Two Vicinal Cysteines Confer a Peculiar Redox Regulation to Low Molecular Weight Protein Tyrosine Phosphatase in Response to Platelet-derived Growth Factor Receptor Stimulation

Abstract

Low molecular weight protein tyrosine phosphatase (LMW-PTP) is an enzyme involved in platelet-derived growth factor (PDGF)-induced mitogenesis and cytoskeleton rearrangement because it is able to bind and dephosphorylate the activated receptor. LMW-PTP presents two cysteines in positions 12 and 17, both belonging to the catalytic pocket; this is a unique feature of LMW-PTP among all protein tyrosine phosphatases. Our previous results demonstrated that in vitro LMW-PTP is oxidized by either H 2 O 2 or nitric oxide with the formation of a disulfide bond between Cys-12 and Cys-17. This oxidation leads to reversible enzyme inactivation because treatment with reductants permits catalytic activity rescue. In the present study we investigated the in vivo inactivation of LMW-PTP by either extracellularly or intracellularly generated H 2 O 2 , evaluating its action directly on its natural substrate, PDGF receptor. LMW-PTP is oxidized and inactivated by exogenous oxidative stress and recovers its activity after oxidant removal. LMW-PTP is oxidized also during PDGF signaling, very likely upon PDGF-induced H 2 O 2 production, and recovers its activity within 40 min. Our results strongly suggest that reversibility of in vivo LMW-PTP oxidation is glutathione-dependent. In addition, we propose an intriguing and peculiar role of Cys-17 in the formation of a S-S intramolecular bond, which protects the catalytic Cys-12 from further and irreversible oxidation. On the basis of our results we propose that the presence of an additional cysteine near the catalytic cysteine could confer to LMW-PTP the ability to rapidly recover its activity and finely regulate PDGF receptor activation during both extracellularly and intracellularly generated oxidative stress.Protein tyrosine phosphorylation plays a key role in the regulation of many cellular processes in eukaryotes such as cellular metabolism, proliferation, differentiation, and oncogenic transformation (1). Accumulating evidence indicates that the contribution of phosphotyrosine protein phosphatases (PTPs) 1 to the control of the cell phosphorylation state is as relevant as that of phosphotyrosine protein kinases. The PTP superfamily is composed of over 70 enzymes that, despite very limited sequence similarity, share a common CX 5 R active site motif and an identical catalytic mechanism. On the basis of their function, structure, and sequence, PTPs can be classified in four main families: 1) tyrosine-specific phosphatases, 2) VH1-like dual specificity PTPs, 3) the cdc25 phosphatases, and 4) the low molecular weight phosphatase (2).The low molecular weight protein tyrosine phosphatase (LMW-PTP) is an 18-kDa enzyme that is expressed in many mammalian tissues (3). Our previous studies on the molecular biology of LMW-PTP in NIH3T3 cells demonstrated a well defined role of this enzyme in platelet-derived growth factor (PDGF)-induced mitogenesis. The most relevant phenotypic effect of LMW-PTP overexpression is a strong reduction of the cell growth rate in response to PDGF stimulation. We ...

Differentiation of normal and cancer cells induced by sulfhydryl reduction: biochemical and molecular mechanisms

“…It is worth noticing that in comparison with these other sulfhydryl reductants, NAC is not toxic. Although also protein tyrosine phosphatases should be activated by sulfhydryl reduction, 3 the lack of Y530 dephosphorylation excludes a role of phosphatases in this NAC-mediated c-Src inactivation. More experiments are in progress to better elucidate this mechanism for c-Src inactivation and to quantitatively determine the oxidized/reduced -SH groups.…”

“…1 In particular, protein tyrosine phosphatases play a pivotal role in the control of cell cycle and differentiation, and were reported to be regulated through the redox state of their cysteine -SH residues. 2,3 Also protein tyrosine kinases have an essential role in the control of cell proliferation and differentiation. For three of them, Yes, Fyn and c-Src, a redox regulation has been recently reported 4,5 and particularly for c-Src, it apparently implies a reversible change in the redox state of a group of four cysteine residues close to the catalytic site.…”

The chemistry of cell signaling by reactive oxygen and nitrogen species and 4-hydroxynonenal

“…The oxidative reaction of cysteine with H 2 O 2 may change structure and function of protein mediating response to changes of redox state in the cell (61,62). An important protein effector by which H 2 O 2 signaling occurs is the family of PTPs (34,(36)(37)(38)63,64). PTPs constitute a large family of important regulatory enzymes that play a key role in several physiological functions.…”

Purification and Characterization of ZmRIP1, a Novel Reductant-Inhibited Protein Tyrosine Phosphatase from Maize

“…A change in a single Cys residue is sufficient to have a profound effect on the molecular architecture of PTPases and thus on their enzymatic activity (Mayadas and Wagner, 1992;Chiarugi et al, 2001;Paget and Buttner, 2003;Tonks, 2005;Zimmermann et al, 2007;Winterbourn and Hampton, 2008;Álvarez et al, 2009;Bucciarelli et al, 2009). With this in mind, we searched the protein homology of ZmRIP1 and found that distinct differences between ZmRIP1 and its homologs exist in the Cys residues at two sites (i.e.…”