INTRODUCTION

The binding of insulin to its cell surface receptor induces phosphorylation of specific tyrosyl residues within the intracellular domain of the receptor -subunit. This autophosphorylation reaction activates the receptor's intrinsic tyrosine kinase activity toward various cellular substrates including IRS-1,¹ IRS-2, and Shc proteins (1, 2, 3) and thereby plays a key role in the metabolic and mitogenic signaling pathways of insulin (2). Dephosphorylation of the insulin receptor by cellular protein- tyrosine phosphatases (PTPases) attenuates the receptor kinase activity and, hence, the effects of insulin (4, 5, 6). Thus, PTPases may oppose tyrosine kinase-mediated insulin signaling and contribute to insulin resistance. Indeed, altered PTPase activity has been noted in different tissues from diabetic rats (7, 8, 9) and humans (10, 11, 12). Therefore, the development of PTPase inhibitors that act as specific modulators of insulin receptor functions may provide novel ways to treat diabetes. It has been reported previously that vanadate and pervanadate cause a marked improvement of glucose homeostasis in streptozotocin-treated rats (13, 14) by exerting insulin-like effects on peripheral tissues. Both compounds are broad spectrum PTPase inhibitors (15, 16) that appear to function via a mechanism distal to the insulin receptor (17, 18, 19). Because vanadate and pervanadate affect various systems under physiological conditions at relatively high doses (20, 21), they are not likely to become useful therapeutic agents. It has become apparent that PTPases are selective among different phosphotyrosine-containing proteins (22, 23) and synthetic peptides (24, 25, 26), indicating an interaction between PTPase and the specific amino acid sequence of the protein substrate. This behavior has provided the basis for the synthesis of non-hydrolyzable phosphotyrosine peptide analogs (27, 28, 29, 30, 31) with the goal of selective inhibition of tyrosine phosphatases acting on specific protein substrates. We have reported previously that a tris-sulfotyrosyl dodecapeptide analogue of the insulin receptor autocatalytic domain (3S-peptide-I) potently inhibits the dephosphorylation of the insulin receptor in vitro (30). The same study has also shown that the conjugation of 3S-peptide-I to stearic acid leads to an enhanced insulin-stimulated receptor autophosphorylation in intact cells (30).

In this study, we extended this observation by comparing the levels of phosphorylation and signaling of the insulin receptor with that of the epidermal growth factor receptor following ligand stimulation in the presence of stearyl 3S-peptide-I. Our results show that while stearyl 3S-peptide-I specifically enhanced the immediate phosphorylation of the insulin receptor at tyrosine residues and subsequent stimulation of PI-3-kinase and MAP kinase activities in insulin-treated cells, it had no effect on the EGF receptor activation and signaling.

EXPERIMENTAL PROCEDURES

All chemicals used were of the highest purity commercially available. EGF, myelin basic protein (MBP), monoclonal and polyclonal anti-phosphotyrosine antibodies, polyclonal anti-EGF receptor antibody, polyclonal MAP kinase antibody, and polyclonal anti-IRS-1 antibody were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Polyvinylidene difluoride (PVDF) membrane and precast 4-12 and 4-20% gradient polyacrylamide gels were purchased from Novex (San Diego, CA), Protein G-plus/Protein A-agarose beads and monoclonal anti-insulin receptor antibody were from Oncogene Science, Inc. (Uniondale, NY), and polyclonal ERK-1 antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Sodium orthovanadate, Tween-20, Nonidet P-40, and Triton X-100 were purchased from Sigma.

The Chinese hamster ovary (CHO) cell line transfected with an expression plasmid encoding the normal human insulin receptor (CHO/HIRc) was a generous gift from Dr. Morris F. White, Joslin Diabetes Center, Boston, MA. CHO cells overexpressing human EGF receptor (CHO/EGF-R) was a generous gift from Dr. Roger J. Davis, University of Massachusetts Medical School, Worcester, MA. The cells were maintained in Ham's F-12 medium containing 10% fetal bovine serum and were cultured to confluence in 35-mm dishes.

Peptide-I (TRDIYETDYYRK-amide) is a dodecapeptide whose primary sequence is identical to the 1142-1153 sequence of the insulin proreceptor (32). The solid phase synthesis of peptide-I, tyrosine sulfation of resin-bound peptide-I, and its modification by the incorporation of stearic acid at it's amino terminus were performed as described by Liotta et al. (30). Stearyl 3S-peptide-I was purified by reverse phase high performance liquid chromatography on a semipreparative column (Vydac, protein and peptide C₁₈ column), and its composition was confirmed by mass spectrometric analyses.

Both reactions were evaluated following permeabilization with digitonin, essentially as described previously (33). Briefly, serum-starved cells were permeabilized with 35 µg/ml digitonin for 20 min at room temperature, transferred at 6 °C, and then treated with 5 nM EGF for 5 min. Thereafter, the phosphorylation reaction was started by the addition of 100 µM ATP and 4 mM MnCl₂. 10 min later, the dephosphorylation of EGF receptors was initiated by the addition of 20 mM EDTA in the presence or absence of vanadate or 3S-peptide-I at the indicated concentrations. The reaction was stopped after 5 min by immersing the dishes in liquid nitrogen. The cells were lysed in radioimmune precipitation buffer containing 20 mM Tris-Cl (pH 7.5), 137 mM NaCl, 1 mM sodium orthovanadate, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 0.02% NaN₃, 0.2 mM PMSF, 1 mM benzamidine, 8 µg/ml aprotinin, and 2 µg/ml leupeptin. The lysates were centrifuged at 17,000 × g for 10 min at 4 °C; the clarified lysates were incubated with a monoclonal anti-phosphotyrosine antibody. After overnight incubation at 4 °C, the immune complexes were precipitated with Protein G-plus/Protein A-agarose beads and subjected to SDS-polacrylamide gel electrophoresis under reducing conditions (34) followed by electrotransfer on PVDF membranes. Western blotting was performed with a polyclonal anti-phosphotyrosine antibody, as described recently (35). The blots were developed using the ECL chemiluminescence detection system (Amersham Corp.), and the amount of tyrosine phosphorylation of the EGF receptor was evaluated quantitatively by densitometry of the autoradiographs using ImageQuant^TM software (version 3.3) on a Molecular Dynamics laser densitometer.

Cells were serum-starved for 16 h, and the medium was replaced with 0.5 ml of Ham's F-12 medium containing a range of concentrations of stearyl 3S-peptide-I or 1 mM sodium orthovanadate. 1 h later, cells were treated with 100 nM insulin (CHO/HIRc cells) or 1 nM EGF (CHO/EGF-R cells) for 1 min, and the incubation was terminated by removing the fluid and immersing the dishes in liquid nitrogen. Cells were then scraped and lysed into RIPA buffer. The clarified lysates from CHO/HIRc cells were incubated with a monoclonal anti-insulin receptor antibody, whereas lysates from CHO/EGF-R cells were incubated with a monoclonal anti-phosphotyrosine antibody. After overnight incubation at 4 °C, the immune complexes were precipitated with Protein G-plus/Protein A beads. Immunoprecipitates were subjected to SDS-polyacrylamide gel electrophoresis, electrotransferred to PVDF membranes, and probed with a polyclonal anti-phosphotyrosine antibody. The blots were developed using the ECL chemiluminescence detection system, and autoradiograms were quantified by laser densitometry.

Serum-starved cells were preincubated for 1 h at 37 °C with varying concentrations of stearyl 3S-peptide-I followed by the addition of 3 nM insulin for 1 min (CHO/HIRc cells) or 5 nM EGF for 2 min (CHO/EGF-R cells). Cells were then lysed in a buffer containing 20 mM Tris-Cl (pH 8.0), 137 mM NaCl, 1 mM MgCl₂, 1 mM CaCl₂, 10% glycerol, 1% Nonidet P-40, 150 µM sodium orthovanadate, 1 mM PMSF, and 2 mM dithiothreitol. The clarified lysates from CHO/HIRc cells were incubated with a polyclonal anti-IRS-1 antibody, while a monoclonal anti-phosphotyrosine antibody was added to CHO/EGF-R cell lysates. After overnight incubation at 4 °C, the immune complexes were collected by mixing with Protein G-plus/Protein A-agarose. The immune pellets were assayed for PI-3-kinase activity by a method described by Ruderman et al. (36). In brief, the immunoprecipitates were incubated in a buffer containing 20 mM Tris-Cl (pH 7.5), 10 mM MgCl₂, 0.4 mM EGTA, 20 µg of phosphatidylinositol, and 40 µM [-³²P]ATP (10 cpm/fmol). After a 20-min incubation at room temperature, the reaction was stopped with 20 µl of 6 M HCl and 160 µl of chloroform/methanol (1:1 (v/v)). After a brief vortex, the lower organic phase was separated by centrifugation at 17,000 × g for 10 min. Phospholipids present in the lower organic phase were separated by thin layer chromatography on silica gel 60 plates (pretreated with 1% potassium oxalate and activated at 100 °C for 1 h) in chloroform/methanol/water/ammonia (60:47:11.3:2 (v/v/v/v)) along with phosphatidylinositol standards. ³²P-Labeled products were detected by autoradiography using Amersham Hyperfilm-MP and quantified on a Packard InstantImager, and the standard lipids were visualized with iodine vapor.

Serum-starved cells were incubated with 50 µM stearyl 3S-peptide-I for 1 h and then treated with 100 nM insulin for 1 min (CHO/HIRc cells) or 0.5 nM EGF for 2.5 min (CHO/EGF-R cells). Cells were lysed in a lysis buffer containing 50 mM Tris-Cl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM NaF, 10 mM sodium pyrophosphate, 0.1 mM PMSF, 1 mM sodium orthovanadate, 20 µg/ml aprotinin, and 10 µg/ml leupeptin. The clarified lysates were subjected to immunoprecipitation with polyclonal anti-MAP kinase antibody. After collecting the immune complexes by the addition of Protein G-plus/Protein A-agarose, the beads were washed twice with lysis buffer, twice with kinase buffer containing 20 mM HEPES (pH 7.4), 10 mM MgCl₂, and 1 mM dithiothreitol, and assayed for MAP kinase activity. The beads were incubated with 20 mM HEPES (pH 7.4), 10 mM MgCl₂, 1 mM dithiothreitol, 8 µg/ml MBP, and 20 µM [-³²P]ATP (10 cpm/fmol) for 15 min at 22 °C. The reaction was stopped by the addition of an equal volume of 2-fold concentrated Laemmli sample buffer (34). Phosphorylated myelin basic proteins were separated by electrophoresis on 4-20% gradient SDS-polyacrylamide gels under reducing conditions, and ³²P content was quantified with a Packard InstantImager.

Significant differences were determined by an analysis of variance coupled to Fisher's least significant difference test for multiple mean comparison using StatView 4.01 (Abacus Concepts Inc.).

RESULTS

We have shown previously (30) that the tris-sulfotyrosyl dodecapeptide 3S-peptide-I potently inhibits insulin receptor dephosphorylation in digitonin-permeabilized CHO/HIRc cells. Under these conditions, membrane architecture remains intact while allowing the rapid entry of ATP, peptides, and small molecules into the intracellular compartment (30). Here, we investigated the effect of 3S-peptide-I on the dephosphorylation of the EGF receptors after permeabilization of CHO/EGF-R cells with digitonin. To study dephosphorylation of the EGF receptor, phosphorylation reaction was carried out in the presence of EGF and ATP and then was stopped by the addition of EDTA. The amount of tyrosine-phosphorylated EGF receptors was analyzed using an anti-phosphotyrosine antibody immunoblotting method. The phosphotyrosine content of the EGF receptor decreased by ~76% after 5 min of dephosphorylation at 6 °C in control cells (Fig. 1). 3S-peptide-I treatment did not alter EGF receptor dephosphorylation; however, vanadate strongly inhibited dephosphorylation of the EGF receptor. These results show that under these experimental conditions, 3S-peptide-I, whose sequence is unrelated to the EGF receptor (37), does not interact with PTPases acting on the EGF receptor.

We next examined the effect of stearyl 3S-peptide-I on ligand-stimulated phosphorylation of insulin receptor and EGF receptor in intact cells. For these studies, CHO cells expressing large numbers of insulin receptors (CHO/HIRc cells) or EGF receptors (CHO/EGF-R cells) were incubated with a range of concentrations of stearyl 3S-peptide-I or vanadate (1 mM) for 1 h and stimulated with their respective ligand. The extent of receptor tyrosine phosphorylation was then determined by Western immunoblotting. Fig. 2A shows that stearyl 3S-peptide-I (100 µM) was capable of increasing phosphorylation of the insulin receptor by 2.4- and 2.7-fold after addition of insulin for 30 s and 1 min, respectively. Also, stearyl 3S-peptide-I produced a dose-dependent stimulation in the insulin receptor phosphorylation when compared with cells incubated with insulin alone (Fig. 2B). Maximal stimulation was observed with 50 µM stearyl 3S-peptide-I and was maintained with concentrations up to 100 µM. The basal phosphorylation of the insulin receptor in intact CHO/HIRc cells was not affected by the presence of 50 µM stearyl 3S-peptide-I (data not shown). When CHO/HIRc cells were stimulated with insulin and vanadate, the extent of tyrosine phosphorylation of the insulin receptor increased severalfold as reported previously (30).

Stearyl 3S-peptide-I had no detectable activity toward the phosphorylation levels of EGF receptors in CHO/EGF-R cells (Fig. 3). However, inhibition of PTPases by vanadate led to an increase in the ligand-stimulated EGF receptor phosphorylation (Fig. 3), indicating that cellular PTPases acting on tyrosine-phosphorylated EGF receptors are not modulated by this tris-sulfated dodecapeptide. Comparable results as in Fig. 3 were obtained when the EGF receptors were immunoprecipitated from CHO/EGF-R cell lysates with a monoclonal EGF receptor antibody and immunoblotted with a polyclonal anti-phosphotyrosine antibody (data not shown). Thus, it appears that stearyl 3S-peptide-I specifically increases the steady state level of insulin receptor phosphorylation in intact cells.

The activated form of the insulin receptor phosphorylates IRS-1, which then binds with various signal transduction proteins containing src homology 2 domains (SH2), namely p85 subunit of PI-3-kinase, syp (SH-PTP2), GRB-2, and nck (2). The binding of p85 to phosphorylated IRS-1 activates PI-3-kinase activity present in the 110-kDa catalytic subunit, thereby mediating some of the metabolic and mitogenic actions of insulin (see Ref. 38 for a review). Activation of PI 3-kinase in response to EGF in anti-phosphotyrosine immunoprecipitates has also been reported (39, 40).

We examined the effect of stearyl 3S-peptide-I on ligand-stimulated PI-3-kinase activity in CHO/HIRc cells and CHO/EGF-R cells. Cells were incubated with stearyl 3S-peptide-I and then treated with their respective ligand. Cell lysate from CHO/HIRc cells was incubated with a polyclonal anti-IRS-1 antibody while monoclonal anti-phosphotyrosine antibody was added to cell lysate from CHO/EGF-R cells. The immune pellets were assayed for PI-3-kinase activity. In CHO/HIRc cells, insulin (3 nM) caused a 4-fold stimulation of PI-3-kinase activity (Fig. 4A). Stearyl 3S-peptide-I treatment (25 or 50 µM) for 1 h caused a further 1.7-fold (p = 0.07) and 2.7-fold (p = 0.0005) increase in insulin-stimulated PI-3-kinase activity, respectively (Fig. 4A); however, no effect was detected in basal PI-3-kinase activity (data not shown). In CHO/EGF-R cells, EGF stimulated PI-3-kinase activity by ~2.6-fold (Fig. 4B). As was true for the EGF receptor autophosphorylation, stearyl 3S-peptide-I treatment did not increase EGF-stimulated PI-3-kinase activity in CHO/EGF-R cells (Fig. 4B). This result suggests that stearyl 3S-peptide-I specifically enhances insulin-stimulated PI-3-kinase activity.

MAP kinase activity is rapidly stimulated in response to insulin and other growth factors (41, 42) via a mechanism that involves both tyrosine and serine/threonine phosphorylation of the enzyme itself (43). MAP kinase activity was assayed in the anti-MAP kinase immunoprecipitates prepared from cells treated or not with stearyl 3S-peptide-I by measuring the level of phosphorylation of an exogenous substrate, MBP. Low levels of MAP kinase activity were detected in the immunoprecipitates prepared from unstimulated cells. When cells were treated with their respective ligand, a large increase in MAP kinase activity was observed (Fig. 5, A and B). In CHO/HIRc cells, stearyl 3S-peptide-I (50 µM) increased insulin-stimulated MAP kinase activity by 2.3-fold (p < 0.0001) while having no significant effect on EGF-induced activation of MAP kinase in CHO/EGF-R cells (Fig. 5, A and B). Thus, it appears again that 3S-peptide-I is capable of interacting with specific components of insulin signal transduction pathways.

DISCUSSION

Protein tyrosine phosphorylation plays a determinant role in regulating many cellular processes. The level of phosphotyrosine in the cell is a balance between the actions of protein tyrosine kinases and PTPases. It has become apparent that there is a large number of PTPases with distinct specificities, some of which for tyrosine kinase-linked receptors. The mechanism that govern PTPase substrate specificity in cells is largely unknown. Cellular compartmentalization (44), interaction with closely associated regulatory proteins, levels of PTPase inhibitors and activators (45, 46), as well as posttranslational modifications (47, 48, 49) might provide such possible mechanism. In addition, features in the primary structure surrounding the dephosphorylation site may contribute to substrate specificity.

The experiments described in this paper were designed to assess whether 3S-peptide-I displays any specificity as an inhibitor for the PTPases acting on the insulin receptor. 3S-Peptide-I has three nonhydrolyzable sulfotyrosine residues inserted during the chemical synthesis and which correspond to the major autophosphorylation site of the insulin receptor. The incorporation of stearyl moieties to 3S-peptide-I enables its entry in intact cells and thereby allows access of the peptide to the intracellular milieu. We have shown that stearyl 3S-peptide-I stimulated insulin-induced autophosphorylation of the insulin receptor -subunit, which, in turn, increased the association of PI-3-kinase activity with IRS-1 and MAP kinase activation in response to insulin. These stimulatory effects of stearyl 3S-peptide-I were specific for insulin signaling, as receptor autophosphorylation levels, PI-3-kinase, and MAP kinase activation in response to EGF were not affected. Whether sulfotyrosine-containing peptides corresponding to the autophosphorylation site of other growth factor receptors (e.g. receptor for EGF, PDGF, and fibroblast growth factor) can show specificity toward their respective receptor phosphorylation and signaling remains to be determined.

Change in the fatty acid composition of membrane phospholipids by the stearyl moiety might provide a mechanism by which stearyl 3S-peptide-I enhances the transmission of insulin signal. Fatty acids are normal constituants of biological membranes, influencing the physicochemical state of lipid domains (50). It has been proposed that exogenous addition of fatty acids perturbs the bilayer structure of the plasma membrane, leading to alterations in membrane-cytoskeleton interactions and the modification of the physical state of transmembrane receptors and regulatory proteins (51). Consistent with this hypothesis are reports demonstrating that fatty acids can inhibit transmembrane signaling within minutes (52, 53) through changes in membrane fluidity or permeability (54, 55). The lack of effect of free stearic acid on both insulin-stimulated tyrosine phosphorylation of the insulin receptor and activation of PI-3-kinase in intact cells (data not shown) suggests that the primary action of stearyl 3S-peptide-I may not be the result of a nonspecific alteration of membrane phospholipid properties.

Our previous study suggests that the effect of 3S-peptide-I is not mediated through direct stimulation of the insulin receptor -subunit autophosphorylation when assayed in vitro (Ref. 30 and data not shown), but rather is the result of an alteration in tyrosine dephosphorylation. Under the same experimental conditions whereby cells were semipermeabilized with digitonin, we did not observed the inhibition of EGF receptor dephosphorylation by 3S-peptide-I. Therefore, the lack of effect of 3S-peptide-I on the level of tyrosine phosphorylation and dephosphorylation of the EGF receptor and its downstream signaling molecules suggests that cellular PTPases can be selectively inhibited by nonhydrolyzable phosphotyrosyl-containing peptide analogs based, in part, on the primary structure of the protein substrate. On the other hand, 3S-peptide-I does not affect the activity of members of serine/threonine phosphatase family or alkaline phosphatase, but has been shown to partially inhibit a recombinant PTPase, PTP-1B (30). This effect may occur due to the accessibility of the sulfotyrosyl residues per se and not because of the nature of the amino acid residues adjacent to the three sulfotyrosines in the above peptide.

This study demonstrates that 3S-peptide-I is an effective agent that increases the action of insulin on two important signaling mediators. This effect appears to occur as a result of action at a step proximal to the tyrosine phosphorylation of the insulin receptor and the IRS-1. Numerous proteins bind to tyrosine-phosphorylated IRS-1 through their SH2 domains after insulin stimulation. This interaction appears to be an activation step for several intracellular enzymes containing SH2 motifs. Using anti-IRS-1 antibody, we showed that stearyl 3S-peptide-I increased insulin-stimulated PI-3-kinase activity. Because of the multifunctional role played by PI-3-kinase within the cell, i.e. in cell growth, activation of pp70 S6 kinase, and in GLUT-4 translocation (38), experiments are being designed in an attempt to elucidate the impact 3S-peptide-I may have on insulin signal transduction in a number of insulin responsive cells. We observed an increase in insulin-induced MAP kinase activation in response to stearyl 3S-peptide-I. MAP kinase participates in a phosphorylation cascade in cells that plays an important role in coordinating the regulation of a number of kinases and phosphatases involved in glycogen synthesis and nuclear signaling. It is known that the association of SH-PTP2 (Syp) to tyrosine-phosphorylated IRS-1 (56) participates in insulin-stimulated MAP kinase activation (57, 58, 59). Because the formation of a SH-PTP2/IRS-1 complex has been shown to function as a potent activator of the Ras-Raf-MAP kinase cascade (60, 61), our results suggest that 3S-peptide-I may not alter the catalytic activity of SH-PTP2. Although more studies are necessary to evaluate the effect of 3S-peptide-I in the activity of several enzymes in vitro and on various metabolic and mitogenic responses of insulin, these data suggest that selective inhibition of the insulin receptor dephosphorylation by 3S-peptide-I causes a specific activation of downstream components of insulin signal transduction pathways.

There are divergent views regarding which of the three phosphotyrosyl residues (1146, 1150, or 1151) contained in the catalytic domain of the insulin receptor is the primary target of the physiologically relevant PTPase(s) action in situ (4, 24, 25, 62-64). The selective sulfation of individual tyrosyl residues within a given peptide sequence is possible by using protected side-chains on the tyrosine(s) that we do not wish to modify. This methodology allows the development of monosulfated analogs of 3S-peptide-I and should demonstrate whether dephophorylation of the insulin receptor is preferentially inhibited by the relative position of sulfotyrosine residues in peptide-I. Likewise, variations of the peptide scanning approach, which include the synthesis of a series of sulfotyrosylated peptides with different length or the construction of peptide libraries where individual amino acids are replaced by alanine (26, 28) shall define the optimal structure needed for the rational design of an insulin receptor-specific PTPase inhibitor.