title: SH2 Binding Site Protection Assay: A Method for Identification of SH2 Domain Interaction Partners by Exploiting SH2 Mediated Phosphosite Protection.
abstract: Over the last two decades there has been a significant effort in the field to characterize the phosphosite binding specificities of SH2 domains with the goal of deciphering the pY signaling code. Although high throughput studies in various formats using most SH2 domains have collectively provided a rich resource of in vitro SH2-pTyr site specificity maps, this data can only be used approximate what is happening in the cell where protein concentrations and localization are not homogenous, as they are for in vitro experiments. Here we describe an in vivo approach, SH2 site protection assay, which can capture the pTyr binding specificity of SH2 domains in the cell. The basis of this approach is SH2-pY site protection, the ability of SH2 domains to prevent the PTP-dependent dephosphorylation of their pY site binding partners. We overexpress a tracer SH2 domain in cells and quantify the change in abundance of tyrosine phosphorylated sites using MS. Since the method is performed in vivo, it has the advantage of identifying SH2-pY interactions as they occur within in the cell.
authors.affiliation: Raymond and Beverly Sackler Laboratory of Genetics and Molecular Medicine, Department of Genetics and Genome Sciences, University of Connecticut School of Medicine, 400 Farmington Avenue, Farmington, CT, 06032, USA. jjadwin@uchc.edu.
authors.forename: Joshua A
authors.initials: JA
authors.lastname: Jadwin
mesh_terms: D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D015151:Immunoblotting / Q000379:methods; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism / Q000494:pharmacology; D013058:Mass Spectrometry / Q000379:methods; D010766:Phosphorylation; D019000:Phosphotyrosine; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping / Q000379:methods*; D040901:Proteomics / Q000379:methods; D018909:src Homology Domains
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D019000:Phosphotyrosine
keywords: MS/MS; SH2 domain; iTRAQ; pTyr protection