The Inactivation Mechanism of Low Molecular Weight Phosphotyrosine-protein Phosphatase by H2O2

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 H 2 O 2 . The enzymes are protected from inactivation by P i , a competitive inhibitor, suggesting that the H 2 O 2 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 H 2 O 2 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 H 2 O 2 . Because a physiological concentration of H 2 O 2 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.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). A fine balancing of cellular protein tyrosine phosphorylation levels is determined by regulating the activities of protein-tyrosine kinases and/or protein-tyrosine phosphatases (PTPs).1 Receptor protein-tyrosine kinases are considered to be the major enzymes regulating mitogenic protein phosphorylation cascades; nevertheless, the presence of SH2 domains in particular PTPs and the receptorlike structure of some membrane PTPs clearly indicate that PTPs are also regulated in the cell. The PTP superfamily consists of four main families: the tyrosine-specific phosphatases, the VH1-like dual specificity phosphatases, the cdc25 phosphatases, and the low molecular weight phosphatases (LMW-PTPs). Despite extremely limited sequence similarity, all share an active site motif consisting of a cysteine and an arginine separated by five residues (CXXXXXR, where X is any amino acid). All PTPs have identical catalytic mechanism, which involves the formation of a cysteinyl-phosphate intermediate (2).Recent papers from our laboratory have demonstrated that LMW-PTP is involved in the regulation of cellular signaling started by the activation of PDGF and insulin receptors (3-5). In fact, the overexpression of the wild type enzyme in NIH/3T3 cells causes decrease of cellular growth rate and of phosphorylation level of the PDGF receptor (3). Furthermore, the overexpression in ...

Prediction of a ligand‐induced conformational change in the catalytic core of Cdc25A

“…Cdc25A is similar to the low molecular weight PTP in that there is an additional cysteine residue that appears able to form a disul¢de bridge with the catalytic cysteine under certain conditions [7,13]. In the latter case, this residue (reduced Cys-17) has been proposed to play a role in the hydrolysis of the phosphoenzyme intermediate by interaction with the water nucleophile [14].…”

Activation of SRC Tyrosine Kinases in Response to ICAM-1 Ligation in Pulmonary Microvascular Endothelial Cells

“…One of the identified downstream targets of ROS is tyrosine phosphatases. ROS cause oxidation of cysteine residues in the tyrosine phosphatase catalytic domain to form a disulfide bond, resulting in the inactivation of tyrosine phosphatases (37)(38)(39)(40)(41). Whether inactivation of tyrosine phosphatases is one of the underlying mechanisms whereby xanthine oxidase-dependent ROS production mediates activation of SRC tyrosine kinases remains to be determined.…”

Toll Receptor-Mediated Regulation of NADPH Oxidase in Human Dendritic Cells

“…Reactive oxygen species are known to participate in cellular signaling through the regulation of transcription factors, such as AP-1 and NF-B (22,23), and various enzyme systems (e.g., protein tyrosine phosphates, protein kinase C, and mitogen-activated protein kinases) (50,51). In DC, oxygen radicals were implicated in both DC differentiation (43) and maturation (26,27) and in IL-8/CXCL8 production (24,25).…”