title: Characterization of a putative phosphorylation switch: adaptation of SPOT synthesis to analyze PDZ domain regulation mechanisms.
abstract: Transient macromolecular complexes are often formed by protein-protein interaction domains (e.g., PDZ, SH2, SH3, WW), which are often regulated (positively or negatively) by phosphorylation. To address the in vitro analysis of PDZ domain regulation by such phosphorylation, we improved the inverted peptide method. This method is based on standard SPOT synthesis, followed by inversion of the peptide under acidic conditions to generate the free C termini necessary for PDZ domain ligand recognition. The benefit of the newly introduced acidic conditions is the preservation of the incorporated phosphate group during peptide synthesis. Furthermore, the improved method is more robust and shows an increased signal-to-noise ratio. As representative examples, we used the AF6, ERBIN, and SNA1 (alpha-1-syntrophin) PDZ domains to analyze the influence of ligand-position-dependent phosphorylation. We could clearly demonstrate severe down-regulation by phosphorylation of the PDZ ligand position -2 (<50 %) and slightly less at position -1 ( approximately 50 %). These results are specific and reproducible for all three PDZ domains. Finally, we confirmed the influence of negative regulation by using the protein kinase BCR as the AF6 PDZ domain ligand. For the first time, this approach allows the SPOT synthesis technique to be used to screen large libraries of phosphorylated peptides in vitro. This should ultimately help in the identification of phosphorylation-dependent regulation mechanisms in vivo.
authors.affiliation: Institut für Medizinische Immunologie, Charité-Universitätsmedizin Berlin, Hessische Strasse 3-4, 10115 Berlin, Germany.
authors.forename: Prisca
authors.initials: P
authors.lastname: Boisguerin
authors.forename: Bernhard
authors.initials: B
authors.lastname: Ay
authors.forename: Gerald
authors.initials: G
authors.lastname: Radziwill
authors.forename: Rafael D
authors.initials: RD
authors.lastname: Fritz
authors.forename: Karin
authors.initials: K
authors.lastname: Moelling
authors.forename: Rudolf
authors.initials: R
authors.lastname: Volkmer
mesh_terms: D005786:Gene Expression Regulation; D046228:Microarray Analysis / Q000379:methods*; D015394:Molecular Structure; D019151:Peptide Library; D010455:Peptides / Q000138:chemical synthesis*; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary
chemical_list: D019151:Peptide Library; D010455:Peptides