Functional Proteomics Identifies Acinus L as a Direct Insulin- and Amino Acid-Dependent Mammalian Target of Rapamycin Complex 1 (mTORC1) Substrate

Mol Cell Proteomics. 2015 Aug;14(8):2042-55. doi: 10.1074/mcp.M114.045807. Epub 2015 Apr 23.

Abstract

The serine/threonine kinase mammalian target of rapamycin (mTOR) governs growth, metabolism, and aging in response to insulin and amino acids (aa), and is often activated in metabolic disorders and cancer. Much is known about the regulatory signaling network that encompasses mTOR, but surprisingly few direct mTOR substrates have been established to date. To tackle this gap in our knowledge, we took advantage of a combined quantitative phosphoproteomic and interactomic strategy. We analyzed the insulin- and aa-responsive phosphoproteome upon inhibition of the mTOR complex 1 (mTORC1) component raptor, and investigated in parallel the interactome of endogenous mTOR. By overlaying these two datasets, we identified acinus L as a potential novel mTORC1 target. We confirmed acinus L as a direct mTORC1 substrate by co-immunoprecipitation and MS-enhanced kinase assays. Our study delineates a triple proteomics strategy of combined phosphoproteomics, interactomics, and MS-enhanced kinase assays for the de novo-identification of mTOR network components, and provides a rich source of potential novel mTOR interactors and targets for future investigation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / metabolism
  • Amino Acid Sequence
  • Amino Acids / metabolism*
  • Gene Knockdown Techniques
  • HeLa Cells
  • Humans
  • Insulin / metabolism*
  • Mechanistic Target of Rapamycin Complex 1
  • Molecular Sequence Data
  • Multiprotein Complexes / metabolism*
  • Nuclear Proteins / chemistry
  • Nuclear Proteins / metabolism*
  • Phosphoproteins / metabolism
  • Phosphorylation
  • Protein Interaction Mapping
  • Proteome / metabolism
  • Proteomics / methods*
  • Regulatory-Associated Protein of mTOR
  • Substrate Specificity
  • TOR Serine-Threonine Kinases / metabolism*

Substances

  • ACIN1 protein, human
  • Adaptor Proteins, Signal Transducing
  • Amino Acids
  • Insulin
  • Multiprotein Complexes
  • Nuclear Proteins
  • Phosphoproteins
  • Proteome
  • RPTOR protein, human
  • Regulatory-Associated Protein of mTOR
  • TOR Serine-Threonine Kinases
  • Mechanistic Target of Rapamycin Complex 1