From the Laboratory of Cell Signaling, NHLBI, National Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
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Stimulation of various cells with growth factors results in a transient increase in the intracellular concentration of H2O2 that is required for growth factor-induced protein tyrosine phosphorylation. The effect of H2O2 produced in response to epidermal growth factor (EGF) on the activity of protein-tyrosine phosphatase 1B (PTP1B) was investigated in A431 human epidermoid carcinoma cells. H2O2 inactivated recombinant PTP1B in vitro by oxidizing its catalytic site cysteine, most likely to sulfenic acid. The oxidized enzyme was reactivated more effectively by thioredoxin than by glutaredoxin or glutathione at their physiological concentrations. Oxidation by H2O2 prevented modification of the catalytic cysteine of PTP1B by iodoacetic acid, suggesting that it should be possible to monitor the oxidation state of PTP1B in cells by measuring the incorporation of radioactivity into the enzyme after lysis of the cells in the presence of radiolabeled iodoacetic acid. The amount of such radioactivity associated with PTP1B immunoprecipitated from A431 cells that had been stimulated with EGF for 10 min was 27% less than that associated with PTP1B from unstimulated cells. The amount of iodoacetic acid-derived radioactivity associated with PTP1B reached a minimum 10 min after stimulation of cells with EGF and returned to base line values by 40 min, suggesting that the oxidation of PTP1B is reversible in cells. These results indicate that the activation of a receptor tyrosine kinase by binding of the corresponding growth factor may not be sufficient to increase the steady state level of protein tyrosine phosphorylation in cells and that concurrent inhibition of protein-tyrosine phosphatases by H2O2 might also be required.
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INTRODUCTION |
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Ligation of a variety of cell surface receptors, including those for growth factors and cytokines, induces a transient increase in the intracellular concentration of H2O2 in mammalian cells (1-3). Inhibition of this effect blocks receptor-mediated signal transduction. For example, inhibition of the accumulation of H2O2 by introducing catalase into NIH 3T3 or A431 cells prevented the induction of tyrosine phosphorylation by platelet-derived growth factor or epidermal growth factor (EGF)1 (2, 3). Direct exposure of cells to H2O2 also increases protein tyrosine phosphorylation and activates signal transduction pathways (2-5). Because the extent of protein tyrosine phosphorylation in a cell reflects an equilibrium between the actions of protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs), either stimulation of PTKs or inhibition of PTPs would be expected to shift the equilibrium toward phosphorylation. PTP activity in crude cell extracts can be inactivated by various oxidants, including H2O2, and this inactivation can be reversed by incubation with thiol compounds such as dithiothreitol (DTT) and GSH (6, 7). These observations suggest that PTPs might undergo H2O2-dependent inactivation in cells, resulting in a shift in the equilibrium with PTKs toward phosphorylation.
PTPs constitute a diverse family of enzymes that can be divided into several subgroups, including receptor PTPs and nonreceptor PTPs (8-11). All PTPs contain an essential cysteine residue in the signature active site motif, HCXXGXXR(S/T). The PTP active site cysteines exhibit low pKa values (5.4 for mammalian PTP1 (12), 5.6 for human dual specific PTP (13), and 4.7 for Yersinia PTP (14)) and are readily ionized at neutral pH, whereas the pKa of a typical cysteine residue is 8.5. The ionized essential sulfhydryl group (thiolate anion) contributes to the formation of a thiol-phosphate intermediate in the catalytic mechanism of PTPs (15). In addition, the essential cysteine is the target of specific modification by various sulfhydryl-alkylating reagents (12-14, 16, 17).
We now demonstrate that H2O2, either added extracellularly or generated intracellularly in response to EGF, can cause reversible inactivation of PTPs in cells, and we identify the most plausible electron donor responsible for the reactivation of such inactivated PTPs. PTP1B, the widely expressed cytosolic enzyme originally purified from human placenta (18), was chosen as the target enzyme and was studied in A431 human epidermoid carcinoma cells.
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EXPERIMENTAL PROCEDURES |
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Materials--
Dulbecco's modified Eagle's medium, fetal
bovine serum, penicillin, and streptomycin were from Life Technologies,
Inc. Rabbit polyclonal antibodies to PTP1B were kindly provided by
B. G. Neel (Harvard University). A monoclonal antibody to PTP1B
was obtained from Oncogene Science. Horseradish peroxidase-conjugated
antibodies to mouse or to rabbit immunoglobulin G were from Amersham
Pharmacia Biotech. Yeast glutathione reductase (GR) and bovine catalase were from Boehringer Mannheim. The synthetic peptide Raytide and the
kinase p43v-abl were from Oncogene Science.
[-33P]ATP was from Amersham Pharmacia Biotech, and
14C- or 3H-labeled iodoacetic acid was from NEN
Life Science Products.
Recombinant PTP1B-- Complementary DNA corresponding to the 37-kDa form (NH2-terminal 321 residues) of PTP1B was obtained by the polymerase chain reaction, placed downstream of the phage T7 RNA polymerase promoter at the NdeI site of pET-14b (Novagen) (thus providing a histidine tag attached to the NH2 terminus of PTP1B by a thrombin-sensitive sequence), and expressed in Escherichia coli strain BL21 (DE3) by standard procedures. The histidine-tagged PTP1B fusion protein was purified from Escherichia coli extract with the use of an immobilized nickel resin (Novagen). The purity of the PTP1B preparation as assessed by SDS-polyacrylamide gel electrophoresis (PAGE) was >95%. A portion of the purified PTP1B was treated with thrombin to cleave the histidine tag and was repurified on a Mono S column. Both the purified histidine-tagged and thrombin-treated enzymes were incubated in the presence of 10 mM DTT for 1 h and then dialyzed in an anaerobic chamber at 4 °C against a deoxygenated solution containing 20 mM Mes-NaOH (pH 6.5) and 0.1 mM EDTA. Portions of the dialyzed enzyme were stored in an anaerobic chamber at 4 °C. Unless otherwise specified, histidine-tagged PTP1B was used throughout this study.
Preparation of Thioredoxin (Trx), Glutaredoxin (Grx), and Trx Reductase (TR)-- Rat Trx cDNA (19) was obtained by the polymerase chain reaction, cloned into the pET-17b expression vector, and expressed in E. coli by standard procedures. Recombinant Trx was purified to homogeneity from the cytosolic fraction of E. coli by heat treatment at 65 °C followed by sequential chromatography on Sephacryl S-100 HR gel filtration (Amersham Pharmacia Biotech) and high performance liquid chromatography (HPLC) DEAE-5PW ion exchange columns. TR and Grx were purified from rat liver as described (20-22).
Determination of Protein Concentration-- The concentrations of recombinant PTP1B and Trx were determined spectrophotometrically, and the A280 values of 0.1% solutions were 1.231 and 0.738, respectively. The concentrations of other proteins were determined with the BCA protein assay reagent (Pierce), with bovine serum albumin as a standard.
Assay of PTP1B Activity-- Two different methods were used to assay PTP1B activity. The activity of recombinant PTP1B was measured spectrophotometrically with p-nitrophenyl phosphate (pNPP) as a substrate in a reaction mixture containing 40 mM Bis-Tris-HCl (pH 7.0), 2 mM EDTA, 50 mM NaCl, and 10 mM pNPP (13). The initial velocity of p-nitrophenol formation was measured by monitoring the change in A405. An assay of PTP1B activity in immune complexes was performed with 33P-phosphorylated Raytide as a substrate (23). Assay mixtures (50 µl) containing the immune complex, 20 mM Tris-HCl (pH 7.5), bovine serum albumin (0.1 mg/ml), 1 mM EDTA, 10 mM DTT, and 90 nM 33P-phosphorylated Raytide were incubated at 30 °C for 30 min, after which the reaction was terminated by the addition of 10 µl of glacial acetic acid, and the radioactivity associated with the peptide was measured as described (24).
Determination of Free SH Groups-- PTP1B (100 µg) that had been treated with DTT and then dialyzed against a DTT-free buffer under anaerobic conditions was incubated at 25 °C with 100 µM H2O2 in a total volume of 100 µl containing 40 mM Bis-Tris-HCl (pH 7.0), 150 mM NaCl, and 1 mM EDTA. After 20 min, the oxidation reaction was stopped by adding 1 µg of catalase. The resulting oxidized and control unoxidized enzymes (100 µg in 100 µl) were separately mixed with 300 µl of a solution containing 266 µM 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), 6 M guanidine hydrochloride, and 50 mM Tris-HCl (pH 8.0). The concentration of thionitrobenzoic acid released was determined spectrophotometrically with a molar extinction coefficient of 13,700 at 412 nm (25).
Identification of the H2O2-sensitive Residue in PTP1B-- PTP1B (64 µg) that had been incubated with 100 µM H2O2 for 10 min and then treated with catalase as described above was incubated with 2 mM [3H]iodoacetic acid in a total volume of 100 µl of 40 mM Bis-Tris-HCl (pH 6.5) containing 0.1 mM EDTA. As a control, unoxidized PTP1B (64 µg) was likewise treated with [3H]iodoacetic acid. After 10 min at room temperature, the reaction was stopped by adding 100 mM 2-mercaptoethanol and the reaction mixtures were subjected to gel filtration chromatography on a Sephadex G-25 column to remove unreacted iodoacetic acid. The PTP1B-containing fractions were pooled and incubated overnight at room temperature with 2.5 µg of endoproteinase Lys-C in a total volume of 400 µl. The resulting digestion products were analyzed by HPLC on a C18 column with a linear gradient (0-60%, v/v) of acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 1 ml/min over 60 min. Fractions corresponding to each peptide peak were collected manually, and a portion (10%) of each fraction was analyzed for 3H radioactivity.
Iodoacetic Acid Labeling and Immunoprecipitation of PTP1B-- A431 cells, maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 units/ml), were allowed to reach 80-90% confluence in 150-mm dishes. The cells were then deprived of serum for 16 h and subsequently stimulated with EGF (200 ng/ml) or H2O2 (1 or 3 mM). The cells were rinsed and then exposed in an anaerobic chamber to 1 ml of O2-free lysis buffer (50 mM Bis-Tris-HCl (pH 6.5), 0.5% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, leupeptin (0.5 µg/ml), aprotinin (0.5 µg/ml), and 0.1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride) containing 2 mM [14C]iodoacetic acid and briefly sonicated. As a control, serum-deprived cells that were not stimulated with EGF or H2O2 were likewise lysed and labeled. After 30 min at room temperature in the dark, the labeling reaction was stopped by adding 0.2 ml of 200 mM cold iodoacetic acid in 0.8 M Tris-HCl (pH 7.5) and followed by the addition of 0.1 ml of 1 M DTT. The reaction mixtures were then centrifuged at 10,000 × g for 20 min. The resulting supernatants were subjected to a G-25 gel filtration chromatography to remove excess radioactivity. Protein-containing fractions were pooled, and protein concentrations were measured. The pooled samples were precleared by incubating for 4 h at 4 °C with 40 µl of goat anti-mouse immunoglobulin-coated immunobeads (25% slurry) (Sigma) and centrifuging at 3000 × g for 30 s. Afterward, 40 µl of goat anti-mouse immunoglobulin immunobeads (25% slurry) that had been absorbed with an excess of monoclonal antibody to PTP1B were added to the resulting supernatants, and incubation was continued for an additional 4 h. The beads were separated; extensively washed once in ice-cold O2-free lysis buffer, twice in O2-free 50 mM Tris-HCl buffer(pH 7.5), and twice in O2-free phosphate-buffered saline (pH 7.4); and subjected either to SDS-PAGE in order to measure the amount of radioactivity incorporated into PTP1B or to assay the amount of PTP activity regenerated by treatment with DTT.
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RESULTS |
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PTP1B was originally isolated from human placenta as a soluble 37-kDa protein (18). It was subsequently shown that the full-length (50-kDa) PTP1B protein contains 435 amino acids and that the 37-kDa protein corresponds to the NH2-terminal 321 residues (26). The COOH-terminal 114 residues of the full-length protein contain a sequence responsible for localization of PTP1B in the endoplasmic reticulum (23). The 37-kDa PTP1B contains six cysteine residues, which do not appear to form disulfide bonds on the basis of the crystal structure of the protein (27). Both Cys121 and Cys215 of PTP1B are conserved among all members of the PTP family (28). Cys215, which has a pKa value of 5.4, is the essential cysteine residue located at the active site. However, mutation of Cys121 in PTP1, the rat homolog of PTP1B, markedly reduces PTP activity (15). The active site cysteines of various PTPs, including Cys215 of PTP1B, are specifically targeted by the sulfhydryl-modifying reagent iodoacetic acid (12-14, 16, 17). The same active site cysteine residues have also been implicated as the site of oxidation by various oxidants (2, 3, 6, 7, 29), in which case one should be able to monitor the extent of H2O2-induced inactivation of PTPs in cells by measuring the amount of radioactivity incorporated into the enzyme after cell lysis in the presence of radiolabeled iodoacetic acid. With this aim, we expressed the 37-kDa PTP1B in E. coli, purified the recombinant protein, and subjected it to modification by H2O2 and iodoacetic acid.
Identification of the H2O2-sensitive Cysteine in PTP1B-- Incubation with H2O2 resulted in the inactivation of the purified recombinant 37-kDa PTP1B in a manner dependent on time and H2O2 concentration (Fig. 1, A and B). When the enzyme samples from the experiment shown in Fig. 1B (which had been inactivated to various extents by incubation with different concentrations of H2O2) were subjected to labeling with [14C] iodoacetic acid at pH 6.5, the extent of labeling decreased in proportion to the extent of inactivation (Fig. 1C), indicating that the site of oxidation by H2O2 is the same as the site of labeling by iodoacetic acid. The data shown in Fig. 1 were obtained with histidine-tagged enzyme, but similar results were obtained with PTP1B lacking the histidine tag.
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Oxidative Inactivation of PTP1B in A431 Cells-- A431 cells were incubated for various times with EGF and then lysed in an anaerobic chamber by exposure to a pH 6.5 buffer containing Triton X-100 and [14C]iodoacetic acid. PTP1B was then immunoprecipitated from the cell lysate with a highly specific monoclonal antibody, and the radioactivity associated with the precipitated 50-kDa PTP1B protein was measured after SDS-PAGE. The extent of incorporation of [14C]iodoacetic acid into PTP1B decreased with time of incubation of the cells with EGF, reaching a minimum at 10 min, and returned to the basal value by 40 min (Fig. 2).
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Reactivation of PTP1B-- Recombinant PTP1B that had been inactivated by exposure to H2O2 was incubated in the presence of various electron donors and reactivation was monitored (Fig. 4). In addition to DTT and GSH, the Trx system (consisting of Trx, TR, and NADPH) and the Grx system (consisting of Grx, GSH, GR, and NADPH) were used as electron donors. In the Trx system, oxidized Trx is reduced by TR with the use of electrons supplied by NADPH. In the Grx system, oxidized Grx is reduced by GSH, which in turn receives electrons from NADPH through the action of GR. The most rapid reactivation was achieved by DTT (4 mM), whereas GSH (4 mM) was the least efficient electron donor. The Trx system was slightly less efficient than DTT, and the Grx system was substantially less effective than the Trx system. The functional efficacy of the Grx and GR preparations was demonstrated by measuring GSH-disulfide transhydrogenase (30) and glutathione peroxidase (31) activities, respectively. Each component of the Trx and Grx systems was used at a saturating concentration; i.e. increasing their concentrations did not increase the rate of reactivation (data not shown).
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DISCUSSION |
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Given the recent observation that H2O2 is required for the growth factor-induced tyrosine phosphorylation of cellular proteins, we investigated whether H2O2 produced in response to EGF is capable of inactivating PTP1B in A431 cells.
First, with the use of the recombinant 37-kDa form of PTP1B, we
demonstrated that the essential residue Cys215 is the site
of oxidation by H2O2. The oxidized products of
cysteine include sulfenic acid, disulfide, sulfinic acid, and sulfonic acid (Cys-SO3H). The disulfide intermediate can be excluded
as the H2O2-modified form of PTP1B on the basis
of our observation that only one out of six DTNB-sensitive residues was
lost after H2O2 oxidation, and the sulfinic and
sulfonic acid intermediates can be excluded on the basis of the
observation that the oxidized PTP1B can be reduced back to its original
state by DTT. Nevertheless, PTP1B was shown to form sulfinic and
sulfonic acid intermediates when oxidized in the presence of
osteoporosis drug alendronate (32) and pervanadate (33), respectively.
Cysteine sulfenic acid is highly unstable and readily undergoes
condensation with a thiol. However, the sulfenic acid intermediate of
PTP1B is probably stabilized by the fact that, according to the x-ray
structure of the 37-kDa form of PTP1B (27), no cysteine residues are
located near Cys215. Furthermore, the sulfenate anion
(Cys-SO) is also probably stabilized by a salt bridge to
Arg221, which was shown to stabilize the thiolate anion of
Cys215 and consequently to reduce its
pKa.
Second, we measured the amount of radiolabeled iodoacetic acid incorporated into PTP1B as a means of monitoring changes in the oxidation state of the protein in A431 cells stimulated with EGF. This approach was based on our observations that iodoacetic acid reacts almost exclusively with Cys215-SH of PTP1B at pH 6.5 and that the oxidation of this cysteine residue by H2O2 prevents its reaction with iodoacetic acid. Exposure of A431 cells to EGF resulted in a decrease in the extent of iodoacetic acid labeling of PTP1B, with the maximal (27%) decrease apparent 10 min after stimulation and the labeling returning to base-line values by 40 min. This reduced labeling is probably due to the oxidation of PTP1B by H2O2, given that EGF elicits H2O2 production and that inhibition of H2O2 accumulation inhibits protein tyrosine phosphorylation in A431 cells (3). Furthermore, direct exposure of recombinant PTP1B to H2O2 also resulted in a decrease in labeling by iodoacetic acid. A similar decrease in labeling of PTP1B with a fluorescein-conjugated iodoacetamide was also observed when PTP1B was immunoprecipitated from EGF- or H2O2-treated A431 cells.2 This observation is consistent with previous data showing that PTP activity measured in crude extracts of HER 14 cells was reduced by 40% as a result of treatment of cells with H2O2 (7).
Third, we compared the abilities of Trx, Grx, and GSH to reactivate oxidized recombinant PTP1B in vitro; Trx at 3.8 µM was markedly more efficient than 3.8 µM Grx or 4 mM GSH. The cellular concentrations of Trx and GSH are approximately 2-14 µM and 1-10 mM, respectively (34, 35). Thus, Trx is predicted to be a major electron donor for PTP1B reduction in cells. Trx was previously shown to be the preferred electron donor for the reduction of glyceraldehyde-3-phosphate dehydrogenase containing an active site cysteine sulfenic acid, whereas Grx was preferred for the reduction of the same enzyme containing a disulfide (36-38).
Although we have demonstrated the sensitivity of PTP1B to H2O2 formed in response to treatment of cells with EGF, other PTP enzymes are also probably susceptible to such inactivation. However, it is possible that the concentration of H2O2 is sufficiently high to inactivate PTPs only in limited regions of the cell in which the H2O2-producing components are recruited and that H2O2 molecules that diffuse away from such regions are readily eliminated by various peroxidases. Such a localized inactivation of PTP1B is one possible explanation for our observation that only 27% of this enzyme was oxidized in EGF-activated A431 cells. Neither the mechanism of H2O2 generation, the site of generation, nor the concentration of H2O2 generated in response to growth factors and cytokines is known.
On the basis of the previous observation that growth factor-induced protein tyrosine phosphorylation requires H2O2 production and our current observation that growth factor-induced generation of H2O2 is sufficient to cause inactivation of PTP1B, we propose that the activation of a receptor PTK by interaction with a growth factor may not be sufficient to increase the steady state level of protein tyrosine phosphorylation in a cell; rather, concurrent inhibition of PTPs by H2O2 may also be required for this effect. The extent of tyrosine phosphorylation of receptor PTKs and their substrates would then return to basal values after degradation of H2O2 and the subsequent reactivation of PTPs by electron donors. Our in vitro data suggest that Trx might be a physiological electron donor for PTP1B. It remains to be determined whether other PTPs also form a sulfenic acid intermediate on oxidation with H2O2 and whether Trx reduces such oxidized intermediates. The low molecular weight PTP, which shows no apparent sequence similarity to other PTPs but which shares several common features in active site architecture (9, 39), forms a disulfide on oxidation with nitric oxide (40).
A scheme depicting the proposed roles of H2O2 and Trx in growth factor-induced protein tyrosine phosphorylation is shown in Fig. 6. This scheme is consistent with the following observations: 1) production of H2O2 via NADPH oxidase results in inhibition of the PTP activity of CD45 in neutrophils, 2) blocking the H2O2 production by treatment with N-acetylcysteine or diphenylene iodonium, an inhibitor of NADPH oxidase, restores its PTP activity (41, 42), and 3) removal of intracellular oxidants by pyrrolidine dithiocarbamate diminishes protein tyrosine phosphorylation (43). The scheme is also consistent with the suggestion that the ligand-independent basal activity of receptor PTKs might be sufficient to increase the extent of protein tyrosine phosphorylation in cells treated with thiol-alkylating agents, such as iodoacetic acid and iodoacetamide, or oxidants, such as ultraviolet light, that cause the inactivation of PTPs (29).
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Previously proposed mechanisms for the regulation of nonreceptor PTP activity include phosphorylation at serine and tyrosine residues (23, 44-48), anchoring via SRC homology 2 domains (49, 50), and proteolysis (51, 52). In contrast to these positive regulation mechanisms of PTP activity, oxidation by H2O2 provides a means for negative regulation of PTP activity. Negative modulation mediated by ligand-induced dimerization has also been proposed for the receptor PTP CD45 (53).
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ACKNOWLEDGEMENTS |
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We thank Edmond H. Fisher for encouragement, Benjamin G. Neel for antibodies to PTP1B and R. Levine and M. Nauman for peptide sequencing.
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FOOTNOTES |
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* A preliminary report of these data was presented at the Seventeenth ASBMB Annual Meeting, San Francisco, August, 1997 (54).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
These authors contributed equally to this work.
§ On leave from the Korea Research Institute of Bioscience and Biotechnology and partly supported by a grant from the Korea Ministry of Science and Technology and a grant from the Office of International Programs, NHLBI, National Institutes of Health.
¶ Present address: Dept. of Biochemistry, College of Medicine, Chungbuk National University, Chungbuk 361-763, Korea.
To whom correspondence should be addressed: Bldg. 3, Rm. 122, National Institutes of Health, Bethesda, MD 20892. Tel.: 301-496-9646; Fax: 301-480-0357; E-mail: sgrhee{at}helix.nih.gov.
1 The abbreviations used are: EGF, epidermal growth factor; PTK, protein-tyrosine kinase; PTP, protein-tyrosine phosphatase; PTP1, protein-tyrosine phosphatase 1; PTP1B, protein-tyrosine phosphatase 1B; DTT, dithiothreitol; GR, glutathione reductase; PAGE, polyacrylamide gel electrophoresis; Trx, thioredoxin; Grx, glutaredoxin; TR, Trx reductase; HPLC, high performance liquid chromatography; pNPP, p-nitrophenyl phosphate; Bis-Tris, [bis(2-hydroxyethyl)imino]-tris(hydroxymethyl) methane; DTNB, 5,5'-dithiobis-(2-nitrobenzoic acid).
2 Y. Wu and S. G. Rhee, unpublished result.
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REFERENCES |
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