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Phosphoproteome analysis reveals the importance of Greatwall-endosulfine pathway to regulate cell-cycle progression upon the rapamycin-induced autophagy

ABSTRACT: Protein phosphorylation plays an important role in the process of autophagy. Autophagy is associated with cell cycle, which is controlled by the balance between the cyclin-dependent kinases and phosphatases activities. However, up to now, the signal transduction pathways that underlied rapamycin-induced autophagy and cell cycle have remained elusive. Here, we used stable isotope labeling of amino acids in cell culture and high-throughput quantitative mass spectrometry to perform a global proteome and phosphoproteome analysis of rapamycin-induced autophagy in vitro. By monitoring proteome and phosphoproteome alterations with rapamycin treatment, we detected downregulation of mTOR signaling pathway and confirmed the occurrence of autophagy. Additionally, spliceosome and nuclear pore complexes were enriched to be regulated, which further validated autophagy as a stress response itself. More importantly, we discovered that mTORC1 could control cell cycle progression though Greatwall-endosulfine-PP2A pathway in HeLa cells. Firstly, we found the elevated profile of cell cycle-related substrates and determined the enrichment of CDK1, MAPK1 and MAPK3 kinases, which implied the activation of these kinases. Secondly, Pathway interrogation using kinase inhibitor treatment confirmed the effect of mTOR inhibition on CDK1 activity and cell cycle progression. Thirdly, we discovered that Greatwall-endosulfine complex was required to mediate mTOR-mediated cell-cycle progression. In conclusion, we presented a high-confidence map of phosphoproteomic of autophagy and unveiled the Greatwall-endosulfine pathway to regulate cell-cycle progression in the rapamycin-induced autophagy.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hela Cell

SUBMITTER: luqi jin

LAB HEAD: Shiping Ding

PROVIDER: PXD014737 | Pride | 2021-04-06

REPOSITORIES: Pride

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Phosphoproteome Profiling Revealed the Importance of mTOR Inhibition on CDK1 Activation to Further Regulate Cell Cycle Progression.

Jin Luqi L Chen Yu Y Yan Chunlan C Guo Xiaoyuan X Jiang Tingting T Guli Ayiding A Song Xinghui X Wan Qun Q Shu Qiang Q Ding Shiping S

Journal of proteome research 20210402 5

The mammalian target of rapamycin (mTOR) functions as a critical regulator of cell cycle progression. However, the underlying mechanism by which mTOR regulates cell cycle progression remains elusive. In this study, we used stable isotope labeling of amino acids in cell culture with a two-step strategy for phosphopeptide enrichment and high-throughput quantitative mass spectrometry to perform a global phosphoproteome analysis of mTOR inhibition by rapamycin. By monitoring the phosphoproteome alte ...[more]

PMID: 33797919

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