Abstract
Low molecular weight phosphotyrosine-protein phosphatase (LMW-PTP) shares no general sequence homology with other PTPs, although it has an active site sequence motif CXXXXXR and a reaction mechanism identical to those of all PTPs. The main function of this enzyme is the down-regulation of platelet-derived growth factor and insulin receptors. Both human LMW-PTP isoenzymes are inactivated by H2O2. The enzymes are protected from inactivation by Pi, a competitive inhibitor, suggesting that the H2O2 reaction is directed to active site. Analysis of free thiols performed on the inactivated enzymes demonstrates that only two out of the eight LMW-PTP cysteines are modified. Time-course high performance liquid chromatography-electrospray mass spectrometry, together with specific radiolabeling and tryptic fingerprint analyses, enables us to demonstrate that H2O2 causes the oxidation of Cys-12 and Cys-17 to form a disulfide bond. Because both residues are localized into the active site region, this modification inactivates the enzyme. Fluorescence spectroscopy experiments suggest that the fold of the enzyme is modified during oxidation by H2O2. Because a physiological concentration of H2O2 produces enzyme inactivation and considering that the activity is restored by reduction with low molecular weight thiols, we suggest that oxidative stress conditions and other processes producing hydrogen peroxide regulate the LMW-PTP in the cell.
Highlights
Protein tyrosine phosphorylation in eucaryotes is a key mechanism for cellular control, because it is involved in several processes, such as cellular metabolism, proliferation, differentiation, and oncogenic transformation [1]
Because a physiological concentration of H2O2 produces enzyme inactivation and considering that the activity is restored by reduction with low molecular weight thiols, we suggest that oxidative stress conditions and other processes producing hydrogen peroxide regulate the Low molecular weight phosphotyrosine-protein phosphatase (LMW-protein-tyrosine phosphatases (PTPs)) in the cell
Immunoprecipitation experiments performed with cells overexpressing the dominant negative enzyme form have demonstrated that the enzyme associates with these receptors only when they are stimulated by their specific growth factors [5, 6]
Summary
Materials—Human recombinant LMW-PTPs (IF1 and IF2 isoenzymes) were prepared as described previously [11]. Protein Digestion and Purification of Peptides—The modified and radiolabeled enzyme (see below) was dissolved in 50 l of 0.2 M ammonium bicarbonate, pH 8.5, containing 6 M guanidinium chloride; 250 l of 0.2 M ammonium bicarbonate buffer, pH 8.5, was added in order to adjust the concentration of guanidinium chloride to 1 M. This solution was incubated for 4 min in a boiling water bath and chilled in ice. The trypsin digestion was initiated by adding a small volume of trypsin solution (final concentration, 5%, w/w). The electrospray mass spectra of LMW-PTPs were obtained under the following conditions: electrospray capillary voltage, 4 kV; fragmentor voltage, 120 V; nebulizer gas (N2), 40 psi; drying gas (N2), 10 liters/min at 300 °C; scan range, 500 –2000 m/z; scan rate, 3 ms/atomic mass unit; resolution, Ͼ1 atomic mass unit
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