title: The SPOT technique as a tool for studying protein tyrosine phosphatase substrate specificities.
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.
authors.affiliation: Serono Pharmaceutical Research Institute, 14, chemin des Aulx, 1228 Plan-Les-Ouates, Geneva, Switzerland.
authors.forename: Xavier
authors.initials: X
authors.lastname: Espanel
authors.forename: Martine
authors.initials: M
authors.lastname: Huguenin-Reggiani
authors.forename: Rob
authors.initials: R
authors.lastname: Hooft van Huijsduijnen
mesh_terms: D020816:Amino Acid Motifs; D019151:Peptide Library; D010748:Phosphopeptides / Q000138:chemical synthesis; D010766:Phosphorylation; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D011972:Receptor, Insulin / Q000378:metabolism; D013379:Substrate Specificity / Q000502:physiology
chemical_list: D019151:Peptide Library; D010748:Phosphopeptides; D011972:Receptor, Insulin; D017027:Protein Tyrosine Phosphatases