©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Osmotic Loading of Neutralizing Antibodies Demonstrates a Role for Protein-tyrosine Phosphatase 1B in Negative Regulation of the Insulin Action Pathway (*)

(Received for publication, March 20, 1995; and in revised form, June 7, 1995)

Faiyaz Ahmad Pei-Ming Li Joseph Meyerovitch (§) Barry J. Goldstein (¶)

From the Dorrance H. Hamilton Research Laboratories, Division of Endocrinology and Metabolic Diseases, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Protein-tyrosine phosphatases (PTPases) have been postulated to balance the steady-state phosphorylation and the activation state of the insulin receptor and its substrate proteins. To explore whether PTP1B, a widely expressed, non-receptor-type PTPase, regulates insulin signaling, we used osmotic shock to load rat KRC-7 hepatoma cells with affinity-purified neutralizing antibodies that immunoprecipitate and inactivate the enzymatic activity of recombinant rat PTP1B in vitro. In cells loaded with PTP1B antibody, insulin-stimulated DNA synthesis and phosphatidylinositol 3`-kinase activity were increased by 42% and 38%, respectively, compared with control cells loaded with preimmune IgG (p < 0.005). In order to characterize the potential site(s) of action of PTP1B in insulin signaling, we also determined that insulin-stimulated receptor autophosphorylation and insulin receptor substrate 1 tyrosine phosphorylation were increased 2.2- and 2.0-fold, respectively, and that insulin-stimulated receptor kinase activity toward an exogenous peptide substrate was increased by 57% in the PTP1B antibody-loaded cells. Osmotic loading did not alter the cellular content of PTP1B protein, suggesting that the antibody acts in the cell by sterically blocking catalytic interactions between PTP1B and its physiological substrates. These studies demonstrate that PTP1B has a role in the negative regulation of insulin signaling and acts, at least in part, directly at the level of the insulin receptor. These results also show that insulin signaling can be enhanced by the inhibition of specific PTPases, a maneuver that has potential clinical relevance in the treatment of insulin resistance and Type II diabetes mellitus.


INTRODUCTION

As recent studies have made great advances in our understanding of reversible tyrosine phosphorylation in the cellular insulin action pathway, interest has also grown in the role of protein-tyrosine phosphatases (PTPases) (^1)in balancing the steady-state level of tyrosine phosphorylation of proteins involved in insulin signal transduction(1, 2) . PTPases can potentially impact on insulin signaling at several levels, including dephosphorylation of the active (autophosphorylated) form of the insulin receptor, which attenuates the receptor kinase activity, and dephosphorylation of the protein-tyrosine residues of insulin receptor substrates such as IRS-1, IRS-2, and Shc, which will modulate postreceptor pathways of insulin action(3) .

The PTPases constitute a large superfamily of transmembrane (receptor-type) and intracellular (non-receptor-type) enzymes involved in a variety of regulatory processes(4, 5) . Recent studies exploring the role of individual PTPases in cellular regulation have demonstrated that their effects may be complex, and various PTPase homologs may have positive or negative effects on cell activation, cell growth, or differentiation. For example, CD45, which activates the p56 tyrosine kinase by dephosphorylation of a specific phosphotyrosine residue (6) and the PTPase Cdc25, which initiates the induction of mitosis by the tyrosine dephosphorylation and activation of p34(7) . These findings have enhanced our view of the cellular roles of PTPases, in considering not only the identification of PTPases that might impact on various signaling pathways, but also whether the influence of their PTPase activity is stimulatory or inhibitory.

Since multiple PTPase enzymes are expressed in insulin-sensitive tissues, there is a need to establish the significance and physiological role of individual PTPases in liver, fat, and muscle tissue. For the insulin signaling pathway, only a few PTPases have been shown to affect insulin action at the receptor or post-receptor sites. In hepatoma cells, using expression of an antisense RNA construct, we have recently demonstrated that the transmembrane PTPase LAR has a negative regulatory effect on insulin receptor autophosphorylation and receptor kinase activity(8) . The SH2 domain containing PTPase, SH-PTP2, or Syp, has also been shown by several groups to have a positive effect on post-receptor insulin signaling, although the mechanism and site of interaction with the insulin action pathway has not been identified(9, 10, 11, 12) .

PTP1B is a particularly important candidate for involvement in insulin signaling since it is an abundant intracellular PTPase that is widely expressed in insulin-sensitive tissues(2) . As the first PTPase to be purified to homogeneity and characterized biochemically, early studies of PTP1B demonstrated that it was able to efficiently dephosphorylate the insulin receptor in vitro(13, 14) . Moreover, microinjection of a truncated form of PTP1B from human placenta into Xenopus oocytes diminished insulin-stimulated oocyte maturation and S6 peptide phosphorylation(15, 16) . In the present work, we used an osmotic loading technique to inhibit PTP1B activity in situ with a neutralizing antibody in order to explore its potential role in the regulation of insulin signaling in hepatoma cells. The results demonstrate that PTP1B has an essential role in the negative regulation of insulin signaling and acts by balancing insulin-stimulated kinase activity at the level of the receptor as well as influencing distal components of the insulin action cascade.


EXPERIMENTAL PROCEDURES

Materials

A well-differentiated subline of the H4-EII-C3 rat hepatoma cell line (KRC-7) was kindly provided by Dr. John Koontz (University of Tennessee)(17) . Cells were maintained in DMEM containing 10% (v/v) fetal bovine serum (Sigma). Prestained molecular size markers were from Bio-Rad. Other materials were obtained as indicated and were of the highest grade available.

Osmotic Loading of PTP1B Antibodies

Antiserum to PTP1B was developed by coupling a peptide antigen corresponding to amino acids 42-56 of the rat PTP1B sequence (18) to keyhole limpet hemocyanin and injecting the conjugate subcutaneously into rabbits(19) . After several cycles of boosting, antibody reactive toward PTP1B was immunopurified by adsorption and elution from an affinity column of recombinant full-length rat PTP1B (14) coupled to Affi-Gel 10 (Bio-Rad)(19) .

Intracellular loading of PTP1B antibodies or control preimmune rabbit IgG was achieved by promoting uptake of extracellular proteins by incubation in a hypertonic medium, followed by lysis of cytoplasmic pinosomes in a hypotonic solution by the method of Okada and Rechsteiner(20) . Briefly, subconfluent KRC-7 cells were washed with phosphate-buffered saline and then incubated for 10 min in a hypertonic medium containing 0.5 M sucrose, 10% (w/v) polyethylene glycol 1000, 10% (v/v) fetal calf serum, and PTP1B antibody or pre-immune rabbit IgG (30 µg/ml) in DMEM buffered with 25 mM HEPES, pH 6.8. The cells were then rapidly rinsed with a hypotonic solution of diluted DMEM:water (6:4) buffered with 25 mM HEPES, pH 6.8, and incubated in the hypotonic medium for 2 min. After rinsing 3 times with normal culture medium, the cells were allowed to recover for 6 h before the assays were performed. Viability of the hepatoma cells was demonstrated by exclusion of 0.4% (w/v) trypan blue dye (Sigma).

Immunoblot Analysis of PTP1B Abundance and Insulin-stimulated Protein-tyrosine Phosphorylation

After recovery from osmotic loading, hepatoma cells were stimulated with 100 nM insulin for 1 min, and the cells were lysed into a buffer consisting of 25 mM HEPES, pH 7.0, with 1% Triton X-100, 1 mM dithiothreitol, 25 µg/ml phenylmethylsulfonyl fluoride, 1 unit/ml aprotinin, and 25 µg/ml leupeptin at 4 °C. The homogenate was cleared by microcentrifugation, and 60 µg of cell lysate protein was fractionated on 10% polyacrylamide gels containing SDS (21) in a minigel apparatus (Bio-Rad). In some experiments, cell lysate proteins were then immunoprecipitated wih PTP1B antibody with and without the addition of goat anti-rabbit IgG (Sigma) and Trisacryl-Protein A (Pierce), prior to fractionation by gel electrophoresis. For the visualization of insulin receptor and IRS-1 phosphorylation, 7.5% polyacrylamide gels were used.

After gel separation, proteins were transferred to nitrocellulose filters (0.45-µ pore size) at 100 V for 3 h in buffer containing 20% (v/v) methanol, 25 mM Tris base and 192 mM glycine at pH 8.3. Nitrocellulose membranes were then incubated in blocking buffer containing 150 mM NaCl, 0.05% (v/v) Nonidet P-40, 5% (w/v) bovine serum albumin, 1% (w/v) ovalbumin, 0.01% (w/v) sodium azide, and 10 mM Tris, pH 7.4, for 1 h at room temperature with rocking. Fresh blocking solution was then applied containing 1.0 µg/ml affinity-purified PTP1B antibody or 1.1 µg/ml anti-Tyr(P) antibodies (22) with rocking for 2 h. Membranes were washed 3 times for 10 min in blotting buffer without antibodies, followed by incubation with 2 µCi of I-Protein A (30 mCi/mg) (ICN Biomedicals Inc., Irvine, CA) for 1 h at room temperature followed by 3 additional 10-min washes with blotting buffer. Immunoreactive proteins were visualized by direct PhosphorImager analysis (Molecular Dynamics). Protein was assayed by the method of Bradford(23) .

Insulin Stimulation of DNA Synthesis

Hepatoma cells were grown to 50-60% confluence and made quiescent by incubation for 48 h in DMEM containing 0.1% (w/v) insulin-free bovine serum albumin. The cells were then loaded with PTP1B antibody or control IgG, allowed to recover for 6 h, and the medium was exchanged for DMEM with 0.1% (w/v) bovine serum albumin with or without 100 nM insulin for 1 h. DNA synthesis was then measured by the addition of [^3H]thymidine (98 Ci/mmol; Amersham) to 1 µCi/ml for a 1-h incubation(24) . Cells were washed 3 times with DMEM, 5% (w/v) ice-cold trichloroacetic acid was added, and the cells were maintained in trichloroacetic acid overnight at 4 °C. Trichloroacetic acid-insoluble material was then recovered by dissolving in 1 M NaOH, neutralizing with 1 M HCl, and the radioactivity was determined by scintillation counting.

Insulin-stimulated Phosphatidylinositol 3`-Kinase Activity

In vitro phosphorylation of phosphatidylinositol was performed in immune complexes as described (25) . After recovery from osmotic loading, hepatoma cells were treated with 100 nM insulin for 5 min, washed once with ice-cold phosphate-buffered saline and twice with 20 mM Tris-HCl, pH 7.5, containing 137 mM NaCl, 1 mM MgCl(2), 1 mM CaCl(2), and 100 µM Na(3)VO(4), before solubilization in the final wash buffer also containing 1% (v/v) Nonidet P-40 and 10% (v/v) glycerol. Insoluble material was removed by centrifugation at 13,000 g for 10 min, and the supernatant was incubated with anti-phosphotyrosine antibody overnight at 4 °C. Immune complexes were precipitated from the supernatant with Protein A-coupled Trisacryl (Pierce), washed, and resuspended in 50 µl of 10 mM Tris-HCl, pH 7.5, containing 100 mM NaCl and 1 mM EDTA. After adding 10 µl of 100 mM MnCl(2) and 10 µl of 2 µg/µl phosphatidylinositol (sonicated in 10 mM Tris-HCl, pH 7.5, containing 1 mM EGTA), the phosphorylation reaction was initiated by adding 10 µl of 440 µM ATP containing 30 µCi of [-P]ATP and continued for 10 min at 22 °C. The reaction was stopped with 20 µl of 8 N HCl and 160 µl of CHCl(3):methanol (1:1), the samples were centrifuged, and the lower organic phase was applied to a silica gel thin layer chromatography plate coated with potassium oxalate. The plates were developed in CHCl(3):CH(3)OH:H(2)O:NH(4)OH (60:47:11.3:2), dried, and assayed by PhosphorImager analysis (Molecular Dynamics).

Insulin Receptor Tyrosine Kinase Activity

Osmotically loaded hepatoma cells were stimulated with 100 nM insulin for 5 min and lysed into 50 mM HEPES buffer, pH 7.4, containing 2 mM sodium orthovanadate, 100 mM NaF, 2 mM EDTA, 1% (v/v) Triton X-100, 0.01% (w/v) aprotinin, and 30 µg/ml phenylmethylsulfonyl fluoride at 4 °C. The lysate was centrifuged at 10,000 g for 20 min, and the supernatant was passed through a wheat germ agglutinin affinity column to partially purify the insulin receptors(26) . Receptor kinase activity was assayed using 100 µl (50 µg of protein) of the affinity column eluate by incubating with [-P]ATP (500 µM, 2 µCi) in the presence of 2.5 mg/ml poly(Glu:Tyr) (4:1) in 50 mM Tris-HCl buffer, pH 7.4, containing 10 mM MgCl(2) and 2.5 mM MnCl(2)(27) . After 20 min at room temperature, the reaction was stopped by adding ice-cold 10% (w/v) trichloroacetic acid and 3 ml of 10% (w/v) bovine serum albumin as carrier. The precipitated proteins were washed in trichloroacetic acid and dissolved in 1 ml of 1 N NaOH, and the radioactivity was determined by liquid scintillation counting. Tyrosine kinase activity was expressed as picomoles of P incorporated per mg of poly(Glu:Tyr) per min per mg of protein.

Statistical Analysis

Quantitative data were calculated from the mean ± S.E. for a minimum of 4 separate determinations. Statistical calculations were based on analysis of variance for comparison of more than two groups with Bonferroni's correction for determination of significance. Student's t test was used for comparing two samples.


RESULTS

Purification and Characterization of PTP1B Antibodies

Anti-peptide PTP1B antibodies were affinity-purified over a column of recombinant rat PTP1B and tested for their reaction specificity with a lysate of KRC-7 cell proteins (Fig. 1). Immunoprecipitation of lysate proteins with the PTP1B antibodies followed by Western blotting of the immunoisolated proteins with the same antibody revealed a single band with an M(r) of approximately 50,000, as we have demonstrated for the full-length PTP1B protein expressed in rat tissues(28, 29) . The affinity-purified antibodies were also able to immunoprecipitate recombinant PTP1B expressed in Escherichia coli. The interaction of the antibody with the enzyme substrate also proved it to be neutralizing in that it completely abolished the in vitro activity of the PTP1B enzyme (data not shown).


Figure 1: Specificity of anti-PTP1B antibody for PTP1B in KRC-7 hepatoma cell lysate proteins. After hepatoma cells were osmotically loaded with affinity-purified PTP1B antibody or preimmune IgG and allowed to recover, aliquots of cell lysates (60 µg of protein) were immunoprecipitated with PTP1B antibody and immunoblotted with the PTP1B antibody as described under ``Experimental Procedures.'' Molecular size markers were used to determine the mass of PTP1B to be approximately 50 kDa. Lanes 1-2 and 3-4 represent replicate samples of immunoprecipitated hepatoma cell proteins.



Cell Viability and Uptake of PTP1B Antibodies following Osmotic Loading

As assessed by exclusion of trypan blue dye, 70-75% of the cells remained viable following the osmotic loading of either PTP1B antibody or preimmune IgG. Furthermore, the ability of the antibody-loaded cells to perform a variety of metabolic functions including macromolecule synthesis and phosphorylation reactions requiring ATP attests to their viability after the osmotic shock (see below).

In order to demonstrate that the PTP1B antibody was taken up by the KRC-7 cells after osmotic loading and was associated with cellular PTP1B protein, cell lysate samples were precipitated with a goat anti-rabbit IgG second antibody and Trisacryl-Protein A and subjected to immunoblotting with the PTP1B antibody (Fig. 2). The second antibody was able to immunoprecipitate the loaded PTP1B antibody still complexed to the enzyme from its intracellular localization only in the cells osmotically loaded with the PTP1B antibody. Since PTP1B was not immunoprecipitated from the cells loaded with the preimmune IgG, the interaction between the loaded antibody and cellular PTP1B protein is shown to be specific and of high affinity and sufficient duration to continue to be present well after the 6-h recovery period.


Figure 2: Demonstration of PTP1B antibody uptake by the hepatoma cells after osmotic loading and recovery. Hepatoma cells were loaded with PTP1B antibody (lanes 5-7) or preimmune IgG (lanes 2-4) and allowed to recover. Aliquots of cell lysates (60 µg of protein) were then incubated with Trisacryl-Protein A alone (lanes 2 and 5) or Trisacryl-Protein A and goat anti-rabbit IgG (lanes 3-4 and 6-7). The immunoprecipitated proteins bound to the Trisacryl beads were washed and subjected to gel electrophoresis and immunoblotting with the PTP1B antibody. Lane 1 represents control cells not loaded with antibody, from which a 60-µg protein lysate was immunoprecipitated with PTP1B antibody along with Trisacryl-Protein A and the second antibody to quantitate the total amount of PTP1B present in comparison with the PTP1B antibody or IgG-loaded cells.



Of the total mass of PTP1B in the KRC-7 cells directly precipitated with PTP1B antibody, 70% of the loaded PTP1B antibody-PTP1B enzyme complex is immunoprecipitable with the second antibody and Protein A (Fig. 2). When total protein lysates from the osmotically loaded cells were directly immunoprecipitated with PTP1B antibody and immunoblotted, there was an apparent decrease of 35-40% in amount of immunoprecipitable PTP1B in the cells loaded with the PTP1B antibody (Fig. 1). In order to determine whether the PTP1B antibody loading affected the cellular content of PTP1B, immunoblotting was also performed with aliquots of total cell protein from the loaded cells that were fractionated on gels without prior immunoprecipitation. The content of PTP1B protein in the cells loaded with PTP1B antibody was not decreased, but on average was 10-15% higher than the PTP1B content of the control cells (Fig. 3). Thus, the inability to immunoprecipitate a portion of the PTP1B mass from the cells loaded with PTP1B antibody results from occupancy of PTP1B epitopes by the neutralizing antibody which remains associated with the PTP1B protein in the cells.


Figure 3: Quantitation of PTP1B mass in hepatoma cells loaded with PTP1B antibody or rabbit IgG. Hepatoma cells were loaded with PTP1B antibody (lanes 4-6) or preimmune IgG (lanes 1-3) and allowed to recover. Replicate samples of cell lysate protein (60 µg of protein) were then fractionated on 10% (w/v) polyacrylamide gels, and immunoblotting with PTP1B antibody was performed.



Effects of PTP1B Antibody Loading on Cellular Insulin Signaling

Thymidine Incorporation

As an effect of insulin on cell growth and mitogenesis, the incorporation of thymidine into DNA was studied in the antibody-loaded KRC-7 cells. In the control cells, thymidine incorporation was stimulated 2.4-fold by 100 nM insulin in serum-free medium (Fig. 4). In the cells loaded with PTP1B antibody, insulin-stimulated thymidine incorporation was increased by 42% compared to the control cells (p = 0.002). No significant change in the basal level of thymidine incorporation was observed.


Figure 4: Effect of PTP1B antibody loading in hepatoma cells on insulin stimulation of DNA synthesis. Hepatoma cells were loaded with PTP1B antibody or control IgG, allowed to recover for 6 h, and the cells were treated with or without 100 nM insulin for 1 h. DNA synthesis was then measured by adding [^3H]thymidine for an additional 1-h incubation as described under ``Experimental Procedures.'' Cells in individual 35-mm sample wells were washed, and the incorporation of ^3H into trichloroacetic acid (TCA)-insoluble material was determined by scintillation counting.



Activation of Phosphatidylinositol 3`-Kinase Activity

As a more proximal effect of insulin action, the activated insulin receptor kinase phosphorylates its major intracellular substrate, IRS-1, which in turn promotes the association of phosphatidylinositol 3`-kinase with IRS-1 and stimulates its enzyme activity(1) . After loading of preimmune IgG or PTP1B antibodies, there was no effect on the level of basal phosphatidylinositol 3`-kinase, which was undetectable in the absence of insulin stimulation (Fig. 5). However, following stimulation of quiescent cells with 100 nM insulin, phosphatidylinositol 3`-kinase activity was significantly increased by 38% in the PTP1B antibody-loaded cells compared to control (p = 0.0038).


Figure 5: Effect of PTP1B antibody loading in hepatoma cells on insulin-stimulated phosphatidylinositol 3`-kinase activity. Hepatoma cells were loaded with PTP1B antibody or control IgG, allowed to recover for 6 h, and the cells were treated with or without 100 nM insulin for 5 min. In vitro phosphorylation of phosphatidylinositol was performed in anti-phosphotyrosine immune complexes of the hepatoma cell lysates as described under ``Experimental Procedures.''



Insulin-stimulated Tyrosine Phosphorylation of Its Receptor and IRS-1

In order to determine whether cellular PTP1B directly affects the phosphorylation of the insulin receptor, which might initiate its negative influence on post-receptor pathways, we examined insulin-stimulated receptor autophosphorylation, activation of the receptor kinase activity toward an exogenous substrate, and phosphorylation of IRS-1 in the antibody-loaded cells. After osmotic loading, cells were treated with insulin for 1 min, and immunoblotting with anti-phosphotyrosine antibodies was performed to visualize the protein phosphotyrosine content of the 95-kDa insulin receptor beta-subunit and the 180-185-kDa IRS-1 protein. As shown in Fig. 6, addition of insulin rapidly increased the phosphorylation of its receptor and activated the receptor kinase activity, as evidenced by a significant increase in the tyrosine phosphorylation of IRS-1. Compared to cells loaded with the preimmune IgG, osmotic loading of PTP1B antibodies prior to insulin stimulation resulted in a 2.2-fold increase in insulin receptor phosphorylation (p = 0.02) along with a commensurate 2.0-fold increase in IRS-1 phosphorylation (p = 0.006). No alteration in the basal level of insulin receptor or IRS-1 tyrosine phosphorylation was observed.


Figure 6: Insulin-stimulated receptor autophosphorylation and IRS-1 phosphorylation in hepatoma cells after osmotic loading of control IgG (lanes 1-3) or PTP1B antibody (lanes 4-6). After recovery from osmotic antibody loading, control KRC-7 hepatoma cells without insulin treatment (lanes 1 and 4) and cells treated with 100 nM insulin for 1 min (duplicate samples in lanes 2-3 and 5-6) were extracted into cell lysis buffer, and aliquots of cell protein were separated by SDS-gel electrophoresis and analyzed by immunoblotting with affinity-purified rabbit polyclonal antiphosphotyrosine antibody, as described under ``Experimental Procedures.'' The migration position of IRS-1 (180 kDa) and the insulin receptor beta-subunit (95 kDa) is shown.



Insulin-stimulated Receptor Kinase Activity

To directly assess whether inhibition of cellular PTP1B activity affected the activation of the insulin receptor kinase enzyme, the receptor kinase activity was determined by measuring the phosphorylation of an exogenous substrate, poly(Glu:Tyr) (4:1). As shown in Fig. 7, the basal tyrosine kinase activity was slightly higher in the cells loaded with the PTP1B antibody compared to preimmune IgG (34.9 ± 0.8 pmol/mg poly(Glu:Tyr)/min/mg of protein) and PTP1B antibody (42.0 ± 3.2 pmol/mg poly(Glu:Tyr)/min/mg of protein), but the difference was not statistically significant. After insulin stimulation, however, the receptor tyrosine kinase activity was significantly elevated in the PTP1B antibody-loaded cells, 57% higher than the insulin-stimulated kinase activity in the cells loaded with preimmune IgG (p = 0.0006). These results demonstrate that inhibition of PTP1B activity in situ has a direct effect on the activity of the insulin receptor kinase, which is translated into further activation of downstream pathways of insulin signal transduction.


Figure 7: Insulin-stimulated tyrosine kinase activity of insulin receptors isolated from hepatoma cells osmotically loaded with control IgG or PTP1B antibody. After loading and cell recovery, hepatoma cells were stimulated with 100 nM insulin for 5 min. Insulin receptors were partially purified by wheat germ agglutinin-agarose lectin chromatography under conditions that preserve the receptor phosphorylation state, and the tyrosine kinase activity of the isolated receptors was assayed using a poly(Glu:Tyr) (4:1) substrate as described under ``Experimental Procedures.'' Tyrosine kinase activity was expressed as picomoles of P incorporated per mg of poly(Glu:Tyr) per min per mg of protein.




DISCUSSION

PTP1B is a widely expressed enzyme that was first identified as a prominent PTPase in the cytosol fraction of placenta(30) . Since it is an abundant enzyme found in a variety of insulin-sensitive tissues(14, 31) , early studies by Cicirelli et al.(16) and Tonks et al.(15) implicated PTP1B as a potential regulator of insulin signaling by demonstrating that microinjection of Xenopus oocytes with a purified, truncated form of the activated PTP1B protein was able to block insulin-stimulated ribosomal S6 peptide phosphorylation and retard insulin-induced oocyte maturation. These studies have prompted further investigations into the potential role of the native PTP1B enzyme in the regulation of tyrosine phosphorylation induced by the insulin receptor and other growth factors.

In recent studies exploring the physiology of PTP1B, this intracellular PTPase has also been shown to have a complex intracellular itinerary which plays an integral role in determining its subcellular localization and might be expected to influence its access to physiological substrates in the cell. Cloning of the PTP1B cDNA revealed that the full-length protein has a C-terminal segment that directs the association of at least a portion of the native protein with intracellular membranes either through a hydrophobic interaction or by attachment to a noncatalytic subunit(18, 31, 32, 33, 34, 35) . The subcellular localization of PTP1B is also determined by cellular mechanisms that regulate the proteolytic cleavage of the C-terminal segment, which releases a soluble, truncated form of the protein as demonstrated in an activated platelet model(36) . A substantial portion of the uncleaved, full-length form of PTP1B, however, is also found in the cytosol of rat tissues and cultured cells(28, 37) , raising the possibility that it interacts with potential substrates in both the soluble and particulate fractions of the cell.

In order to closely examine the potential involvement of PTP1B in insulin signaling, in the present study we disrupted the activity of PTP1B in situ in intact cells and evaluated in detail its influence on potential cellular targets and certain metabolic pathways. We focused on insulin-sensitive hepatoma cells, in which the intracellular activity of PTP1B was inhibited by osmotic loading of neutralizing antibodies. Various aspects of insulin receptor signaling were then evaluated to determine at which points along the insulin signaling cascade PTP1B might affect cellular responses to insulin. This approach is complementary to the approach taken by Lammers et al.(38) who showed in a transfection model that overexpression of PTP1B in situ almost completely dephosphorylated insulin and insulin-like growth factor 1 receptor beta-subunits in the basal state and also reduced the phosphotyrosine content of the ligand-activated receptor beta-subunits to less than 50% of the control level. Our method of inhibiting the endogenous PTP1B activity would be expected to directly reduce the effects of PTP1B on cellular signaling processes. By inhibiting the activity of PTP1B in situ, these experiments avoid potential problems in studies employing high levels of PTPase overexpression which may alter their normal subcellular distribution or affect the post-translational regulation of the expressed enzymes by saturating protein processing enzymes or associated binding proteins.

Our results show that inhibition of PTP1B in situ has the initial effect of enhancing insulin signaling at the level of the insulin receptor kinase itself. Studies on the activation of the insulin receptor kinase have shown that phosphorylation of two tyrosines in the kinase domain, involving tyrosine 1158 and either tyrosine 1162 or 1163 occurs first, and that the partially phosphorylated receptors with mono- or bisphosphorylated kinase domains exhibit minimal activation of the beta-subunit kinase activity. Phosphorylation of the third tyrosyl residue in this so-called ``regulatory domain'' rapidly follows the bisphosphorylation stage and leads to full activation of the receptor kinase toward exogenous substrates(1) . In this way, the transition between bis- and trisphosphorylation in the receptor regulatory domain may be considered to be a discrete molecular ``switch,'' in which the steady-state level of phosphorylation in this region can determine the overall degree of receptor kinase activation(3) . The recently described crystal structure of the insulin receptor kinase domain has provided a structural picture that is consistent with these functional data(39) . In the basal (unphosphorylated) state, tyrosine 1162 is held in the active site and blocks the binding of both substrate and ATP. trans-Autophosphorylation of tyrosines in the regulatory domain can occur when tyrosine 1162 is disengaged by insulin binding and the activity of the receptor kinase is dramatically enhanced. Dephosphorylation of the activated receptor by PTPase action then allows tyrosine 1162 to return to its autoinhibitory position, and the receptor kinase is deactivated. By reducing the influence of PTP1B on insulin receptor dephosphorylation, the loading of neutralizing antibodies appears to augment the relative abundance of receptors in the fully activated, trisphosphorylated state, which results in enhanced receptor autophosphorylation and increased kinase activity toward the exogenous peptide substrate as well as increased IRS-1 phosphorylation in vivo.

The increased activation of the insulin signaling pathway in the PTP1B antibody-loaded cells also extends to several post-receptor effects, including enhanced IRS-1 phosphorylation, insulin-stimulated phosphatidylinositol 3`-kinase activity, and incorporation of thymidine into DNA. The increased tyrosine phosphorylation of IRS-1 could result from the enhanced insulin receptor kinase activation, diminished IRS-1 tyrosine dephosphorylation, or a combination of these two potentially independent effects in the PTP1B antibody-loaded cells. Augmentation of the activation of the phosphatidylinositol 3`-kinase most likely results from the increased IRS-1 tyrosyl phosphorylation with increased or prolonged association of the phosphatidylinositol 3`-kinase enzyme with cellular IRS-1, which activates the enzyme(40, 41) . The activation of cellular DNA synthesis by insulin is thought to proceed through parallel pathways that involve the activation of p21, mediated by binding of a Grb2-Sos complex to phosphorylated IRS-1 or Shc with activation of downstream serine/threonine kinases(1) , as well as the possible direct involvement of the activated phosphatidylinositol 3`-kinase itself in stimulating DNA synthesis(42) . The quantitative contribution of each of these pathways toward DNA synthesis may depend on the cell type under study. Inhibition of PTP1B activity in situ may influence DNA synthesis by enhancing the tyrosine phosphorylation of IRS-1, with increased distal signaling mediated by phosphatidylinositol 3`-kinase or p21 activation. Alternatively, a decrease in inhibition by PTP1B may affect the tyrosine phosphorylation state and activation of additional downstream signaling components, such as MAP kinase, which is activated at least in part by tyrosine phosphorylation(43, 44) .

While the present work focused on the regulation of insulin action by PTP1B, other studies have suggested that PTP1B may influence signaling pathways elicited by a number of growth factor receptors and oncogenic cellular tyrosine kinases. Transfection of PTP1B in vivo into fibroblasts along with a panel of protein-tyrosine kinases showed that, when highly overexpressed, PTP1B can dephosphorylate a wide range of receptors including the epidermal growth factor receptor, insulin-like growth factor 1 receptor, platelet-derived growth factor receptors (alpha and beta), the c-kit kinase, and the colony-stimulating factor 1 receptor as well as the insulin receptor (38) . Catalytically inactive PTP1B has also been shown to physically associate with specific autophosphorylation sites on the epidermal growth factor receptor(45) . In addition, transformation by overexpression of the neu oncogene can be suppressed by transfection of PTP1B into NIH3T3 cells(46) . Since the available data suggest that PTP1B influences a number of cellular pathways, it may have a more general role in the dephosphorylation of a variety of protein-tyrosine residues, in addition to its demonstrated effect on balancing reversible tyrosine phosphorylation in insulin signaling. Additional studies are necessary to explore the relative specificity of action of PTP1B on physiological substrates in various signaling pathways. In addition, the importance of cellular mechanisms that may modulate the activity of PTP1B on its cellular targets, including association of PTP1B with regulatory subunits, proteolysis of the C terminus, and serine phosphorylation of PTP1B, need to be studied in more detail(47, 48, 49) .

SH-PTP2 (Syp) is a widely expressed, intracellular PTPase with SH2 domains that has recently been shown to have a positive role in mitogenic signaling induced by insulin, insulin-like growth factor 1, and epidermal growth factor in studies employing dominant-negative enzymes or microinjection of reagents that block complex formation between its SH2 domains and endogenous substrates(9, 10, 11, 12) . These data are consistent with the recognition of SH-PTP2 as the mammalian homolog of the Drosophila csw gene product, which potentiates the action of the Drosophila c-raf homolog to transmit positively signals downstream of the torso receptor tyrosine kinase(50) . Although SH-PTP2 can associate with the insulin receptor in recombinant in vitro systems(51, 52) , the site of its involvement in insulin signaling in situ is unclear since it does not appear to interact directly with the insulin receptor in intact cells, and overexpression of catalytically active SH-PTP2 protein does not affect insulin signaling(10, 11, 53, 54) . SH-PTP2 complexes with IRS-1 by its SH2 domains, in a process that activates its intrinsic PTPase activity and is likely to play a role in the cellular effects of SH-PTP2(9, 55, 56) . However, the available data would suggest that the signaling potential of SH-PTP2 requires the formation of a complex that brings the active enzyme into close proximity with downstream signaling molecules and substrates in addition to IRS-1(9, 10) .

The present work provides strong evidence for the involvement of PTP1B in the negative regulation of insulin signaling in a physiologically relevant cell type. Our recent studies (^2)have also demonstrated that the transmembrane PTPase LAR negatively modulates insulin signal transduction by balancing the steady-state level of receptor kinase activity at the cell membrane(8) . Thus, insulin signaling is balanced at multiple levels by a number of PTPases, including the negative influence of PTP1B and LAR at the level of the receptor itself (and possibly also involving post-receptor pathways) and the positive influence of SH-PTP2, which acts at a downstream site that remains to be identified. Further studies will help elucidate the exact mechanism of the regulation of insulin action by these two enzymes as well as their possible involvement in defective insulin action in disease states. The results of the present study also demonstrate that insulin signaling can be enhanced by the specific inhibition of PTP1B, a maneuver that has potential clinical relevance in the treatment of insulin resistance and Type II diabetes mellitus.


FOOTNOTES

*
These studies were supported by National Institutes of Health Grant DK43396 (to B. J. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Div. of Pediatric Endocrinology, Chaim Sheba Medical Center, Tel Hashomer, 56261 Israel.

To whom correspondence and reprint requests should be addressed: Director, Div. of Endocrinology and Metabolic Diseases, Jefferson Medical College, Rm. 349 Alumni Hall, 1020 Locust St., Philadelphia, PA 19107-6799. Tel.: 215-955-1272; Fax: 215-923-7932; b\_goldstein{at}lac.jci.tju.edu.

(^1)
The abbreviations used are: PTPase, protein-tyrosine phosphatase; DMEM, Dulbecco's minimal essential medium; IRS-1, insulin receptor substrate 1; LAR, leukocyte common antigen-related.

(^2)
D. T. Kulas, B. J. Goldstein, and R. A. Mooney, submitted for publication.


ACKNOWLEDGEMENTS

We are grateful to Dr. Jon Backer for helpful discussions regarding the osmotic loading procedure and the phosphatidylinositol kinase assays and to Dr. C. Ronald Kahn for his support during the initial development of the PTP1B antibodies. We also thank Dr. John Koontz for generously providing the KRC-7 hepatoma cells.


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