Early Signaling Events Triggered by Peroxovanadium [bpV(phen)] Are Insulin Receptor Kinase (IRK)-Dependent: Specificity of Inhibition of IRK-Associated Protein Tyrosine Phosphatase(s) by bpV(phen)
Christian J. Band,
Barry I. Posner,
Victor Dumas and
Jean-Olivier Contreres
The Polypeptide Hormone Laboratory and the Departments
of Medicine and Physiology McGill University Montreal,
Quebec, Canada H3A 2B2
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ABSTRACT
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Peroxovanadiums (pVs) are potent protein tyrosine
phosphatase (PTP) inhibitors with insulin-mimetic properties in
vivo and in vitro. We have established the existence
of an insulin receptor kinase (IRK)-associated PTP whose inhibition by
pVs correlates closely with IRK tyrosine phosphorylation, activation,
and downstream signaling. pVs have also been shown to activate various
tyrosine kinases (TKs) that could participate in activation of the
insulin-signaling pathway. In the present study we have sought to
determine whether pV-induced IRK tyrosine phosphorylation requires the
intrinsic kinase activity of the IRK, and whether IRK activation is
necessary to realize the early steps in the insulin-signaling cascade.
To address this we evaluated the effect of a pure pV compound, bis
peroxovanadium 1,10-phenanthroline [bpV(phen)], in HTC rat hepatoma
cells overexpressing normal (HTC-IR) or kinase-deficient (HTC-M1030)
mutant IRKs. We showed that at a dose of 0.1
mM, but not 1 mM,
bpV(phen) induced IRK-dependent events. Thus, 0.1
mM bpV(phen) increased tyrosine phosphorylation
and IRK activity in HTC-IR but not HTC-M1030 cells. Tyrosine
phosphorylation of insulin signal-transducing molecules was promoted in
HTC-IR but not HTC-M1030 cells by bpV(phen). The association of p185
and p60 with the src homology-2 (SH2) domains of Syp and the
p85-regulatory subunit of phosphatidylinositol 3'-kinase was induced by
bpV(phen) in HTC-IR, but not in HTC-M1030 cells, as was insulin
receptor substrate-1-associated phosphatidylinositol 3'-kinase
activity. Thus autophosphorylation and activation of the IRK by
bpV(phen) is effected by the IRK itself, and the early events in the
insulin- signaling cascade follow from this activation event. This
establishes a critical role for PTP(s) in the regulation of IRK
activity. bpV(phen) could be distinguished from insulin only in its
ability to activate ERK1 in HTC-M1030 cells, thus indicating that this
event is IRK independent, consistent with our previous hypothesis that
bpV(phen) inhibits a PTP involved in the negative regulation of
mitogen-activated protein kinases.
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INTRODUCTION
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Vanadium salts stimulate lipogenesis, antilypolysis, glucose
uptake (1), and glycogen synthase activity (2, 3, 4), induce mitogenesis
in insulin-responsive cells (5, 6), and reduce blood glucose in
diabetic rats when orally administered (7). The mechanism of the
insulin mimetic action of vanadate has been suggested to involve
activation of the insulin receptor kinase (IRK) (4, 8); however, little
or no increase in IRK tyrosine phosphorylation has been observed in
vanadate-treated cells (9, 10). Rather, the insulin mimetic action of
vanadate appears to derive from activation of a cytosolic tyrosine
kinase (TK) (1).
Hydrogen peroxide (H2O2) has also been shown to
mimick insulin (11, 12). Mixing vanadate and
H2O2 in solution results in the formation of
peroxovanadium (pV) species, which are more powerful insulin-mimetics
than either agent alone (10, 13) and have a broader range of
insulin-like effects (14). Moreover, pVs are 1000 times more potent
than vanadate as inhibitors of protein tyrosine phosphatase (PTP)
activity (15) and induce IRK autophosphorylation and activation in
liver (16), adipocytes (1, 10), and Chinese hamster ovary (CHO) cells
(17) without activating the vanadate-sensitive cytosolic TK (1). It is
noteworthy that treatment of cells with pVs leads to increased tyrosine
phosphorylation of p185 insulin receptor substrate-1 (IRS-1) (17, 18),
a key intermediate in insulin signaling, as well as Raf-1 and
mitogen-activated protein (MAP) kinase (19, 20).
In previous work we demonstrated the existence of an IRK-associated
PTP(s) (15) and suggested that, in the absence of insulin stimulation,
a futile cycle operates in which low-level IRK autophosphorylation is
countered by the dephosphorylating activity of this PTP(s), thus
preventing net autophosphorylation and hence IRK activation in the
basal state (21, 22). We proposed that pVs promote IRK tyrosine
phosphorylation and activation by inhibiting this closely associated
PTP(s) based on the following: 1) the addition of pV to partially
purified IRK had no effect on activation in the absence or presence of
insulin (10, 13); 2) pVs are potent inhibitors of IRK dephosphorylation
in endosomes, which appear to be the major site at which IRK
dephosphorylation occurs (15, 21); 3) a close relationship exists
between IRK activation and IRK-associated PTP inhibition (22).
On the other hand it has been shown that pVs can activate various TKs
(23, 24) probably by inhibiting a range of different PTPs. If
pV-induced IRK tyrosine phosphorylation and activation are due to
autophosphorylation rather than phosphorylation by another TK, we would
not anticipate seeing pV-induced tyrosine phosphorylation of a
kinase-negative IRK. Additionally, if pVs entrain the insulin-signaling
pathway by effecting substrate phosphorylation independently of IRK, we
would expect to see tyrosine phosphorylation of the same substrates in
cells expressing both normal and mutant IRKs. To determine whether
pV-induced IRK tyrosine phosphorylation and proximal downstream
signaling events require activation of the IRK, we have studied the
effect of the previously synthesized pure pV compound,
bis-peroxovanadium 1,10-phenanthroline [bpV(phen)] in HTC-IR rat
hepatoma cells overexpressing normal (HTC-IR) and kinase-negative
(HTC-M1030) IRKs. Our observations indicate that the activation of IRK
by bpV(phen) is effected by the IRK itself, and that early events in
the insulin-signaling cascade follow from this activation event.
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RESULTS
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Dose-Dependent Effect and Synergy of bpV(phen) and Insulin on IRK
Activation in HTC-IR and HTC-M1030 Cells
bpV(phen) activates the IRK in intact rat liver and cultured
H4IIEC3 hepatoma cells (22). We have investigated the level of tyrosine
kinase activity (TKA) in preparations of partially purified insulin
receptors (IRs) from HTC rat hepatoma cells treated with a range of
bpV(phen) doses with or without insulin. TKA was determined in
vitro using the synthetic substrate poly
Glu4Tyr1. In HTC-IR cells, which express 4
times more IR than HTC-WT (data not shown), a saturating dose of
insulin (100 nM) produced a 4-fold increase in TKA relative
to untreated cells (Fig. 1A
). A
comparable response was obtained after treatment with 0.1
mM bpV(phen). The effect of bpV(phen) was dose-dependent,
the 1 mM dose producing a 16-fold increase in TKA (Fig. 1A
). In contrast, in HTC-M1030, which express high levels of
kinase-deficient IRs, neither insulin (100 nM) nor
bpV(phen) at 0.01 and 0.1 mM stimulated TKA to a
significant extent (Fig. 1B
). Thus, at these doses of bpV(phen), the
native form of the IR is necessary for IRK activation, suggesting that
the latter is the predominant TKA stimulated by bpV(phen).

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Figure 1. Dose Response of IRK Activation by bpV(phen), Alone
or Combined with Insulin, in HTC-IR and HTC M1030 Cells
HTC-IR cells (panel A) and HTC-M1030 cells (panel B) were incubated in
HEPES- buffered Krebs-Ringer bicarbonate solution containing 1% BSA
with the indicated concentrations of bpV(phen) alone (open
bars), or in the presence of 100 nM insulin
(solid bars). After 30 min of stimulation, the cells
were washed and solubilized in the presence of phosphatase inhibitors.
IRs were partially purified by lectin chromatography, and kinase
activities present in the eluates were measured, in
vitro, using poly (Glu4:Tyr1) as
substrate. Each assay was performed with an equal amount of IR (10 fmol
of insulin binding). Exogenous kinase activities were expressed as
picomoles of phosphate incorporated into poly
(Glu4:Tyr1)/10 min/10 fmol of insulin
binding.
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When the dose-response of bpV(phen) was carried out in the presence of
100 nM insulin, a synergistic effect on IRK activity was
observed in HTC-IR cells. At bpV(phen) doses greater than 0.01
mM, the level of IRK was greatly above that obtained with
either insulin or bpV(phen) individually (Fig. 1
). The synergistic
effect also manifest in HTC-M1030 [except at the high 1 mM
dose of bpV(phen)], implies that insulin and bpV(phen) activate the
IRK by different mechanisms. Insulins action is well documented (25)
and involves hormone binding to the extracellular
-subunit of the
IR, resulting in a conformational change, leading to receptor
autophosphorylation and activation of the intrinsic tyrosine kinase
activity (TKA) of the ß-subunit of the IR. Studies have suggested
that bpV(phen) acts by preventing the dephosphorylation, and hence
inactivation, of the IRK through the inhibition of an IRK-associated
PTP(s) (10, 15, 22, 26). Our results are consistent with this proposed
mechanism of action and, in addition, strongly suggest that
IRK-associated PTP(s) maintain a quiescent IRK in the absence of
insulin and modulate the extent of IRK activity in its presence. The
observation in HTC-M1030 cells that IRK activation by 1 mM
bpV(phen), (albeit weak in comparison to that seen in HTC-IR cells),
was not further augmented by insulin (Fig. 1B
) suggests that in this
circumstance, TKAs other than the IRK may be activated. No appreciable
differences in the pattern or extent of protein tyrosine
phosphorylation was observed in HTC-IR vs. HTC-M1030 cells
in response to 1 mM bpV(phen) treatment (Fig. 2
). In contrast, at a dose of 0.1
mM bpV(phen), proteins underwent significantly greater
tyrosine phosphorylation in HTC-IR compared with HTC-M1030 cells (Fig. 2
). These data demonstrate relative specificity of bpV(phen) action and
are consistent with preferential inhibition of the IRK-associated
PTP(s) at the 0.1 mM dose of bpV(phen). At this latter dose
there is a requirement for the IRK to observe tyrosine phosphorylation
of proteins, whereas at the higher dose of 1.0 mM the
requirement for IRK activation is lost. Due to this dose-dependent
specificity of action we have carried out subsequent studies with 0.1
mM concentrations of bpV(phen).

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Figure 2. Differential Effect of 1 mM and 0.1
mM bpV(phen) on Tyrosine Phosphorylation of Proteins in
HTC-IR vs HTC-M1030 Cells
Cells were incubated with 1 mM or 0.1 mM
bpV(phen) for 20 min and solubilized in the presence of phosphatase
inhibitors. Total cell extracts containing 50 µg of protein were
subjected to SDS-PAGE on 7.5% gels and immunoblotted with PY
antibodies. The blots were incubated with horseradish
peroxidase-conjugated GAM antibodies, and labeled proteins were
detected by enhanced chemiluminescence (ECL, Amersham).
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Specificity of bpV(phen) for IRK Activation
We assessed whether the TKA measured in partially purified IR
preparations from bpV(phen)-treated (0.1 mM) cells was
predominantly the IRK. This was accomplished by determining the total
TKA and [125I]insulin binding in supernatants of wheat
germ agglutinin (WGA) eluates in which IRs were immunoprecipitated with
960 antibodies (or non-immune IgG). The percentage of IRs
immunoprecipitated was determined by subtracting supernatant values for
960 from those derived with control IgG, as previously described
(22). As shown in Table 1
, almost
complete immunoprecipitation of IRs, assessed by
[125I]insulin binding (B), was effected by
960 in
HTC-IR (98100%) and HTC-M1030 cells (8599%). In the absence of
stimulation, low levels of total TKA were present, which probably
reflect IRK activity, since 91 and 100% of this activity was
immunoprecipitated by
960 in HTC-IR and HTC-M1030 cells,
respectively. In HTC-IR cells treated with insulin, bpV(phen), or both
agents, IRK accounted for the large majority of the total TKA, as
judged by the percentage of total TKA immunoprecipitated by
960
(Table 1
). Thus, 0.1 mM bpV(phen) does not substantially
activate tyrosine kinases other than the IRK in HTC cells since in
HTC-M1030 cells, total TKA was the same after bpV(phen) and insulin
treatment, and in both cases 100% of this activity was
immunoprecipitated by
960. These findings are in line with specific
activation of IRK through an inhibitory action of bpV(phen) on an
IRK-associated PTP(s).
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Table 1. Percentage of Insulin Binding and Tyrosine
Kinase Activity Precipitated by Anti-IR Antibodies Following bpV(phen)
and/or Insulin Treatment of HTC Cells
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Tyrosine Phosphorylation of Endogenous Proteins by bpV(phen)
Treatment of HTC-IR cells with bpV(phen) resulted in the
appearance of five major tyrosine-phosphorylated proteins with
molecular masses of 55, 6874, 94, 114, and 122 kDa, as well as
species of 48, 60, 131, 161, and 185 kDa, and this effect was
potentiated by insulin, the addition of which did not generate new
phosphotyrosine (PY) species (Fig. 3
).
These data strongly suggest that proteins tyrosine phosphorylated in
response to bpV(phen) are substrates of the IRK, which may include Shc
(55 kDa) (27), SH-PTP2 (68 kDa) (28), IRS-1 (185 kDa) (29), and the
ß-subunit of the IR (94 kDa). The duration of exposure of the blot to
film was insufficient to reveal a pattern of PY-containing proteins
from cells treated with insulin only (Fig. 3
). Longer exposure times
revealed proteins with molecular masses of 185, 94, and 60 kDa (data
not shown). Thus, bpV(phen) enabled the visualization of
tyrosine-phosphorylated IRK substrates, which are not seen with insulin
alone. This presumably reflects sustained IRK activation due to the
inhibition of IRK dephosphorylation. In HTC-WT and HTC-M1030 cells,
bpV(phen) produced a weak phosphorylation of the 55-, 68- to 74-, 114-,
and 122-kDa proteins, and addition of insulin induced the appearance of
p185. These responses are consistent with the activation of low levels
of endogenous IRKs.

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Figure 3. Substrate Phosphorylation in HTC Cells After
bpV(phen) and/or Insulin Treatment
Cells were incubated with 100 nM insulin for 5 min, 0.1
mM bpV(phen) for 20 min, or 0.1 mM bpV(phen)
for 15 min before the addition of 100 nM insulin for 5 min
and solubilized in the presence of phosphatase inhibitors. Total cell
extracts containing 50 µg of protein were subjected to SDS-PAGE on
7.5% gels and immunoblotted with PY antibodies. The blots were
incubated with horseradish peroxidase-conjugated GAM antibodies, and
labeled proteins were detected by enhanced chemioluminescence (ECL,
Amersham).
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bpV(phen) Stimulates Tyrosine Phosphorylation of the IR ß-Subunit
in HTC-IR, but not HTC-M1030 Cells
The level of autophosphorylation of the IR in response to
treatment of cells with insulin and/or bpV(phen) was determined by
assessing the PY content of the ß-subunit in equal amounts of
immunopurified IRs. As expected, insulin stimulated autophosphorylation
of the IRK in HTC-IR but not HTC-M1030 cells. Of interest, bpV(phen)
also stimulated IRK autophosphorylation in HTC-IR cells while failing
to do so in HTC-M1030 cells. In general, there was a correlation
between the extent of IRK autophosphorylation and activation.
Our results show that although an equal level of IRK activity was
produced by insulin and 0.1 mM bpV(phen) (Fig. 1A
), the
latter mediated a much greater level of IR autophosphorylation (Fig. 4
). The effect of insulin on
autophosphorylation was barely detectable, but was dramatically
increased when the hormone was added to the cells together with
bpV(phen). In this circumstance, a synergistic effect was observed,
consistent with bpV(phen) and insulin acting through different
mechanisms to promote autophosphorylation of the ß-subunit. These
data strengthen the hypothesis that bpV(phen) acts by preventing the
dephosphorylation of the IRK through the inhibition of an associated
PTP(s). In HTC-WT cells, the pattern of IRK autophosphorylation was
essentially the same as in HTC-IR cells, but of lower magnitude. Since
experiments were carried out on equal amounts of immunopurified IRK we
have no ready explanation for this interesting observation.
Association of bpV(phen) and Insulin-Induced
Tyrosine-Phosphorylated Proteins with Syp src Homology-2 (SH2)
Domains
The PTP Syp (SH-PTP2) associates, via its SH2 domains, with
tyrosine-phosphorylated IRS-1 in response to insulin stimulation (30).
We tested whether bpV(phen) could mimic this effect by examining the
association of proteins from HTC cell lysates to a
glutathione-S-transferase (GST)-SypSH2 fusion protein (28).
The proteins bound to GST-SypSH2 were exhibited using
PY antibodies.
In lysates from HTC-IR cells treated with bpV(phen), a
tyrosine-phosphorylated protein of 185 kDa, corresponding to the
expected mobility of IRS-1, complexed with the GST fusion protein, as
did a 60-kDa protein, and a 94 kDa protein that probably corresponds to
the ß-subunit of the IRK (Fig. 5
).
Again, insulin combined with bpV(phen) increased the signal intensity
compared with bpV(phen) alone, particularly with respect to the 94- kDa
species (Fig. 5
). Qualitatively similar results were observed in HTC-WT
cell lysates. In contrast, in lysates from HTC-M1030 cells treated with
bpV(phen) with or without insulin, p185, p94, and p60 were barely
detectable (Fig. 5
).

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Figure 5. Tyrosine-Phosphorylated Proteins Associated with
Syp After bpV(phen) and/or Insulin Treatment
Cells were incubated with 100 nM insulin for 5 min, 0.1
mM bpV(phen) for 20 min, or 0.1 mM bpV(phen)
for 15 min before the addition of 100 nM insulin for 5 min
and solubilized in the presence of phosphatase inhibitors. Total cell
extracts containing 0.5 mg protein were incubated with 5 µg
GST-SypSH2 fusion protein bound to glutathione-Sepharose beads. After
washing, the proteins bound to the beads were subjected to SDS-PAGE on
7.5% gels and immunoblotted with PY antibodies. The blots were then
incubated with [125I]GAM antibodies, and labeled proteins
were visualized by autoradiography.
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Coimmunoprecipitation of IRK Substrates with the Regulatory Subunit
of Phosphatidylinositol 3'-Kinase (PI3-Kinase) in Response to
bpV(phen)
In response to insulin, PI3-kinase associates, via SH2 domains in
the p85-regulatory subunit, with specific PY residues of the IRK (31)
and substrates of the IRK including IRS-1 (32), p62 GTPase-activating
protein (GAP)-associated protein (33), and a 60-kDa phosphoprotein of
unknown function (28). We tested whether bpV(phen) could induce similar
associations by examining PY-containing proteins present in p85
immunoprecipitates from HTC cells. In HTC-IR cells treated with
insulin, a low but detectable signal was observed at 185 kDa. In
bpV(phen)-treated cells, this signal was of much higher intensity
although a strong signal at 60 kDa was present. Insulin added with
bpV(phen) promoted an increase in the PY content of p185, but decreased
the PY content of the 60-kDa species (Fig. 6
). This effect, also noted in HTC-WT
cells, probably reflects a lower level of p60 associated with p85
rather than a reduction in p60 PY content, because in
PY Western
blots of total cell extracts (Fig. 3
) and of GST-SypSH2-associated
proteins (Fig. 5
), insulin combined with bpV(phen) increased the PY
content of the 60-kDa species. We speculate that p185 competes with and
displaces p60 from p85. Although in this study we have not further
examined the 60-kDa protein, this hypothesis is consistent with the
behavior of a 60-kDa protein in HTC-IR cells that is tyrosine
phosphorylated in response to insulin and exhibits preferential binding
to Syp and other IRK substrates than to p85 (28). In cells expressing
mutant IRKs, the 185-kDa species was barely detectable in response to
insulin or bpV(phen), but was phosphorylated to a greater extent when
both agents were combined. However, the signal remained well below that
observed in similarly treated HTC-IR and WT cells (Fig. 6
).

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Figure 6. Tyrosine-Phosphorylated Proteins
Coimmunoprecipitating with the p85 Subunit of PI3-Kinase After Insulin
and/or bpV(phen) Stimulation of HTC Cells
Cells were incubated with 100 nM insulin for 5 min, 0.1
mM bpV(phen) for 20 min, or 0.1 mM bpV(phen)
for 15 min before the addition of 100 nM insulin for 5 min
and solubilized in the presence of phosphatase inhibitors. Total cell
extracts containing 1 mg of protein were immunoprecipitated with an
-p85 antibody, and the immunoprecipitates were subjected to SDS-PAGE
on 7.5% gels and immunoblotted with PY antibodies. Blots were
incubated with [125I]GAM antibodies, and labeled proteins
were visualized by autoradiography. The mol wt markers are shown on the
left.
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bpV(phen) Stimulation of IRS-1-Associated PI3-Kinase Activity Is
IRK-Dependent
In response to insulin, IRS-1 becomes tyrosine phosphorylated and
recruits PI3-kinase activity (29, 32, 34). We assayed PI3-kinase
activity in IRS-1 immunoprecipitates of HTC cells with and without
insulin and/or bpV(phen) treatment. Insulin and bpV(phen) individually
increased IRS-1-associated PI3-kinase activity 10- to 14-fold over
basal levels in HTC-IR, but only 4-fold in M1030 cells. Thus PI3-kinase
activation is largely IRK-dependent in HTC cells (Fig. 7
). Insulin potentiated the effects of
bpV(phen) on IRS-1-associated PI3-kinase activity, particularly in
IR-overexpressing cells in which a marked synergistic effect was
observed. In both cell lines the level of IRS-1-associated PI3-kinase
activity in response to a given treatment was consistent with the
degree of tyrosine phosphorylation of p185 promoted by that treatment,
as assessed in p85 immunoprecipitates (Fig. 6
). PI3-kinase activity in
WT cells was slightly greater for each treatment compared with M1030
cells (data not shown).

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Figure 7. Effect of Insulin and bpV(phen) on PI3-Kinase
Activity Associated with IRS-1
Cells were incubated with 100 nM insulin for 5 min, 0.1
mM bpV(phen) for 20 min, or 0.1 mM bpV(phen)
for 15 min before the addition of 100 nM insulin for 5 min.
The cells were solubilized in the presence of phosphatase inhibitors.
Total cell extracts containing 1 mg of protein were subjected to
immunoprecipitation with anti-IRS-1 antibodies. PI3-kinase activity
coimmunoprecipitating with IRS-1 was determined by an in
vitro assay described in Materials and Methods
and was quantitated as the amount of radioactivity (cpm) incorporated
into phosphatidylinositol in 3 min. The results are the mean of three
separate experiments, and the error bars represent
SEs.
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ERK1 Stimulation by bpV(phen) Is Largely IRK-Independent
The activation by insulin of MAP kinases follows IRK-stimulated
tyrosine phosphorylation of IRS-1 and Shc, which associate with
Grb-2-Sos (27, 35, 36), resulting in Ras activation and the
sequential activation of Raf and MAP kinase kinase (MEK).
MEK, a dual specific kinase immediately upstream of MAP kinases,
activates the latter by phosphorylation on specific threonine and
tyrosine residues (37, 38). We recently described an ability of
bpV(phen) to activate MAP kinases in primary rat hepatocyte cultures
independently of MEK (19). In the present study we have tested whether
this was dependent on IRK activation by evaluating the effect of
bpV(phen) on ERK1 activity in HTC-IR vs. HTC-M1030 cells. As
expected, insulin stimulation of ERK1 activity was higher in HTC-IR
than in WT cells and was almost absent in M1030 cells (Fig. 8
). In contrast, bpV(phen) induced a
fairly constant level of ERK1 activation in all three cell types.
Insulin slightly augmented ERK1 activity when added in combination with
bpV(phen) to HTC-IR cells only (Fig. 8
). These results indicate that
bpV(phen) activation of ERK1 is not only independent of MEK but also of
IRK activation. As previously discussed (19) the activity of MAP
kinases is negatively regulated by selective dephosphorylation by
specific tyrosine/threonine phosphatases (39, 40) that may be inhibited
by bpV(phen).

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Figure 8. Effect of Insulin and bpV(phen) on ERK1 Activity
Cells were incubated with 100 nM insulin for 5 min, 0.1
mM bpV(phen) for 20 min, or 0.1 mM bpV(phen)
for 15 min before the addition of 100 nM insulin for 5 min.
ERK1 immunoprecipitated from lysates from HTC-IR (solid
bars), HTC-WT (hatched bars), and HTC-M1030
(open bars) cells was assayed for activity as described
in Materials and Methods. ERK1 activity is expressed
relative to that measured in untreated cells (basal), which was
normalized to 100%. The results are the mean of three separate
experiments, and the error bars represent
SEs.
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DISCUSSION
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Peroxovanadiun species are produced by combining
H2O2 with vanadate (10, 13) as well as by
synthetic procedures (22). The synthetic compounds, consisting of a
vanadium atom linked to an oxyanion, one or two peroxyanions, and an
ancillary ligand within its inner coordination sphere, are the most
powerful PTP inhibitors described to date (14, 22). When added to cells
or administered in vivo, they activate IRK and mimick
insulin effects (14, 22). We have suggested that pV-induced IRK
activation arises from the ability of these compounds to inhibit an
IRK-associated PTP, which normally functions to prevent IRK
autoactivation in the absence of insulin (21, 22). Various studies have
shown that pVs can effect tyrosine phosphorylation of a wide range of
cellular proteins presumably by inhibiting a range of PTPs (20, 23, 24). In the present study we sought to determine to what extent
pV-induced IRK tyrosine phosphorylation was dependent on the intrinsic
kinase activity of IRK itself; as well as the extent to which
downstream signaling events were dependent on IRK activation. Our
approach was to compare the responses of HTC cells overexpressing
normal IRKs (HTC-IR), and kinase-negative mutant IRKs (HTC-M1030) to
bpV(phen) and insulin.
The effect of bpV(phen) on cellular TKA and protein tyrosine
phosphorylation was dose-dependent. Thus 0.1 mM bpV(phen)
and insulin stimulated TKA in HTC-IR cells but had minimal to no effect
in HTC-M1030 cells. In contrast, 1.0 mM bpV(phen) weakly
activated TKA in HTC-M1030 compared with the activation observed in
HTC-IR cells. The addition of insulin further activated the IRK in
HTC-IR but not in HTC-M1030 cells, suggesting that, in the latter, 1.0
mM bpV(phen) activated TKAs other than IRK. A promiscuous
effect of 1.0 mM bpV(phen) was substantiated by the finding
that the pattern of tyrosine-phosphorylated proteins did not differ in
HTC-IR vs. HTC-M1030 cells treated with this dose of
compound. However, at 0.1 mM bpV(phen),
tyrosine-phosphorylated proteins were readily observed only in HTC-IR
cells, indicating relative specificity of bpV(phen) action in this cell
line. Earlier work had shown that distinct synthetic pV compounds
inhibited PTPs associated with the IRK and the epidermal growth factor
receptor kinase (14, 22). Although our data are consistent with
relative specificity of inhibition by pVs of IRK-associated PTP(s), the
cellular context may be equally important. Thus the effect of pVs may
depend not only on the nature and dose of the compound studied but also
on the tissue or cell line examined.
In subsequent studies we have focused our attention on the 0.1
mM dose of bpV(phen). We found that bpV(phen) significantly
increased the PY content of the IR ß-subunit in cells expressing
kinase-active IRKs (HTC-IR) but not in those expressing kinase-negative
IRKs (HTC-M1030) (Fig. 4
). Thus bpV(phen)-induced IRK tyrosine
phosphorylation reflects receptor autophosphorylation rather than the
action of other tyrosine kinases. Interestingly, although insulin and
0.1 mM bpV(phen) comparably activated IRK in HTC-IR cells
(Fig. 1A
), the latter produced a greater degree of ß-subunit tyrosine
phosphorylation than the former (Fig. 4
). This may reflect the
phosphorylation of tyrosine residues irrelevant to IRK activation as
previously reported by Drake et al. (41) in their studies of
the in vivo effects of insulin and bpV(phen). Indeed they
showed that phosphorylation of some IRK tyrosine residues may even have
an inhibitory or restrictive effect on the attained level of IRK
activation. Also of note is the greater level of tyrosine
phosphorylation seen for receptors of HTC-IR compared with those of
HTC-WT cells. Further studies are needed to determine whether,
consequent to pV stimulation, a higher proportion of receptors undergo
tyrosine phosphorylation in HTC-IR vs. HTC-WT cells or
whether the level of tyrosine phosphorylation per activated IRK is
different in the two cell lines.
We previously demonstrated the existence of an IRK-associated PTP(s)
that is dynamically engaged in regulating the level of tyrosine
phosphorylation of activated endosomal IRKs (15). This led to the
suggestion that low level IRK autophosphorylation, measurable in the
absence of insulin (15), does not result in net IRK autophosphorylation
by virtue of the dephosphorylating activity of the IRK-associated
PTP(s), thus preventing IRK activation in the basal state (15). In the
present study the failure of bpV(phen) to activate IRKs in HTC-M1030
cells indicates that pV-induced tyrosine phosphorylation of the IRK is
dependent on its intrinsic TKA and not that of other tyrosine kinases.
Since pVs do not stimulate IRK activity in vitro, it seems
unlikely that they stimulate IRK in vivo by directly
interacting with the receptor (10, 13). The observation that pV-induced
IRK activation closely correlates with the extent of inhibition of
IRK dephosphorylation has suggested a causal relationship between the
two processes (22). Our current data strongly support this proposal and
argue for a key role of IRK-associated PTP(s) in restraining IRK
activation. The PTP(s) involved in IRK dephosphorylation is being
intensively sought since its identification should facilitate the
design of potent and specific inhibitors which, as insulin mimetics,
could improve the clinical management of diabetes (22).
Subsequent to IRK activation and internalization, early events in
insulin signaling involve tyrosine phosphorylation of the p185
substrate proteins, IRS-1 and IRS-2 (32, 42, 43, 44), as well as other
substrates (e.g. p60) whose functional significance has yet
to be fully elucidated (28, 45, 46). In the present study, bpV(phen)
induced the appearance of a range of tyrosine-phosphorylated
proteins in HTC-IR but not in HTC-M1030 cells (Figs. 3
, 5
, and 6
). Thus
activated IRK is necessary for the appearance of these proteins, few of
which were seen on incubating cells with insulin alone. Nevertheless,
the augmentation of their tyrosine phosphorylation with the addition of
insulin to bpV(phen) supports the idea that they constitute a range of
IRK substrates. Those that are not detected in the presence of insulin
alone become evident in the presence of the magnifying effect of
bpV(phen)-induced PTP inhibition.
Specific phosphotyrosine motifs within IRS-1 and -2 recruit SH2
domain-containing transducing proteins (27, 30, 47, 48). Prinicipal
among these molecules is PI3-kinase, which is recruited, via the SH2
domain of its p85 subunit, to YXXM motifs on IRS-1 (27, 30, 47, 48).
bpV(phen) promoted the association of PI3-kinase with IRS-1 in HTC-IR
cells to the same extent as insulin (Fig. 6
). The synergy observed
between bpV(phen) and insulin on IRS-1-associated PI3-kinase activity
could be effected via bpV(phen) inhibition of PTPs involved in
dephosphorylating PY residues in YXXM motifs of IRS-1, although no such
regulatory phosphatases have been described to date. The fact that
bpV(phen) stimulated IRS-1 associated PI3-kinase activity only weakly
in HTC-M1030 cells (Fig. 7
) suggests that the critical event in
bpV(phen)-induced activation of PI3-kinase is the activation of
IRK.
The signaling pathways that mediate insulin activation of MAP kinases
have been well defined. The activated IRK tyrosine phosphorylates IRS-1
(35, 36) and, in certain cell lines Shc (27, 36), each of which
associates with Grb-2-Sos leading to the exchange of GDP for GTP on
p21ras, resulting in p21ras activation and the sequential activation,
by serine/threonine phosphorylation, of Raf and MEK. MEK
activates MAP kinases by phosphorylation on specific threonine and
tyrosine residues (37, 38). Conversely, MAP kinase activity can be
negatively regulated by dual specific tyrosine/threonine phosphatases
(39, 40), which have been identified in a wide range of cell types (39, 49). Recently, pervanadate was shown to activate Raf, MEK,
and MAP kinase in HeLa cells by a mechanism suggested to involve
receptor tyrosine kinase and p21ras activation (20). We recently showed
that ERK1 can be activated by bpV(phen), but not insulin, in the
presence of the specific MEK inhibitor PD98059 (19) and suggested that
this MEK-independent activation involved the inhibition of the dual
specific phosphatase(s) that inactivates MAP kinase. However, this
study could not rule out a role for IRK activation in contributing to
bpV(phen)-induced ERK1 stimulation. The present study enables an
assessment of the contribution of IRK activation in realizing bpV(phen)
stimulation of ERK1. We conclude that this contribution is minimal
since in HTC-IR and WT cells, insulin did not significantly augment
bpV(phen)-stimulated ERK1 activity, nor was the ERK1 activity much
greater in response to bpV(phen) in HTC-IR vs. WT and M1030
cells (Fig. 8
). In addition, ERK1 activity was considerably higher in
response to bpV(phen) vs. insulin in M1030 cells (Fig. 8
),
an effect that cannot be attributed to activation of other receptor
tyrosine kinases, since in bpV(phen)-treated M1030 cells the IRK
represented 100% of the total cellular kinase activity (Table 1
).
Taken together, these data suggest that bpV(phen) acts predominantly
through IRK and Ras-independent mechanisms to activate ERK1
and thus strengthen our hypothesis that it inhibits a PTP involved in
the negative regulation of MAP kinases.
In summary, the observation that bpV(phen) promotes tyrosine
phosphorylation of intact but not kinase-negative IRKs is best
explained by pV-mediated inhibition of the IRK-associated PTP(s).
Furthermore, the early downstream events in the insulin-signaling
cascade are activated by bpV(phen) in an IRK-dependent manner, again
arguing for the key involvement of receptor-associated PTP(s) in
dephosphorylating and hence terminating IRK activity. The only effect
of bpV(phen) that was independent of IRK activation was its ability to
stimulate ERK1. It is intriguing to consider the possibility that
bpV(phen) activates both IRK and ERK by inhibiting a common PTP(s).
 |
MATERIALS AND METHODS
|
---|
Materials
Porcine insulin was a gift from Lilly Research Laboratories
(Indianapolis, IN). DMEM, FCS, penicillin, and streptomycin were
purchased from GIBCO BRL Life technologies (Burlington, Ontario,
Canada). Protein A-Sepharose (PAS), and WGA-Sepharose 6MB were from
Pharmacia (Montreal, Quebec, Canada). MgCl2 was from BDH
(St Laurent, Quebec, Canada). ATP was from Boehringer Manheim (Laval,
Quebec, Canada). Carrier-free sodium-[125I] and
[
32P]ATP (3000 Ci/mmol) were purchased from NEN-DuPont
(Wilmington, DE). NaCl, Mg2SO4, trichloroacetic
acid, and glycerol were from Anachemia Ltd. (Lachine, Quebec, Canada).
Antibodies for IRS-1 and p85 were purchased from Upstate Biotechnology
Inc. (Lake Placid, NY). Polyglutamic acid-tyrosine (4:1) [(poly
(Glu4Tyr1)], goat anti-rabbit (GAR) and goat
anti-mouse (GAM) antiphosphotyrosine (
PY) antibodies, aprotinin,
leupeptin, pepstatin, benzamidine, glutamine, BSA,
N-acetyl-D-glucosamine, Triton X-100, HEPES,
Tris, NaF, Na3VO4, EGTA, EDTA,
phenylmethylsulfonyl fluoride (PMSF), dithiothreitol (DTT), and
reagents for gel electrophoresis were obtained from Sigma (St Louis,
MO). Immobilon-P transfer membranes were from Millipore Canada Ltd.
(Mississauga, Ontario, Canada). The peroxovanadium compound bpV(phen)
was synthesized and purified as previously reported (22).
Cell Culture
Wild type HTC rat hepatoma cells and HTC cells transfected with
expression plasmids containing either human IR cDNA or human IR cDNA
mutated at amino acid 1030 (lysine to methionine, M1030) in the kinase
domain of the IR were a generous gift of Dr. Goldfine (University of
California, San Francisco, CA) (50). HTC-IR and M1030 cells express,
respectively, 4 and 8 times the insulin-binding capacity of the wild
type cells. Importantly, HTC-M1030 cells contain all the tyrosine
residues of the wild type IRK. Cells were grown to 75% confluency in
150-cm2 flasks in DMEM with 10% FCS, as previously
described (51). In all experiments, cells were serum-starved for
36 h in DMEM, containing 0.5% BSA before stimulation with
insulin, or bpV(phen).
Preparation of Cell Lysates
After stimulation with the agents, as described in the figure
legends, the cells (4 x 107) were rinsed in ice-cold
PBS (20 mM sodium phosphate, pH 7, 4, 150 mM
NaCl) and lysed in 5 ml solubilization buffer (50 mM HEPES,
pH 7.4, 150 mM NaCl, 10% glycerol, 1.5 mM
MgCl2, 1.5% Triton X-100, 10 mM NaPPi, 100
mM NaF, 1 mM EGTA, 2 mM
Na3VO4, 1 mM PMSF, 10 µg/ml
aprotinin, and 10 µg/ml leupeptin) for 30 min at 4 C. Lysates were
centrifuged at 10,000 x g for 20 min, and supernatants
were assayed for protein content by the method of Bradford (52) using
BSA as a standard.
Lectin-Affinity Purification of IRs and Insulin Binding
Supernatants, obtained as described above, were recycled five
times through 1 ml WGA Sepharose columns preequilibrated with 50
mM HEPES buffer, pH 7.6, containing 150 mM
NaCl, 1 mM benzamidine, 1 mM PMSF, 0.1%
Triton-X 100, and 2 mM Na3VO4. To
remove receptor-bound insulin, columns were washed first with 50 ml of
50 mM HEPES buffer, pH 6.0, containing 150 mM
NaCl, 10 mM Mg2SO4, 1 mM
benzamidine, 1 mM PMSF, 0.1% Triton-X 100, and 2
mM sodium orthovanadate, then with 10 ml of the above
buffer adjusted to pH 7.6 and lacking Mg2SO4. IRs were
eluted from WGA columns with 1 ml 50 mM HEPES buffer, pH
7.6, containing 0.3 M
N-acetyl-D-glucosamine; 20 µM
leupeptin; 20 µM pepstatin A, and 10 µg/ml of
aprotinin. Specific binding of [125I]insulin, to lectin
purified IRs, was determined as described previously (53).
IRK Assay
Aliquots of WGA eluates containing IR (1015 fmol of insulin
binding) were added to a reaction mixture containing 87.5
mM HEPES, pH 7.4; 40 mM MgCl2; 25
µM [
32P]ATP (5 µCi/assay) and 5 mg/ml
Poly (Glu4:Tyr1) in a final volume of 100 µl.
After a 10-min incubation at room temperature, 50 µl of reaction
mixture were spotted on Whatman 3MM filters and air dried. The filters
were immersed in a solution of ice-cold 10% trichloroacetic acid-10
mM pyrophosphate before washing and counting as described
previously (54).
Immunoprecipitation of IRs
WGA eluates (0.100.15 pmol of insulin binding) were incubated
with mild agitation for 4 h at 4 C with 50 µl of
960
antibody, which recognizes residues 942969 of the juxtamembrane
region of the IR
-subunit (53), preadsorbed to PAS beads. Samples
were centrifuged at 12,000 x g for 5 min at 4 C, and
supernatants were assayed for IRK activity. Immune complexes were
resuspended in 100 µl of Laemmli buffer (2.5% SDS, 10% glycerol, 5
mM DTT, and 25 mM Tris HCl, pH 6.8), boiled for
5 min, subjected to SDS-PAGE, and Western blotted with
960, or
PY
antibodies as indicated in the figure legends.
Association of Proteins to Syp SH2 Domains
A GST fusion protein containing the SH2 domains of Syp
(GST-SypSH2) (55) was adsorbed to Glutathione-Sepharose 4B beads.
GST-SypSH2 revealed a single band of expected size (46 kDa) after
SDS-PAGE and Coomassie blue staining. Glutathione-Sepharose beads
containing 5 µg of bound GST-SypSH2 were incubated with cell lysates
(1 mg protein) for 4 h at 4 C, with 4 mM DTT present
in the incubation mixture to maintain the reduced state of the GST
fusion protein.
Immunoprecipitation of the p85-Regulatory Subunit of
PI3-Kinase
Cell lysates (1 mg protein) were incubated for 3 h at 4 C
with 5 µl of
-p85 antibody preadsorbed to PAS beads. Immune
complexes, obtained by centrifugation at 12,000 x g
for 5 min, were resuspended in 100 µl Laemmli buffer.
Immunoprecipitated proteins were subjected to SDS-PAGE and analyzed by
Western blotting with
PY.
Western Blotting
After the addition of Laemmli buffer, samples containing equal
amounts of insulin binding, or protein, were boiled for 5 min and
subjected to SDS-PAGE under reducing conditions before electrophoretic
transfer of proteins onto Immobilon-P membranes. The membranes were
placed in blocking solution (50 ml PBS containing 20% FCS) for 1
h at room temperature and then in blocking solution containing
960
(1:100), or
PY (1:2500) for 2 h at room temperature. The blots
were then washed three times for 10 min in washing buffer (50 ml PBS
containing 0.1% Tween-20) and incubated in 50 ml PBS, with 5% FCS and
[125I]GAR, or [125I]GAM antibodies (700,000
cpm/electrophoretic lane transferred). After a 1-h incubation, the
membranes were washed in 50 ml washing buffer three times for 10 min
each time, air dried, and exposed to Kodak X-AR film (Eastman Kodak
Co., Rochester, NY) at -80 C for varying lengths of time. Signal
intensities were quantitated using an LKB Ultrascan XL enhanced
densitometer (LKB, Rockville, MD).
IRS-1-Associated PI3-Kinase Activity
IRS-1 was immunoprecipitated from total cell lysates (1 mg
protein) with a rat IRS-1 antibody, preadsorbed to PAS under conditions
identical to those described for immunoprecipitations with
-p85 (see
above). IRS-1 immunoprecipitates were extensively washed (56),
resuspended in 50 µl of PI3-kinase reaction buffer (20 mM
Tris-HCl, pH 7.5, 100 mM NaCl, 0.5 mM EGTA)
containing 0.2 mg/ml L-
-phosphatidylinositol (Avanti
Polar Lipids, Inc. Alabaster, AL), and assayed for PI3-kinase activity,
as described elsewhere (56). The reaction was initiated by adding 10
µCi [
-P32]ATP and 20 mM
MgCl2 and terminated after a 3-min incubation at room
temperature by adding 150 µl of chloroform/methanol/11.6
N HCl (100:200:2). Chloroform (100 µl) was added, and the
organic phase was separated and washed twice with methanol/11.6
N HCl (1:1). The lipids were concentrated in
vacuo, spotted onto Silica Gel 60 TLC plates (Merck and Co. Inc.,
Rathway NJ), and developed in chloroform/methanol/28% ammonium
hydroxide/H2O (43:38:5:7). The phosphorylated products were
visualized by autoradiography, and phosphatidylinositol 3'-phosphate
was identified as the species that comigrated with nonradioactive
phosphatidylinositol 4'-phosphate (Avanti Polar Lipids, Inc.), which
was spotted on the plates and revealed by reaction with potassium
iodide vapor. The silica containing the reaction product was collected,
resuspended in scintillation fluid, and counted.
MAP Kinase Activity Assay
The activity of ERK1 was analyzed by an immune complex kinase
assay using myelin basic protein (MBP) as a substrate (57) with slight
modifications. Cell lysates were incubated with mild agitation for 90
min at 4 C with 5 µl of ERK1 (C-16) (Santa Cruz Biotechnology, Inc.)
antiserum preadsorbed to PAS beads. The beads were washed three times
with lysis buffer and twice with MAP kinase assay buffer (50
mM HEPES, pH 7.4, 5 mM magnesium acetate, 2
mM DTT, 1 mM EGTA, 0.2 mM
Na3VO4). The phosphorylation of MBP was assayed
by resuspending the beads in a total final volume of 100 µl MAP
kinase assay buffer containing 25 µg/ml MBP, 50 µM ATP,
and 1 µCi [
-32P]ATP. Reactions, initiated upon
addition of ATP, were carried out at 30 C for 30 min, and terminated by
the addition of 25 µl 5 x Laemmli sample buffer and boiling for
5 min. Samples were subsequently subjected to SDS-PAGE on 12.5% gels
after which gels were incubated for 3 h in 5% acetic acid/17%
methanol/78% H2O, dried under vacuum, and exposed to x-ray
film. Quantitative assessment of ERK1 activity was achieved by
scintillation counting of phosphorylated MBP bands excised from the
gels.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Barry I. Posner, Polypeptide Laboratory, Strathcona Anatomy Building, 3640 University Street, Room W315, Montreal, Quebec, Canada H3A 2B2.
This work was supported by a grant from the Medical Research Council of
Canada (B.I.P.)
Received for publication August 1, 1997.
Revision received September 25, 1997.
Accepted for publication September 29, 1997.
 |
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