mTORC1 phosphorylation sites encode their sensitivity to starvation and rapamycin

Abstract

The mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) protein kinase promotes growth and is the target of rapamycin, a clinically useful drug that also prolongs life span in model organisms. A persistent mystery is why the phosphorylation of many bona fide mTORC1 substrates is resistant to rapamycin. We find that the in vitro kinase activity of mTORC1 toward peptides encompassing established phosphorylation sites varies widely and correlates strongly with the resistance of the sites to rapamycin, as well as to nutrient and growth factor starvation within cells. Slight modifications of the sites were sufficient to alter mTORC1 activity toward them in vitro and to cause concomitant changes within cells in their sensitivity to rapamycin and starvation. Thus, the intrinsic capacity of a phosphorylation site to serve as an mTORC1 substrate, a property we call substrate quality, is a major determinant of its sensitivity to modulators of the pathway. Our results reveal a mechanism through which mTORC1 effectors can respond differentially to the same signals.

Figure 1. Rapamycin differentially inhibits mTORC1 phosphorylation sites

(A) Responses of known mTORC1 phosphorylation sites in HEK-293E cells and mouse embryonic fibroblasts (p53^−/− MEFs) to 1 hr treatments with 100 nM rapamycin, 250 nM Torin1, or vehicle control. Cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum (FBS) and antibiotics. Subsequently, cell lysates were analyzed by immunoblotting for the levels and phosphorylation states of the specified proteins. (B) Sequence alignment of known and putative mTORC1 phosphorylation sites. Positions are numbered relative to the central phosphoacceptor serine or threonine and known rapamycin-resistant sites are indicated. (C) Dephosphorylation of mTORC1 phosphorylation sites in response to Torin1. p53^−/− MEFs were treated with 1 μM Torin1, lysed at the indicated time points, and lysates analyzed as in (A). (D) Quantitation by densitometry of immunoblots shown in (A) and (C).

Figure 2. The kinase activity of mTORC1 toward peptides encompassing its phosphorylation sites correlates with their resistance to rapamycin

(A) In vitro kinase activity of mTORC1 toward a set of short synthetic peptides, each containing an established mTORC1 phosphorylation site, was analyzed by autoradiography (representative example shown). Phosphorylation levels of the specified peptides were quantified by densitometry. Data are means ± S.D. (n = 3 to 5). (B) In vitro kinase activity of mTORC1 toward indicated peptide substrates. Results are displayed as the relative kinase activity of mTORC1 for each peptide and the rapamycin sensitivity of each site is indicated. The sequences of all indicated phosphorylation sites are available in Fig. S2. (C) In vitro kinase activity of mTORC1 towards peptides containing indicated LARP1 sites was analyzed as in (A). (D) Rapamycin sensitivity of LARP1 phosphorylation sites in cells. FLAG immunoprecipitates from HEK-293E cells stably expressing FLAG-LARP1 and treated with 100 nM rapamycin, 250 nM Torin1 or vehicle control for 1 hr were analyzed by mass spectrometry and phosphorylation ratios determined from chromatographic peak intensities. Data are means ± S.D. (n = 3). *P < 0.05 for differences between treated and non-treated conditions (Mann-Whitney t test). (E) Effects of rapamycin-FKBP12 on mTORC1 activity towards peptide substrates. Experiment was performed and analyzed as in (A), in the presence of recombinant FKBP12 or the complex of FKBP12 and 100 nM rapamycin, using purified mTORC1 (bottom panel). Activity normalized to mTORC1 treated with FKBP12 alone for individual peptide substrates (top panel). (F) In vitro kinase activity of the truncation mutant of mTOR towards peptide substrates. Experiment was performed and analyzed as in (A) using the mTOR truncation mutant (amino acid positions 1295–2549) in a complex with mLST8. (G) Effects of rapamycin-FKBP12 on the activity of truncated mTOR towards peptide substrates. Experiment was performed and analyzed as in (E) using the mTOR truncation mutant. (H) Binding of the peptide substrates to the mTOR kinase domain in the absence and presence of FKBP12-rapamcyin. A pull-down assay using streptavidin agarose was performed from the mixture of biotinylated peptides encompassing established mTORC1 phosphorylation sites and the FLAG-tagged mTOR truncation mutant in the presence of AMP-PNP and analyzed by immunoblotting for the FLAG tag. For pull-down assays with FKBP12-rapamycin, 100 nM rapamycin was preincubated with 50 ng FKBP12 for 30 min and added to the assay mixtures. (I) Steady-state kinetic measurements of mTORC1 kinase activity towards individual peptides encompassing mTORC1 phosphorylation sites. K_m and k_cat values of mTORC1 activity towards indicated peptide substrates were determined by measuring the rate of mTORC1 phosphorylation over a range of peptide substrate concentrations (0, 10, 100, 250, 500 and 1000 μM) at a non-limiting ATP concentration, 500 μM. For kinetic measurements in the presence of FKBP12-rapamycin, 100 nM rapamycin was preincubated with 50 ng FKBP12 for 30 min and added to reaction mixtures. The steady-state kinetic parameters were obtained by fitting the reaction rates to the Michaelis-Menten equation. *Note: K_m exceeds the highest concentration tested for the S6K1 T389 peptide.

Figure 3. Conservative modifications to mTORC1 phosphorylation sites are sufficient to alter their sensitivity to rapamycin within cells

(A) In vitro kinase activity of mTORC1 towards peptides containing indicated modifications to the Grb10 S150 site were analyzed by autoradiography (representative example shown). Phosphorylation levels of the specified peptides were quantified by densitometry. Data are means ± S.D. (n = 3 to 5). (*P < 0.05) (B) In vitro kinase activity of mTORC1 toward peptides encompassing the hydrophobic motif phosphorylation sites of indicated kinases was analyzed by autoradiography as in (A). Data are means ± S.D. (*P < 0.05) (C) Time-dependent responses of wild-type and mutant T389S S6K1 to rapamycin. S6K1^−/−S6K2^−/− MEFs stably expressing wild-type or T389S S6K1 were treated with 5 nM rapamycin up to 2 hr. Cell lysates were analyzed by immunoblotting for the levels and phosphorylation states of the specified proteins. Phosphorylation levels of the specified proteins were quantified by densitometry (graphs). Experiment was performed at least three times and a representative example is shown. (D) Concentration-dependent responses of wild-type and mutant S6K1 to rapamycin. S6K1^−/−S6K2^−/− MEFs stably expressing FLAG-tagged wild-type or T389S S6K1 were treated with increasing concentrations of rapamycin for 20 min and analyzed as in (C). (E) Effects of rapamycin on in vitro kinase activities of wild-type and mutant T389S S6K1. HEK-293T cells expressing FLAG-tagged wild-type or T389S S6K1 were treated with 50 nM rapamycin or vehicle for 15 min and the recombinant protein was purified from lysates using FLAG M2 agarose. Subsequently, in vitro kinase activity of wild-type or T389S S6K1 toward a S6 peptide containing the T235/236 phosphorylation sites was analyzed by autoradiography and quantified by densitometry. Data are means ± S.D. (n = 3). (*P < 0.05) (F) S6K1^−/−S6K2^−/− MEFs stably expressing wild-type or T389S S6K1 were treated with 1 μM Torin1 for indicated time points. Cell lysates were analyzed as in (C) (representative example shown). Data are means ± S.D. (n = 3). (G) In vitro kinase activity of mTOR towards peptides containing indicated modifications to the ULK1 S758 site were analyzed as in (A). The high level of activity of mTORC1 towards the wild-type ULK1 peptide reflects the fact that it contains more than one site phosphorylated by mTORC1. Data are means ± S.D. (n = 3). (*P < 0.05) (H) Time-dependent responses of wild-type and mutant S758T ULK1 to rapamycin. Experiment was performed and analyzed as in (C) with ULK1^−/− MEFs stably expressing an shRNA against endogenous ULK2 as well as FLAG-tagged wild-type or S758T ULK1. (I) Concentration-dependent responses of wild-type and mutant S758T ULK1 to rapamycin. Experiment was performed and analyzed as in (D) for 2 hr with ULK1^−/−ULK2^−/− MEFs stably FLAG-tagged wild-type or S758T ULK1. Note: For all peptide sequences, phosphoacceptor sites are in red text and modified residues in yellow highlight.

Figure 4. The sequence composition of mTORC1 phosphorylation sites encodes their sensitivity to physiological signals that regulate mTORC1

(A) Differential responses of established mTORC1 phosphorylation sites to partial amino acid or serum starvation. p53^−/− MEFs were placed in media with 100, 20, or 0% of the normal levels of amino acids or 10, 2, or 0% FBS for 30 min. Cell lysates were analyzed by immunoblotting for the levels and phosphorylation states of the specified proteins. (B) Differential responses of established mTORC1 phosphorylation sites to complete amino acid starvation. p53^−/− MEFs were placed in 0% amino acid media for the indicated time points. Cell lysates were analyzed as in (A). Phosphorylation levels of the specified proteins were quantified by densitometry (graph). (C) Differential concentration-dependent responses of wild-type and mutant T389S S6K1 to partial amino acid starvation. S6K1^−/−S6K2^−/− MEFs stably expressing FLAG-tagged wild-type or T389S S6K1 were placed in media with 100, 50, 20, 10, 5, or 0% of the normal levels of amino acids for 20 min. Cell lysates were analyzed as in (A). Phosphorylation levels of the specified proteins were quantified by densitometry (graph). (D) Differential concentration-dependent responses of wild-type and mutant S758T ULK1 to partial amino acid starvation. Experiment was performed and analyzed as in (C) for 2 hr under partial amino acid starvation with ULK1^−/−ULK2^−/− MEFs stably expressing FLAG-tagged wild-type or S758T ULK1. (E) A conservative change to the mTORC1 phosphorylation site S6K1 T389 is sufficient to alter the proliferation rate of cells cultured under partial amino acid starvation. S6K1^−/−S6K2^−/− MEFs stably expressing barcoded wild-type and T389S S6K1 were mixed in equal number and cultured in either 100% amino acid RPMI with 10% FBS and antibiotics or 20% amino acid RPMI with 10% dialyzed FBS and antibiotics. After 32 population doublings, cells were harvested and genomic DNA was isolated and analyzed by quantitative real-time PCR. (F) Model for the role of substrate quality in the regulation of mTORC1 phosphorylation sites. Substrate quality is an important determinant of how mTORC1 substrates respond to pharmacological and natural regulators of the kinase.