Phospho‐tyrosine dependent protein–protein interaction network

Abstract

Post‐translational protein modifications, such as tyrosine phosphorylation, regulate protein–protein interactions ( PPI s) critical for signal processing and cellular phenotypes. We extended an established yeast two‐hybrid system employing human protein kinases for the analyses of phospho‐tyrosine ( pY )‐dependent PPI s in a direct experimental, large‐scale approach. We identified 292 mostly novel pY ‐dependent PPI s which showed high specificity with respect to kinases and interacting proteins and validated a large fraction in co‐immunoprecipitation experiments from mammalian cells. About one‐sixth of the interactions are mediated by known linear sequence binding motifs while the majority of pY ‐ PPI s are mediated by other linear epitopes or governed by alternative recognition modes. Network analysis revealed that pY ‐mediated recognition events are tied to a highly connected protein module dedicated to signaling and cell growth pathways related to cancer. Using binding assays, protein complementation and phenotypic readouts to characterize the pY ‐dependent interactions of TSPAN 2 (tetraspanin 2) and GRB 2 or PIK 3R3 (p55γ), we exemplarily provide evidence that the two pY ‐dependent PPI s dictate cellular cancer phenotypes. image A modified yeast two‐hybrid approach employed on a large scale generates a network of 292 human phospho‐tyrosine ( pY )‐dependent protein–protein interactions. Conditional interactions are validated, and pY ‐dependent interaction specificity and network features are assessed. A pY ‐dependent protein interaction data set is generated using a modified yeast two‐hybrid approach. Network analyses assess the extent of known linear motif‐based pY recognition, pointing toward the importance of context for interaction specificity, and reveal a highly connected pY ‐recognition module in the human proteome. A large fraction of PPI s is validated by co‐immunoprecipitation with good success rate. pY ‐dependent TSPAN 2 interactions are related to cancer phenotypes.