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Originally published In Press as doi:10.1074/jbc.M412424200 on December 28, 2004

J. Biol. Chem., Vol. 280, Issue 11, 10298-10304, March 18, 2005
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Differential Oxidation of Protein-tyrosine Phosphatases*

Arnoud Groen{ddagger}, Simone Lemeer{ddagger}§, Thea van der Wijk{ddagger}, John Overvoorde{ddagger}, Albert J. R. Heck§, Arne Ostman¶, David Barford||, Monique Slijper§, and Jeroen den Hertog{ddagger}**

From the {ddagger}Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands, §Utrecht University, Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands, the Karolinska Institute, Cancer Center Karolinska, Dept. of Pathology-Oncology, SE-171 76 Stockholm, Sweden, and the ||Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London, SW3 6JB, United Kingdom

Received for publication, November 3, 2004 , and in revised form, December 8, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Oxidation is emerging as an important regulatory mechanism of protein-tyrosine phosphatases (PTPs). Here we report that PTPs are differentially oxidized, and we provide evidence for the underlying mechanism. The membrane-proximal RPTP{alpha}-D1 was catalytically active but not readily oxidized as assessed by immunoprobing with an antibody that recognized oxidized catalytic site cysteines in PTPs (oxPTPs). In contrast, the membrane-distal RPTP{alpha}-D2, a poor PTP, was readily oxidized. Oxidized catalytic site cysteines in PTP immunoprobing and mass spectrometry demonstrated that mutation of two residues in the Tyr(P) loop and the WPD loop that reverse catalytic activity of RPTP{alpha}-D1 and RPTP{alpha}-D2 also reversed oxidizability, suggesting that oxidizability and catalytic activity are coupled. However, catalytically active PTP1B and LAR-D1 were readily oxidized. Oxidizability was strongly dependent on pH, indicating that the microenvironment of the catalytic cysteine has an important role. Crystal structures of PTP domains demonstrated that the orientation of the absolutely conserved PTP loop arginine correlates with oxidizability of PTPs, and consistently, RPTPµ-D1, with a similar conformation as RPTP{alpha}-D1, was not readily oxidized. In conclusion, PTPs are differentially oxidized at physiological pH and H2O2 concentrations, and the PTP loop arginine is an important determinant for susceptibility to oxidation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Phosphorylation of proteins on tyrosine residues has an important role in many cellular processes like proliferation, differentiation, and migration. Tyrosine phosphorylation is mediated by the balanced action of protein-tyrosine kinases and protein-tyrosine phosphatases (PTPs)1 (1). Not only protein-tyrosine kinases but also PTPs have important roles in diseases, including cancer and diabetes (2–5). The human genome encodes 103 cysteine-based PTP family members, of which 38 are strictly phosphotyrosine-specific (4, 5). These "classical" PTPs are subdivided into transmembrane, receptor PTPs (RPTPs) (21 genes) and intracellular, nonreceptor PTPs (17 genes). The cytoplasmic PTPs encode a single PTP domain, whereas most RPTPs contain two catalytic domains. The membrane-proximal domain (D1) contains most catalytic activity (6, 7), whereas the membrane-distal domain (D2) has a regulatory function (8). The PTPs, including the RPTP-D2s, are highly conserved in sequence and three-dimensional structure (9–11). Only two amino acid residues that are conserved in all active PTPs, but not in RPTP-D2s, are responsible for the lack of catalytic activity in RPTP-D2s, because mutation of these residues renders RPTP-D2s active (10, 12, 13).

Relatively little is known about the regulation of PTPs. Reversible oxidation of the absolutely conserved catalytic site cysteine that is essential for catalysis (14) is emerging as an important regulatory mechanism (15). The catalytic cysteines are susceptible to oxidation because of their low pKa (16, 17). Oxidation of cysteine residues to sulfenic acid is reversible, whereas further oxidation to sulfinic (doubly oxidized) or sulfonic (triply oxidized) forms is irreversible (18). Oxidized PTP1B shows a newly identified bond, termed cyclic sulfenamide, in which the sulfur of the cysteine is covalently linked to the main chain nitrogen of the neighboring serine (19, 20). Cyclic sulfenamide, like sulfenic acid, can be reduced by thiols, and importantly, it suppresses oxidation to sulfinic and sulfonic acids.

There are many physiological stimuli that induce reactive oxygen species (ROS) production. For instance, ROS are produced in response to stimuli like UV light, growth factors, and insulin, leading to inactivation of PTPs (21–26). ROS-induced inhibition of PTP activity may be essential for stimulus-induced signaling. For instance, platelet-derived growth factor induces oxidation of Shp2, and N-acetyl cysteine, a ROS quencher, blocks Shp2 oxidation and reduces platelet-derived growth factor signaling (25). Interestingly, Reynolds et al. (27) demonstrated in a mathematical model that epidermal growth factor-induced epidermal growth factor receptor activation, together with ROS-mediated inactivation of PTPs is sufficient to cause lateral signal propagation.

PTPs are emerging as important redox sensors in cells. Recently, we found that RPTP{alpha} is regulated by oxidation in an unexpected way. Whereas RPTP{alpha}-D1 contains most of the catalytic activity, it appears that RPTP{alpha}-D2 is much more sensitive to oxidation than RPTP{alpha}-D1 in vitro and in cells in response to UV irradiation (28). Yet RPTP{alpha} is inactivated in response to H2O2 treatment, because RPTP{alpha}-D2 undergoes a conformational change in response to oxidation, leading to stabilization of an inactive dimeric conformation (8, 29). Mutation of the catalytic cysteine in RPTP{alpha}-D2 renders RPTP{alpha} less sensitive to oxidation, in that H2O2 and UV treatment of living cells do not lead to complete inactivation of RPTP{alpha}-C723S, whereas wild type RPTP{alpha} is inactivated completely (8, 28).

In the present study, we investigated differential oxidation of PTPs. oxPTP immunoprobing and matrix-assisted laser desorption ionization time-of-flight (MALDI-ToF) mass spectrometry indicated that the mutations in the Tyr(P) loop and the WPD loop that are responsible for the difference in catalytic activity between RPTP{alpha}-D1 and RPTP{alpha}-D2 were also involved in the difference in oxidizability. Furthermore, we demonstrate that PTP1B and LAR-D1 were highly oxidizable. The susceptibility of PTPs to oxidation was strongly dependent on pH. Comparison of the crystal structures suggested that the orientation of the PTP loop arginine is important for the oxidizability of PTPs. Taken together, the subtle differences in the microenvironment of the catalytic cysteines determine the oxidizability of PTPs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Constructs—pGEX-based bacterial expression vectors encoding GST fusion proteins of RPTP{alpha}-D1 contained the region from amino acids 167 to 503 and RPTP{alpha}-D2 from amino acids 504 to 793 (30). D2-E690D, D2-V555Y,and D2-E690D/V555Y have been described (13). RPTP{alpha}-D1-Y262V and D1-D401E were made by site-directed mutagenesis and verified by sequencing. The pGEX construct encoding zebrafish PTP1B (residues 1–282) has been described (31). pGEX-LAR-D1 and pGEX-LAR-D2 encoded human LAR residues 1275–1608 and 1609–1897, respectively. pGEX-RPTPµ-D1 encoded human RPTPµ residues 765–1165. These constructs were derived by PCR and verified by sequencing.

Analysis of Oxidized PTPs Using the oxPTP Antibody—Reversible oxidation was assayed using an antibody, oxPTP, that specifically recognizes the sulfonic acid form of catalytic site cysteines of PTPs (28, 32). As outlined in Fig. 1A, following treatment, the sample is alkylated to protect reduced cysteines. Subsequently, the sample is treated with dithiothreitol (DTT) to reduce reversibly oxidized cysteines and treated with pervanadate to convert reduced cysteines to the sulfonic acid form. Finally, the sulfonic acid form is detected using the oxPTP antibody by immunoblotting. The procedure has been described in detail (28). Briefly, GST fusion proteins, purified using standard procedures, were bound to glutathione beads. All of the incubations and washes were done with 20 mM Tris, pH 7.5, unless otherwise stated. The proteins were reduced for 30 min in 10 mM DTT. The beads were washed twice and were incubated with varying concentrations of H2O2 for 20 min. Immediately after oxidation proteins were alkylated, using 40 mM iodoacetic acid for 30 min. The proteins were washed extensively, reduced with 10 mM DTT, washed twice, and incubated for 1 h with 0.1 mM pervanadate (1 mM pervanadate, 1 mM Na3VO4 mixed with 5 mM H2O2, final concentrations, and left for at least 5 min at room temperature) to convert reduced cysteines irreversibly into the sulfonic acid form. Laemmli sample buffer was added, and the samples were boiled for 5 min and run on a 10% SDS-polyacrylamide gel (5 µg of protein/lane). The samples were transferred to polyvinylidene difluoride membrane by semi-dry blotting. For detection with oxPTP antibody, the blots were blocked in 0.1% bovine serum albumin, 0.1% Tween in 50 mM Tris, pH 7.5, 150 mM NaCl for 1 h or overnight and incubated with oxPTP antibody (1.5 h at room temperature or overnight at 4 °C). Subsequently, horseradish peroxidase-coupled goat-anti-rabbit (1:10,000; BD Pharmigen) was used, and the antibody signals were visualized by ECL. In some cases, the membranes were stripped and reprobed with anti-RPTP{alpha} antibody (30) and subsequently developed using ECL.



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  		FIG. 1.
Differential oxidation of RPTP{alpha}-D1 and RPTP{alpha}-D2. A, reduced (thiolate anion form, S–), reversibly oxidized (sulfenic acid or sulfenamide; SOH), and irreversibly oxidized (sulfinic and sulfonic acid; SO2/3H) PTP catalytic cysteines were detected as described in detail under "Materials and Methods" by subsequent alkylation with iodoacetic acid or iodoacetamide, reduction with DTT and detection by MALDI-ToF mass spectrometry (MS) or peroxidation with pervanadate (PV), and detection with oxPTP antibody. B and C, RPTP{alpha}-D1 (B) and RPTP{alpha}-D2 (C) were treated with increasing concentrations of H2O2 (31, 62, 125, 250, 500, and 1000 µM) and processed for detection of reversible oxidation, using the oxPTP antibody. The negative control (nc) was treated with DTT for 20 min. The positive control (pc) was not alkylated with iodoacetic acid, resulting in complete peroxidation in response to pervanadate. The samples were run on SDS-PAGE gels and blotted, and the immunoblots were probed with oxPTP antibody (upper panels). Equal amounts of fusion protein were loaded per lane, which was monitored by Coomassie Blue staining (data not shown) and by reprobing of the blots with anti-RPTP{alpha} antibodies (lower panels). Representative immunoblots, developed with ECL, are depicted here. D, GST fusion proteins encoding RPTP{alpha}-D1 (top panel) and RPTP{alpha}-D2 (bottom panel) were treated with different concentrations of H2O2 ranging from 31 to 250 µM. PTP activity was assayed using p-nitrophenylphosphate as a substrate and is depicted as a percentage of the maximal activity.

 
Phosphatase Assays—Fusion proteins were bound to GST beads in 20 mM Tris-HCl, pH 7.5, 10 mM DTT. The beads were incubated with varying concentrations of H2O2 for 20 min at pH 7.5 and then directly incubated in 20 mM MES buffer, pH 6.0, 1 mM EDTA, 150 mM NaCl, 10 mM p-nitrophenylphosphate for 45 min at 30 °C. The reactions were quenched with 0.4 M NaOH, and optical density was measured with a spectrophotometer at 440 nm (wavelength).

MALDI-ToF Mass Spectrometry—All of the reactions and washing steps were performed in 20 mM Tris-HCl, pH 7.5. GST fusion proteins of RPTP{alpha}-D2 and mutated RPTP{alpha}-D2 (V555Y, E690D, and V555Y/E690D) were immobilized on glutathione-Sepharose beads and incubated with 10 mM DTT. The beads were washed and incubated with H2O2 for 20 min at room temperature. Subsequently, the beads were treated with 100 mM iodoacetamide for 30 min at room temperature to derivatize the unaffected cysteines. Finally, the beads were washed and incubated with 10 mM DTT for 10 min to reduce singly oxidized cysteines. The fusion protein was cleaved off by thrombin protease (4 h at room temperature), and the protein of interest was subjected to overnight tryptic digestion. For desalting, the peptides were adsorbed on C18 ZipTips (Millipore) and washed with 0.1% trifluoroacetic acid. The peptides retained on the columns were eluted using {alpha}-cyano-4-hydroxycinnamic acid (10 mg/ml) in 10% acetonitrile, 0.1% trifluoroacetic acid. Peptide mass fingerprint spectra were recorded on a MALDI-ToF Voyager DE-STR (Applied Biosystems) mass spectrometer operated in positive ion Reflectron mode. After time-delayed extraction, the ions were accelerated to 20 kV for ToF mass spectrometric analysis. A total of 150 shots were acquired, and the signal was averaged per spectrum. MALDI-ToF spectra were analyzed using the Applied Biosystems Data Explorer software.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Differential Oxidation of RPTP{alpha}-D1 and RPTP{alpha}-D2—We investigated oxidation of PTPs using the oxPTP antibody (Fig. 1A). Consistent with a previous report (28), RPTP{alpha}-D1 was oxidized at 250 µM H2O2 but not at lower concentrations (Fig. 1B), and RPTP{alpha}-D2 was already detectably oxidized at the lowest concentration used (31 µM H2O2) (Fig. 1C). The negative control in our oxPTP experiments represents the fully reduced PTP, achieved by treatment with 10 mM DTT for 20 min. For the positive control, the alkylation step was omitted, leading to complete triple oxidation of the catalytic cysteine upon pervanadate treatment (Fig. 1).

In parallel with the oxPTP immunoprobing experiments, we performed PTP activity assays using p-nitrophenylphosphate as a substrate. RPTP{alpha}-D1 retained much of its activity at low concentrations of H2O2 (31 µM) (Fig. 1D). RPTP{alpha}-D2, although much less active than D1, is more susceptible to H2O2 than RPTP{alpha}-D1 and is already inactivated at low concentrations of H2O2 (31 µM; Fig. 1D). Together, these results show that RPTP{alpha}-D2 is more susceptible to oxidation than RPTP{alpha}-D1 as detected by the oxPTP antibody and by PTP activity assays.

Two Residues Determine the Difference between RPTP{alpha}-D1 and RPTP{alpha}-D2—Previously, we have shown that the difference in catalytic activity between RPTP{alpha}-D1 and RPTP{alpha}-D2 is caused by two residues in the Tyr(P) loop (Tyr262 in D1 and Val555 in D2) and WPD loop (Asp401 in D1 and Glu690 in D2) that are always conserved in catalytically active PTPs and never in inactive RPTP-D2s. The single mutants in RPTP{alpha}-D2, E690D and V555Y, regain some of their activity, and the double mutant V555Y/E690D is as active as RPTP{alpha}-D1 (12, 13, 33). These mutants were tested for their susceptibility to H2O2 using the oxPTP antibody. Whereas wild type RPTP{alpha}-D2 was oxidized at 31 µM H2O2, RPTP{alpha}-D2-V555Y was only oxidized at 125 µM H2O2 and above (Fig. 2A). Even higher levels of H2O2 were required for RPTP{alpha}-D2-E690D oxidation (Fig. 2A). Moreover, RPTP{alpha}-D2-V555Y/E690D oxidation was only detected at 1 mM H2O2 (Fig. 2A).



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  		FIG. 2.
The Tyr(P) loop and the WPD loop are important for oxidizability. A and B, RPTP{alpha}-D2 and mutants, V555Y, E690D, and V555Y/E690D (VY/ED) (A) and RPTP{alpha}-D1 and mutants, Y262V and D401E (B), were treated with increasing concentrations of H2O2 (31–1000 µM). Negative and positive controls were included for all mutants, and oxPTP immunoprobing was performed as described in the legend to Fig. 1. C, PTP activity of RPTP{alpha}-D1, RPTP{alpha}-D2, and their mutants was assayed using p-nitrophenylphosphate and quantified spectrophotometrically. The experiments were done in triplicate, and the standard deviations are indicated.

 
To test whether the Tyr(P) loop and the WPD motif were responsible for low oxidizability of RPTP{alpha}-D1, Tyr262 was mutated to valine, and Asp401 was mutated to glutamate. RPTP{alpha}-D1-Y262V and RPTP{alpha}-D1-D401E were much more sensitive to oxidation than wild type RPTP{alpha}-D1, in that appreciable oxidation was already detected at 31–62 µM H2O2 (Fig. 2B), similar to wild type RPTP{alpha}-D2. These results support the hypothesis that the Tyr(P) loop and WPD loop are important for susceptibility to oxidation of RPTP{alpha}-D1 and RPTP{alpha}-D2.

We determined the catalytic activity of RPTP{alpha}-D1, RPTP{alpha}-D2, and their mutants, allowing direct comparison of PTP activity and oxidizability. Consistent with previous results (12, 13, 33), the single point mutations in RPTP{alpha}-D2 enhanced its activity to some extent, whereas RPTP{alpha}-D2-V555Y/E690D exhibited catalytic activity similar to RPTP{alpha}-D1 (Fig. 2C). Mutation of Asp401 and Tyr262 completely abolished RPTP{alpha}-D1 activity (Fig. 2C), demonstrating that the Tyr(P) loop and WPD loop are essential for catalytic activity.

Mass spectrometry experiments were performed to further investigate oxidation of RPTP{alpha}-D1 and RPTP{alpha}-D2. Unfortunately, the Cys433 containing peptide of RPTP{alpha}-D1 did not resolve well in the MALDI-ToF spectra, and detection of Cys433 oxidation failed. The peptide containing Cys723 of RPTP{alpha}-D2 and its oxidized derivatives were readily detectable by MALDI-ToF (Fig. 3). At 1 mM H2O2, four peaks were evident in the spectra. As a result of the alkylation/reduction procedure, the reversibly oxidized Cys723 peptide was detected in its reduced form (m/z 2075.98), whereas alkylated Cys723 peptide corresponds to reduced Cys723 peptide (m/z 2133.00). The other two peaks, m/z 2107.98 and 2123.98, represent irreversibly oxidized RPTP{alpha}-D2, with relative mass shifts of +32 and +48 corresponding to the formation of the sulfinic and sulfonic acid species, respectively. Reversibly oxidized Cys723 peptide was already detected at the lowest concentrations of H2O2 (31 µM), and the fraction of reversibly oxidized Cys723 peptide increased gradually with increasing H2O2 concentrations (Fig. 3). The sulfinic acid species were only detected at high H2O2 concentrations (250 µM H2O2 and higher) and sulfonic acid only at 1 mM H2O2.



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  		FIG. 3.
MALDI-ToF analysis of oxidation of RPTP{alpha}-D2 and mutants. Purified RPTP{alpha}-D2 was treated with H2O2, and MALDI-ToF spectra were acquired as described under "Materials and Methods." The m/z range that contains the active site Cys723 peptide (residues 710–729) is depicted. Reversibly oxidized Cys723 peptide was detected in reduced form (m/z 2075.98, *), the sulfinic acid containing peptide shifted +32.00 to m/z 2107.98 (**), and the sulfonic acid containing peptide +48.00 to m/z 2123.98 (***). The reduced Cys723 containing peptide was detected in alkylated form with a shift in relative molecular mass of + 57.02 (m/z 2133.00, ****). Peak intensities of reduced and reversibly oxidized Cys723 peptides were determined from the MALDI-ToF spectra (panels on the right). Relative oxidation (y axes run from 0 to 100%) is plotted against increasing H2O2 concentrations (0, 31, 62, 125, 250, 500, and 1000 µM, left to right) for wild type (WT) and mutant RPTP{alpha}-D2 (as indicated).

 
Based on the MALDI-ToF spectra, we calculated the fraction of reversibly oxidized Cys723 peptide, relative to the total amount of Cys723 peptide. Wild type RPTP{alpha}-D2 was highly sensitive to oxidation, whereas RPTP{alpha}-D2-E690D was moderately sensitive, showing low levels of reversible oxidation at low H2O2 concentrations (Fig. 3). RPTP{alpha}-D2-V555Y and RPTP{alpha}-E690D/V555Y were the least sensitive to oxidation, because for both mutants significant oxidation was detected only at high concentrations of H2O2 (250 µM and higher; Fig. 3). The sulfinic and sulfonic acid form of Cys723 peptides in the MALDI-ToF spectra of RPTP{alpha}-D2 were not detected in any of the mutants (data not shown), again pointing toward a decreased sensitivity to oxidation for all mutants. The MALDI-ToF results are consistent with the oxPTP antibody data.

The oxidation and PTP activity results (Figs. 2 and 3) demonstrate that the two residues that determine the difference in catalytic activity between RPTP{alpha}-D1 and RPTP{alpha}-D2 are also responsible for the difference in oxidizability, which may suggest that PTP catalytic activity and oxidizability are linked.

Oxidizability of PTP1B and LAR—Our results suggest an inverse relation between catalytic activity and oxidizability. However, other PTPs that are catalytically active have been reported to be oxidized. Therefore, we investigated oxidation of PTP1B in parallel to RPTP{alpha}-D1 and RPTP{alpha}-D2. Oxidation of zebrafish PTP1B in which the oxPTP epitope (VHCSAG) is fully conserved (31) was readily detected at very low levels of H2O2 (31 µM), and PTP1B oxidation was maximal at 125–250 µM H2O2 (Fig. 4), similar to RPTP{alpha}-D2 (Fig. 1C). PTP1B is catalytically active (data not shown), arguing against an inverse correlation between catalytic activity and oxidation.



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  		FIG. 4.
Differential oxidation of other PTPs. PTP1B, LAR-D1, LAR-D2, RPTP{alpha}-D1-Q474G, RPTP{alpha}-D2-H764G, and PTPµ-D1 were treated with increasing concentrations of H2O2 (31–1000 µM), and reversible oxidation was detected as in Fig. 1. nc, negative control; pc, positive control.

 
Next, we investigated oxidizability of the two catalytic domains of the RPTP, LAR. Both LAR-D1 and LAR-D2 were readily oxidized at low H2O2 concentrations (31 µM) with maximal oxidation levels at 125–250 µM, like RPTP{alpha}-D2 and PTP1B (Fig. 4). PTP activity assays that were done in parallel demonstrate that LAR-D1 is catalytically active and LAR-D2 is not (data not shown), consistent with previous reports (6). Taken together, the finding that the catalytically active PTP1B and LAR-D1 are readily oxidized indicates that there is not a strict inverse correlation between catalytic activity and oxidizability.

Because the Tyr(P) loop and WPD loop are conserved in PTP1B and LAR-D1, other features of the PTPs must play a role in determining the sensitivity to oxidation. Gly259 in PTP1B (Gln474 in RPTP{alpha}-D1 and His764 in RPTP{alpha}-D2) is an important determinant of substrate specificity (34) and determines the flexibility of Gln262 in the Q loop of the catalytic pocket (35). Gly259 might therefore be involved in the difference in oxidizability between PTP1B and RPTP{alpha}-D1. We mutated Gln474 in RPTP{alpha}-D1 and His764 in RPTP{alpha}-D2 to glycine, the residue at the corresponding position in PTP1B. RPTP{alpha}-D1-Q474G was poorly oxidized (Fig. 4), like wild type RPTP{alpha}-D1 (Fig. 1B). RPTP{alpha}-D2-H764G was readily oxidized (Fig. 4), like wild type RPTP{alpha}-D2 (Fig. 1C). Taken together, these results indicate that Gly259, a determinant of substrate specificity in PTP1B and RPTP{alpha}, is not involved in differential oxidation of PTPs.

Extensive comparison of the primary sequences of the PTPs involved did not lead to the identification of residues that might be responsible for the difference in oxidizability, except for three residues that are conserved in RPTP{alpha}-D1 (331TNL333) and RPTP{alpha}-D2 (624TEL626) but not in PTP1B (109NRV111). Thr331 is buried in the catalytic pocket of RPTP{alpha}-D1 close to the catalytic Cys433 and therefore might have a role in oxidizability of Cys433. We mutated Thr331 and the two following residues to the corresponding residues in PTP1B (T -> N, TN -> NR, and TNL -> NRV) and investigated oxidizability and catalytic activity of the mutants. However, these mutants were not catalytically active, and oxidation was not detectable using the oxPTP antibody (data not shown), suggesting that these mutations severely disrupted the conformation of the catalytic pocket.

pH-dependent PTP Oxidation—Cysteine residues are most vulnerable to oxidation in the thiolate anion form. Therefore, the pH may be an important determinant for oxidation of PTPs.

It is well known that the catalytic activity of PTPs is strongly dependent on pH (36, 37). We assayed the PTP activity of RPTP{alpha}-D1, RPTP{alpha}-D2, RPTP{alpha}-D2-V555Y/E690D, and PTP1B at different pH. PTPs are highly active at low pH (optimum activity at pH 5.5–6.0 for all PTPs tested). PTP activity decreased rapidly with increasing pH to 10% of the maximum activity at pH 8.0. It is noteworthy that although the absolute activity of the four PTPs tested is different (PTP1B > RPTP{alpha}-D1 > RPTP{alpha}-D2-V555Y/E690D >> RPTP{alpha}-D2), the pH-dependent decrease in activity is very similar (Fig. 5A).



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  		FIG. 5.
PTP oxidizability is pH-dependent. A, PTP activity was assayed at pH 5.5–8.0 and is depicted relative to the maximal activity. x, RPTP{alpha}-D1; {blacktriangleup}, RPTP{alpha}-D2; {diamondsuit}, RPTP{alpha}-D2-V555Y/E690D; •, PTP1B. B, PTPs were treated with increasing concentrations of H2O2 (31, 62, or 125 µM) at pH 6.5–9.0, and reversible oxidation was assessed as in Fig. 1. nc, negative control; pc, positive control.

 
To investigate whether oxidation is a pH-dependent process, H2O2 treatment was done at a range of different pHs. Three H2O2 concentrations were used that are indicative of oxidizability, 31, 62, and 125 µM. Strikingly, the oxidizability of RPTP{alpha}-D1 was clearly dependent on pH in that RPTP{alpha}-D1 was oxidized in response to low levels of H2O2 (31 µM) at pH 8.5 and 9.0 (Fig. 5B). RPTP{alpha}-D2 oxidation was not affected by elevating the pH, but lowering the pH to 7.0 and 6.5 gradually reduced oxidation of RPTP{alpha}-D2 (Fig. 5B). PTP1B was sensitive to oxidation at pH 7.5 and, like RPTP{alpha}-D2, lost its sensitivity when the pH was lowered to 6.5. RPTP{alpha}-D2-V555Y/E690D responded to different pHs much like RPTP{alpha}-D1, in that RPTP{alpha}-D2-V555Y/E690D was oxidized at high pH (pH 8.5 and 9.0). Taken together, these results demonstrate that oxidation of PTPs is strongly dependent on the pH, suggesting that the microenvironment of the catalytic cysteines has an important role.

PTP loop Arginine Is Important for Oxidizability—The crystal structures of the PTPs involved have all been solved: RPTP{alpha}-D1 (38), RPTP{alpha}-D2 (11), PTP1B (9), and LAR-D1 and LAR-D2 (10). Comparison of the microenvironment of the catalytic cysteine of these five PTPs indicated that the absolutely conserved PTP loop arginine is oriented differently in RPTP{alpha}-D1 than in RPTP{alpha}-D2, PTP1B, or LAR (Fig. 6). We determined the distance between the S{gamma} atom of the catalytic cysteine and the three N atoms of the guanidinium group of the arginine (Table I). Strikingly, the N{eta}1 atom was much closer to the S{gamma} atom of the catalytic cysteine in RPTP{alpha}-D1 than in the other PTPs (4.9 Å versus 7.1–8.0 Å; Table I). The N{epsilon} atoms were located at similar distances in all five PTPs (5.2–6.1 Å), whereas the N{eta}2 atoms were partially buried in most structures at similar distances to the S{gamma} atom (6.1–7.3 Å). The proximity of the N{eta}1 atom to the S{gamma} atom may account for the difference in oxidizability between RPTP{alpha}-D1 and other PTPs, because the guanidinium group of Arg439 may sterically hinder the reaction of H2O2 with the thiolate group of Cys433.



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  		FIG. 6.
Structural basis for differential oxidation of PTPs. Shown is a comparison of the microenvironment of the catalytic cysteine in RPTP{alpha}-D1, RPTP{alpha}-D2, LAR-D1, LAR-D2, PTP1B, and RPTPµ-D1. The PTP loop is in red, the Tyr(P) loop is in green, the Q loop is in blue, and the WPD loop is in olive green. Note the difference in orientation of the guanidinium group of Arg439 in RPTP{alpha}-D1 and Arg1101 in RPTPµ-D1, compared with the PTP loop arginine in the other PTPs.

 


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  		TABLE I
Difference in microenvironment of the catalytic cysteine in RPTP{alpha} -D1 and RPTPµ -D1 compared to other PTPs

The distances between the catalytic cysteine S{gamma} atom and the PTP loop arginine guanidinium group N atoms (Å) were determined.

 
We analyzed the crystal structures of other classical PTPs, RPTPµ-D1, TC-PTP, Shp-1, Shp-2, PTP-SL, and YOP51. We found that only in RPTPµ-D1, was the guanidinium group of the PTP loop arginine oriented similarly as in RPTP{alpha}-D1 (Fig. 6), and the N{eta}1 atom was relatively close to the S{gamma} atom (5.5 Å; Table I). Indeed, RPTPµ-D1 was only oxidized at high H2O2 concentrations (Fig. 4), similar to RPTP{alpha}-D1. Our results are consistent with a crucial role for the PTP loop arginine in the oxidizability of PTPs.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Oxidation is an attractive regulatory mechanism for PTPs, because ROS are readily formed in response to various stimuli, and oxidation is reversible. Here, we report that PTPs were oxidized differentially, which may be important for fine-tuning of the cellular responses to stimuli. We demonstrate that oxidizability and catalytic activity of RPTP{alpha}-D1 and RPTP{alpha}-D2 were reversed upon mutation of two residues in the Tyr(P) loop and WPD loop that are absolutely conserved in catalytically active PTPs but not in RPTP-D2s. However, these two residues cannot explain the difference in oxidizability between RPTP{alpha}-D1 on the one hand and the catalytically active PTPs, PTP1B, and LAR-D1 on the other, because these residues are conserved in all active PTPs. We found that oxidation of PTPs was highly dependent on pH, and we identified the PTP loop arginine as an important determinant in oxidizability.

oxPTP immunoprobing, PTP activity assays, and MALDI-ToF mass spectrometry consistently showed differential oxidation of purified RPTP{alpha}-D1 and RPTP{alpha}-D2 in vitro. We have previously shown that Cys723, the catalytic cysteine of RPTP{alpha}-D2, is preferentially oxidized in living cells in response to UV treatment (28). Mutation of Cys723, but not Cys433, abolished oxidation of full-length RPTP{alpha} in living cells. Moreover, differential oxidizability of RPTP{alpha}-D1 and RPTP{alpha}-D2 is consistent with previous data that RPTP{alpha}-D2 rather than RPTP{alpha}-D1 is the redox sensor. Mutation of Cys723 renders full-length RPTP{alpha} at least in part insensitive to oxidation, in that H2O2 treatment or UV treatment of living cells completely inactivates wild type RPTP{alpha}, but not RPTP{alpha}-C723S (8, 28). H2O2-induced inactivation of RPTP{alpha} is due to a conformational change in RPTP{alpha}-D2, which leads to stabilization of an inactive RPTP{alpha} dimer conformation (8, 29). How RPTP{alpha}-D2 changes conformation in response to H2O2 remains elusive and will require elucidation of the crystal structure of oxidized RPTP{alpha}-D2. The catalytic Cys723 is required for the conformational change (8), and recently we found evidence that none of the other cysteines in RPTP{alpha} is involved in H2O2-induced stable dimer formation (39), ruling out the possibility of intramolecular disulfide bond formation, which has been observed in nonclassical Cys-based PTPs, including LMW-PTP (40), PTEN (41), and CDC25 (42). It is likely that the conformational change in RPTP{alpha}-D2 is initiated by cyclic sulfenamide formation, which leads to extensive reorganization of the catalytic site of PTP1B (19, 20).

Although oxidizability of RPTP{alpha}-D1 and RPTP{alpha}-D2 was reversed upon mutation of only two residues in the Tyr(P) loop and WPD loop (Figs. 2 and 3), this cannot explain the difference in oxidizability between PTP1B, LAR-D1, and RPTP{alpha}-D1 (Fig. 4), because the Tyr(P) loop and WPD loop are conserved in all three active PTPs. Oxidation of PTPs is strongly dependent on pH (Fig. 5), suggesting that the microenvironment of the catalytic cysteine is an important determinant for oxidizability. Comparison of the crystal structures of the classical PTPs suggested that the orientation of the PTP loop arginine was important. The orientation of the PTP loop arginine in RPTPµ-D1 suggested that RPTPµ-D1 would not be highly susceptible to oxidation. Indeed, like RPTP{alpha}-D1, RPTPµ-D1 was relatively insensitive to oxidation (Fig. 4). The proximity of the PTP loop arginine to the catalytic cysteine may sterically hinder the reaction of H2O2 with the thiolate anion group. To directly test the role of the PTP loop arginine in oxidizability, we mutated Arg439 to lysine in RPTP{alpha}-D1. Unfortunately, GST-RPTP{alpha}-D1-R439K was not stable, and we did not obtain sufficient material to determine the oxidation of this fusion protein.

It is not clear why the guanidinium group of Arg439 is oriented differently in RPTP{alpha}-D1 than in most other PTPs. Unlike the other crystal structures, RPTP{alpha}-D1 formed dimers in the crystals in which a helix loop-helix wedge-like structure inserts into the catalytic site of the dyad-related monomer (38), which may affect the orientation of the guanidinium group. However, the position of the PTP loop arginine and particularly its guanidinium group in RPTPµ-D1, which did not form dimers like RPTP{alpha}-D1 in crystals (43), is similar to that in RPTP{alpha}-D1, indicating that this conformation is not driven by dimerization.

It is noteworthy that differential oxidation of PTPs is especially obvious at relatively low H2O2 concentrations. Cellular stimuli induce H2O2 concentrations in a range, similar to the H2O2 concentrations we have used for our experiments. For instance, platelet-derived growth factor induces ROS levels in cells that are comparable with the levels in cells resulting from exogenously added H2O2 concentrations of 0.1–1.0 mM (21). Differential oxidation of PTPs is important for fine-tuning of the cellular response to ROS production. Apparently, different inactivating mechanisms have evolved in the RPTPs: direct oxidation of the catalytic cysteine in LAR and indirect inactivation through oxidation of the catalytic cysteine in RPTP{alpha}-D2, resulting in stabilization of an inactive dimer conformation (8, 29). LAR-D2 does undergo a conformational change in response to H2O2, like RPTP{alpha}-D2, and H2O2 induces heterodimer formation between full-length RPTP{alpha} and LAR (44). Whether oxidation of LAR-D2 is required for full inactivation of LAR remains to be determined.

In conclusion, we demonstrate that PTPs are differentially oxidized, and we provide evidence for the underlying mechanism. It will be interesting to see whether other PTP family members, including the nonclassical Cys-based PTPs, are differentially oxidized as well.


   FOOTNOTES
 
* This work was supported in part by the Netherlands Proteomics Centre and grants from the Research Council for Earth and Life Sciences (Aarden Levenswetenschappen) and Aspasia with financial aid from the Netherlands Organisation for Scientific Research (Nederlandse Organisatie voor Wetenschappelijk Onderzoek) and the Dutch Cancer Society/Koningin Wilhelmina Fonds. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

** To whom correspondence should be addressed. Tel.: 31-30-2121800; Fax: 31-30-2516464; E-mail: hertog{at}niob.knaw.nl.

1 The abbreviations used are: PTP, protein-tyrosine phosphatase; RPTP, receptor PTP; D1, membrane-proximal PTP domain; D2, membrane-distal PTP domain; LAR, leukocyte common antigen related; ROS, reactive oxygen species; GST, glutathione S-transferase; DTT, dithiothreitol; MALDI-ToF, matrix-assisted laser desorption ionization time-of-flight; oxPTP antibody, antibody that recognizes oxidized catalytic site cysteines of PTPs; MES, 4-morpholineethanesulfonic acid. Back


   ACKNOWLEDGMENTS
 
We thank Tony Hunter for critical reading of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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