Insights into the suppressor of T-cell receptor (TCR) signaling-1 (Sts-1)-mediated regulation of TCR signaling through the use of novel substrate-trapping Sts-1 phosphatase variants.
Insights into the suppressor of T-cell receptor (TCR) signaling-1 (Sts-1)-mediated regulation of TCR signaling through the use of novel substrate-trapping Sts-1 phosphatase variants.
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DOI：
10.1111/febs.12615
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发表时间：
2014-02
期刊：
影响因子：
--
通讯作者：
Carpino N
中科院分区：
文献类型：
--
作者：
Luis BS;Carpino N
高亲和力底物捕获蛋白酪氨酸磷酸酶已被广泛用于研究许多磷酸酶的内源靶标并解决底物特异性问题。在此，我们将底物捕获磷酸酶的概念扩展到包括组氨酸磷酸酶超家族的酶。这是首次描述应用于组氨酸磷酸酶家族成员的底物捕获技术。最近有报道称，T 细胞受体信号传导的磷酸酶抑制剂 (Sts)-1 可以负向调节 T 细胞受体下游的信号传导。我们通过对磷酸酶催化结构域内的关键活性位点残基进行诱变，生成了 Sts-1 的高亲和力底物捕获变体。亲核 His380 和通用酸性 Glu490 的突变产生了 Sts-1 酶，这些酶催化失活，但对 T 细胞中已知 Sts-1 调节的重要酪氨酸激酶 Zap-70 显示出高亲和力。 Sts-1 底物捕获突变体从活化 T 细胞裂解液中分离出酪氨酸磷酸化的 Zap-70，验证了 Zap-70 作为 Sts-1 的可能底物，并强调了突变体作为底物捕获剂的功效。钒酸盐对 Zap-70 相互作用的抑制表明底物捕获效应是通过 Sts-1 磷酸酶活性位点发生的。最后，T 细胞中 Sts-1 底物捕获突变体的过度表达阻断了 T 细胞受体信号传导，证实了 Sts-1 对 Zap-70 的抑制作用。
High affinity substrate-trapping protein tyrosine phosphatases have been widely used both to investigate the endogenous targets of many phosphatases and to address questions of substrate specificity. Herein, we extend the concept of a substrate-trapping phosphatase to include an enzyme of the histidine phosphatase superfamily. This is the first description of substrate-trapping technology applied to a member of the histidine phosphatase family. The phosphatase suppressor of T-cell receptor signaling (Sts)-1 has recently been reported to negatively regulate signaling downstream of the T-cell receptor. We generated high-affinity substrate-trapping variants of Sts-1 by mutagenesis of key active site residues within the phosphatase catalytic domain. Mutation of both the nucleophilic His380 and the general acid Glu490 yielded Sts-1 enzymes that were catalytically inactive but showed high affinity for an important tyrosine kinase in T cells that Sts-1 is known to regulate, Zap-70. Sts-1 substrate-trapping mutants isolated tyrosine-phosphorylated Zap-70 from lysates of activated T cells, validating Zap-70 as a possible substrate for Sts-1 and highlighting the efficacy of the mutants as substrate-trapping agents. Inhibition of the Zap-70 interaction by vanadate suggests that the substrate-trapping effect occurred via the Sts-1 phosphatase active site. Finally, overexpression of Sts-1 substrate-trapping mutants in T cells blocked T-cell receptor signaling, confirming the inhibitory effect of Sts-1 on Zap-70.
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DOI：
10.1042/bj20091383
发表时间：
2010-04-14
期刊：
The Biochemical journal
影响因子：
--
作者：
Hinton SD;Myers MP;Roggero VR;Allison LA;Tonks NK
通讯作者：
Tonks NK
影响因子：
2.9
作者：
Chen, Yunting;Jakoncic, Jean;Nassar, Nicolas
通讯作者：
Nassar, Nicolas
影响因子：
64.5
作者：
Deindl, Sebastian;Kadlecek, Theresa A.;Kuriyan, John
通讯作者：
Kuriyan, John
影响因子：
64.5
作者：
Wang, Zhigao;Jiang, Hui;Wang, Xiaodong
通讯作者：
Wang, Xiaodong
影响因子：
56.9
作者：
Plas, DR;Johnson, R;Thomas, ML
通讯作者：
Thomas, ML