Src-同源性-2(SH2)结构域选择性结合位于靶结合蛋白中的磷酸酪氨酸(pTyr)残基;因此,它们是pTyr介导的信号通路的关键要素。SH2结构域与pTyr的结合作为一种对接机制,吸引蛋白质进入信号中枢,在某些情况下,它还可以调节信号酶如蛋白激酶或蛋白磷酸酶的催化活性。因此,选择性结合SH2结构域的化合物可潜在地用于调节此类含SH2结构域的酶的活性。本章介绍了如何通过肽与SH2结构域的变构结合来测量蛋白酪氨酸磷酸酶活性的调节,并以从细菌中纯化的人重组蛋白酪氨酸磷酸酶SHP2(含Src同源-2结构域的蛋白酪氨酸磷酸酶2)为例。在存在以不同浓度选择性结合和激活SHP2的肽的情况下,随时间测量针对人工底物DiFMUP(6,8-二氟-4-甲基伞形基磷酸酯)的磷酸酶活性,以确定半数最大有效浓度(EC50)。
Src-homology-2 (SH2) domains bind selectively to phosphotyrosine (pTyr) residues located in target binding proteins; therefore, they are key elements in pTyr-mediated signaling pathways. The binding of an SH2 domain to a pTyr acts as a docking mechanism that attracts proteins into signaling hubs, and in some cases, it can also regulate the catalytic activity of signaling enzymes such as protein kinases or protein phosphatases. Therefore, compounds that selectively bind SH2 domains can be potentially used to modulate the activity of such SH2 domain-containing enzymes. This chapter describes how to measure the regulation of protein tyrosine phosphatase activity through allosteric binding of peptides to SH2 domains, and uses human recombinant protein tyrosine phosphatase SHP2 (Src homology-2 domain-containing protein tyrosine phosphatase 2) purified from bacteria as a case example. The phosphatase activity against the artificial substrate
DiFMUP (6, 8-Difluoro-4-Methylumbelliferyl Phosphate) is measured over time in the presence of a peptide that selectively binds and activates SHP2 at different concentrations to determine the half maximal effective concentration (EC50).