The SPOT technique as a tool for studying protein tyrosine phosphatase substrate specificities.
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.
Espanel
Xavier
X
Serono Pharmaceutical Research Institute, 14, chemin des Aulx, 1228 Plan-Les-Ouates, Geneva, Switzerland.
Huguenin-Reggiani
Martine
M
Hooft van Huijsduijnen
Rob
R
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
D019151:Peptide Library; D010748:Phosphopeptides; D011972:Receptor, Insulin; D017027:Protein Tyrosine Phosphatases