Insulin Receptor Substrate (IRS)-2 Is Dephosphorylated More Rapidly than IRS-1 via Its Association with Phosphatidylinositol 3-Kinase in Skeletal Muscle Cells*

(Received for publication, October 24, 1996, and in revised form, January 27, 1997)

Takehide Ogihara , Bo-Chul Shin Dagger , Motonobu Anai §, Hideki Katagiri , Kouichi Inukai §, Makoto Funaki , Yasushi Fukushima , Hisamitsu Ishihara , Kuniaki Takata Dagger , Masatoshi Kikuchi §, Yoshio Yazaki , Yoshitomo Oka and Tomoichiro Asano par

From the Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113, the Dagger  Laboratory of Molecular and Cellular Morphology, Institute for Molecular and Cellular Regulation, Gumma University, 3-39-15, Showa-cho, Maebashi, Gumma 371, the § Institute for Adult Disease, Asahi Life Foundation, 1-9-14, Nishishinjuku, Shinjuku-ku, Tokyo 160, and the  Third Department of Internal Medicine, Yamaguchi University School of Medicine, 1144, Kogushi, Ube, Yamaguchi 755, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES


ABSTRACT

Insulin receptor substrate (IRS)-2 is structurally and functionally similar to IRS-1. Indeed, stimulation with insulin or insulin-like growth factor I led to the rapid tyrosine phosphorylation of both IRS-1 and IRS-2, which in turn activated phosphatidylinositol (PI) 3-kinase in L6 cells and rat skeletal muscle. However, IRS-2 was rapidly dephosphorylated (3-10 min after the addition of insulin/insulin-like growth factor I), whereas IRS-1 phosphorylation continued for at least 60 min. The time courses of the PI 3-kinase activity associated with IRS-1 and IRS-2 paralleled the tyrosine phosphorylation of these proteins. Preincubation with sodium orthovanadate, an inhibitor of protein tyrosine phosphatase, blocked the rapid dephosphorylation of IRS-2, suggesting the involvement of tyrosine phosphatase. The activation of PI 3-kinase apparently plays an important role in the rapid dephosphorylation of IRS-2, as IRS-2 dephosphorylation was inhibited markedly by suppressing PI 3-kinase activity with wortmannin or overexpression of the dominant negative p85 subunit of PI 3-kinase, which cannot bind the p110 catalytic subunit. In addition, platelet-derived growth factor stimulation prior to insulin stimulation decreased IRS-associated PI 3-kinase and significantly inhibited the dephosphorylation of IRS-2. Taken together, these observations suggest that IRS-2 plays a unique role in mediating the signals from the insulin receptor to downstream molecules and that this effect is more transient than that of IRS-1. Tyrosine phosphatase and IRS-associated PI 3-kinase activity thus contribute to the rapid dephosphorylation of IRS-2.


INTRODUCTION

Insulin binding to its receptor induces activation of the receptor tyrosine kinase followed by the phosphorylation of several cytosolic substrates. A major substrate of the insulin receptor is a 165-185-kDa protein termed insulin receptor substrate (IRS)1-1 (1). After insulin stimulation, IRS-1 is rapidly phosphorylated on multiple tyrosine residues and binds to several Src-homology 2 domains containing proteins (SH2 proteins) (2), which include regulatory subunits for phosphatidylinositol (PI) 3-kinase (p85 (3), p55gamma (4, 5), and p55alpha (5)), Grb2 (6), SHPTP2 (7), and Nck (8). Consequently, IRS-1 mediates activation of PI 3-kinase, p21ras and mitogen-activated protein kinase (9), resulting in the promotion of glucose uptake, glycogen synthesis, mitogenesis, or gene expression. Therefore, IRS-1 was originally thought to play important roles in insulin signaling.

However, studies using IRS-1-deficient mice, derived from targeted gene disruption, have demonstrated that a 180-190-kDa protein functions as an alternative substrate for the insulin receptor (10, 11). Cloning of this protein revealed a structure similar to that of IRS-1, which led this protein to be designated IRS-2 (12). IRS-2 has multiple conserved tyrosine phosphorylation sites that can bind to various SH2 proteins (12), indicating that IRS-2 is functionally similar to IRS-1. Herein, we investigated the functional differences between IRS-1 and IRS-2. We found that IRS-2 is dephosphorylated much more rapidly and activates PI 3-kinase more transiently than IRS-1 in skeletal muscle cells. Furthermore, important roles of its associated PI 3-kinase in the rapid dephosphorylation of IRS-2 are discussed.


EXPERIMENTAL PROCEDURES

Antibodies

Polyclonal anti-IRS-1 and -IRS-2 antibodies were prepared by immunizing rabbits with synthetic peptides derived from amino acids based on the unique COOH termini of mouse, rat, and human IRS-1 (RRSSEDLSNYASINFQKQPEDRQ, corresponding to residues 1211-1233 (mouse), 1213-1235 (rat), and 1220-1242 (human)) and mouse IRS-2 (TYASIDFLSHHLKEATVVKE, residues 1302-1321). Antibody against the p85 subunit of PI 3-kinase was raised as described (5). The antibodies were affinity purified and concentrated as described (14). The anti-phosphotyrosine (alpha PY) monoclonal antibody (4G10) was purchased from Upstate Biotechnology Inc. Anti-hemagglutinin epitope antibody (12CA5) was from Boehringer Mannheim. Anti-IRS-1, -IRS-2, -p85, and -PY antibodies immunoprecipitated 70-90% of each protein, as assessed by immunoblotting the lysate before and after immunoprecipitation. The efficiencies of immunoprecipitation with anti-IRS-1 and anti-IRS-2 antibodies were comparable (80-90%).

Cell Culture

L6 myoblasts were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum at 37 °C in 5% (v/v) CO2 in air. The cells were seeded onto 10-cm-diameter plastic culture dishes at a density of 3,000 cells/cm2. L6 cells were rendered quiescent in Dulbecco's modified Eagle's medium containing 2% serum for 10 days to promote fusion into myotubes. Myotube formation was determined as the percentage of nuclei present in multinucleated myotubes. In this experiment, 80-90% of myoblasts were fused into myotubes.

Construction of Dominant Negative p85 Mutant (Delta p85)

A full-length p85alpha cDNA was isolated by screening a cDNA library from MIN6, a mouse insulinoma cell line (15), on the basis of the reported sequence (16). Comparison with the reported sequence revealed codon CCC (Pro46) to have been replaced with CAG (Gln46) in our p85alpha cDNA. To construct a Delta p85 mutant that cannot bind to the p110 catalytic subunit, we synthesized two oligonucleotide primers as follows: sense, TTCGACTCTATACAGAACAC corresponding to nucleotides 994-1013 of p85alpha ; antisense, GCCGGCATAGTCTGGAACATCGTATGGATATTGGATTTCCTGGGAAGTACG containing an NaeI site (GCCGGC), the antisense sequence of the influenza virus hemagglutinin epitope tag (YPYDVPDYA) and nucleotides 1970-1990 of p85alpha . Polymerase chain reaction amplification was carried out using the two primers and p85alpha cDNA as a template. A fragment containing nucleotides 994-1990 of p85alpha , as well as nucleotide sequences for the hemagglutinin tag and NaeI site at its 3'-end, was obtained. The PstI-DraI fragment (nucleotides 1033-2092) of p85alpha was replaced with the PstI-NaeI fragment of the polymerase chain reaction product, such that amino acids 479-512 of p85alpha , i.e. those responsible for binding with p110 (17), were replaced with a hemagglutinin tag sequence.

Construction of Recombinant Adenovirus

The recombinant adenoviruses Adex1CALacZ (18) and Adex1CADelta p85, which encode the Escherichia coli lacZ (beta -galactosidase) gene and Delta p85, respectively, were constructed as described (19, 20). For protein production, L6 cells were infected with these adenoviruses at a multiplicity of infection of 10~30 and incubated for 48 h. lacZ gene expression was confirmed by histochemical staining with 5-bromo-4-chloro-3-indolyl beta -D-galactopyranoside (0.5 mg/ml). When adenovirus Adex1CALacZ was applied, lacZ expression gene was observed in more than 90% of L6 cells (data not shown). In addition, L6 cells infected with Adex1CALacZ exhibited no significant difference in the expression of p85 protein or in PI 3-kinase activity compared with noninfected cells (data not shown). Therefore, in this study, L6 cells infected with Adex1CALacZ were used as a control. The expression of Delta p85 protein was confirmed by immunoblotting the anti-p85 antibody immunoprecipitate with anti-p85 and anti-hemagglutinin antibodies.

Animals

Male Sprague Dawley rats (7 weeks, 244-272 g) were fasted for 12 h before the experiments and anesthetized with sodium pentobarbital. The portal vein was exposed, and 4 ml of normal saline with or without 10-5 M insulin was injected. After the indicated periods, the soleus muscle was removed and homogenized immediately with a Polytron aggregate generator (Kinematica) operated at maximum speed for 30 s in ice-cold solubilization buffer (10 ml/g tissue), composed of 50 mM Hepes at pH 7.5, 137 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM sodium pyrophosphate, 10 mM sodium fluoride, 2 mM EDTA, 2 mM sodium orthovanadate (Na3VO4), 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, and 10 mg/ml aprotinin. The homogenates were centrifuged at 15,000 × g at 4 °C for 30 min to remove insoluble material, and the supernatant was immunoprecipitated (as described below).

Immunoprecipitation and Immunoblotting

Confluent monolayers of L6 cells were incubated for 3 h in serum-free Dulbecco's modified Eagle's medium prior to insulin stimulation. In some experiments, the cells were first incubated with 2 mM Na3VO4 for 15 min or 10 nM wortmannin (Sigma) for 10 min. The cells were stimulated with the indicated concentrations of insulin for the indicated periods at 37 °C. In some experiments, the cells were stimulated with 10-7 M insulin-like growth factor I (IGF-I). In other experiments, the cells were stimulated with 50 ng/ml platelet-derived growth factor (PDGF) (Upstate Biotechnology Inc.) for 30 min before insulin stimulation. After stimulation, the cells were lysed at 4 °C with ice-cold phosphate-buffered saline (2 ml/10-cm dish of L6 cells) containing 10% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 100 µM Na3VO4. Insoluble material was removed by centrifugation at 15,000 × g for 10 min at 4 °C. One-ml quantities of the cell lysates (~1 mg of total protein) were incubated with anti-IRS-1, anti-IRS-2, or alpha PY antibody (10 µg each) for 1 h and precipitated by incubation with 20 µl of protein A-Sepharose (Pharmacia Biotech Inc.) for 1 h. The immunocomplexes were washed five times with phosphate-buffered saline containing 10% Triton X-100 and boiled in 30 µl of Laemmli sample buffer containing 10 mM dithiothreitol. The proteins were resolved on 7.5% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Schleicher & Schuell). After blocking with TBS-T (10 mM Tris, pH 7.6, 150 mM NaCl, 0.05% Tween 20) containing 3% bovine serum albumin for 1 h, the membranes were incubated with the appropriate antibodies. The proteins were visualized by blotting with 125I-protein A or by enhanced chemiluminescence (ECL) using horseradish peroxidase-labeled anti-rabbit or mouse IgG (Amersham Corp.). In some experiments using 125I-protein A, the signals were analyzed stoichiometrically using Bioimage analyzer BAS2000 (Fuji).

PI 3-Kinase Activity

The L6 cells (6.0 × 105 cells) in 10-cm dishes were solubilized in 6 ml of ice-cold lysis buffer containing 20 mM Tris, pH 7.5, 137 mM NaCl, 1 mM CaCl2, 1% Nonidet P-40, 10 mg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 100 µM Na3VO4. Lysates were extracted by centrifugation at 15,000 × g for 10 min, and 1-ml quantities of the lysates (~0.3 mg of total protein) were incubated with anti-IRS-1 or anti-IRS-2 antibody (10 µg each) for 1 h. Immunocomplexes were precipitated with 20 µl of protein A-Sepharose for 1 h. PI 3-kinase was assayed in the immunoprecipitates as described (4).


RESULTS

L6 Cells Express IRS-1 and IRS-2, Which Are Tyrosine-phosphorylated in Response to Insulin Stimulation

We initially examined the expression and insulin-induced tyrosine phosphorylation of IRS-1 and IRS-2 in L6 cells. Cell lysates were immunoprecipitated with anti-IRS-1 or anti-IRS-2 antibody and immunoblotted with anti-IRS-1 or anti-IRS-2 antibody. As shown in Fig. 1A, anti-IRS-1 and anti-IRS-2 antibodies specifically recognize IRS-1 and IRS-2, respectively (lanes 1-4). After the incubation with 10-7 M insulin for 3 min, cell lysates were immunoprecipitated with anti-IRS-1 or anti-IRS-2 antibody. Immunoblotting with alpha PY antibody revealed tyrosine phosphorylation of IRS-1 and IRS-2 (lanes 5 and 6). The migration of IRS-2 being slightly behind of that of IRS-1 on SDS-polyacrylamide gel electrophoresis is consistent with the apparent molecular mass of IRS-2 being about 10 kDa higher than that of IRS-1, as reported (12). These results demonstrated that L6 cells express IRS-1 and IRS-2, both of which are tyrosine-phosphorylated in response to insulin stimulation. In this experiment, the IRS-1 immunoprecipitate derived from 5 µl of cell lysate and the IRS-2 immunoprecipitate derived from 15 µl of cell lysate yielded equivalent PY signals in the alpha PY immunoblotting (data not shown). As the immunoprecipitation efficiencies of anti-IRS-1 and anti-IRS-2 antibodies were comparable, the relative amount of the PY form of IRS-2 was roughly 30% of that of IRS-1.


Fig. 1. IRS-1 and IRS-2 expression, insulin-induced tyrosine phosphorylation, and PI 3-kinase activation in L6 cells. Panel A, IRS-1 and IRS-2 expression, insulin-induced tyrosine phosphorylation. L6 cells were incubated with 10-7 M insulin for 3 min. Cell lysates were immunoprecipitated with anti-IRS-1 (alpha IRS-1) or anti-IRS-2 (alpha IRS-2) antibody. The immunoprecipitates were resolved by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and immunoblotted with the indicated antibodies. Panel B, insulin concentration dependence of insulin-induced IRS-1 and IRS-2 tyrosine phosphorylation. L6 cells were incubated with the indicated concentrations of insulin for 3 min. Cell lysates were immunoprecipitated with alpha IRS-1 or alpha IRS-2 antibody and immunoblotted with alpha PY antibody (upper panel) and with alpha IRS-1 or alpha IRS-2 antibody (lower panel). Panel C, insulin concentration dependence of IRS-1- and IRS-2-associated PI 3-kinase activity. PI 3-kinase activities in the immunoprecipitates derived with alpha IRS-1 or alpha IRS-2 antibody were assayed as described (4). Fold increases in basal PI 3-kinase activity are presented. Bars indicate S.E. from three independent experiments.
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L6 cells were then stimulated with the indicated concentrations of insulin for 3 min (Fig. 1B). The tyrosine phosphorylations of IRS-1 and IRS-2 were increased by insulin in a concentration-dependent manner without altering the amounts of either protein. IRS-1- and IRS-2-associated PI 3-kinase activities were also increased in a similar insulin concentration-dependent manner (Fig. 1C).

IRS-2 Is Dephosphorylated More Rapidly than IRS-1 in L6 Cells and Rat Skeletal Muscle

Fig. 2A shows the time courses of IRS-1 and IRS-2 tyrosine phosphorylation. IRS-1 and IRS-2 were both tyrosine-phosphorylated within 1 min after the addition of insulin/IGF-I, with peaks at 3 min. However, IRS-2 was rapidly dephosphorylated, and phosphorylation was essentially undetectable beyond 10 min, whereas IRS-1 phosphorylation persisted for more than 60 min. The amounts of IRS-1 and IRS-2 proteins did not change until at least 60 min. To quantitate the sensitivity of alpha PY blotting, we did a PY blot of 0.5 volume (1/2 ×, 1/4 ×, 1/8 ×) of the samples of IRS-1 and IRS-2 immnunoprecipitated at 3 min after the addition of insulin, which were those most heavily tyrosine-phosphorylated. At 1/2 ×, 1/4 ×, and 1/8 ×, PY signals of IRS-1 and IRS-2 immunoprecipitates were detectable, but no signal was seen at 1/16 × (data not shown). These results indicate that the amount of IRS-2 phosphorylation decreased to less than one-eighth of the maximal level beyond 10 min.


Fig. 2. Time courses of insulin/IGF-I-induced IRS-1 and IRS-2 tyrosine phosphorylation and associated PI 3-kinase activity. Panel A, time courses of insulin/IGF-I-induced IRS-1 and IRS-2 tyrosine phosphorylation in L6 cells. L6 cells were stimulated with 10-7 M insulin or IGF-I for the indicated periods. Cell lysates were immunoprecipitated with alpha IRS-1 or alpha IRS-2 antibody and subsequently immunoblotted with alpha PY antibody (upper panel) and with alpha IRS-1 or alpha IRS-2 antibody (lower panel). Panel B, time courses of the insulin-induced tyrosine phosphorylation of IRS-1 and IRS-2 in rat skeletal muscle. Rats were fasted for 12 h before experiments, and 4 ml of normal saline with or without 10-5 M insulin was injected through the portal vein. After the indicated periods, the soleus muscle was removed and homogenized as described under "Experimental Procedures." Cell lysates were immunoprecipitated with alpha IRS-1 or alpha IRS-2 antibody and immunoblotted with alpha PY antibody (upper panel) and with alpha IRS-1 or alpha IRS-2 antibody (lower panel). The data are representative of three independent experiments. Panel C, time courses of IRS-1- and IRS-2-associated PI 3-kinase activity in L6 cells. After stimulation with 10-7 M insulin or IGF-I, PI 3-kinase activities in the immunoprecipitates derived with alpha IRS-1 or alpha IRS-2 antibody were assayed as described. The relative PI 3-kinase activity, as a percent of the maximal increase from the basal level, is indicated. Bars indicate S.E. from three experiments.
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Fig. 2B illustrates the time courses of IRS-1 and IRS-2 tyrosine phosphorylation in rat skeletal muscle. Although IRS-1 and IRS-2 were both tyrosine-phosphorylated within 3 min after the injection of insulin into the portal vein, tyrosine phosphorylation of IRS-2 was attenuated much more rapidly than that of IRS-1. Thus, IRS-2 is more transiently phosphorylated by insulin/IGF-1 stimulation than IRS-1 in L6 cells and rat skeletal muscle.

Next, we compared insulin-induced PI 3-kinase association with IRS-1 and IRS-2. Relative PI 3-kinase activities were expressed as the percentage of the maximal increase from the basal level. The 100% values of PI 3-kinase associated with IRS-1 and IRS-2 (derived from 1.0 × 105 cells) were 2199 and 302 cpm, respectively. Thus, the relative amount of PI 3-kinase associated with IRS-2 was 13.6% that of IRS-1. Fig. 2C (left panel) shows that whereas IRS-1-associated PI 3-kinase activity was attenuated to 48% of the maximal increase from the basal level at 60 min after the addition of insulin, that of IRS-2 decreased to 12% of the maximum at 60 min. Similar results were obtained with IGF-I stimulation (Fig. 2C, right panel). This rapid decrease in IRS-2-associated PI 3-kinase activity is considered to be reflected by the rapid dephosphorylation of IRS-2 shown in Fig. 2A. Thus, it is likely that IRS-2 mediates signals from the insulin/IGF-I receptor to PI 3-kinase more transiently than does IRS-1 in skeletal muscle cells.

Sodium Orthovanadate Inhibits the Faster Dephosphorylation of IRS-2

To examine whether the rapid dephosphorylation of IRS-2 occurs via protein tyrosine phosphatase, we incubated L6 cells with Na3VO4, an inhibitor of tyrosine phosphatase, prior to insulin stimulation. After incubation with 2 mM Na3VO4 for 15 min, insulin-induced phosphorylation of IRS-2 continued for at least 60 min after the addition of insulin (Fig. 3). This result suggests the presence of tyrosine phosphatase(s), which is involved in the rapid dephosphorylation of IRS-2.


Fig. 3. Effects of Na3VO4 and wortmannin on the phosphorylation of IRS-1 and IRS-2. L6 cells were incubated with 2 mM Na3VO4 for 15 min or 10 nM wortmannin (WT) for 10 min, then stimulated with 10-7 M insulin for the indicated periods. Cell lysates were immunoprecipitated with alpha IRS-1 or alpha IRS-2 antibody and immunoblotted with alpha PY antibody.
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Wortmannin and Overexpression of Delta p85 Mutant Inhibit the Faster Dephosphorylation of IRS-2

Furthermore, we attempted to identify the molecular event that constitutes the signal leading to the dephosphorylation of IRS-2. We examined the possible involvement of PI 3-kinase in IRS-2 dephosphorylation utilizing two independent procedures. First, we incubated L6 cells with 10 nM wortmannin, a potent inhibitor of PI 3-kinase, for 10 min prior to insulin stimulation. The wortmannin treatment markedly inhibited the rapid dephosphorylation of IRS-2, whereas no significant effect was observed for IRS-1 (Fig. 3).

Second, the Delta p85 mutant of PI 3-kinase, which cannot bind to the p110 catalytic subunit and contains a hemagglutinin epitope tag, was overexpressed using an adenovirus expression system. The expression of Delta p85 protein was confirmed by immunoprecipitation with anti-p85 antibody and immunoblotting with anti-hemagglutinin antibody (Fig. 4A). Fig. 4A (left panel) shows the anti-p85 immunoblotting of anti-p85 immunoprecipitates from the control cells (overexpressing lacZ, lane 1) and the cells overexpressing Delta p85 (lane 2). Delta p85 overexpression reached a level approximately 8-fold that of the wild type p85 protein, which was assessed by blotting with 125I-protein A and quantitated with BAS2000. The expression of Delta p85 protein was also confirmed by immunoprecipitation with anti-p85 antibody followed by immunoblotting with anti-hemagglutinin antibody (Fig. 4A, right panel). Delta p85 protein was detected by anti-hemagglutinin blotting in L6 cells overexpressing Delta p85 (lane 4), but not in control cells (lane 3). The PI 3-kinase activities of alpha PY antibody immunoprecipitates from control cells and from those expressing Delta p85 were then compared. The PI 3-kinase activity was suppressed by Delta p85 overexpression to 60 and 15% of that of control cells in the absence and presence of insulin, respectively (Fig. 4B).


Fig. 4. Overexpression of Delta p85 and the effect of Delta p85 on phosphorylation of IRS-1 and IRS-2. Panel A, the expression of Delta p85 protein was confirmed by immunoprecipitation with anti-p85 (alpha p85) antibody followed by immunoblotting with anti-p85 and anti-hemagglutinin (alpha HA) antibody. Lanes 1 and 3, lacZ-overexpressing L6 cells as a control; lanes 2 and 4, Delta p85-overexpressing cells. Panel B, PI 3-kinase activities in immunoprecipitates derived with alpha PY antibody from control cells and cells overexpressing Delta p85 with or without 10-7 M insulin stimulation for 3 min. Panel C, L6 cells overexpressing Delta p85 were stimulated with 10-7 M insulin for the indicated periods. Cell lysates were immunoprecipitated with alpha IRS-1 or alpha IRS-2 antibody and immunoblotted with alpha PY antibody.
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We observed that overexpression of Delta p85 affected neither the binding of Grb2 to IRS-1 and IRS-2 nor mitogen-activated protein kinase activation in response to insulin stimulation in L6 cells (data not shown). These observations were consistent with those made in 3T3-L1 adipocytes (13).

The time courses of tyrosine phosphorylation of IRS-1 and IRS-2 in L6 cells overexpressing Delta p85 are shown in Fig. 4C. With Delta p85 overexpression, IRS-2 dephosphorylation was markedly inhibited, whereas no change was observed in IRS-1. These results suggest that PI 3-kinase activation is necessary for the rapid dephosphorylation of IRS-2.

PDGF Stimulation Prior to Insulin Stimulation Prevents PI 3-Kinase from Binding IRSs and Delays Dephosphorylation of IRS-2

In addition, we performed an experiment to determine whether it is PI 3-kinase activation alone or the binding of active PI 3-kinase to IRS-2 which induces the rapid dephosphorylation of IRS-2. L6 cells were stimulated with 50 ng/ml PDGF for 30 min and then with 10-7 M insulin for the indicated periods. Fig. 5A shows that the PI 3-kinase activity associated with phosphotyrosine was increased 10-24-fold by PDGF stimulation prior to insulin stimulation compared with insulin stimulation alone, whereas insulin-induced IRS-associated PI 3-kinase activations were remarkably decreased. This is considered to be attributable to most of the PI 3-kinase having associated with autophosphorylated PDGF receptor due to the preceding treatment with PDGF, resulting in a decrease in the PI 3-kinase associated with IRSs. Under these conditions, IRS-2 dephosphorylation was significantly inhibited (Fig. 5B, lower right). These observations support the notion that, rather than total PI 3-kinase activity, it is IRS-associated PI 3-kinase activity that is important for the rapid dephosphorylation of IRS-2.


Fig. 5. Effect of PDGF stimulation on associated PI 3-kinase activity and tyrosine phosphorylation of IRS-1 and IRS-2. L6 cells were stimulated with 50 ng/ml PDGF for 30 min, then stimulated with 10-7 M insulin for the indicated periods. Panel A, PI 3-kinase activities in the immunoprecipitates derived with alpha PY, alpha IRS-1, and alpha IRS-2 antibodies were assayed as described. The relative PI 3-kinase activity as a percent of the maximal increase from the basal level or fold increase is indicated. Bars indicate S.E. from three experiments. Panel B, cell lysates were immunoprecipitated with alpha IRS-1 or alpha IRS-2 antibody and immunoblotted with alpha PY antibody.
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DISCUSSION

The binding of insulin to its receptor activates receptor tyrosine kinase. The activated insulin receptor phosphorylates several cytosolic substrates, which contribute to the divergent biological effects of insulin. These include a major insulin receptor substrate, IRS-1, and the recently cloned IRS-2. These two proteins had been considered to be structurally and functionally similar (12, 22). To date, little information has been available on the different characteristics of IRS-1 and IRS-2. It was reported that IRS-2 binds to the insulin receptor through its newly identified domain, which is absent in IRS-1 (23, 24). A recent report showed that after treatment with tumor necrosis factor-alpha , IRS-1 inhibits insulin receptor tyrosine kinase activity, whereas IRS-2 does not (25). In this study, we demonstrated that IRS-2 is dephosphorylated more rapidly and activates PI 3-kinase more transiently than IRS-1 in skeletal muscle cells. This is the first report detailing the different features of IRS-1 and IRS-2 in tyrosine phosphorylation and signal transduction to downstream molecules.

Next, we attempted to clarify the molecular mechanisms leading to rapid dephosphorylation of IRS-2. When the cells were incubated with Na3VO4 to block tyrosine phosphatase, IRS-2 tyrosine phosphorylation was prolonged. The rapid dephosphorylation of IRS-2 occurs via protein tyrosine phosphatase, as expected. However, when the cells were incubated with 10 nM wortmannin to inhibit PI 3-kinase, the dephosphorylation was also markedly inhibited. In addition, when the Delta p85 mutant was overexpressed, IRS-2 dephosphorylation was markedly inhibited, suggesting that PI 3-kinase activation is necessary for rapid dephosphorylation of IRS-2. It is unlikely that Delta p85 binds to phosphotyrosine residues of IRS-1 and IRS-2 in a nonspecific manner because the overexpression of Delta p85 affected neither the binding of Grb2 to IRS-1 and IRS-2 nor mitogen-activated protein kinase activation. When IRS-1- and IRS-2-associated PI 3-kinase was reduced, despite PI 3-kinase activity with phosphotyrosine being increased, by sequential addition of PDGF and insulin, IRS-2 dephosphorylation was reduced. We have come to the conclusion that IRS-associated PI 3-kinase activity rather than total PI 3-kinase activity is important for rapid dephosphorylation of IRS-2.

Although the mechanism by which IRS-associated PI 3-kinase triggers the rapid dephosphorylation of IRS-2 remains unknown, the evidence supports two possibilities. First, IRS-1-associated PI 3-kinase leads to phosphorylation of serine residues of IRS-1. PI 3-kinase possesses not only lipid kinase, but also serine kinase activity (26). In vitro kinase assays of anti-p85 immunoprecipitates have shown that IRS-1 is serine- phosphorylated by IRS-1-associated PI 3-kinase (27, 28). We speculate that IRS-2-associated PI 3-kinase phosphorylates serine residues of IRS-2 as well. Meanwhile, serine/threonine phosphorylation of IRSs affects their tyrosine phosphorylation (29, 30). When the serine/threonine phosphorylation of IRS-1 is augmented by okadaic acid (29) or calyculin A (30), which are serine/threonine phosphatase inhibitors, insulin-induced tyrosine phosphorylation of IRS-1 is impaired. It is possible that serine/threonine phosphorylation of IRSs modulates not only their tyrosine phosphorylation but also dephosphorylation by tyrosine phosphatase. Different serine/threonine phosphorylation sites in IRS-1 and IRS-2 might be responsible for the different degree of accessibility to tyrosine phosphatase. We speculate that IRS-2-associated PI 3-kinase, which phosphorylates serine residues of IRS-2, modulates rapid tyrosine dephosphorylation of IRS-2.

Second, a role for PI 3-kinase in protein sorting has been suggested by the finding that a yeast PI 3-kinase homolog, VSP34, is involved in sorting proteins to vacuoles (31). PI 3-kinase binding to the PDGF receptor is reportedly essential for the postendocytic sorting of this receptor (32). We speculated that PI 3-kinase binding to IRS-1 and IRS-2 is necessary for their intracellular trafficking and that they are sorted and dephosphorylated differently.

In conclusion, IRS-2 is dephosphorylated more rapidly than IRS-1, and IRS-2 dephosphorylation is regulated to some extent by IRS-associated PI 3-kinase activity. Based on the results of this study, we have devised a model for the roles of IRS-1 and IRS-2. We propose that IRS-1 transmits continuous signals from the insulin receptor, whereas IRS-2 mediates transient signals regulated by its associated PI 3-kinase activity. The continuous IRS-1 pathway and the duration-regulated IRS-2 pathway might operate in combination to fine tune the acute and chronic actions of insulin.


FOOTNOTES

*   This work was supported by a grant-in-aid for scientific research from the Japanese Ministry of Education and by a grant from TMFC.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
par    To whom correspondence should be addressed. Tel.: 81-3-3815-5411 (ext. 3133); Fax: 81-3-5803-1874; E-mail: asano-tky{at}umin.u-tokyo.ac.jp.
1   The abbreviations used are; IRS, insulin receptor substrate; PI, phosphatidylinositol; alpha PY, anti-phosphotyrosine; Delta p85, dominant negative p85 mutant; Na3VO4, sodium orthovanadate; IGF-I, insulin-like growth factor I; PDGF, platelet-derived growth factor.

ACKNOWLEDGEMENT

We are grateful to Dr. Izumu Saito (Institute of Medical Science, University of Tokyo) for the generous gift of the recombinant Adex1CALacZ and the cassette cosmid for constructing recombinant adenovirus.


REFERENCES

  1. Sun, X. J., Rothenberg, P., Kahn, C. R., Backer, J. M., Araki, E., Wilden, P. A., Cahill, D. A., Goldstein, B. J., and White, M. F. (1991) Nature 352, 73-77 [CrossRef][Medline] [Order article via Infotrieve]
  2. Sun, X. J., Crimmins, D. L., Myers, M. G., Jr., Miralpeix, M., and White, M. F. (1993) Mol. Cell. Biol. 13, 7418-7428 [Abstract]
  3. Backer, J. M., Myers, M. G., Jr., Shoelson, S. E., Chin, D. J., Sun, X. J., Miralpeix, M., Hu, P., Margolis, B., Skolnik, E. Y., Schlessinger, J., and White, M. F. (1992) EMBO J. 11, 3469-3479 [Abstract]
  4. Pons, S., Asano, T., Glasheen, E., Miralpeix, M., Zhang, Y., Fisher, T. L., Myers, M. G., Jr., Sun, X. J., and White, M. F. (1995) Mol. Cell. Biol. 15, 4453-4465 [Abstract]
  5. Inukai, K., Anai, M., Van Breda, E., Hosaka, T., Katagiri, H., Funaki, M., Fukushima, Y., Ogihara, T., Yazaki, Y., Kikuchi, M., Oka, Y., and Asano, T. (1996) J. Biol. Chem. 271, 5317-5320 [Abstract/Free Full Text]
  6. Lowenstein, E. J., Daly, R. J., Batzer, A. G., Li, W., Margolis, B., Lammers, R., Ullrich, A., Skolnik, E. Y., Bar-Sagi, D., and Schlessinger, J. (1992) Cell 70, 431-442 [Medline] [Order article via Infotrieve]
  7. Feener, E. P., Plutzky, J., and Neel, B. G. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 11239-11243 [Abstract]
  8. Lee, C.-H., Li, W., Nishimura, R., Zhou, M., Batzer, A., Myers, M. G., Jr., White, M. F., Schlessinger, J., and Skolnik, E. Y. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11713-11717 [Abstract]
  9. Skolnik, E. Y., Lee, C.-H., Batzer, A., Vicentini, L. M., Zhou, M., Daly, R., Myers, M. J., Jr, Backer, J. M., Ullrich, A., White, M. F., and Schlessinger, J. (1993) EMBO J. 12, 1929-1936 [Abstract]
  10. Tamemoto, H., Kadowaki, T., Tobe, K., Yagi, T., Sakura, H., Hayakawa, T., Terauchi, Y., Ueki, K., Kaburagi, Y., Satoh, S., Sekihara, H., Yoshioka, S., Horikoshi, H., Furuta, Y., Ikawa, Y., Kasuga, M., Yazaki, Y., and Aizawa, S. (1994) Nature 372, 182-186 [CrossRef][Medline] [Order article via Infotrieve]
  11. Araki, E., Lipes, M. A., Patti, M.-E., Brüning, J. C., Haag, B., III, Johnson, R. S., and Kahn, R. C. (1994) Nature 372, 186-189 [CrossRef][Medline] [Order article via Infotrieve]
  12. Sun, X. J., Wang, L.-M., Zhang, Y., Yenush, L., Myers, M. G., Jr., Glasheen, E., Lane, W. S., Pierce, J. H., and White, M. F. (1995) Nature 377, 173-177 [CrossRef][Medline] [Order article via Infotrieve]
  13. Katagiri, H., Asano, T., Inukai, K., Ogihara, T., Ishihara, H., Shibasaki, Y., Murata, T., Terasaki, J., Kikuchi, M., Yazaki, Y., and Oka, Y. (1997) Am. J. Physiol 272, E326-E331 [Abstract/Free Full Text]
  14. Oka, Y., Asano, T., Shibasaki, Y., Kasuga, M., Kanazawa, Y., and Takaku, F. (1988) J. Biol. Chem. 263, 13432-13439 [Abstract/Free Full Text]
  15. Katagiri, H., Terasaki, J., Murata, T., Ishihara, H., Ogihara, T., Inukai, K., Fukushima, Y., Anai, M., Kikuchi, M., Miyazaki, J., Yazaki, Y., and Oka, Y. (1995) J. Biol. Chem. 270, 4963-4966 [Abstract/Free Full Text]
  16. Escobedo, J. A., Navankasattusas, S., Kacanaugh, W. M., Milfay, D., Fried, V. A., and Williams, L. T. (1991) Cell 65, 75-82 [Medline] [Order article via Infotrieve]
  17. Dhand, R., Hara, K., Hiles, I., Bax, B., Gout, I., Panayotou, G., Fry, M. J., Yonezawa, K., Kasuga, M., and Waterfield, M. D. (1994) EMBO J. 13, 511-521 [Abstract]
  18. Kanegae, Y., Lee, G., Sato, Y., Tanaka, M., Nagai, M., Sakaki, T., Sugano, S., and Saito, I. (1995) Nucleic Acids Res. 23, 3816-3821 [Abstract]
  19. Miyake, S., Makimura, M., Kanegae, Y., Harada, S., Sato, Y., Takamori, K., Tokuda, C., and Saito, I. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1320-1324 [Abstract/Free Full Text]
  20. Katagiri, H., Asano, T., Ishihara, H., Inukai, K., Shibasaki, Y., Kikuchi, M., Yazaki, Y., and Oka, Y. (1996) J. Biol. Chem. 271, 16987-16990 [Abstract/Free Full Text]
  21. Deleted in proofDeleted in proof
  22. Patti, M.-E., Sun, X.-J., Brüning, J. C., Araki, E., Lipes, M. A., White, M. F., and Kahn, C. R. (1995) J. Biol. Chem. 270, 24670-24673 [Abstract/Free Full Text]
  23. Sawka-Verhelle, D., Tartare-Deckert, S., White, M. F., and Van Obberghen, E. (1996) J. Biol. Chem. 271, 5980-5983 [Abstract/Free Full Text]
  24. He, W., Craparo, A., Zhu, Y., O'Neill, T. J., Wang, L.-M., Pierce, J. H., and Gustafson, T. A. (1996) J. Biol. Chem. 271, 11641-11645 [Abstract/Free Full Text]
  25. Peraldi, P., Hotamisligil, G. S., Buurman, W. A., White, M. F., and Spiegelman, B. M. (1996) J. Biol. Chem. 271, 13018-13022 [Abstract/Free Full Text]
  26. Dhand, R., Hiles, I., Panayotou, G., Roche, S., Fry, M. J., Gout, I., Totty, N. F., Truong, O., Vicendo, P., Yonezawa, K., Kasuga, M., Courtneidge, S. A., and Waterfield, M. D. (1994) EMBO J. 13, 522-533 [Abstract]
  27. Lam, K., Carpenter, C. L., Ruderman, N. B., Friel, J. C., and Kelly, K. L. (1994) J. Biol. Chem. 269, 20648-20652 [Abstract/Free Full Text]
  28. Tanti, J.-F., Gremeaux, T., Van Obberghen, E., and Le Marchand-Brustel, Y. (1994) Biochem. J. 304, 17-21 [Medline] [Order article via Infotrieve]
  29. Tanti, J.-F., Gremeaux, T., Van Obberghen, E., and Le Marchand-Brustel, Y. (1994) J. Biol. Chem. 269, 6051-6057 [Abstract/Free Full Text]
  30. Kanety, H., Feinstein, R., Papa, M. Z., Hemi, R., and Karasik, A. (1995) J. Biol. Chem. 270, 23780-23784 [Abstract/Free Full Text]
  31. Schu, P. V., Takegawa, K., Fry, M. J., Stack, J. H., Waterfield, M. D., and Emr, S. D. (1993) Science 260, 88-91 [Medline] [Order article via Infotrieve]
  32. Joly, M., Kazlauskas, A., Fay, F. S., and Corvela, S. (1994) Science 263, 684-687 [Medline] [Order article via Infotrieve]

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