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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1AGo). 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. 1AGo). 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. 1BGo). 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).



View larger version (15K):
[in this window]
[in a new window]
 
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.

 
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. 1Go). 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. Insulin’s action is well documented (25) and involves hormone binding to the extracellular {alpha}-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. 1BGo) 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. 2Go). 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. 2Go). 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).



View larger version (39K):
[in this window]
[in a new window]
 
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 {alpha}PY antibodies. The blots were incubated with horseradish peroxidase-conjugated GAM antibodies, and labeled proteins were detected by enhanced chemiluminescence (ECL, Amersham).

 
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 {alpha}960 antibodies (or non-immune IgG). The percentage of IRs immunoprecipitated was determined by subtracting supernatant values for {alpha}960 from those derived with control IgG, as previously described (22). As shown in Table 1Go, almost complete immunoprecipitation of IRs, assessed by [125I]insulin binding (B), was effected by {alpha}960 in HTC-IR (98–100%) and HTC-M1030 cells (85–99%). 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 {alpha}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 {alpha}960 (Table 1Go). 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 {alpha}960. These findings are in line with specific activation of IRK through an inhibitory action of bpV(phen) on an IRK-associated PTP(s).


View this table:
[in this window]
[in a new window]
 
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

 
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, 68–74, 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. 3Go). 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. 3Go). 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.



View larger version (53K):
[in this window]
[in a new window]
 
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 {alpha}PY antibodies. The blots were incubated with horseradish peroxidase-conjugated GAM antibodies, and labeled proteins were detected by enhanced chemioluminescence (ECL, Amersham).

 
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. 1AGo), the latter mediated a much greater level of IR autophosphorylation (Fig. 4Go). 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.



View larger version (24K):
[in this window]
[in a new window]
 
Figure 4. Phosphotyrosine Content of the ß-Subunit After bpV(phen) and/or Insulin Treatment 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. IRs were partially purified by lectin chromatography. Immunoprecipitations were performed with anti-IR antibodies ({alpha}960) on lectin eluates containing 100 fmol [125I]insulin binding. The immunoprecipitated proteins were subjected to SDS-PAGE on 7.5% gels and Western blotted with {alpha}960 or {alpha}PY antibodies. Blots were incubated with [125I]GAR antibodies, and labeled proteins were visualized by autoradiography. Top panel, Autoradiogram of {alpha}PY Western blot. Bottom panel, Labeling of the ß-subunit of IRs in {alpha}PY and {alpha}960 immunoblots were quantitated by densitometry of autoradiograms and expressed as ratios of PY per ß-subunit.

 
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 {alpha}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. 5Go). Again, insulin combined with bpV(phen) increased the signal intensity compared with bpV(phen) alone, particularly with respect to the 94- kDa species (Fig. 5Go). 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. 5Go).



View larger version (39K):
[in this window]
[in a new window]
 
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 {alpha}PY antibodies. The blots were then incubated with [125I]GAM antibodies, and labeled proteins were visualized by autoradiography.

 
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. 6Go). 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 {alpha}PY Western blots of total cell extracts (Fig. 3Go) and of GST-SypSH2-associated proteins (Fig. 5Go), 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. 6Go).



View larger version (47K):
[in this window]
[in a new window]
 
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 {alpha}-p85 antibody, and the immunoprecipitates were subjected to SDS-PAGE on 7.5% gels and immunoblotted with {alpha}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.

 
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. 7Go). 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. 6Go). PI3-kinase activity in WT cells was slightly greater for each treatment compared with M1030 cells (data not shown).



View larger version (15K):
[in this window]
[in a new window]
 
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.

 
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. 8Go). 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. 8Go). 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).



View larger version (29K):
[in this window]
[in a new window]
 
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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 4Go). 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. 1AGo), the latter produced a greater degree of ß-subunit tyrosine phosphorylation than the former (Fig. 4Go). 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. 3Go, 5Go, and 6Go). 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. 6Go). 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. 7Go) 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. 8Go). In addition, ERK1 activity was considerably higher in response to bpV(phen) vs. insulin in M1030 cells (Fig. 8Go), 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 1Go). 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 [{lambda}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 ({alpha}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 (10–15 fmol of insulin binding) were added to a reaction mixture containing 87.5 mM HEPES, pH 7.4; 40 mM MgCl2; 25 µM [{gamma} 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 (0.10–0.15 pmol of insulin binding) were incubated with mild agitation for 4 h at 4 C with 50 µl of {alpha}960 antibody, which recognizes residues 942–969 of the juxtamembrane region of the IR {alpha}-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 {alpha}960, or {alpha}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 {alpha}-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 {alpha}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 {alpha}960 (1:100), or {alpha}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 {alpha}-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-{alpha}-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 [{gamma}-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 [{gamma}-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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Shisheva A, Shechter Y 1993 Mechanism of pervanadate stimulation and potentiation of insulin-activated glucose transport in rat adipocytes: dissociation from vanadate effect. Endocrinology 133:1562–1568[Abstract]
  2. Dubyak GR, Kleinzeller A 1980 The insulin-mimetic effects of vanadate in isolated rat adipocytes: dissociation from effects of vanadate as a (Na+-K+)ATPase inhibitor. J Biol Chem 255:5306–5312[Free Full Text]
  3. Shechter Y, Karlish JDK 1980 Insulin-like stimulation of glucose oxidation in rat adipocytes by vanadyl (IV) ions. Nature 284:556–558[Medline]
  4. Tamura S, Brown TA, Whipple JH, Yamaguchi YF, Dubler RE, Cheng K, Larner J 1984 A novel mechanism for the insulin-like effect of vanadate on glycogen synthase in rat adipocytes. J Biol Chem 259:6650–6658[Abstract/Free Full Text]
  5. Smith JB 1983 Vanadium ions stimulate DNA synthesis in Swiss mouse 3T3 and 3T6 cells. Proc Natl Sci USA 80:6162–6166[Abstract]
  6. Wice B, Milbrandt J, Glaser L 1987 Control of muscle differentiation in BC3H1 cells by fibroblast growth factor and vanadate. J Biol Chem 262:1810–1817[Abstract/Free Full Text]
  7. Meyerovitch J, Rothenberg P, Shechter Y, Bonner-Weir S, Kahn CR 1991 Vanadate normalizes hyperglycemia in two mouse models of non-insulin-dependent diabetes mellitus. J Clin Invest 87:1286–1294[Medline]
  8. Roth RA, Steele-Perkins G, Hari J, Stover C, Pierce S, Turner J, Edman JC, Rutter WJ 1988 Insulin and insulin-like growth factor receptors and responses. Cold Spring Harbor Symp Quant Biol 53:537–543[Medline]
  9. Venkatesan N, Avidan A, Davidson MB 1991 Antidiabetic action of vanadyl in rats independent of in vivo insulin-receptor kinase activity. Diabetes 40:492–498[Abstract]
  10. Fantus GI, Kadota S, Deragon G, Foster B, Posner BI 1989 Pervanadate [peroxide(s) of Vanadate] mimics insulin action in rat adipocytes via activation of the insulin receptor tyrosine kinase. Biochemistry 28:8864–8871[Medline]
  11. Czech MP, Lawrence JC Jr, Lynn WS 1974 Hexose transport in isolated brown fat cells. A model system for investigating insulin action on membrane transport. J Biol Chem 249:5421–5427[Abstract/Free Full Text]
  12. Krieger-Brauer HI, Kather H 1992 Human fat cells possess a plasma membrane-bound H2O2-generating system that is activated by insulin via a mechanism bypassing the receptor kinase. J Clin Invest 89:1006–1013[Medline]
  13. Kadota S, Fantus GI, Deragon G, Guyda HJ, Hersh B, Posner BI 1987 Peroxide(s) of vanadium: a novel and potent insulin-mimetic agent which activates the insulin receptor kinase. Biochem Biophys Res Commun 147:259–266[Medline]
  14. Bevan PA, Drake, PG, Yale JF, Shaver A, Posner BI 1995 Peroxovanadium compounds: biological actions and mechanism of insulin-mimesis. Mol Cell Biochem 153:49–58[Medline]
  15. Faure R, Baquiran G, Bergeron JJM, Posner BI 1992 The dephosphorylation of insulin receptor and epidermal growth factor receptors: role of endosome-associated phosphotyrosine phosphatase(s). J Biol Chem 267:11215–11221[Abstract/Free Full Text]
  16. Hadari YR, Tzahar E, Nadiv O, Rothenberg P, Roberts CTJ, LeRoith D, Yarden Y, Zick Y 1992 Insulin and insulinomimetic agents induce activation of phosphatidylinositol 3'-kinase upon its association with pp185 (IRS-1) in intact rat livers. J Biol Chem 267:17483–17486[Abstract/Free Full Text]
  17. Wilden PA, Broadway D 1995 Combination of insulinomimetic agents H2 O2 and vanadate enhances insulin receptor mediated tyrosine phosphorylation of IRS-1 leading to IRS-1 association with the phosphatidylinositol 3-kinase. J Cell Biochem 58:279–291[Medline]
  18. Hadari YR, Geiger B, Nadiv O, Sabanay I, Roberts CT, LeRoith D, Zick Y 1993 Hepatic tyrosine-phosphorylated proteins identified and localized following in vivo inhibition of protein tyrosine phosphatases: effects of H2O2 and vanadate administration into rat livers. Mol Cell Endocrinol 97:9–17[CrossRef][Medline]
  19. Band CJ, Posner BI 1997 Phosphatidylinositol 3'-kinase and p70s6k are required for insulin but not bisperoxovanadium 1,10-phenanthroline (bpV(phen)) inhibition of insulin-like growth factor binding protein gene expression: evidence for MEK-independent activation of mitogen-activated protein kinase by bpV(phen). J Biol Chem 272:138–145[Abstract/Free Full Text]
  20. Zhao Z, Tan Z, Diltz CD, You M, Fischer EH 1996 Activation of mitogen-activated protein (MAP) kinase pathway by pervanadate, a potent inhibitor of tyrosine phosphatases. J Biol Chem 271:22251–22255[Abstract/Free Full Text]
  21. Bevan AP, Burgess JW, Yale JF, Drake PG, Lachance D, Baquiran G, Shaver A, Posner BI 1995 Selective activation of the rat hepatic endosomal insulin receptor kinase. Role for the endosome in insulin signaling. J Biol Chem 270:10784–10791[Abstract/Free Full Text]
  22. Posner BI, Faure R, Burgess JW, Bevan AP, Lachance D, Zhang-Sun G, Fantus G, Ng JB, Hall DA, Soo Lum B, Shaver A 1994 Peroxovanadium compounds: a new class of potent phosphotyrosine phosphatase inhibitors which are insulin mimetics. J Biol Chem 269:4596–4604[Abstract/Free Full Text]
  23. Imbert V, Peyron JF, Farahi Far D, Mari B, Auberger P, Rossi B 1994 Induction of tyrosine phosphorylation and T-cell activation by vanadate peroxide, an inhibitor of protein tyrosine phosphatases. Biochem J 297:163–173[Medline]
  24. Harrison ML, Isaacson CC, Burg DL, Geahlen RL, Low PS 1994 Phosphorylation of human erythrocyte band 3 by endogenous p72syk. J Biol Chem 269:955–959[Abstract/Free Full Text]
  25. Cheatham B, Kahn CR 1995 Insulin action and the insulin signaling network. Endocr Rev 16:117–142[Medline]
  26. Cummings C, Zhu L, Sorisky A, Liu XJ 1996 A peroxovanadium compound induces Xenopus oocyte maturation: inhibition by a neutralizing anti-insulin receptor antibody. Dev Biol 175:338–346[CrossRef][Medline]
  27. Skolnik EY, Lee CH, Batzer A, Vicentini LM, Zhou M, Daly R, Myers MJ Jr, Backer JM, Ullrich A, White MF, Schlessinger J 1993 The SH2/SH3 domain-containing protein GRB2 interacts with tyrosine-phosphorylated IRS1 and Shc: implications for insulin control of ras signaling. EMBO J 12:1929–1936[Abstract]
  28. Zhang-Sun G, Yang CR, Viallet J, Feng GH, Bergeron JJM, Posner BI 1996 A 60-kilodalton protein in rat hepatoma cells overexpressing insulin receptor was tyrosine phosphorylated and associated with Syp, phosphatidylinositol 3-kinase, and Grb2 in an insulin-dependent manner. Endocrinology 137:2649–2658[Abstract]
  29. Sun XJ, Rothenberg P, Kahn CR, Backer JM, Araki E, Wilden PA, Cahill DA, Goldstein BJ, White MF 1991 Structure of the insulin receptor substrate IRS-1 defines a unique signal transduction protein. Nature 352:73–77[CrossRef][Medline]
  30. Kuhne MR, Pawson T, Lienhard GE, Feng G-S 1993 The insulin receptor substrate 1 associates with the SH2-containing phosphotyrosine phosphatase Syp. J Biol Chem 268:11479–11481[Abstract/Free Full Text]
  31. Van Horn DJ, Myers MG Jr, Backer JM 1994 Direct activation of the phosphatidylinositol 3'-kinase by the insulin receptor. J Biol Chem 269:29–32[Abstract/Free Full Text]
  32. Backer JM, Myers MG Jr, Sun XJ, Chin DJ, Shoelson SE, Miralpeix M, White MF 1993 Association of IRS-1 with the insulin receptor and the phosphatidylinositol 3'-kinase. J Biol Chem 268:8204–8212[Abstract/Free Full Text]
  33. Sung CK, Sanchez-Margalet V, Goldfine ID 1994 Role of p85 subunit of phosphatidylinositol-3-kinase as an adaptor molecule linking the insulin receptor, p62, and GTPase-activating protein. J Biol Chem 269:12503–12507[Abstract/Free Full Text]
  34. Backer JM, Myers MG Jr, Shoelson SE, Chin DJ, Sun XJ, Miralpeix M, Hu P, Margolis B, Skolnik EY, Schlessinger J, White MF 1992 Phosphatidylinositol 3'-kinase is activated by association with IRS-1 during insulin stimulation. EMBO J 11:3469–3479[Abstract]
  35. Tobe K, Matuoka K, Tamemoto H, Ueki K, Kaburagi Y, Asai S, Noguchi T, Matsuda M, Tanaka S, Hattori S, Fukui Y, Akanuma Y, Yazaki Y, Takenawa T, Kadowaki T 1993 Insulin stimulates association of insulin receptor substrate-1 with the protein abundant src homology/growth factor receptor-bound protein 2. J Biol Chem 268:11167–11171[Abstract/Free Full Text]
  36. Yonezawa K, Ando A, Kaburagi Y, Yamamoto-Honda R, Kitamura T, Hara K, Nakafuku M, Okabayashi Y, Kadowaki T, Kaziro Y, Kasuga M 1994 Signal transduction pathways from insulin receptors to Ras: analysis by mutant insulin receptors. J Biol Chem 269:4634–4640[Abstract/Free Full Text]
  37. Seger R, Krebs EG 1995 The MAPK signaling cascade. FASEB J 9:726–735[Abstract/Free Full Text]
  38. Marshall CJ 1994 MAP kinase kinase kinase, MAP kinase kinase and MAP kinase. Curr Opin Genet Dev 4:82–89[Medline]
  39. Muda M, Boschert U, Dickinson R, Martinou J-C, Martinou I, Camps M, Schlegel W, Arkinstall S 1996 MKP-3, a noval cytosolic protein-tyrosine phosphatase that exemplifies a new class of mitogen-activated protein kinase phosphatase. J Biol Chem 271:4319–4326[Abstract/Free Full Text]
  40. Sun H, Tonks NK, Bar-Sagi D 1994 Inhibition of Ras-induced DNA synthesis by expression of the phosphatase MKP-1. Science 266:285–288[Medline]
  41. Drake PG, Bevan AP, Burgess JW, Bergeron JJM, Posner BI 1996 A role for tyrosine phosphorylation in both activation and inhibition of the insulin receptor tyrosine kinase in vivo. Endocrinology 137:4960–4968[Abstract]
  42. Patti ME, Sun XJ, Bruening JC, Araki E, Lipes MA, White MF, Kahn CR 1995 4PS/insulin receptor substrate (IRS)-2 is the alternative substrate of the insulin receptor in IRS-1-deficient mice. J Biol Chem 270:24670–24673[Abstract/Free Full Text]
  43. Tobe K, Tamemoto H, Yamauchi T, Aizawa S, Yazaki Y, Kadowaki T 1995 Identification of a 190-kDa protein as a novel substrate for the insulin receptor kinase functionally similar to insulin receptor substrate-1. J Biol Chem 270:5698–5701[Abstract/Free Full Text]
  44. White MF, Kahn CR 1994 The insulin signaling system. J Biol Chem 269:1–4[Free Full Text]
  45. Milarski KL, Lazar DF, Wiese RJ, Saltiel AR 1995 Detection of a 60 kDa tyrosine-phosphorylated protein in insulin-stimulated hepatoma cells that associates with the SH2 domain of phosphatidylinositol 3-kinase. Biochem J 308:579–583[Medline]
  46. Danielsen AG, Roth RA 1996 Role of the juxtamembrane tyrosine in insulin receptor-mediated tyrosine phosphorylation of p60 endogenous substrates. Endocrinology 137:5326–5331[Abstract]
  47. Myers MG Jr, Backer JM, Sun XJ, Shoelson SE, Hu P, Schlessinger J, Yoakim M, Schaffhausen B, White MF 1992 IRS-1 activates phosphatidylinositol 3'-kinase by associating with src homology 2 domains of p85. Proc Natl Acad Sci USA 89:10350–10354[Abstract]
  48. Sun XJ, Wang LM, Zhang Y, Yenush L, Myers MJ Jr, Glasheen E, Lane WS, Pierce JH, White MF 1995 Role of IRS-2 in insulin and cytokine signalling. Nature 377:173–177[CrossRef][Medline]
  49. Misra-Press A, Rim CS, Yao, H, Roberson MS, Stork PJ 1995 A novel mitogen-activated protein kinase phosphatase. Structure, expression, and regulation. J Biol Chem 270:14587–14596[Abstract/Free Full Text]
  50. Sbraccia P, Wong KY, Brunetti A, Rafaeloff R, Trischitta V, Hawley DM, Goldfine ID 1990 Insulin down-regulates insulin receptor number and up-regulates insulin receptor affinity in cells expressing a tyrosine kinase-defective insulin receptor. J Biol Chem 265:4902–4907[Abstract/Free Full Text]
  51. Iwamoto Y, Wong KY, Goldfine ID 1981 Insulin action in cultured HTC and H35 rat hepatoma cells: receptor binding and hormone sensitivity. Endocrinology 108:44–51[Abstract]
  52. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal Biochem 72:248–254[CrossRef][Medline]
  53. Burgess JW, Wada I, Ling N, Khan MN, Bergeron JJM, Posner BI 1992 Decrease in ß-subunit phosphotyrosine correlates with internalization and activation of the endosomal insulin receptor kinase. J Biol Chem 267:10077–10086[Abstract/Free Full Text]
  54. Khan MN, Baquiran G, Brule C, Burgess J, Foster B, Bergeron JJM, Posner BI 1989 Internalization and activation of the rat liver insulin receptor kinase in vivo. J Biol Chem 264:12931–12940[Abstract/Free Full Text]
  55. Feng G-S, Hui C-C, Pawson T 1993 SH2-containing phosphotyrosine phosphatase as a target of protein-tyrosine kinases. Science 259:1607–1611[Medline]
  56. Fukui Y, Hanafusa H 1989 Phosphatidylinositol kinase activity associates with viral p60src protein. Mol Cell Biol 9:1651–1658[Medline]
  57. Meloche S 1995 Cell cycle reentry of mammalian fibroblasts is accompanied by the sustained activation of p44mapk and p42mapk isoforms in the G1 phase and their inactivation at the G1/S transition. J Cell Physiol 163:577–588[Medline]