Abstract

BackgroundOur 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.Methodology/Principal FindingsWe 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, TiO2 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.Conclusions/SignificanceIn 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.

Highlights

  • The mammalian target of rapamycin is a well conserved serine/threonine kinase that plays a key physiological role in the control of cell growth [1]. mTOR is a component of two distinct multiprotein complexes [2,3,4]. mTOR complex 1 regulates temporal control of cell growth while mTOR complex 2 regulates the organization of the actin cytoskeleton. mTORC1 signaling is sensitive to rapamycin, a macrolide antibiotic and anti-cancer drug, whereas mTORC2 mediates rapamycin-insensitive signaling [5]. mTORC1 signaling is stimulated by nutrients, growth factors, and high levels of cellular energy

  • The constituents of mTORC1 include mTOR, raptor, mLST8/G-protein b-subunit like protein (GbL), Proline-Rich Akt Substrate of 40 kDa (PRAS40), and Deptor [7]. This signaling complex exerts at least some of its biological effects by phosphorylating p70 ribosomal protein S6 kinase (S6K) at a single site, and eukaryotic initiation factor 4E binding protein 1 (4E-BP1) at multiple sites. mTORC1 phosphorylation of critical residues involved in the activation of S6K1 (Thr389) and 4E (eIF4E) binding protein 1 (4E-BP1) (Thr37 and Thr46) are generally sensitive to the inhibitory effects of rapamycin [8,9]

  • Though much research has been devoted to understanding the mTORC1 pathway, the mechanisms underlying many of its biological functions remain poorly understood

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Summary

Introduction

The mammalian target of rapamycin (mTOR) is a well conserved serine/threonine kinase that plays a key physiological role in the control of cell growth [1]. mTOR is a component of two distinct multiprotein complexes [2,3,4]. mTOR complex 1 (mTORC1) regulates temporal control of cell growth while mTOR complex 2 (mTORC2) regulates the organization of the actin cytoskeleton. mTORC1 signaling is sensitive to rapamycin, a macrolide antibiotic and anti-cancer drug, whereas mTORC2 mediates rapamycin-insensitive signaling [5]. mTORC1 signaling is stimulated by nutrients, growth factors, and high levels of cellular energy. MTORC1 signaling is stimulated by nutrients, growth factors, and high levels of cellular energy. The constituents of mTORC1 include mTOR, raptor, mLST8/G-protein b-subunit like protein (GbL), Proline-Rich Akt Substrate of 40 kDa (PRAS40), and Deptor [7]. This signaling complex exerts at least some of its biological effects by phosphorylating p70 ribosomal protein S6 kinase (S6K) at a single site, and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) at multiple sites. 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. The mechanisms involved in many mTORC1 biological functions remain poorly understood

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