Chemical Knockdown of Protein-tyrosine Phosphatase 1B by 1,2-Naphthoquinone through Covalent Modification Causes Persistent Transactivation of Epidermal Growth Factor Receptor

Abstract

1,2-Naphthoquinone (1,2-NQ), an atmospheric contaminant, causes the contraction of guinea pig trachea through the activation of epidermal growth factor receptor (EGFR) by inhibiting protein-tyrosine phosphatases (PTPs). Phosphorylation of EGFR is negatively regulated by PTPs, but details of the mechanism by which 1,2-NQ inhibits PTPs have not been elucidated. Results described in this report demonstrate that 1,2-NQ forms covalent bonds with PTP1B after exposure to human epithelial A431 cells. In this study, a concentration-dependent phosphorylation of EGFR was found to be coupled to the reduction of PTP activity in the cells. The reduction in PTP activity was due to the irreversible modification of PTP1B, and when PTP1B was overexpressed by the cells, the 1,2-NQ-mediated EGFR phosphorylation was suppressed. Studies with purified PTP1B and 1,2-NQ showed that the reduction in enzyme activity was due to a nucleophilic attack by the quinone on the enzyme, to form covalent bonds. Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry analysis and mutation experiments revealed that PTP1B inactivation was primarily due to covalent attachment of the quinone to Cys-121 of the enzyme, with binding to His-25 and Cys-215 as well. Collectively, the results show that covalent attachment of 1,2-NQ to PTP1B is at least partially responsible for the reduction of PTP activity, which leads to prolonged transactivation of EGFR in the cells.

Covalent binding of quinones activates the Ah receptor in Hepa1c1c7 cells

“…*Significantly different from control at p < 0.05 (ANOVA). through Cys151, Cys253, Cys273, Cys288, and Cys489, thereby activating EGFR and Nrf2 (Iwamoto et al, 2007;Kobayashi et al, 2009) because PTP1B and Keap1 are known to be negative regulators for EGFR and Nrf2, respectively (Itoh et al, 1999;Barford et al, 1995). These findings strongly indicate that 1,2-NQ is a bi-aromatic hydrocarbon that potentially activates EGFR and Nrf2 following covalent modification of PTP1B and Keap1 protein, respectively, through these thiol groups.…”

The Antioxidant Defense System Keap1-Nrf2 Comprises a Multiple Sensing Mechanism for Responding to a Wide Range of Chemical Compounds

“…Instead, the interesting possibility exists that each compound reacts with specific target proteins unrelated to the Keap1-Nrf2 system through specific cysteine residues. Indeed, the class 2 compound 1,2-NQ was reported to target Cys-121 in protein tyrosine phosphatase 1B (PTP1B) (14). Emitted as one of combustion products of fossil fuels and also formed in the atmosphere by photochemical reactions of aromatic hydrocarbons, 1,2-NQ is considered to create a variety of hazardous effects in vivo, including acute cytotoxicity, immunotoxicity, and carcinogenesis.…”

“…Peptides were mixed with ␣-cyano-4-hydroxycinnamic acid (2.5 mg/ml) containing 50% acetonitrile and 0.1% trifluoroacetic acid and dried on stainless steel targets at room temperature. Analyses were performed using an AXIMA-TOF2 (Shimadzu) with a nitrogen laser, as described previously (14). All analyses took place in the positive ion mode, and the instrument was calibrated immediately prior to each series of studies.…”

Polycyclic Aromatic Hydrocarbon Quinones as Redox and Electrophilic Chemicals Contaminated in the Atmosphere

“…To address this issue, we prepared a specific antibody against 1,2-NQ. 18) This antibody can recognize 1,2-NQ, 1,2-dihydroxynaphthalene, 1,2-NQ-4-mercaptoethanol adduct and 1,2-NQ-4-sulfonate, but not 1,4-NQ, and found that there were cellular proteins modified by 1,2-NQ. This suggests that 1,2-NQ exhibits not only redox but also electrophilic characteristics.…”

“…As expected, a concentration-dependent phosphorylation occurring during exposure of A431 cells to 1,2-NQ was found to be coupled to the reduction of PTP activity in the cells; under these conditions, 1,2-NQ did bind to PTP1B among cellular PTPs. 18) In cell-free systems, matrix-assisted laser desorption and ionization time-of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that 1,2-NQ is covalently bound to Cys121, thereby causing reduction of the catalytic activity. Thus, we concluded that covalent modification of 1,2-NQ to PTP1B is at least partially responsible for the reduction of PTP activity, which leads to prolonged phosphorylation of EGFR in A431 cells.…”