Defining Human Tyrosine Kinase Phosphorylation Networks Using Yeast as an In Vivo Model Substrate

Abstract

Systematic assessment of tyrosine kinase-substrate relationships is fundamental to a better understanding of cellular signaling and its profound alterations in human diseases such as cancer. In human cells, complex signaling networks, feedback loops, conditional activity and intra-kinase redundancy combine to confound systematic attempts to address this topic. We leveraged the model organism S. cerevisiae to individually express human non-receptor tyrosine kinases (NRTKs) and exploited the full yeast proteome as an in vivo model substrate. For 16 NRTKs, we recorded 3,279 kinase-substrate relationships involving 1,351 yeast pY-sites. From this data we generated a set of new linear kinase motifs and assigned ~1,300 known human pY-sites to specific NRTKs. Furthermore, experimentally defined pY-sites for each individual kinase were shown to cluster within the yeast interactome network irrespective of linear motif information. We therefore applied a network inference approach to predict kinase-substrate relationships for more than 3,500 human proteins, marking a substantial step forward in our understanding of kinase biology.. Corwin et al., revised3 7/25/2017 3

multiSLIDE is a web server for exploring connected elements of biological pathways in multi-omics data

“…When visualizing omics modalities where the resident molecules are mutually exclusive, e.g., proteome and metabolome, multiSLIDE allows users to input the inter-omics relationship data for linked visualization. Further, externally curated biological networks, such as transcription factor (TF) regulatory networks 25 – 30 and kinase-substrate networks 31 – 33 , can be integrated and visualized through linkers in multiSLIDE.…”

“…Second, we use multiSLIDE to simultaneously visualize the kinase proteins from the proteomics data and the substrate sites from the phosphoproteome data, with lines connecting known kinase-substrate pairs. In this instance, we uploaded the “custom” network from externally curated 31 – 33 kinase-substrate map to the tool as a user (see Methods for details).…”

“…The custom “Upload” option is used to select the molecules here. Kinase-substrate interactions were curated from PhosphoSitePlus 31 , PhosphoNetworks 32 , and a predictive network inference approach 33 to build a kinase-substrate map, which was uploaded into multiSLIDE using the upload network feature. The connecting lines show these curated relationships, with the highlighted (brown) lines connecting cyclin-dependent kinases CDK1 and CDK2 with known substrates.…”

Proteome-wide Prediction of Lysine Methylation Reveals Novel Histone Marks and Outlines the Methyllysine Proteome

“…The size of the predicted methyl-Lys proteome is comparable to that of the annotated phospho-Tyr proteome. This is not surprising given the comparable number of Lys methyltransferases (KMTs) and Tyr kinases (Biggar and Li, 2015;Corwin et al, 2017;Paul and Mukhopadhyay, 2004). However, the functions of the methyl-Lys proteome and phospho-Tyr proteome are markedly different.…”

Protein Phosphorylation Dynamics: Unexplored Because of Current Methodological Limitations

“…Because the current curated lists of P‐sites based on experimental procedures have a poor match with the computationally searched optimal motifs, we might question the degrees of relaxation in the 3D conformational changes that might drastically modify the affinity parameters for the kinases. Consequently, researchers have tried to pinpoint protein substrates for foreign transfected kinases in yeast systems, [ 39,40 ] and have computationally searched for potential phosphorylatable domains instead of focusing on linear motifs. [ 41–43 ] Finally, a feedback resource (ProteomeXchange) that is constantly updated by experimenters is publicly available.…”