| First Author | Luis BS | Year | 2014 |
| Journal | FEBS J | Volume | 281 |
| Issue | 3 | Pages | 696-707 |
| PubMed ID | 24256567 | Mgi Jnum | J:205199 |
| Mgi Id | MGI:5544362 | Doi | 10.1111/febs.12615 |
| Citation | Luis BS, et al. (2014) 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. FEBS J 281(3):696-707 |
| abstractText | 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. STRUCTURED DIGITAL ABSTRACT: Sts-1 physically interacts with Zap-70 by anti tag coimmunoprecipitation (1, 2, 3) Zap-70 physically interacts with Sts-1 by anti bait coimmunoprecipitation (1, 2) Zap-70 physically interacts with Sts-1 by pull down (1, 2, 3, 4, 5). |
