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Post‐translational protein modifications, such as tyrosine phosphorylation, regulate protein–protein interactions (PPIs) 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 PPIs in a direct experimental, large‐scale approach. We identified 292 mostly novel pY‐dependent PPIs 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‐PPIs 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 TSPAN2 (tetraspanin 2) and GRB2 or PIK3R3 (p55γ), we exemplarily provide evidence that the two pY‐dependent PPIs dictate cellular cancer phenotypes. Synopsis 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 PPIs is validated by co‐immunoprecipitation with good success rate. pY‐dependent TSPAN2 interactions are related to cancer phenotypes. Graphical Abstract 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 high-sensitivity, high-quality yeast two-hybrid screen using short-read sequencing as its readout is used to map the interactome of human methyltransferases. To accelerate high-density interactome mapping, we developed a yeast two-hybrid interaction screening approach involving short-read second-generation sequencing (Y2H-seq) with improved sensitivity and a quantitative scoring readout allowing rapid interaction validation. We applied Y2H-seq to investigate enzymes involved in protein methylation, a largely unexplored post-translational modification. The reported network of 523 interactions involving 22 methyltransferases or demethylases is comprehensively annotated and validated through coimmunoprecipitation experiments and defines previously undiscovered cellular roles of nonhistone protein methylation.

The ability of the glucocorticoid receptor (GR) to regulate the transcriptional output of genes relies on its interactions with transcriptional coregulators. However, which coregulators are required for GR-dependent activation is context-dependent and can be influenced by the sequence of the DNA bound by GR and by the nature of the GR isoform responsible for the regulation of a gene. Here, we screened for GR-interacting proteins for which the interaction signal differed between two GR isoforms GRα and GRγ. These isoforms diverge by a single amino acid insertion in a domain, the lever arm, which adopts DNA sequence-specific conformations. We identify Basic Leucine Zipper ATF-Like Transcription Factor 3 (BATF3), an AP-1 family transcription factor, as a GR coregulator whose interaction with GR is modulated by the lever arm. Further, a combination of experiments uncovered that BATF3 acts as a gene-specific coactivator of GR whose coactivator potency is influenced by the sequence of the GR binding site. Together, our findings suggest that GR isoform and the sequence of GR binding site influence the interaction of GR with BATF3, which might direct the assembly of gene-specific regulatory complexes to fine-tune the expression of individual GR target genes.