analyzed.
Results shown are means ± S.E. from three experiments normalized
to the amount of activity present in the precipitates from cells
treated with only insulin (i.e. maximum activation).
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Fig. 6.
Dose-dependent activation of
MER-Akt and its inhibition of the insulin-stimulated association of
IRS-1 with PI 3-kinase. A, IRS-1-associated PI 3-kinase
activity. 3T3-L1-expressing MER-Akt cells were pretreated with the
indicated concentrations of HT for 4 h and then stimulated with
100 nM insulin for 5 min as indicated. The cells were
lysed, and the IRS-1-associated PI 3-kinase activity was analyzed as
described above. B, activation of Akt. 3T3-L1 cells were
treated as in A, lysed, and the MER-Akt was precipitated and
assayed for enzymatic activity by utilizing a GSK-3-based peptide. A
representative autoradiograph of the phosphorylation of the peptide is
shown as well as the mean -fold activations ± S.E. from three
experiments.
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To test whether the inhibition observed via Akt activation was mediated
via the mTOR pathway, we utilized the mTOR inhibitor rapamycin. As was
observed for PDGF inhibition, rapamycin significantly blocked the
inhibitory response observed with Akt activation (Fig. 7A).

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Fig. 7.
Akt modulation of the insulin-stimulated
association of IRS-1 with PI 3-kinase: effect of rapamycin and the
mutation of serines 612, 632, 662, and 731. A, effect
of rapamycin. 3T3-L1-expressing MER-Akt cells were pretreated as
indicated with 200 nM rapamycin for 30 min, then 1 µM HT for 3.5 h and then stimulated with 100 nM insulin for 5 min as indicated. The cells were then
lysed, and the amount of IRS-1-associated PI 3-kinase activity was
measured. Results shown are means ± S.E. from three experiments
normalized to the amount of activity present in the precipitates from
cells treated with only insulin (i.e. maximum activation).
B, effect of serine mutations. 3T3-L1-expressing MER-Akt
cells were infected with a retrovirus encoding either the wild type
IRS-1 (wt) or the quadruple mutant IRS-1 (quad
mut). The cells were pretreated with 1 µM HT
for 4 h, then stimulated with 100 nM insulin for 5 min
as indicated. The cells were lysed and the Myc-tagged IRS-1-associated
PI 3-kinase activity was analyzed as described above. Results
shown are means ± S.E. from three experiments normalized to the
amount of activity present in the precipitates from cells treated with
only insulin (i.e. maximum activation).
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Since the PDGF inhibition of the insulin-stimulated association of PI
3-kinase and IRS-1 was dependent upon the presence of specific serine
residues in the phosphorylation domain (Fig. 4), we tested whether a
similar requirement was also present for the inhibition via Akt. To
this end, the quadruple IRS-1 mutant (S612A/S632A/S662A/S731A) as well
as the epitope-tagged wild type IRS-1 were expressed in the 3T3-L1
cells containing the MER-Akt. These cells were stimulated with insulin
either with or without a prior activation of the MER-Akt and lysed, and
the amount of IRS-1-associated PI 3-kinase was measured. The activation
of the MER-Akt was found to inhibit the insulin-stimulated association
of PI 3-kinase with the wild type expressed IRS-1 but not with the
expressed quadruple IRS-1 mutant (Fig. 7B).
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DISCUSSION |
Prior studies have demonstrated that increased Ser/Thr
phosphorylation of IRS-1 can be stimulated by a number of factors
including activators of protein kinase C, Ser/Thr phosphatase
inhibitors like okadaic acid, PDGF, insulin, or activation of cellular
stress pathways by tumor necrosis factor and other cytokines (8-17). This serine phosphorylation may be a contributing factor in the development of insulin resistance since an increase in serine phosphorylation interferes with the tyrosine phosphorylation of IRS-1
by the insulin receptor and its subsequent association with PI
3-kinase. Thus, it is important to identify the kinase cascades responsible for the serine phosphorylation of IRS-1.
Prior studies have identified several kinases that can phosphorylate
IRS-1. These include casein kinase II (25), glycogen synthase kinase 3 (26), MAP kinase (17), and even a lipid kinase, the
phosphatidylinositol 3-kinase (27, 28). In the case of MAP kinase, a
particular serine (serine 612) has been identified in the IRS-1
molecule as being responsible for causing the subsequent inhibition of
insulin-stimulated association with PI 3-kinase (17). In this prior
work, the MAP kinase was activated by phorbol esters, a potent but
non-physiological activator of protein kinase C. In the present work,
endothelin-1, a normal regulator of protein kinase C (19), was also
found to inhibit the ability of insulin to stimulate the IRS-1
association with PI 3-kinase. This inhibition also appeared to be
mediated via the activation of MAP kinase since the MEK inhibitor
PD98059 completely blocked the response to endothelin-1. Moreover, this
inhibition required the phosphorylation of Ser-612 since endothelin-1
did not inhibit the insulin-stimulated association of a Ser-612 mutant of IRS-1 with the PI 3-kinase (data not shown). Since endothelin-1 can
inhibit insulin-stimulated glucose uptake in vivo as well as
in vitro (23, 29) and the levels of endothelin-1 are
elevated in obesity and diabetes mellitus (30, 31), this pathway may contribute to insulin resistance in these conditions.
In the present studies, we have also examined the pathway that is
involved in the PDGF-induced negative regulation of the insulin
signaling cascade. Although PDGF did induce the activation of MAP
kinase in the 3T3-L1 cells, this pathway did not appear to be required
for the negative regulation of the insulin signaling cascade. In
contrast to the results with endothelin-1, the MEK inhibitor had
essentially no effect on the PDGF-induced inhibition in
IRS-1-associated PI 3-kinase, although it completely inhibited the
ability of PDGF to stimulate MAP kinase in these cells. This finding of
a lack of inhibition by the MEK inhibitor of the PDGF response was also
recently reported by Staubs et al. (15).
These results indicate that PDGF is most likely regulating the
insulin-induced increase in IRS-1-associated PI 3-kinase by a pathway
independent of the MAP kinase cascade. Further evidence to support this
hypothesis is provided by the finding that the IRS-1 mutant lacking
Ser-612 is still negatively regulated by PDGF. However, a mutant IRS-1
lacking three other serines in the same domain (Ser-632, -662, and
-731) was found to lose its ability to be negatively regulated by PDGF
treatment of cells. These results are consistent with the hypothesis
that the negative regulation occurs via the serine phosphorylation of
IRS-1; however, the sites required for this effect appear to differ
from that required for the negative regulation by MAP kinase.
In addition to activating the MAP kinase cascade, PDGF also stimulates
the PI 3-kinase/Akt/70-kDa S6 kinase cascade in cells (18). Prior
studies have shown that the activation of the PI 3-kinase is important
in mediating the negative regulation of the insulin response (14, 15).
Consistent with this prior work, wortmannin, an inhibitor of the PI
3-kinase, did block the PDGF inhibitory effect on the
insulin-stimulated association of IRS-1 with PI 3-kinase. However,
wortmannin had no effect on the endothelin response, further indicating
that these two hormones exert their effects through distinct signaling
cascades. To determine whether molecules further downstream in this
kinase cascade could also induce a negative regulation of the
insulin-signaling pathway, we have utilized a conditionally active form
of Akt (21). This kinase can be turned on independently of other
signals by treatment of cells with the estrogen antagonist,
hydroxytamoxifen. Activation of this chimeric Akt by treatment of cells
with hydroxytamoxifen was found to inhibit the insulin-stimulated
increase in IRS-1-associated PI 3-kinase. Control experiments verified
that no significant inhibition was induced by hydroxytamoxifen in cells
not expressing this chimeric Akt. The inhibition induced by
hydroxytamoxifen in MER-Akt-expressing cells paralleled the increase in
Akt enzymatic activity of this chimera. This inhibition by Akt also
appeared to require the same serines as those required for the negative regulation by PDGF since activation of Akt did not inhibit the insulin-induced association of PI 3-kinase and a mutant IRS-1 lacking
these serines.
The finding that Akt can negatively regulate the insulin-induced
increase in IRS-1-associated PI 3-kinase is important since it suggests
that, during the insulin-stimulated activation of Akt, there is also a
feedback inhibition. This would also explain the insulin resistance
that occurs during periods of hyperinsulinemia (32). Finally, it would
explain the decrease in insulin-stimulated biological responses that is
observed in cells overexpressing various Akt constructs (21, 33).
One explanation of these results is that PDGF activates Akt and Akt
itself directly phosphorylates IRS-1, thereby inhibiting its subsequent
tyrosine phosphorylation and association with PI 3-kinase. However, the
sites identified as the important regulatory ones (serines 612, 632, 662, and 731) do not fit the consensus Akt phosphorylation site (34).
Alternatively, it is possible that a kinase activated by Akt is
responsible for this phosphorylation. One such candidate is the enzyme
called the mammalian target of rapamycin, mTOR, or FRAP (24). The
kinase activity of this enzyme has been shown to be regulated by Akt,
insulin, and PDGF (33, 35). In addition, the sites phosphorylated by
this enzyme in one of its substrates, PHAS-I (for properties of heat
and acid stability-I), are similar to the regulatory sites identified
in IRS-1 in that both have a (Ser/Thr)-Pro motif (36). Finally, the
finding that rapamycin at least partly inhibits the negative regulation
by both PDGF and Akt is consistent with this hypothesis.