Chan Wai Cheung, Knowlton Gregory S, Bishop Anthony C
Amherst College, Department of Chemistry, Amherst, MA, 01002, USA.
Chembiochem. 2017 Oct 5;18(19):1950-1958. doi: 10.1002/cbic.201700253. Epub 2017 Aug 23.
Methods for activating signaling enzymes hold significant potential for the study of cellular signal transduction. Here we present a strategy for engineering chemically activatable protein tyrosine phosphatases (actPTPs). To generate actPTP1B, we introduced three cysteine point mutations in the enzyme's WPD loop. Biarsenical compounds were screened for the capability to bind actPTP1B's WPD loop and increase its phosphatase activity. We identified AsCy3-EDT as a robust activator that selectively targets actPTP1B in proteomic mixtures and intact cells. Introduction of the corresponding mutations in T-cell PTP also generates an enzyme (actTCPTP) that is strongly activated by AsCy3-EDT . Given the conservation of WPD-loop structure among the classical PTPs, our results potentially provide the groundwork of a widely generalizable approach for generating actPTPs as tools for elucidating PTP signaling roles as well as connections between dysregulated PTP activity and human disease.
激活信号酶的方法在细胞信号转导研究中具有巨大潜力。在此，我们提出一种构建化学可激活蛋白酪氨酸磷酸酶（actPTP）的策略。为生成actPTP1B，我们在该酶的WPD环中引入了三个半胱氨酸点突变。对双砷化合物进行筛选，以确定其结合actPTP1B的WPD环并提高其磷酸酶活性的能力。我们鉴定出AsCy3-EDT是一种强大的激活剂，它能在蛋白质组混合物和完整细胞中选择性地靶向actPTP1B。在T细胞磷酸酶中引入相应突变也会产生一种被AsCy3-EDT强烈激活的酶（actTCPTP）。鉴于经典蛋白酪氨酸磷酸酶中WPD环结构的保守性，我们的结果可能为生成actPTP提供一种广泛通用方法的基础，actPTP可作为阐明蛋白酪氨酸磷酸酶信号作用以及失调的蛋白酪氨酸磷酸酶活性与人类疾病之间联系的工具。
