Molecular and Cellular Biochemistry 221: 147–154, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.

147

Phosphorylation and activation of protein tyrosine phosphatase (PTP) 1B by insulin receptor Shrikrishna Dadke1, Anasua Kusari1 and Jyotirmoy Kusari1,2 1

Department of Physiology, 2Molecular and Cellular Biology Program, Tulane University School of Medicine, New Orleans, LA, USA

Received 1 February 2001; accepted 9 April 2001

Abstract We have previously reported a direct in vivo interaction between the activated insulin receptor and protein-tyrosine phosphatase1B (PTP1B), which leads to an increase in PTP1B tyrosine phosphorylation. In order to determine if PTP1B is a substrate for the insulin receptor tyrosine kinase, the phosphorylation of the Cys 215 Ser, catalytically inactive mutant PTP1B (CS-PTP1B) was measured in the presence of partially purified and activated insulin receptor. In vitro, the insulin receptor tyrosine kinase catalyzed the tyrosine phosphorylation of PTP1B. 53% of the total cellular PTP1B became tyrosine phosphorylated in response to insulin in vivo. Tyrosine phosphorylation of PTP1B by the insulin receptor was absolutely dependent upon insulin-stimulated receptor autophosphorylation and required an intact kinase domain, containing insulin receptor tyrosines 1146, 1150 and 1151. Tyrosine phosphorylation of wild type PTP1B by the insulin receptor kinase increased phosphatase activity of the protein. Intermolecular transdephosphorylation was demonstrated both in vitro and in vivo, by dephosphorylation of phosphorylated CS-PTP1B by the active wild type enzyme either in a cell-free system or via expression of the wild type PTP1B into Hirc-M cell line, which constitutively overexpress the human insulin receptor and CS-PTP1B. These results suggest that PTP1B is a target protein for the insulin receptor tyrosine kinase and PTP1B can regulate its own phosphatase activity by maintaining the balance between its phosphorylated (the active form) and dephosphorylated (the inactive form) state. (Mol Cell Biochem 221: 147–154, 2001) Key words: insulin receptor, PTP1B, tyrosine phosphorylation Abbreviations: IR – insulin receptor; IRS-1 – insulin receptor substrate-1; PTKase – protein tyrosine kinase; PTPase – protein tyrosine phosphatase; PBS – phosphate buffered saline; SDS-PAGE – sodium dodecyl sulfate polyacrylamide gel electrophoresis

Introduction The involvement of PTP1B in insulin signaling has been suggested in numerous reports. In initial studies, microinjection of purified placental PTP1B into Xenopus oocytes was shown to block insulin-induced S6 peptide phosphorylation and inhibit insulin-induced oocyte maturation [1, 2]. Subsequent studies have demonstrated that PTP1B is expressed at relatively high levels in insulin sensitive tissues [3]. We have

shown in rat L6 myotubes that insulin increases total cellular PTPase activity in a time and dose dependent manner. Increased activity is due mainly, if not entirely, to increased PTP1B activity, following increased PTP1B mRNA and protein expression [4]. Recently we have demonstrated that elevated activity and protein expression of PTP1B in the skeletal muscle of insulin resistant, type II diabetic Goto–Kakizaki rats lead to the inhibition of insulin-stimulated insulin receptor autophosphorylation, the phosphorylation of insulin re-

Address for offprints: S. Dadke, Fox Chase Cancer Center, Room # W451, 7701 Burholme Avenue, Philadelphia, PA 19111, USA Present addresses: S. Dadke, Fox Chase Cancer Center, Room # W451, 7701 Burholme Avenue, Philadelphia, PA 19111, USA; A. Kusari, Department of Developmental and Cell Biology, University of California at Irvine, 5450 BioSci II, Irvine, CA 92687-2300, USA; J. Kusari, Retinoid Research, Mail code RD-2C, Allergan, Inc. 2525 Dupont Drive P.O. Box 19534 Irvine, CA 92623-9534

148 ceptor substrate-1 (IRS-1) protein [5] and it was shown that over-expression of catalytically active PTP1B inhibited insulin-stimulated glucose incorporation into glycogen [6]. It has been demonstrated that deletion of the ptp1B gene in mice causes marked insulin sensitivity and prolonged IR autophosphorylation [7] and that PTP1B can dephosphorylate IRS-1 [8]. Recently we have shown that down regulation of insulin signaling by PTP1B is mediated by an N terminal binding region [9]. Taken together these data suggest that PTP1B acts as a negative regulator of insulin signaling. Previously, we have demonstrated a direct in vivo interaction between the insulin receptor and PTP1B, using a mutant derivative of PTP1B in which the critical active site a cysteine residue at amino acid position 215, has been mutated to serine (CS-PTP1B). We have also shown that the interaction of the insulin receptor with PTP1B is absolutely dependent upon insulin-stimulated autophosphorylation of the receptor. Insulin receptor tyrosine residues 1146, 1150 and 1151 are essential for the PTP1B:insulin receptor interaction. Tyrosine residues 66, 152 and/or 153 of PTP1B are phosphorylated by the activated insulin receptor and are also necessary for formation of the PTP1B:insulin receptor complex [9, 10]. In this study, we extend the results from our previous study by providing evidence that PTP1B is a direct substrate of the insulin receptor tyrosine kinase in both intact cell and cell-free systems. Furthermore, tyrosine phosphorylation of PTP1B by the insulin receptor tyrosine kinase increases the catalytic activity of PTP1B. In addition, PTP1B appears to modulate its own tyrosine phosphorylation state, thereby providing a mechanism for regulating its own enzymatic activity.

Materials and methods Insulin was kindly provided by Eli Lilly and Company (Indianapolis, IN, USA). Fetal bovine serum (FBS), cell culture media, geneticin, gentamycin, and glutamax were purchased from Life Technologies, Inc. (Gaithersburg, MD, USA). Methotrexate and hygromycin B were from Calbiochem (San Diego, CA, USA). Monoclonal anti-phosphotyrosine antibody (PY-20) used for immunoblotting was from Transduction Laboratories (Lexington, KY, USA). Wheat germ agglutinin Agarose required for the purification of insulin receptor was purchased from Vector Laboratories (Burlingame, CA, USA). Polyclonal anti-PTP1B antibody was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY, USA). Tween-20, protein molecular weight standards, acrylamide and TEMED were purchased from Bio-Rad (Hercules, CA, USA). Non-fat dry milk was from Nestle Foods Co. (Glendale, CA, USA). Anti-mouse and anti-rabbit IgGs conjugated with HRP and

ECL reagent were purchased from Amersham Life Science (Arlington Heights, IL, USA). Nitrocellulose membrane was from Schleicher and Schuell (Keene, NH, USA). All other reagents were purchased from Sigma (St. Louis, MO, USA) and were the highest quality available.

Cell culture The cell lines HircB and Hirc-M were used in this study. Briefly, HircB is a rat fibroblast cell line overexpressing human insulin receptors [11]. Hirc-M is a rat-1 fibroblast cell line overexpressing human insulin receptors and the CSPTP1B [6]. Hirc-M cells were propagated in Dulbecco’s modified Eagles media (DMEM F12), containing 10% FBS, 50 µg/ml Gentamycin, 400 µg/ml hygromycin and 500 nM methotrexate. HircB cells were cultured in the same media except hygromycin.

Purification and activation of insulin receptors Insulin receptors were partially purified using wheat germ agglutinin-Agarose [6] from HircB cells, and cells expressing various mutant insulin receptors [11, 12]. The partially purified insulin receptors were activated and phosphorylated by incubating the receptor preparation with insulin at 4°C for 30 min, and then in the presence of manganese chloride, ATP and sodium vanadate at 4°C for another 10 min.

Transient transfection For transient transfection, 60% confluent Hirc-M cells were transfected using Lipofectamine (Gibco Life Sciences) according to the manufacturer’s recommendations with different plasmids as indicated. Forty-eight hours after transfection, the cells were serum starved with 0.1% FBS for 16 h. Following serum starvation, cells were incubated in the absence or presence of 100 nM insulin for 5 min at 37°C.

Immunoprecipitation and immunoblotting Following insulin stimulation, the cells were washed with 5 ml of ice cold phosphate-buffered saline (PBS). The cells were then lysed in the NP-40 lysis buffer (50 mM Tris-HCl, pH 8.0, 137 mM sodium chloride, 10% glycerol, 1% Nonidet P-40, 50 mM sodium fluoride, 10 mM β-glycerophosphate, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride and 1 µg/ ml aprotinin) for 5 min on ice. The lysates were spun at 14,000 rpm for 10 min at 4°C. The supernatants were removed and assayed for total protein content using bicinchoninic acid

149 (Pierce). Two hundred µg of whole cell lysate was immunoprecipitated with 10 µl of anti-phosphotyrosine monoclonal antibody. The immunocomplexes were washed 5 times with ice cold lysis buffer and boiled for 5 min in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. Samples were fractionated by SDS-PAGE and transferred to nitrocellulose membrane. Immunoblots were developed by using the ECL (Amersham) detection system.

In vitro PTP1B phosphorylation and dephosphorylation For phosphorylation, 10 µg of full length GST-CS-PTP1B fusion protein was incubated at 4°C in the presence of activated insulin receptors and [γ-32P]ATP (1 µCi/µl, 3000 Ci/ mmol). Phosphorylated GST-CS-PTP1B was then isolated from the reaction mixture using glutathione-sepharose 4B beads. Pellets were washed 5 times with ice-cold PBS, and suspended in SDS-PAGE sample buffer. Samples were analyzed by SDS-PAGE and autoradiography was performed. To follow the dephosphorylation of the phosphorylated GST-CSPTP1B in vitro, previously phosphorylated GST-CS-PTP1B was mixed with catalytically active PTP1B. Incubation was carried out at 30°C in low salt buffer (25 mM imidazole, pH 7.0, 1 mM EDTA, 1 mM EGTA, 0.1% β-ME, 2 mM MgCl2, 0.025% PMSF, 1 µg/ml leupeptin, 0.1 nM benzamidine, and 250 mM sucrose). At various time intervals, aliquots were withdrawn, boiled in SDS sample buffer, electrophoresed on a 10% SDS-polyacrylamide gel, and extent of PTP1B phosphorylation was evaluated by autoradiography. In vivo dephosphorylation of CS-PTP1B was examined by transiently transfecting Hirc-M cells with catalytically active PTP1B [6]. In control (cells not transfected with catalytically active PTP1B) and cells transfected with catalytically active PTP1B, phosphorylation of CS-PTP1B was measured under basal and insulin-stimulated conditions by immunoprecipitation with antiphosphotyrosine monoclonal antibody, and subsequent immunoblotting with anti-PTP1B polyclonal antibody.

PTPase activity assay Protein tyrosine phosphatase activity in whole-cell lysates was determined by procedure described previously [13]. Briefly, cell lysates were incubated with the 32P-labeled IR peptide at 30°C in low salt buffer for various time points. The reaction was terminated by adding a 3-fold excess volume of ice cold 10% trichloroacetic acid, and the mixture was centrifuged at 14,000 rpm for 5 min at 4°C. 32Pi released from the labeled substrate was measured after organic extraction using a mixture of isobutanol:toulene (1:1) and 5 mM silico-

tungstic acid + 1 mM H2SO4, and then 5% ammonium molybdate in 2M H2SO4. The mixture was centrifuged at 1000 rpm for 4 min at 4°C. The upper phase was taken for liquid scintillation counting. In order to determine activity of phosphorylated PTP1B, full length GST fusion protein of catalytically active PTP1B was incubated overnight at 30°C in presence of activated insulin receptor and [γ-thio]-ATP. PTP1B was then precipitated from the reaction mixture using glutathione-sepharose 4B as described earlier. Tyrosine phosphatase activity of PTP1B in the pellet was determined as described earlier [14]. Briefly, 96 µl of 10 mM Tris-HCl, pH 7.0 was added per well to a 96well microtitre plate. Ten µg of catalytically active and previously phosphorylated PTP1B was added to it. The substrate, 3-0-methylfluorescin phosphate (OMFP) was freshly prepared by sonication in dimethylsulphoxide for 10 min and 2 µl of the substrate was added to the reaction mixture. The plates were shaken for 15 min at room temperature. The absorbance was measured at 477 nm using a Spectra Max 340 plate reader.

Results PTP1B is a substrate of the insulin receptor tyrosine kinase The insulin receptor possesses intrinsic tyrosine kinase activity which becomes activated upon ligand-binding by autophosphorylation. The activated receptor then in turn phosphorylates several cellular proteins. Previously we have shown that PTP1B complexes in vivo with the activated insulin receptor and is tyrosine phosphorylated [10]. It is possible that tyrosine phosphorylation of PTP1B is mediated by the activated insulin receptor, or occurs via an indirect mechanism. To differentiate between these possibilities, 10 µg of full length CS-PTP1B was incubated overnight with [γ32 P]ATP either in the absence or presence of the activated insulin receptor. There was no detectable phosphorylation of CS-PTP1B in absence of the activated insulin receptor (Fig. 1, lane 1). However, addition of the activated insulin receptor resulted in the phosphorylation of PTP1B (Fig. 1, lane 2). These data indicate that PTP1B is a substrate of the activated insulin receptor, at least in vitro.

The activated insulin receptor tyrosine kinase phosphorylates a majority of the intracellular PTP1B Although we have demonstrated that PTP1B is phosphorylated by the IR in vitro (Fig. 1), it becomes necessary to determine if this phenomenon occurs in vivo. In addition, the

150

Fig. 1. Insulin receptor phosphorylates CS-PTP1B in vitro. Ten µg full length GST-CS-PTP1B protein was added to a kinase reaction mixture either in the absence (lane 1) or presence (lane 2) of activated insulin receptor together with [γ-32P]ATP. GST-CS-PTP1B was precipitated using glutathione sepharose 4B. The samples were fractionated by 10% SDS-PAGE. The gel was dried and exposed to a Kodak x-ray film. This is a representative experiment independently performed 3 times. Lane 1: No IR; lane 2: 4 µg IR. Arrow indicates the phosphorylated CS-PTP1B.

extent to which PTP1B is phosphorylated in vivo is unknown. To address these issues, we employed the Hirc-M cell line (rat-1 fibroblasts that overexpress both the human insulin receptor and CS-PTP1B) as our model system. Hirc-M cells were stimulated with 100 nM insulin for 5 min, and 200 µg of total cell lysate was subjected to immunoprecipitation with anti-phosphotyrosine monoclonal antibody, followed by immunoblotting with an anti-PTP1B polyclonal antibody (Fig. 2, lane 5). The initial immunoprecipitation was followed by a second round of immunodepletion using the anti-phosphotyrosine antibody, and the second immunoprecipitate was immunoblotted with the anti-PTP1B polyclonal antibody (Fig. 2, lane 6). The relative recovery of PTP1B by immunoprecipitation with anti-phosphotyrosine antibody was then compared with the amount of total cellular PTP1B, as determined by immunoblot analysis of increasing amounts of total cell lysate with the anti-PTP1B polyclonal antibody (Fig. 2, lanes 1–4). As expected, the level of detectable PTP1B protein increased with increasing amounts of cell lysate. Assuming that immunoprecipitation with anti-phosphotyrosine antibody resulted in complete recovery of tyrosine-phosphorylated PTP1B (Fig. 2, lanes 5 and 6), comparison between

PTP1B protein levels recovered with anti-phosphotyrosine antibody and that observed for total cell lysate revealed that 53% of the total cellular PTP1B content was tyrosine phosphorylated in insulin-stimulated Hirc-M cells. These results provide evidence that the insulin receptor mediates the tyrosine phosphorylation of PTP1B in vivo and that after insulin stimulation, a significant amount of the total cellular PTP1B becomes tyrosine phosphorylated. Furthermore, the majority of PTP1B tyrosine phosphorylated after insulin stimulation suggests that this enzyme plays an important role in insulin signal transduction.

Insulin receptor tyrosine residues 1146, 1150 and 1151 are required for tyrosine phosphorylation of PTP1B Previously, we have demonstrated that insulin receptor tyrosine residues 1146, 1150 and/or 1151 are required for the interaction between the insulin receptor and PTP1B and complete replacement of these tyrosine residues with phenylalanine greatly reduces the interaction of the activated insulin receptor with PTP1B [10]. In this study, we extend our find-

Fig. 2. Stoichiometry of tyrosine phosphorylation of CS-PTP1B induced by insulin. Increasing amounts of total Hirc-M cell lysate (25–200 µg of protein) were subjected to immunoblot analysis with anti-PTP1B antibodies (lane 1 = 25 µg; lane 2 = 50 µg; lane 3 = 100 µg; lane 4 = 200 µg). Insulin-stimulated Hirc-M cell lysate (200 µg) was immunoprecipitated with anti-phosphotyrosine antibodies (lane 5). After the first immunoprecipitation, the supernatant was further subjected to immunoprecipitation with anti-phosphotyrosine antibodies (lane 6). Both immunoprecipitates were then immunoblotted with anti-PTP1B antibodies. The percentage of cellular PTP1B that was tyrosine phosphorylated was estimated by comparing the intensity of the PTP1B signal recovered with anti-phosphotyrosine antibodies (lane 5 plus lane 6) with the intensity of those observed for total cell lysate. This is a representative experiment independently performed 3 times. Arrow indicates CS-PTP1B.

151 ings by investigating more closely the role of these individual tyrosine residues in mediating the tyrosine phosphorylation of PTP1B. In order to accomplish this, several cell lines expressing various insulin receptor mutations were used. These included: (1) IR Tyr1146Phe (Y1146F) in which tyrosine residue 1146 (by the numeric assignment of Ullrich et al. [15]) has been replaced with phenylalanine, (2) IR Tyr1150Phe (Y1150F) in which tyrosine residue 1150 has been replaced with phenylalanine, (3) IR Tyr1151Phe (Y1151F) in which tyrosine residue 1151 has been replaced with phenylalanine and (4) IR Lys1018Ala (K1018A) in which lysine residue at 1018 has been replaced with alanine. Equal amounts of partially purified wild type and various mutant insulin receptor preparations were phosphorylated and activated in vitro as described earlier. GST-CS-PTP1B was then incubated with individual insulin receptor preparations in presence of [γ-32P]ATP. GST-CS-PTP1B was purified using glutathione sepharose 4B. The phosphotyrosine content of CS-PTP1B was assessed by electrophoresing the samples and subsequent autoradiography. Compared to the wild type receptor, the Y1146F receptor exhibited a substantial decrease in kinase activity towards CSPTP1B (Table 1). Replacement of Tyr 1150 or Tyr 1151 abolishes or greatly reduces the tyrosine kinase activity of the insulin receptor towards CS-PTP1B. In fact, the lack of tyrosine kinase activity of the Y1150F mutant receptor towards CS-PTP1B was similar to that found with the kinase dead K1018A insulin receptor [16]. Thus, the importance of each of these tyrosine residues in mediating the tyrosine kinase activity of the activated insulin receptor towards PTP1B is as follows: Tyr1150 > Tyr 1151 > Tyr1146. Previously, we have shown very little interaction between PTPase and the Y1146F insulin receptor. There was no interaction of PTP1B with the insulin receptor in which twin tyrosines at positions 1150 and 1151 had been replaced with phenylalanine [10].

These along with our present observations indicate that phosphorylation of PTP1B by the insulin receptor depends on interaction of the protein with the receptor.

Tyrosine phosphorylation of PTP1B increases its enzymatic activity The significance of PTP1B tyrosine phosphorylation is not entirely clear. However, increasing evidence suggests that this could modulate PTP1B’s enzymatic activity [17–25]. To determine whether insulin-stimulated tyrosine phosphorylation of PTP1B regulates its phosphatase activity, we had to obtain a phosphorylated form of the enzyme which is resistant to phosphatase activity. To address this issue, catalytically active PTP1B was phosphorylated by the activated insulin receptor in presence of [γ-thio]ATP. The resulting thio-phosphorylated residues were relatively resistant to phosphotyrosine phosphatase activity [26]. The activity of the thio-phosphorylated PTP1B was assayed at various times during insulin receptor mediated kinase reaction using OMFP as a substrate [14]. Catalytic activity of thiophosphorylated PTP1B increased 38-fold over that of basal by 30 min and then remained almost unaltered until 16 h (Fig. 3).

Tyrosine phosphorylated PTP1B can dephosphorylate itself in vitro and in vivo The high percentage of tyrosine phosphorylation of PTP1B induced by insulin receptor (Fig. 2) suggested the possibil-

Table 1. Tyrosine phosphorylation of CS-PTP1B by wild type and various mutants of insulin receptor Insulin receptor

Wild type

Y1146F

Y1150F

Y1151F

K1018A

Tyrosine phosphorylation of PTP1B*

13.69

5.99

0.0

1.79

0.0

*Values are expressed in arbitrary densitometric units. Ten µg full length GST-CS-PTP1B was added to a kinase reaction mixture including either wild type or different mutant insulin receptors carrying point mutations such as Y1146F, Y1150F, Y1151F, or K1018A, and [γ-32P]ATP. After overnight incubation, CS-PTP1B was precipitated using glutathione sepharose 4B. The protein was subjected to 10% SDS-PAGE. The gel was dried and exposed to a Kodak x-ray film. The autoradiograms were quantified by densitometric scanning and values are expressed in arbitrary densitometric units. This is a representative experiment independently performed 3 times.

Fig. 3. Activity enhancement of phosphorylated PTP1B. Catalytically active PTP1B was phosphorylated by partially purified insulin receptors in the presence of [γ-thio]ATP. At 0 min, 30 min, 2 h, 6 h, 12 h and 16 h, aliquots were sampled from the reaction mixture and assayed for their phosphatase activity using OMFP as a substrate. The release of the fluorescin was measured at 477 nm using a Spectra MAX 340 plate reader. This figure provides the mean ± S.D. for 3 experiments.

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Fig. 4. PTP1B auto-dephosphorylation. Full length GST-CS PTP1B was phosphorylated and purified as described earlier. It was then mixed with catalytically active PTP1B and incubated at 30°C for different time points. At 0 min (lane 1), 15 min, 30 min, 60 min and 120 min (lanes 3–6, respectively), aliquots were withdrawn and subjected to 10% SDS-PAGE. The gel was dried and exposed to a Kodak x-ray film. The control reaction mixture (lane 2) did not include the active PTP1B and was stopped at 120 min. This is a representative experiment independently performed 3 times. Arrow indicates CS-PTP1B.

ity that the phosphatase activity of PTP1B may modulate its own state of tyrosine phosphorylation. To investigate this possibility further, under in vitro conditions, CS-PTP1B was phosphorylated at first as described earlier, and was then incubated with catalytically active PTP1B for varying periods of time. The phosphotyrosine content of CS-PTP1B was determined by autoradiography. In the presence of catalytically active PTP1B, phosphotyrosine content of CS-PTP1B decreased rapidly by 30 min (Fig. 4, lane 4), and reached a minimum at 120 min (Fig. 4, lane 6). However, there was no change in tyrosine phosphorylation of CS-PTP1B in the absence of catalytically PTP1B (Fig. 4, lane 2). These results suggest that PTP1B can dephosphorylate itself under in vitro conditions. In order to determine in vivo dephosphorylation of CSPTP1B by catalytically active PTP1B, Hirc-M cells were transiently transfected with catalytically active PTP1B. Fortyeight hours after transfection, cells were incubated in the absence or presence of 100 nM insulin for 5 min and whole cell lysates were prepared. A portion of the total whole cell lysates was used to measure phosphatase activity. The remain-

ing whole cell lysate was used to determine phosphotyrosine content of PTP1B by immunoprecipitation and immunoblotting as described earlier. Compared to the cells which were not transfected with catalytically active PTP1B, both basal and insulin-stimulated phosphatase activities were higher in cells transfected with catalytically active PTP1B (Fig. 5A). The phosphotyrosine content of PTP1B was also decreased (Fig. 5B). These results further indicate that PTP1B can dephosphorylate itself under in vivo conditions.

Discussion Protein tyrosine phosphorylation plays an important role in regulating many cellular processes. The level of phosphotyrosine in the cell is a balance between the action of PTKases and PTPases. As the role of PTKases in signal transduction is now understood in some detail, increasing attention is being focused on the role of PTPases in regulating signaling pathways.

Fig. 5. Dephosphorylation of CS-PTP1B. Hirc-M cells were transiently transfected with catalytically active PTP1B. Forty-eight h after transfection, cells were serum starved for 16 h. Following serum starvation, cells were incubated without and with 100 nM insulin at 37°C for 5 min, and then lysed in lysis buffer. (A) Whole cell lysates were spun at 14,000 rpm for 10 min at 4°C and supernatant was assayed for phosphatase activity as described under Materials and methods. Experiments were performed in triplicate (mean ± S. D.); (B) 200 µg of total cell lysate was immunoprecipitated with anti-phosphotyrosine antibody. The proteins were fractionated on a 10% SDS-PAGE and immunoblotted with anti-PTP1B antibody. This is a representative experiment independently performed 3 times.

153 Growing evidence suggests that PTP1B is an important negative regulator of insulin signal transduction [1–10]. Previously, we have reported that PTP1B complexes in vivo with the activated insulin receptor and subsequently becomes phosphorylated on Tyr66, Tyr152, and/or Tyr153, and down regulation of insulin signaling by PTP1B is mediated by an N-terminal binding region [9, 10]. The purpose of this study was to investigate the biochemical and physiological significance of this tyrosine phosphorylation of PTP1B. We report that PTP1B is a direct substrate of the insulin receptor tyrosine kinase. In addition, a majority of the intracellular PTP1B becomes tyrosine phosphorylated after insulin treatment. This provides further evidence for the potential importance of PTP1B in insulin action. Although PTP1B is tyrosine phosphorylated by the activated insulin receptor, the significance of this biochemical event remained unclear. It now appears that the increase in the tyrosine phosphorylation of many phosphatases enhance their catalytic activity. The phosphatase activity of SHP2 is modulated by tyrosine/threonine phosphorylation resulting from growth factor activation in cells transformed by the Rous sarcoma virus [17–21]. The in vivo phosphorylation of PTP1C in response to CSF-1, insulin, or pp60v-src results in 4-fold activation of the enzymatic activity [22–24]. Low Mr phosphotyrosine-protein phosphatase is tyrosine-phosphorylated by pp60v-src both in vivo and in vitro, correlating with an increase in its catalytic activity [25]. It has also been previously shown that a GST-CS-PTP1B fusion protein was heavily tyrosine-phosphorylated when incubated with 3T3/ hEGFR cell lysates [27] and EGFR tyrosine phosphorylates and increases the catalytic activity of PTP1B [28]. Insulin-induced upregulation of the catalytic activity of PTP1B through enhanced tyrosine phosphorylation might result in facilitating the dephosphorylation of target molecules of PTP1B, such as the activated insulin receptor, IRS-1 and thereby potentially terminating insulin signal transduction [5, 7–10]. Additionally, insulin-stimulated increased tyrosine phosphorylation of PTP1B may serve to modulate its own enzymatic activity as suggested by the decreased phosphotyrosine content of CS-PTP1B in the presence of catalytically active PTP1B. It seems to suggest that PTP1B is capable of dephosphorylating itself, however, we can not rule out the involvement of other protein-tyrosine phosphatases in the dephosphorylation of CS-PTP1B. Stein-Gerlach et al. have reported similar findings for PTP-1D [29]. These results suggest a possibility that the enzyme may modulate its own activity by maintaining a balance between its phosphorylated (active form) and dephosphorylated (inactive form) states. In summary, our data provide a model for PTP1B action in the insulin signal transduction pathway. After insulinstimulation, the insulin receptor undergoes autophosphorylation on multiple tyrosine residues which increases intrinsic

tyrosine kinase activity of the receptor. The phosphorylated and activated insulin receptor transiently binds and phosphorylates tyrosine residues of PTP1B. Tyrosine phosphorylation of PTP1B increases its catalytic activity. Catalytically active PTP1B can then interact with and dephosphorylate its target proteins such as insulin receptor, IRS-1 thus attenuating the insulin action. Finally, PTP1B undergoes auto-dephosphorylation, probably via an intermolecular mechanism and returns to its inactive, basal state. Further studies are warranted to explore this possibility.

Acknowledgments This work was supported by National Institute of Health Grant DK 46490 (to J.K.). We thank Drs. C.R. Kahn and J.M. Olefsky for providing cells overexpressing either wild type insulin receptor or various mutant forms of the receptor. We also thank Dr. J. Chernoff for providing catalytically active or inactive PTP1B – glutathione-S-transferase (GST) fusion protein.

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