Effects of metal ions on the activity of protein tyrosine phosphatase VHR: highly potent and reversible oxidative inactivation by Cu2+ ion.
The posttranslational regulation of protein tyrosine phosphatases (PTPs) has been suggested to have a crucial role in maintaining the phosphotyrosine level in cells. Here we examined the regulatory effects of metal ions on human dual-specificity vaccinia H1-related protein tyrosine phosphatase (VHR) in vitro. Among various metal ions examined, Fe3+, Cu2+, Zn2+, and Cd2+ exerted their inactivational effects on VHR, and Cu2+ is the most potent inactivator. The VHR activity inactivated by the metal ions except Cu2+ was significantly restored by EDTA. The efficacy of Cu2+ for the VHR inactivation was about 200-fold more potent than that of H2O2. Cu2+ also inactivated other PTPs including PTP1B and SHP-1. The Cu2+-mediated inactivation at the submicromolar range was eradicated by dithiothreitol treatment. The loss of VHR activity correlated with the decreased [14C]iodoacetate labeling of active-site cysteine, suggesting that Cu2+ brought about the oxidation of the active-site cysteine. On the contrary, Zn2+ that exerted an inactivational effect at millimolar concentrations appeared not directly linked to the active-site cysteine, as indicated by the fact that [14C]iodoacetate labeling was unaffected and that the effect of Zn2+ on the Y78F mutant was increased. The reduction potential of VHR was estimated to be -331 mV by utilizing the reversibility of the redox state of VHR. Thus, we conclude that the highly potent Cu2+ inactivation of VHR is a consequence of the oxidation of the active-site cysteine and the mode of Zn2+ inactivation is distinct from that of Cu2+.
Kim
J H
JH
School of Chemistry and Molecular Engineering and Center for Molecular Catalysis, Seoul National University, South Korea.
Cho
H
H
Ryu
S E
SE
Choi
M U
MU
D001665:Binding Sites; D003300:Copper / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D054640:Dual Specificity Phosphatase 3; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005978:Glutathione / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007477:Ions; D013058:Mass Spectrometry; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000378:metabolism*; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013997:Time Factors; D015032:Zinc / Q000378:metabolism
D007477:Ions; D003300:Copper; D006861:Hydrogen Peroxide; C517025:DUSP3 protein, human; D054640:Dual Specificity Phosphatase 3; D017027:Protein Tyrosine Phosphatases; D005978:Glutathione; D015032:Zinc; D003545:Cysteine; D010100:Oxygen; D004229:Dithiothreitol