Product(s) used in this publication: PepStar™ Peptide Microarrays
Cellular organization and response to internal and external stimuli are mediated by an intricate web of protein interactions. Some of these interactions are regulated by covalent posttranslational modifications such as phosphorylation and acetylation. These modifications can change the chemical nature of the interaction interfaces and modulate the binding affinity of the interacting partners. In signal transduction, the most frequent modification is reversible phosphorylation of tyrosine, serine or threonine residues. Protein phosphorylation may modulate the activity of enzymes by modifying their conformation, or regulate the formation of complexes by creating docking sites to recruit downstream effectors. Families of modular domains, such as SH2, PTB, and 14-3-3, act as "readers" of the modification event. Specificity between closely related domains of the same family is mediated by the chemical properties of the domain binding surface that, aside from offering a hydrophilic pocket for the phosphorylated residue, shows preference for specific sequences. Although the protein structure and the cell context are also important to ensure specificity, the amino acid sequence flanking the phosphorylation site defines the accuracy of the recognition process, and it is therefore essential to define the binding specificity of phosphopeptide binding domains in order to understand and to infer the interaction web mediated by phosphopeptides. Methods commonly used to discover new interactions (such as yeast two hybrid and phage display) are not suited to study interactions with phosphorylated proteins. On the other hand, peptide arrays are a powerful approach to precisely identify the binding preference of phosphopeptide recognition domains. Here we describe a detailed protocol to assemble arrays of hundreds to thousands phospho-peptides and to screen them with any modular domain of interest.
14-3-3; Binding domains; Interaction networks; Phosphopeptide; SH2; Spot synthesis