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.
Highlights
Protein-tyrosine phosphatases (PTPs),2 these dephosphorylation enzymes play important roles in the signal transduction controlled by receptor tyrosine kinases
Phosphorylation of epidermal growth factor receptor (EGFR) Coupled to Reduction of protein-tyrosine phosphatases (PTPs) Activity by 1,2-NQ in the Cellular System—Exposure of A431 cells to 1,2-NQ for 10 min caused a concentration-dependent phosphorylation of EGFR (Fig. 1A)
H2O2-mediated phosphorylation of EGFR was blocked by pretreatment of A431 cells with either catalase conjugated with polyethylene glycol or Trolox, a scavenging agent for reactive oxygen species (ROS), whereas EGFR activation caused by 1,2-NQ was not
Summary
Materials—1,2-NQ was purchased from Tokyo Kasei Industries, Ltd. (Tokyo, Japan). Naphthalene, Dulbecco’s modified Eagle’s medium, penicillin streptomycin solution, imidazole, p-nitrophenol (pNP), and 3,5-dimethoxy-4-hydroxycinnamic acid were obtained from Sigma. trans-1,2-Dihydroxy-1,2-dihydronaphthalene was kindly donated by Dr S. Cell lysates were incubated with Affi-gel 10 (Bio-Rad) cross-linked with anti-37-kDa PTP1B IgG (8.2 mg of protein/ml of gel) which was prepared according to the manufacturer’s instructions. The gels were centrifuged at 13,000 ϫ g for 1 min and washed with 1 ml of ice-cold radioimmune precipitation assay lysis buffer three times, and the PTP1B from the cell lysates was eluted with 20 l of 0.1 M glycine-HCl (pH 3.0) at 25 °C. The incubation mixture (0.2 ml) for the first stage consisted of 37-kDa PTP1B (0.1 mg), different concentrations of 1,2-NQ and 50 mM potassium phosphate buffer (pH 7.5), 0.1 mM EDTA, unless other noted. Data were fitted by nonlinear regression analysis according to a Michaelis-Menten kinetic model (GraphPad Prism version 4.0, San Diego, CA)
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