Dataset Information

Time-resolved Phosphoproteomic Analysis Elucidates Hepatic 11,12-Epoxyeicosatrienoic Acid Signaling Pathways

ABSTRACT: Epoxyeicosatrienoic acids (EETs) are potent lipid mediators with well-established effects in vascular tissues. Recent studies indicated an emerging role of these eicosanoids in metabolic diseases and the EET signaling pathway was shown to be involved in hepatic insulin sensitivity. However, compared to vascular tissues, there is only limited knowledge about the underlying signaling pathways in the liver. Therefore, we employed an LC-MS/MS-based time-resolved phosphoproteomics approach to characterize 11,12-EET-mediated signaling events in the liver cell line Hepa 1-6. 11,12-EET treatment resulted in the time-dependent regulation of phosphopeptides involved in processes as yet unknown to be affected by EETs, including RNA processing, splicing and translation regulation. Pathway analysis combined with western blot-based validation revealed enhanced AKT/mTOR/p70S6K signaling as demonstrated by increased acute phosphorylation of AKT (Ser473) and p70S6K (Thr389). In addition, 11,12-EET treatment led to differential regulation of phosphopeptides including important mediators of the DNA damage response and we observed a prolonged induction of the etoposide-induced DNA damage marker γH2AX in response to 11,12-EET. In summary, our findings extend current knowledge of 11,12-EET signaling events and emphasize the importance of the AKT/mTOR/p70S6K pathway in hepatic 11,12-EET signaling. Based on the results presented in this study, we furthermore propose a novel role of EET signaling in the regulation of the DNA damage response.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Hepatocyte, Liver

SUBMITTER: Marco Rahm

LAB HEAD: Stefanie M. Hauck

PROVIDER: PXD015245 | Pride | 2019-11-06

REPOSITORIES: Pride

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Publications

Time-resolved phosphoproteomic analysis elucidates hepatic 11,12-Epoxyeicosatrienoic acid signaling pathways.

Rahm Marco M Merl-Pham Juliane J Adamski Jerzy J Hauck Stefanie M SM

Prostaglandins & other lipid mediators 20191024

Epoxyeicosatrienoic acids (EETs) are potent lipid mediators with well-established effects in vascular tissues. Recent studies indicated an emerging role of these eicosanoids in metabolic diseases and the EET signaling pathway was shown to be involved in hepatic insulin sensitivity. However, compared to vascular tissues, there is only limited knowledge about the underlying signaling pathways in the liver. Therefore, we employed an LC-MS/MS-based time-resolved phosphoproteomics approach to charact ...[more]

PMID: 31669255

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Project description:Understanding the mechanism of resistance in platinum-based regimens for the treatment of high-grade serous ovarian cancer (HGSOC) is important for identifying new therapeutic targets to improve the clinical outcome of ovarian cancer patients. Mass spectrometry-based proteomic strategy was applied to spheroidal cisplatin sensitive and resistant HGSOC generated cell lines in the absence and presence of cisplatin drug. A complete expressed HGSOC proteome and phosphoproteome was characterized in cisplatin sensitive and resistant HGSOC cell lines providing insight into the mechanism of resistance development. PCA analysis showed that phosphorylation of a few proteins provides better classification than the whole proteome of the cellular subtypes. Specifically, a distinctive phosphoproteomic signature between cisplatin sensitive and resistant cell lines in the absence of drug was observed. This same phosphoproteomic signature was observed in our cisplatin sensitive cell line in the absence and presence of drug, indicating a vital role for phosphorylation of proteins in resistance development to cisplatin. The most phosphorylated protein was sequestosome (p62/SQSTM1). Differential expressions of apoptosis by the prognostic factor ratio of Bcl-2/Bax and autophagy, known to be regulated by p62/SQSTM1, was validated in the proteome data and by western blot analysis. A significant increase in apoptosis in the presence of cisplatin was observed in only the sensitive cell line while autophagy revealed increased expression in the resistant relative to sensitive cell line. Furthermore, site specific phosphorylation on 20 modified residues of sequestosome was characterized. Elevated expression of phosphorylation of sequestosome in resistant HGSOC cell lines was validated with western blot analysis. Here, we propose phosphorylation of sequestosome to be a marker and key in cisplatin resistance development in HGOSC ovarian cancers by shuttling ubiquitinated proteins to the autophagy pathway and influencing down-regulation of apoptosis.

Dataset Information

Time-resolved Phosphoproteomic Analysis Elucidates Hepatic 11,12-Epoxyeicosatrienoic Acid Signaling Pathways

Publications

Time-resolved phosphoproteomic analysis elucidates hepatic 11,12-Epoxyeicosatrienoic acid signaling pathways.

Similar Datasets

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