Quantitative phosphoproteomic analysis of IL-33-mediated signaling.

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IL-33 exerts its effects through a heterodimeric receptor complex resulting in the production and release of proinflammatory cytokines. A detailed understanding of the signaling pathways activated by IL-33 is still unclear. To gain insights into the IL-33-mediated signaling mechanisms, we carried out a SILAC-based global quantitative phosphoproteomic analysis that resulted in the identification of 7191 phosphorylation sites derived from 2746 proteins. We observed alterations in the level of phosphorylation in 1050 sites corresponding to 672 proteins upon IL-33 stimulation. We report, for the first time, phosphorylation of multiple protein kinases, including mitogen-activated protein kinase activated protein kinase 2 (Mapkapk2), receptor (TNFRSF) interacting serine-threonine kinase 1 (Ripk1), and NAD kinase (Nadk) that are induced by IL-33. In addition, we observed IL-33-induced phosphorylation of several protein phosphatases including protein tyrosine phosphatase, nonreceptor-type 12 (Ptpn12), and inositol polyphosphate-5-phosphatase D (Inpp5d), which have not been reported previously. Network analysis revealed an enrichment of actin binding and cytoskeleton reorganization that could be important in macrophage activation induced by IL-33. Our study is the first quantitative analysis of IL-33-regulated phosphoproteome. Our findings significantly expand the understanding of IL-33-mediated signaling events and have the potential to provide novel therapeutic targets pertaining to immune-related diseases such as asthma where dysregulation of IL-33 is observed. All MS data have been deposited in the ProteomeXchange with identifier PXD000984 (http://proteomecentral.proteomexchange.org/dataset/PXD000984).", "claims": [{"id": "claim-method", "statement": "To gain insights into the IL-33 mediated signaling mechanisms, we carried out a SILAC-based global quantitative phosphoproteomic analysis that resulted in the identification of 7,191 phosphorylation sites derived from 2,746 proteins.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:25367039", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "To gain insights into the IL-33 mediated signaling mechanisms, we carried out a SILAC-based global quantitative phosphoproteomic analysis that resulted in the identification of 7,191 phosphorylation sites derived from 2,746 proteins."}], "state": "complete"}, {"id": "claim-data-access", "statement": "The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium [30] (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD000984.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:25367039", "locator": "sections[10] \"2.8 Data availability\" paragraph 2", "quote": "The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium [30] (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD000984."}], "state": "complete"}, {"id": "claim-target-result", "statement": "We observed alterations in the level of phosphorylation in 1,050 sites corresponding to 672 proteins upon IL-33 stimulation.", "kind": "qualitative", "target": null, "evidence": [{"asset": "source:pmid:25367039", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "We observed alterations in the level of phosphorylation in 1,050 sites corresponding to 672 proteins upon IL-33 stimulation."}], "state": "complete"}], "gaps": []}, "experiment": {"state": "complete", "objective": "We observed alterations in the level of phosphorylation in 1,050 sites corresponding to 672 proteins upon IL-33 stimulation.", "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:25367039"], "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": "complete", "blocked_on": null, "description": "The GEO series accession resolves and its sample accessions are listed. 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