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

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

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Free PMC article

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

Jennifer Jasmin Schwarz et al. Mol Cell Proteomics. .
Free PMC article

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.

Figures

Fig. 1.
Fig. 1.
Experimental setup to study the mTORC1- and insulin/amino acid-dependent phosphoproteome. A, Inducible shRaptor HeLa cells were differentially labeled by SILAC. Raptor knockdown was induced with doxycycline, as indicated. All cells were starved for serum and amino acids (aa) for 16 h. Cells were stimulated for 30 min with aa and insulin, as indicated. Lysates were mixed equally and tryptic digests were subjected to SCX fractionation. Twenty-eight fractions per biological replicate were collected and a small aliquot of each fraction was analyzed by LC/MS. The remaining samples were subjected to titanium dioxide (TiO2)-based enrichment of phosphopeptides and analyzed by LC/MS. Data of four biological replicates were quantitatively analyzed using MaxQuant. B, Immunoblot analysis was performed for all samples subjected to MS analysis. To control for insulin+aa stimulation, the mTORC2 readouts Akt-pS473 and mTOR-pS2481, as well as the mTORC1 readout S6K-pT389 were detected. shRaptor induction was confirmed by raptor detection.
Fig. 2.
Fig. 2.
The phosphoproteome after insulin and amino acid stimulation with and without raptor knockdown. A, Numbers of identified and quantified (phospho-)proteins and (phospho-)peptides obtained by SILAC-MS analyses of four independent biological replicates. The number of phosphopeptides was calculated based on the number of MaxQuant peptide IDs in the Phospho(STY)sites table. B, Localization probabilities calculated for all 4,099 phosphorylation sites reported by MaxQuant. Only sites with a localization probability of ≥ 0.75 were considered localized. C, D, Mean log10 ratios of phosphopeptides in starved versus restimulated samples and in restimulated cells with and without raptor knockdown plotted against negative log10 p values (Student's t test). Phosphopeptides were considered regulated with a regulation factor of at least 1.5 and a p value below 0.05 for the insulin- and aa-dependent phosphoproteome (gray sector). For raptor-dependent changes of protein phosphorylation levels, a minimum fold change of 1.3 (yellow and gray sector) or 1.5 (gray sector) with a p value < 0.05 was applied to define two sets of candidates.
Fig. 3.
Fig. 3.
The mTOR interactome. A, Experimental setup of the mTOR interactome analysis. HeLa cells were differentially labeled with SILAC amino acids and subjected to mock or mTOR immunopurification (IP) experiments. Eluates were mixed in equal ratios and proteins were separated by SDS-PAGE followed by in-gel digestion with trypsin and LC/MS analysis. Data obtained from three biological replicates including a SILAC label switch were analyzed using MaxQuant. B, Immunoblot analysis of samples obtained from mock and mTOR IPs as depicted in A. The mTOR IP yielded intact mTOR complexes as shown by co-immunoprecipitation of the known interaction partners raptor, rictor, and PRAS40. C, Mean log10 ratios of proteins detected in mTOR versus mock IP experiments and quantified in at least two out of three biological replicates were plotted against the negative log10 p value (Student′s t test). Proteins were considered significantly enriched with a mean ratio > 5 and a p value < 0.01 (sector highlighted in gray). Core components of mTOR complex 1 and 2 are marked in orange (mTOR, raptor, rictor, mLST8, and mSIN1). Proteins that were also found regulated in our phosphoproteome analysis are marked in blue. Raptor was found regulated in both analyses (marked in blue +orange) D, Overlap of proteins enriched with mTOR, and with changes in phosphorylation levels following restimulation and raptor knockdown. Proteins marked in light blue are sensitive to amino acid and insulin treatment, whereas proteins depicted in orange are responsive to raptor knockdown. E, Gene Ontology (GO) enrichment analysis of proteins enriched with mTOR for the three main GO domains cellular compartment, molecular function, and biological process. The length of the bar represents the log10 Benjamini-Hochberg corrected p value. The numbers represent the percentage of associated genes for each term.
Fig. 4.
Fig. 4.
Acinus L is directly phosphorylated by mTOR at S240/S243. A, B, Immunoblots of the mTOR kinase assay. To purify mTORC1 kinase, raptor IPs were performed. GST-tagged acinus NT-myc-FLAG served as substrate, and 4EBP1, known to be phosphorylated by mTOR on T37 and T46, served as a positive control. To test the specificity of the kinase reaction, the mTOR inhibitor Torin 1 was used in two different concentrations as indicated. The mock IP served as an additional negative control. C, Relative intensities of the monophosphorylated peptide LSEGSQPAEEEEDQETPSR of acinus L for all indicated conditions in two independent replicates. The dashed line indicates the cut-off for background phosphorylation as determined by the mock IP with acinus NT-myc-FLAG. D, E, Annotated MS/MS spectra of the monophosphorylated peptide LSEGSQPAEEEEDQETPSR of acinus L with localization of the phosphate moiety to S240 and S243, respectively. The zoom-in shown in D highlights the presence of the y16 ion (m/z 1788.91) specific for pS240, which is absent in E. *b, *y: fragment ions with neutral loss of H3PO4 (97.9768 u).
Fig. 5.
Fig. 5.
Acinus L is a novel mTOR interactor. A, Immunoblot analysis of mTOR and mock immunopurifications (IPs). The mTOR IP yielded intact mTOR complex 1 as shown by copurification of raptor. Acinus L specifically copurified with mTOR. B, Overview of the acinus constructs used in C. The FLAG-tag is shown in orange, the myc-tag in blue. C, Immunblot analysis of IPs with anti-FLAG antibody. The immunblots show an interaction of mTOR and raptor with acinus L and acinus NT-myc-FLAG. Acinus L was detected with an antibody against the N terminus of acinus L and the FLAG epitope, respectively.

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