title: 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.
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.
authors.affiliation: Program in Molecular and Cellular Biology, Stony Brook University, NY, USA.
authors.forename: Boris S
authors.initials: BS
authors.lastname: Luis
authors.forename: Nick
authors.initials: N
authors.lastname: Carpino
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D020134:Catalytic Domain; D002460:Cell Line; D002478:Cells, Cultured; D006801:Humans; D008213:Lymphocyte Activation / Q000187:drug effects; D051379:Mice; D018345:Mice, Knockout; D050505:Mutant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013047:Specific Pathogen-Free Organisms; D013379:Substrate Specificity; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D050505:Mutant Proteins; D010446:Peptide Fragments; D047428:Protein Kinase Inhibitors; D011948:Receptors, Antigen, T-Cell; D011994:Recombinant Proteins; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human; C497262:Zap70 protein, mouse; D017027:Protein Tyrosine Phosphatases; C553497:TULA-2 protein, mouse
keywords: T-cell receptor (TCR) signaling; histidine phosphatase superfamily; suppressor of T-cell receptor signaling (Sts) proteins