More than 200 proteins copurify with spliceosomes, the compositionally dynamic RNPs catalyzing pre-mRNA splicing. To better understand protein - protein interactions governing splicing, we systematically investigated interactions between human spliceosomal proteins. A comprehensive Y2H interaction matrix screen generated a protein interaction map comprising 632 interactions between 196 proteins. Among these, 242 interactions were found between spliceosomal core proteins and largely validated by coimmunoprecipitation. To reveal dynamic changes in protein interactions, we integrated spliceosomal complex purification information with our interaction data and performed link clustering. These data, together with interaction competition experiments, suggest that during step 1 of splicing, hPRP8 interactions with SF3b proteins are replaced by hSLU7, positioning this second step factor close to the active site, and that the DEAH-box helicases hPRP2 and hPRP16 cooperate through ordered interactions with GPKOW. Our data provide extensive information about the spliceosomal protein interaction network and its dynamics.

A 'writer-reader-eraser' post-translational modification regulatory system consisting of a large number of methyltransferases 8,9 , methyl-recognition domain-containing proteins 10 and putative demethylases 11 are expressed in different subcellular locations in humans, an indication that protein methylation is involved in processes other than epigenetic regulation.We prepared 82 Y2H bait strains spanning human R-methyltransferases (PRMT1-PRMT8) 8 , 16 SET domaincontaining K-methyltransferases (PKMTs) 9 , 9 members of the JMJD domain-containing protein family of protein demethylases and AOF2 (LSD1) 11 (Supplementary Table 1). In our current matrix screening protocol 4,12 , we perform four replicates, testing every set of baits individually against each of the ~13,000 prey contained in the matrix. Interacting prey are identified by their position in the matrix. To increase the sensitivity of the approach while also reducing the workload, we used a pooled strategy to test each protein pair substantially more than four times. Baits were pooled with all prey strains and then assayed for interaction in more than 100,000 separate spots ( Fig. 1a and Supplementary Fig. 1). Using Y2H-seq, we obtained 4-10 times the number of positive colonies obtained with the matrix approach. To reveal the prey identities, we collected all colonies and performed a 36-base parallel sequencing run. More than 20 million reads mapped perfectly to human RefSeq coding sequences (open reading frames, ORFs), corresponding to more than 500,000 unique 36-base reads (Supplementary Table 2). To rank the potentially interacting proteins for subsequent interaction retesting, we calculated a 'SeqScore' that incorporates the number of total mappings and the number of unique reads matching the ORF ( Supplementary Fig. 2). Notably, >99.7% of the RefSeq mappings matched to the 400 top-ranked genes, thus allowing the identification of potentially interacting ORFs with an extremely high signal-tobackground ratio (Supplementary Table 2).We performed four biological replicates and demonstrated in statistical pairwise comparisons that they result in very similar ranked prey orders (Supplementary Fig. 3). Top-ranked prey in at least two replicate screens were retested against all baits in a pairwise manner (Supplementary Fig. 4) and yielded 463 protein interactions (Supplementary Table 3). The success rate of the retest-that is, the probability that the prey is interactingdecreased with decreasing SeqScore (Fig. 1b).We also performed a matrix screen in quadruplicate with a subset of the protein methyltransferase (PMT) and protein demethylase (PDeM) baits for direct method comparison. With the matrix approach, we found 151 interactions (Supplementary 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 in...

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.

TCR stimulation leads to an increase in cellular adhesion among other outcomes. The adhesion and degranulation promoting adapter protein (ADAP) is known to be rapidly phosphorylated after T cell stimulation and relays the TCR signal to adhesion molecules of the integrin family. While three tyrosine phosphorylation sites have been characterized biochemically, the binding capabilities and associated functions of several other potential phosphotyrosine motifs remain unclear. Here, we utilize in vitro phosphorylation and mass spectrometry to map novel phosphotyrosine sites in the C-terminal part of human ADAP (486–783). Individual tyrosines were then mutated to phenylalanine and their relevance for cellular adhesion and migration was tested experimentally. Functionally important tyrosine residues include two sites within the folded hSH3 domains of ADAP and two at the C-terminus. Furthermore, using a peptide pulldown approach in combination with stable isotope labeling in cell culture (SILAC) we identified SLP-76, PLCγ, PIK3R1, Nck, CRK, Gads, and RasGAP as phospho-dependent binding partners of a central YDDV motif of ADAP. The phosphorylation-dependent interaction between ADAP and Nck was confirmed by yeast two-hybrid analysis, immunoprecipitation and binary pulldown experiments, indicating that ADAP directly links integrins to modulators of the cytoskeleton independent of SLP-76.