Chemical knockdown of protein-tyrosine phosphatase 1B by 1,2-naphthoquinone through covalent modification causes persistent transactivation of epidermal growth factor receptor
Chemical knockdown of protein-tyrosine phosphatase 1B by 1,2-naphthoquinone through covalent modification causes persistent transactivation of epidermal growth factor receptor
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DOI：
10.1074/jbc.m705224200
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发表时间：
2007-11-16
影响因子：
4.8
通讯作者：
Kumagai, Yoshito
中科院分区：
文献类型：
--
作者：
Iwamoto, Noriko;Sumi, Daigo;Kumagai, Yoshito
1，2-萘醌（1，2-NQ）是一种大气污染物，通过抑制蛋白酪氨酸磷酸酶（PTPs）激活表皮生长因子受体（EGFR），引起豚鼠气管收缩。EGFR的磷酸化受PTP负调控，但1，2-NQ抑制PTP的机制细节尚未阐明。本报告中描述的结果表明，暴露于人上皮A431细胞后，1，2-NQ与PTP 1B形成共价键。在这项研究中，发现EGFR的浓度依赖性磷酸化与细胞中PTP活性的降低相关联。PTP活性的降低是由于PTP 1B的不可逆修饰，当PTP 1B被细胞过表达时，1，2-NQ介导的EGFR磷酸化被抑制。用纯化的PTP 1B和1，2-NQ的研究表明，酶活性的降低是由于醌对酶的亲核攻击，形成共价键。基质辅助激光解吸电离飞行时间质谱分析和突变实验表明，PTP 1B失活主要是由于醌共价连接到酶的Cys-121，结合His-25和Cys-215以及。总的来说，结果表明，1，2-NQ与PTP 1B的共价连接至少部分地导致PTP活性的降低，这导致细胞中EGFR的延长的反式激活。
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.