Study On Molecular Mechanism And Function Of Crosstalk Between Phosphorylation And Acylation Based On Proteomics Techniques

Post-translational modification of proteins is a fine regulation of protein structure and function,and is a key regulatory mechanism in the life activities of cells.More and more evidences show that there are crosstalks between post-translational modifications,and this mechanism can delicately regulate complex cell signal transduction networks.Phosphorylation is the most common and most widely studied post-translational modification in cell,but at present there is still a lack of global understanding of the crosstalk between phosphorylation and lysine acylation.This article uses the advantages of mass spectrometry-based proteomics technology,such as high throughput,high sensitivity,and high quantitative accuracy,to conduct in-depth discussions on crosstalk between the phosphorylation and histone acylation,and the crosstalk between phosphorylation and ubiquitination.First,the molecular mechanism of AMPK?Adenosine 5?-monophosphate-activated protein kinase?-mediated phosphorylation in DNA damage response and the function of its crosstalk with histone acetylation were investigated.AMPK-mediated phosphorylation is widely regarded as the core regulation of energy metabolism,but its role in tumor is still unclear.Studies have shown that AMPK can participate in the tumorigenesis by regulating the DNA damage response,and repair of DNA damage requires histone modification-mediated chromatin regulation.Based on this,we first used TALENs technology to establish a biological model of DNA damage in MEF?mouse embryo fibroblasts?cells under conditions of AMPK deficiency.Combining the quantitative phosphoproteomics technology and global histone modification analysis based on biological mass spectrometry,a platform for studying the interactive regulation behavior of phosphorylation and histone modification in the biological model was established.The result of protein phosphorylation modification revealed a series of substrate proteins regulated by AMPK in DNA damage.Subsequent bioinformatics analysis suggested that the regulatory network of AMPK in the DNA damage response is related to DNA damage repair,gene transcription,chromatin regulation,and histone modification.Further systematic analysis of histone modifications of this biological model reflects that AMPK can indirectly regulate histone acetylation through phosphorylated substrate proteins during DNA damage.This part of the research is the first to combine large-scale omics technology to place AMPK in DNA damage to investigate its regulatory function.The aspect also provides new ideas for exploring phosphorylation and histone acylation.Secondly,the phosphorylation-dependent SCF?SKP1-CUL1-F-box?E3 ubiquitin ligase-mediated degradation mechanism of ubiquitin was systematically studied.In the classical SCF recognition mechanism,the phosphorylation of the substrate is often a prerequisite for further ubiquitin degradation,so phosphorylation-dependent ubiquitin degradation is essentially a crosstalk between phosphorylation and ubiquitination.However,the previous SCF substrate discovery process is based on the known SCF substrate recognition pattern.This limitation has led to the fact that phosphorylation-dependent degradation substrates have not been identified to a large extent.We innovatively analyzed the dynamic changes of protein expression profile,phosphorylation profile and transcriptome during the degradation process of phosphorylation-dependent ubiquitination,combined with the existing public database information,to identify a variety of potential phosphorylation-dependent degrade substrate.Biochemical experiments confirmed that some of these substrates are novel degradation substrates that are potentially dependent on SCF phosphorylation.Bioinformatics analysis of the degradation-related phosphorylation sequences on these substrates revealed a variety of potential novel phosphorylation degradation determinants?phosphodegron?.We selected BAG3?Bcl-2-associated athanogene 3?protein,which is closely related to tumor development,and conducted in-depth verification of its degradation mechanism.Combined with molecular biology experiments such as interaction proteome analysis and co-immunoprecipitation of BAG3,we determined that FBXO22?F-box only protein 22?mediates its ubiquitination degradation by directly binding BAG3.Based on literature and phosphoproteomic results,as well as point mutation experiments and kinase inhibitor experiments,it was proved that the ubiquitin degradation of BAG3 regulated by FBXO22 depends on the phosphorylation of S377 and is regulated by ERK?Extracellular signal-regulated kinase?.Combining the analysis of the amino acid sequence near this site and the simulated structural biology analysis,we found that SCF^FBXO22 targeted the degradation substrate by identifying the conserved phosphorylated amino acid sequence"XXPpSPXPXX".Finally,in HeLa cells,we experimented with FBXO22 knockout or exogenous expression of the BAG3 S377A mutant,demonstrating that FBXO22's defective regulation of BAG3 phosphorylation-dependent ubiquitin degradation mechanisms can cause apoptosis and cause disturbances in the cell cycle process promote tumor growth.In conclusion,through this part of the study,we confirmed that the interactive regulation of substrate phosphorylation on ubiquitination is universal in SCF-mediated degradation of ubiquitin,and we also identified a broad phosphorylation-dependent SCF substrate.A new phosphodegron recognized by FBXO22 and a new ERK-FBXO22-BAG3 regulatory axis involved in tumorigenesis were also discovered.In summary,we use quantitative modification omics based on biological mass spectrometry to systematically study the crosstalk between phosphorylation and two types of protein acylation in two different biological models,and further explore biological effects of regulatory mechanisms in tumors,revealing the important role of crosstalk in life activities.This study not only hints at the universality and importance of the interactive regulation of post-translational modifications in organisms,but also provides a valuable reference strategy for other systematic research strategies of interactive regulation between modifications.