Investigation of Proteomic and Phosphoproteomic Responses to Signaling Network Perturbations Reveals Functional Pathway Organizations in Yeast

Abstract

SUMMARY Governance of protein phosphorylation by kinases and phosphatases constitutes an essential regulatory network in eukaryotic cells. Network dysregulation leads to severe consequences and is often a key factor in disease pathogenesis. Previous studies revealed multiple roles for protein phosphorylation and pathway structures in cellular functions from different perspectives. We seek to understand the roles of kinases and phosphatases from a protein homeostasis point of view. Using a streamlined tandem mass tag (SL-TMT) strategy, we systematically measure proteomic and phosphoproteomic responses to perturbations of phosphorylation signaling networks in yeast deletion strains. Our results emphasize the requirement for protein normalization for more complete interpretation of phosphorylation data. Functional relationships between kinases and phosphatases were characterized at both proteome and phosphoproteome levels in three ways: (1) Gene Ontology enrichment analysis, (2) Δgene-Δgene correlation networks, and (3) molecule covariance networks. This resource illuminates kinase and phosphatase functions and pathway organizations.

Post-translational modification analysis of Saccharomyces cerevisiae histone methylation enzymes reveals phosphorylation sites of regulatory potential

“…4 C ). Notably, two phosphorylation sites previously reported on Set1p ( 35 ), at threonine 1001 and threonine 1006, are situated at its interface with Swd1p ( Fig. 4 C , inset ).…”

“…A comprehensive catalog of modification types and sites on an enzyme is requisite to understanding its post-translational regulation. Although several enrichment-based proteomic studies have identified many PTMs on histone MTases and DMases in S. cerevisiae ( 33 , 34 , 35 , 36 , 37 , 38 ), there has been no systematic characterization of any specific methylation enzymes carried out to date. To this end, we homologously overexpressed and purified all yeast histone MTases and DMases and performed a combinatorial mass spectrometric pipeline ( 39 ) to comprehensively identify high-confidence PTM sites of various types.…”

H3 relaxin protects against calcium oxalate crystal‐induced renal inflammatory pyroptosis

“…Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Gene and Genome (KEGG) pathway enrichment analyses were further performed based on Fisher's exact test to map the DEPs 38,39 . The protein‐protein interaction (PPI) networks were predicted using STRING (https://string-db.org) and Cytoscape software 40 …”

“…38,39 The protein-protein interaction (PPI) networks were predicted using STRING (https://string-db.org) and Cytoscape software. 40…”

Maximized quantitative phosphoproteomics allows high confidence dissection of the DNA damage signaling network

“…Phosphoproteomics, the systematic and unbiased mapping of phosphorylation events, is achieved mainly using mass spectrometry (MS)-based approaches. Both instrumentation and bioinformatic tools applied for phosphopeptide identification have been continuously evolving 15 – 17 , culminating in large phosphoproteomic datasets in recent years 18 – 24 . In addition to in depth coverage of the phosphoproteome, comprehensive mapping of kinase-mediated signaling also requires quantitative analysis of each phosphopeptide or phosphorylation site to monitor its abundance in conditions of active kinase compared to conditions in which the kinase of interest is chemically and/or genetically ablated 11 , 25 , 26 .…”