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2 Serono Pharmaceutical Research Institute, 14, chemin des Aulx, 1228 Plan-Les-Ouates, Geneva, Switzerland.
Reprint requests to: Rob Hooft van Huijsduijnen, Serono Pharmaceutical Research Institute, 14, chemin des Aulx, 1228 Plan-Les-Ouates, Geneva, Switzerland; e-mail: rob.hooft{at}serono.com; fax: +41 22 794 6965
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-ß 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.
Keywords: PTP; protein-tyrosine phophatase; SPOT; protein-protein interactions
Abbreviations: aa, amino acid(s) • GST, glutathione-S-transferase • IR, insulin receptor • PBS, phosphate-buffered saline • PTP, protein tyrosine phosphatase • STAT, signal transducer and activator of transcription • TC-PTP, T-cell protein tyrosine phosphatase
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