Bioinformatic Study Of Protein Phosphorylation In The Regulation Of Phase Separation

Phorein phosphorylation is catalyzed by protein kinases(PKs)through covalently attaching phosphate groups to specific amino acid residues in substrates.Protein phosphorylation is reversible,and dephosphorylation reaction is mediated by protein phosphatases(PPs).Numerous proteins containing phosphoprotein-binding domains(PPBDs)can recognize phosphorylation residues in specific short linear motifs to ensure the fidelity of phospho-signalling in vivo.Phase separation or liquid-liquid phase separation is a ubiquitous and crucial mechanism for spatiotemporal organization of various biochemical reactions,by creating membraneless organelles in eukaryotic cells.Many proteins are involved in the phase separation process and recent studies have shown that phosphorylation can dynamically regulate the phase separation process.How to collect,curate and analyze these data and extract useful information for further experiment research has become a big challenge in this field.Protein phospho-regulators composed of PKs,PPs and proteins containing PPBDs are key molecules in phosphorylation-dependent signalling networks and a better understanding of the protein phospho-regulators is fundermental for studying the phosphorylation.Through literature mining and database integration,we have collected 2,643 experimentally verified protein phospho-regulators.By integrating the existing classification information,we classified the protein phospho-regulators into 26 groups and 208 families.Together with HMM-and orthology-based identifications,we identified 197,348 phospho-regulators in 164 eukaryotic species.In particular,we provided rich annotations for the regulators and developed iEKPD which contains about 99.8 GB of data.We anticipate that iEKPD represents a more useful resource for further study of phospho-regulators.Protein phosphorylation frequently exert their functional effects through following recognition by proteins containing PPBDs and how to accurately predict the binding relationship is key to study the function of phosphorylation.Based on the classification information in iEKPD,we collected 4,458 known the binding relationships as a benchmark data set from the literature,significantly improved our previously developed algorithm of group-based prediction system(GPS),and implemented a deep learning plus transfer learning strategy for model training.Then we developed a novel online service named GPSPBS,which can hierarchically predict PPBD-specific binding sites of 122 individual PPBDs belonged to 16 families.Liquid-liquid phase separation(LLPS)is driven by multivalent interactions among macromolecules and underlies the formation of membraneless compartments in cells.According to recent studies,post-translational modifications of proteins,especially phosphorylation modification,can regulate phase separation by affecting multivalent interactions.The identification of proteins undergoing LLPS is the foundation of understanding the molecular mechanisms of LLPS.We first collected 9,285 experimentally identified LLPS-associated proteins from the literature.These proteins were classified into 40 distinct biomolecular condensates,and we computationally identified potential orthologs of these known proteins in other eukaryotes.In total,the data resource of LLPS(DrLLPS)contained 437,887 known and potential LLPS-associated proteins with rich annotations.Using the phosphorylation regulators and phosphorylation modification information of substrates in DrLLPS and combining i GPS and GPS-PBS tools,we reconfigured and analyzed the phosphorylation network in the phase separation process and predicted the important PKs,proteins containing PPBDs and their substrates which are in part consistent with previous studies.Taken together,through the identification of phospho-regulators,the identification of proteins undergoing LLPS,and the reconfiguration the phosphorylation network in phase separation process,we systematically studied the LLPS-associated proteins and their phosphorylation moditification.Our study provides useful resources for further analyzing the molecular mechanisms of phase separation and the regulation of phosphorylation during phase sepation process.