Phosphoproteomic Profiling of In Vivo Signaling in Liver by the Mammalian Target of Rapamycin Complex 1 (mTORC1)

Texas A&M University, United States of America
PLoS ONE (Impact Factor: 3.23). 06/2011; 6(6):e21729. DOI: 10.1371/journal.pone.0021729
Source: PubMed


Our understanding of signal transduction networks in the physiological context of an organism remains limited, partly due to the technical challenge of identifying serine/threonine phosphorylated peptides from complex tissue samples. In the present study, we focused on signaling through the mammalian target of rapamycin (mTOR) complex 1 (mTORC1), which is at the center of a nutrient- and growth factor-responsive cell signaling network. Though studied extensively, the mechanisms involved in many mTORC1 biological functions remain poorly understood.
We developed a phosphoproteomic strategy to purify, enrich and identify phosphopeptides from rat liver homogenates. Using the anticancer drug rapamycin, the only known target of which is mTORC1, we characterized signaling in liver from rats in which the complex was maximally activated by refeeding following 48 hr of starvation. Using protein and peptide fractionation methods, TiO(2) affinity purification of phosphopeptides and mass spectrometry, we reproducibly identified and quantified over four thousand phosphopeptides. Along with 5 known rapamycin-sensitive phosphorylation events, we identified 62 new rapamycin-responsive candidate phosphorylation sites. Among these were PRAS40, gephyrin, and AMP kinase 2. We observed similar proportions of increased and reduced phosphorylation in response to rapamycin. Gene ontology analysis revealed over-representation of mTOR pathway components among rapamycin-sensitive phosphopeptide candidates.
In addition to identifying potential new mTORC1-mediated phosphorylation events, and providing information relevant to the biology of this signaling network, our experimental and analytical approaches indicate the feasibility of large-scale phosphoproteomic profiling of tissue samples to study physiological signaling events in vivo.

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    • "In a parallel set of analyses, total phosphopeptides were enriched with Titan-sphere Phos-TiO reagents as described [33]. The TiO method is necessary to ascertain changes in the phosphoproteome of peptides containing serine or threonine phosphorylation sites. "
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    • "In order to maximize and be able to detect p70S6 phosphorylation [37,38], six C57Bl/6J mice were food-deprived for 18 hrs. At the end of the fasting period, animals received either Rapatar (0.5 mg/kg) or PBS via gavage and 15 minutes later were allowed access to food. "
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