Insulin Receptor Kinase-Associated Phosphotyrosine Phosphatases in Hepatic Endosomes: Assessing the Role of Phosphotyrosine Phosph
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《内分泌学杂志》
Polypeptide Hormone Laboratory (C.L., G.B., B.I.P.) and Department of Anatomy and Cell Biology (A.F., J.J.M.B., B.I.P.), McGill Cancer Center
Department of Biochemistry, McGill University (F.G., M.L.T.), Montreal, Quebec, Canada H3A 2B2
Abstract
Previous work has shown that bisperoxo(1,10-phenanthroline)-oxovanadate(v) anion [bpV(phen)] induces potent insulin-mimicking effects in the rat, selectively activates the endosomal (EN) insulin receptor kinase (IRK) in liver, and markedly abolishes endosomal IRK-associated phosphotyrosine phosphatase (PTP) activity while reducing that of total ENs by approximately 30%. In this study we examined the relatively selective effect of bpv(phen) on endosomal PTP activities for the purpose of defining IRK-associated PTP(s). Using an in-gel PTP assay, we detected multiple (20) species of endosomal PTP (30 to >220 kDa), with five that were markedly inhibited after in vivo bpV(phen) administration. Using a combination of Mono Q anionic exchange chromatography and immunoblotting, we demonstrated that LAR (leukocyte common antigen-related), PTP-, and PTP-1B were present in endosomal subfractions not significantly inhibited by bpv(phen). PTP-1B activity was assayed in immunoprecipitates from hepatic ENs of control and bpV(phen)-treated rats and was found to be inhibited by approximately 30% after bpv(phen) treatment. To clarify the role of PTP-1B in dephosphorylating IRK, we prepared hepatic ENs from wild-type and PTP-1B-null mice. We found that the phosphotyrosine content of IRK was similar in these two types of ENs, and that IRK dephosphorylation was not affected in ENs from PTP-1B-null mice compared with that in ENs from wild-type mice. These data suggest that LAR , PTP-, and PTP-1B are not candidates for the IRK-associated PTP in hepatic ENs, and that IRK dephosphorylation in ENs may result from the concerted actions of several PTPs.
Introduction
THE INSULIN RECEPTOR kinase (IRK) is a heterotetrameric protein consisting of two extracellular -subunits containing the insulin-binding site and two transmembrane -subunits possessing intrinsic tyrosine kinase activity (1). After the binding of insulin to its receptor, there is autophosphorylation on tyrosine residues of the -subunits leading to IRK activation (2) and the phosphorylation of insulin receptor substrates, especially insulin receptor substrate-1 and -2 (3). Concomitantly, there is rapid internalization of the activated IRK into endosomes (ENs) (4, 5). These events are important for insulin signal transduction and the realization of its physiological actions (6). Within ENs, insulin is dissociated from the IRK and degraded by endosomal acid insulinase (7), whereas IRKs are dephosphorylated (8) and largely recycled to the cell surface, with a small proportion undergoing degradation in late ENs-lysosomes (9).
The tyrosine phosphorylation state of the IRK reflects a balance between its intrinsic kinase activity and the action of protein tyrosine phosphatases (PTPs) (10). In previous work we demonstrated an IRK-associated PTP activity in rat liver ENs, whose significance was highlighted by the study of its inhibition (8). Thus, the peroxovanadium compounds (pVs), potent PTP inhibitors, were shown to mimic insulin action through their capacity to promote IRK tyrosine phosphorylation by inhibiting the IRK-associated PTP(s) in ENs (11) (12). Interestingly, we observed relative specificity for this inhibitory effect, because an in vivo dose of bisperoxo(1,10-phenanthroline)-oxovanadate(v) anion [bpV(phen)], which completely inhibited the dephosphorylation of endosomal IRK, only modestly inhibited (30%) total endosomal PTP activity (13).
Considerable work has been directed at identifying PTP(s) that dephosphorylate IRK as possible targets for insulin mimetic drug development (14). Several PTPs [viz. PTP-, leukocyte common antigen-related (LAR), and PTP-1B] have been considered candidates based on studies of their overexpression, substrate-trapping mutants, mouse knockout models, and other approaches (15). Although PTP-1B has emerged as being of particular interest, the key endosomal IRK-associated PTP(s) remains to be identified. In this study we have used bpv(phen)-induced PTP inhibition to characterize possible endosomal IRK-associated PTPs and a knockout mouse model to assess the role of PTP-1B in this.
Materials and Methods
Animals
Female Sprague Dawley rats (160–180 g body weight) were purchased from Charles River Laboratories, Inc. (St. Constant, Canada). PTP-1B-knockout mice were generated as previously described (16). All animal procedures were performed in accordance with the standards of the Canadian animal care committee.
Reagents
Porcine insulin was a gift from Eli Lilly Co. (Indianapolis, IN). Kodak X-OMAT AR film, phenylmethylsulfonylfluoride (PMSF), aprotinin, leupeptin, pepstatin A, HEPES (free acid), polyglutamic acid-tyrosine (4:1) (pGT), RIA grade BSA, and most other chemicals were purchased from Sigma-Aldrich Corp. (St. Louis, MO). The Mono Q HR 5/5 column, wheat-germ agglutinin-Sepharose 6MB (WGA-Sepharose) and protein A-Sepharose CL-4B were obtained from Pharmacia Biotech (Uppsala, Sweden). 1,4-Dithiothreitol (DTT) and ATP (disodium salt) were obtained from Roche Molecular Biochemicals (Laval, Canada). [-32P]ATP (3000 Ci/mmol) was purchased from NEN Life Science Products (Lachine, Canada). Drs. Jesse Ng and Alan Shaver (Department of Chemistry, McGill University, Quebec, Canada) prepared bpV(phen) as described previously (11). Reagents for electrophoresis were obtained from Bio-Rad Laboratories, Inc. (Richmond, CA). Polyvinylidene difluoride Immobilon-P transfer membranes were obtained from Millipore Corp. (Mississauga, Canada).
Antibodies
Polyclonal anti-PTP-1B was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Polyclonal PTP- was a gift from Dr. Frank R. Jirik (University of Calgary, Alberta, Canada). Anti-LAR antibody was raised by immunizing rabbits with a glutathione-S-transferase-LAR fusion protein containing the cytoplasmic domain of the rat LAR sequence provided by Dr. B. J. Goldstein (Department of Medicine, Jefferson Medical College of Thomas Jefferson University, Philadelphia, PA). A monoclonal antiphosphotyrosine antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). An antibody to the triphosphorylated activation domain of the IRK (IR/IGF1R; pYpYpY1158/1162/1163) was purchased from BioSource International, Inc. (Camarillo, CA).
Preparation of hepatic ENs
Animals (rats or mice) were fasted overnight (16–18 h), anesthetized, and killed by decapitation at the indicated times after intrajugular injections of the agents indicated in the figure legends. Livers were rapidly excised and minced at scissor point in ice-cold 0.25 M sucrose solution/5 mM Tris-HCl buffer (pH 7.4) containing 1 mM benzamidine, 1 mM PMSF, and 1 mM MgCl2. ENs were prepared as previously described (4). The protein content of cell fractions was measured using a Bio-Rad Protein Assay Reagent (catalog no. 500-0006) with BSA as standard.
Mono Q anion exchange chromatography
ENs (6 mg protein) were suspended in a final volume of 12 ml solubilization buffer [0.5 M HEPES (pH 7.4), 1% Triton X-100, 1 mM DTT, 1 mM benzamidine, 1 mM PMSF, 20 μM leupeptin, 20 μM pepstatin A, and 0.3 trypsin inhibitory units/ml aprotinin] and rotated at 4 C for 1 h. The mixture was centrifuged at 40,000 rpm (202,000 x g) for 30 min in a Beckman SW40 rotor (Beckman Coulter, Palo Alto, CA). The supernatant was passed through an 0.22-μM pore size syringe filter and loaded onto a Mono Q HR 5/5 column that had been washed with 10 ml equilibration buffer [0.5 M HEPES buffer (pH 7.4) containing 1% Triton X-100, 1 mM Na2 EDTA, 1 mM benzamidine, 1 mM PMSF, 1 mM DTT, and 5% glycerol]. After sample application, the column was washed with 10 ml equilibration buffer, and 50 fractions (1 ml each) were eluted with a linear NaCl gradient (0–500 mM) in equilibration buffer.
Measurement of PTP activity
In vitro assay using [32P]pGT as substrate.
The PTP activity of eluted fractions from Mono Q chromatography was measured using [32P]pGT as substrate, as previously described (13). Briefly, 10-μl aliquots of each fraction were incubated with [32P]pGT [1.5–5 x 10–2 μCi)] in a final volume of 100 μl reaction mixture [25 mM Na2HPO4-NaH2PO4 buffer (pH 7.4), 1 mM EDTA, 1 mM DTT, and 0.005% BSA] at 30 C for 7 min. The reaction was terminated by trichloroacetic acid precipitation, followed by centrifugation at 12,000 x gav. The [32P]phosphate released from [32P]pGT was measured by scintillation counting of the supernatant in a 1219 RACKBETA liquid scintillation counter (90% counting efficiency; PerkinElmer, Wellesley, MA).
In vitro assay using [32P]IRK as substrate.
IRK was partially purified from hepatic microsomes by WGA-Sepharose column chromatography, as described previously (17), and labeled with 32P by incubating the WGA-Sepharose-purified material with [-32P]ATP (100 μCi) in 900 μl 50 mM HEPES buffer (pH 7.4; final concentrations, 300 nM insulin, 50 μM ATP, 5 mM MnCl2, and 0.1% Triton X-100) at 30 C for 1 h. Labeling was stopped by adding 900 μl 20 mM EDTA, 2 mM DTT, and 1 mM ATP, and the mixture was passed through an Econo-Pac 10DG column (Bio-Rad Laboratories, Inc.) to remove free [-32P]ATP from [32P]IRK. PTP activity in fractions eluted from Mono Q columns was assayed by incubating [32P]IRK (10 μl) with 10 μl of each fraction and 20 μl buffer (10 mM EDTA, 1 mM DTT, and 0.5 mM ATP) at 30 C for 30 min. Dephosphorylation was stopped by adding Laemmli sample buffer and boiling for 5 min, followed by SDS-PAGE (7.5% gel) and autoradiography at –80 C. PTP activity was reflected by the loss of [32P]IRK content quantified by densitometry analysis (Bio-Rad GS-700 Imaging Densitometer) of the 94-kDa band (IRK -subunit) on the autoradiogram. In situ IRK dephosphorylation in intact ENs was conducted as described in detail previously (8).
In vitro assay of triphosphorylated IRK.
Endosomes (25 μg) were suspended in solution (90 μl) containing a final concentration of 50 mM HEPES (pH 7.4), 150 mM KCl, 5 mM NaCl, 5 mM MnCl2, and 1 mM DTT. IRK autophosphorylation was initiated by adding 10 μl unlabeled ATP (final concentration, 1 mM) and incubating at 37 C for 5 min. The reaction was stopped by adding 10 μl prewarmed (37 C) stopping buffer containing 500 mM HEPES (pH 7.4) and 100 mM EDTA. Continuing the incubation at 37 C resulted in dephosphorylation, which was assayed at the times noted in Fig. 5C by adding a stopping solution of 50 μl 150 mM Tris (pH 6.8), 6.9% sodium dodecyl sulfate, 30% glycerol, and 100 mM DTT, followed by immediate immersion at 100 C for 2 min. The solution was then subjected to PAGE, followed by transfer to nitrocellulose membranes and immunoblotting with antibody to the triphospho-IRK (IR/IGF1R; pYpYpY1158/1162/1163) as described by the supplier (BioSource International, Inc.).
PTP-1B activity.
ENs (300 μg protein) were suspended in solubilization buffer [0.5 M HEPES (pH 7.4), 1% Triton X-100, 1 mM DTT, 1 mM benzamidine, 1 mM PMSF, 20 μM leupeptin, 20 μM pepstatin A, and 0.3 trypsin inhibitory units/ml aprotinin] in a final volume of 500 μl, rotated (Clay Adams Nutator mixer, a division of Becton Dickinson, Parsippany, NY) at 4 C for 1 h, and centrifuged at 4 C for 15 min at a maximum speed of 13,000 rpm in a Biofuge Pico centrifuge (Heraeus Biofuge Pico, DJB Labcare, Buckinghamshire, UK). The supernatant was incubated with 2 μg anti-PTP-1B antibody and rotated overnight at 4 C, followed by the addition of 100 μl of a 50% slurry of protein A-Sepharose. The suspension was rotated for an additional 4 h at 4 C, then centrifuged for 20 sec at maximum speed in the Biofuge centrifuge. The pellet was washed three times with solubilization buffer, followed by gently vortexing and spinning as described above. After the final centrifugation, the pellet was resuspended by gently vortexing in 50 μl 50 mM HEPES (pH 7.4). To measure PTP activity, the suspension (20 μl) was incubated with [32P]pGT (1.5–5 x 10–2 μCi) in 100 μl reaction buffer at 30 C for 7 min as described above.
In-gel assays were performed as described in detail by Burridge and Nelson (18) with minor modification. [32P]pGT was synthesized as noted above and incorporated into a 7.5% sodium dodecyl sulfate-polyacrylamide gel (acrylamide/bisacrylamide, 30:0.8). Instead of Tween 40, Tween 20 was used throughout the protocol.
Immunoblotting
Aliquots of pooled PTP peaks (30 μl) were boiled in Laemmli buffer for 5 min, resolved by SDS-PAGE (7.5% gel), and transferred to Immobilon-P membranes at a constant current of 400 mA for 4 h. Membranes were probed with the indicated first antibody against specific PTPs for 2 h, followed by 1-h incubation with horseradish peroxidase-labeled goat antirabbit IgG. Immunoreactive proteins were detected using enhanced chemiluminescence (ECL, Amersham Biosciences, Indianapolis, IN) according to the manufacturer’s manual and quantified by densitometry (GS-700 Imaging Densitometer, Bio-Rad Laboratories, Inc.). For detection of IRK-phosphotyrosine (PY), a 50-μg aliquot of mouse ENs was subjected to SDS-PAGE and immunoblotted with PY and 125I-labeled goat antimouse IgG as second antibody, as described previously (4, 5).
Results
Pattern of endosomal PTP inhibition by bpV(phen)
In previous work we showed that 15 min after the administration of bpV(phen) (0.6 μmol/100 g body weight), IRK-associated PTP activity was almost completely inhibited (8). In the present study we determined whether there were endosomal PTPs selectively inhibited by bpV(phen) and, therefore, possible candidates for the IRK-associated PTP(s) in ENs. In initial studies we resolved endosomal PTP activities using Mono Q anionic liquid chromatography (Fig. 1). Solubilized hepatic ENs, from rats injected with insulin 2 min before killing were loaded onto Mono Q anionic columns, which were eluted with a linear NaCl gradient (0–500 mM). Each eluted fraction (1 ml) was assayed for its ability to remove labeled phosphate from 32P-labeled pGT. We consistently observed that PTP activity was eluted from the Mono Q column in three major peaks (Fig. 1A). The protein concentrations in the individual peaks were difficult to assess with the Bio-Rad technique due to interference from Triton X-100. Therefore, we estimated the relative protein concentration by subjecting equal peak volumes to SDS-PAGE and silver staining, followed by scanning densitometry of the individual lanes. The densities of peaks 1, 2, and 3 were 1.3, 0.69, and 1.0, respectively, which approximately paralleled their relative PTP activities, suggesting that the PTP reaction was occurring in a linear range with respect to protein.
To evaluate whether bpv(phen) effected a selective inhibition of endosomal PTP(s), we isolated hepatic ENs 15 min after bpV(phen) administration and 2 min after insulin injection. We observed selective inhibition of PTP activity (24–30%) eluting in peak 1 compared with that in peaks 2 and 3 using either 32P-labeled, partially purified IRK or 32P-labeled pGT as substrate for the assay of PTP activity (Fig. 1B and inset). These data are consistent with previous work showing that IRK-associated PTP(s) is selectively inhibited by bpV(phen) (13) and suggest that the PTPs that are inhibited are concentrated in peak 1 rather than in peaks 2 and 3.
Selective inhibition of multiple endosomal PTPs by bpV(phen)
To assess the differential inhibition of endosomal PTPs by bpV(phen), total hepatic ENs from control and insulin-treated rats with and without previous bpv(phen) treatment were subjected to SDS-PAGE in gels impregnated with 32P-labeled pGT. After in-gel renaturation of the resolved proteins, autoradiograms of these gels were developed. As can be seen, approximately 20 bands of desphosphorylating activity could be identified in ENs from control rats (Fig. 2, lanes 1 and 2). Insulin treatment 2 min before death and preparation of ENs had no observable effect on either the number or the intensity of these bands (Fig. 2, lanes 3 and 4). In contrast, the administration of bpv(phen) 15 min before the preparation of ENs resulted in a noticeable reduction or abolition of at least five bands (demarcated by arrows at the right margin) corresponding to entities with molecular masses of approximately 220, 110, 62, 52, and 43 kDa. It is noteworthy that the intensities of the other PTP bands were minimally influenced or even increased. We conclude that the in vivo administration of bpV(phen) selectively inhibits a discrete number of endosomal PTPs. Analysis of in-gel PTP activity effected by the PTPs in peak 1 also demonstrated selective inhibition of in-gel PTP activity (data not shown).
Evaluation of PTP-1B, PTP-, and LAR as possible IRK-associated PTPs
Because PTP-1B, PTP-, and LAR have all been considered candidates for the IRK-associated PTP, we wanted to determine whether any or all of these PTPs were selectively localized to peak 1. Therefore, we pooled peak fractions of each of peaks 1–3 eluted from the Mono Q chromatograms and subjected these to immunoblotting using specific antibodies against PTP-1B, PTP-, and LAR, respectively. As shown in Fig. 3, all three of these PTPs were detected largely in peak 3, suggesting that PTP-1B, PTP-, and LAR are not major bpv(phen)-inhibited PTPs and, hence, are unlikely candidates for the IRK-associated PTP(s) in hepatic ENs.
This view is strengthened vis-a-vis PTP-1B by direct assay of PTP-1B immunoprecipitated from hepatic ENs. Hepatic ENs prepared from rats that had received insulin with or without prior bpV(phen) treatment were solubilized and incubated with a specific PTP-1B antibody to immunoprecipitate PTP-1B. As shown in Fig. 3B, bpV(phen) inhibited PTP-1B activity in the immunoprecipitates by 30.4 ± 5.9% (mean ± SE; n = 3) under conditions where IRK dephosphorylation was almost completely inhibited (13). This observation also suggests that endosomal PTP-1B is unlikely by itself to constitute the IRK-associated PTP.
Phosphorylation of IRK in PTP-1B-null mice
PTP-1B-null mice were created and bred as previously described (16). ENs were prepared from PTP-1B-null and wild-type mouse livers 2 min after the injection of insulin at a subsaturating dosage (1.5 μg/100 g body weight). As expected, the PTP-1B content in ENs from null mice was barely, if at all, detectable, whereas that in wild-type was readily demonstrated (Fig. 4, top panel). As shown (Fig. 4, middle panel), the phosphotyrosine content of the IRK -subunit, detected by immunoblotting with antibody against PY, was comparable in preparations of ENs from both types of mouse, as was the yield of IRK -subunit determined using the IRK -subunit-specific antibody 960 (13).
Dephosphorylation of IRK in PTP-1B-null mice
The dephosphorylation of endosomal IRK was assessed in situ using the procedure developed previously for intact ENs from rat liver (8). A typical experiment is depicted in Fig. 5A, where a time-dependent loss of 32P from the IRK -subunit in ENs from both null and wild-type mice is shown. The 32P content of IRK in hepatic ENs was normalized by plotting this relative to the initial 32P content of the IRK -subunit. As is evident in Fig. 5B, the rates of IRK -subunit dephosphorylation were essentially identical in ENs from both PTP-1B-null and wild-type mice (Fig. 5B). This observation suggests that PTP-1B has minimal or limited influence on the phosphorylation state of IRK in hepatic ENs.
Because IRK triphosphorylated (pYpYpY 1158/1162/1163) in the activation domain is fully activated as a kinase, dephosphorylation of this form of the receptor was assayed using an antibody specific to the triphosphorylated activation domain of the IRK. It is shown in Fig. 5C that there was no appreciable difference in the loss of this form of the receptor in ENs from either control or PTP-1B-null mice. Thus, the absence of PTP-1B in hepatic ENs results in neither a blunting of overall IRK dephosphorylation or the more specific dephosphorylation of the activation domain of the IRK.
Discussion
In previous work on the mechanism of action of pV compounds, we demonstrated that ENs constitute an important site for IRK signaling and dephosphorylation (12). We also observed that bpV(phen) administration in vivo could inhibit IRK dephosphorylation completely while inhibiting overall endosomal PTP activity by only approximately 30% (13). In this study we have confirmed this observation. Thus, we subjected ENs from control and bpV(phen)-treated rats to mono Q anionic chromatography and observed that PTP activity was resolved into three fractions, of which fraction 1 demonstrated inhibition relative to either fraction 2 or 3. Selective inhibition of endosomal PTP activity was also indicated by the observation that an in-gel phosphatase assay of hepatic ENs from animals pretreated with bpv(phen) demonstrated that five of the approximately 20 species of activity were inhibited. We then went on to exploit this finding by directly analyzing endosomal PTP activities differentially inhibited by bpv(phen).
Previous studies have focused on PTP, LAR, and PTP-1B as possible PTPs involved in dephosphorylating the IRK in vivo (15). It was therefore of particular interest that PTP, LAR, and PTP-1B were found in fraction 3 and not in the fraction in which PTP inhibition was most marked. This observation suggested that these PTPs are probably not among those associating with the IRK in ENs. Interestingly, when we examined PTP-1B activity in immunoprecipitates of ENs, we found approximately 30% inhibition after bpV(phen) treatment. Although unlikely, we could not exclude the possibility that a selected subfraction of PTP-1B, associated with IRK, was fully inhibited by bpV(phen) treatment.
Of these enzymes, PTP-1B has received particular attention, and a range of studies have suggested that it can attenuate insulin signaling. Thus, PTP 1B, inactivated by mutation at the active site (C215S), was shown to associate with the IRK (19, 20). Furthermore, crystal structure and kinetic studies demonstrated preferential dephosphorylation of IRK residues 1162 and 1163 by PTP-1B (21). Other support comes from the microinjection of PTP 1B in Xenopus oocytes, which resulted in decreased insulin-induced IR phosphorylation, S6 kinase activity, and meiosis (22, 23). In hepatoma cells, cellular loading of PTP-1B antibodies increased IRK phosphorylation and insulin signaling (24), and overexpression in L6 myocytes and Fao hepatoma cells decreased downstream insulin-signaling events (25). Knockout studies confirmed these observations, in that insulin increased IRK phosphorylation in liver and muscle of knockout mice that were hypersensitive to insulin administration (16, 26). In contrast, knockout models of either PTP- or LAR were unremarkable (27, 28).
We therefore analyzed the dephosphorylation of IRK in ENs from control and PTP-1B-null mice. IRK dephosphorylation occurred at the same rate in ENs from both control and null mice, and there was no difference in the rate at which the activation domain of the IRK was dephosphorylated in ENs from control vs. null mice. Together, these observations argue that PTP-1B is not associated with endosomal IRK. These findings are consistent with several other studies. Thus, in rat McA-RH7777 hepatoma cells, there was a direct correlation between the concentrations of PTP-1B, LAR, and SHP2 (maximum, >10-fold) and cell density, but no corresponding decrease in insulin sensitivity or IRK phosphorylation (29). Also, leptin treatment of ob/ob mice increased hepatic sensitivity to insulin, augmented IRK activation, and correspondingly increased hepatic levels of PTP-1B (30), the opposite of that expected for a negative regulator of IRK. Because it was observed that PTP-1B negatively regulates leptin signaling by dephosphorylating Janus kinase-2 (31, 32), an alternative explanation for the hypersensitivity of PTP-1B-null mice to insulin, especially in the liver, is enhanced hepatic leptin signaling in this circumstance.
Various studies have demonstrated that PTP-1B is largely localized on the cytoplasmic face of the endoplasmic reticulum (ER) via its C-terminal 35 amino acids (33, 34). Interestingly, it has been demonstrated that internalized epidermal growth factor receptor and platelet-derived growth factor receptor appear to be associated with ER-anchored PTP-1B in cells expressing D181A mutant, but not in wild-type PTP 1B, at later times (30 min) after exposure to ligand (35). Similarly, in HEK 293 cells, it was found that the PTP-1B D181A mutant associated with IRK under both insulin-independent and -dependent conditions (36). More recently, it has been suggested that this association occurred to some degree in a perinuclear endosomal compartment (37). Therefore, it appears likely that the interaction between internalized receptor tyrosine kinases (RTKs) and PTP-1B is predominantly with the ER-associated enzyme. The participation of the ER in the formation of the phagasome indicates that there are mechanisms allowing for the association of internalized molecules with ER-associated molecules (38). It is also possible that a portion of the internalized IRK in ENs is directed to the ER. This would be consistent with the observation that RTKs-activated phosphotidylinositol 3-kinases occur on various endomembranes in the course of cell signaling (39, 40). Also, there is evidence indicating that PTP-1B dephosphorylates ligand-independent tyrosine phosphorylation of IRK precursors and -subunits and may thereby play an important role to prevent inappropriate RTK activation during biogenesis and cellular processing (41). Finally, we cannot rule out the possibility that PTP-1B may be brought to the plasma membrane along with newly synthesized IRK and play a role in rapidly dephosphorylating cell surface IRK after insulin stimulation.
Using an in-gel PTP assay, we found approximately 20 PTPs in ENs, a subfraction of which was inhibited by bpV(phen). One or more of these entities might be the relevant PTP associated with the IRK in ENs. It is also worth noting that IRK dephosphorylation may be effected by the coordinate action of several PTPs, in which case a deficiency of one might be largely compensated by the function of the others.
In summary, the present study identifies a number of PTPs in ENs, a subgroup of which is significantly inhibited by bpV(phen) treatment. Our data indicate that PTP-1B may not be the important endosomal IRK-associated PTP. However, the interaction between IRK and PTP-1B in other intracellular compartments may well be involved in aspects of IRK signaling, as might the impact of PTP-1B on other signaling components (viz. leptin) and on downstream targets of the activated IRK (42).
Acknowledgments
We thank Drs. Simon S. Wing and Rose Oughtred for help with the setup and use of the Mono Q column chromatogram.
Footnotes
This work was supported by a grant from the Canadian Institutes for Health Research.
First Published Online November 3, 2005
Abbreviations: bpV(phen), Bisperoxo(1,10-phenanthroline)-oxovanadate(v) anion; DTT, 1,4-dithiothreitol; EN, endosome; ER, endoplasmic reticulum; IR, insulin receptor; IRK, insulin receptor kinase; LAR, leukocyte common antigen-related; pGT, polyglutamic acid-tyrosine (4:1); PMSF, phenylmethylsulfonylfluoride; PTP, phosphotyrosine phosphatase; pV, peroxovanadium; PY, phosphotyrosine; RTK, receptor tyrosine kinase; WGA, wheat-germ agglutinin.
Accepted for publication October 21, 2005.
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Department of Biochemistry, McGill University (F.G., M.L.T.), Montreal, Quebec, Canada H3A 2B2
Abstract
Previous work has shown that bisperoxo(1,10-phenanthroline)-oxovanadate(v) anion [bpV(phen)] induces potent insulin-mimicking effects in the rat, selectively activates the endosomal (EN) insulin receptor kinase (IRK) in liver, and markedly abolishes endosomal IRK-associated phosphotyrosine phosphatase (PTP) activity while reducing that of total ENs by approximately 30%. In this study we examined the relatively selective effect of bpv(phen) on endosomal PTP activities for the purpose of defining IRK-associated PTP(s). Using an in-gel PTP assay, we detected multiple (20) species of endosomal PTP (30 to >220 kDa), with five that were markedly inhibited after in vivo bpV(phen) administration. Using a combination of Mono Q anionic exchange chromatography and immunoblotting, we demonstrated that LAR (leukocyte common antigen-related), PTP-, and PTP-1B were present in endosomal subfractions not significantly inhibited by bpv(phen). PTP-1B activity was assayed in immunoprecipitates from hepatic ENs of control and bpV(phen)-treated rats and was found to be inhibited by approximately 30% after bpv(phen) treatment. To clarify the role of PTP-1B in dephosphorylating IRK, we prepared hepatic ENs from wild-type and PTP-1B-null mice. We found that the phosphotyrosine content of IRK was similar in these two types of ENs, and that IRK dephosphorylation was not affected in ENs from PTP-1B-null mice compared with that in ENs from wild-type mice. These data suggest that LAR , PTP-, and PTP-1B are not candidates for the IRK-associated PTP in hepatic ENs, and that IRK dephosphorylation in ENs may result from the concerted actions of several PTPs.
Introduction
THE INSULIN RECEPTOR kinase (IRK) is a heterotetrameric protein consisting of two extracellular -subunits containing the insulin-binding site and two transmembrane -subunits possessing intrinsic tyrosine kinase activity (1). After the binding of insulin to its receptor, there is autophosphorylation on tyrosine residues of the -subunits leading to IRK activation (2) and the phosphorylation of insulin receptor substrates, especially insulin receptor substrate-1 and -2 (3). Concomitantly, there is rapid internalization of the activated IRK into endosomes (ENs) (4, 5). These events are important for insulin signal transduction and the realization of its physiological actions (6). Within ENs, insulin is dissociated from the IRK and degraded by endosomal acid insulinase (7), whereas IRKs are dephosphorylated (8) and largely recycled to the cell surface, with a small proportion undergoing degradation in late ENs-lysosomes (9).
The tyrosine phosphorylation state of the IRK reflects a balance between its intrinsic kinase activity and the action of protein tyrosine phosphatases (PTPs) (10). In previous work we demonstrated an IRK-associated PTP activity in rat liver ENs, whose significance was highlighted by the study of its inhibition (8). Thus, the peroxovanadium compounds (pVs), potent PTP inhibitors, were shown to mimic insulin action through their capacity to promote IRK tyrosine phosphorylation by inhibiting the IRK-associated PTP(s) in ENs (11) (12). Interestingly, we observed relative specificity for this inhibitory effect, because an in vivo dose of bisperoxo(1,10-phenanthroline)-oxovanadate(v) anion [bpV(phen)], which completely inhibited the dephosphorylation of endosomal IRK, only modestly inhibited (30%) total endosomal PTP activity (13).
Considerable work has been directed at identifying PTP(s) that dephosphorylate IRK as possible targets for insulin mimetic drug development (14). Several PTPs [viz. PTP-, leukocyte common antigen-related (LAR), and PTP-1B] have been considered candidates based on studies of their overexpression, substrate-trapping mutants, mouse knockout models, and other approaches (15). Although PTP-1B has emerged as being of particular interest, the key endosomal IRK-associated PTP(s) remains to be identified. In this study we have used bpv(phen)-induced PTP inhibition to characterize possible endosomal IRK-associated PTPs and a knockout mouse model to assess the role of PTP-1B in this.
Materials and Methods
Animals
Female Sprague Dawley rats (160–180 g body weight) were purchased from Charles River Laboratories, Inc. (St. Constant, Canada). PTP-1B-knockout mice were generated as previously described (16). All animal procedures were performed in accordance with the standards of the Canadian animal care committee.
Reagents
Porcine insulin was a gift from Eli Lilly Co. (Indianapolis, IN). Kodak X-OMAT AR film, phenylmethylsulfonylfluoride (PMSF), aprotinin, leupeptin, pepstatin A, HEPES (free acid), polyglutamic acid-tyrosine (4:1) (pGT), RIA grade BSA, and most other chemicals were purchased from Sigma-Aldrich Corp. (St. Louis, MO). The Mono Q HR 5/5 column, wheat-germ agglutinin-Sepharose 6MB (WGA-Sepharose) and protein A-Sepharose CL-4B were obtained from Pharmacia Biotech (Uppsala, Sweden). 1,4-Dithiothreitol (DTT) and ATP (disodium salt) were obtained from Roche Molecular Biochemicals (Laval, Canada). [-32P]ATP (3000 Ci/mmol) was purchased from NEN Life Science Products (Lachine, Canada). Drs. Jesse Ng and Alan Shaver (Department of Chemistry, McGill University, Quebec, Canada) prepared bpV(phen) as described previously (11). Reagents for electrophoresis were obtained from Bio-Rad Laboratories, Inc. (Richmond, CA). Polyvinylidene difluoride Immobilon-P transfer membranes were obtained from Millipore Corp. (Mississauga, Canada).
Antibodies
Polyclonal anti-PTP-1B was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Polyclonal PTP- was a gift from Dr. Frank R. Jirik (University of Calgary, Alberta, Canada). Anti-LAR antibody was raised by immunizing rabbits with a glutathione-S-transferase-LAR fusion protein containing the cytoplasmic domain of the rat LAR sequence provided by Dr. B. J. Goldstein (Department of Medicine, Jefferson Medical College of Thomas Jefferson University, Philadelphia, PA). A monoclonal antiphosphotyrosine antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). An antibody to the triphosphorylated activation domain of the IRK (IR/IGF1R; pYpYpY1158/1162/1163) was purchased from BioSource International, Inc. (Camarillo, CA).
Preparation of hepatic ENs
Animals (rats or mice) were fasted overnight (16–18 h), anesthetized, and killed by decapitation at the indicated times after intrajugular injections of the agents indicated in the figure legends. Livers were rapidly excised and minced at scissor point in ice-cold 0.25 M sucrose solution/5 mM Tris-HCl buffer (pH 7.4) containing 1 mM benzamidine, 1 mM PMSF, and 1 mM MgCl2. ENs were prepared as previously described (4). The protein content of cell fractions was measured using a Bio-Rad Protein Assay Reagent (catalog no. 500-0006) with BSA as standard.
Mono Q anion exchange chromatography
ENs (6 mg protein) were suspended in a final volume of 12 ml solubilization buffer [0.5 M HEPES (pH 7.4), 1% Triton X-100, 1 mM DTT, 1 mM benzamidine, 1 mM PMSF, 20 μM leupeptin, 20 μM pepstatin A, and 0.3 trypsin inhibitory units/ml aprotinin] and rotated at 4 C for 1 h. The mixture was centrifuged at 40,000 rpm (202,000 x g) for 30 min in a Beckman SW40 rotor (Beckman Coulter, Palo Alto, CA). The supernatant was passed through an 0.22-μM pore size syringe filter and loaded onto a Mono Q HR 5/5 column that had been washed with 10 ml equilibration buffer [0.5 M HEPES buffer (pH 7.4) containing 1% Triton X-100, 1 mM Na2 EDTA, 1 mM benzamidine, 1 mM PMSF, 1 mM DTT, and 5% glycerol]. After sample application, the column was washed with 10 ml equilibration buffer, and 50 fractions (1 ml each) were eluted with a linear NaCl gradient (0–500 mM) in equilibration buffer.
Measurement of PTP activity
In vitro assay using [32P]pGT as substrate.
The PTP activity of eluted fractions from Mono Q chromatography was measured using [32P]pGT as substrate, as previously described (13). Briefly, 10-μl aliquots of each fraction were incubated with [32P]pGT [1.5–5 x 10–2 μCi)] in a final volume of 100 μl reaction mixture [25 mM Na2HPO4-NaH2PO4 buffer (pH 7.4), 1 mM EDTA, 1 mM DTT, and 0.005% BSA] at 30 C for 7 min. The reaction was terminated by trichloroacetic acid precipitation, followed by centrifugation at 12,000 x gav. The [32P]phosphate released from [32P]pGT was measured by scintillation counting of the supernatant in a 1219 RACKBETA liquid scintillation counter (90% counting efficiency; PerkinElmer, Wellesley, MA).
In vitro assay using [32P]IRK as substrate.
IRK was partially purified from hepatic microsomes by WGA-Sepharose column chromatography, as described previously (17), and labeled with 32P by incubating the WGA-Sepharose-purified material with [-32P]ATP (100 μCi) in 900 μl 50 mM HEPES buffer (pH 7.4; final concentrations, 300 nM insulin, 50 μM ATP, 5 mM MnCl2, and 0.1% Triton X-100) at 30 C for 1 h. Labeling was stopped by adding 900 μl 20 mM EDTA, 2 mM DTT, and 1 mM ATP, and the mixture was passed through an Econo-Pac 10DG column (Bio-Rad Laboratories, Inc.) to remove free [-32P]ATP from [32P]IRK. PTP activity in fractions eluted from Mono Q columns was assayed by incubating [32P]IRK (10 μl) with 10 μl of each fraction and 20 μl buffer (10 mM EDTA, 1 mM DTT, and 0.5 mM ATP) at 30 C for 30 min. Dephosphorylation was stopped by adding Laemmli sample buffer and boiling for 5 min, followed by SDS-PAGE (7.5% gel) and autoradiography at –80 C. PTP activity was reflected by the loss of [32P]IRK content quantified by densitometry analysis (Bio-Rad GS-700 Imaging Densitometer) of the 94-kDa band (IRK -subunit) on the autoradiogram. In situ IRK dephosphorylation in intact ENs was conducted as described in detail previously (8).
In vitro assay of triphosphorylated IRK.
Endosomes (25 μg) were suspended in solution (90 μl) containing a final concentration of 50 mM HEPES (pH 7.4), 150 mM KCl, 5 mM NaCl, 5 mM MnCl2, and 1 mM DTT. IRK autophosphorylation was initiated by adding 10 μl unlabeled ATP (final concentration, 1 mM) and incubating at 37 C for 5 min. The reaction was stopped by adding 10 μl prewarmed (37 C) stopping buffer containing 500 mM HEPES (pH 7.4) and 100 mM EDTA. Continuing the incubation at 37 C resulted in dephosphorylation, which was assayed at the times noted in Fig. 5C by adding a stopping solution of 50 μl 150 mM Tris (pH 6.8), 6.9% sodium dodecyl sulfate, 30% glycerol, and 100 mM DTT, followed by immediate immersion at 100 C for 2 min. The solution was then subjected to PAGE, followed by transfer to nitrocellulose membranes and immunoblotting with antibody to the triphospho-IRK (IR/IGF1R; pYpYpY1158/1162/1163) as described by the supplier (BioSource International, Inc.).
PTP-1B activity.
ENs (300 μg protein) were suspended in solubilization buffer [0.5 M HEPES (pH 7.4), 1% Triton X-100, 1 mM DTT, 1 mM benzamidine, 1 mM PMSF, 20 μM leupeptin, 20 μM pepstatin A, and 0.3 trypsin inhibitory units/ml aprotinin] in a final volume of 500 μl, rotated (Clay Adams Nutator mixer, a division of Becton Dickinson, Parsippany, NY) at 4 C for 1 h, and centrifuged at 4 C for 15 min at a maximum speed of 13,000 rpm in a Biofuge Pico centrifuge (Heraeus Biofuge Pico, DJB Labcare, Buckinghamshire, UK). The supernatant was incubated with 2 μg anti-PTP-1B antibody and rotated overnight at 4 C, followed by the addition of 100 μl of a 50% slurry of protein A-Sepharose. The suspension was rotated for an additional 4 h at 4 C, then centrifuged for 20 sec at maximum speed in the Biofuge centrifuge. The pellet was washed three times with solubilization buffer, followed by gently vortexing and spinning as described above. After the final centrifugation, the pellet was resuspended by gently vortexing in 50 μl 50 mM HEPES (pH 7.4). To measure PTP activity, the suspension (20 μl) was incubated with [32P]pGT (1.5–5 x 10–2 μCi) in 100 μl reaction buffer at 30 C for 7 min as described above.
In-gel assays were performed as described in detail by Burridge and Nelson (18) with minor modification. [32P]pGT was synthesized as noted above and incorporated into a 7.5% sodium dodecyl sulfate-polyacrylamide gel (acrylamide/bisacrylamide, 30:0.8). Instead of Tween 40, Tween 20 was used throughout the protocol.
Immunoblotting
Aliquots of pooled PTP peaks (30 μl) were boiled in Laemmli buffer for 5 min, resolved by SDS-PAGE (7.5% gel), and transferred to Immobilon-P membranes at a constant current of 400 mA for 4 h. Membranes were probed with the indicated first antibody against specific PTPs for 2 h, followed by 1-h incubation with horseradish peroxidase-labeled goat antirabbit IgG. Immunoreactive proteins were detected using enhanced chemiluminescence (ECL, Amersham Biosciences, Indianapolis, IN) according to the manufacturer’s manual and quantified by densitometry (GS-700 Imaging Densitometer, Bio-Rad Laboratories, Inc.). For detection of IRK-phosphotyrosine (PY), a 50-μg aliquot of mouse ENs was subjected to SDS-PAGE and immunoblotted with PY and 125I-labeled goat antimouse IgG as second antibody, as described previously (4, 5).
Results
Pattern of endosomal PTP inhibition by bpV(phen)
In previous work we showed that 15 min after the administration of bpV(phen) (0.6 μmol/100 g body weight), IRK-associated PTP activity was almost completely inhibited (8). In the present study we determined whether there were endosomal PTPs selectively inhibited by bpV(phen) and, therefore, possible candidates for the IRK-associated PTP(s) in ENs. In initial studies we resolved endosomal PTP activities using Mono Q anionic liquid chromatography (Fig. 1). Solubilized hepatic ENs, from rats injected with insulin 2 min before killing were loaded onto Mono Q anionic columns, which were eluted with a linear NaCl gradient (0–500 mM). Each eluted fraction (1 ml) was assayed for its ability to remove labeled phosphate from 32P-labeled pGT. We consistently observed that PTP activity was eluted from the Mono Q column in three major peaks (Fig. 1A). The protein concentrations in the individual peaks were difficult to assess with the Bio-Rad technique due to interference from Triton X-100. Therefore, we estimated the relative protein concentration by subjecting equal peak volumes to SDS-PAGE and silver staining, followed by scanning densitometry of the individual lanes. The densities of peaks 1, 2, and 3 were 1.3, 0.69, and 1.0, respectively, which approximately paralleled their relative PTP activities, suggesting that the PTP reaction was occurring in a linear range with respect to protein.
To evaluate whether bpv(phen) effected a selective inhibition of endosomal PTP(s), we isolated hepatic ENs 15 min after bpV(phen) administration and 2 min after insulin injection. We observed selective inhibition of PTP activity (24–30%) eluting in peak 1 compared with that in peaks 2 and 3 using either 32P-labeled, partially purified IRK or 32P-labeled pGT as substrate for the assay of PTP activity (Fig. 1B and inset). These data are consistent with previous work showing that IRK-associated PTP(s) is selectively inhibited by bpV(phen) (13) and suggest that the PTPs that are inhibited are concentrated in peak 1 rather than in peaks 2 and 3.
Selective inhibition of multiple endosomal PTPs by bpV(phen)
To assess the differential inhibition of endosomal PTPs by bpV(phen), total hepatic ENs from control and insulin-treated rats with and without previous bpv(phen) treatment were subjected to SDS-PAGE in gels impregnated with 32P-labeled pGT. After in-gel renaturation of the resolved proteins, autoradiograms of these gels were developed. As can be seen, approximately 20 bands of desphosphorylating activity could be identified in ENs from control rats (Fig. 2, lanes 1 and 2). Insulin treatment 2 min before death and preparation of ENs had no observable effect on either the number or the intensity of these bands (Fig. 2, lanes 3 and 4). In contrast, the administration of bpv(phen) 15 min before the preparation of ENs resulted in a noticeable reduction or abolition of at least five bands (demarcated by arrows at the right margin) corresponding to entities with molecular masses of approximately 220, 110, 62, 52, and 43 kDa. It is noteworthy that the intensities of the other PTP bands were minimally influenced or even increased. We conclude that the in vivo administration of bpV(phen) selectively inhibits a discrete number of endosomal PTPs. Analysis of in-gel PTP activity effected by the PTPs in peak 1 also demonstrated selective inhibition of in-gel PTP activity (data not shown).
Evaluation of PTP-1B, PTP-, and LAR as possible IRK-associated PTPs
Because PTP-1B, PTP-, and LAR have all been considered candidates for the IRK-associated PTP, we wanted to determine whether any or all of these PTPs were selectively localized to peak 1. Therefore, we pooled peak fractions of each of peaks 1–3 eluted from the Mono Q chromatograms and subjected these to immunoblotting using specific antibodies against PTP-1B, PTP-, and LAR, respectively. As shown in Fig. 3, all three of these PTPs were detected largely in peak 3, suggesting that PTP-1B, PTP-, and LAR are not major bpv(phen)-inhibited PTPs and, hence, are unlikely candidates for the IRK-associated PTP(s) in hepatic ENs.
This view is strengthened vis-a-vis PTP-1B by direct assay of PTP-1B immunoprecipitated from hepatic ENs. Hepatic ENs prepared from rats that had received insulin with or without prior bpV(phen) treatment were solubilized and incubated with a specific PTP-1B antibody to immunoprecipitate PTP-1B. As shown in Fig. 3B, bpV(phen) inhibited PTP-1B activity in the immunoprecipitates by 30.4 ± 5.9% (mean ± SE; n = 3) under conditions where IRK dephosphorylation was almost completely inhibited (13). This observation also suggests that endosomal PTP-1B is unlikely by itself to constitute the IRK-associated PTP.
Phosphorylation of IRK in PTP-1B-null mice
PTP-1B-null mice were created and bred as previously described (16). ENs were prepared from PTP-1B-null and wild-type mouse livers 2 min after the injection of insulin at a subsaturating dosage (1.5 μg/100 g body weight). As expected, the PTP-1B content in ENs from null mice was barely, if at all, detectable, whereas that in wild-type was readily demonstrated (Fig. 4, top panel). As shown (Fig. 4, middle panel), the phosphotyrosine content of the IRK -subunit, detected by immunoblotting with antibody against PY, was comparable in preparations of ENs from both types of mouse, as was the yield of IRK -subunit determined using the IRK -subunit-specific antibody 960 (13).
Dephosphorylation of IRK in PTP-1B-null mice
The dephosphorylation of endosomal IRK was assessed in situ using the procedure developed previously for intact ENs from rat liver (8). A typical experiment is depicted in Fig. 5A, where a time-dependent loss of 32P from the IRK -subunit in ENs from both null and wild-type mice is shown. The 32P content of IRK in hepatic ENs was normalized by plotting this relative to the initial 32P content of the IRK -subunit. As is evident in Fig. 5B, the rates of IRK -subunit dephosphorylation were essentially identical in ENs from both PTP-1B-null and wild-type mice (Fig. 5B). This observation suggests that PTP-1B has minimal or limited influence on the phosphorylation state of IRK in hepatic ENs.
Because IRK triphosphorylated (pYpYpY 1158/1162/1163) in the activation domain is fully activated as a kinase, dephosphorylation of this form of the receptor was assayed using an antibody specific to the triphosphorylated activation domain of the IRK. It is shown in Fig. 5C that there was no appreciable difference in the loss of this form of the receptor in ENs from either control or PTP-1B-null mice. Thus, the absence of PTP-1B in hepatic ENs results in neither a blunting of overall IRK dephosphorylation or the more specific dephosphorylation of the activation domain of the IRK.
Discussion
In previous work on the mechanism of action of pV compounds, we demonstrated that ENs constitute an important site for IRK signaling and dephosphorylation (12). We also observed that bpV(phen) administration in vivo could inhibit IRK dephosphorylation completely while inhibiting overall endosomal PTP activity by only approximately 30% (13). In this study we have confirmed this observation. Thus, we subjected ENs from control and bpV(phen)-treated rats to mono Q anionic chromatography and observed that PTP activity was resolved into three fractions, of which fraction 1 demonstrated inhibition relative to either fraction 2 or 3. Selective inhibition of endosomal PTP activity was also indicated by the observation that an in-gel phosphatase assay of hepatic ENs from animals pretreated with bpv(phen) demonstrated that five of the approximately 20 species of activity were inhibited. We then went on to exploit this finding by directly analyzing endosomal PTP activities differentially inhibited by bpv(phen).
Previous studies have focused on PTP, LAR, and PTP-1B as possible PTPs involved in dephosphorylating the IRK in vivo (15). It was therefore of particular interest that PTP, LAR, and PTP-1B were found in fraction 3 and not in the fraction in which PTP inhibition was most marked. This observation suggested that these PTPs are probably not among those associating with the IRK in ENs. Interestingly, when we examined PTP-1B activity in immunoprecipitates of ENs, we found approximately 30% inhibition after bpV(phen) treatment. Although unlikely, we could not exclude the possibility that a selected subfraction of PTP-1B, associated with IRK, was fully inhibited by bpV(phen) treatment.
Of these enzymes, PTP-1B has received particular attention, and a range of studies have suggested that it can attenuate insulin signaling. Thus, PTP 1B, inactivated by mutation at the active site (C215S), was shown to associate with the IRK (19, 20). Furthermore, crystal structure and kinetic studies demonstrated preferential dephosphorylation of IRK residues 1162 and 1163 by PTP-1B (21). Other support comes from the microinjection of PTP 1B in Xenopus oocytes, which resulted in decreased insulin-induced IR phosphorylation, S6 kinase activity, and meiosis (22, 23). In hepatoma cells, cellular loading of PTP-1B antibodies increased IRK phosphorylation and insulin signaling (24), and overexpression in L6 myocytes and Fao hepatoma cells decreased downstream insulin-signaling events (25). Knockout studies confirmed these observations, in that insulin increased IRK phosphorylation in liver and muscle of knockout mice that were hypersensitive to insulin administration (16, 26). In contrast, knockout models of either PTP- or LAR were unremarkable (27, 28).
We therefore analyzed the dephosphorylation of IRK in ENs from control and PTP-1B-null mice. IRK dephosphorylation occurred at the same rate in ENs from both control and null mice, and there was no difference in the rate at which the activation domain of the IRK was dephosphorylated in ENs from control vs. null mice. Together, these observations argue that PTP-1B is not associated with endosomal IRK. These findings are consistent with several other studies. Thus, in rat McA-RH7777 hepatoma cells, there was a direct correlation between the concentrations of PTP-1B, LAR, and SHP2 (maximum, >10-fold) and cell density, but no corresponding decrease in insulin sensitivity or IRK phosphorylation (29). Also, leptin treatment of ob/ob mice increased hepatic sensitivity to insulin, augmented IRK activation, and correspondingly increased hepatic levels of PTP-1B (30), the opposite of that expected for a negative regulator of IRK. Because it was observed that PTP-1B negatively regulates leptin signaling by dephosphorylating Janus kinase-2 (31, 32), an alternative explanation for the hypersensitivity of PTP-1B-null mice to insulin, especially in the liver, is enhanced hepatic leptin signaling in this circumstance.
Various studies have demonstrated that PTP-1B is largely localized on the cytoplasmic face of the endoplasmic reticulum (ER) via its C-terminal 35 amino acids (33, 34). Interestingly, it has been demonstrated that internalized epidermal growth factor receptor and platelet-derived growth factor receptor appear to be associated with ER-anchored PTP-1B in cells expressing D181A mutant, but not in wild-type PTP 1B, at later times (30 min) after exposure to ligand (35). Similarly, in HEK 293 cells, it was found that the PTP-1B D181A mutant associated with IRK under both insulin-independent and -dependent conditions (36). More recently, it has been suggested that this association occurred to some degree in a perinuclear endosomal compartment (37). Therefore, it appears likely that the interaction between internalized receptor tyrosine kinases (RTKs) and PTP-1B is predominantly with the ER-associated enzyme. The participation of the ER in the formation of the phagasome indicates that there are mechanisms allowing for the association of internalized molecules with ER-associated molecules (38). It is also possible that a portion of the internalized IRK in ENs is directed to the ER. This would be consistent with the observation that RTKs-activated phosphotidylinositol 3-kinases occur on various endomembranes in the course of cell signaling (39, 40). Also, there is evidence indicating that PTP-1B dephosphorylates ligand-independent tyrosine phosphorylation of IRK precursors and -subunits and may thereby play an important role to prevent inappropriate RTK activation during biogenesis and cellular processing (41). Finally, we cannot rule out the possibility that PTP-1B may be brought to the plasma membrane along with newly synthesized IRK and play a role in rapidly dephosphorylating cell surface IRK after insulin stimulation.
Using an in-gel PTP assay, we found approximately 20 PTPs in ENs, a subfraction of which was inhibited by bpV(phen). One or more of these entities might be the relevant PTP associated with the IRK in ENs. It is also worth noting that IRK dephosphorylation may be effected by the coordinate action of several PTPs, in which case a deficiency of one might be largely compensated by the function of the others.
In summary, the present study identifies a number of PTPs in ENs, a subgroup of which is significantly inhibited by bpV(phen) treatment. Our data indicate that PTP-1B may not be the important endosomal IRK-associated PTP. However, the interaction between IRK and PTP-1B in other intracellular compartments may well be involved in aspects of IRK signaling, as might the impact of PTP-1B on other signaling components (viz. leptin) and on downstream targets of the activated IRK (42).
Acknowledgments
We thank Drs. Simon S. Wing and Rose Oughtred for help with the setup and use of the Mono Q column chromatogram.
Footnotes
This work was supported by a grant from the Canadian Institutes for Health Research.
First Published Online November 3, 2005
Abbreviations: bpV(phen), Bisperoxo(1,10-phenanthroline)-oxovanadate(v) anion; DTT, 1,4-dithiothreitol; EN, endosome; ER, endoplasmic reticulum; IR, insulin receptor; IRK, insulin receptor kinase; LAR, leukocyte common antigen-related; pGT, polyglutamic acid-tyrosine (4:1); PMSF, phenylmethylsulfonylfluoride; PTP, phosphotyrosine phosphatase; pV, peroxovanadium; PY, phosphotyrosine; RTK, receptor tyrosine kinase; WGA, wheat-germ agglutinin.
Accepted for publication October 21, 2005.
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