Search Organism: All OrganismsArabidopsis thalianaBacillus subtilis 168Bos taurusCaenorhabditis elegansCanis familiarisDanio rerioDrosophila melanogasterEquus caballusEscherichia coli K12Gallus gallusHomo sapiensMacaca mulattaMus musculusNematostella vectensisOryza sativaPan troglodytesRattus norvegicusSaccharomyces cerevisiaeSchizosaccharomyces pombeStrongylocentrotus purpuratusTakifugu rubripesXenopus laevis home help / support contribute downloads mirrors about us Protein Sci. Oct (2002); 11(10):2326-34 The SPOT technique as a tool for studying protein tyrosine phosphatase substrate specificities. Espanel X, Huguenin-Reggiani M, Van Huijsduijnen RH Serono Pharmaceutical Research Institute, 14, chemin des Aulx, 1228 Plan-Les-Ouates, Geneva, Switzerland. Abstract: The activity of protein tyrosine phosphatases (PTPs) is restricted by their substrate specificities. The analysis of PTP specificity was greatly helped by the discovery that "substrate-trapping" PTP mutants, such as PTP-1B D181A, stably and specifically bind their substrates. We have set up a PTP substrate specificity assay based on the SPOT technique, which involves the microsynthesis of (phospho)peptides on membranes. To validate this approach, substrate trapping PTP-1B was tested on its cognate ligand, the autophosphorylated insulin receptor (IR). On SPOT membranes, IR peptides with phosphotyrosine 1163 were efficiently bound by PTP1B D181A, and dephosphorylated by PTP-1B. Phosphotyrosine 1163 was preferred over the neighboring 1158 and 1162 phosphotyrosines. PTP-1B also recognized IR-like motifs in Trk autophosphorylation domains, and STAT 5 phosphopeptides. Using a gridded 20-by-20 SPOT library, we show that peptides with the YZM motif (Z: phosphotyrosine) are the strongest ligands for PTP-1B D181A, but not the optimal substrates for dephosphorylation by wild-type PTP1B. In addition we show that PTP-1B and PTP-beta dephosphorylation efficiency is strongly modulated by the introduction of phospho-serine or phospho-threonine in their cognate phospho-tyrosine substrates. Altogether our data illustrate that the SPOT technique is a highly efficient tool for the study of PTP substrate specificity. [PUBMED: 12237455] Copyright © 2009, Tyers Lab, The Samuel Lunenfeld Research Institute, Mount Sinai Hospital, All Rights Reserved. terms and conditions - privacy policy - Osprey Network Visualization System BioGRID: A General Repository for Interaction Datasets. Chris Stark, Bobby-Joe Breitkreutz, Teresa Reguly, Lorrie Boucher, Ashton Breitkreutz, Mike Tyers. Nucleic Acids Res. Jan 1;34:D535-9.