Quantitative phosphoproteomics reveals crosstalk between phosphorylation and O-GlcNAc in the DNA damage response pathway.

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The addition and removal of O-GlcNAc is catalyzed by the O-GlcNAc transferase (OGT) and O-GlcNAcase, respectively. One mechanism by which O-GlcNAc is thought to mediate proteins is by regulating phosphorylation. To provide insight into the pathways regulated by O-GlcNAc, we have utilized SILAC-based quantitative proteomics to carry out comparisons of site-specific phosphorylation in OGT wild-type and Null cells. Quantitation of the phosphoproteome demonstrated that of 5529 phosphoserine, phosphothreonine, and phosphotyrosine sites, 232 phosphosites were upregulated and 133 downregulated in the absence of O-GlcNAc. Collectively, these data suggest that deletion of OGT has a profound effect on the phosphorylation of cell cycle and DNA damage response proteins. Key events were confirmed by biochemical analyses and demonstrate an increase in the activating autophosphorylation event on ATM (Ser1987) and on ATM's downstream targets p53, H2AX, and Chk2. Together, these data support widespread changes in the phosphoproteome upon removal of O-GlcNAc, suggesting that O-GlcNAc regulates processes such as the cell cycle, genomic stability, and lysosomal biogenesis. All MS data have been deposited in the ProteomeXchange with identifier PXD001153 (http://proteomecentral.proteomexchange.org/dataset/PXD001153).", "claims": [{"id": "claim-target-result", "statement": "The paper used a Student’s unpaired t-test with a p-value below 0.05 to compare groups, and its source proteomics data were deposited in PRIDE under ProteomeXchange identifier PXD001153; the headline finding was that OGT deletion caused widespread phosphoproteomic changes, including increased ATM activation and phosphorylation of downstream DNA-damage targets.", "kind": "qualitative", "target": null, "evidence": [{"asset": "source:pmid:25263469", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "Quantitation of the phosphoproteome demonstrated that out of 5,529 phosphoserine, phosphothreonine and phosphotyrosine sites, 232 phosphosites were upregulated and 133 downregulated in the absence of O-GlcNAc."}, {"asset": "source:pmid:25263469", "locator": "sections[1] \"1. Introduction\" paragraph 5", "quote": "Here, we applied a stable isotope labeling of amino acids in cell culture (SILAC)-based strategy to study phosphoproteomic changes induced by the deletion of OGT in cells [17, 27]."}, {"asset": "source:pmid:25263469", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "Together, these data support widespread changes in the phosphoproteome upon removal of O-GlcNAc, suggesting that O-GlcNAc regulates processes such as the cell cycle, genomic stability, and lysosomal biogenesis."}], "state": "complete"}], "gaps": []}, "experiment": {"state": "partial", "objective": "The paper used a Student’s unpaired t-test with a p-value below 0.05 to compare groups, and its source proteomics data were deposited in PRIDE under ProteomeXchange identifier PXD001153; the headline finding was that OGT deletion caused widespread phosphoproteomic changes, including increased ATM activation and phosphorylation of downstream DNA-damage targets.", "steps": [{"id": "read_published_estimate", "order": 1, "name": "Read the published estimate from the source", "kind": "other", "state": "complete", "blocked_on": null, "description": "The claim, method and evidence span published with the source article.", "duration_hours": null, "parameters": {}, "inputs": ["source:pmid:25263469"], "outputs": ["the published estimate and its evidence"], "instruments": [], "reagents": []}, {"id": "obtain_source_data", "order": 2, "name": "Obtain the source data from the named repository", "kind": "data-preparation", "state": "blocked", "blocked_on": "data:accession_absent", "description": "The GEO series accession resolves and its sample accessions are listed. 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