Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei 112, Taiwan.
Biochem Biophys Res Commun. 2010 Jan 1;391(1):230-4. doi: 10.1016/j.bbrc.2009.11.037. Epub 2009 Nov 10.
Src homology-2 (SH2) domain-containing phosphatase 2 (SHP2) is known to participate in several different signaling pathways to mediate cell growth, survival, migration, and differentiation. However, due to the lack of proper analytical tools, it is unclear whether the phosphatase activity of SHP2 is activated in most studies. We have previously developed an activity-based probe LCL2 that formed covalent linkage with catalytically active protein tyrosine phosphatases (PTPs). Here, by combining LCL2 with a SHP2 specific antibody, we established an assay system that enables the direct monitoring of SHP2 activity upon cisplatin treatment of cancer cells. The protocol is advantageous over conventional colorimetric or in-gel PTP assays as it is specific and does not require the use of radioisotope reagents. Using this assay, we found SHP2 activity was selectively activated by cisplatin. Moreover, the activation of SHP2 appeared to be specific for cisplatin as other DNA damage agents failed to activate the activity. Although the role of SHP2 activation by cisplatin treatments is still unclear to us, our results provide the first direct evidence for the activation of SHP2 during cisplatin treatments. More importantly, the concept of using activity-based probe in conjunction with target-specific antibodies could be extended to other enzyme classes.
Src 同源物-2（SH2）结构域结合磷酸酶 2（SHP2）已知参与几种不同的信号通路，以介导细胞生长、存活、迁移和分化。然而，由于缺乏适当的分析工具，目前尚不清楚在大多数研究中 SHP2 的磷酸酶活性是否被激活。我们之前开发了一种基于活性的探针 LCL2，它与催化活性的蛋白酪氨酸磷酸酶（PTP）形成共价键。在这里，我们通过将 LCL2 与 SHP2 特异性抗体结合，建立了一种测定系统，可在顺铂处理癌细胞时直接监测 SHP2 活性。该方案优于传统的比色或凝胶 PTP 测定法，因为它是特异性的，并且不需要使用放射性同位素试剂。使用该测定法，我们发现 SHP2 活性可被顺铂选择性激活。此外，SHP2 的激活似乎是顺铂特异性的，因为其他 DNA 损伤剂未能激活该活性。虽然我们尚不清楚顺铂处理激活 SHP2 的作用，但我们的结果为顺铂处理过程中 SHP2 的激活提供了第一个直接证据。更重要的是，使用活性探针与靶标特异性抗体结合的概念可以扩展到其他酶类。
