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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
Author(s) -
Luis Boris S.,
Carpino Nick
Publication year - 2014
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.12615
Subject(s) - phosphatase , protein tyrosine phosphatase , microbiology and biotechnology , biochemistry , biology , mutant , phosphorylation , t cell receptor , receptor tyrosine kinase , receptor , proto oncogene tyrosine protein kinase src , chemistry , t cell , immune system , genetics , gene
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 abstractSts-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 )