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J. Biol. Chem., Vol. 275, Issue 24, 18318-18326, June 16, 2000

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Overexpression of Protein-tyrosine Phosphatase-1B in Adipocytes Inhibits Insulin-stimulated Phosphoinositide 3-Kinase Activity without Altering Glucose Transport or Akt/Protein Kinase B Activation^*

Carol L. Venable^§¶, Ernst U. Frevert^§, Young-Bum Kim^**, Britta M. Fischer, Shubhangi Kamatkar^§§, Benjamin G. Neel^§§¶¶, and Barbara B. Kahn

From the  Diabetes Unit, Division of Endocrinology and ^§§ Cancer Biology Program, Division of Hematology-Oncology, Department of Medicine, Beth Israel Deaconess Medical Center, and Harvard Medical School, Boston, Massachusetts 02215

Received for publication, October 15, 1999, and in revised form, March 2, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Previous studies suggested that protein-tyrosine phosphatase 1B (PTP1B) antagonizes insulin action by catalyzing dephosphorylation of the insulin receptor (IR) and/or other key proteins in the insulin signaling pathway. In adipose tissue and muscle of obese humans and rodents, PTP1B expression is increased, which led to the hypothesis that PTP1B plays a role in the pathogenesis of insulin resistance. Consistent with this, mice in which the PTP1B gene was disrupted exhibit increased insulin sensitivity. To test whether increased expression of PTP1B in an insulin-sensitive cell type could contribute to insulin resistance, we overexpressed wild-type PTP1B in 3T3L1 adipocytes using adenovirus-mediated gene delivery. PTP1B expression was increased ~3-5-fold above endogenous levels at 16 h, ~14-fold at 40 h, and ~20-fold at 72 h post-transduction. Total protein-tyrosine phosphatase activity was increased by 50% at 16 h, 3-4-fold at 40 h, and 5-6-fold at 72 h post-transduction. Compared with control cells, cells expressing high levels of PTP1B showed a 50-60% decrease in maximally insulin-stimulated tyrosyl phosphorylation of IR and insulin receptor substrate-1 (IRS-1) and phosphoinositide 3-kinase (PI3K) activity associated with IRS-1 or with phosphotyrosine. Akt phosphorylation and activity were unchanged. Phosphorylation of p42 and p44 MAP kinase (MAPK) was reduced ~32%. Overexpression of PTP1B had no effect on basal, submaximally or maximally (100 nM) insulin-stimulated glucose transport or on the EC₅₀ for transport. Our results suggest that: 1) insulin stimulation of glucose transport in adipocytes requires 45% of maximal tyrosyl phosphorylation of IR or IRS-1 and <50% of maximal activation of PI3K, 2) a novel PI3K-independent pathway may play a role in insulin-induced glucose transport in adipocytes, and 3) overexpression of PTP1B alone in adipocytes does not impair glucose transport.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Insulin stimulates glucose transport and metabolism by activating a cascade of signaling events (1-3). Insulin binds to its transmembrane receptor, causing receptor trans-phosphorylation on tyrosyl residues and activating receptor tyrosine kinase activity toward a variety of substrates including IRS-1-4,¹ shc, and Gab1 (2, 4, 5). These tyrosyl-phosphorylated proteins provide high affinity binding sites for the Src homology 2 domains of several signal relay molecules, such as the regulatory subunit of phosphoinositide 3-kinase (PI3K), which leads to kinase activation and stimulation of several downstream mediators, including the serine/threonine kinase Akt/protein kinase B (6, 7).

Insulin stimulates glucose transport primarily by eliciting translocation of the major insulin responsive glucose transporter GLUT4 from intracellular vesicles to the plasma membrane (2, 3, 8). In classic insulin responsive target tissues, such as adipose tissue and muscle, PI3K activity is necessary and partially sufficient for insulin-induced glucose transport (9-11). Whether Akt, atypical protein kinase C isoforms, or other, as yet unknown, downstream mediators are necessary for the effect of insulin-induced PI3K activation on GLUT4 translocation remains controversial (12-15).

Impaired insulin-stimulated glucose transport in muscle and adipose tissue is a major contributor to the pathogenesis of insulin-resistant states such as obesity and type 2 diabetes. Substantial efforts have been made to identify proteins that impair critical steps in insulin signal transduction in individuals with insulin resistance. The central role of tyrosine phosphorylation of the insulin receptor and its substrates in insulin action has focused attention on protein-tyrosine phosphatases (PTPs) as candidate molecules implicated in the pathogenesis of insulin resistance. Indeed, increased expression and activity of several PTPs has been observed in skeletal muscle and adipose tissue in insulin-resistant, obese states in rodents and humans (16-20). In obese humans, the increased PTP activity in skeletal muscle and adipose tissue has been reported to be due mainly to increased expression of two PTPs, the transmembrane (receptor-like) leukocyte antigen related phosphatase (LAR) and the nontransmembrane tyrosine phosphatase PTP1B (17, 18). Whereas Src homology phosphatase 2 expression is also increased, immunodepletion (17, 18) and substrate specificity (21) studies suggest it may be less important. Consistent with the notion that increased PTP activity may contribute to insulin resistance, weight loss in obese humans results in a decrease in the level of these PTPs, concomitant with an increase in the glucose disposal rate (22). However, such studies provide only correlative suggestions that one or more of these PTPs plays a causal role in insulin resistance.

Some studies using cultured cells support the notion that PTP1B participates in regulation of insulin receptor signaling. Overexpression of PTP1B in rat fibroblasts that also overexpress insulin receptors at high levels results in decreased insulin-stimulated insulin receptor and IRS-1 phosphorylation (23). However, fibroblasts do not express GLUT4, the major physiologically relevant glucose transporter, so the significance of these findings for glucose transport in insulin responsive tissues remains unclear. In transient transfection studies in primary rat adipocytes, overexpression of PTP1B was reported to decrease cell surface GLUT4 in the presence of insulin (24). However, surface levels of GLUT4 were decreased also in the absence of insulin (24), such that the overall increment of cell surface GLUT4 in response to insulin was normal in PTP1B-overexpressing cells. Moreover, the transient transfection approach does not permit any assessment of the consequences of PTP1B overexpression on protein phosphorylation or activation of intracellular signal transduction. The most compelling evidence that PTP1B could play an important role in the physiologic regulation of insulin receptor signaling comes from recent work characterizing mice rendered PTP1B-deficient by homologous recombination (25). These mice exhibit increased insulin sensitivity in terms of glucose homeostasis and enhanced insulin-induced tyrosyl phosphorylation of the IR and IRS-1 in muscle and liver. Notably, however, insulin-induced tyrosyl phosphorylation in adipose tissue appears unaffected (25). Thus, although it now seems likely that PTP1B is an important negative regulator of insulin signaling in muscle and liver, its role in normal insulin action in the adipocyte remains uncertain. Moreover, it remains unclear whether increased PTP1B expression in adipocytes, at levels comparable to those found in insulin-resistant rodents and humans, can impair insulin-induced signal transduction and lead to insulin resistance.

To address these questions, we used adenovirus-mediated gene transfer to achieve greater than 90% efficient expression of human PTP1B in the highly insulin responsive target cell, the 3T3-L1 adipocyte. With PTP1B overexpression ranging from 3-fold (at 4-8 h after viral transduction) to 20-fold (with longer time after transduction) over endogenous levels, neither maximal nor submaximal glucose transport was altered despite blunted upstream insulin signaling, reflected by 50-60% impairments in IR and IRS-1 tyrosyl phosphorylation and PI3K activation. Insulin-stimulated Akt activity also remained unaffected, whereas MAPK phosphorylation in response to insulin was reduced ~32%. Our data demonstrate that, in 3T3L1 adipocytes, <50% of maximal PI3K activation is necessary for maximal activation of glucose transport and Akt/protein kinase B by insulin. Furthermore, our results suggest that the modest increase in PTP1B activity observed in adipose tissue of obese, insulin-resistant humans and rodents is, by itself, unlikely to impair insulin signaling to a pathophysiologically significant degree such that it would interfere with glucose transport. Conceivably, however, increased PTP1B activity in other tissues or in combination with elevation of other protein-tyrosine phosphatases could alter insulin action and lead to the development of insulin resistance.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell Culture-- 293 cells and COS-7 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Life Technologies, Inc.) with 10% fetal calf serum (Atlanta Biologicals, Norcross, GA), 50 units/ml penicillin, and 50 µg/ml streptomycin (Life Technologies, Inc.) at 37 °C, 5% CO₂. 3T3-L1 fibroblasts (obtained from the ATCC, catalog no. CCL 92.1, Rockville, MD) were grown in DMEM with 10% newborn calf serum, 50 units/ml penicillin, and 50 µg/ml streptomycin (all from Life Technologies, Inc.) at 37 °C, 5% CO₂. Two days after reaching confluence, fibroblasts were induced to differentiate by treatment with 0.5 mM 3-isobutyl-1-methyl-xanthine, 0.25 µM dexamethasone (both from Sigma), and 1 µg/ml insulin (porcine, crystalline, gift from R. Chance, Eli Lilly, Indianapolis, IN) for 3 days. During and following differentiation, DMEM was supplemented with 10% fetal calf serum. Cells were used for experiments at least 10 days after induction of differentiation and only if greater than 90% of cells showed fat droplets.

Transfection of COS-7 Cells-- COS-7 cells were transfected at 80-90% confluence in serum-free Optimem (Life Technologies, Inc.), using 2.5 µg of DNA and 12.5 µl of LipofectAMINE reagent (Life Technologies, Inc.)/35-mm diameter dish with pJ3H constructs containing the respective mouse and human PTP1B cDNA. The pJ3H vector places an N-terminal HA tag on expressed proteins. Transfected cells were incubated overnight, and the medium was changed to standard DMEM with 10% fetal calf serum in the morning. After 24 h, the cells were collected as described below.

Generation of Recombinant Wild Type PTP1B Adenovirus-- Human PTP1B cDNA (3.2 kilobases) was cloned as an EcoRI fragment (26) into pACCMV·pLpA (27) (a gift of C. Newgard, University of Texas, Southwestern, Dallas, TX), resulting in the plasmid pACCMV·pLpA-PTP1B. Viral recombination was achieved as described previously (10, 27). Briefly, pACCMV·pLpA-PTP1B (0.7 µg/35-mm diameter dish) was co-transfected with the plasmid pJM-17 (1 µg/35-mm diameter dish, gift of C. Newgard) into 80% confluent 293 cells (Life Technologies, Inc.). Recombination, as indicated by cell lysis, occurred 1-2 weeks after transfection. Six clones of recombinant virus were assayed by immunoblotting transduced 293 cell lysates with anti-PTP1B antibodies. All six clones revealed successful integration of PTP1B into adenovirus, and one clone was amplified further in 293 cells. The virus was purified from 40 15-cm diameter plates by two sequential cesium chloride gradient centrifugations. A recombinant adenovirus encoding -galactosidase (gift of C. Newgard) was amplified and purified in the same way as the PTP1B-encoding virus.

Transduction of 3T3-L1 Adipocytes-- 3T3-L1 adipocytes were transduced with recombinant adenovirus overnight with constant agitation on a rocking platform in DMEM with 10% fetal calf serum. PTP1B- and -galactosidase-encoding recombinant adenoviruses were used at a concentration of 1 × 10¹² plaque forming units/ml, as determined by spectrophotometry (corresponding to 1 × 10⁹ plaque forming units/ml by limiting dilution). Experiments were performed at 16, 40, or 72 h after transduction. For experiments at 40 and 72 h, adenovirus was removed at 16 h, and cells were incubated for an additional 24-56 h in DMEM with or without serum starvation. For glucose transport, experiments were also performed after only 4 or 8 h of transduction to assess the effects of low level overexpression of PTP1B.

Preparation of COS-7 and 3T3-L1 Cell Lysates-- 3T3-L1 adipocytes were incubated in DMEM with 0.1% or 10% fetal calf serum for 16 h prior to study, stimulated with insulin (0 or 100 nM) for 1 or 5 min, washed once in phosphate-buffered saline, and collected in 200 (16-mm diameter dish) or 500 µl (35-mm diameter dish) of ice-cold lysis buffer (pH 7.4) (20 mM HEPES, 100 mM NaF, 10 mM NaPP, 1% Nonidet P-40, 0.1% SDS, 2 mM EDTA, 2 mM vanadate, 1 µg/ml aprotinin and leupeptin (28)). The lysates were then homogenized by 20 strokes with a Potter homogenizer and centrifuged for 15 min in a microfuge at 4 °C. COS-7 cells were collected in the same manner except without insulin stimulation.

Protein-tyrosine Phosphatase Assays-- PTP activity was measured in lysates of 3T3-L1 adipocytes that were not transduced or were transduced with -galactosidase or PTP1B adenoviruses. Lysates from cells at the indicated times post-transduction were collected in a buffer containing 20 mM Tris (7.5), 140 mM NaCl, 1% Nonidet P-40, and 1 µl/ml aprotinin and leupeptin. Aliquots containing 10 µg of cellular protein were incubated with a solution containing 20 mM Tris (pH 7.5), 10 mM dithiothreitol, and 10 mM pNPP for 30 min or 1 h at 37 °C. Reactions were terminated by the addition of 0.2 M NaOH. PTP activity was then determined by absorbance at 410 nm.

Immunoblotting-- SDS-PAGE and immunoblotting were performed as described previously (10, 29, 30) using 1.5-mm thick minigels (Novex, San Diego, CA), nitrocellulose membranes (pore size 0.45 µm, Schleicher & Schuell), and the Mini Trans-blot Transfer cell (Bio-Rad) with Towbin buffer, 20% (v/v MeOH), and 0.02% SDS. Membranes were blocked in either Tris-buffered saline (TBS) with 0.05% Tween 20 and 5% low fat dry milk or TBS with 0.05% Tween 20 and 4% bovine serum albumin (Sigma) for 1 h at room temperature or overnight at 4 °C. The membranes were then incubated for 11/2 h at room temperature or overnight at 4 °C with one of the following primary antibodies: monoclonal PY20 (Santa Cruz Biotechnology, Santa Cruz, CA, 1:200), polyclonal anti-IRS-1 (gift of C. R. Kahn, Joslin Diabetes Center, Boston, MA, 1:200), polyclonal anti-insulin receptor (gift of K. Siddle, University of Cambridge, Cambridge, United Kingdom, 1:1000), polyclonal anti-phospho-MAP kinase (Promega, Madison, WI, 1:10,000), polyclonal anti-MAP kinase (gift of J. Blenis, Harvard Medical School, Boston, MA, 1:5000), monoclonal anti-HA tag (Roche Molecular Biochemicals, 1:1000), polyclonal anti-phospho-Akt (New England Biolabs, Beverly, MA, 1:1000), polyclonal anti-PTP1B (polyclonal, 1:1000) (31), polyclonal anti-GLUT1 (gift of B. Thorens, University of Lausanne, Lausanne, Switzerland, 1:100), or polyclonal anti-GLUT4 (gift of H. Haspel, Henry Ford Hospital, Detroit, MI, 1:400). Membranes were washed in TBS with 0.05% Tween 20 for 15 min at room temperature and incubated with the appropriate horseradish peroxidase-coupled secondary antibodies (1:2000 dilution in TBS with 0.05% Tween 20, Amersham Pharmacia Biotech) for 1-2 h. The membranes were then washed for 25 min in TBS with 0.05% Tween 20, and bands were visualized with enhanced chemiluminescence (Amersham Pharmacia Biotech) and quantified by densitometry using ImageQuant software (Molecular Dynamics, Sunnyvale, CA).

PI3K Assays-- Lysates from 3T3-L1 adipocytes (80-100 µg of protein) were collected as described above with the exception that buffer A (20 mM Tris, pH 7.5, 5 mM EDTA, 10 mM Na₄P₂O₇, 100 mM NaF, 2 mM Na₃VO₄) 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin were used. Lysates were subjected to immunoprecipitation with 5 µl of a polyclonal IRS-1 antiserum (gift of M. White, Joslin Diabetes Center, Boston, MA) or PY20 monoclonal phosphotyrosine antibodies (Santa Cruz) coupled to protein A-Sepharose (Sigma). Immune complexes were washed as described (32) and resuspended in 50 ml of Tris-NaCl buffer (10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, and 100 µM Na₃VO₄). PI3K activity was measured as reported (32). Radioactivity in the spots corresponding to PI3-phosphate was quantified using densitometry and ImageQuant software (Molecular Dynamics).

Akt Assays-- Lysates from 3T3-L1 adipocytes (80-100 µg of protein) were collected in buffer A (above). Samples were immunoprecipitated for 4 h at 4 °C with 4 µg of a polyclonal Akt antiserum (recognizing both Akt1 and Akt2) (Upstate Biotechnology, Lake Placid, NY), coupled to protein G-Sepharose beads (Amersham Pharmacia Biotech). Immune pellets were washed as described (33), resuspended in 50 µl of kinase buffer (50 mM Tris, pH 7.5, 10 mM MgCl₂, 1 mM dithiothreitol, 5 µM ATP, 1 µM protein kinase inhibitor, 30 µM Crosstide, and 2 µCi of [-³²P]ATP) (34), and incubated at 30 °C for 30 min. Aliquots (40 µl) of the reaction mixture were spotted onto phosphocellulose p81 paper (Whatman, Clifton, NJ) and washed, and the incorporated counts were determined by scintillation counting (33).

Glucose Transport Assays-- Glucose transport was measured after cells were incubated following two protocols. In protocol 1, fully differentiated adipocytes were not transduced or were transduced with -galactosidase or PTP1B adenoviruses for 4, 8, or 16 h followed by a 3-4-h serum starvation. In protocol 2, cells were left nontransduced or were transduced for 16 h with -galactosidase or PTP1B virus. The medium was then changed to standard DMEM with 10% fetal calf serum without virus, and cells were incubated for another 24 or 56 h at 37 °C, 5% CO₂, so that experiments could be performed at 40 and 72 h after viral transduction. In both protocols, cells were starved for 3-4 h in serum-free DMEM (25 mM glucose) at 37 °C, 5% CO₂ and then washed once with glucose-free minimum Eagle's medium. Insulin (0, 0.1, 0.3, 0.6, 1.0, or 100 nM) was added for 30 min, followed by the addition of 100 µM [³H]2-deoxy-D-glucose, 0.33 µCi per 35-mm diameter well (New England Nuclear, Boston, MA). Cells were incubated for a further 10 min in a gently shaking waterbath at 37 °C. Transport was subsequently stopped by placing the cells on ice and adding 1:1 (v/v) ice-cold phloretin solution (82 µg/liter in phosphate-buffered saline) (Sigma). Cells were washed once with cold phosphate-buffered saline, solubilized in 1 N NaOH at 37 °C for 30 min, and [³H]2-deoxy-D-glucose incorporation was measured by liquid scintillation counting.

Statistical Analysis-- Data are presented as mean ± S.E. Statistical analysis was performed by analysis of variance (ANOVA) using Statview software. Curve fitting was performed with Origin 35 (MicroCal Software, Inc., Northampton, MA).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Overexpression of PTP1B in 3T3-L1 Adipocytes-- We overexpressed human PTP1B in mouse adipocytes. Because there is no available PTP1B antibody with equal affinity for mouse and human PTP1B, to enable quantification of the relative level of PTP1B expression in transduced cells, we first determined the relative affinity of our antibodies for mouse and human PTP1B. The antibody used was raised against a glutathione S-transferase fusion protein containing the N-terminal 150 amino acids of human PTP1B (GST-N) (31). This region is 93% identical between human and mouse PTP1B. We transiently transfected COS-7 cells with HA-tagged mouse and human PTP1B cDNA and compared anti-HA reactivity with anti-PTP1B reactivity. When transfected COS-7 cell lysates were immunoblotted with the anti-HA antibody, approximately equal amounts of mouse and human PTP1B were detected (Fig. 1A, left panel). However, when the same cell lysates were immunoblotted with anti-PTP1B antiserum, there was a substantially higher intensity signal for human PTP1B (Fig. 1A, right panel). Quantifying these results reveals an ~7-fold greater sensitivity of the anti-PTP1B antiserum for human, compared with mouse PTP1B; hence signals derived from endogenous mouse PTP1B must be multiplied by seven to compare with signals derived from the exogenous human PTP1B protein.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Expression of PTP1B in COS-7 cells (A) and 3T3-L1 adipocytes (B and C). A, cell lysates were collected from COS-7 cells 40 h after transfection with vector only or with cDNA encoding HA-tagged mouse or human PTP1B. Proteins were separated by SDS-PAGE and immunoblotted with HA antibody or PTP1B antiserum as described under "Materials and Methods." A representative blot is shown with corresponding quantitation. B, cell lysates were prepared from 3T3-L1 adipocytes at 16, 40, or 72 h after transduction with adenovirus encoding -galactosidase or human PTP1B. Proteins were separated by SDS-PAGE and immunoblotted with PTP1B antiserum. This blot is representative of four separate experiments. C, corrected PTP1B overexpression levels in 3T3-L1 adipocytes. Immunoreactivity was quantitated by densitometry or phosphorimaging. Optical density units for human PTP1B measured on blots such as that shown in B were divided by 7 to correct for the lower affinity of the PTP1B antiserum for mouse (endogenous) compared with human (transduced) PTP1B. Bars in C show means ± S.E. for four separate experiments. Data are expressed as increase in PTP1B expression over endogenous levels in control adipocytes transduced with adenovirus encoding -galactosidase.

For example, Fig. 1B shows results of 3T3-L1 adipocytes transduced with adenovirus encoding -galactosidase or human PTP1B. Transduction with -galactosidase-encoding adenovirus had no effect on the relatively low level of endogenous PTP1B protein compared with nontransduced cells (not shown). The PTP1B expression did not change over 72 h after transduction with -galactosidase-encoding virus; hence only the level at 72 h is shown. The PTP1B-transduced cells show an increase in the immunoreactive signal of ~20-35-fold at 16 h, ~98-fold at 40 h, and ~140-fold at 72 h after transduction compared with cells transduced with the -galactosidase-encoding adenovirus (Fig. 1B). However, normalizing for the difference in antiserum sensitivity for human and mouse PTP1B demonstrated with COS cell transfections shown in Fig. 1A, the corrected increase in PTP1B expression in PTP1B-transduced adipocytes is approximately 3-5-fold at 16 h, ~14-fold at 40 h, and ~20-fold at 72 h (Fig. 1C). We carried out the biochemical studies described below at these different time points to compare low level overexpression as seen in insulin-resistant humans and rodents with higher level overexpression.

Overexpression of PTP1B Increases Total Protein-tyrosine Phosphatase Activity-- Total PTP activity was measured in lysates of 3T3-L1 adipocytes at 16, 40, and 72 h after transfection with -galactosidase or PTP1B adenovirus (Fig. 2). Sixteen hours post-transduction, total PTP activity was increased by ~50% in PTP1B-transduced cells compared with either nontransduced or -galactosidase-transduced cells. By 40 h post-transduction, PTP activity was increased ~3-4-fold, and at 72 h it was increased 5-6-fold. These results indicate that the PTP1B-encoding adenovirus was overexpressing active PTP1B in transduced 3T3-L1 adipocytes. The discrepancy between the level of PTP1B protein overexpression (Fig. 1) and the increase in total PTP activity is because of the fact that other PTPs contribute significantly to total PTP activity in 3T3-L1 adipocytes. However, the observation that an ~20-fold overexpression at 72 h results in an ~5-6-fold increase in total PTP activity suggests that PTP1B is a significant source of PTP activity in 3T3L1 adipocytes, possibly accounting for as much as 25% of paranitrophenyl phosphatase activity in total lysates.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Total protein-tyrosine phosphatase activity in 3T3-L1 adipocytes that were not transduced (no virus, open bar) or were transduced with adenovirus encoding -galactosidase (striped bar) or human PTP1B (black bar). At 16, 40, or 72 h after transduction, cell lysates were prepared, and protein-tyrosine phosphatase activity was measured using pNPP as the substrate as described under "Materials and Methods." Samples were read in a spectrophotometer at absorbance 410 nm. Results are mean ± S.E. for four separate experiments. *, different from cells with no virus and -galactosidase (-gal) at p < 0.05.

Overexpression of PTP1B Decreases Insulin Receptor and IRS-1 Phosphorylation-- We examined the effects of PTP1B overexpression on components of the IR signaling pathway. In control, nontransduced cells, maximal insulin stimulation for 5 min increased IR tyrosyl phosphorylation by 7-8-fold (Fig. 3A) and IRS-1 tyrosyl phosphorylation by 14-fold (Fig. 3B). Adenovirus-directed -galactosidase expression had no significant effect on basal or insulin-stimulated IR or IRS-1 tyrosyl phosphorylation. In PTP1B-overexpressing cells, basal tyrosyl phosphorylation of the IR and IRS-1 was not significantly affected, but there was a 60% decrease in insulin-stimulated IR phosphorylation (Fig. 3A) and a 55% decrease in insulin-stimulated IRS-1 phosphorylation (Fig. 3B) compared with control cells transduced with -galactosidase. Because total amounts of IR and IRS-1 protein levels were not altered by PTP1B overexpression (Fig. 3C), the reduction in phosphorylation of IR and IRS-1 when normalized for the amount of IR or IRS-1 protein was also 60 and 55%, respectively. Because acute insulin stimulation had no effect on IR or IRS-1 protein levels, the bars in Fig. 3C show total IR and IRS-1 protein in the basal- and insulin-stimulated states combined.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Insulin receptor (A) and IRS-1 (B) tyrosine phosphorylation in 3T3-L1 adipocytes that were not transduced or were transduced with adenovirus encoding -galactosidase or PTP1B as described under "Materials and Methods." Cells were incubated for 16 h with 0.1% fetal calf serum and stimulated for 5 min with 0 () or 100 nM (+) insulin prior to being lysed at 40 or 72 h after transduction. Lysates (40 µg) were run on SDS-PAGE and immunoblotted with an antiphosphotyrosine antibody. Bands representing the -subunit of the insulin receptor and IRS-1 were quantitated by densitometry. This Western blot is representative of eight blots for insulin receptor and six for IRS-1 performed on a total of three different sets of cells. The bar graph shows the mean ± S.E. for quantitation of Western blots from three separate sets of cells, each blotted in duplicate or triplicate. * indicates the difference from insulin-stimulated values in cells with no virus at p < 0.004 or with -galactosidase at p < 0.0001 for insulin receptor (A) and at p < 0.02 compared with no virus and p < 0.03 compared with -galactosidase for IRS-1 (B). C, quantitation of insulin receptor and IRS-1 protein levels in lysates from 3T3-L1 adipocytes treated as described above. Lysates were run on SDS-PAGE and immunoblotted with either insulin receptor (left panel) or IRS-1 (right panel) antibody. Protein levels were quantitated by densitometry. Results are mean ± S.E. for three separate sets of cells. Because there was no difference in IR or IRS-1 levels in basal- and insulin-treated cells, data from these two conditions are combined. D, insulin receptor and IRS-1 phosphorylation in 3T3-L1 adipocytes that were not transduced or transduced with adenovirus encoding -galactosidase or PTP1B and treated as described for A and B except insulin stimulation was for 1 min in D. Each lysate was run twice on adjacent lanes, as 32 (lanes 1, 3, and 5) and 43 µg (lanes 2, 4, and 6). The position of molecular mass markers is shown on the left, and arrows indicating tyrosine-phosphorylated IRS-1 and IR are shown on the right. This Western blot is representative of three separate blots.

We considered the possibility that overexpression of PTP1B might not interfere with the initial phosphorylation of IR and IRS-1 but instead result in a more rapid dephosphorylation of these proteins. In this case, the initial phosphorylation could potentially be sufficient to fully stimulate downstream biologic effects such as glucose transport. Therefore, to determine whether the initial phosphorylation reached normal levels, we also measured insulin-stimulated phosphorylation of the insulin receptor and IRS-1 at 1 min of insulin stimulation. Fig. 3D shows that overexpression of PTP1B had as great an inhibitory effect on phosphorylation of the insulin receptor and IRS-1 at 1 min of insulin stimulation as at 5 min (Fig. 3, A and B). At 1 min, both insulin receptor phosphorylation and IRS-1 phosphorylation were decreased 50-70%. On the blot shown, each sample was run twice at two different concentrations. Lanes 1, 3, and 5 contain 32 µg of protein, and lanes 2, 4, and 6 contain 43 µg.

Overexpression of PTP1B Decreases Insulin-stimulated PI3K Activity-- We next assessed the consequences of PTP1B overexpression on the PI3K pathway. There was no significant difference in the basal levels of IRS-1-associated PI3K activity in cells overexpressing PTP1B compared with nontransduced cells or cells transduced with -galactosidase virus (Fig. 4). As expected, insulin dramatically stimulated PI3-kinase activity (11-14-fold) in nontransduced cells, and transduction with -galactosidase virus had no significant effect on this stimulation (Fig. 4). However, in cells overexpressing PTP1B, insulin-stimulated PI3K activity was reduced by 50%. A similar (~50%) decrease in PI3K activity recovered in anti-phosphotyrosine immune complexes was also observed in PTP1B overexpressing cells (data not shown).

View larger version (22K): [in this window] [in a new window]   Fig. 4.   IRS-1-associated PI3-kinase activity in 3T3-L1 adipocytes that were not transduced or were transduced with adenovirus encoding -galactosidase or PTP1B as described under "Materials and Methods." Following overnight serum starvation, cells were stimulated with 0 () or 100 nM (+) insulin for 5 min, and lysates were prepared as described under "Materials and Methods." PI3-kinase activity was measured in IRS-1 immunoprecipitates. Activity was quantitated by densitometry. A representative autoradiogram of thin layer chromatography and the quantitation of PI3-kinase activity from four different experiments are shown. Data are mean ± S.E. * indicates the difference from no virus and -galactosidase insulin-stimulated values at p < 0.005 and p < 0.01, respectively.

PTP1B Overexpression Has No Effect on Akt Activity-- We assessed Akt activation by immune complex kinase assays and by immunoblotting with an antibody that specifically recognizes Akt phosphorylated on serine 473. For immune complex assays, we employed an antibody that recognizes Akt-1 and Akt-2 and utilized Crosstide as the substrate. Insulin stimulated Akt activity 7-8-fold in nontransduced 3T3L1 adipocytes (Fig. 5, bar graph). Interestingly, neither -galactosidase nor PTP1B expression affected either basal or insulin-stimulated Akt activity. Consistent with these findings, insulin-stimulated phosphorylation of Akt (at Ser-473) was unaltered in cells overexpressing PTP1B compared with -galactosidase-expressing cells (Fig. 5, autoradiogram).

View larger version (27K): [in this window] [in a new window]   Fig. 5.   Akt activity and phosphorylation in 3T3-L1 adipocytes that were not transduced or were transduced with adenovirus encoding -galactosidase or PTP1B as described under "Materials and Methods." Following overnight serum starvation, cells were stimulated with 0 () or 100 nM (+) insulin for 5 min, and lysates were prepared as described under "Materials and Methods." Akt activity was measured after immunoprecipitation with an antibody that recognizes both Akt1 and Akt2 and with Crosstide as the substrate. The graph shows the quantitation of Akt activity from three different experiments. Data are mean ± S.E. In addition, lysates of -galactosidase- and PTP1B-transduced cells were run on SDS-PAGE and immunoblotted with an antibody that recognizes phosphorylated Akt (Western blot at the top). This is representative of three blots on three separate sets of cells.

PTP1B Overexpression Decreases Insulin-induced MAPK Activation-- In addition to activating PI3K, insulin stimulates other downstream signaling pathways, such as the Erk MAPKs. We assessed the effect of PTP1B overexpression on insulin-stimulated Erk activation by performing immunoblots with phospho-specific antibodies that recognize activated Erks. Erk phosphorylation was decreased by about 32% in PTP1B-overexpressing cells at 40 h after transduction (p < 0.04) (Fig. 6), the same time at which PI3K activity was reduced ~50% (Fig. 4). The total amounts of Erk1 and Erk2 protein were unaltered by PTP1B overexpression (not shown). Thus, distinct downstream pathways from the insulin receptor may be affected to somewhat different extents by PTP1B overexpression and its consequent effects on IR and IRS-1 tyrosyl phosphorylation.

View larger version (25K): [in this window] [in a new window]   Fig. 6.   p42 and p44 MAP kinase phosphorylation in 3T3-L1 adipocytes that were not transduced or were transduced with adenovirus encoding -galactosidase or PTP1B as described under "Materials and Methods." Cells were incubated for 16 h with 0.1% fetal calf serum and stimulated for 5 min with 0 () or 100 nM (+) insulin prior to being lysed at 40 or 72 h after transduction. Lysates were run on SDS-PAGE and immunoblotted with an antibody specific for phosphorylated MAP kinase. Bands were quantitated by densitometry. This Western blot is representative of four separate experiments for which the mean ± S.E. are shown in the bar graphs. * indicates the difference from insulin-stimulated values in cells with no virus or -galactosidase at p < 0.05.

Overexpression of PTP1B Has No Effect on Glucose Transport-- First we measured glucose transport in 3T3L1 adipocytes at 40 and 72 h after transduction with adenoviruses. At these times, total PTP activity was increased up to 5-6-fold, and IR and IRS-1 tyrosyl phosphorylation were reduced 55-60% in cells transduced with PTP1B-expressing adenoviruses. Subsequently, we performed glucose transport experiments at 4, 8, and 16 h post-transfection to assess the effects of lower levels of PTP1B overexpression (levels closer to those found in adipose tissue and the muscle of insulin-resistant rodents and humans), as well as to account for the possibility that cells might be able to compensate for chronic overexpression of PTP1B. Adipocytes were stimulated for 30 min with a range of insulin concentrations (0-100 nM). Fig. 7A shows the effects of short term exposure to the virus and hence relatively low levels of PTP1B overexpression. There was no difference in basal, submaximally (0.3 and 0.6 nM), or maximally (100 nM) insulin-stimulated glucose transport in cells overexpressing PTP1B compared with control cells, which were not transduced, or cells transduced with -galactosidase adenovirus (Fig. 7A). The results were the same at 4, 8, and 16 h post-transduction (as well as at 40 and 72 h post-transduction when PTP1B levels were much higher).

View larger version (24K): [in this window] [in a new window]   Fig. 7.   2-Deoxyglucose uptake in 3T3-L1 adipocytes that were not transduced (no virus) or were transduced with adenovirus encoding -galactosidase or PTP1B as described under "Materials and Methods." Cells were studied at 4, 8, 16, 40, and 72 h after transduction, and the results were similar at all time points. For all experiments, cells were serum-starved for 3-4 h before a 30-min stimulation with insulin (0-100 nM). [3H]2-deoxy-D-glucose uptake was measured as described under "Materials and Methods." A shows a representative experiment performed at 16 h after adenovirus transduction. Results are mean ± S.E. for triplicate samples. This experiment is representative of four separate experiments. B shows dose-response curves performed at 40 h after viral transduction. There was no difference in the EC50 among groups (see "Results"). Mean glucose transport values for multiple experiments expressed as pmol/well/min for no virus,  galactosidase, and PTP1B, respectively, were: basal 24 ± 5, 32 ± 11, and 19 ± 7 (p = NS) and maximally insulin-stimulated 122 ± 9, 129 ± 19, and 122 ± 20 (p = NS).

Fig. 7B shows more complete dose-response curves performed in cells at 40 h after transduction with adenoviruses. Even with higher levels of expression, there is no significant effect of PTP1B on insulin-stimulated glucose transport. The dose-response curve for cells overexpressing PTP1B is superimposable on the curve for cells that are not transduced (no virus). Whereas there may be a very slight tendency for the dose response in cells transduced with -galactosidase-encoding virus to be shifted leftward, there are no significant differences in the EC₅₀ among the groups. The EC₅₀ values are: for cells which are not transduced 0.83 ± 0.37 nM, for cells transduced with -galactosidase adenovirus 0.39 ± 0.01 nM, and for cells transduced with PTP1B adenovirus 0.84 ± 0.11 nM (p = NS). Basal and maximally insulin-stimulated glucose transport at 40 and 72 h after transduction, expressed as pmol/well/min are also not different in PTP1B overexpressing cells compared with either no virus or -galactosidase-expressing control cells (see values in figure legend). Importantly, this lack of effect of PTP1B overexpression on glucose transport is observed despite a 55-60% reduction in IR and IRS-1 tyrosyl phosphorylation (Fig. 3) and a 50% reduction in insulin-stimulated PI3K activity (Fig. 4). GLUT1 and GLUT4 protein levels were unaltered by PTP1B overexpression (not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The expression and activity of PTP1B, as well as certain other PTPs, has been reported to be elevated in adipose tissue and skeletal muscle of obese humans and rodents (16-19, 22). These findings led to the hypothesis that increased activity of specific PTPs may play a role in the pathogenesis of insulin resistance. Because obesity is a major risk factor for type 2 diabetes, this hypothesis has been extended to suggest that increased PTPs could possibly contribute to the development of type 2 diabetes (even though this overexpression may reverse after diabetes is fully established (20)). Such correlative studies cannot assess directly whether overexpression of phosphatases plays a causal role in insulin resistance nor can they elucidate the biochemical consequences of overexpression of individual PTPs in insulin responsive tissues. Furthermore, they cannot distinguish which of the several PTPs, which are overexpressed in insulin-resistant states, are important in impairing insulin action in specific insulin target tissues. To address these questions, we overexpressed PTP1B in 3T3L1 adipocytes and determined the effects on insulin-stimulated signaling events and glucose transport. Our results show that high level overexpression of PTP1B leads to substantial impairment of tyrosyl phosphorylation of IR and IRS1, a comparable decrease in insulin-stimulated PI3K activity and a slightly smaller impairment of MAPK activation. However, these biochemical consequences were not sufficient to affect insulin-stimulated glucose transport over a wide range of insulin concentrations. Thus, our results suggest that PTP1B overexpression alone is unlikely to have significant effects on glucose disposal in fat cells. Moreover, our results support other recent studies using very different approaches that show that relatively small amounts of insulin-stimulated PI3K activity are sufficient for maximal insulin stimulation of glucose uptake (35, 36).

We chose to study PTP1B because of the PTPs that are overexpressed in insulin-resistant states, PTP1B is perhaps the most promising candidate for a mediator of the pathogenesis of insulin resistance. In overexpression studies, PTP1B interacts directly with the IR (37), and in vitro it interacts with IRS-1 (38). The interaction of PTP1B and IRS-1 appears to be facilitated by the binding of PTP1B to the adaptor protein Grb2 (38). Studies in rat fibroblasts demonstrate that PTP1B overexpression decreases the phosphorylation of IR and IRS-1 (23), again suggesting an inhibitory action on insulin-stimulated pathways. Transient transfection studies in rat adipocytes argued that PTP1B overexpression may decrease cell surface GLUT4 (24). Most compellingly, Elchebly et al. (25) recently reported that PTP1B knockout mice exhibit significantly enhanced whole body insulin sensitivity, which correlates with enhanced and/or sustained (depending on the tissue studied) tyrosyl phosphorylation of the IR and IRS1 in response to insulin stimulation. Interestingly, in contrast to the enhanced insulin sensitivity in muscle and liver, insulin-stimulated IR and IRS-1 tyrosyl phosphorylation were unaffected in adipocytes of PTP1B knockout mice (25). Moreover, in studies in mice bearing a different targeted mutation of the PTP1B gene (39) we found enhanced whole body insulin-stimulated glucose disposal but no effect on insulin-stimulated glucose uptake in isolated adipocytes (data not shown). Taken together, these findings suggest that PTP1B may not be a physiologically important mediator of normal insulin signaling in fat cells, whereas it appears to be rate-limiting for insulin action in other insulin target tissues such as liver and muscle (25, 39). Nevertheless, it remained possible that when overexpressed as in obese humans and rodents, PTP1B might significantly antagonize insulin action even in fat cells. Our current results demonstrate that overexpression of PTP1B significantly impairs multiple components of the insulin signal transduction pathway in fat cells. However, these effects do not result in impaired insulin-stimulated glucose uptake.

The lack of effect of PTP1B overexpression on insulin-evoked glucose uptake, even though it diminishes insulin-stimulated IR and IRS-1 tyrosyl phosphorylation and PI3K activation by 50-60%, has several interesting implications. First, <45% of IR and IRS-1 phosphorylation appears to be required for maximal insulin-stimulated glucose transport in adipocytes. Several studies have reported greater than 90% "spare" IRs in adipocytes, which could account for the fact that a near complete decrease in insulin receptor phosphorylation may be necessary to impair maximally insulin-stimulated glucose transport (40). However, spare receptors are unlikely to explain the lack of effect on glucose transport at submaximal insulin concentrations. Furthermore, the observation that less than half of maximal IRS-1 phosphorylation is required for maximal effects of insulin on glucose transport is a newer concept (41). Similar conclusions were reached in a recent study in which the protein tyrosine binding or Shc and IRS-1 NPX4 binding domains of IRS-1 were expressed in HIRcB fibroblasts and 3T3 L1 adipocytes, leading to an 80-90% decrease in IRS-1 tyrosyl phosphorylation but no effect on insulin-stimulated glucose transport (35).

Our data, together with that of Staubs et al. (36), also suggest that <50% of maximal IRS-1-associated PI3K activity is required for maximal insulin-stimulated glucose transport. Indeed, in the latter study, even in the presence of a 90% decrease in insulin-stimulated PI3K activity, insulin fully stimulated glucose uptake. Conversely, Krook et al. (42) expressed mutant insulin receptors in Chinese hamster ovary cells and found significantly increased insulin-stimulated IRS-1 tyrosine phosphorylation and activation of PI3K activity but markedly impaired metabolic and mitogenic effects of insulin. Taken together, these studies suggest that phosphorylation of IRS-1 and activation of PI3K may be neither necessary nor sufficient for many of the biologic effects of insulin. In this study, we considered the possibility that another insulin-stimulated pathway not involving IRS1 could be important for the metabolic effects of insulin. Therefore, we also measured antiphosphotyrosine-associated PI3K and found a 50-60% reduction in adipocytes overexpressing PTP1B, similar to the effect we saw on IRS-1-associated PI3K activity. Furthermore, our preliminary results in isolated adipocytes from mice with targeted disruption of IRS1 or IRS2 indicate that IRS1, and not IRS2, is important for a normal glucose transport response to insulin in adipocytes (43).

Our current data and the studies cited above (35, 36) indicate that only minimal levels of PI3K activation appear to be sufficient to evoke maximal insulin-stimulated glucose transport. Potentially, a specific pool of PI3K, perhaps operating at a specific intracellular location, may be needed to stimulate glucose transport. Such a location does not appear to be the GLUT4 vesicle itself but is more likely to be another compartment in the microsomal or cytoskeletal fractions (44-48).

Our data also demonstrate that submaximal IRS-1-associated PI3K activity is sufficient to maximally activate Akt. These findings concur with our recent study in humans with type 2 diabetes. In vivo insulin administration resulted in a 50% lower stimulation of PI3K activity in muscle of diabetic subjects compared with controls, but activation of Akt isoforms was normal (49). Also in agreement, overexpression of a dominant negative mutant of dynamin in H4IIE hepatoma cells resulted in a 50% decrease in insulin-stimulated IRS-1-associated PI3K activity, yet Akt activity remained unchanged (50). Thus, in multiple insulin target cells, partial stimulation of PI3K activity is sufficient for full activation of Akt.

It is possible that overexpression of PTP1B might lead to an increased rate of insulin receptor dephosphorylation when insulin is removed. However, because PI3 kinase activity peaks rapidly and transiently before maximal effects on IR phosphorylation or tyrosine kinase activity occur (34, 51, 52) and because little PI3 kinase stimulation appears to be necessary for full stimulation of glucose transport or Akt activation, the potential physiological effects of an enhanced rate of IR dephosphorylation would be best studied in vivo. Importantly, in the current study, even when insulin signaling is substantially impaired because of PTP1B overexpression, there is no impact on glucose transport or Akt activation.

Accumulating evidence strongly suggests that the IR and IRS-1 appear to be substrates for PTP1B in vitro (38) and in vivo (25). However, the effectiveness of PTP1B to reduce IR and IRS-1 tyrosyl phosphorylation and/or insulin-stimulated PI3K activation appears to differ in different cell types. When expressed at normal levels, PTP1B may not play a physiologically important role in dephosphorylation of IR and IRS1 in adipocytes in contrast to muscle and liver (25). However, it is surprising that, even in the presence of massive overexpression of PTP1B in the current study (e.g. in adipocytes transduced for longer time periods with the PTP1B adenovirus), there is only an ~55-60% decline in IR and IRS-1 tyrosyl phosphorylation in adipocytes. Under these conditions, total cellular PTP activity is increased by 5-6-fold, indicating that the overexpressed PTP1B now constitutes a major cellular PTP activity in transduced cells. These data suggest that factors other than the mere ability of PTP1B to dephosphorylate the IR and/or IRS1 determine whether, and the extent to which, PTP1B plays this role in intact insulin target cells. The intracellular location of PTP1B is one possible explanation. Previous work indicates that PTP1B resides predominantly in intracellular membranes, especially the endoplasmic reticulum, in tissue culture cells (31). It is unclear how IR molecules on the plasma membrane gain access to endoplasmic reticulum-bound PTP1B following insulin binding. However, perhaps the IR trafficking pathway is different in adipocytes and other insulin responsive tissues.

Finally, although several studies have found that PTP1B levels and activity are increased in adipose tissue from insulin-resistant rodents or humans (see Introduction), our data indicate that overexpression of PTP1B alone most likely does not impair insulin-stimulated glucose transport in adipocytes. Our results contrast with those of Chen et al. (24), who transiently overexpressed PTP1B in rat adipocytes and noted a decrease in insulin-stimulated glucose uptake. Beyond the considerable differences in methodology between the two studies, one possible explanation for the different conclusions relates to the effects of PTP1B on basal glucose transport in the transient transfection studies. In those studies (24), cell surface GLUT4 in PTP1B-overexpressing cells was decreased significantly in the basal state, such that the increment in insulin-stimulated GLUT4 was very similar between the control and PTP1B overexpressing cells. Thus, it appears that the major effect of PTP1B in transiently transfected rat adipocytes is on basal rather than insulin-stimulated GLUT4 translocation.

Our data do not preclude the possibility that PTP1B overexpression may decrease insulin sensitivity in other insulin responsive tissues, such as muscle and/or liver. Also, because PTP1B overexpression, at least at high levels, can diminish important aspects of insulin signaling in fat cells, it remains possible that in the presence of other overexpressed PTPs, PTP1B may contribute to insulin resistance even in fat cells. Because other PTPs (e.g. leukocyte antigen related and Src homology phosphatase 2) have also been found to be overexpressed in adipocytes (and skeletal muscle) in insulin-resistant states, studies in which combinations of PTPs are expressed in various insulin responsive tissues will be required to fully determine the contribution of this up-regulation of PTPs to the pathogenesis of insulin resistance.

	ACKNOWLEDGEMENTS

We thank Drs. C. Newgard for adenovirus plasmids and C. R. Kahn, M. F. White, J. Blenis, K. Siddle, B. Thorens, and H. Haspel for antibodies.

	FOOTNOTES

^* This work was supported by the National Institutes of Health Grants RO1 DK-43051 and RO1 CA 49152.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.

^§ Contributed equally to this work.

^¶ Supported by a Sarnoff Foundation Fellowship and a grant from the American Diabetes Association.

Supported by the Deutsche Forschungsgemeinschaft Grant Fr 1095/11 and Physician Scientist Award AG 00294 from the NIA, National Institutes of Health.

^** Supported by the Uehara Memorial Foundation Research Fellowship and a mentor based fellowship from the American Diabetes Association.

^¶¶ To whom correspondence may be addressed: Beth Israel Deaconess Medical Center, 330 Brookline Ave., Boston, MA 02215. Tel.: 617-667-2823; Fax: 617-667-0610.

To whom correspondence may be addressed: Diabetes Unit, Beth Israel Deaconess Medical Cntr., 99 Brookline Ave., Boston, MA 02215. Tel.: 617-667-5422; Fax: 617-667-2927.

Published, JBC Papers in Press, April 4, 2000, DOI 10.1074/jbc.M908392199

	ABBREVIATIONS

The abbreviations used are: IRS, insulin receptor substrate; PI3K, phosphoinositide 3-kinase; PTP, protein-tyrosine phosphatase; IR, insulin receptor; MAPK, mitogen-activated protein kinase; DMEM, Dulbecco's modified Eagle's medium; HA, hemagglutinin; pNPP, p-nitrophenyl phosphate; PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; MAP, mitogen-activated protein; PI3, phosphoinositide 3; GST, glutathione S-transferase; Erk, extracellular signal-regulated kinase; NS, not significant.

	REFERENCES

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