The mTOR-regulated phosphoproteome reveals a mechanism of mTORC1-mediated inhibition of growth factor signaling

Abstract

The mammalian target of rapamycin (mTOR) protein kinase is a master growth promoter that nucleates two complexes, mTORC1 and mTORC2. Despite the diverse processes controlled by mTOR, few substrates are known. We defined the mTOR-regulated phosphoproteome by quantitative mass spectrometry and characterized the primary sequence motif specificity of mTOR using positional scanning peptide libraries. We found that the phosphorylation response to insulin is largely mTOR dependent and that mTOR exhibits a unique preference for proline, hydrophobic, and aromatic residues at the +1 position. The adaptor protein Grb10 was identified as an mTORC1 substrate that mediates the inhibition of phosphoinositide 3-kinase typical of cells lacking tuberous sclerosis complex 2 (TSC2), a tumor suppressor and negative regulator of mTORC1. Our work clarifies how mTORC1 inhibits growth factor signaling and opens new areas of investigation in mTOR biology.

Fig. 1. Identification of the mTOR-regulated phosphoproteome

(A) Phosphopeptide abundances were determined from two sets of samples: HEK-293E cells serum starved for 4 hrs, treated with 100 nM rapamycin, 250 nM Torin1, or vehicle control for 1 hr, and then stimulated with 150 nM insulin for 20 min and TSC2^+/+ and TSC2^−/− MEFs treated with 100 nM Torin1 or vehicle control for 1 hr. (B and C) Distributions of robust z-scores (median absolute deviations (MADs) away from the median (B) log₂(Torin1/Insulin) for HEK-293Es or (C) log₂(TSC2^−/− Torin1/TSC2^−/− vehicle) for MEFs). p-values associated with enrichment for known mTOR-modulated sites among the −2.5 MAD Torin1-sensitive phosphopeptides were determined by Fisher’s exact test. Phosphopeptides detected in both replicates had to meet the −2.5 MAD threshold both times to be considered mTOR-regulated. (D, E, and F) Correspondence between (D) Torin1 treatment and serum deprivation in HEK-293Es, (E) Torin1 and rapamycin treatment in HEK-293Es, and (F) Torin1 treatment and upregulation in TSC2^−/− MEFs. The relevant robust z-scores for both replicates, phosphopeptides corresponding to known mTOR-modulated sites, Spearman’s rank correlation coefficient (ρ), and associated p-values are indicated. Outliers were excluded to aid in visualization.

Fig. 2. Characterization of a consensus mTOR phosphorylation motif

(A) The position-specific scoring matrix (PSSM) resulting from quantification of the in vitro phosphorylation of a position scoring peptide library (PSPL) by mTORC1. (B) The visualized mTOR consensus motif. Letter height is proportional to the PSSM score. Only those selected residues with scores greater than a standard deviation from the average PSSM score within a row are shown. (C and D) Classification of the mTOR-regulated phosphopeptides in (C) HEK-293E and (D) MEFs organized by rapamycin sensitivity (−2.5 MAD (log₂ (Rapamycin/Insulin)) or TSC2 upregulation (+2.5 MAD log₂(TSC2^−/− vehicle/TSC2^+/+ vehicle)), consistency with the mTOR motif (5^th percentile by Scansite), or presence of an AGC motif ((R/K)X(R/K)XX(S*/T*)). The numbers represent the number of unique phosphopeptides or proteins. Refer to Figs. S5, S6 and Table S4 for more details.

Fig. 3. Grb10 as an mTORC1 substrate with rapamycin-sensitive and -insensitive sites

(A) HEK-293E cells were deprived of serum for 4 hrs, treated with 100 nM rapamycin or 250 nM Torin1 for 1 hr, and then stimulated with 150 nM insulin for 15 min. Cell lysates were analyzed by immunoblotting. (B) TSC2^+/+ MEFs stably expressing FLAG-Grb10 were serum deprived for 4 hours, treated with 250 nM Torin1 for 1 hr, and then stimulated with 150 nM insulin for 15 min. All FLAG-tagged Grb10 constructs correspond to isoform c of human Grb10. FLAG-immunoprecipitates were incubated in buffer, CIP, or heat-inactivated CIP and analyzed by immunoblotting. (C) HEK-293E cells were deprived of amino acids or both amino acids and serum for 50 min, and then stimulated with either amino acids or serum for 10 min and analyzed by immunoblotting. (D) TSC2^+/+ and TSC2^−/− MEFs were treated and analyzed as in (A). (E) mTORC1 in vitro kinase assays with substrates in the presence of the indicated inhibitors and radiolabeled ATP were analyzed by autoradiography. (F) Schematic representation of Grb10 protein structure with the phosphorylation sites from vertebrate orthologs aligned below. Numbering is according to human isoform a. (G) The phosphorylation state of Grb10 from kinase assays performed similarly to (E) were analyzed by targeted mass spectrometry (MS) and phosphorylation ratios determined from chromatographic peak intensities. (H) FLAG-immunoprecipitates from HEK-293E cells stably expressing FLAG-Grb10 treated as in (A) were analyzed as in (G). Data are means ± s.e.m (n=2–6). *Mann-Whitney t-test p-values < 0.05 for differences between stimulated and treated conditions. (I) A summary of (F), (G), and (H) for each Grb10 phosphorylation site. (J) FLAG-immunoprecipitates from TSC2^−/− MEFs stably expressing FLAG-Grb10 treated with 100 nM rapamycin or 250 nM Torin1 for 1 hr were analyzed by immunoblotting with Grb10 phospho-specific antibodies. (K) TSC2^−/− MEFs stably expressing FLAG-Grb10, 5A (S150A T155A S158A S474A S476A), or 9A (5A + S104A S426A S428A S431A) mutants treated with 250 nM Torin1 for 1 hr were analyzed by immunoblotting.

Fig. 4. mTORC1 inhibits PI3K-Akt signaling by regulating Grb10 function and stability

(A) S6K1^−/− S6K2^−/− or control cells expressing short hairpin RNA (shRNA) constructs against GFP or raptor were treated with 250 nM Torin1 for 1 hr, and lysates were analyzed by immunoblotting. (B) TSC2^−/− MEFs expressing shRNAs against GFP or Grb10 were deprived of serum for 4 hrs and then stimulated with 100 nM insulin for 15 min as indicated and analyzed by immunoblotting. (C) TSC2^−/− MEFs expressing a control shRNA or shRNA against Grb10 were treated as in (B). IRS1 and IRS2 immunoprecipitates and cell lysates were analyzed by immunoblotting. (D) TSC2^−/− MEFs coexpressing an shRNA against the mouse Grb10 3’UTR and an empty vector, FLAG-Grb10, or 5A cDNA expression construct were treated and analyzed as in (B). (E) TSC2^−/− MEFs stably expressing FLAG-Grb10 were labeled for 2 hours with [³⁵S]cysteine and methionine and then chased for the indicated times in the presence of vehicle control, 100 nM rapamycin, or 100 nM Torin1. FLAG-immunoprecipitates were analyzed by autoradiography. Data are means ± s.e.m (n=3). *Two-way ANOVA p-values < 0.05 for differences between vehicle and inhibitor treatment. (F) TSC2^−/− MEFs stably expressing FLAG-Grb10 or 9A mutant were treated and analyzed as in (E) but without inhibitor treatment. (G) mTORC1 orchestrates feedback inhbition of PI3K-Akt signaling by activating and stabilizing Grb10 while inhibiting and destabilizing IRS proteins.