Quantitative Proteomics And Phosphoproteomics Identify The Role Of Rictor In The Regulation Of Cell Adhesion

Background and Objective:The mammalian target of rapamycin complex2?mTORC2?plays critical roles in various biological processes,such as GTPase activity regulation,actin cytoskeleton reorganization,formation of stress-fibers or F-actin,and the phosphorylation of a series of proteins.Rictor,rapamycin insensitive companion of mTOR,is a subunit of mTORC2 and has been reported to regulate the growth and survival of cells after stimulated by the extracellular signals.To better understand the functions of mTORC2 and the underlying molecular mechanisms,we established a stable cell line depleted of Rictor,a specific component in mTORC2,and investigated the quantitative changes of the cellular proteome and phosphoproteome.Methods:First of all,293T cells were infected with shRictor lentivirus so that the virus could be amplified.The amplified virus was transfected into human renal cell carcinoma?ACHN?and a stable cell line depleted of Rictor was screened out.Then,cellular proteins were extracted by 8M urea.The proteins were reducted and alkylated and digested by the mean of filter aided proteome preparation?FASP?.Most of the digested peptides were used for enriching phosphorylation sites using TiO₂ magnetic beads.For the quantification of the cellular proteome,TMT labeling was carried out for the remaining peptides.The labeled peptides were mixed with equal amount and fractionated by high PH reverse chromatography columns.The TMT labeled peptides?8 fractions?and phosphorylation enriched peptides were detected with LC-MS/MS.Finally,the Proteome Discoverer?PD?software was used to analyze and quantify the mass spec raw datas,then the differentially expressed proteins were screened out.Results:For TMT labeled samples,5 096 proteins were identified from eight fractions,and 4864 of these proteins were quantified.2 175 proteins were observed with a P value less than 0.05 and 151 proteins were detected with more than 1.5 fold expression changes,included 101 down-regulated proteins and 50 up-regulated proteins in Rictor knockdown cells.In addition,98 phosphorylation sites from 69proteins were observed to be down-regulated in the Rictor knockdown cells,and 13phosphorylation sites from 8 proteins were up-regulated.Conclusion:The combination of quantitative proteomics and phosphoproteomics analyses revealed that Rictor/mTORC2 was involved in various cellular processes.Intriguingly,both the proteome and phosphoproteome regulated by Rictor/mTORC2were significantly involved with cell adhesion and screened out multiple associated differential proteins,e.g.JUP,CD9,LAMB2 etc.In addition,the phosphorylation of tight junction protein 2?TJP2?,an important component of the tight junction,was reduced upon Rictor knockdown(Ser⁹⁷⁹,Ser⁹⁸⁶,Tyr¹¹⁵).Further investigation revealed that Rictor/mTORC2 regulated the function of tight junction through TJP2.Taken together,this study comprehensively maps the proteome and phosphoproteome regulated by Rictor/mTORC2,and reveals its role in promoting renal cancer cell migration and invasion through modulating cell adhesion.