ABSTRACT
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.
