Differential Modulation of the Tyrosine Phosphorylation State of the Insulin Receptor by IRS (Insulin Receptor Subunit) Proteins
Barbara T. Solow,
Shuko Harada,
Barry J. Goldstein,
Judith A. Smith,
Morris F. White and
Leonard Jarett
Department of Pathology and Laboratory Medicine (B.T.S., S.H.,
J.A.S., L.J.) University of Pennsylvania School of Medicine
Philadelphia, Pennsylvania 19104
Dorrance H. Hamilton
Research Laboratories (B.J.G.) Division of Endocrinology, Diabetes
and Metabolic Diseases Department of Medicine Jefferson Medical
College of Thomas Jefferson University Philadelphia, Pennsylvania
19107
Joslin Diabetes Center (M.F.W.) Boston,
Massachusetts 02215
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ABSTRACT
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In response to insulin, tyrosine kinase activity
of the insulin receptor is stimulated, leading to autophosphorylation
and tyrosine phosphorylation of proteins including insulin receptor
subunit (IRS)-1, IRS-2, and Shc. Phosphorylation of these proteins
leads to activation of downstream events that mediate insulin action.
Insulin receptor kinase activity is requisite for the biological
effects of insulin, and understanding regulation of insulin receptor
phosphorylation and kinase activity is essential to understanding
insulin action. Receptor tyrosine kinase activity may be altered by
direct changes in tyrosine kinase activity, itself, or by
dephosphorylation of the insulin receptor by protein-tyrosine
phosphatases. After 1 min of insulin stimulation, the insulin receptor
was tyrosine phosphorylated 8-fold more and Shc was phosphorylated 50%
less in 32D cells containing both IRS-1 and insulin receptors
(32D/IR+IRS-1) than in 32D cells containing only insulin receptors
(32D/IR), insulin receptors and IRS-2 (32D/IR+IRS-2), or insulin
receptors and a form of IRS-1 that cannot be phosphorylated on tyrosine
residues (32D/IR+IRS-1F18). Therefore, IRS-1
and IRS-2 appeared to have different effects on insulin receptor
phosphorylation and downstream signaling. Preincubation of cells with
pervanadate greatly decreased protein-tyrosine
phosphatase activity in all four cell lines. After
pervanadate treatment, tyrosine phosphorylation of insulin receptors in
insulin-treated 32D/IR, 32D/IR+IRS-2, and
32D/IR+IRS-1F18 cells was markedly increased,
but pervanadate had no effect on insulin receptor phosphorylation in
32D/IR+IRS-1 cells. The presence of tyrosine-phosphorylated IRS-1
appears to increase insulin receptor tyrosine phosphorylation and
potentially tyrosine kinase activity via inhibition of protein-tyrosine
phosphatase(s). This effect of IRS-1 on insulin receptor
phosphorylation is unique to IRS-1, as IRS-2 had no effect on insulin
receptor tyrosine phosphorylation. Therefore, IRS-1 and IRS-2 appear to
function differently in their effects on signaling downstream of
the insulin receptor. IRS-1 may play a major role in regulating insulin
receptor phosphorylation and enhancing downstream signaling after
insulin stimulation.
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INTRODUCTION
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Insulin activates the insulin receptor tyrosine kinase, leading to
autophosphorylation of the insulin receptor ß-subunit on multiple
tyrosines (reviewed in Ref. 1). Autophosphorylation of the insulin
receptor increases its tyrosine kinase activity, leading to
phosphorylation of intracellular substrates, such as insulin receptor
substrate (IRS)-1, IRS-2, and Shc. Activation of these molecules and
the subsequent activation of other intracellular molecules leads to the
biological responses associated with insulin. Dephosphorylation of the
insulin receptor ß-subunit by protein-tyrosine phosphatases reduces
tyrosine kinase activity of the insulin receptor, and inhibition of
these protein-tyrosine phosphatases by vanadate or pervanadate may
cause increases in receptor tyrosine phosphorylation and in downstream
signaling responses (2, 3). The importance of protein-tyrosine
phosphatases in regulation of insulin signaling can be seen in
transgenic mice deficient in either the protein-tyrosine phosphatase
LAR (leukocyte common-antigen-related protein-tyrosine phosphatase) or
protein-tyrosine phosphatase-1B (PTP-1B) (4, 5). Mice deficient in LAR
had a 47% reduction in hepatic phosphatidylinositol 3'-kinase activity
and significant resistance to insulin-stimulated glucose disposal (4).
PTP-1B+/+ mice rapidly gained weight and became insulin
resistant when fed a high-fat diet, whereas, mice deficient in PTP-1B
(PTP-1B-/-) were resistant to weight gain and remained
insulin sensitive (5). Taken together, these findings demonstrate that
protein-tyrosine phosphatases regulate insulin signaling.
IRS-1 is one of the major proteins phosphorylated in response to
insulin, and the tyrosine phosphorylated form of IRS-1 binds to several
cytoplasmic signaling proteins through their SH2 domains (6). Lower
than normal levels of IRS-1 or variations in the amino acid sequence of
IRS-1 have been associated with some cases of non-insulin-dependent
diabetes mellitus (7, 8, 9, 10, 11), and IRS-1- deficient mice are mildly insulin
resistant (12, 13). Therefore, it appears that IRS-1 signaling is an
integral part of normal insulin signaling, and deficiencies in IRS-1
signaling may contribute to non-insulin-dependent diabetes
mellitus.
IRS-2 is another major protein phosphorylated in response to insulin
and is structurally similar to IRS-1 (reviewed in Ref. 14). These two
proteins have an amino-terminal pleckstrin homology domain followed
immediately by a phosphotyrosine-binding domain that binds to
phosphorylated NPXY motifs. The carboxy-terminal regions of IRS-1 and
IRS-2 contain multiple src homology 2 (SH2)- and SH3-protein-binding
sites. Some of the proteins that are known to bind to these sites are
phosphatidylinositol 3'-kinase, Grb-2, and SHP-2 (Syp, SH-PTP2).
Although IRS-1 and IRS-2 are structurally similar, recent research
suggests that they are differentially expressed and mediate distinct
responses.
Mouse 32D cells contain no IRS-1, IRS-2, or IGF-I receptors and very
few insulin receptors (15). By transfecting cells with genes encoding
the insulin receptor and either IRS-1 or IRS-2, differences in receptor
interactions with IRS-1 and IRS-2 may be examined. Previously, we
demonstrated that insulin receptor tyrosine phosphorylation was greater
in 32D cells transfected with both the insulin receptor and IRS-1 genes
(32D/IR+IRS-1) than in cells transfected with genes encoding the
insulin receptor alone (32D/IR) (16). Although phosphorylation of the
insulin receptor ß-subunit was less in 32D/IR cells, the number of
insulin receptors in these cells was equal to or greater than the
number in 32D/IR+IRS-1 cells, and the number of insulin receptors that
immunoprecipitated with antiphosphotyrosine antibodies was similar to
that in 32D/IR+IRS-1 cells (16). These findings suggested that the
level of tyrosine phosphorylation per insulin receptor was less in
32D/IR cells than in 32D/IR+IRS-1 cells. Therefore, the lower level of
insulin receptor tyrosine phosphorylation observed in 32D/IR cells was
not due to a lower number of tyrosine-phosphorylated insulin receptors
but rather less tyrosine phosphorylation per insulin receptor.
Interestingly, more Shc tyrosine phosphorylation was observed in 32D/IR
cells than in 32D/IR+IRS-1 cells (16). These results were supported by
results obtained by others that Shc and IRS-1 compete for binding to
the insulin receptor (17, 18, 19).
The goal of the present study is to further examine why the insulin
receptor is tyrosine phosphorylated more and Shc is phosphorylated less
in the presence of IRS-1. We also looked at the role of IRS-2 in
regulation of insulin receptor and Shc phosphorylation. Our data
demonstrated that the presence of tyrosine-phosphorylated IRS-1, but
not IRS-2, decreased Shc phosphorylation and increased insulin receptor
phosphorylation, indicating that IRS-1 and IRS-2 have different
functions and different effects on signaling downstream of the insulin
receptor.
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RESULTS
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Effect of Insulin on Tyrosine Phosphorylation of the Insulin
Receptor and Downstream Signaling Molecules
To determine whether tyrosine phosphorylation of IRS-1 was a
prerequisite for increased insulin receptor phosphorylation,
phosphorylation of the insulin receptor, Shc, IRS-1, or IRS-2 was
analyzed in insulin-stimulated 32D mouse myeloid progenitor cells
expressing either the human insulin receptor gene (32D/IR), insulin
receptor and IRS-1 genes (32D/IR+IRS-1), insulin receptor and IRS-2
genes (32D/IR+IRS-2), or genes encoding the insulin receptor and IRS-1
with 18 tyrosine substituted with phenylalanine
(32D/IR+IRS-1F18) (20). Cells were incubated with insulin,
and cell lysates were subjected to Western blot analysis using
antiphosphotyrosine antibodies or antiinsulin receptor antibodies.
After incubation with insulin, the insulin receptor was tyrosine
phosphorylated to a greater extent in 32D/IR+IRS-1 cells than in
32D/IR, 32D/IR+IRS-1F18, or 32D/IR+IRS-2 cells (Fig. 1
). Increased phosphorylation of the
insulin receptor was not attributed to an increase in the number of
insulin receptors in 32D/IR+IRS-1 cells since Western blot analysis of
proteins in cell extracts using antiinsulin receptor antibodies
detected similar levels of insulin receptors in all four cell types
(Fig. 1
, bottom).

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Figure 1. Effect of Insulin on Tyrosine Phosphorylation of
the Insulin Receptor, IRS-1, and Shc in 32D Cell Clones
32D/IR, 32D/IR+IRS-1, 32D/IR+IRS-1F18, and 32D/IR+IRS-2
cells were incubated without or with 100 nM insulin for 1
or 5 min. Cells were lysed, and equal amounts of protein were subjected
to SDS-PAGE and Western blot analysis with antiphosphotyrosine
antibodies (top) or antiinsulin receptor ß-subunit
antibodies (bottom).
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The length of time after addition of insulin until the peak in
phosphorylation of the insulin receptor was also different in
32D/IR+IRS-1 cells from that in 32D/IR, 32D/IR+IRS-1F18, or
32D/IR+IRS-2 cells. In 32D/IR+IRS-1 cells, the insulin receptor had the
greatest level of tyrosine phosphorylation 1 min after insulin
stimulation, being 8-fold greater than the peaks in insulin receptor
phosphorylation in the other cell types (Fig. 2
). Over the next 14 min, insulin
receptor tyrosine phosphorylation rapidly declined to a level that was
only 3-fold higher than insulin receptor phosphorylation in the other
cell types. The rate of decline in insulin receptor dephosphorylation
then slowed to a rate that was similar to the rate of dephosphorylation
in the other cell types after 5 min incubation with insulin. Tyrosine
phosphorylation of IRS-1 followed the same time course as insulin
receptor phosphorylation. In 32D/IR, 32D/IR+IRS-1F18, and
32D/IR+IRS-2 cells, insulin receptor tyrosine phosphorylation increased
1 min after insulin stimulation and continued to increase until 5 min
after insulin stimulation but to a lesser degree than the peak of
phosphorylation observed in 32D/IR+IRS-1 cells. After 5 min of insulin
incubation, tyrosine phosphorylation of the insulin receptor started to
decline.

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Figure 2. Tyrosine Phosphorylation of the Insulin Receptor in
32D Cell Clones Treated with Insulin for 030 min
32D/IR, 32D/IR+IRS-1, 32D/IR+IRS-1F18, and 32D/IR+IRS-2
cells were incubated with 100 nM insulin for 030 min.
Cells were lysed, and equal amounts of protein were subjected to
SDS-PAGE and Western blot analysis with antiphosphotyrosine antibodies.
The degree of insulin receptor tyrosine phosphorylation from two
independent experiments was quantitated using a PhosphorImager and
ImageQuant software and expressed in ImageQuant Units (IMU) (mean
± SD). The inset graph depicts the degree
of insulin receptor phosphorylation in 32D/IR,
32D/IR+IRS-1F18, and 32D/IR+IRS-2 cells and is scaled from
030,000 IMU.
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No tyrosine phosphorylation of IRS-1 was detected in 32D/IR cells,
32D/IR+IRS-1F18, or 32D/IR+IRS-2 cells after insulin
stimulation, but IRS-1 was prominently phosphorylated in 32D/IR+IRS-1
cells (Fig. 1
). After Western blot analysis of cellular proteins with
anti-IRS-1 antibodies, similar levels of IRS-1 or IRS-1F18
were found in 32D/IR+IRS-1 and 32D/IR+IRS-1F18 cells,
respectively; but no IRS-1 was detected in 32D/IR cells or 32D/IR+IRS-2
cells (Fig. 3
). Likewise, Western blot
analysis of cellular proteins with anti-IRS-2 antibody detected IRS-2
only in 32D/IR+IRS-2 cells (Fig. 3
). These results indicated that the
observed increase in insulin receptor phosphorylation in the presence
of IRS-1 is an IRS-1-specific effect. Since IRS-1F18 has no
detectable tyrosine phosphorylation and did not increase insulin
receptor phosphorylation, tyrosine phosphorylation of IRS-1 appears to
be a prerequisite for increased insulin receptor phosphorylation.

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Figure 3. Levels of IRS-1, IRS-2, and Shc in 32D/IR,
32D/IR+IRS-1, 32D/IR+IRS-1F18, and 32D/IR+IRS-2 Cells
Cell extracts from cells treated without or with 100 nM
insulin for 1 min were subjected to SDS-PAGE followed by Western blot
analysis with either anti-IRS-1, anti-IRS-2, or anti-Shc antibodies.
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The 52-kDa isoform of Shc is tyrosine phosphorylated by the insulin
receptor and competes with IRS-1 for insulin receptor binding (17, 18, 19).
The 52-kDa isoform of Shc was phosphorylated in all four cell types but
was phosphorylated more in 32D/IR, 32D/IR+IRS-1F18, and
32D/IR+IRS-2 cells than in 32D/IR+IRS-1 cells (Fig. 1
). However, levels
of Shc were similar in all four cell types, as determined by Western
blot analysis of cellular proteins using anti-Shc antibodies (Fig. 3
).
When Shc phosphorylation levels were standardized by taking a ratio of
Shc phosphorylation to insulin receptor expression levels as shown in
Fig. 4
, less Shc phosphorylation in the
32D/IR+IRS-1 cells was not the result of lower levels of insulin
receptors in these cells than in the other three cell types. The lower
levels of Shc tyrosine phosphorylation in 32D/IR+IRS-1 cells, but not
in 32D/IR cells, 32D/IR+IRS-1F18 cells, and 32D/IR+IRS-2
cells, suggested that tyrosine-phosphorylated IRS-1 competed with Shc
for binding to the insulin receptor. In the presence of
IRS-1F18 or IRS-2, however, Shc bound to and was
phosphorylated by the insulin receptor to the same degree as in their
absence.

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Figure 4. Ratio of Shc Tyrosine Phosphorylation to Insulin
Receptor Levels in 32D/IR, 32D/IR+IRS-1, 32D/IR+IRS-1F18,
and 32D/IR+IRS-2 Cells
Cells were incubated without or with 100 nM insulin for 1
or 5 min. Cells were lysed, and equal amounts of protein were subjected
to SDS-PAGE and Western blot analysis with antiphosphotyrosine
antibodies or antiinsulin receptor ß-subunit antibodies. Shc tyrosine
phosphorylation and insulin receptor levels were quantitated from three
experiments using a PhosphorImager and ImageQuant software and
expressed as the ratio of Shc tyrosine phosphorylation to insulin
receptor levels (mean ± SD).
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Although Shc was tyrosine phosphorylated less in 32D/IR+IRS-1 cells, a
number of proteins were tyrosine phosphorylated in these cells that did
not appear to be tyrosine phosphorylated in 32D/IR,
32D/IR+IRS-1F18, or 32D/IR+IRS-2 cells (Fig. 1
). These
proteins had molecular masses of approximately 120 and 70 kDa.
Insulin-stimulated tyrosine phosphorylation of these proteins suggested
that a protein-tyrosine kinase or kinases may be activated after
tyrosine phosphorylation of IRS-1 but not IRS-2 or Shc. The 120-kDa
proteins are most likely a complex of proteins potentially consisting
of FAK, JAK, c-Cbl, and/or SHPS-1. These proteins are known to be
tyrosine phosphorylated after insulin stimulation (21, 22, 23, 24). SHP-2 is
approximately 70 kDa and may be tyrosine phosphorylated in response to
insulin (2). In addition, SHP-2 is known to bind to IRS-1 and
potentially IRS-2 (25). To examine tyrosine phosphorylation of SHP-2,
SHP-2 was immunoprecipitated from 32D/IR or 32D/IR+IRS-1 cells
incubated with or without insulin. Western blot analysis of
immunoprecipitated SHP-2 using anti-SHP-2 antibodies showed that SHP-2
precipitated in equal quantities from cells incubated with or without
insulin (data not shown). However, Western blot analysis with
antiphosphotyrosine antibodies did not detect any tyrosine
phosphorylation of SHP-2 in either cell type. Therefore, the 70-kDa
tyrosine-phosphorylated protein is not SHP-2.
Pervanadate Inhibits Protein-Tyrosine Phosphatase Activity
To study the mechanisms by which IRS-1 increased insulin receptor
phosphorylation, we examined the effects of pervanadate, which is an
irreversible inhibitor of protein-tyrosine phosphatases (26), on
protein-tyrosine phosphatase activity in 32D cell clones. The
potency of pervanadate in inhibiting phosphatase activity in 32D/IR,
32D/IR+IRS-1, 32D/IR+IRS-1F18, and 32D/IR+IRS-2 cells was
determined by measuring protein-tyrosine phosphatase activity in cell
lysates from cells treated with different levels of pervanadate for 20
min at 37 C. In cell extracts from 32D/IR, 32D/IR+IRS-1,
32D/IR+IRS-1F18, and 32D/IR+IRS-2 cells, protein-tyrosine
phosphatase activities before addition of pervanadate were similar when
either [32P]myelin basic protein or
[32P]RCM (carboxymethylated-maleylated, reduced
form)-lysozyme was used as substrate (Fig. 5
). Protein-tyrosine phosphatase activity
in cell extracts from cells treated with 5 µM pervanadate
followed by cell lysis was approximately 50% of that observed in cells
incubated without pervanadate. As the pervanadate concentration was
increased to 20 µM, protein-tyrosine phosphatase activity
in the cell extracts declined to less than 20% of that observed in
cells incubated without pervanadate. Pervanadate is formed by mixing
hydrogen peroxide with sodium orthovanadate (27), but hydrogen peroxide
(20 µM) alone had no effect on protein-tyrosine
phosphatase activity. These results demonstrated that
protein-tyrosine phosphatase activity in the four 32D cell
cloneswas similar and that pervanadate was an effective
inhibitor of protein-tyrosine phosphatase activity.

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Figure 5. Effect of Pervanadate on Protein-Tyrosine
Phosphatase Activity in 32D/IR, 32D/IR+IRS-1,
32D/IR+IRS-1F18, and 32D/IR+IRS-2 Cells
Cells were incubated with 0, 5, 10, or 20 µM of
pervanadate for 20 min. Cells were then lysed, and protein-tyrosine
phosphatase activity was measured in the cell extracts as described in
Materials and Methods using either
[32P]RCM-lysozyme (left) or
[32P]myelin basic protein (right) as the
substrate. Experiments were conducted in triplicate and expressed as
picomoles/min/mg protein (mean ± SD).
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Effect of Pervanadate on Tyrosine Phosphorylation of the Insulin
Receptor
To examine whether IRS-1 increased insulin receptor tyrosine
phosphorylation via inhibition of protein-tyrosine phosphatases, the
effect of preincubating cells with pervanadate on insulin receptor
phosphorylation was studied. 32D/IR, 32D/IR+IRS-1,
32D/IR+IRS-1F18, or 32D/IR+IRS-2 cells were incubated with
10 µM pervanadate for 20 min followed by addition of
100 nM insulin. 32D/IR, 32D/IR+IRS-1F18,
or 32D/IR+IRS-2 cells incubated with pervanadate had much greater
levels of insulin receptor tyrosine phosphorylation than cells
incubated with insulin in the absence of pervanadate, as determined by
Western blot analysis of cellular proteins using antiphosphotyrosine
antibodies (Figs. 6
and 7
). In 32D/IR+IRS-1 cells, the insulin
receptor was phosphorylated to the same degree in the presence or
absence of pervanadate. In the absence of insulin, 10 µM
pervanadate had no detectable effect on insulin receptor
phosphorylation in any of the four cell types (Figs. 6
and 7
, 0 min
time points). The effect of hydrogen peroxide on the phosphorylation
state of the insulin receptor was also examined. Incubation of cells
with 20 µM hydrogen peroxide had no effect on basal or
insulin-stimulated insulin receptor tyrosine phosphorylation in any of
the four cell types. These results suggest that phosphatases
dephosphorylate the insulin receptor in the absence of
tyrosine-phosphorylated IRS-1 (32D/IR cells,
32D/IR+IRS-1F18, and 32D/IR+IRS-2 cells), but when IRS-1 is
present, these phosphatases are inhibited from dephosphorylating the
insulin receptor.

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Figure 6. Insulin Receptor Phosphorylation in Cells Treated
with or without Pervanadate
2D/IR, 32D/IR+IRS-1, 32D/IR+IRS-1F18, or 32D/IR+IRS-2 cells
were incubated with or without 10 µM pervanadate for 20
min at 37 C followed by addition of 100 nM insulin for 0,
1, or 5 min. Cells were lysed, and equal amounts of protein were
subjected to SDS-PAGE followed by Western blot analysis with
antiphosphotyrosine antibodies.
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Figure 7. Quantitation of Insulin Receptor Tyrosine
Phosphorylation
32D/IR, 32D/IR+IRS-1, 32D/IR+IRS-1F18, or 32D/IR+IRS-2
cells were incubated with or without 10 µM pervanadate
for 20 min at 37 C followed by addition of 100 nM insulin
for 0, 1, or 5 min. Cells were lysed, and equal amounts of protein were
subjected to SDS-PAGE followed by Western blot analysis with
antiphosphotyrosine antibody. Insulin receptor tyrosine phosphorylation
was quantitated from three experiments using ImageQuant software and
expressed as percent phosphorylation (mean ± SD) with
tyrosine phosphorylation of insulin receptors in 32D/IR+IRS-1 cells
after 1 min of insulin stimulation designated as 100%.
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As a consequence of protein-tyrosine phosphatase inactivation, tyrosine
phosphorylation of cellular proteins after pervanadate treatment
increased with increasing concentrations of pervanadate. At
concentrations of pervanadate ranging from 2.5 µM to 10
µM, insulin receptors had higher levels of tyrosine
phosphorylation in only the 32D/IR, 32D/IR+IRS-1F18, and
32D/IR+IRS-2 cells after treatment of cells with insulin when compared
with cells not treated with pervanadate (Fig. 8
). This effect did not appear to be due
to oxidation and activation of the insulin receptor since insulin
receptors in cells incubated with pervanadate, but not insulin, were
tyrosine phosphorylated to the same degree as insulin receptors from
cells incubated without insulin or pervanadate (data not shown). As the
concentration of pervanadate was increased to 20 µM, both
basal and insulin-stimulated tyrosine phosphorylation of insulin
receptors started to increase in all four cell types including
32D/IR+IRS-1 cells. This increase in insulin receptor phosphorylation
in 32D/IR+IRS-1 cells treated with 20 µM pervanadate
demonstrated that insulin receptor tyrosine phosphorylation was not
saturated in 32D/IR+IRS-1 incubated with insulin but not with
pervanadate. Therefore, the reason that lower concentrations of
pervanadate increased receptor phosphorylation in the other three cell
types, but not in 32D/IR+IRS-1 cells, was not because receptor
phosphorylation was already saturated in 32D/IR+IRS-1 cells.

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Figure 8. Insulin Receptor Tyrosine Phosphorylation in 32D
Cell Clones Treated with Different Concentrations of Pervanadate
32D/IR, 32D/IR+IRS-1, 32D/IR+IRS-1F18, or 32D/IR+IRS-2
cells were incubated with 020 µM pervanadate for 20 min
at 37 C followed by addition of 100 nM insulin for 5 min.
Cells were lysed, and equal amounts of protein were subjected to
SDS-PAGE followed by Western blot analysis with antiphosphotyrosine
antibodies. Insulin receptor tyrosine phosphorylation was quantitated
from duplicate experiments using ImageQuant software and expressed in
ImageQuant Units (IMU) (mean ± scap]sd).
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Vanadate is also an inhibitor of protein-tyrosine phosphatases, but
unlike pervanadate, which is an irreversible inhibitor, vanadate is a
competitive inhibitor (26). When 32D cells were incubated with 500
µM vanadate for 2 h and then stimulated with
insulin, tyrosine phosphorylation of the insulin receptor increased in
vanadate-treated 32D/IR cells, 32D/IR+IRS-1F18 cells, and
32D/IR+IRS-2 cells, but not in vanadate-treated 32D/IR+IRS-1 cells
(data not shown). These results were similar to those observed with
pervanadate-treated cells and supported the idea that IRS-1 inhibits
protein-tyrosine phosphatases from dephosphorylating the insulin
receptor.
Effect of Clonal Variation on Insulin Receptor Tyrosine
Phosphorylation
32D/IR+IRS-1 cells had higher levels of insulin receptor tyrosine
phosphorylation than 32D/IR, 32D/IR+IRS-1F18, or
32D/IR+IRS-2 cells at insulin concentrations ranging from 10
nM to 200 nM (data not shown). This increase in
insulin receptor phosphorylation in 32D/IR+IRS-1 cells was not due to
clonal variation. Cell lines transfected at different times and
expressing slightly different levels of insulin receptor and/or IRS-1
were examined. In all cases, 32D/IR+IRS-1 cells had higher levels of
insulin receptor tyrosine phosphorylation and lower levels of Shc
tyrosine phosphorylation than 32D/IR cells, 32D/IR+IRS-1F18
cells, or 32D/IR+IRS-2 cells (Fig. 9
).
Furthermore, pervanadate had no effect on insulin receptor
tyrosine phosphorylation levels in any of the 32D/IR+IRS-1 cell
lines.

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Figure 9. Effect of Insulin on Tyrosine Phosphorylation of
the Insulin Receptor, IRS-1, and Shc in 32D Cell Clones
32D/IR and three different clones of 32D/IR+IRS-1 cells (clones A, B,
and C) were incubated without or with 100 nM insulin for 1
or 5 min. Cells were lysed, and equal amounts of protein were subjected
to SDS-PAGE and Western blot analysis with antiphosphotyrosine
antibodies (top) or antiinsulin receptor ß-subunit
antibodies (bottom).
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Amount of Insulin Bound to Insulin Receptors in 32D/IR,
32D/IR+IRS-1, and 32D/IR+IRS-1F18
Cells
Another explanation for the increase in insulin receptor tyrosine
phosphorylation observed in 32D/IR+IRS-1 cells could be that insulin
was bound to insulin receptors more in 32D/IR+IRS-1 cells than in the
other three cell types. This increase in insulin binding might be
caused by either an increase in the affinity of the insulin receptor
for insulin or an increase in the number of insulin receptors on the
cell surface in 32D/IR+IRS-1 cells. Insulin binding studies
demonstrated that the amount of total cell associated
[125I]insulin was similar in 32D/IR, 32D/IR+IRS-1, and
32D/IR+IRS-1F18 cells. After subtracting the amount of
intracellular insulin, [125I]insulin bound on the cell
surface in 32D/IR cells was actually higher than 32D/IR+IRS-1 cells
since the amount of intracellular insulin in 32D/IR cells was lower
than in the other cell types (data not shown). Therefore, the increase
in insulin receptor tyrosine phosphorylation in 32D/IR+IRS-1 cells was
not due to greater levels of insulin bound to insulin receptors.
Role of Phosphatidylinositol 3'-Kinase in Increasing Insulin
Receptor Tyrosine Phosphorylation
Phosphatidylinositol 3'-kinase is activated after tyrosine
phosphorylation of IRS-1 (28). To rule out regulation of insulin
receptor phosphorylation by phosphatidylinositol 3'-kinase or a
signaling molecule downstream of phosphatidylinositol 3'-kinase, we
examined the effect of the phosphatidylinositol 3'-kinase inhibitor
Wortmannin on insulin receptor tyrosine phosphorylation (29). 32D/IR or
32D/IR+IRS-1 cells were incubated with or without 0.5 µM
Wortmannin for 30 min at 37 C and then stimulated with insulin. Very
little change in insulin receptor tyrosine phosphorylation was observed
in cells incubated with Wortmannin when compared with cells not
incubated with Wortmannin (data not shown). These results indicated
that phosphatidylinositol 3'-kinase and signaling proteins downstream
of phosphatidylinositol 3'-kinase were not involved in increasing
insulin receptor phosphorylation in the presence of IRS-1.
Effect of Deletion of SAIN or Pleckstrin Homology Domain of
IRS-1 on Insulin Receptor Tyrosine Phosphorylation
To examine the role of specific domains of IRS-1 on insulin
receptor tyrosine phosphorylation, insulin receptor tyrosine
phosphorylation was examined in 32D/IR, 32D/IR+IRS-1,
32D/IR+IRS-1
SAIN, 32D/IR+IRS-1
PH, and
32D/IR+IRS-1F18 cells. IRS-1
SAIN has amino
acids 309555 deleted corresponding to a region of IRS-1 called SAIN,
which may mediate interaction between the insulin receptor and IRS-1.
However, the SAIN region has not been shown to be essential for
interaction between the insulin receptor and IRS-1 in 32D cells (30).
IRS-1
PH is missing amino acids 6155, which comprise
the pleckstrin homology domain. This region of IRS-1 is essential for
proper interaction of IRS-1 with the insulin receptor. After treatment
of 32D/IR, 32D/IR+IRS-1, 32D/IR+IRS-1
SAIN,
32D/IR+IRS-1
PH, and 32D/IR+IRS-1F18 cells
with insulin, the insulin receptor was phosphorylated more in
32D/IR+IRS-1 cells than in the other cell types (Fig. 10a
). However,
32D/IR+IRS-1
SAIN cells had approximately 70% of the
level of insulin receptor phosphorylation in 32D/IR+IRS-1 cells after 1
min of insulin treatment. The other three cell types, including
32D/IR+IRS-1
PH, had much lower levels of insulin
receptor tyrosine phosphorylation as would be expected since
IRS-1
PH interacts poorly with the insulin receptor and
is tyrosine phosphorylated less by the insulin receptor than wild-type
IRS-1 when expressed in 32D/IR cells (30). As shown in Fig. 10b
, our
results were consistent with these findings. IRS-1 phosphorylation was
much less in 32D/IR+IRS-1
PH cells, but about 80% in
32D/IR+IRS-1
SAIN cells, compared with the one in
32D/IR+IRS-1 cells. These results support the concept that
tyrosine-phosphorylated IRS-1 with the capacity to bind to the insulin
receptor increases insulin receptor tyrosine phosphorylation.

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Figure 10. Insulin Receptor and IRS-1 Tyrosine
Phosphorylation in 32D/IR, 32D/IR+IRS-1,
32D/IR+IRS-1 SAIN, 32D/IR+IRS-1 PH, and
32D/IR+IRS-1F18 Cells
Cells were incubated with 100 nM insulin for 0, 1, or 5
min. Cells were lysed, and equal amounts of protein were subjected to
SDS-PAGE followed by Western blot analysis with antiphosphotyrosine
antibody. Tyrosine phosphorylation of the insulin receptor or IRS-1 was
quantitated from two experiments using ImageQuant software and
expressed as percent phosphorylation of the insulin receptor or IRS-1
(mean ± SD), respectively, with tyrosine
phosphorylation in 32D/IR+IRS-1 cells after 1 min of insulin
stimulation designated as 100%. Insulin receptor tyrosine
phosphorylation is shown in panel a, and IRS-1 tyrosine phosphorylation
is shown in panel b.
|
|
Effect of IRS-1 on in Vitro Phosphorylation of the
Insulin Receptor
To confirm that IRS-1 increased insulin receptor phosphorylation,
in vitro studies were conducted. IRS-1 used in these studies
was either commercially purchased or purified from recombinant
baculovirus. Either wheat-germ agglutinin enriched insulin receptors
from 32D/IR cells, insulin receptors, and IRS-1, or insulin receptors,
IRS-1, and cell extract as a source of protein-tyrosine phosphatases
were combined, and insulin-stimulated insulin receptor tyrosine
phosphorylation was examined using Western blot analysis with
antiphosphotyrosine antibodies. An increase in insulin-stimulated
insulin receptor autophosphorylation (50% ± 10%) was observed when
exogenous IRS-1 was added to the assay in the presence or absence of
cell extract (Fig. 11
). As the amount
of cell extract was increased above 2 µg protein, tyrosine
phosphorylation of the insulin receptor and IRS-1 declined until IRS-1
and the insulin receptor were no longer tyrosine phosphorylated (data
not shown). Protein-tyrosine phosphatase activity associated with wheat
germ agglutinin-enriched insulin receptors was approximately 400
pmol/min/mg protein of [32P] phosphate hydrolyzed from
[32P] myelin basic protein. Protein-tyrosine phosphatase
activity in cell extracts was about 500 pmol/min/mg protein of
[32P] phosphate hydrolyzed from [32P]
myelin basic protein. Additional in vitro studies were then
conducted. In one set of these studies, insulin receptors were
incubated with or without insulin for 30 sec at 37 C followed by
addition of IRS-1 to certain samples. The incubations were continued
for an additional period of time and then terminated. In the absence of
insulin but in the presence of IRS-1, little phosphorylation of IRS-1
or the insulin receptor was observed (Fig. 12
, right lane), and the
level of insulin receptor phosphorylation was similar to that in assays
containing insulin receptor but no IRS-1 or insulin (basal level, shown
in Fig. 11
, left lane). In the presence of insulin and in
the absence of IRS-1, insulin receptor tyrosine phosphorylation
increased, but then remained at a relative constant level from 40330
sec of insulin incubation (Fig. 12
). In the presence of IRS-1 and
insulin, insulin receptor tyrosine phosphorylation peaked at 1 min.
However, after 30 sec incubation with IRS-1 (60 sec with insulin),
insulin receptor phosphorylation was greater in the presence of IRS-1
than in the absence of IRS-1. Interestingly, insulin receptor tyrosine
phosphorylation did not increase until 30 sec after addition of IRS-1.
This apparent delayed effect of IRS-1 on insulin receptor
phosphorylation suggests that IRS-1 first needs to be phosphorylated by
the insulin receptor before affecting insulin receptor phosphorylation.
These results are in agreement with the results observed in
insulin-treated 32D cells containing insulin receptors and IRS-1 or
IRS-1F18, which also suggested that IRS-1 needs to be
phosphorylated for insulin receptor tyrosine phosphorylation to
increase.

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Figure 11. Phosphorylation of Wheat Germ Agglutinin-Enriched
Insulin Receptors in the Presence or Absence of IRS-1 and/or Cell
Extract
Wheat germ agglutinin-enriched insulin receptors were tyrosine
phosphorylated in buffer containing 100 nM insulin and 200
µM ATP for 2 min at 37 C. In certain experiments, cell
extract and/or IRS-1 was then added. The mixtures were incubated for an
additional 5 min, and the reactions terminated. Proteins in the
mixtures were subjected to SDS-PAGE followed by Western blot analysis
with antiphosphotyrosine antibodies.
|
|

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Figure 12. Phosphorylation of Wheat Germ Agglutinin-Enriched
Insulin Receptors at Different Time Points after Insulin Addition in
the Presence or Absence of IRS-1
Wheat germ agglutinin-enriched insulin receptors were tyrosine
phosphorylated in buffer containing 100 nM insulin and 200
µM ATP at 37 C. In certain experiments, IRS-1 was then
added 30 sec after the addition of insulin. The mixtures were incubated
for an additional period of time, and the reactions were terminated.
Proteins in the mixtures were subjected to SDS-PAGE followed by Western
blot analysis with antiphosphotyrosine antibodies.
|
|
Preparations of wheat-germ agglutinin-enriched insulin receptors also
contain other cellular proteins. To identify protein-tyrosine
phosphatases that may be present in the wheat-germ agglutinin-enriched
insulin receptor preparations, these preparations were analyzed for the
presence of the protein-tyrosine phosphatases SHP-2, PTP1B, and CD45.
These phosphatases were chosen because they are known to be present in
hematopoietic cells and they have been shown to play a role in insulin
signaling. As shown in Fig. 13
, all
three of these phosphatases were present in 32D/IR cell extracts, but
only SHP-2 and CD45 were present in the wheat germ-enriched insulin
receptor preparations. These results confirm that in the in
vitro studies, protein-tyrosine phosphatases are present in the
wheat germ-enriched insulin receptors. In the in vitro
studies, the presence of SHP-2 and CD45 in the wheat germ-enriched
insulin receptors suggests that one or both of the PTPases may be
involved in dephosphorylating the insulin receptor in the absence but
not the presence of IRS-1. Although the presence of these two
phosphatases in the in vitro assays does not rule out
involvement of other PTPases, including PTP1B, that may interact with
IRS-1 and the insulin receptor in situ.

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Figure 13. Presence of Protein-Tyrosine Phosphatases in Wheat
Germ Agglutinin-Enriched Insulin Receptors
Protein in 32D/IR cell extracts (A) or in wheat germ
agglutinin-enriched insulin receptor preparations (B) were separated by
SDS-PAGE and subjected to Western blot analysis using anti-SHP-2
antibodies, anti-CD45 antibodies, or anti-PTP1B antibodies.
|
|
 |
DISCUSSION
|
---|
Characterization of the role of proteins such as IRS-1 that
interact with the insulin receptor and mediate insulin receptor
signaling is important in elucidating the regulation of insulin action.
The 32D cells make a good model for studying specific interactions
between IRS proteins and the insulin-signaling pathway because these
cells are devoid of IRS proteins (15). By transfecting these cells with
IRS-1 or IRS-2, the specific roles of these proteins in insulin
signaling can be examined. Previously, we had found that the insulin
receptor was tyrosine phosphorylated more in the presence of IRS-1 than
in the absence of IRS-1 (16). This increase in insulin receptor
tyrosine phosphorylation appeared to be due to an increase in tyrosine
phosphorylation of receptors themselves and not due to differences in
receptor number. In the present studies, we demonstrated that the
insulin receptor was phosphorylated more in the presence of IRS-1 than
in the absence of IRS-1, in the presence of IRS-2, or in the presence
of a nonphosphorylated form of IRS-1 (Fig. 1
). From studies with
pervanadate (Figs. 6
and 7
), this increase in insulin receptor
phosphorylation was attributed to inhibition of a protein-tyrosine
phosphatase(s) by IRS-1, but we could not rule out a direct effect of
IRS-1 on the insulin receptor either by increasing tyrosine kinase
activity or increasing autophosphorylation. Since the presence of IRS-2
did not result in an increase in insulin receptor tyrosine
phosphorylation or tyrosine kinase activity, this effect appeared to be
IRS-1 specific. In vitro experiments further
demonstrated that IRS-1 increased insulin receptor tyrosine
phosphorylation (Figs. 11
and 12
).
The insulin receptor ß-subunit is phosphorylated on seven tyrosines
(reviewed in Ref. 1). Three of the phosphorylated tyrosines comprise
the kinase-regulatory domain (31). When one or all of these three
residues are substituted with phenylalanine, tyrosine kinase activity
is greatly reduced both in vitro and in whole cells
(32, 33, 34, 35). Receptors with these phenylalanine for tyrosine substitutions
have an impaired ability to phosphorylate IRS-1 and are unable to
modulate most insulin-regulated biological functions. Insulin
binding to the insulin receptor increases insulin receptor tyrosine
phosphorylation and receptor tyrosine kinase activity. After insulin
activates the receptor tyrosine kinase, a means must exist for
regulating the duration and intensity of the signal. One potential
means of regulation is dephosphorylation of the phosphotyrosines in
the kinase-regulatory domain of the insulin receptor (2).
Dephosphorylation of these phosphotyrosines by protein-tyrosine
phosphatases greatly reduces receptor tyrosine kinase activity.
However, if the protein-tyrosine phosphatases are inhibited from
dephosphorylating the insulin receptor, then the insulin receptor
retains its full kinase activity. Thus, signaling downstream of the
insulin receptor can be amplified by inhibition of protein-tyrosine
phosphatases as well as by activation of the insulin receptor tyrosine
kinase. In our studies, IRS-1 appeared to inhibit protein-tyrosine
phosphatases until it was fully phosphorylated, which happened within
the first minute of insulin addition. This resulted in increased
phosphorylation of the insulin receptor ß-subunit for at least 29
more minutes. However, the rate of decline in insulin receptor tyrosine
phosphorylation for 14 min following the peak of insulin receptor
tyrosine phosphorylation in 32D/IR+IRS-1 cells was much greater than
the rate of decline in the other cell types, indicating that after
IRS-1 is fully phosphorylated IRS-1 may recruit and/or activate
additional phosphatases.
In the in vitro experiments (Fig. 11
), as the amount of cell
extract was increased, phosphorylation of the insulin receptor and the
insulin receptor and IRS-1 declined until no tyrosine phosphorylation
was observed. Yet, in vivo, the insulin receptor and IRS-1
are tyrosine phosphorylated in the cellular milieu. The differences in
tyrosine phosphorylation observed in vivo and in
vitro may be due to compartmentalization of the insulin receptor
and IRS-1 or targeting of specific phosphatases to the insulin receptor
and IRS-1 (36, 37). Cells contain a large number of protein-tyrosine
phosphatases, and some of these phosphatases have specific substrates.
The mechanisms controlling phosphatase substrate specificity in cells
include cellular compartmentalization (38), interaction with regulatory
proteins (39), activators and inhibitors (40, 41), and
posttranslational modification (42). In addition, both the amino acid
sequence immediately adjacent to the dephosphorylation site and the
three-dimensional structure of the region surrounding the
dephosphorylation site may contribute to specificity (43).
Protein-tyrosine phosphatases that have been found to dephosphorylate
the insulin receptor include LAR (leukocyte common-antigen related
protein-tyrosine phosphatase) (38, 44, 45), PTP1B (protein-tyrosine
phosphatase 1B) (46, 47), rPTP
(receptor protein-tyrosine
phosphatase-
) (48), and CD45 (49). SHP-2 has also been implicated in
dephosphorylating the insulin receptor, but conflicting results have
been published regarding the role of SHP-2 in insulin receptor
dephosphorylation (reviewed in Ref. 2). In vitro, SHP-2
dephosphorylates the insulin receptor and IRS-1 (50, 51, 52). In intact
cells, overexpression of the gene encoding SHP-2 does not effect
insulin receptor tyrosine phosphorylation, and studies have not
demonstrated a direct interaction between SHP-2 and the insulin
receptor (53, 54, 55). In mouse 32D cells, one or more of these
phosphatases may dephosphorylate the insulin receptor ß-subunit.
Also, the presence of IRS-1 may specifically inhibit one or more of
these phosphatases from dephosphorylating the insulin receptor. In our
in vitro studies, both SHP-2 and CD45 were detected in the
wheat germ agglutinin-enriched insulin receptors. These results
indicated that in vitro SHP-2 and/or CD45 may be involved in
dephosphorylating the insulin receptor in the absence, but not the
presence, of IRS-1. However, these phosphatases may not have the same
role in dephosphorylating the insulin receptor in vitro as
in intact cells because in intact cells, but not in vitro,
signaling proteins including protein-tyrosine phosphatases and
insulin receptors are in specific three-dimensional orientations at
specific locations within the cell.
IRS-1 may inhibit protein-tyrosine phosphatases from dephosphorylating
the insulin receptor via one of several potential mechanisms. When
IRS-1 is associated with the insulin receptor, it may physically block
protein-tyrosine phosphatases from access to the three tyrosines in the
regulatory domain. Alternatively, tyrosine-phosphorylated IRS-1 may
bind to protein-tyrosine phosphatases through SH2 domains, thereby
restricting access to the insulin receptor or serve as an alternative
substrate targeting the phosphatases away from the insulin receptor.
However, it is unlikely that IRS-1 serves as an alternative substrate
because tyrosine phosphorylated IRS-2 should also serve as an
alternative substrate, but our studies showed that the presence of
IRS-2 did not increase insulin receptor phosphorylation (Fig. 1
).
Although studies with vanadate and pervanadate indicate that IRS-1
inhibited protein-tyrosine phosphatases from dephosphorylating the
insulin receptor, our experiments did not exclude IRS-1 from directly
increasing insulin receptor tyrosine phosphorylation either by
increasing receptor kinase activity or increasing receptor
autophosphorylation. In the latter case, receptor autophosphorylation
may be increased if IRS-1 blocks other substrates from binding to and
being phosphorylated by the insulin receptor. However, if some
mechanism other than inhibition of protein-tyrosine phosphatases
was involved in increasing insulin receptor tyrosine phosphorylation in
the presence of IRS-1, then pervanadate should have an additive effect
on insulin receptor tyrosine phosphorylation in the presence of IRS-1.
As depicted in Fig. 6
, this was not the case. Therefore, the most
plausible mechanism involved in increasing insulin receptor tyrosine
phosphorylation in the presence of IRS-1 appears to be inhibition of
protein-tyrosine phosphatases.
Two regions of IRS-1 appear to be important for interaction with the
insulin receptor. One, called the pleckstrin homology domain, is
located at the amino terminus of IRS-1 (56), and the other, a
phosphotyrosine-binding domain, is located near the pleckstrin homology
domain (57). In IRS-1F18 and IRS-2, both of these regions
are intact, permitting interaction with the insulin receptor. Shc,
IRS-1, and IRS-2 bind to the same site on the insulin receptor,
tyrosine 960, and should compete for phosphorylation by the insulin
receptor (14, 17, 18, 19). However, in our studies, Shc was phosphorylated
to the same degree in 32D/IR+IRS-1F18 and 32D/IR+IRS-2 as
in 32D/IR cells and phosphorylated less in 32D/IR+IRS-1 cells (Fig. 1
).
Increased Shc phosphorylation in the 32D/IR+IRS-1F18 cells
and 32D/IR+IRS-2 cells was not due to differences in the levels of Shc
or insulin receptors in each of the cell types or to differences in the
levels of IRS-1 in the 32D/IR+IRS-1F18 cells and
32D/IR+IRS-1 cells (Figs. 3
and 4
). These results indicate that
IRS-1F18 and IRS-2 do not compete as well as IRS-1 against
Shc for binding to the insulin receptor. Siemester et al.
(58) have reported that a 262-amino acid IRS-1 region comprising five
tyrosine phosphorylation sites within YXXM motifs is an excellent
substrate of the insulin receptor; and after this IRS-1 domain is
tyrosine phosphorylated, it binds more tightly to the insulin receptor.
Our findings suggest that in its dephosphorylated form,
IRS-1F18 may only transiently associate with the insulin
receptor, thus allowing phosphorylation of Shc by the insulin receptor
and dephosphorylation of the insulin receptor by protein-tyrosine
phosphatases. However, while IRS-1 is being phosphorylated, it binds
with high affinity to the insulin receptor, thus competing with Shc for
tyrosine phosphorylation and blocking dephosphorylation of the insulin
receptor by protein-tyrosine phosphatases.
The amino termini of IRS-1 and IRS-2 are highly conserved in that both
IRS-1 and IRS-2 have a pleckstrin homology domain and a
phosphotyrosine-binding domain that are involved in binding to the
insulin receptor ß-subunit (14). A third domain on IRS-2, which is
not present on IRS-1, is located between residues 591 and 786 and is
called the KRLB domain (59, 60). The KRLB domain has been found to
contribute significantly to the interaction between IRS-2 and the
insulin receptor. Binding of the KRLB domain of IRS-2 to the insulin
receptor results in tyrosine phosphorylation of the KRLB domain
(Tyr624 and Tyr628), which leads to decreased
binding of IRS-2 to the insulin receptor. This observation suggests
that phosphorylation of the KRLB domain causes the release of IRS-2
from the receptor. Therefore, tyrosine phosphorylation of the KRLB
domain of IRS-2 may decrease its affinity for the insulin receptor
ß-subunit, whereas tyrosine phosphorylation of IRS-1 may increase its
affinity for the insulin receptor ß-subunit.
We showed in this study that IRS-1 has a new function; it increases
tyrosine phosphorylation of the insulin receptor ß-subunit. This
function may be unique to IRS-1 as IRS-2 had no effect on tyrosine
phosphorylation of the ß-subunit. Likewise, the presence of IRS-1
decreased Shc tyrosine phosphorylation and possibly the pathways that
are activated downstream of Shc. This function is also unique to IRS-1,
as IRS-2 had no effect on Shc tyrosine phosphorylation by the insulin
receptor tyrosine kinase. Although advances have been made in
understanding the role of reversible tyrosine phosphorylation of the
insulin receptor in insulin action, this process is not totally
understood, and molecular defects in this process that may lead to
insulin resistance have yet to be discovered. Thus, an increase in
insulin receptor tyrosine phosphorylation and tyrosine kinase activity
in the presence of IRS-1 may enhance and prolong downstream signaling
events. Defects in IRS-1, as are found in some diabetic patients, could
contribute to insulin resistance and type II diabetes mellitus
(7, 8, 9, 10, 11).
 |
MATERIALS AND METHODS
|
---|
Materials
Mouse monoclonal antibodies against phosphotyrosine (4G10) and
rabbit polyclonal antibodies against IRS-1, IRS-2, and PTP1B were
obtained from Upstate Biotechnology, Inc. (Lake Placid,
NY). Rabbit polyclonal antibodies against phosphotyrosine, the insulin
receptor ß-subunit, or Shc were obtained from Transduction Laboratories, Inc. (Lexington, KY). Rabbit polyclonal antibodies
against SHP-2 and mouse monoclonal antibodies against CD45 were from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit
antimouse Ig was from Rockland Inc. Porcine insulin was a gift from Dr.
R. E. Chance (Eli Lilly & Co. Research Laboratory).
[125I]Protein A (>30 µCi/µg) was from ICN Biomedicals, Inc. (Costa Mesa, CA) and
[
-32P] ATP (10 mCi/ml, 3000 Ci/mmol) was from
Amersham Pharmacia Biotech (Arlington Heights, IL). IRS-1
was either from Upstate Biotechnology, Inc. or purified
from recombinant baculovirus. Chemicals were purchased from either
Sigma Chemical Co. (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA). Pervanadate was prepared by mixing
equal volumes of freshly prepared 0.1 M
H2O2 and 0.1 M
Na3VO4 (17). The mixture was incubated for 10
min before use.
Cell Culture and Treatments
32D mouse myeloid progenitor cells expressed either the human
insulin receptor gene (32D/IR), insulin receptor and IRS-1 genes
(32D/IR+IRS-1), insulin receptor and IRS-2 genes (32D/IR+IRS-2), or
genes encoding the insulin receptor and IRS-1 with specific deletions
or substitutions (32D/IR+IRS-1
SAIN,
32D/IR+IRS-1
PH, 32D/IR+IRS-1F18)(30, 61).
32D/IR cells expressed insulin receptors but not IRS-1 or IRS-2,
32D/IR+IRS-1 cells expressed insulin receptors and IRS-1, 32D/IR+IRS-2
expressed insulin receptors and IRS-2, 32D/IR+IRS-1
SAIN
expressed insulin receptors and IRS-1 with amino acids 309555 deleted
corresponding to a region of IRS-1 called SAIN, and
32D/IR+IRS-1
PH expressed insulin receptors and IRS-1
with amino acids 6155 deleted. Cells were cultured in RPMI 1640
medium as previously described (16, 20). Before addition of insulin,
cells were cultured in DMEM with 0.1% BSA for 34 h (serum
deprivation). Cells were incubated with or without insulin, washed in
ice-cold PBS, and lysed in lysis buffer (10 mM Tris, pH
7.4, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA,
5 mM EGTA, 20 mM
Na4P2O7, 20 mM NaF, 1
mM Na3VO4, 1 mM
phenylmethylsulfonyl fluoride (PMSF), 8 µg/ml aprotinin, and 8
µg/ml leupeptin) (16). Insoluble material was removed by
centrifugation. Protein was measured using the BCA assay (Pierce Chemical Co., Rockford, IL) and adjusted to equal
concentrations.
Western Blot Analyses
Lysates containing equal amounts of protein were solubilized in
Laemmli buffer, and subjected to SDS-PAGE and electrotransfer onto
polyvinylidene difluoride membrane (Immobilon-P, Millipore Corp., Milford, MA). Western blot analyses with antibodies
against phosphotyrosine (0.5 µg/ml), the insulin receptor ß-subunit
(1 µg/ml), Shc (1 µg/ml), IRS-1 (1 µg/ml), or IRS-2 (1 µg/ml)
were performed as described previously (16), and radiolabeled proteins
were detected by a PhosphorImager using ImageQuant software
(Molecular Dynamics, Inc., Sunnyvale, CA).
Activity Assays of Protein-Tyrosine Phosphatases
[32P] myelin basic protein or [32P]
RCM-lysozyme was phosphorylated as previously described (62). For the
pervanadate experiments, 32D/IR, 32D/IR+IRS-1, 32D/IR+IRS-2, or
32D/IR+IRS-1F18 cells were incubated with 0 to 20
µM pervanadate for 20 min. Cells were washed twice in
buffer containing 50 mM HEPES, pH 7.4, 125 mM
NaCl, 1 mM EDTA, 1 mM dithiothreitol, and 1
mM PMSF. Cells were lysed in the same buffer containing 1%
Triton X-100. Insoluble material was removed by centrifugation at
15,000 x g for 5 min at 4 C. Protein-tyrosine
phosphatase assays (30 µl final volume) were conducted by incubating
cell extract with 2 µM [32P]myelin basic
protein or [32P]RCM-lysozyme in buffer containing 50
mM imidazole, pH 7.2, 1% BSA, and 0.1%
ß-mercaptoethanol for 20 min at 37 C. The reactions were terminated
by adding 70 µl of 20% trichloroacetic acid and subjected to
centrifugation at 15,000 x g for 5 min. A portion of
the supernatant, 50 µl, was counted in a liquid scintillation
counter. Control assays without cell extract were run in parallel to
measure unincorporated [32P] phosphate, and this value
was subtracted from protein tyrosine phosphatase activity in each
sample.
For in vitro assays, a portion of either the wheat germ
agglutinin-enriched insulin receptors (63) or cell extract from 32D/IR
cells lysed in lysis buffer in the absence of phosphatase inhibitors
was assayed for protein-tyrosine phosphatase activity using the same
assay as described for measuring phosphatase activity in cell extract
from pervanadate-treated cells.
Insulin Binding in 32D/IR,
32D/IR+IRS-1F18, and 32D/IR+IRS-1
Cells
Insulin binding to 32D/IR, 32D/IR+IRS-1F18 or
32D/IR+IRS-1 cells was assessed in triplicate after incubating the
cells with [125I]iodoinsulin in Krebs-Ringer
3-[N-morpholino]propane sulfonic acid buffer, as
described by Smith and Jarett (64). Briefly, cells were incubated with
0.75 nM [125I]insulin for 30 min at 37 C to
measure total cell-associated insulin including insulin bound to the
membrane and intracellular insulin or for 60 min at 4 C to measure
insulin bound to the membrane. This concentration of insulin (0.75
nM) was used to specifically detect binding to the insulin
receptor. However, nonspecific binding was measured by incubating with
excess unlabeled insulin (4 µM), and then specific
binding was determined by subtracting nonspecific binding from total
binding. Iodoinsulin bound to the cells was separated from unbound
insulin by centrifugation through 10 mM phosphate buffer
containing 0.25 M sucrose. The amount of intracellular
[125I]insulin was determined by centrifugation of cells
through 0.25 M sucrose after removal of cell surface
insulin by an acid wash. Results of the insulin-binding experiments
were quantitated using a
-counter.
In Vitro Phosphorylation of Insulin Receptors in the
Presence or Absence of IRS-1
Wheat germ agglutinin-enriched insulin receptors were prepared
from 32D/IR cells as previously described (63). Insulin receptors were
phosphorylated for 2 min at 37 C in a reaction containing wheat germ
agglutinin-enriched insulin receptors (1 µg of protein), 50
mM HEPES, pH 7.4, 125 mM NaCl, 1 mM
EDTA, 10 mM MgCl2, 5 mM
MnCl2, 5 mM dithiothreitol, 200
µM ATP, 100 nM insulin, and 1 mM
PMSF. In certain experiments, cell extract (010 µg of protein)
and/or 0.8 µg of IRS-1 was then added. The mixtures (50 µl) were
then incubated for an additional 5 min, and the reactions terminated by
adding 5x Laemmli sample buffer and boiling for 3 min. Tyrosine
phosphorylation of the insulin receptor and IRS-1 was analyzed by
subjecting the protein in the mixtures to SDS-PAGE followed by Western
blot analysis with antiphosphotyrosine antibodies.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Leonard Jarett, M.D., Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, B103 Richards Building, 37th and Hamilton Walk, Philadelphia, Pennsylvania 19104.
This work was supported in part by NIH Diabetes Training Grant
5T32DK-07314 (to B.T.S.), NIH R01 43396 (to B.J.G.), and a grant from
the Lucille P. Makey Charitable Trust (to S.H.).
Received for publication February 11, 1999.
Revision received July 7, 1999.
Accepted for publication July 12, 1999.
 |
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