Dataset Information

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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Publications

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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Project description:Autophagy has been implicated as a host defense mechanism against intracellular pathogens. However, certain intracellular pathogens such as Leishmania can manipulate the host’s autophagy to promote their survival. Recently, our findings regarding the regulation of autophagy by Leishmania donovani indicate that this pathogen induces non-classical autophagy in infected macrophages, independent of mammalian target of rapamycin complex 1 regulation. This occurs in the background of enhanced mTOR activity, which suggests the fine-tuning of autophagy to optimally promote parasite survival and may involve the sequestration or modulation of specific autophagosome-associated proteins. To investigate how Leishmania potentially manipulates the composition of host-cell autophagosomes, we undertook a quantitative proteomic study of the human monocytic cell line THP-1 following infection with L. donovani. We used stable isotope labeling by amino acid in cell culture and liquid chromatography-tandem mass spectrometry to compare expression profiles between autophagosomes isolated from THP-1 cells infected with L. donovani or treated with known autophagy inducers. Select proteomics results were validated by immunoblotting. In this study, we showed that L. donovani modulates the composition of macrophage autophagosomes during infection when compared to autophagosomes induced by either rapamycin (selective autophagy) or starvation (non-selective autophagy). Among 1787 proteins detected in Leishmania-induced autophagosomes, 146 were significantly modulated compared to the proteome of rapamycin-induced autophagosomes while 57 were significantly modulated compared to starvation-induced autophagosomes. Strikingly, 23 Leishmania proteins were also detected in the proteome of Leishmania-induced autophagosomes. Together, our data provide the first comprehensive insight into the proteome dynamics of host autophagosomes in response to Leishmania infection and demonstrate the complex relations between the host and pathogen at the molecular level.

Project description:Although malignant gliomas frequently show aberrant activation of the mammalian target of rapamycin (mTOR), mTOR inhibitors have performed poorly in clinical trials. Besides regulating cell growth and translation, mTOR controls the initiation of autophagy. By recycling cellular components, autophagy can mobilize energy resources, and has thus been attributed cancer-promoting effects. Here, we asked whether the activation of autophagy represents an escape mechanism to pharmacological mTOR inhibition, and explored co-treatment with mTOR and autophagy inhibitors as a therapeutic strategy. Mimicking conditions of the glioma microenvironment, glioma cells were exposed to nutrient starvation and hypoxia. Following treatment with mTOR inhibitor torin2 or rapamycin, and autophagy inhibitors bafilomycin A1 or MRT68921, we analyzed autophagic activity, cell growth, viability and oxygen consumption. Changes in global proteome were quantified by mass spectrometry. In the context of hypoxia and starvation, autophagy was strongly induced in glioma cells, and further increased by mTOR inhibition. While torin2 enhanced glioma cell survival, co-treatment with torin2 and bafilomycin A1 failed to promote cell death. Importantly, treatment with bafilomycin A1 alone also protected glioma cells from cell death. Mechanistically, both compounds significantly reduced glioma growth and oxygen consumption. Mass spectrometry analysis showed that bafilomycin A1 induced broad changes in the cellular proteome. More specifically, proteins downregulated by bafilomycin A1 were associated with the mitochondrial respiratory chain and ATP synthesis. Taken together, our results show that activation of autophagy does not account for the cytoprotective effects of mTOR inhibition in our in vitro model of the glioma microenvironment. Our proteomic findings suggest that the pharmacological inhibition of autophagy induces extensive changes in the cellular proteome that can support glioma cell survival under nutrient-deplete and hypoxic conditions. These findings provide a novel perspective on the complex role of autophagy in gliomas and may have implications for the design of future trials.

Dataset Information

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

Publications

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

Similar Datasets

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