Expression of dominant negative mutant SHPTP2 attenuates phosphatidylinositol 3'-kinase activity via modulation of phosphorylation of insulin receptor substrate-1.

To clarify the role of protein-tyrosine phosphatase (PTPase) containing Src homology 2 regions (SHPTP2) in insulin signaling, either wild-type or mutant SHPTP2 (ΔPTP; lacking full PTPase domain) was expressed in Rat 1 fibroblasts overexpressing human insulin receptors. In response to insulin, phosphorylation of insulin receptor substrate 1 (IRS-1), IRS-1-associated PTPase activities and phosphatidylinositol (PI) 3′-kinase activities were slightly enhanced in wild-type cells when compared with those in the parent cells transfected with hygromycin-resistant gene alone. In contrast, introduction of ΔPTP inhibited insulin-induced association of IRS-1 with endogenous SHPTP2 and impaired both insulin-stimulated phosphorylation of IRS-1 and activation of PI 3′-kinase. Furthermore, decreased content of p85 subunit of PI 3′-kinase was also found in mutant cells. Consistently, the insulin-stimulated mitogen-activated protein kinase activities and DNA synthesis were also enhanced in wild-type cells, but impaired in mutant cells. Thus, the interaction of SHPTP2 with IRS-1 may be associated with modulation of phosphorylation levels of IRS-1, resulting in the changes of PI 3′-kinase and mitogen-activated protein kinase activity. Furthermore, an impaired insulin signaling in mutant cells may be partly reflected in a decreased content of p85 protein of PI 3′-kinase.

The phosphorylation state of tyrosine residue is regulated by both protein-tyrosine kinase (PTK) 1 and protein-tyrosine phos-phatase (PTPase) and is critical for cell growth, differentiation and metabolism (1,2). Insulin binding to its receptor results in the sequential autophosphorylation of tyrosine residues in the cytoplasmic region of the receptor, stimulation of receptor PTK, then the phosphorylation of insulin receptor substrate-1 (IRS-1). IRS-1 contains several potential phosphorylation sites at tyrosine residues (3), and tyrosine-phosphorylated IRS-1 binds to phosphatidylinositol (PI) 3Ј-kinase and Grb2 through the association of their Src homology 2 (SH2) regions (4). SH2 domains, at first identified in Src family PTK, are regions of about 100 amino acids, and they can directly interact with proteins containing phosphotyrosine. Thus, SH2 domains are thought to play important roles in signal transduction via tyrosine phosphorylation (5,6).
The novel non-transmembrane PTPase, SHPTP2 (7,8) (also known as Syp (9), PTP1D (10), and PTP2C (11)), which contains a single phosphatase domain and two adjacent copies of SH2 regions at its amino terminus, has been cloned in mammalian cells. SH-PTP2 is widely expressed and is probably the mammalian homologue of Drosophila Corkscrew (12). The Corkscrew gene product potentiates the Drosophila homologue of mammalian c-Raf to positively transmit signals downstream of the Torso receptor PTK. These data suggest an important role for SHPTP2 downstream of the receptor PTK in mammalian cells. In fact, activated platelet-derived growth factor and epidermal growth factor receptors bind directly to the SH2 domains of SHPTP2, leading to the phosphorylation of the PTPase on tyrosine residues, which further stimulates its catalytic activity and positively regulates mitogenic signaling of these growth factor receptors via SHPTP2⅐Grb2⅐Sos complex formation (9,10,(13)(14)(15)(16).
Although it has been reported that SHPTP2 also binds to tyrosine-phosphorylated IRS-1 in vitro and in vivo (13,(17)(18)(19), the precise role of SHPTP2 in insulin signal transduction remains unclear. Recently, Xiao et al., have reported that microinjection of either anti-Syp antibody or the GST-SH2 fusion protein of Syp into Rat 1 fibroblasts over expressing human insulin receptors (HIRc) blocks insulin-stimulated DNA synthesis (20). Furthermore, Milarski et al. (21) have reported that the introduction of a catalytically inactive mutant Syp (Cys-459 3 Ser) in NIH-3T3 cells leads to impairment of insulin-stimulated mitogen-activated protein (MAP) kinase activity and thymidine uptake. Milarski's results have been confirmed by Noguchi (22) and Yamauchi (23), who showed that the introduction of either catalytically inactive (Cys-459 3 Ser) or a SH2 mutant of SHPTP2 into Chinese hamster ovary cells overexpressing the human insulin receptor (CHO-IR) attenuates insulin-stimulated MAP kinase activity (22,23), but not insulin-stimulated PI 3Ј-kinase activity (22). Furthermore, overex-pressing wild-type SHPTP2 provides no additional increment in insulin-stimulated MAP kinase (22,23) and PI 3Ј-kinase activities in CHO-IR cells (22).
However, in our preliminary study, the introduction of mutant SHPTP2, which completely lacked the catalytic domain (⌬PTP), attenuated insulin-stimulated PI 3Ј-kinase activity in HIRc cells. Furthermore, overexpression of wild-type SHPTP2 enhanced MAP kinase activity. The contrast of this stimulation to the inhibition observed in CHO-IR cells suggests that the influence of introduction of wild-type or mutant SHPTP2 differs between CHO cells and Rat 1 fibroblasts. Therefore, we examined insulin signaling in cells overexpressing either wildtype (WT) or ⌬PTP (MT) to clarify the role of SHPTP2 in insulin signal transduction. We found that insulin-stimulated PI 3Ј-kinase activities were enhanced in WT cells, but attenuated in MT cells, resulting in modulation of DNA synthesis via the regulation of IRS-1 phosphorylation. This is the first direct evidence that SHPTP2 positively regulates both the insulinstimulation of MAP kinase cascade and PI 3Ј-kinase pathway.  (24). A polyclonal anti-SHPTP2 antibody (␣Syp) and anti-IRS-1 antibody (␣IRS-1) for Western blotting were from Upstate Biotechnology Inc. (Lake Placid, NY). Antiserum against either GST-IRS-1 or GST-SHPTP2 fusion protein for the immunoprecipitation study was raised in a rabbit against corresponding GST fusion proteins (17,25). Protein G-Sepharose was purchased from Pharmacia PL Biochemical (Uppsala, Sweden). Aprotinin, phenylmethylsulfonyl fluoride (PMSF), para-nitrophenylphosphate (p-NPP), protein kinase inhibitor, and phosphatidylinositol were purchased from Sigma Bromodeoxyuridine (BrdUrd), a monoclonal anti-BrdUrd antibody, and enhanced chemiluminescence reagents were from Amersham (Buckinghamshire, United Kingdom). Lipofectin reagent was from Life Technologies, Inc. Hygromycin B was from Wako Chemical Inc. (Osaka, Japan). All other reagents were of analytical grade from Nakarai Chemicals (Kyoto, Japan).

Materials-Purified
Preparation of Expression Constructs-The eukaryotic expression vector, pCAGGS was a gift from Dr. J. Miyazaki (Tokyo University). This vector consists of a strong promoter based upon that of ␤-actin (26). A cDNA from pBluescript SHPTP3 (8) was digested with EcoRI and ligated into the EcoRI site in the expression vector pCAGGS (pCAGGS-SHPTP2). For the mutant SH-PTP2 vector, PCR primers were designed to amplify a pair of SH2 domains of SHPTP2 (amino acids 1-216), attached to an EcoRI cleavage site. The full-length cDNA served as the template as described (25). The PCR product was subcloned, sequenced, and finally ligated into the EcoRI site in the expression vector, pCAGGS-⌬PTP.
Generation of Stable Cell Lines-Rat 1 fibroblasts those expressed human insulin receptors (HIRc) (27) and mutant insulin receptors (HIRY/F2) (28), provided by Dr. J. M. Olefsky (University of California, San Diego), were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Either wild-type or mutant expression constructs and pHyg, which contains the hygromycin B phosphotransferase gene, were co-transfected into HIRc or HIRY/F2 cells using Lipofectin according to the manufacturer's recommendations. Stable cell lines were selected in 400 g/ml hygromycin B. Clonal cell lines were isolated by limiting dilution, then screened by immunoblotting with ␣Syp. Parent cells transfected with pHyg vector alone were used for control in our study (pHyg).
Insulin Binding Assay-Insulin binding to each cell line was determined as described (29). In brief, 125 I-insulin binding to cells (5 ϫ 10 3 cells) was measured at 8.3 nM insulin concentration in Eagle's medium containing 1% bovine serum albumin and 20 mM HEPES (pH 7.6) at 4°C for 16 h.
Measurement of PTPase Activity-PTPase activity was measured using p-NPP as the substrate as described by Sugimoto et al. (30). The immunoprecipitate with GST-SH2-SHPTP2 antiserum was incubated with 10 mM p-NPP at 30°C for 15 min in 50 l of 50 mM 3,3-dimethylglutarate (pH 5.6), containing 50 mM NaCl, 10 mM dithiothreitol, and 2 mM EDTA. The reactions were terminated with 950 l of 1 N NaOH. The amount of para-nitrophenol produced was determined by measuring the absorbance at 405 nm.
Association of GST-SHPTP2 Fusion Proteins with Phosphorylated Proteins in Response to Insulin-Insulin-stimulated association of GST-SHPTP2 fusion proteins with phosphorylated proteins were analyzed according to method of Staubs et al. (31). In brief, GST fusion proteins containing either full-length SHPTP2 or only SH2 domains of SHPTP2 (⌬PTP) were made as described (25,32). To prepare whole cell lysates, cells were starved for 24 h. Then, cells were stimulated with 100 nM insulin for 1 min at 37°C. Following insulin stimulation, cell lysates (500 g of protein) were incubated with either GST-SHPTP2 or GST-SH2 fusion protein coupled to glutathione-Sepharose beads in the presence of phosphatase inhibitors for 90 min at 4°C. After extensively washing, bound proteins to either GST-SHPTP2 or GST-SH2 fusion protein were resolved by SDS-PAGE, electrotransferred to membrane, and immunoblotted with ␣PY20.
Insulin-stimulated Tyrosine Phosphorylation and Association of IRS-1 with SH-PTP2-Cells were starved in serum-free Dulbecco's modified Eagle's medium for 24 h, then stimulated with insulin at 37°C for 5 min. Thereafter, the cells were lysed in 20 mM Tris-Cl (pH 7.5) containing 1 mM EDTA, 140 mM NaCl, 1% Nonidet P-40, 1 mM sodium orthovanadate, 1 mM PMSF, 50 mM NaF, 50 g/ml aprotinin at 4°C for 20 min. The cell lysates were centrifuged to remove insoluble materials at 15,000 ϫ g for 20 min. The supernatants were immunoprecipitated at 4°C with ␣IRS-1. The bound proteins were then resolved by SDSpolyacrylamide gel electrophoresis (SDS-PAGE), electrotransferred to Immobilon-P, and immunoblotted with specific antibodies (␣PY20, ␣IRS-1, and ␣Syp). The blots were then incubated with horseradish peroxidase-linked second antibody, followed by enhanced chemiluminescence detection according to the manufacturer's instructions.
Relationship between Degree of Insulin-stimulated Phosphorylation of IRS-1 and Its Association of p85 in pHyg and MT Cells-After insulin stimulation (1-100 nM), whole cell lysates (1 mg of protein) were immunoprecipitated with ␣IRS-1. Bound proteins were resolved by SDS-PAGE, and electrotransferred to membrane, and immunoblotted with either ␣PY20 or ␣p85 antibody. Then, we stoichiometrically analyzed the relationships between the degree of phosphorylation of IRS-1 and degree of its association of p85 in pHyg and MT cells using densitometric scanning.
Western Blot Analysis of p85 Subunit of PI 3Ј-Kinase in the Cells-The cells were homogenized and the cell lysate (40 g of protein) was then resolved by SDS-PAGE, electrotransferred to Immobilon-P, and immunoblotted using two different specific antibodies (polyclonal ␣p85 and monoclonal anti-p85 antibody).
MAP Kinase Activity-MAP kinase activity was assayed in vitro using bovine myelin basic protein as the substrate as described (34). In brief, confluent cells in six-well plates were starved for 24 h in serumfree Dulbecco's modified Eagle's medium, then stimulated with insulin at 37°C for 10 min. Thereafter, the cells were lysed in 250 l of 25 mM Tris-Cl (pH 7.4) containing 25 mM NaCl, 80 mM ␤-glycerophosphate, 1 mM sodium orthovanadate, 10 mM NaF, 10 mM sodium pyrophosphate, 1 mM EGTA, 1 mM PMSF, 10 g/ml leupeptin. After a brief sonication, the cell lysates were centrifuged and 10 l of the supernatants were assayed for kinase activity, in a final volume of 40 l containing 1 M protein kinase inhibitor, 50 M ATP, 2 Ci of [␥-32 P]ATP, and 20 g of myelin basic protein at 25°C for 15 min. Twenty-five l of the reaction was transferred onto P81 phosphocellulose paper, a 2.5-cm diameter (Whatman). The papers were washed with 180 mM phosphoric acid five times and then rinsed with 95% ethanol. Phosphorylation was quantified by scintillation counting.
BrdUrd Incorporation-The cells were grown on glass coverslips, starved for 48 h, and then stimulated with 10 nM insulin. Fifteen hours later, the cells were pulsed for 30 min with BrdUrd. The cells were fixed and stained using a monoclonal anti-BrdUrd antibody and peroxidase-linked second antibody according to the manufacturer's recommendations.
Statistical Analysis-Data are means Ϯ S.E. as indicated. The p values were determined by Scheffe's multiple comparison test, and p Ͻ 0.05 was considered statistically significant.

The Expression of Wild-type and ⌬P-SHPTP2 in HIRc
Cells-We transfected either full-length SHPTP2 cDNA or cDNA encoding only the SH2 domains (⌬PTP) of SHPTP2 into HIRc cells and established several cell lines overexpressing either wild-type (WT) or ⌬PTP (MT). Immunoblot with the ␣Syp antibody showed that WT11 expressed 5-fold increases in SHPTP2 protein when compared with pHyg cells as shown in Fig. 1A. In agreement with the results of Western blotting, total cellular PTPase activity immunoprecipitated with ␣GST-SH2-SHPTP2 antiserum also increased in WT11 cells as summarized in column 1 in Table I. In contrast, although the mutant clone (MT15) expressed 2-3-fold more endogenous rat SHPTP2 as shown in Fig. 1B, total PTPase activity immunoprecipitated with ␣GST-SH2-SHPTP2 antiserum was similar to that of pHyg cells. To evaluate the effects of overexpression of wild-type and ⌬PTP on insulin signaling, we first measured the level of insulin binding. Percent insulin binding to 5 ϫ 10 3 cells at a concentration of 8.3 nM was 43.6 Ϯ 1.3%, 45.8 Ϯ 1.3%, and 47.8 Ϯ 1.0% in pHyg, WT, and MT cells, respectively, suggesting that all clones had the same numbers of insulin receptors.
Association of GST-SHPTP2 Fusion Proteins with Phosphorylated Proteins in Response to Insulin-After insulin stimulation, whole cell lysates from pHyg cells were incubated with GST fusion proteins containing either full-length SHPTP2 or its SH2 domains alone (⌬PTP), then tyrosine-phosphorylated proteins bound to GST fusion proteins were analyzed by Western blotting using ␣PY20. As shown in Fig. 2, four tyrosinephosphorylated proteins were detected, and two major tyrosine-phosphorylated proteins bound to both GST proteins in response to insulin. A 185-kDa tyrosine-phosphorylated protein was thought to be IRS-1, and a 115-kDa tyrosine-phosphorylated protein might be a SHPTP2-binding protein as reported by Yamauchi et al. (35). A 95-kDa tyrosine-phosphorylated protein (a ␤ subunit of insulin receptor) more efficiently bound to GST-full-length SHPTP2 rather than to GST-SH2 in response to insulin, although its association with both GST proteins was weaker when compared with two other major tyrosine-phosphorylated proteins. Furthermore, an undefined protein (135 kDa) was also bound to both GST proteins. Moreover, we could not find any qualitative differences in profile of binding proteins between two GST fusion proteins.
Changes in Tyrosine Phosphorylation of IRS-1 in Response to Insulin-To determine whether the overexpression of either wild-type or ⌬PTP could affect the insulin-stimulated tyrosine phosphorylation of cellular proteins, we tested the phosphorylation levels of insulin receptors and IRS-1. As shown in Fig. 3A, the insulin-stimulated phosphorylation levels of insulin receptor and IRS-1 were increased in WT11 cells and decreased in MT15 cells when compared with those of pHyg cells. When the insulin-stimulated phosphorylation levels of IRS-1 were assessed using densitometric scanning, the stimulation in WT11 cells and MT15 cells was 110 and 67% of that in pHyg cells, respectively. When ␣IRS-1-immunoprecipitates were blotted with ␣PY20, we confirmed that phosphorylation levels of IRS-1 were increased in WT11 cells, and decreased in MT15 cells (Fig.  3B). However, the content of IRS-1 proteins was comparable among these cells (Fig. 3C).
Since SHPTP2 was associated with IRS-1 both in vitro and in vivo as reported previously (13,(17)(18)(19), we tested whether this association could be observed in HIRc cells. Cells were stimulated with insulin, and then the cell lysates were immunopre- was resolved by SDS-PAGE and transferred to an Immobilon membrane using the standard procedures. Immunoblotting proceeded using ␣Syp, and the blots were visualized using anti-rabbit antiserum and enhanced chemiluminescence. Several clones expressed wild-type SHPTP2 (68 kDa). Clone WT11 was used for the present study. B, screening for positive clones expressing mutant SHPTP2. ⌬PTP (24 kDa) were expressed in some clones, and MT15 was used for the present study.

TABLE I
Effects of ␣IRS-1 immunoabsorption on SHPTP2-specific PTPase activity after insulin stimulation in various cell lines Cells were incubated with or without 100 nM insulin, and then PTPase activity of SHPTP2 in the resultant supernatant fraction after removal of PTPase associated with IRS-1 using ␣GST-IRS-1 antiserum was measured as described under "Experimental Procedures." All data indicate the SH-PTP2 specific PTPase activities in the fraction immunoprecipitated with ␣GST-SH2-SHPTP2 and are expressed as means Ϯ S.E. of four different experiments. Difference of the residual SH-PTP2specific PTPase activity between insulin-untreated and insulin-treated cells was determined in each experiment. The data represent the incremental SH-PTP2-specific PTPase activity associated with IRS-1 after insulin stimulation. cipitated with ␣IRS-1 and immunoblotted with ␣Syp as shown in Fig. 4. In WT11 cells, a significant amount of SHPTP2 associated with IRS-1 was found in the basal state and was remarkably increased in response to insulin. In contrast, when the immunoprecipitate of ␣Syp was immunoblotted with ␣PY20, a 185-kDa tyrosine-phosphorylated IRS-1 protein was co-immunoprecipitated with SHPTP2 only after insulin stimulation, but not in the basal state (data not shown). Thus, this association of IRS-1 with SHPTP2 in the basal state was not consistent by different means. In contrast to WT11 cells, basal and insulin-stimulated association of IRS-1 with rat endogenous SHPTP2 was not detected in MT15 cells.
To assess how much SHPTP2 was associated with IRS-1 after insulin stimulation, we measured total cellular SHPTP2specific PTPase activity and studied the effects of ␣IRS-1 immunoabsorption on the PTPase activity in these cells. The total cellular PTPase activity of SHPTP2 was not stimulated by insulin (data not shown). In response to insulin, PTPase activity was decreased due to immunoabsorption by ␣IRS-1 antibody by 0.005 and 0.169 ⌬A 405 nm /15 min/10 7 cells in pHyg cells and WT11 cells, respectively, but was not significantly affected in MT15 cells as shown in Table I. The activity reductions are explained by a portion of SHPTP2 binding to IRS-1 in response to insulin. Even with more than 5-fold greater SHPTP2 activity in WT11 cells than in pHyg cells, a 6-fold higher proportion of this elevated PTPase activity was removed. These results strongly suggest that overexpression of native SHPTP2 acts to enhance insulin-facilitated IRS-1 binding of the SHPTP2 and that overexpression of the SH2 region blocks this enhancement.
PI 3Ј-Kinase Activity Associated with IRS-1 and p85 Subunit-In response to insulin, PI 3Ј-kinase is activated through IRS-1-p85 complex formation and this pathway is thought to be independent of the MAP kinase cascade. We therefore measured the PI 3Ј-kinase activities in these cells. As shown in Fig.  5 (A and B), the levels of PI 3Ј-kinase activities associated with IRS-1 in all cell lines increased in response to insulin. However, the magnitude of activation in WT11 cells was greater than that in pHyg cells (141% of pHyg cells). On the other hand, the stimulation of the PI 3Ј-kinase in MT cells was attenuated (60% of pHyg cells).
It is possible that ⌬PTP mutant, which consists only SH2 domains, nonspecifically inhibits p85 binding to IRS-1, resulting in the impaired PI 3Ј-kinase activation in MT cells. To rule out this possibility, we have performed the stoichiometric analysis to determine relationships between the degree of phosphorylation of IRS-1 and degree of its association of p85 in pHyg and MT cells. In response to insulin, IRS-1 was less phosphorylated in MT cells compared to that in pHyg cells as shown in Fig. 6A. However, when association of IRS-1 with p85 in MT cells was adjusted to the value of pHyg cells, degree of tyrosinephosphorylation of IRS-1 in MT cells was only 20% of pHyg cells, suggesting that IRS-1 more efficiently binds p85 in MT cells (Fig. 6B). These results clearly indicate that impairment of insulin-stimulated PI 3Ј-kinase activity in MT cells may be mainly due to decreased levels of IRS-1 phosphorylation, but not due to nonspecifically inhibition of p85 binding to IRS-1 by   FIG. 2. Association of GST-SHPTP2 fusion proteins with phosphorylated proteins in response to insulin. After being starved for 24 h, cells were stimulated with 100 nM insulin for 1 min at 37°C. Following insulin stimulation, whole cell lysates (500 g of protein) were incubated with either GST-SHPTP2 (right) or GST-SH2 (left) fusion protein coupled to glutathione-Sepharose beads for 90 min at 4°C. After extensive washing, bound proteins to either GST-SHPTP2 or GST-SH2 fusion protein were resolved by SDS-PAGE, electrotransferred to membrane, and immunoblotted with ␣PY20.

FIG. 3. Insulin-stimulated phosphorylation of insulin receptor and IRS-1 in WT11, MT15, and pHyg cells.
A, tyrosine-phosphorylated proteins in the cell lysate (200 g of protein) from the basal and 10 nM insulin-stimulated cells were analyzed by Western blotting using ␣PY20. B, after immunoprecipitated with ␣IRS-1 antibody, bound proteins were analyzed by Western blotting using ␣PY20. Both basal and 10 nM insulin-stimulated phosphorylation levels of IRS-1 were presented. C, to quantify IRS-1 protein content, equal amount of cell lysates (40 g of protein/line) was analyzed by Western blotting using ␣IRS-1 antibody .   FIG. 4. Association of IRS-1 with SHPTP2 in WT11, MT15, and pHyg cells. After cells were incubated with or without 100 nM insulin for 5 min, lysed, and immunoprecipitated with ␣IRS-1. Bound proteins were resolved by SDS-PAGE. The immunoprecipitate was analyzed by Western blotting using ␣Syp.

mutant protein expression.
To further analyze the total PI 3Ј-kinase activity in the cells, PI 3Ј-kinase activity immunoprecipitated with ␣p85 antibody was measured, we found that total PI 3Ј-kinase activities immunoprecipitated with ␣p85 subunits was decreased in MT cells, but unchanged in WT11 cells (Fig. 5, C and D). To rule out the possibility that the decreased PI 3Ј-kinase activity in HIRc cells which expressed the ⌬PTP originated from clonal variation or from specific characteristics of the parent HIRc cells, we tested the effects of ⌬PTP expression on PI 3Ј-kinase activity in another independent cell line, HIRY/F2 cells, where Y/F2 mutant insulin receptors were expressed (28). We have cloned HIRY/F2 cells, which overexpress either wild-type (WT1) or ⌬PTP (MT6) at expression levels similar to those for HIRc cells (WT11 and MT15). In HIRY/F2 cells, PI 3Ј-kinase activities immunoprecipitated with ␣IRS-1 antiserum were also decreased in MT6 cells but increased in WT1 cells as shown in Fig. 7 (A and B). Furthermore, PI 3Ј-kinase activity immunoprecipitated with ␣p85 antibody was decreased in only MT6 cells but increased in WT1 cells. To study the reason for decreased total PI 3Ј-kinase activity, we next assessed the protein content of p85 subunit in these cell lines. As shown in Fig. 8, Western blot analysis using polyclonal ␣p85 antibody showed that the content of p85 protein was decreased in MT cells in both HIRc and HIRY/F2 cells. Using another monoclonal p85 antibody that recognizes a different epitope, we confirmed that the protein content of the p85 subunit was decreased in MT cells, suggesting that expression of ⌬PTP might induce decreased protein content of p85 subunits in both HIRc and HIRY/F2 cells. On the other hand, p85 protein content in WT cells was comparable to that in pHyg cells.
Insulin-stimulated MAP Kinase Cascade-To assess the changes in insulin signaling in the cells expressing wild-type and ⌬PTP, we measured insulin-stimulated MAP kinase activity. MAP kinase activity in the basal state was identical among these cell lines and increased depending upon the insulin concentration in all cell lines. In WT11 cells, insulin-stimulated MAP kinase activity was enhanced compared with that of pHyg cells. Enhanced insulin action was mainly due to responsiveness (percent over basal; 600% in WT11 cells and 290% in pHyg cells, p Ͻ 0.01) with a smaller increase in insulin sensitivity (ED 50 value 2.1 nM in WT11 cells and 3.2 nM in pHyg cells). On the other hand, in MT15 cells, MAP kinase activity was attenuated compared with that in pHyg cells (percent over basal 180% in MT cells, p Ͻ 0.01). Similar results were obtained in HIRY/F2 cell lines (data not shown).
Insulin-stimulated DNA Synthesis-We examined insulinstimulated DNA synthesis by measuring the incorporation of BrdUrd in both HIRc (Fig. 9A) and HIRY/F2 (Fig. 9B) cells and found that the percentage of BrdUrd-labeled cells was increased in WT cells and decreased in MT cells when compared with those in pHyg cells in both cell lines, even though the percentage of labeled cells was comparable in the basal state among these cell lines.
As shown in Fig. 10, we found that insulin-stimulated PI 3Ј-kinase activity was well correlated with IRS-1 phosphorylation levels in WT and MT cells (r ϭ 0.953, p Ͻ 0.05). Furthermore, changes in PI 3Ј-kinase activity were well correlated with those in DNA synthesis (r ϭ 0.986, p Ͻ 0.01), as well as in the case of MAP kinase (r ϭ 0.91, p Ͻ 0.05) in HIRc cells. DISCUSSION We have established cell lines overexpressing either wildtype or mutant SHPTP2 (⌬PTP) originated from HIRc and HIRY/F2 cells. In response to insulin, IRS-1-associated SHPTP2 activity in WT11 cells was significantly greater than that in pHyg cells. On the other hand, SHPTP2 activities bound to IRS-1 in MT15 cells was not affected by insulin treatment. Thus, introduction of ⌬PTP inhibited the association of IRS-1 with native endogenous SHPTP2 and overexpression of wildtype SHPTP2 enhanced its association with IRS-1 compared to that in pHyg cells in response to insulin.
Does the change in association between SHPTP2 and IRS-1 modulate insulin signaling? In our current study, we found that both insulin-stimulated levels of tyrosine phosphorylation of insulin receptors and IRS-1 were increased in WT cells and decreased in MT cells. Thus, we speculate that SHPTP2 potentiates the phosphorylation levels of IRS-1 and regulates the insulin signal through either the inhibition of an undefined PTPase(s) or the activation of undefined PTK(s). As shown in Fig. 2, in addition to IRS-1 and insulin receptor ␤ subunit, two undefined tyrosine-phosphorylated proteins bound to SH2 domains of SHPTP2 in response to insulin. One was a 115-kDa tyrosine-phosphorylated protein and thought to be a SHPTP2binding protein as reported in several studies (21)(22)(23)35). The other high molecular weight tyrosine-phosphorylated protein (pp135) remains undefined. Although the precise roles of these proteins for regulation of insulin signaling are unclear, they may be substrate(s) for SHPTP2. Further characterization of these proteins may have important implications for clarifying the role of SHPTP2 in the signal transduction of insulin.
However, previous studies (21)(22)(23) reported that introduction of dominant negative mutant SHPTP2 did not modulate the phosphorylation level of IRS-1. Noguchi et al. (22) showed that introduction of negative SHPTP2 inhibited Ras-MAP kinase pathway but that the phosphorylation of IRS-1 was unchanged. In the present study, our ⌬PTP mutant consists only  SH2 domains of SHPTP2 and tertiary structure of its SH2 domains may differ from those of native SHPTP2 and their catalytically inactive mutant (Cys 3 Ser). Therefore, it may be important to rule out the possibility that this SH2 mutant nonspecifically interacts with other SH2 binding sites rather than endogenous SHPTP2 binding sites, leading to inhibit functions of other SH2-containing molecules. However, SH2 domains of SHPTP2 are reported to behave differently compared with those of PTP1C in vitro (25) and in vivo (36), even though they have 60 -70% of homology in SH2 regions of both PTPases. Moreover, overexpression of wild-type SHPTP2 induces the opposite effects when compared with MT cells. Furthermore, we could not find any qualitative difference in the profile of binding proteins between GST fusion proteins containing full-length SHPTP2 and ⌬PTP as shown in Fig. 2. Thus, we speculate that ⌬PTP mutant interfere with native SHPTP2 to associate endogenous SHPTP2 binding sites including IRS-1. In the present study, we confirmed that the introduction of mutant SHPTP2 attenuated insulin-stimulated MAP kinase activity as reported (21)(22)(23). However, we also found that ⌬PTP impaired the insulin-stimulated activation of PI 3Ј-kinase associated with IRS-1 in MT cells. Furthermore, we found that insulin-stimulated PI 3Ј-kinase activity was well correlated with IRS-1 phosphorylation levels that was evaluated using three cell lines, as shown in Fig. 10A. Thus, we speculate that changes in phosphorylation levels of IRS-1 might be associated with both changes in PI 3Ј-kinase and MAP kinase activities in HIRc cells.
Although ⌬PTP may act by nonspecifically inhibiting between IRS-1 and p85 subunit of PI 3Ј-kinase in MT cells, this seem unlikely based on the following. First, our SH2 protein was not able to bind a GST-IRS-1 fusion protein that includes binding motif for p85 of PI 3Ј-kinase and no SHPTP2 binding site (17). Furthermore, p85 was more efficiently bound to phosphorylated IRS-1 in MT cells, when compared with that in pHyg cells (Fig. 6). Considering these data together, we believe that decreased levels of phosphorylation of IRS-1 may cause impairment of its association with p85, resulting in decrease in PI 3Ј-kinase activity rather than unphysiological inhibition of IRS-1 association with p85 of PI 3Ј-kinase. Therefore, the reason for this discrepancy between Noguchi's study and ours still remains unclear. However, one can speculate that different cell lines (CHO-IR versus HIRc) may respond differently to SHPTP2 depending on its concentration in the cells.
Interestingly, the content of the p85 subunit of PI 3Ј-kinase was significantly decreased in MT cells, and its content in WT cells was comparable with that of pHyg cells. Since the protein content of IRS-1 (Fig. 3C) was not decreased in MT cells compared with that in pHyg cells, the decrease in protein content of p85 subunit might be specific. The decrease in p85 content in MT cells was confirmed in two different parent cell lines. Therefore, it may be possible that SHPTP2 regulates expression of a p85 subunit of PI 3Ј-kinase. Similarly, Hausdorff et al. (37) reported that microinjection of either GST-SH2 fusion protein or anti-SHPTP2 antibody into 3T3-L1 adipocytes inhibited insulin-stimulated expression of Glut1 and that SHPTP2 was necessary for insulin-stimulated expression of Glut1 protein (37). They speculate that this may be caused by inhibition of p21 ras activation (38). However, McGuire et al. (39) have reported that expression of p85 is not affected in NIH3T3 cells after a 40-h treatment with lovastatin, which inhibits farnesylation of p21 ras , suggesting that p21 ras does not modulate the expression of p85 (39). Although the mechanism for regulation of p85 expression remains unclear, it has been reported that differentiation of preadipocyes to adipocytes leads to increased levels of the expression and phosphorylation of insulin receptor and IRS-1, and resulted in increased expression of p85 (40). Therefore, based upon these data, SHPTP2 may modulate expression of p85 via activation of insulin signaling.
In conclusion, overexpression of SHPTP2 enhanced the insulin-stimulated activities of both MAP kinase and PI 3Ј-kinase through increased IRS-1 phosphorylation and introduction of dominant negative mutant SHPTP2 (⌬PTP) attenuated these pathways through impaired IRS-1 phosphorylation. This is the first direct evidence that levels of SHPTP2 activity positively regulate the stimulation of both MAP kinase cascade and PI 3Ј-kinase pathway via modulation of phosphorylation of IRS-1 in Rat 1 fibroblasts expressing human insulin receptors.