Dataset Information

Large-scale phosphoproteomics reveals Shp-2 phosphatase-dependent regulators of Pdgf-receptor signaling

ABSTRACT: Despite its low cellular abundance, tyrosine phosphorylation (pTyr) regulates numerous cell signaling pathways in health and disease. We applied comprehensive phosphoproteomics to unravel differential regulators of receptor tyrosine kinase (RTK)–initiated signaling networks upon activation by Pdgf-ββ, Fgf-2, or Igf-1, and identified over 40,000 phosphorylation sites, including many phosphotyrosine sites without additional enrichment. The analysis revealed RTK-specific regulation of hundreds of pTyr sites on key signaling molecules. We found the tyrosine phosphatase Shp2 to be the master regulator of Pdgfr pTyr signaling. Application of a recently introduced allosteric Shp2 inhibitor revealed global regulation of the Pdgf-dependent tyrosine phosphoproteome, which significantly impaired cell migration. Additionally, we present a list of hundreds of Shp2-dependent targets and putative substrates including Rasa1 and Cortactin with increased tyrosine phosphorylation, and Gab1 and Erk1/2 with decreased tyrosine phosphorylation. Our study demonstrates that large-scale quantitative phosphoproteomics can precisely dissect tightly regulated kinase-phosphatase signaling networks.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture, Fibroblast

SUBMITTER: Tanveer Batth

LAB HEAD: Jesper Velgaard Olsen

PROVIDER: PXD005803 | Pride | 2018-03-07

REPOSITORIES: Pride

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Large-Scale Phosphoproteomics Reveals Shp-2 Phosphatase-Dependent Regulators of Pdgf Receptor Signaling.

Batth Tanveer S TS Papetti Moreno M Pfeiffer Anamarija A Tollenaere Maxim A X MAX Francavilla Chiara C Olsen Jesper V JV

Cell reports 20180301 10

Despite its low cellular abundance, phosphotyrosine (pTyr) regulates numerous cell signaling pathways in health and disease. We applied comprehensive phosphoproteomics to unravel differential regulators of receptor tyrosine kinase (RTK)-initiated signaling networks upon activation by Pdgf-ββ, Fgf-2, or Igf-1 and identified more than 40,000 phosphorylation sites, including many phosphotyrosine sites without additional enrichment. The analysis revealed RTK-specific regulation of hundreds of pTyr s ...[more]

PMID: 29514104

Similar Datasets

Project description:Dynamic tyrosine phosphorylation is fundamental to a myriad of cellular processes. However, the inherently low abundance of tyrosine phosphorylation in the proteome and the inefficient enrichment of phosphotyrosine(pTyr)-containing peptides has led to poor pTyr peptide identification and quantitation, critically hindering researchers’ ability to elucidate signaling pathways regulated by tyrosine phosphorylation in systems where cellular material is limited. The most popular approaches to wide-scale characterization of the tyrosine phosphoproteome use pTyr enrichment with pan-specific, anti-pTyr antibodies from a large amount of starting material. Methods that decrease the amount of starting material and increase the characterization depth of the tyrosine phosphoproteome while maintaining quantitative accuracy and precision would enable the discovery of tyrosine phosphorylation networks in rarer cell populations. To achieve these goals, a novel method leveraging the multiplexing capability of tandem mass tags (TMT) and the use of trigger channels – cells treated with the promiscuous tyrosine phosphatase inhibitor pervanadate (PV) – selectively increased the relative abundance of pTyr-containing peptides. After PV facilitated selective fragmentation of pTyr-containing peptides, TMT reporter ions delivered sensitive quantitation of each peptide for the experimental samples while the quantitation from PV trigger channels was ignored. This method yielded up to 6.3-fold boost in quantification depth of statistically significant data derived from contrived ratios, compared to TMT without trigger channels or intensity-based label-free (LF) quantitation while maintaining quantitative accuracy and precision, allowing quantitation of over 2300 unique pTyr peptides from only 1 mg of T cell receptor-stimulated Jurkat T cells. The trigger channel strategy can potentially be applied in analyses of other post-translational modifications where treatments that broadly boost the levels of those modifications across the proteome are available.

			Action	DRS
	MaxQuant-PDGF-SHP099-SILAC.zip	Other
	Phospho-MQ-output.zip	Other
	RTKPhospho.zip	Other
	SILAC-pSTY-SHP099-rawfiles.zip	Other
	tables.pdf	Pdf