Amherst College, Department of Chemistry, Amherst, Massachusetts, 01002, USA.
Dana-Farber Cancer Institute, Department of Cancer Biology, Boston, MA, 02215, USA.
Sci Rep. 2019 Nov 6;9(1):16148. doi: 10.1038/s41598-019-52002-1.
Strategies for the direct chemical activation of specific signaling proteins could provide powerful tools for interrogating cellular signal transduction. However, targeted protein activation is chemically challenging, and few broadly applicable activation strategies for signaling enzymes have been developed. Here we report that classical protein tyrosine phosphatase (PTP) domains from multiple subfamilies can be systematically sensitized to target-specific activation by the cyanine-based biarsenical compounds AsCy3 and AsCy5. Engineering of the activatable PTPs (actPTPs) is achieved by the introduction of three cysteine residues within a conserved loop of the PTP domain, and the positions of the sensitizing mutations are readily identifiable from primary sequence alignments. In the current study we have generated and characterized actPTP domains from three different subfamilies of both receptor and non-receptor PTPs. Biarsenical-induced stimulation of the actPTPs is rapid and dose-dependent, and is operative with both purified enzymes and complex proteomic mixtures. Our results suggest that a substantial fraction of the classical PTP family will be compatible with the act-engineering approach, which provides a novel chemical-biological tool for the control of PTP activity and the study of PTP function.
直接化学激活特定信号蛋白的策略可为细胞信号转导的研究提供强大的工具。然而，靶向蛋白激活在化学上具有挑战性，并且很少有针对信号酶的广泛适用的激活策略。在这里，我们报告说，来自多个亚家族的经典蛋白酪氨酸磷酸酶（PTP）结构域可以通过基于菁染料的双砷化合物 AsCy3 和 AsCy5 被系统地敏化，以实现针对特定靶标的激活。可激活的 PTP（actPTP）的工程设计是通过在 PTP 结构域的保守环内引入三个半胱氨酸残基来实现的，并且敏化突变的位置可以从一级序列比对中轻松识别。在本研究中，我们已经从受体和非受体 PTP 的三个不同亚家族中生成并表征了 actPTP 结构域。双砷诱导的 actPTP 的刺激是快速且剂量依赖性的，并且可用于纯化酶和复杂的蛋白质混合物。我们的结果表明，经典 PTP 家族的很大一部分将与 act 工程化方法兼容，这为控制 PTP 活性和研究 PTP 功能提供了一种新的化学生物学工具。
