Large 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. 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 brokers. Inhibition of the Zap-70 conversation by vanadate suggests that the substrate-trapping effect occurred via the Sts-1 phosphatase active site. Finally overexpression of Sts-1 substrate-trapping mutants MLN4924 in T cells blocked T-cell receptor signaling confirming the inhibitory effect of Sts-1 on Zap-70. [8]. In order to further address the question of whether Zap-70 could be a authentic Sts-1 target we sought to develop high-affinity ‘substrate-trapping’ variants of Sts-1 and determine whether these mutants could interact stably with Zap-70. Substrate-trapping techniques have been widely used to identify target substrates of PTPs [11]. They involve the use of mutant PTPs in which the catalytic cysteine that serves as a nucleophile and/or the proton-donating aspartate found within the WPD loop have been changed to a serine or an alanine respectively [12]. These mutants are catalytically inactive but retain the ability to bind their native substrates [13 14 Substrate-trapping can also serve as an innate regulatory mechanism for sequestering specific components away from signaling circuits as in the case of the pseudo- phosphatase MK-STYX targeting an effector for stress granule formation Ras-GTPase-activating protein-binding protein [15]. In this study we used INHA antibody the development of high-affinity Sts-1 mutants to investigate the possibility of Zap-70 being a substrate for Sts-1. Our results suggest that Sts-1 can directly target Zap-70 in T cells. Results Development of Sts-1 substrate-trapping mutants With the aim of generating a catalytically inactive Sts-1 phosphatase as a substrate-trapping mutant we targeted three residues in the active site of Sts-1PGM for mutation: the nucleophilic His380 and two additional basic residues that are proposed to undergo crucial electrostatic interactions with the substrate’s phosphate moiety Arg462 and His565 (Fig. 1A). We hypothesized that altering these residues could yield catalytically inactive phosphatase enzymes that would nonetheless interact stably MLN4924 with substrates. Speculating that removal of an acidic residue within the active site might decrease the electrostatic repulsion of the incoming phosphate group and thus increase the substrate-binding affinity we also targeted Glu490 for mutation. A series of single and compound mutations were introduced into the Sts-1PGM to generate a total of 15 candidate high-affinity mutants (Fig. 1A). To evaluate catalytic activity we expressed wild-type and mutant Sts-1PGM as Flag-tagged proteins in HEK293T cells and performed immune complex phosphatase activity assays on anti-Flag immunoprecipitates. Although not all of the Sts-1PGM mutants were expressed well (Fig. 1B: H565A/E490A H380C/H565A R462A/ H565A and H380C/E490Q/H464A) those that were lacked measurable catalytic activity (Fig. 1 Fig. 1 Development of Sts-1 high-affinity mutants. (A) Representation of conserved active site residues in Sts-1PGM MLN4924 MLN4924 generated in PYMOL with the crystal structure of Sts-1PGM complexed with phosphate (Protein Data Bank ID: 2IKQ). A total of 15 candidate Sts-1 … To identify potential substrate-trapping Sts-1PGM variants we took advantage of two observations: first the T-cell tyrosine kinsase Zap-70 has been identified as a potential in vivo Sts-1 substrate; and second Sts-1PGM can efficiently dephosphorylate Zap-70 and Zap-70-derived phosphopeptides in vitro [7 8 We began by assessing the conversation between a Zap-70-derived phosphopeptide (GSVYESPpYSDPEEL) and the different Sts-1PGM mutants explained above. Pulldown assays were performed in which phosphopeptide-coupled beads were added to lysates prepared from cells expressing wild-type or mutant.